oligomerization of ABCG2 in membrane extracts from tumour cells and human ...... bypass the ABC transporter mediated efflux, thereby âevadingâ the drug resistance .... concentrations in tissues to have a marked result on patient survival and ...
CHARACTERIZATION OF ABC TRANSPORTERS IN BOTH MAMMALIAN CELLS (ABCG2, ABCC2) AND PLASMODIUM FALCIPARUM (Pgh1)
Mara L. Leimanis
Institute of Parasitology McGill University, Montreal, Canada
A thesis submitted to McGill University in the partial fulfillment of the requirements of the degree of Doctor of Philosophy
© Mara L. Leimanis March 2008
This thesis is dedicated to my immediate family, Andris, Inara, Daina, and Karina, who all patiently endured the completion of this thesis.
ii
Thesis Abstract In the first part of the thesis two ABC transporters in mammalian cells are explored. Initially, the expression of members of the ABC family of transporters in erythrocytes was measured. It was found that ABCG2 (also known as the breast cancer resistance protein, BCRP, the mitoxantrone resistance protein, and ABC placenta) was expressed in mature human erythrocytes.
This work concentrated on characterizing the
oligomerization of ABCG2 in membrane extracts from tumour cells and human erythrocytes. Given the ability of ABCG2 to transport protoporphyrin IX or heme, these findings may shed some light on the normal function of erythrocytes. The second chapter of the thesis attempts to elucidate the drug binding characteristics of ABCC2 (MRP2). A radiolabelled photoreactive analogue of LTC4 (IAALTC4) was synthesized and used to carry out photoaffinity labelling experiments; a technique used to predict drug binding to a target protein. LTC4 is an endogenous substrate of ABCC2 since previous reports have shown LTC4 transport by ABCC2. Our binding studies revealed specific photoaffinity labelling of IAALTC4 to ABCC2 transfected cells. This work shows for the first time the direct binding of LTC4 to ABCC2, and further expands on the current biochemical knowledge of ABCC2. The long-standing drug of choice to treat malaria, chloroquine (CQ), is no longer effective due to increasing drug resistance. The lack of both new drug development and a clear understanding of the mechanism(s) of drug resistance have made achieving the global initiative to halve the malaria burden by the year 2010 more problematic; this aim now requires alternative methods of treatment to CQ. Therefore, the second main objective of this thesis was to address the growing problem of malaria drug resistance by exploring alternative therapies and a potential modulator of CQ resistance. In the third chapter, modulators of MRP1-mediated resistance were explored in chloroquine-sensitive and -resistant parasites. Multidrug resistance protein 1 (MRP1, ABCC1) is an ATP-binding cassette (ABC) transporter in the MRP family of transporters, and has been implicated in a multidrug resistance (MDR) phenotype in cancerous mammalian cell lines. In an attempt to explore ABCC1-like proteins in iii
parasites, we exposed both CQ-sensitive and -resistant Plasmodium falciparum (P. falciparum) strains to several well-characterized ABCC1-specific inhibitors. MK571 (an LTD4 receptor antagonist) alone was found to be more toxic to the CQ-resistant strain (FCR-3) than to the CQ-sensitive strain (3D7). Lastly, novel candidate compounds were screened for antimalarial activity.
We
demonstrated for the first time the efficacy of novel 1,2,4-trioxolane derivatives and their antimalarial efficacy in vitro. These novel compounds were synthesized with a greater cost effectiveness than previously characterized 1,2,4-trioxolane derivates. Of 11 compounds tested against both CQ-sensitive (3D7) and CQ-resistant (FCR-3) strains of P. falciparum, two candidate leads (IC50 in low µM) were identified. In summary, the work presented in this thesis involved drug discovery, biochemical characterization of proteins (ABCG2, ABCC2, Pgh1), and the examination of modulators of drug resistance. Primarily, our current findings build on a growing body of knowledge in alternative treatments for CQ-resistant malaria. Secondly, we explored the oligomerization of an erythrocyte membrane protein, ABCG2 that may imply a role in the normal function of the cell.
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Résumé Dans la première partie de la thèse, deux transporteurs ABC ont été explorés. Initialement, nous avons fait l’analyse des protéines ABC (ATP-binding cassette) dans le globules rouges, en examinant leur niveau d’expression au niveau de leur membrane. Nous avons observé que ABCG2 aussi appelé : “breast cancer resistant protein,-BCRP”, “mitoxantrone resistant protein”, et “ABC placental”, était exprimé dans les globules rouges matures. Ce travaille s’est concentré sur la caractérisation de l’oligomérization de ABCG2 dans la membrane des cellules cancéreuses et dans les globules rouges humaines. Nous savons déjà que ABCG2 transporte la protoporphyrin IX ou hème, alors nous souhaitons que ces résultats ajoutent à la connaissance de la fonction normale des globules rouges. Dans le deuxième chapitre, nous avons exploré les caractéristiques de liaison de ABCC2 (MRP2) avec les substrats. En utilisant un analogue photoréactif de LTC4 (IAALTC4) marqué pour faire des études de photoaffinités, une technique fut utilisée pour prédire la liaison d’un composé sur une protéine. La LTC4 est un substrat naturel (endogène) de ABCC2 et des résultats établis antérieurement ont montré le transport de LTC4 par ABCC2. Nos études de liaisons en photoaffinité demontrent spécifiquement que IAALTC4 trace les cellules transfectées avec ABCC2. Ces résultats montrent pour la première fois, la liaison de LTC4 à ABCC2, tout en nous apportant plus d’information biochimique sur ABCC2. La Chloroquine (CQ) est l’agent chimiothérapeutique le plus rèpandu pour combattre et traiter la malaria; toutefois son efficacité est en constante décroissance due à une résistance du parasite rependue mondialement. Les organismes mondiaux de santé préconisent l’utilisation de type de traitements visant une réduction de 50% du paludisme pour l’année 2010.
Toutefois, le manque de nouvelles molécules et
l’absence d’une compréhension claire des mécanismes de résistance nuisent à l’atteinte de ce but. L’objectif de cette deuxième partie de la thèse est d’essayer de trouver des nouvelles thérapies pour l’énorme problème de paludisme tout en
explorant un
potentiel modulateur de résistance à la CQ. v
Troisièmement, nous avons étudié un des modulateurs de résistance contre des composés quinoline chez les parasites. La protéine “Multidrug resistance associated protein 1 (MRP1, ABCC1)” est un des transporteurs “ATP-binding cassette (ABC)” et est associé avec le phénotype de résistance multiple aux drogues (MDR) chez les cellules cancéreuses des mammifères. Dans un effort d’explorer les protéines “ABCC1like” chez le parasite, nous avons exposé des souches CQ-sensibles et résistantes de P. falciparum à plusieurs inhibiteurs de ABCC1 bien connues. Nos résultats ont démontré que l’inhibiteur de ABCC1, MK571 (un antagoniste du récepteur de LTD4), avait plus d’activité anti-paludique contre les parasites CQ-résistants (FCR-3) qu’avec les CQsensibles (3D7). Le dernier objectif était d’examiner l’activité anti-paludique de nouvelles drogues. Nous avons démontré pour la première fois, en utilisant des essaies in vitro, que de nouveaux dérivés du 1,2,4-trioxolane possédaient une activité anti-paludique.
Les
nouveaux composés ont été synthétisés de façon plus économique que les dérivées des anciennes générations. Des 11 composés analysés pour leur toxicité sur des souches CQ-sensibles (3D7) et CQ-résistants (FCR-3) de Plasmodium falciparum (P. falciparum) et deux composés candidats (IC50 dans les bas µM) ont été trouvés. En résumé, le travail présenté dans cette thèse inclus la découverte de nouveaux composés, la caractérisation biochimique des protéines (ABCG2, ABCC2, Pgh1), et l’exploration de modulateurs de résistance aux drogues. Nos résultats décrivent le potentiel de nouveaux traitements contre le parasite de la malaria résistant à la CQ. Deuxièmement, la characterisation d’une protéine ABC dans les globules rouges peut impliquer qu’elle joue un rôle dans la fonction des globules rouges.
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Table of Contents TITLE PAGE
i
DEDICATION
ii
ABSTRACT
iii
RÉSUMÉ
v
TABLE OF CONTENTS
vii
ACKNOWLEDGMENTS
xii
CONTRIBUTION OF AUTHORS
xii
STATEMENT OF ORIGINALITY
xiii
STATEMENT FROM THESIS OFFICE
xv
LIST OF FIGURES
xvi
LIST OF TABLES
xviii
LIST OF COMMONLY USED ABBREVIATIONS
xix
INTRODUCTION
1
REFERENCES
4
Chapter 1-Literature Review
6
Part I: ABC Transporters (ABCG2, ABCC2) in Mammalian Cells
7
ATP Binding Cassette (ABC) Transporters
7
Diversity of ABC Transporters
8
Structure of ABC Transporters
8
Multidrug Resistance Transporters in Cancer
11
P-glycoprotein (Pgp1, ABCB1)
13
Multidrug Resistance Protein 1 (MRP1, ABCC1)
14
MRP Family of Transporters
16
ABCG2 (BCRP, MXR, ABCP)
17
ABCG2 as a Homodimer
18
ABCG2 in Maintenance of Cellular Homeostasis
20
ABCG2: Substrate Specificity, Role in MDR, Inhibition of ABCG2
20 vii
ABCC2
22
ABCG2: Substrate Specificity, Role in MDR, Inhibition of ABCC2
22
Photoaffinity Labelling: Techniques and Significance
23
Clinical Multidrug Resistance
24
Part II: Drug Resistance in Plasmodium falciparum
26
Malaria Infection in Humans
26
Malaria Biology
27
Asexual Development: Malarial Invasion and Hemoglobin Degradation 30 Malaria Pathology, Diagnosis and Immunity
31
Malaria Treatment, Chemoprophylaxis and Clinical Complications
33
Antimalarial Drugs Class I: Quinoline-based Drugs
34
Antimalarial Drugs Class II, III: Atovaquone/Proguanil, Primaquine
35
Artemisinin and Derivatives
36
Chloroquine Drug Resistance in Malaria
38
Multidrug Resistance
39
Similarities Between MDR in Mammalian Cells and in P. falciparum
41
The Mechanisms of Multidrug Resistance in P. falciparum
42
ABC Transporter Genes in Plasmodium falciparum
43
Additional Transporter Genes Involved with CQR: pfcrt
45
Recent Developments and Future Outlook for Malaria Control
46
References
49
Chapter 2- ABCG2 Membrane Transporter in Mature Human Erythrocytes is Exclusively Homodimer
71
Abstract
72
Introduction
73
Materials and Methods
75
Results & Discussion
77
Acknowledgments
84 viii
References
85
CONNECTING STATEMENT 1
88
Chapter 3- Characterization of LTC4 Binding to ABCC2 Membrane Transporter
89
Abstract
90
Introduction
91
Materials and Methods
93
Results
96
Discussion
104
Acknowledgments
106
References
107
CONNECTING STATEMENT 2
111
Chapter 4-Chloroquine-resistant Plasmodium falciparum Are Hypersensitive to MK571, a leukotriene LTD4 receptor antagonist
112
Abstract
113
Introduction
114
Materials and Methods
116
Results & Discussion
118
Acknowledgments
124
References
125
CONNECTING STATEMENT 3
130
Chapter 5- Preliminary Synthesis and In Vitro Characterization of Novel Ozonides (trioxolanes) as Antimalarial Drugs
131
Abstract
132
Introduction
133
Materials and Methods
135
Results & Discussion
137
Acknowledgments
141
References
142
CONCLUDING STATEMENT
144 ix
References
148
Appendix I
149
Appendix II
151
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Acknowledgments Firstly, I would like to take a moment to communicate to my parents without whom this thesis would likely not have been written, and who together allowed me to explore my interests and develop myself as an individual, my deepest gratitude. Together, they supported my decision to pursue studies in scientific research; individually, my mother has offered strength and confidence to be on a specific career path, and my father has offered never ending encouragement to remain persistent and resilient in achieving my goals. Secondly, a few words to my two sisters Daina, and Karina, thank-you for your ongoing support and love. Your patience over the years has been vital to me during this process. To Elias, I would like to thank for allowing me to work rather independently and freely, offering advice at seemingly always just the right time. You have been an endless source of ideas and solutions. My time spent in the lab and the opportunities my experience has created will be reflected in the many years to come. To Tim I would like to give a special thanks for seeing me through the writing of my thesis, and for all of your generosity and patience, you truly are one of a kind! To my present and former lab mates Remi, Omar, Roni I would like to thank-you for your friendship, and help in the lab. Especially, to my former lab mate and good friend Dr. Joel Karwatsky, I would like to extend a very great thank-you for not only teaching me “good laboratory practices”, but also for your deep friendship over the years. To the staff the students of the Institute of Parasitology namely Gordie and Shirley thank-you for your support and smiley faces! In other acknowledgements my dear friends Stephanie B, Michelle W, Bradley K, Debbie Z, Janina F, Grietina M, Laila J, David P-B, Mikelis S, Yesim I, Andra L, Maris P, and to others who have influenced me along the way Anne S, Alex K, Sebastian S, Perry C, Naim O, Rya B, Cat I, Aws A-W, Peter L, Eddy S and finally, Jonah K. I would like to express a sincere gratitude for your care over the years. Lastly, to my grandmothers Zelma and Rasma, I would like to thank for your many life lessons. xi
Contribution of Authors The experimental work presented in this thesis was designed and performed by the author, under the supervision of Dr. Elias Georges. In the second manuscript MRP2 (ABCC2) expressing transfectants were provided by Dr. Marcel de Haas from Dr. P. Borst laboratory at the Department of Molecular Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands. Additionally for this paper, the drug AALTC4 was initially synthesized by Dr. Joel Karwatsky, a former student in Dr. Elias George’s lab.
In the fourth manuscript drugs were synthesized by Sabine Thielges, and Oleg
Shirobokov, under the supervision of Dr. George Just and Dr. Nicolas Moitessier in the Department of Chemistry, McGill University, and Montreal, Quebec, Canada.
xii
Statement of Originality The following findings are considered original contributions to the fields of drug resistance in both malaria and cancer research. Manuscript I (Chapter 2): Mara, L. Leimanis and Elias Georges. (2007). ABCG2 Membrane Transporter in Mature Human Erythrocytes is Exclusively a Homodimer. Biochemical and Biophysical Research Communications 354(2):345-50. In the first manuscript, an ABC transporter was explored in mature erythrocytes. ABCG2 has been well characterized to play a role in both drug resistance and to play a protective role for cells under hypoxia. Additionally, ABCG2 has been shown to be expressed in hematopoietic stem cells. It was of interest to detect the presence of ABCG2 in erythrocytes. In the study, we were able to determine the expression of ABCG2 in high abundance relative to MCF7 resistance to mitoxantrone resistant cell line. The 140-kDa dimer was present in high abundance even under denaturing conditions. Further exploration revealed that at higher concentrations of denaturing agent the dimer was reduced to its 70-kDa monomer form. This was the first work demonstrating high abundance of higher oligomeric forms of ABCG2 in human erythrocytes. Manuscript II (Chapter 3):
Mara L. Leimanis, Joel Karwatsky, Elias Georges.
Characterization of LTC4 binding to ABCC2 Membrane Transporter (In preparation). In the second manuscript we studied the photoaffinity binding of IAALTC4 to ABCC2. Previous work in our lab has used this technique extensively for biochemical analysis of protein-drug interactions. We demonstrated the specific and direct interaction of IAALTC4 with ABCC2. Drug binding was inhibited by the potent ABCC1 inhibitor MK571, which has also been shown to inhibit drug efflux by ABCC2. Additionally, we demonstrated, based on digestion mapping studies, that the third transmembrane region xiii
was labelled. Based on previous work with other ABC transporters, and the work presented in this manuscript, we hypothesize that transmembrane domains 16 and 17 are involved at least in part with this drug interaction.
Manuscript III (Chapter 4): Mara L. Leimanis and Elias Georges. Chloroquineresistant Plasmodium falciparum Are Hypersensitive to MK571, a leukotriene LTD4 receptor antagonist (In preparation). In the third manuscript we report that the previously characterized and clinically tested drug MK571 shows toxicity towards chloroquine-resistant parasites. The drug had previously been characterized to be a potent anti-asthmatic agent in humans. It is a specific inhibitor of MRP1 (ABCC1), a well studied ABC transporter ubiquitously expressed in human tissues and associated with multidrug resistance in tumour cell lines. Further exploration revealed that the Pgp homologue in Plasmodium falciparum, Pgh1 may be directly or indirectly involved with this hypersensitivity of MK571 in CQresistant parasites. Manuscript IV (Chapter 5): Mara L. Leimanis, Sabine Thielges, Oleg Shirobokov, George Just, Nicolas Moitessier, Elias Georges. Synthesis and Characterization of Novel Ozonides (trioxolanes) as Antimalarial Drugs, (Unpublished to date). Lastly, we explored the synthesis and characterization of novel antimalarials. Novel drug development in malaria is crucial due to the extensive drug resistance towards chloroquine in the field. The drugs synthesized as described in this manuscript fall under the category of ozonides (or trioxolanes), based on their molecular composition. The study design was based on the similarly structured drug artemisinin, which is currently one of the few drugs available to treat chloroquine-resistant parasites. We found several novel compounds with antimalarial activity in the low µM range. This study provides a framework from which further work to characterize additional drug leads may be conducted.
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Statement from Thesis Office As stated in the "Guidelines for Thesis Preparation" a thesis may be prepared to include a collection of manuscripts whereby “all components must be integrated into a cohesive unit with a logical progression from one chapter to the next. In order to ensure that the thesis has continuity, connecting texts that provide logical bridges preceding and following each manuscript are mandatory.” In addition the thesis must include: “1-a table of contents; 2-a brief abstract in both English and French; 3-an introduction which clearly states the rational and objectives of the research; 4-a comprehensive review of the literature (in addition to that covered in the introduction to each paper); 5-a final conclusion and summary; 6-a thorough bibliography;
7-Appendix containing an ethics certificate in the case of research
involving human or animal subjects, microorganisms, living cells, other biohazards and/or radioactive material.”
Any “additional material must be provided (e.g., in
appendices) in sufficient detail to allow a clear and precise judgment to be made of the importance and originality of the research reported in the thesis.” In the event there are co-authored papers “an explicit statement in the thesis” is required “as to who contributed to such work and to what extent.” This is stated in a separate section titled "Contributions of Authors", and this should appear “as a preface to the thesis.” “The supervisor must attest to the accuracy of this statement at the doctoral oral defence.” This creates difficulty to any examiners and therefore “it is in the candidate's interest to clearly specify the responsibilities of all the authors of the coauthored papers.”
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List of Figures Chapter 1
Page
Figure 1: Structural diversity of ABC transporters. Where TM domain typically contains six predicted membrane-spanning helices. Color codes are used to show the lack of similarity between structural categories and families (as defined by sequence similarity).
9
Figure 2: ATP-switch mechanism of ABC transporters.
11
Figure 3: The topology of ABCC1-like and ABCC4-like ABC Transporters.
15
Figure 4: Schematic illustration of inter- and intramolecular disulfide bonds assumed in ABCG2 (WT) proteins. The disulfide bond at Cys-603 may link two ABCG2 proteins to form a homodimer. Cys-592 and Cys-608 are considered to form an intramolecular disulfide bond.
19
Figure 5: Life cycle of human malaria parasites.
28
Figure 6: Drug resistance in P. falciparum from studies in sentinel sites (up to 2004).
40
Figure 7: (A) MDR in P. falciparum is believed to be multifactorial, this model also incorporates the potential role of drug transporters and CQR. (B) Predicted structure and polymorphisms in P. falciparum multidrug resistance-1. Pgh1 (PfMDR1) has two homologous halves, each with six predicted TM domains and a NBD.
44
Chapter 2 Figure 1: Detection of ABCG2 homodimer in mature erythrocytes. MCF7/Mitox (drug resistant) and mature erythrocytes by immunoblot and Coomassie blue staining using the Fairbanks gel system .
78
Figure 2: Expression and analysis of ABCG2 in erythrocytes from different ethnic groups and blood types by immunoblot.
80
Figure 3: The effects of increasing concentrations of DTT on homodimer/monomer pools of ABCG2 in MCF7/Mitox and erythrocyte membrane ghosts as resolved on 7.5% Laemmli polyacrylamide gels.
82
Figure 4: Modulation of Pheophorbide A (PheA) transport in drug
83 xvi
sensitive and Mitox resistant MCF7 tumour cells and erythrocytes in the presence or absence of inhibitor.
Chapter 3 Figure 1: Chemical structure of IAALTC4.
96
Figure 2: Photolabelling of MDCKII parental and MDCKII/ABCC2 with IAALTC4.
97
Figure 3: Specificity of IAALTC4 photolabelling as shown using a non-specific antibody for Na+/K+ ATPase.
99
Figure 4: Competition of IAALTC4 binding to ABCC2 with several drugs.
100
Figure 5: Photolabelling of ABCC2 digestion products demonstrating C-terminal labelling, and schematic diagram of potential drug binding domain in the last TM segment of ABCC2.
102
Chapter 4 Figure 1: Organic structure of MK571. Figure 2: Cytotoxicity assays of CQ with CQS 3D7 and CQR W2, FCR-3 strains of Plasmodium falciparum. Figure 3: Cytotoxicity assays of CQ with CQS D10(D10), 7G8K(D10) and CQR 7G8K strains of Plasmodium falciparum.
118 119,120 121
Chapter 5 Figure 1: Reactions at the 1, 4-dioxane yielding compounds 1-13.
138
Figure 2: Structures of synthesized compounds.
139
Appendix 1 Chapter 5 Supplemental Figure 1: Cytotoxicity assays of CQ in CQS (3D7) and CQR (FCR-3) strains of Plasmodium falciparum.
149
Chapter 5 Supplemental Figure 2: Cytotoxicity assays of novel 150 trioxolanes in CQS (3D7) and CQR (FCR-3) strains of Plasmodium falciparum.
xvii
List of Tables Chapter 1
Page
Table 1: Spectrum of clinically useful antimalarial drugs for either prophylaxis or treatment, according to stage-specific activity.
35
Chapter 4 Table 1: In vitro antimalarial activity of compounds chloroquine and MK571 against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum.
123
Chapter 5 Table 1: In vitro antimalarial activity of compounds artemisinin, chloroquine and compounds 3-13 against the chloroquine-sensitive 3D7 and chloroquine-resistant FCR-3 strains of Plasmodium falciparum.
140
xviii
List of Commonly Used Abbreviations ABC
ATP Binding Cassette
ABCG2/BCRP/
ATP Binding Cassette G2/Breast Cancer Resistance Protein/
MXR/ABCP
Mitoxantrone Resistance Protein/ABC Placenta
ATP
Adenine triphosphate
CQ
Chloroquine
CQR
Chloroquine resistant
CQS
Chloroquine sensitive
EE
Exoerythrocytic
FV
Food vacuole
FP
Ferriprotoporphyrin IX
GSH
Glutathione
HIF
Hypoxia-inducible transcription factor
IAALTC4
[125I]iodoaryl azido-leukotriene C4
IE
Intraerythrocytic
LTC4
Leukotriene C4
MDR
Multidrug resistance
MK571
[3-{2-(7-chloro-2-quinolinyl)ethenyl}phenyl][{3(dimethylamino-3-oxopropyl)thio}methyl]thio propanoic acid
MRP1/ABCC1
Multidrug Resistance Protein 1/ATP Binding Cassette C1
MRP2/ABCC2/ cMOAT
Multidrug Resistance Protein 2/ATP Binding Cassette C2/ canalicular Multi-specific Organic Anion Transporter
MSD
Membrane spanning domain
NBD
Nucleotide binding domain
pfmdr1
Plasmodium falciparum multidrug resistance 1
PfCRT
Plasmodium falciparum chloroquine resistance transporter
Pgp1/ABCB1
P-glycoprotein1/ATP Binding Cassette B1
Pgh1
P-glycoprotein homologue 1
TMD
Trans membrane domain xix
Introduction More than half of the world’s population lives in an area where they are at risk of infection from malaria. Malaria is becoming an increasingly important subject of study due to an increase in global traveling, and the development of resistance to insecticides and antimalarial drugs. The majority of deaths due to Plasmodium falciparum (P. falciparum) occurs in African children, and is associated with severe anemia or cerebral malaria. The World Health Organization (WHO) estimates that 1-3 million deaths per annum are due to malaria; 2% of these deaths are attributed to multidrug resistant P. falciparum infections [1]. In order to address the urgent need for novel treatment methods, work towards developing antimalarials is underway.
The main drugs under development are
derivatives from existing families of drugs. The most prominent are, the artemisinins, derived from an ancient family of compounds, and have recently been described as very attractive candidates from which to make derivatives. Specifically, due to activity against blood stage parasites (the asexual cycle and gametocytes), and the lack of widespread clinical resistance, these drugs (trioxolane derivatives) have been increasingly studied. Artemisinin and derivatives such as artesunate, artemether and artheether act as prodrugs, in that the trioxane group must be activated once inside the parasite. Useful derivatives require an inexpensive synthesis method and must maintain stability in order to reach the drug target [2, 3]. One such derivative is in clinical trials, but additional synthetic modifications should be explored with the goal of improving intrinsic activity and stability to hydrolysis. A better understanding of existing mechanisms of resistance towards quinoline-based drugs (including chloroquine (CQ), amodiaquine, quinidine, quinine and mefloquine) may also lead to improvements in treatment.
There is increasing evidence that
resistance to antimalarial drugs in P. falciparum may be mediated in part by overexpression of membrane transport proteins which cause the efflux of drugs in an energy-dependent manner. The role of these drug efflux mechanisms in anticancer drug resistance in tumour cells is now well established and appears to be clinically 1
significant in certain cancers (as reviewed by [4-6]).
Studies to identify and
characterize similar roles for homologous membrane proteins in resistance to antimalarial drugs in P. falciparum have been inconsistent [7, 8]. A recent study by Vezmar et al. [9] determined that multidrug resistance protein 1 (MRP1/ABCC1) mediates the transport of several quinoline-based antimalarial drugs through direct binding [10]. ABCC1 is an ATP-dependent drug efflux pump that is a member of the ABC (ATP Binding Cassette) family of trafficking proteins [11, 12]. ABCC1 causes a multidrug resistance (MDR) phenotype and is believed to be associated with drug resistance in tumour cells [13]. Interestingly, we and others have demonstrated the expression of ABCC1 in membranes from human erythrocytes [14]. This supports the hypothesis that ABC transporters may be involved in the drug resistance to CQ and other quinoline-based drugs, and may contribute to our understanding of drug resistance in P. falciparum.
Further characterization of additional membrane transporters in
human erythrocytes is required. In addition to host erythrocyte membrane ABC transporters, it has also been found that malaria parasites express similar transporters, which may be exploited as drug targets in the parasite. Specifically, an ABC transporter, and Pgp homologue termed, Pgh1 is found on the food vacuole (FV) membrane of drug resistant parasites [7]. The food vacuole (FV) is of particular relevance as it is the site of hemoglobin digestion and CQ action [15]. Extensive work has been done to characterize this transporter, and the phenomenon of drug resistance in parasites [16]. It has however been found, that the mechanism(s) involve much more than simply the modulation of one ABC transporter. Drug resistance (or the multidrug resistance) is both multifactorial (i.e., involves additional transporters present on the FV) and polymorphic, and therefore requires multiple genetic mutations. Despite the discovery of these resistance mechanisms, they have not led to new treatments and new strategies.
Methods to overcome drug
resistance are still urgently needed. Improving our knowledge of drug transport and resistance mechanisms of ABC transporters can be gained through biochemical characterization, which includes efforts to elucidate drug binding characteristics of transporters. Current working models of the 2
mechanism of transport exist for ABC transporters.
This model outlines the
combination of both transmembrane ligand translocation and ATP hydrolysis, during which multiple conformational changes of the protein take place in a dynamic process. This transport is largely an ATP-driven mechanism, as substrate binding alone does not control translocation [17, 18]. ABCC2, one of the five clinically relevant members of the ABC family to date (MRP1/ABCC1-MRP5/ABCC5), is associated with various biliary pathologies in humans. ABCC2 shares high sequence homology with ABCC1, but no work has been reported on the drug binding characteristics of this protein. To facilitate this work specific inhibitors of ABCC1-mediated transport are also effective on ABCC2, though to a lesser potency. A traditional means of examining drug binding in ABC transporters involves photoaffinity labelling [19]. This technique requires the novel synthesis of drugs with a photoreactive side-chain. The drug binding properties determined through photoaffinity labeling techniques has been shown to complement and support the current drug transport data [9, 20, 21]. The drug binding characteristics of ABCC2 can be explored using this technique. Overall, the first chapter looks to describe the literature as reviewed for both ABC transporters in mammalian cells (1), as well as a general overview of malaria and chloroquine drug resistance (2). Given the problems associated with drug resistance, there is still a need to understand ABC transporter expression and function in both mammalian and parasite systems, as presented in the second, third and fourth chapters, respectively. The fifth chapter is a brief study on antimalarial drug discovery. The subjects of this thesis will attempt to clarify and expand in our understanding of these drug resistance proteins and to demonstrate the interplay and dependency of host and parasite ABC transporters. Save the last chapter of the thesis, the first four provide structural and functional information on two ABC transporters that is important to our understanding of host-parasite interactions.
3
References [1] Bjorkman A, Bhattarai A. Public health impact of drug resistant Plasmodium falciparum malaria. Acta Trop 2005; 94:163-9. [2] Klayman DL. Qinghaosu (artemisinin): an antimalarial drug from China. Science 1985; 228:1049-55. [3] Vennerstrom JL, Arbe-Barnes S, Brun R et al. Identification of an antimalarial synthetic trioxolane drug development candidate. Nature 2004; 430:900-4. [4] Leonard GD, Fojo T, Bates SE. The role of ABC transporters in clinical practice. Oncologist 2003; 8:411-24. [5] Robey RW, Polgar O, Deeken J et al. ABCG2: determining its relevance in clinical drug resistance. Cancer Metastasis Rev 2007; 26:39-57. [6] Szakacs G, Paterson JK, Ludwig JA et al. Targeting multidrug resistance in cancer. Nat Rev Drug Discov 2006; 5:219-34. [7] Cowman AF, Karcz S, Galatis D, Culvenor JG. A P-glycoprotein homologue of Plasmodium falciparum is localized on the digestive vacuole. J Cell Biol 1991; 113:1033-42. [8] Duraisingh MT, Cowman AF. Contribution of the pfmdr1 gene to antimalarial drugresistance. Acta Trop 2005; 94:181-90. [9] Vezmar M, Georges E. Direct binding of chloroquine to the multidrug resistance protein (MRP): possible role for MRP in chloroquine drug transport and resistance in tumour cells. Biochem Pharmacol 1998; 56:733-42. [10] Vezmar M, Deady LW, Tilley L, Georges E. The quinoline-based drug, N-[4-[1hydroxy-2-(dibutylamino)ethyl] quinolin-8-yl]-4-azidosalicylamide, photoaffinity labels the multidrug resistance protein (MRP) at a biologically relevant site. Biochem Biophys Res Commun 1997; 241:104-11. [11] Hyde SC, Emsley, P., Hartshorn, M.J., Mimmack, M.M., Gileadi, U., Pearce, S.R., Gallagher, M.P., Gill, D.R., Hubbard, R.E., and C.F. Higgins. Structural model of ATPbinding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport. Nature 1990; 346:362-365. 4
[12] Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol 1992; 8:67-113. [13] Cole SP, Bhardwaj G, Gerlach JH et al. Overexpression of a transporter gene in a multidrug-resistant human lung cancer cell line. Science 1992; 258:1650-4. [14] Pulaski L, Jedlitschky G, Leier I et al. Identification of the multidrug-resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes. Eur J Biochem 1996; 241:644-8. [15] Sullivan DJ, Jr., Gluzman IY, Russell DG, Goldberg DE. On the molecular mechanism of chloroquine's antimalarial action. Proc Natl Acad Sci U S A 1996; 93:11865-70. [16] Reed MB, Saliba KJ, Caruana SR et al. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 2000; 403:906-9. [17] Hollenstein K, Dawson RJ, Locher KP. Structure and mechanism of ABC transporter proteins. Curr Opin Struct Biol 2007; 17:412-8. [18] Linton KJ. Structure and Function of ABC Transporters. Physiology 2007; 22:122130. [19] Karwatsky JM, Georges E. Drug binding domains of MRP1 (ABCC1) as revealed by photoaffinity labeling. Curr Med Chem Anticancer Agents 2004; 4:19-30. [20] Daoud R, Julien M, Gros P, Georges E. Major photoaffinity drug binding sites in multidrug resistance protein 1 (MRP1) are within transmembrane domains 10-11 and 16-17. J Biol Chem 2001; 276:12324-30. [21] Karwatsky J, Leimanis M, Cai J et al. The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain. Biochemistry 2005; 44:340-51.
5
Chapter 1 Literature Review
6
PART I: ABC TRANSPORTERS (ABCG2, ABCC2) IN MAMMALIAN CELLS
"Cancer is a huge problem. We can bury our heads in the sand and say it is too complicated. But I think any problem that seems to be non-solvable tests a society... if we can be logical and passionate about it, we can go much further here than anybody thinks."
VICTOR LING
ATP Binding Cassette (ABC) Transporters ABC transporters are found in all cells. The physiological function of ABC transporters is to facilitate transmembrane transport of ligands, which is critical for maintaining the normal cellular physiology of the cell. This may include elimination of waste products or toxic compounds out of cells.
Some ABC transporters play a key role in the
elimination of toxic compounds that may arise due to dietary toxins or cytotoxic drugs (e.g. cancer chemotherapy treatment). This has led to a clear understanding that certain ABC transporters play an essential role in the chemical defense of organisms. In addition to transporting toxins and various substrates, ABC transporters also transport sugars, amino acids, peptides and salts [1]. 7
Diversity of ABC Transporters The ABC drug transporters can be divided into two main groups: the P-glycoprotein (Pgp)-like group and the Multidrug resistance associate protein (MRP) group. This division is based on alignment of amino acid residues in the nucleotide binding domains (NBD), combined with the specific protein topology and the drug specificity of the transporters. In addition to humans, Pgp-type multidrug transporters are found in P. falciparum (pfMDR1, Pgh1), Entamoeba histolytica (ehPgp), and Leishmania donovani (ldMDR1). MRP type multidrug transporters are found in Saccharomyces cerevisiae (Ycf1p, Yor1) , and in Leishmania species (lmPgpA). ABC transporters have also been found in plants, including a Pgp homologue from Arabidopsis thaliana (see review [2]). The diversity of these proteins spans mammalian, microbial and plant systems, and the characterization of these proteins is of ongoing interest, specifically in the areas of drug resistance.
Structure of ABC Transporters The minimum architecture of ABC transporters contain two copies of two structural units: a highly hydrophobic transmembrane domain (TMD), and a peripherally located ATP binding domain, termed the nucleotide binding domain (NBD).
Figure 1
illustrates the various domain organizations found among members of the ABC transporter family [3] as divided into subfamilies labeled A-G [4]. Both units are necessary to mediate transport; however, the TMD domain forms the pathway by which the substrate crosses the membrane and therefore may contribute to substrate specificity and ligand binding. And in ABC transporters located in the plasma membrane the NBDs, are oriented toward the cytoplasmic side of the membrane, and couple nucleotide hydrolysis for transport. Within the NBD is a conserved region of approximately 200 amino acids that constitutes the Walker A and B boxes. The Walker A and B boxes are separated by the ABC signature motif, which is characteristic of all ABC transporters. In total, seven conserved regions in the NBDs are homologous throughout all of the ABC transporter families; within this 200 amino acid stretch is an aromatic residue (~14), the Walker A box (~36-44), Q-loop (~83-86), ABC signature 8
(~140-44), Walker B motif (~160-67), D-loop (~170-171), and the H-loop (~195-97) [5]. The signature motif also distinguishes the ABC transporters from other ATP binding proteins, such as kinases, which also contain Walker sequences [6, 7]. Although, overall homology between different ABC transporters may be low, the conserved NBD domain is typically 30-40% homologous between family members. It is worthwhile to take a closer look at the structure and function of ABC transporters.
The most frequent arrangement of the eukaryotic ABC transporters is a fused single polypeptide of the TMD preceding the NBD as with Pgp (ABCB1) [8] and ABCC1 [9]. This structure is illustrated in Figure 1 and follows the sequence of TMD-NBD-TMDNBD in the case of both Pgp (ABCB1) and ABCC1. Other eukaryotic ABC proteins, such as the ABCG2 or Breast Cancer Resistance Protein (BCRP), exist as “halftransporters” [10], in that the proteins only have one TMD rather than two as seen with Pgp and ABCC1. The secondary structure of Pgp is predicted to contain a tandem 9
repeat of six transmembrane helices, each set of which is followed by an ATP binding domain; this is termed the “two-times-six paradigm” [11-13]. The structure of ABCC1 differs from Pgp in that it has an additional hydrophobic N-terminal extension that spans 5 transmembrane regions attached to the two-times-six TM core in the C-terminal [14]. A great deal of work has gone into examining the molecular mechanism underlying the function of ABC transporters, and to understand how the four key domains interact with each other. The NBDs interact with each other in an allosterical manner for drug transport [15] and ATPase activity [16], since both are essential for function. Mutational studies revealed that both NBD domains are essential for function and that activity is inhibited by orthovanadate (Vi) [17], which maintains a NBD transition state intermediate by occupying the ternary complex of Pgp with the γ-phosphate of ADP. These early clues gave rise to the “two-step mechanism” of transport [18]. What came next was the understanding that likely two molecules of ATP were required to form a binding pocket, and that although only a transient complex is formed [19], that the NBD likely formed a dimeric structure. To elucidate the mechanism with greater detail, structural data was required. Structural data exists, for three full-length bacterial ABC transporters: BtuCD from E. coli (which transports vitamin B12) [20], HI1470-1 from Haemophilus influenzae (which transports a metal-chelate) [21], and Sav1866, a drug transporter from Staphylococcus aureus [22]. From this work it was determined that of the two distinct subfamilies of ABC transporters, namely Sav1866 (12 TMD) and BtuCD/HI1470-1 (20 TMD) have similar NBD dimers, albeit distinct TMDs (reviewed in [23]). This work was done in addition to the low-medium resolution data that already existed for Pgp, the only mammalian multidrug transporter with structural data available [24-27]. Together, these data have led to the “ATP-switch model” for transport (Figure 2) [28]. Schematic representation of “ATP-switch model” is outlined in recent reviews by Higgins and Linton [5, 23, 29]. In this outline of the molecular operation of ABC transporters and drug resistance mechanisms, four distinct steps are described: 1) the “open dimer” is formed, whereby the ligand binds to the TMDs in the open NBD dimer 10
conformation (which is associated with an increased affinity for ATP), 2) the “closed dimer” is formed in which bound ATP is trapped, resulting in a large conformational change in the TMDs initiating substrate translocation, 3) ATP is hydrolyzed resulting in the NBD transitioning from a “closed dimer” to an “open dimer”, which returns the transporter to its basal state, and lastly 4) Pi and ADP are released which completes the transport cycle and returns the protein to a high affinity state ready for ligand binding. Transport or efflux of drugs out of the cell is therefore an energy-dependent process requiring ATP.
The use of non-hydrolysable ATP-analogues in place of ATP
inactivates the transport function [30].
Although binding sites remain to be clearly defined, this model provides a general framework for understanding the “switch” that takes place between “open dimer” and “closed dimer” transport mechanisms of ABC transporters. This information enhances our understanding of the mechanisms underlying drug resistance. It also provides potential frameworks for reversing drug resistance.
This may include the co-
administration of inhibitors and cytotoxic agents, or the use of cytotoxic agents that bypass the ABC transporter mediated efflux, thereby “evading” the drug resistance mechanism. The use of specific inhibitors with direct clinical application is discussed in later sections.
11
Multidrug Resistance Transporters in Cancer Cancer may be defined by three distinct properties of cells: 1) cells are aggressive, in their growth and cell division, not respecting normal growth limits, 2) invasive and capable of invading and destroying neighboring tissues, and 3) may be metastatic and spread to other locations in the body. Diseases causing cancer are differentiated from benign tumours, which are self-limiting, non-invasive and not metastatic. One method to combat cancer is using chemotherapy.
Chemotherapy is effective in several
childhood and adult cancers, including some leukemias, lymphomas, sarcomas, choriocarcinoma, and testicular cancers. Most metastatic cancers exhibit resistance to chemotherapy (intrinsic resistance), and other cancers may initially respond to chemotherapy but later recur as cancers that have acquired chemotherapy resistance (acquired resistance) [31, 32].
A form of acquired drug resistance is multidrug
resistance (MDR). This drug resistance is associated with reduced accumulation of multiple structurally distinct drugs in tumour cells. In addition, multidrug resistant tumour cells that are treated with a particular class of natural products display crossresistance to these and other agents, by an energy-dependent mechanism. It is believed that the processes used to export intracellular metabolites can be co-opted to export xenobiotic metabolites from cells [33]. This MDR phenotype can be conferred by changes in expression or sequence of a number of transport proteins. The end result is the translocation of drugs from the inner to the outer leaflet of the lipid bilayer; the drug thus does not render the cell nonfunctional, and the cancer survives. In the last 25 years this increased drug efflux from cells has been linked predominantly to two integral membrane proteins, P-glycoprotein (Pgp, ABCB1) [33] and Multidrug resistanceassociated protein (MRP1, ABCC1) [9]. Both Pgp and ABCC1 appear to bind and efflux drugs out of cells. Most recently, a third integral membrane protein ABCG2 (BCRP) has also been shown to play a role in drug resistance [10]. Briefly, the human multidrug resistance protein family consists of five clinically relevant members. ABCC1 has been well characterized as conferring MDR, although there are many additional homologues termed ABCC2, 3, 4 and 5 [34]. Although, both ABCC1 and Pgp belong to the ATP-binding cassette (ABC) superfamily of transport 12
proteins, they only share 15% amino acid homology [9]. Anti-cancer drug specificity for ABCC1 and Pgp suggest that cytotoxic natural products can be segregated into three classes: the anthracyclines, vinca alkaloids, and etoposide. ABCC1 also confers lowlevel resistance to completely synthetic drugs such as methotrexate and cisplatin [35]. Other structurally diverse compounds have been identified that reverse the Pgpmediated MDR phenotype.
Such reversing agents, or "chemosensitizers", include:
calmodulin inhibitors, steroids, calcium channel blockers, and other lipophilic agents [36].
Agents such as the calcium channel blocker verapamil do not however,
completely reverse ABCC1-mediated drug resistance [37, 38]. In addition to the drug efflux phenomenon as described above, two other major mechanism of drug resistance exist for cancerous cells, including 1) decreased drug uptake (i.e. drugs may require transporters to enter the cell) and, 2) changes in the capacity of the drug to effectively terminate the cancerous cell (i.e. alteration in cell cycle, increased repair of damaged DNA, reduced apoptosis, or altered drug metabolism) [34]. However, for the purpose of this review ABC transporters implicated in drug resistance in cancer (Pgp, ABCC1, ABCC2, and ABCG2) will be discussed.
P-glycoprotein (Pgp1, ABCB1) The first reported MDR protein was Pgp [33], which was shown to contribute to the protection of human cells against hydrophobic xenobiotics.
The discovery of the
human Pgp gene, MDR1 advanced understanding of the MDR phenotype in tumour cells, as it was found to be over-expressed in many drug-resistant cell lines (reviewed by Ueda [39]). Of critical importance is the expression of Pgp in the liver, kidney, gastrointestinal tract and at pharmacological barrier sites, as well as adult stem cells [34, 40]. The expression of the 170-kDa Pgp correlates with organs involved with drug excretion, therefore a physiological role in chemoprotection is likely. Pgp, the first ABC transporter to be characterized also has the broadest spectrum of substrates. A more complete list of Ppg substrates includes: vinca alkaloids [41, 42], anthracyclines [43], epipodophyllotoxins and taxanes [44], actinomycin D [31], mitoxantrone [45] and other drugs (bisantrene, colchicines, methotrexate) (reviewed in 13
[34]). In contrast, Pgp transport may be inhibited by various structurally unrelated compounds termed pharmacological chemosensitizers [36, 46]. Early studies revealed the possibility of reversing the drug resistance with a calcium channel blockers verapamil, both in vitro and in vivo, in vincristine resistant P388 cells.
This
immediately demonstrated the possible therapeutic use of inhibitors [47]. This “first generation” of inhibitors included verapamil, cyclosporine and quinine, and were drugs already in use for other medical purposes. Toxicity and side effects of the compounds at the concentrations necessary for reversal of drug resistance were common and therefore additional compounds were identified. A “second generation” of inhibitors, with fewer side effects was developed in hopes of improving the outcomes of clinical trials. This included a cyclosporine D analogue PSC-833 (Valspodar) that showed little effect in clinical trials. Due to limited drug clearance of PSC-833 and toxicity, this ultimately resulted in its discontinued development.
A “third generation” of Pgp
inhibitors has been developed to overcome issues of toxicity, however clinical trials are ongoing to determine efficacy (reviewed in [34]). In light of the efforts to overcome Pgp-mediated drug resistance it has proven challenging to achieve high cellular concentrations in tissues to have a marked result on patient survival and improved overall outcome. Work is ongoing to develop new-generation inhibitors that are more potent and specific.
Multidrug Resistance Protein 1 (MRP1, ABCC1) Another ABC transporter found to confer an MDR phenotype is the multidrug resistance protein 1 (ABCC1). It was first identified in small cell lung cancer (SCLC) cells displaying clinical drug resistance with normal levels of Pgp while overexpressing the ABCC1 protein [9]. ABCC1 is expressed in a wide range of tissues and clinical tumours [48]. This 190-kDa integral membrane phosphoglycoprotein consists of a hydrophobic N-domain with five potential membrane-spanning domains linked to an MDR1-like core (MSD1-NBD1-L1-MSD2-NBD2) by an intracellular linker domain (Lo) (Figure 3). The amino acid sequence of ABCC1 predicts a molecular weight of 171 kDa, however due to three integral N-glycosylations of high mannose 14
oligosaccharides at Asn19, Asn 23, Asn 1006 the resulting full-length glycosylated protein is shown to be 190 kDa [49, 50]. ABCC1 may participate indirectly in active transport of drugs into subcellular organelles or influence intracellular drug distribution. Daoud et al. [51] recently determined the possible drug binding site(s) of ABCC1 using photoreactive probes:
N-2-hydroxy-4-azido-5-iodophenylacetyl-dihydro-7-chloro-8-
amino-cin-choanan-9R-ol (IACI) and iodoaryl azido-Rh123 (IAARh123) photolabelled the TMD 10-11 and 16-17, indicating that these regions are possibly involved in the binding and transport of drugs out of the cell [51]. ABCC1 transport is believed to occur in three of the following possible ways: 1) drugs are conjugated to glutathione (GSH), glucoronide, or sulfate [51, 52], 2) drugs are co-transported together with GSH [30, 52-55], and 3)
drugs may be transported alone, neither conjugated nor co-
transported [56, 57].
15
The substrate specificity of ABCC1 is similar to that of Pgp, in that it also transports anthracylines, vinca alkaloids and etoposide; however taxanes are not substrates of ABCC1. ABCC1 differs from Pgp in that it is able to transport LTC4 and other endogenous substrates [48], this difference is attributed in part to the N-terminal extension of ABCC1, which is thought to play a role in the interaction of anionic substrates [58, 59]. In terms of chemosensitizing agents, ABCC1 specific inhibitors have not been as readily identified as for Pgp. In a recent study by Loe et al. the effect of verapamil on ABCC1, in mammalian cells, was more closely examined [60]. In this study it was found that verapamil was shown to inhibit the transport of an arachidonic acid metabolite, leukotriene C4 (LTC4). The inhibition of LTC4 transport into inside-out membrane vesicles prepared from ABCC1-transfected cells occurred in a competitive manner, but only in the presence of 1 mM reduced glutathione (GSH) or its nonreducing S-methyl derivative. It was deduced that verapamil may not inhibit ABCC1 directly but that the drug may cause the preferential transport of GSH (a physiological antioxidant helping to maintain intracellular oxidative homeostasis) by means of other substrates of ABCC1 [61]. One specific inhibitor, MK571, a leukotriene receptor antagonist (LTD4), has been used as a research tool to inhibit ABCC1 [62]. The issues concerning the reversal of Pgp-specific resistance, has resulted in only very limited development of ABCC1 as a possible clinical target.
MRP Family of Transporters MRP1-5 (ABCC1-5) proteins are divided into two groups based on sequence homology. ABCC1 (MRP1), ABCC2 (MRP2) and ABCC3 (MRP3) are grouped together due to a shared N-terminal domain extension (Figure 3) [63, 64]. ABCC4 (MRP4) and ABCC5 (MRP5) constitute a shorter group of MRPs that lack TMDo. The first group is more closely related to ABCC1 in amino acid identity (45%-58% overall; 61%-74% identity in the NBD). The second group is less closely related to ABCC1 in amino acid identity (36%-39% overall; 57%-62% NBD), and does not have the third TMD. All of the MRPs share a greater homology to one another than to Pgp and therefore have been grouped separately from other classes of ABC transporters (Figure 1). ABCC2 shares 16
49% homology with ABCC1 and is thought to transport a similar range of GSH conjugates as ABCC1. Other isoforms of the ABCC family have been identified and the ABCC (MRP) subfamily consists of 12 members, most of which transport organic anions (reviewed in [34, 48, 63, 65, 66]). In summary, ABCC1, ABCC2 and ABCC3 [67-72] have been functionally characterized as conjugate export pumps implicated in drug resistance to certain anti-cancer drugs and organic anions; where as ABCC4 and ABCC5 have been shown to be resistant to nucleoside analogues [67-72].
ABCG2 (BCRP, MXR, ABCP) In recent years, this protein, variously named ABCG2/BCRP/MXR/ABCP, has been implicated in many areas of biology and medicine. ABCG2 is one of the most recently characterized ABC transporters first described in 1998, in a non-ABCC1, non-Pgp expressing cell line shown to confer drug resistance [10] .
The cDNA encoding
ABCG2 was initially cloned from a doxorubicin-resistant MCF7 breast cancer cell line (MCF-7/AdrVp) and was termed the breast cancer resistance protein (BCRP). Since then, it has been found that the expression of the gene is not limited to either breast tissue or to cancer cells. Other groups that have separately cloned the BCRP cDNA have named the gene MXR (mitoxantrone resistance protein) and ABCP (placental ABC protein), for it’s resistance to mitoxantrone and the high levels of expression in the placenta, respectively. ABCG2 influences the absorption, distribution and excretion of drugs as well as cytotoxins. Tissues with the highest ABCG2 mRNA levels include the placenta, liver and intestine, however, the biological functions of this protein are incompletely understood. ABCG2 has been further implicated not only in cancer drug resistance, but also plays a protective role against both exogenous and endogenous toxins in stem cells (reviewed in [73]). The substrate specificity of ABCG2 includes both chemotherapeutic agents such as doxorubicin and methotrexate and naturally occurring compounds such as flavonoids and porphyrins.
ABCG2 expression is upregulated in low oxygen environments
(hypoxia), and the protein has been shown to interact with heme and porphyrins. This may result in a protective effect for cells or tissues in the event of an accumulation of 17
either heme or porphyrins due to hypoxia (defined as a state of reduced oxygen supply to tissues despite adequate perfusion). Unlike ABCC1 and Pgp, ABCG2 requires the formation of two “half-transporters” to homodimerize to be fully active. Phylogenetic analysis reveals that ABCG2 is closely related to ABCG1, an orthologue of the human Drosophila white gene, and is distantly related to ABCC1 and Pgp [74]. ABCG2, like all ABC transporters in this G subfamily, differs from both the ABCB and ABCC family of transporters as illustrated by its “reverse domain” arrangement (Figure 1). In the reverse domain arrangement, the NBD is located N-terminally rather than Cterminally. The evolutionary significance of “half-transporters” (that form homo or heterodimers), as well as the specific amino acids believed to play a role in the formation of homodimers of ABCG2, will be discussed.
ABCG2 as a Homodimer ABCG2 has been previously characterized as a functional dimer [75, 76]. In addition, evidence of higher oligomerization has been suggested and may have evolutionary significance [77].
Similar to ABCG2, the Drosophila eye pigment precursor
transporters white, brown and scarlet, have been shown to function as dimers [78], as well as the human homologues ABCG5 and ABCG8, which form an obligate heterodimer [79]. The complex formed between ABCG5 and ABCG8 is crucial for the excretion of sterols. Mutations in this complex results in sitosterolemia [80]. These findings led to the hypothesis that ABCG2 may share similarities in structure status. An initial study revealed ABCG2 homodimerization, with evidence that the dimer was formed through a disulfide bond [76]. This was demonstrated in a study using reducing agents in which 140-kDa ABCG2 protein was exposed to 2-mercaptoethanol resulting in a monomer migrating to 70-kDa range. Further analysis revealed the presence of 11 cysteine residues that could contribute to the formation of the disulfide linkage. From this analysis, Cys-603 was found to be critical for the stable oligomer formation of ABCG2 [81]. Additional mutagenesis work demonstrated that Cys-603 is necessary for the formation of a symmetrical intermolecular disulfide-bridge, but is not critical for either expression or function of ABCG2. This work also led to the characterization of 18
additional intramolecular disulfide-bridges through Cys-592 and Cys-608 that together play a role in the structure and function of ABCG2 [82]. All three cysteines are found in a large 68-residue extracellular loop connecting TM helices 5 and 6. Most recently, Wakabayashi et al. found that an intramolecular disulfide-bridge between Cys-592 and Cys-608 resulted in ubiquitin-mediated protein degradation in proteasomes, and therefore is critical for the proper folding of ABCG2. Mutation of these residues are critical as wild-type ABCG2 is degraded in lysosomes [83]. This work supports the conclusions of previous investigations that both intra- and intermolecular bonds are necessary for the normal functions of ABCG2 (Figure 4).
Interestingly, this region has undergone further scrutiny as residue 553 in TM5 has been implicated in the homodimerization of ABCG2. This is a highly conserved region and 19
has been shown to be critical for the formation of stable Drosophila white-brown eye pigment transporter heterodimers [84]. Mutations in amino acid 553 disrupted transport in ABCG2, raising further speculation on the contribution of additional residues necessary for homodimerization [85]. An additional conserved region of interest includes the GXXXG motif in TM1, thought to be involved in proper folding of the protein [86]. In summary, it is unknown whether the dimerization of ABCG2 is a regulated process or simply pertinent to p roper protein folding and localization of the membrane protein.
ABCG2 in Maintenance of Cellular Homeostasis In addition to expression in placenta, mammary gland, testis, the blood-brain barrier and the gastrointestinal tract (reviewed in [87]), ABCG2 is expressed in endothelial cells of venules and capillaries, but not arterioles [88].
The expression of ABCG2 may
therefore be regulated at least in part, by oxygen levels in blood. Hematopoietic stem cells also express ABCG2, and at greater levels than Pgp [89, 90]. This expression was found in a population of cells (side population (SP)), using FACS analysis. These SP cells contained a high proportion of hematopoietic stem cells [91, 92]. However, ABCG2 is down-regulated in differentiated hematopoeitic cells, which may imply a preferential functional role for ABCG2 in un-differentiated cells [93].
In one
exception, mature erythroid cells have been demonstrated to reexpress ABCG2 which might suggest a biological function for ABCG2 in these cells that differs from its possible roles in other differentiated blood cells [94]. One such function for ABCG2 might include maintaining cellular homeostasis.
Of interest in this regard is the
response of erythrocytes to an accumulation of heme; this was explored in stem cells that expressed ABCG2 under hypoxic conditions [95].
It was demonstrated that
ABCG2 enhances cellular survival through interactions with heme; ABCG2 knock-out mice had a decreased cellular survival. Furthermore, it was demonstrated that ABCG2 expression is regulated through the hypoxia-inducible transcription factor complex (HIF-1). In light of these results, the activity of ABCG2 in mature erythroid cells (erythrocytes) warrants further exploration. 20
ABCG2: Substrate Specificity, Role in MDR, Inhibition of ABCG2 ABCG2 has been shown to transport a wide range of substrates, some of which are also substrates of Pgp and ABCC1 (reviewed in [34, 96]). Substrates for ABCG2 include anthracyclins (daunorubicin, doxorubicin, epirubicin) [10, 97], epipodophyllotoxins (etoposide, teniposide) [98], kinase inhibitors, imantinib (Gleevec) [97] and flavopiridol [99], camptothecins, (irinotecan (CPT-11), SN-38) [100], topotecan [101], bisantrene [99], methotrexate [102], mitoxantrone [10, 103], and the antiretroviral drug azidothymidine [104]. ABCG2 is also known to transport key physiological substrates. As discussed previously, ABCG2 plays a critical role in hypoxia due to transport capacity of ABCG2 for the physiological substrates poryphyrin and porphyrin-like molecules [95].
Additional substrates for ABCG2 include natural substrates:
flavonoids [105-107], Pheophorbide A [108], PhiP (2-amino-1-methyl-1-6-phenylimidazo [4,5-b]pyridine) [109, 110], sulfated estrogens [111] and fluorescent compounds such as Hoechst 33342 [92, 93], Lysotracker [112] and rhodamine 123 [10, 113, 114]. Due to the wide range of substrates and the significant overlap in that regard with Pgp and ABCC1, it was speculated that ABCG2 could contribute to drug resistance in cancer cells. However, in order to determine the role of a transporter in clinical drug resistance, it must first be shown that there is a distinct correlation between the protein and a resistance phenotype in a malignancy. Secondly, it is important to examine the effectiveness of a protein-specific inhibitor on patient survival in clinical trials. Successful trials would reflect the potential of the protein as a clinically relevant drug target. Studies of this sort have mainly examined both hematological malignancies and solid tumours. These have shown a distinct association between expression of ABCG2 and reduced response in certain cancers (namely Acute Myeloid Leukemia’s (AML), or Acute Lymphoblastic Leukemia’s (ALL)) [115-117].
More recently, ABCG2
expression was directly correlated with a clinically resistant phenotype in elderly patients (≥60 years) with secondary AML [118]. Overall, the role of ABCG2 in solid tumours has been only minimally explored (reviewed in [87]).
21
Efforts to explore ABCG2 as a target for reversing the resistance phenotype and improving survival rate require protein-specific inhibitors. ABCG2 has been shown to be inhibited by both first generation cyclosporin [119], second generation VX-710 (biricodar) [120] and GF120918 (elacridar) [121]) and third generation XR-9576 (tariquidar) [108]) inhibitors as well as fumitrimorgin C (FTC) [122]. Since the initial discovery of ABCG2 there has been fervent progress in characterizing its role in MDR and especially in the substrates it transports. Future work will involve determining the clinical significance of this transporter and a greater understanding of the control mechanisms of expression in vivo. Ongoing work in ABCG2-knockout animal models should help to expand on this knowledge.
ABCC2 ABCC2 (MRP2, cMOAT) is largely expressed in the canalicular membrane of hepatocytes [123-125], the kidney and intestine. Similar to ABCC1 (see topological representation in Figure 3), ABCC2 is thought to contain 17 TM regions grouped into three TMDs, and two cytosolic NBDs. ABCC2 transports both structurally diverse xenobiotics and endogenous molecules such as bilirubin. In addition, ABCC2 can confer drug resistance in tumour cells. The initial characterization of ABCC2 (or the canalicular multispecific organic anion transporter, cMOAT) came from the observation that rats suffering from chronic conjugated hyperbilirubinemia were defective for ABCC2 [126]. Similarly, human patients suffering with Dubin-Johnson syndrome (an autosomal recessive disease) and lacking ABCC2 suffer from hyperbilirubinemia; the transfer of both endogenous and exogenous anionic conjugates from hepatocytes into the bile is defective in these patients. This defect was found to be caused by mutations in the ABCC2 gene [127-130]. Based on this knowledge, it was determined that ABCC2 was a key transporter in the hepato-biliary elimination process (reviewed in [66, 131, 132]). The following sections, explore the role of ABCC2 in drug resistance and the techniques used for biochemical characterization of this clinically relevant ABC transporter.
22
ABCC2: Substrate Specificity, Role in MDR, Inhibition of ABCC2 The substrates for ABCC2 closely overlap those of ABCC1. These include a wide range of both conjugated and unconjugated exogenous molecules such as:
vinca
alkaloids (vinblastine [65], vincristine [133]), anthracyclins (doxorubicin, epirubicin) [133], epipodophyllotoxins (etoposide) [133], taxanes (docetaxel, paclitaxel) [134], camptothecins (irinotecan (CPT-11) [135-137], SN-38 [135, 136], topotecan [135]), cisplatin [133], arsenite [138], methotrexate [56, 57], mitoxantrone [65], and saquinivir [139].
ABCC2 is also known to transport glucuronate-, sulfate- and glutathione-
conjugated physiological substrates [140], as well as reduced and oxidized glutathione [141, 142], leukotrienes C4, D4, and E4 [143], glucuronide conjugates of bilirubin [144, 145], estradiol [146], and glucuronate and sulfate conjugates of bile salts.
An
endogenous substrate effluxed by ABCC2 is LTC4, a potent mediator of smooth muscle contraction and an important mediator in inflammation. Up to 77% of [3H]LTC4 is excreted in the bile in a rat model, with slightly less (50%) found in the bile of primates. This study demonstrates that LTC4 is primarily metabolized by the liver and eliminated in the bile [147]. These results emphasize the importance of LTC4 as an endogenous substrate of ABCC2. This wide substrate specificity of ABCC2 highlights its role in drug transport and chemoprotection. To support this a study revealed that increased expression of ABCC2 in human colorectal carcinoma was associated with resistance to cisplatin [148]. Furthermore, other studies have shown that ABCC2 together with either ABCC1 or ABCC3 may be responsible for multidrug resistance in acute myeloid leukemia [149], and hepatocarcinoma [150], respectively. ABCC2 was also detected in ovarian cancers [151], although its expression was not associated with overall survival time [152]. Specific inhibitors of ABCC2 include both first and second generation inhibitors cyclosporin and PSC-833, as well as MK571 [153, 154]. These inhibitors can greatly aid in the understanding of drug transport and protein function.
23
Photoaffinity Labeling: Techniques and Significance Photoaffinity labeling studies have helped support site-directed mutagenesis and transport studies in revealing both drug binding domains and drug binding sites (reviewed in [155]).
ABCC1 drug binding sites have been characterized using
photoreactive drug analogues, including the quinoline derivatives IAAQ [156] and IACI [157], a tricyclic isoxazole LY475776 [158], rhodamine 123 (Rh123) [51], glutathione (GSH) [159] and leukotriene C4 [58, 160-162]. These binding studies have identified distinct binding sites of ABCC1 in TM regions 10-11 and 16-17 as summarized in [51]. No photoaffinity labeling studies have been reported for ABCC2. This is made more difficult, as no selective inhibitors for ABCC2 are available. MK571 inhibits the transport of LTC4, although about five times less potently than for ABCC1 [160, 162, 163]. Moreover, ABCC2 transports LTC4 but with a lower affinity than ABCC1. Additional ABCC2 inhibitors have been shown to inhibit transport, namely: probenecid, cyclosporine A [153], vinblastine [154], and flavanoid (Quercetin) [164-166]. However, most of these inhibitors also inhibit ABCC1, and therefore lack transporter specificity. Despite this lack of photoaffinity studies to reveal potential drug binding sites in ABCC2, site-directed mutagenesis studies have revealed information regarding TM regions implicated in transport activity and drug export [167-169]. In addition the availability of multiple site-directed antibodies [170] may enable future studies mapping the drug binding domains of ABCC2.
Clinical Multidrug Resistance Clinical trials have been undertaken with a collection of various drugs targeting different cancers, as summarized in a recent review by Szakacs [34], and of the 20 phase III clinical trials with ABC transporter inhibitors over a 10 year period, four reported improved overall survival rates [171-175]. The translation of work from “bench to bedside” has been a seemingly labored effort, without great success. This has caused speculation about the validity of ABC transporters as drug targets [176]. The lack of positive clinical results has not been due to a lack of translational research efforts, but major obstacles include the lack of validated assays and diagnostic tools (i.e. 24
PCR, immunohistochemistry and flow cytommetry) as well as the difficulty of defining the parameters of the clinical trials themselves. In addition, multiple ABC transporters may be working in concert in tumour cells, confusing results and the respective conclusions drawn from those studies. Recently, phase I and phase II clinical trials were undertaken to determine the increase in oral bioavailability of topotecan when administered in combination with the ABCG2 inhibitor GF120918 [177]. Topotecan is used for the treatment of SCLC and metastatic ovarian cancer (after the failure of first-line chemotherapy), and has previously been shown to have moderate oral bioavailability [178, 179]. This study revealed that coadministration of topotecan with GF120918 resulted in complete oral bioavailability of topotecan. No phase III clinical trials have been reported for ABCG2 inhibitors. It is known that ABCC2 plays a key role in the regulation of drug efflux in the liver. This key physiological role in detoxification is important as metabolites are secreted into the bile (reviewed in [180]). In addition to the liver, drugs are also excreted by the kidneys, organs where ABCC2 is also expressed.
The over-expression of ABCC2 is
implicated in multidrug resistance, whereas under-expression (or mutations) of ABCC2 may result in clinical disorders such as hyperbilirubinemia. Clearly, expression of the protein and its regulation are critical in maintaining normal homeostasis in humans. Regulation of ABCC2 expression is affected by both endogenous and exogenous factors (reviewed in [132]).
25
PART II: DRUG RESISTANCE IN PLASMODIUM FALCIPARUM
“The first finding I tried to establish was whether malaria and poverty were intertwined because poor countries lacked the means to fight malaria, or also because malaria contributes to poverty. The evidence suggests both directions of causation.”
JEFFERY D. SACHS
-The End of Poverty-
Malaria Infection in Humans Malaria is one of the most widespread and persistent diseases of mankind. This ancient disease was recognized by Hippocrates about 400 BC. He was able to describe the three stages of a malarial attack: chilly rigor, high fever and profuse sweating. In the nineteenth 19th century, malaria was widespread throughout the Afrotropical, Oriental and Palaearctic regions and was also present in Northern Australia, and Eastern Europe. Additional endemic regions included: South and Central America (north of 32oS), a narrow range along the west coast up to Vancouver, Canada, and a broader range along the east coast of North America which extended into the south-eastern region of Canada. In the twentieth century malaria was almost entirely eradicated in certain countries such as India by 1976 (reviewed in [181]), however the disease persisted in 26
Africa and other countries in Southeastern Asia where eradication made little headway. Present day mortality caused by the disease is a grave problem in Africa amongst young children. Although the disease may not be fatal in all cases, malaria still causes chronic suffering to individuals by lowering their resistance to other diseases, and thereby reducing their life expectancy [181]. Plasmodium falciparum (P. falciparum) is one of the four species in the genus of Plasmodium that causes disease in humans. Also known as malignant tertian malaria (i.e. a fever that recurs at 48 hr intervals), P. falciparum, is the greatest killer and yet in the absence of drug resistance is the most easily cured form of malaria. The pathology associated with P. falciparum is associated with several severe reactions: cerebral malaria, blackwater fever and hemolytic anemia in children. The most dangerous of reactions is cerebral malaria that may induce coma which is most grave in both children and pregnant woman. Hemoglobinuria (blackwater fever) may be attributed to renal failure or irregular treatment with quinine; treatment of malaria with other antimalarial drugs however, prevents the development of blackwater fever. Hemolytic anemia is a fatal condition that occurs largely amongst African infants in areas where there is holoendemic malaria; and it is this anemia that causes the greatest amount of morbidity [182].
Additional pathologies include:
splenomegaly, severe headache, cerebral
ischemia, hepatomegaly and hypoglycemia. The spread, treatment and mechanisms of drug resistant malaria are of great interest with significant clinical implications.
Malaria Biology The protozoan organism that causes malaria is an obligate intracellular parasite classified in the phylum Apicomplexa [183]. The genus Plasmodium includes four species which cause disease in humans: P.falciparum, P. ovale, P. malariae and P. vivax. P. falciparum is distinguishable from the other species of malaria based on the disappearance of ring stage parasitized erythrocytes (PE) from the peripheral circulation of the host [184]. The disappearance of PE from the peripheral circulation is due to changes in the adhesion properties of the parasite, as encoded by the polymorphic var gene family for the P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 27
appears on the surface of mature IE stage parasites resulting in the sequestration of parasites in microvasculature (reviewed in [185]). This contributes to the difficulty in the diagnosis of malaria caused by P. falciparum. P. falciparum causes the greatest morbidity and mortality; and therefore poses the greatest challenge to develop better therapeutic treatments for this species.
To complete its life cycle P. falciparum requires two hosts:
a vertebrate and a
mosquito. Asexual development occurs in the vertebrate, with sexual development in the female Anopheles mosquito. These developmental processes are also referred to as schizogony and sporogony, respectively (Figure 5). During a blood meal, the female Anopheles mosquito injects P. falciparum sporozoites from its salivary gland into the human host, initiating the exoerythrocytic (EE) stage. 28
The slender and motile sporozoites migrate to the liver where they penetrate a liver parenchymal cell and multiply to form a schizont.
After a period of 8-15 days,
thousands of merozoites form within the schizont and are released into the bloodstream when the liver cell bursts, initiating the intraerythrocytic (IE) stage. Once merozoites invade red blood cells (RBCs), a series of growth phases take place. These include development of the merozoites into early, mature and late trophozoites, which eventually mature into merozoite- filled schizonts. The schizonts rupture, releasing the freely circulating merozoites that proceed to invade new neighboring RBCs. Small proportions of the merozoites however, re-infect RBCs, but differentiate into male and female gametocytes which propagate the sexual stage of the life cycle. When a mosquito takes a second blood meal from an infected human host, the gametocytes, unlike any asexual parasites (which are digested), remain viable within the mosquito.
Male
gametocytes
(microgametocytes)
and
female
gametocytes
(macrogametocytes) develop into microgametes and macrogametes, respectively. The key phase of the fertilization process takes place when the microgamete enters the macrogamete, resulting in the formation of a zygote. The zygote develops into a wormlike ookinete that penetrates the mosquito gut, where it matures into an oocyste. It is within the oocyste that the sporozoites develop. Once the oocyst is fully mature, it bursts, releasing sporozoites, that migrate to the salivary gland of the mosquito. During the next blood meal, completing the malaria life cycle, the mosquito injects sporozoites into the human host, thereby initiating the EE stage of the life cycle [184]. Malaria is limited in its geographical distribution largely due to a temperature dependency of the parasite and the range of the vector (Anopheles mosquitoes). During the sexual development of the parasite (sporogony) the temperature must not exceed 33o Celsius (C) and must not go below 20o C, thereby restricting the geographical distribution of the parasite to temperate tropical climates [181]. The mosquito vector largely defines the success of malaria transmission. Based on both ecology and climate, various Anopheles species are distributed throughout different malarious regions globally [186]. In addition, the malaria vector may have a large impact on the prevalence and severity of the infection. As stated in a publication by J.L 29
Gallup et al. [187], vectorial capacity is defined as a measure of efficiency for a mosquitoes to transmit the malaria parasites from one human to another. This results in a number of secondary cases of malaria from one primary case. The vectorial capacity of different species of Anopheles varies greatly, as in the case of Sub-Saharan Africa where the most efficient vector found is Anopheles gambiae (A. gambiae). Strategies to control the transmission are greatly impacted by the fecundity of the vector and its ability to support the development of the parasite. One explanation for this difference in fecundity in the case of A. gambiae can be found in the co-evolution of the mosquito with humans. A. gambiae sensu stricto (s.s.) evolved with humans during the time when African agriculture developed, and as a result the biting habits of A. gambiae are almost exclusively human (anthropophilic) versus other species of Anopheles, which may be equally or exclusively attracted to livestock (zoophilic) [188]. Sub-saharan Africa also carries the greatest burden of malaria globally due to the great prevalence of the most pathogenic malaria species, P. falciparum which is thought to have originated in Africa [188].
Together, all of these factors markedly impact vector control strategies,
especially in Africa, a continent which carries 90% of the global malaria disease burden [189].
Asexual Development: Malarial Invasion and Hemoglobin Degradation The merozoite must successfully invade RBCs to ensure survival and propagation of the parasite. The RBC is unique in part due the elastic properties of the RBC membrane, the lack of a transcellular cytoskeleton, intermediate filaments or microtubules, and a dense solution of hemoglobin, all characteristics that contribute to the ability for the cell to transform shape [190].
The resulting environment is conducive for merozoite
invasion and establishment in the host RBC (reviewed in [191]). Following entry into the host RBC, the parasite degrades hemoglobin, which provides the parasite with the amino acids necessary for survival [192]. A more recent theory suggests that the digestion of hemoglobin provides space for the parasite to grow [193], and that only 16% of all amino acids are used for protein synthesis with the remaining 84% exported 30
to the extracellular medium as waste [194, 195]. Serum concentrations of amino acids are high enough to permit parasite survival by simple diffusion into RBCs as previously shown by Divo et al [196-198]. Hemoglobin degradation occurs predominantly during the trophozoite stage of maturation in the IE stage of the life cycle is mediated by aspartic and cysteine proteases [199-201], and takes place in the food vacuole (FV) of the parasite [202, 203]. Heme released by hemoglobin degradation (hemoglobin is degraded to heme and globin, respectively) is oxidized to a toxic ferric intermediate, that can damage biological membranes and inhibit parasite protease activity [204]. In defense against this toxic molecule, the parasite has devised a mechanism through which the ferric intermediate is polymerized into a crystalline form termed hemozoin (or malarial pigment) [205, 206]. This has recently been confirmed using synthetic beta-hematin (FeIII-protoporphyrin-IX)2 which has been shown to be identical (chemically, spectroscopically and crystallographically) to hemozoin. Ferriprotoporphyrin IX (FP) also known as heme is detoxified by incorporating the FP dimers (β-hematin) into inert hemozoin through the formation of dimers (iron-carboxylate bonds) to one of the propionic side chains of each porphyrin.
The dimers then form chains through
hydrogen bonding to generate the hemozoin crystals [207].
It is believed that
antimalarial drugs (such as CQ) interfere with hemozoin formation, thereby causing the accumulation of toxic cellular byproducts [208].
Malaria Pathology, Diagnosis and Immunity The malarial paroxysm (sudden recurrence or intensification of symptoms) begins with a cold-like stage of a few hours while the body temperature begins to mount rapidly. As the temperature peaks, there may be symptoms of vomiting and headache. The symptoms of malaria in the patient correspond roughly to the stages of the 48 hr life cycle of P. falciparum. In the event that the paroxysms occur at regular intervals, this may indicate that an asexual cycle has become synchronized resulting in all of the schizonts bursting simultaneously. It is this simultaneous bursting of the infected RBCs that causes a sharp increase in fever, along with debilitating anemia. 31
Diagnosis is made primarily by microscopic examination of specific morphological features of the parasite in both thin and thick blood smears [184]. It is during the microscopic examination that speciation of the parasite may be evaluated to confirm the presence of one or more species of Plasmodium. Additional diagnostic methods include immunochromatographic or rapid diagnostic tests (RDTs) (including dip-stick and teststrip methods), molecular techniques (PCR) and most recently by laser desorption mass spectrophotometry (LDMS) [209, 210]. Despite advances in diagnostic technologies, available, misdiagnosis or overdiagnosis (and overtreatment) of malaria is still a common occurrence [211-213]. The proper diagnosis and treatment of a patient infected with malaria is absolutely crucial in the event of a sudden onset of symptoms. However, it has been shown that patients living in endemic areas may carry a parasite burden but remain asymptomatic [214]. In this special case, the patient has acquired partial immunity to the parasite. This type of immunity is a partial immunity, as opposed to sterile immunity, since the individual may still carry a low parasite burden [215]. Partial immunity has been compared to passive immunity whereby sera from “immune” malaria patients, has been shown to be effective in protecting patients from infection [216-218]. Similarly, there may be passive transfer of maternal antibodies during pregnancy [219]. Together these observations construct a framework for vaccine development, in which either whole parasites or specific Plasmodium antigens can be used to elicit a protective immune response. Malaria vaccine development has posed a tremendous challenge for scientists [220]. Generally speaking, the main vaccine candidates currently being explored include: preerythrocytic vaccines (PEV) directed against sporozoites or intra-hepatic parasite stages, erythrocytic vaccines (EV) targeted to blood stages (especially merozoite invasion), and transmission blocking vaccine (TBV) that aim to block human-to-human transmission. One successful whole parasite vaccine is currently in preclinical stages using irradiated sporozoites.
This was the first and still the greatest protective immune response
generate initially shown in a rodent malaria model [221] and later in humans [222]. Many vaccine candidates are currently in clinical trials based on three antigens: the 32
circumsporozoite protein (CSP), the merozoite surface protein (MSP), and the apical membrane antigen (AMA) [223]. The most advanced of the vaccine candidates, termed RTS,S/AS02A, is based on the fusion of the CSP with the hepatitis B surface antigen (HBsAg), formulated with the AS02A adjuvant. In 2003, phase IIb clinical trial data demonstrated impressive results in which, P. falciparum clinical cases were reduced by 30% and severe cases by up to 58% [224]. Subsequent follow-up examination revealed equal protection up to 18 months later [225], and has recently entered phase III clinical trials throughout Africa [226].
Malaria Treatment, Chemoprophylaxis and Clinical Complications Malaria causes profound pathology in human victims. It is estimated that almost half of the world’s population is at risk of contracting malaria, and drug resistance reported in many parts of the world has raised alarm in health communities (map of global distribution Figure 6). This great concern is both in endemic and holoendemic regions, as well as in communities normally not exposed to malaria, such as tourists traveling to malarious regions [227, 228]. Treatment of malaria has traditionally relied on the use of antimalarial drugs. However, resistance has limited the number of effective drugs available and novel drug development has been stagnant. The lack of global initiative is reflected in the fact that from 1976 to 1996, only three of some 1,223 new drugs developed, were new antimalarials. The drug of choice since the 1950s has been chloroquine (CQ) as it was first introduced because it was highly effective, affordable, and non-toxic. Currently, CQ is ineffective in many regions [229-231]. This mainstay drug has largely lost its efficacy due to chloroquine drug resistance (CQR) in the treatment of P. falciparum. In response to this, a recent report released by the World Health Organization stated that the use of all artemisinin monotherapies be halted for the treatment of malaria [232]. Unfortunately, with so few new antimalarial drugs being developed, and the older commonly used drugs becoming ineffective, communities have resorted to the use of multiple drugs (or co-administration of drugs) as a means of treatment. Additional concern about drug
33
treatment has resulted from counterfeit drugs being used in endemic areas, resulting in additional unnecessary casualties [233].
Antimalarial Drugs Class I: Quinoline-based Drugs It is suggested that infusion from bark of the cinchona tree was used in South America dating back as far as 1638, when bark from the cinchona tree was used to cure nobility in Peru. This was commonly used as a treatment for fever before it was imported to Europe and introduced as an antimalarial in the 1800s. The active compound later extracted from the bark was found to be quinine. The most widely used antimalarial drugs to date are the class I quinoline-containing compounds:
chloroquine (CQ),
amodiaquine, quinine, quinidine (used only for severe cases of malaria due to its cardiac pharmacology), mefloquine and halofantrine. Their mode of action has been a long debated topic in the study of malaria biology [234-238]. The most widely accepted mechanism of CQ action is the interference with the detoxification of the heme moiety [239, 240]. Normally, IE malaria parasites detoxify the heme (FP) in the FV; CQ however interferes with this process, creating a toxic environment for the parasite [241, 242]. More recently, it has been shown that CQ forms a noncovalent complex with hematin [243]. Whether this action is shared completely with the other drugs in this class has not been definitively shown. CQ and amodiaquine are dibasic drugs that accumulate to very high concentrations in the acidic FV (Figure 7) [244], whereas mefloquine, quinine and halofantrine are monobasic and do not exhibit this same accumulation phenomenon. Class I drugs are not reliable against primary or latent liver stages or the gametocyte stage of P. falciparum, but will however treat or prevent the clinical symptomatic stages of malaria. Generally, the drugs must be taken for several weeks after exposure until the parasite has completed the liver stage and is susceptible to the therapy. Similarly, quinoline derivatives such as mefloquine and halofantrine are also schizonticides that share the same mechanism of action and interfere with the detoxification of the toxic heme group. Mefloquine, a quinoline methanol derivative, is used for oral prophylactic treatment of malaria caused by CQ-resistant (CQR) and multidrug-resistant strains of P. 34
falciparum [245]. Halofantrine is used to treat acute attacks of malaria [246]. These drugs are also effective against the blood stages of the other species of malaria that infect humans.
Antimalarial Drugs Class II, III: Atovaquone/Proguanil, Primaquine The drugs in class II widen the range of treatment, as these target not only the asexual erythrocytic forms but also the primary liver stages of P. falciparum and other species. This includes atovaquone and proguanil also known as Malarone, the only effective drugs left in parts of Asia where multidrug resistance is widespread [247]. Targeting various maturation stages of the parasite alleviates the extended treatment time, as required for class I drugs. Primaquine is also gametocytocidal for P. falciparum, and is one of the few drugs that are effective at this stage of the infection [248]. However, the side effects of the drug have been rather well documented and is not recommended for patients suffering from glucose-6-phosphate dehydrogenase deficiency [249].
35
It must be noted that information in Table 1 includes several generalizations. Firstly, prophylaxis is not fully achieved as none of the drugs kill sporozoites, it is therefore not truly possible to prevent infection, but rather prevent the development of symptomatic malaria, which is caused by the IE phase of the parasite. Secondly, in the treatment of an established infection, none of the drugs are effective against all liver and IE stages, and therefore the infection may require more than one drug to completely cure the infection.
Artemisinin and Derivatives As earlier noted, over the preceding decades the mainstay drug CQ has largely lost its efficacy for the treatment of P. falciparum due to CQR. There has therefore been a strong global push for the use of artemisinin multidrug treatments [250]. In addition to its use as an antimalarial drug, artemisinin has recently been shown to demonstrate anticancer properties, and therefore may have multiple uses clinically [251-255]. There is an urgent need for novel artemisinin derivatives. The global demand in 2005 for artemether-lumefantrine or ACT (artemisinin combination therapy) was 120 million adult treatments [256]. This translates into 114 tons of artemisinin, constituting ~70% of the current treatment cases. In light of this there has been a stress placed on the extraction of the artemisinin from Artemisia annua (A. annua) by attempting to improve horticulture practices, or by converting artemisinic acid into artemisinin using a largescale biotechnological method [257]. Artemisinin, as extracted from the Chinese herbal sweet wormwood A. annua, is active against malaria in the low nanomolar range. However, due to its poor extraction yield, and its poor solubility, three derivatives have been synthesized with even greater antimalarial activity. Artesunate, artemether, and arteether (reviewed in [258]), all possess greater bioavailability or improved oral potency.
All of the artemisinin
derivatives, however, have the major drawback of short half-lives (3-5 hrs), and therefore require multiple treatments over the course of a few days to achieve cure.
36
The search for novel artemisinin derivatives (trioxanes, trioxolane, tetraoxanes) has resulted in many novel drug candidates [259]. Many of these compounds demonstrate increased metabolic stability, and potency in vitro. Trioxolanes, also termed secondary ozonides, result from an ozonolysis reaction when exposing alkenes to ozone. The trioxolanes also termed next-generation endoperoxides have shown great antimalarial potency as described by Vennerstrom et al. [260]. This drug class is produced by fusing an adamatane ring onto the basic ozonide ring, and is active both in vitro and in vivo as demonstrated using the Plasmodium berghei mouse model. There has been some concern about the neurotoxic effect of the active metabolite, dihydroartemisinin, however in vivo data has not supported this speculation of a toxic side effect [261, 262]. The mechanism of action of artemisinin has been attributed to the 1,2,4 trioxane pharmacophore containing the endoperoxide bridge [263].
Consistent with this
mechanism of action, the antimalarial activity was abolished in the artemisinin derivatives void of the endoperoxide bridge [264]. Once inside the parasite, drugs are metabolized and decompose chemically in the presence of free heme-Fe(II), which results from parasite digestion of hemoglobin. In this process carbon-centered free radicals are created [265-267]. This irreversible redox reaction is believed to lead to the alkylation of heme [268] and several enzymes, including the P. falciparum sercoplasmic (sarco/endoplasmic reticulum) calcium-transporter ATPase (SERCA), PfATP6 [269]. The inhibition of PfATP6 activity by artemisinin and other synthetic endoperoxides is thought to lead to the death of the parasite. More recently Uhlemann et al., [270] have demonstrated that a single amino acid residue in TM segment 3 of PfATP6 is responsible for susceptibility to artemisinins.
This suggests a possible
mechanism of artemisinin resistance in vivo involving a single amino acid change in PfATP6. Experience with CQ and other antimalarials have taught us that caution must be used when administering any monotherapy. Recent reports have shown in vitro selection of artemisinin drug resistance [271, 272], which questions the long term efficacy for this highly potent drug, and the use of artemisinin-combination therapies (ACT’s) in the fight against malaria [273, 274].
37
Chloroquine Drug Resistance in Malaria The emergence of CQR strains of P. falciparum malaria appeared in the late 1950's and the phenotype has spread to all parts of the world where malaria is endemic (Figure 6) [230, 231, 275]. The evolution and spread of CQR has been described as a public health disaster [276]. Overall CQR has been found to be associated with the incomplete elimination of parasites from the blood stream and may be treated by simply increasing the dosage of drugs given, or administering combination antimalarials (e.g. ACT’s as described earlier).
In endemic areas where CQ is the main drug administered to
patients, resistance may occur due to prolonged subtherapeutic blood drug concentrations or irregular drug administration. In extreme cases of frequent relapses of malaria and evidence of multidrug resistance, patient treatment may be altogether unsuccessful.
CQR in P. falciparum is believed to have originated from four
independent loci in Asia and South America. Specifically, two mutational events are believed to have occurred, one in South America, and one in Southeast Asia and Papua New Guinea [230, 277]. Interestingly, Africa which absorbs the greatest number of fatalities to the disease is not believed to have originated CQR, as it is speculated that CQR was imported to Africa from Southeast Asia in the 1970’s. The complex genetics surrounding CQR leads to the understanding that several mutations are required to confer resistance. This is in sharp contrast with the resistance for other drugs, such as pyrimethamine, which only requires a single amino acid substitution in dihydrofolate reductase to confer drug resistance [278]. It bears mentioning that CQR exist for other species of malaria that infect humans, namely Plasmodium vivax (P. vivax). P. vivax is believed to cause as many cases of malaria as P. falciparum and resistance has been documented in both Southeast Asia [279] and South America [280]. This is less of a concern, however, as P. vivax is relatively benign as compared to P. falciparum which causes far greater human morbidity and mortality.
None the less, recent global
assessments have shown that the spread of P. vivax is far greater than anticipated, and may pose a greater threat in the future [281]. It is believed that the spread of CQR P. falciparum in Asia and South America is linked to the emergence of metapopulations (a population of populations where small 38
populations live in patches) vs. in Sub-Saharan Africa where communities already live in this population structure [282]. It is therefore speculated that the spread of CQR is facilitated by specific types of population structures.
This population structure
hypothesis includes multiple variables that affect the transmission of malaria, including the genetics of the parasite population, drug pressure, population size, inoculation rate (or transmission intensity), and the transmission pattern. Together, such data may be used to model the transmission of malaria in a population and trace the spread of CQR [283]. This facilitates the interpretation of field studies, as in French Guyana, where there is weak malaria transmission but high CQR P. falciparum, including multidrug resistant P. falciparum [284]. This is in contrast to rural Sub–Saharan Africa, where despite high transmission rates, the CQR frequency does not exceed 50 % [230, 285]. The spread of drug resistance may be due to additional vector variablities (vector numbers and species, and vector attractiveness to humans), switching of immunologically variable var genes in the parasite, and the selection of spontaneous resistant mutations. An additional source of mutations in CQR (or drug resistance) is mis-matching the half-lives of drugs administered as in the example of artesunate and sulphadoxine-pyrimethamine (SP) (half-lives of a few hours and 50 days respectively). This results in SP as a monotherapy for the remaining 50 days selecting for mutations in growing blood stage parasites [286, 287].
Multidrug Resistance With an increase in CQR, new compounds were introduced to endemic areas, including; mefloquine and halofantrine. Treatment of malaria with halofantrine in patients that had been administered mefloquine, was found to be less effective or failed completely due to cross-resistance to the two drugs (see Figure 6 for complete map of global drug resistance) [288, 289]. In a study by Draper et al. [290], patients exhibited relapses of malaria indicative of multidrug resistance. In this study the resistance to mefloquine and to amodiaquine (a 4-aminoquinoline) developed over the course of seven years. Initially, CQ was administered at a dosage of 5 mg/kg in 1977 but this increased to 10 mg/kg by 1979. No re-emergence of the parasites was noted until 1982, at which time 39
alternative drugs were given to patients. The quinoline-derivatives given as substitutes to CQ were effective only at higher doses (i.e. the minimum inhibitory concentration increased). This was indicative of a malaria parasite population that had developed a lower sensitivity to the drugs in the absence of selective drug pressure. This resistance to CQ increased the likelihood of the patients developing cross-resistance to other antimalarials [290]. Drug resistance however, does not come without consequences to the parasite, as CQR has been correlated with a fitness cost in reproductive capacity. Specific mutations in pfmdr1 a gene widely explored for its contribution to CQR affects parasite growth by up to 25%, as compared to wild-type. These and other results demonstrate that CQR negatively affects asexual parasite growth, gametocytogenesis and/or transmissibility [291-293].
40
Similarities Between MDR in Mammalian Cells and in P. falciparum Antimalarial drug resistance has been found to share similarities with the drug resistance phenotype displayed in cancer cell lines [294, 295]. These findings have led researchers to believe that there may be a common mechanism of drug resistance that exists. The characteristics found across a spectrum of CQR parasites include: 1) a decrease in drug accumulation (as compared to CQ sensitive [CQS] parasites); 2) an energy dependent drug efflux process [296]; and, 3) CQR may be reversed in vitro by the multidrug resistance (MDR) modulator verapamil [295].
The first two
characteristics are also found in cancer drug resistance and verapamil (a calcium channel blocker) reverses drug resistance in tumour cells expressing Pgp [47]. These similarities support the idea that the mechanisms of drug resistance in P. falciparum are similar to tumour cells. The relevance of this finding was called into question by experiments that showed that a loss of P. falciparum mdr overexpression did not increase CQ sensitivity. Additional evidence that link MDR in mammalian cancer cell lines and drug resistant P. falciparum is the presence of homologous ABCC1 or Pgp-like protein(s) in CQR parasites. Furthermore, both mammalian tumour cells and drug resistant strains of malaria display cross-resistance to drugs that differ structurally and functionally [288, 290]. However, it is not clear if resistance is acquired simultaneously or sequentially to different drug classes in malarial parasites. An early model proposed the mechanism of CQ action based on the weak-base trapping of diprotic CQ in the FV of the parasite [202, 244]. However, this model may not adequately explain the efficacy of different CQ side chain derivatives on CQR as well as CQS parasites [297-299]. Side chain functional groups are protonated upon entry into the acidic parasite FV (because of the reduced hydrophobicity of the drug), inhibiting the rate of diffusion of CQ back across the FV membrane. In mammalian cells, CQ diffuses across the endosomal membrane due to a pH gradient [300], a process which may also cause CQ to enter the parasite FV. The parasite FV and mammalian endosomal vesicle are both able to buffer a prototypical weak base, such as CQ, although the parasite is less efficient in buffering the intravesicular CQ. It is 41
believed that this results in a concentration and accumulation of CQ in the parasite FV (Figure 7). CQR is suggested to involve an ATP-dependent drug efflux mechanism since CQ efflux is successfully reversed when glucose is removed from culture or by the addition of the ATPase inhibitor vanadate. This suggests that CQ efflux is an energy-dependent process, requiring both the generation and the hydrolysis of ATP [301]. As earlier stated, parasite resistance to CQ and other antimalarial drugs is due to an increased efflux of drug out of the parasitized RBC.
It has been suggested that
transmembrane proteins causing drug resistance in cancer patients were also involved in drug- resistance in malaria parasites [275]. Other proposed models of drug resistance include:
1)
altered CQ uptake or efflux at the cytoplasmic membrane of the
intraerythrocytic parasite, 2)
altered H+ flux or CQ uptake at the parasite FV
membrane, 3) reduced CQ access to its receptor, hematin (formed in the FV after hemoglobin degradation); and 4) increased glutathione-mediated detoxification of CQhematin complexes. These models are believed to involve either: a Pgp homologue, a Cl- channel regulator, a Na+/H+ exchanger, a FV H+ pump (reduces the CQ access to hematin), and glutathione-S-transferase or a related molecule involved in heme detoxification [240, 302-304]. It is eminently possible that the trait is multi-genic, with some combination of all the above. The shared characteristics between the MDR phenomenon in tumour cells and drug resistant P. falciparum require further exploration; the objectives of this thesis will attempt to address some of these issues with a more in-depth examination of ATPdependent drug efflux mechanisms of drug resistance in malaria to follow.
The Mechanisms of Multidrug Resistance in P. falciparum As discussed in earlier sections, pleiotropic multidrug resistance (MDR) caused by ABC transporters in mammalian cells is characterized by a decreased drug accumulation, increased drug efflux and thirdly, the cells display cross-resistance to structurally different substrates [1, 305]. These same characteristics are seen in CQR strains of P. falciparum. Since Juliano and Ling first reported multidrug resistant 42
Chinese hamster ovary cells and linked this to an increase in Pgp levels [33], the protein has been found in many intrinsically drug-resistant tumours from the colon, kidney and adrenal gland [306-308]. In reflection of this work it was proposed that one of the mechanisms of CQR involved MDR homologues in parasites. Since then, studies have identified the presence of a Pgp homologues in P. falciparum [309, 310]. In total, three full-length ABC transporter genes have been characterized: two of which are Pgp-like homologues, P. falciparum multidrug resistance 1 (pfmdr1) [309, 310], pfmdr2 [311] and the third, a homologue of the yeast GCN20 gene, pfgcn20 [312].
ABC Transporter Genes in P. falciparum Of the three ABC transporter genes, pfmdr1 gene has been found to encode a 162-kDa protein termed P-glycoprotein homologue 1 (Pgh1). This protein product has been demonstrated to have a 54% genomic sequence similarity [309], and 33% amino acid identity with mammalian Pgp [313]. The protein shares a similar structure with Pgp in that it contains two homologous halves, each with 6 TMDs and a NBD. Localization studies have found that Pgh1 is located primarily in the membranes of the FV and to a lesser extent on plasma membranes [275]. It was hypothesized that the over expression and amplification of pfmdr1 would be found in CQR-strains of malaria, however this was not consistently the result in all CQR isolates [275, 309]; in vitro studies found a loss of pfmdr1 amplification in CQR-strains, resulting in an increased susceptibility to mefloquine, halofantrine, and quinine [314, 315]. Certain isolates of CQR parasites were shown to have increased pfmdr1 transcript levels, but later studies did not confirm the observation and revealed a second key transporter in CQR [316-318]. Sequence polymorphisms in pfmdr1 have been identified which are associated with CQR phenotypes in vitro, including the K1 allele at codon 86 involving a single amino acid change (Asn86Tyr), and the 7G8 allele which involves four amino acid changes (Tyr184Phe, Ser
1034Cys,
Asn
1042Asp, and
Asp
1246Tyr) [231]. The last three changes in the 7G8 allele
occur in TMD 11, which has been recognized as an important region for determining substrate specificity in mammalian Ppg (Figure 7) [319]. This variation in mutations is
43
believed to be linked to the various regions from which samples were obtained and how CQR might have evolved depending on the region of the field isolate.
44
Similar to all ABC transporters, Pgh1 shares a conserved structure of two-times-six TM segments coupled to a NBD fold and connected by a linker region [1, 275]. The above mentioned mutations are believed to have contributed to the multidrug resistance more than gene amplification since this is a more efficient means of transmitting the resistance genotype through a population.
Mefloquine resistance has clearly been
associated with pfmdr1 amplification and copy number, and additional cross-resistance to lumefantrine, halofantrine, quinine, and artemisinin [320, 321]. These results lead to the speculation that, although there may be an incomplete association of pfmdr1 and drug resistance, there in not an absence of association. In summary, pfmdr1 does not confer resistance, but rather may modulate resistance. To date, there is little evidence suggesting that pfmdr2 is involved in CQR in P. faliciparum. However, based on its structural similarity to heavy metal tolerance protein (HTM1), it is suggested that it may play a role in metal homeostasis in the parasite [322]. The plasmodial pfgcn20 gene is located on chromosome 11 and encodes a 95.5 kDa protein termed PFGCN20. The protein contains two ATP-binding folds, Walker A and B consensus regions, and an ABC signature sequence although no TMD’s have been found [312]. No data to date suggests the involvement in CQR, rather the proposed function of PFGCN20 is as a subunit of a multiunit ABC transporter or as a component of a plasmodial translation regulatory pathway [322].
Additional Transporter Genes Involved with CQR: pfcrt The correlation between Pgh1 polymorphisms and CQR is a debated topic due to the variation in mutations from parasite samples from different regions of the world. In field isolate studies of CQR-strains from Africa [323], Nigeria [324], Malaysia [325], Guinea-Bissau [326], Indonesia [327] and Thailand [328] there was some association between polymorphisms and CQR. Conversely, studies from Sudan [329], South America [330] and Southeast Asia [331-333], failed to identify any association between the intra-allelic variations and CQR. This great variation in genetic mutations of field isolates was further examined by a study from Wellems et al. [334] in which a genetic 45
cross between a CQS clone (HB3) and a CQR clone (Dd2) mapped CQR to a 36-kb locus on chromosome 7 [318]. Two genes found in this region, cg1 and cg2, exhibited polymorphisms that were proposed to account for the CQR phenotype. Further analysis of cg2 strongly suggested that this gene is involved in CQR, but the implicated allele was also found in one CQS parasite [317]. This indicated that there are additional mutations necessary for CQR, i.e. that the CQR phenotype is likely a polymorphic trait. Further analysis of the 36-kb locus of chromosome 7 revealed a novel, complex 13-exon polymorphic gene named P. falciparum CQR transporter (pfcrt).
The gene encodes an integral membrane protein PfCRT, with 10 TMDs,
localized to the FV of the parasite, but containing no motifs typical of an ABC transporter. It is believed that the 48.6 kDa protein is strongly associated with CQR [335, 336]. Specifically, a critical mutation in PfCRT
Lys
76Thr in the predicted first
TMD alters CQ efflux [337] or reduces the drugs binding to hematin [338]. Transfection studies reveal mutations in PfCRT confer verapamil-reversible chloroquine resistance in vitro [339, 340]. Additional bio-informatics studies have determined PfCRT to be a member of the drug/metabolite transporter (DMT) superfamily [341, 342], and has revealed that the protein may function as a dimer. This Lys
76Thr mutation located in the first TMD is also a region associated with substrate
specificity. Together, it has been demonstrated that PfCRT is a functional transporter that directly mediates the efflux of chloroquine from the digestive vacuole [336, 343].
Recent Developments and Future Outlook for Malaria Control Recent advances in malaria research have included the complete sequencing of the P. falciparum genome in November of 2002 [344]. This result was obtained despite the obstacle of the unusual compositional bias of malaria DNA (at >80% A+T), which creates instability of genomic fragments and difficulty in the assembly of contigs [345]. The Plasmodium data have been organized into PlasmoDB, a public database, that includes all recent sequencing outputs (see [346, 347]). The summary all of this work has resulted in a new initiative termed the World-Wide In Silico Docking On Malaria (WISDOM) project, launched in mid-2005 [348].
Genome analysis has already 46
revealed a number of novel candidate drug targets, many of which are shared with bacteria and/or plants but not mammals. One target in particular, is the apicoplast, (which has been found to be a specialized organelle of algal origin), which is important for lipid and heme biosynthesis [349]. An inhibitor of apicoplast DoxP isoprenoid biosynthesis, fosmidomycin, developed as an antibacterial drug, is showing efficacy in combination with clindamycin against P. falciparum malaria in limited trials [350]. Inhibition of additional pathways of interest, include:
phospholipid metabolism,
proteases involved in hemoglobin degradation, protein prenylation, and others (see [351, 352]). This information comes at a time when novel targets are greatly needed, since none of the four novel antimalarial drugs in phase III trials:
pyronaridine-
artesunate (PYROMAX®), artemether–lumefantrine (Coartem®), chloroproguanildapsone (LapdapTM)-artesunate (CDA), and dihydroartemisinin-piperaquine, affect novel targets (for full drug profiles and recent reports see http://mmv.org). No one can dispute the tremendous human and economic loss that is attributed to malaria. The situation is particularly grave in Sub-Saharan Africa where 1 million lives and 1 percentage point of economic growth is lost due to malaria every year [353]. Additionally, malaria rates fifth in disease burden of communicable diseases with DALYs (Disability adjusted life years: represents the loss of one year of equivalent full health) of 46.5 million [354]. The global fight to control malaria is however, well underway. The newly developed United Nations (UN) Millenium Development Goals (MDG) have outlined eight different goals targeting the eradication of hunger and poverty, the lack of primary education,
health,
environmental
issues,
women’s
rights
and
others
(http://www.unmilleniumproject.org). Specifically, goal number six aims to combat the “big three”: AIDS, tuberculosis, and malaria. Within this target, a sub-goal states that, “by 2015 we will have begun to reverse the incidence of malaria and other major diseases”. More specifically, this target includes providing insecticide-treated bed-nets, periodic indoor spraying with insecticides, and providing artemisinin combination treatment to those populations in need. There has been no time in history where there has been such a holistic approach taken to control malaria. It is with great optimism
47
that we enter the 21st century, exercising novel approaches to controlling the global incidence of malaria.
48
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[331] Basco LK, de Pecoulas, P.E., Le Bras, J., and C.M. Wilson. Plasmodium falciparum: molecular characterization of multidrug-resistant Cambodian isolates. Exp Parasitol 1996; 82:97-103. [332] Mungthin M, Bray PG, Ward SA. Phenotypic and genotypic characteristics of recently adapted isolates of Plasmodium falciparum from Thailand. Am J Trop Med Hyg 1999; 60:469-74. [333] Chaiyaroj SC, Buranakiti A, Angkasekwinai P et al. Analysis of mefloquine resistance and amplification of pfmdr1 in multidrug-resistant Plasmodium falciparum isolates from Thailand. Am J Trop Med Hyg 1999; 61:780-3. [334] Wellems TE, Panton LJ, Gluzman IY et al. Chloroquine resistance not linked to mdr-like genes in a Plasmodium falciparum cross. Nature 1990; 345:253-5. [335] Fidock D, Nomura T., Talley, A., et al. Identification of a Plasmodium falciparum gene (PFCRT) encoding a putative membrane protein linked to chloroquine resistance. In: Molecular Parasitology Meeting. Woods Hole, MA: 1999. pp. 12-16. [336] Fidock DA, Nomura T, Talley AK et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell 2000; 6:861-71. [337] Cooper RA, Ferdig MT, Su XZ et al. Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum. Mol Pharmacol 2002; 61:35-42. [338] Bray PG, Mungthin M, Hastings IM et al. PfCRT and the trans-vacuolar proton electrochemical gradient: regulating the access of chloroquine to ferriprotoporphyrin IX. Mol Microbiol 2006; 62:238-51. [339] Lakshmanan V, Bray PG, Verdier-Pinard D et al. A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance. Embo J 2005; 24:2294-305. [340] Sidhu AB, Verdier-Pinard D, Fidock DA. Chloroquine resistance in Plasmodium falciparum malaria parasites conferred by pfcrt mutations. Science 2002; 298:210-3. [341] Martin RE, Kirk K. The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Mol Biol Evol 2004; 21:193849. [342] Tran CV, Saier MH, Jr. The principal chloroquine resistance protein of Plasmodium falciparum is a member of the drug/metabolite transporter superfamily. Microbiology 2004; 150:1-3. [343] Bray PG, Martin RE, Tilley L et al. Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance. Mol Microbiol 2005; 56:323-33. [344] Gardner MJ, Hall N, Fung E et al. Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 2002; 419:498-511. [345] Hall NaGM. Malaria parasites: genome and molecular biology. England: Caister Academic Press; 2004. 7-31 p. [346] Birkholtz LM, Bastien O, Wells G et al. Integration and mining of malaria molecular, functional and pharmacological data: how far are we from a chemogenomic knowledge space? Malar J 2006; 5:110.
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[347] Stoeckert CJ, Jr., Fischer S, Kissinger JC et al. PlasmoDB v5: new looks, new genomes. Trends Parasitol 2006; 22:543-6. [348] [http://public.eu-egee.org/]. EGEE: Enabling Grid for E-sciencE. [349] Ralph SA, van Dooren GG, Waller RF et al. Tropical infectious diseases: metabolic maps and functions of the Plasmodium falciparum apicoplast. Nat Rev Microbiol 2004; 2:203-16. [350] Wiesner J, Borrmann S, Jomaa H. Fosmidomycin for the treatment of malaria. Parasitol Res 2003; 90 Suppl 2:S71-6. [351] Ridley RG. Medical need, scientific opportunity and the drive for antimalarial drugs. Nature 2002; 415:686-93. [352] Rosenthal PJ, Sijwali PS, Singh A, Shenai BR. Cysteine proteases of malaria parasites: targets for chemotherapy. Curr Pharm Des 2002; 8:1659-72. [353] Sachs J, Malaney P. The economic and social burden of malaria. Nature 2002; 415:680-5. [354] WHO. World Health Report 2004: Changing history. Geneva: World Health Organization. 2004; http://www.who.int/whr/2004/en.
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Chapter 2 ABCG2 Membrane Transporter in Mature Human Erythrocytes is Exclusively Homodimer
Mara L. Leimanis and Elias Georges Institute of Parasitology, McGill University, Quebec, Canada
Reproduced with permission from Biochemical and Biophysical Research Communications. 2007 Mar 9;354(2):345-50. Copyright 2007 Elsevier B.V.
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ABSTRACT The Human ABCG2 protein, a member of ABC transporter family, was shown to transport anticancer drugs and normal cell metabolites.
Earlier studies have
demonstrated the expression of ABCG2 in hematopoietic stem cells and erythroid cells; however little is known about the expression and activity of ABCG2 in mature erythrocytes.
In this report, we show that ABCG2 in mature human erythrocytes
migrates with an apparent molecular mass of 140-kDa, under reducing conditions, on Fairbanks SDS gel system.
In contrast, tumour cells expressing higher levels of
ABCG2 show no detectable homodimers, when resolved under identical reducing conditions. Analysis of the same membrane extracts from tumour cells and human erythrocytes on Laemmli SDS gel system, where samples are boiled in the presence of increasing concentrations of disulfide reducing conditions and then analyzed, migrate with an apparent molecular mass of 70-kDa or a monomer. Drug transport studies using Pheophorbide A, a substrate of ABCG2, show the protein to be active in erythrocytes. Furthermore, Fumitremorgin C, a specific inhibitor of ABCG2 increases the accumulation of Pheophorbide A in erythrocytes and drug resistant cells but not in the parental drug sensitive cells.
Given the ability of ABCG2 to transport
protoprophyrin IX or heme, these findings may have implications on the normal function of erythrocytes.
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INTRODUCTION The breast cancer resistance protein (or ABCG2, also known as the mitoxantrone resistant protein, and ABC placenta) is a member of the ATP-dependent binding cassette (ABC) family of transporters.
Similar to other well-characterized ABC
transporters, namely P-glycoprotein (P-gp1 or ABCB1) and Multidrug Resistance Protein 1 (MRP1 or ABCC1), the ABCG2 was initially discovered in a multidrugresistant cell line, MCF7/AdrVp [1]. ABCG2 has been shown to confer drug resistance in tumour cells, and to mediate the transport of anti-cancer drugs, including mitoxantrone, methotrexate, camptothecans (SN-38, topotecan) and flavopiridol (for recent reviews see [2, 3].
In normal tissue, ABCG2 is found in the canalicular
membrane of the liver, in the epithelia of the small intestine, colon, kidney, placenta, and sweat glands. More recently, ABCG2 expression has been found in hematopoietic stem cells. This latter “side population” of progenitor cells is representative of pluripotent stem cells. It has been suggested that the expression of ABCG2 protects this “side population” from cytotoxic substrates [4, 5]. Furthermore, increased expression of ABCG2 has been demonstrated in erythroid maturation and was shown to decrease intracellular protoporphyrin IX, a natural substrate of ABCG2 [6]. Consistent with these findings, erythrocytes from ABCG2 knock-out mice showed significant increase in intracellular protoprophyrin IX and a decrease in survival confirming further the protective role of ABCG2 in normal tissue [7]. Recently, ABCG2 has been implicated in the transport of heme, and was shown to enhance hypoxic cell survival through interactions with heme [8]. The human ABCG2, with a molecular mass of 70-kDa, encodes one transmembrane domain (TMD, with 6 transmembrane alpha-helices) and one nucleotide binding domain (NBD) all within a 655 amino acid primary sequence. This is in contrast with other ABC transporters (e.g., ABCB1 and ABCC1), which has led some to label ABCG2 “a half-transporter”. Consistent with the latter, two monomers are necessary to form a fully active ABCG2 transporter. Several reports have described ABCG2 as a functional homodimer, possibly as a homotetramer [9-14]. These latter studies have suggested that homodimerization occurs through inter-disulfide bonds between two
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ABCG2 monomers. Mutational analysis of all 11 cysteine residues in human ABCG2, including the three extracellular cysteines, Cys-592, 603 and 608, suggested that interdisulfide bridge at Cys-603 in the 3rd extracellular domain between two monomers is important, but not essential, for oligomerization of ABCG2 [15, 16]. The expression and protective effect of ABCG2 in hematopoietic stem cells and erythroid cells in normal tissues against toxic agents, is similar in action to other ABC transporters. Moreover, the enhanced expression of ABCG2 in hematopoietic stem cells has led some to speculate that a role for ABCG2 in preventing the accumulation of a differentiating factor in stem cells exists [4].
While others have observed the
expression of ABCG2 in Ter119+ erythroid precursors and natural killer lymphocytes [4]; however little is known about the expression and activity of ABCG2 in mature erythrocytes. In this report we demonstrate that ABCG2 is expressed in mature human erythrocytes isolated from at least eight different adults.
In these cells, ABCG2
migrated almost exclusively as 140-kDa protein on Fairbanks SDS gel system under reducing conditions. By contrast, ABCG2 from mitoxantrone-resistant tumour cells (MCF7/Mitox) migrated exclusively as a 70-kDa under identical Fairbanks gel system. Interestingly, analysis of ABCG2 from mature erythrocytes and MCF7/Mitox tumour cells using the Laemmli gel system, in the presence of reducing agent, migrates as a monomer (or 70-kDa protein). The significance of these finding in the normal transport functions of ABCG2 in erythrocytes is discussed.
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MATERIALS AND METHODS Materials - ABCG2-specific antibodies BXP-21 were purchased from Kamiya Biomedical Co. (Seattle, WA). Na+/K+-ATPase-specific monoclonal antibody were purchased from Sigma. All other chemicals were of the highest grade available Cell culture and plasma membrane preparation - Human breast carcinoma cells MCF7 and their drug-resistant clone, MCF7/Mitox, were grown in α-MEM media containing 10 % fetal calf serum (Bio Media). Plasma membranes were prepared as described previously [17]. Protein concentrations were determined by the Lowry method [18]. Eythrocytes ghosts were prepared as described previously [19]. Briefly, freshly drawn erythrocytes in the presence of sodium-citrate were first washed with PBS three times, and then run on a Ficoll gradient to remove leukocytes. Erythrocytes were washed three more times with ice-cold PBS prior to lysis in hypotonic buffer 5P8 (5 mM sodium phosphate, pH 8.0). Hemolysis was initiated by rapid mixing in the presence of protease inhibitors (2 mM PMSF). Lysed erythrocytes were centrifuged at 4 oC for 10 min at 14,000 rpm (16,000 x g) and the supernatant removed by aspiration. The latter wash was repeated four more times and the final pellet suspended in PBS with protease inhibitors. Western Blots - Plasma membranes from tumour cells (MCF7 and MCF7/Mitox cells) or erythrocytes (10-100 μg) were resolved by SDS-PAGE using the Fairbanks gel system [20] and transferred to nitrocellulose membranes using wet electroblotting as outlined by Towbin et al. [21]. The nitrocellulose membranes were blocked in 5 % fetal bovine serum, 5 % skim milk and 7.5 mM NaN3 in PBS, and incubated with various ABCG2-specific antibodies at varying dilutions (1:1000-1:3000 v/v) overnight at 4 oC. Membranes were washed, and incubated with varying dilutions (1:3000-1:6000 v/v) of goat anti-rabbit or mouse antibody conjugated to horseradish peroxidase. Immunoreactive proteins were visualized by chemiluminescence using Pico or Femto SuperSignal Substrate (Pierce). 75
Flow Cytometry Analysis - ABCG2 activity in MCF7 mammalian tumour cells was assessed by FACS analysis as previously described [22] with some modifications. Briefly, cells were washed twice with incubation buffer (HEPES 10 mM, NaCl 150 mM, KCl 5 mM, CaCl2 × 2H20 1.8 mM, MgCl2 × 6H20 1 mM, glucose 10 mM, pH 7.4). Freshly drawn human erythrocytes were collected by finger prick in EDTA-filled 0.5 mL eppendorf tubes and re-suspended in PBS and run on an equal volume Ficoll gradient (to remove leukocytes), and subsequently washed twice with incubation buffer. Cells MCF7 (0.5×106-1.0×106 cells/ml) and whole erythrocytes (0.5 % hematocrit) were incubated with 2 µl PhA with or without ABCG2 specific or non-specific inhibitors: 1-10 µM of FTC, 50 µM verapamil, 50 µM cisplatin, and incubated in 37 oC hot water bather for 30 min. Cells were then washed once with ice-cold incubation buffer and then incubated for 1 hr at 37 oC in PhA-free medium with ABCG2 specific or non-specific inhibitors. Following the efflux phase cells are washed once again with ice cold incubation buffer, resuspended in 1 mL of incubation buffer and kept on ice in the dark and analyzed immediately (within 30 min), using the FACS Aria flow cytometer (Becton Dickinson, CA). PhA fluorescence was measured with a 488-nm argon laser and a 670-nm filter. At least 10,000 events were collected for all of the flow cytometry studies, and by gating forward versus side scatter from a dot blot we were able to determine cellular debris and dead cells from our target population. Results are representative of at least two separate experiments done in triplicate, and calculated as % increase of mean channel fluorescence (MCF).
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RESULTS AND DISCUSSION Several reports have now shown that ABCG2 mediates the transport of normal cell metabolites, including protoprophyrin IX and heme [6, 7]. Given these latter findings, it was of interest to examine the expression of ABCG2 in normal tissue that contains high levels of heme, such as mature human erythrocytes. Figure 1A shows a Western blot of plasma membrane extracts from drug-sensitive (MCF7) or -resistant (MCF7/Mitox) tumour cells and human erythrocytes probed with ABCG2 specific monoclonal antibody, the BXP-21. The results in Figure 1A show a BXP-21 reactive polypeptide with an apparent molecular mass of 70-kDa in drug resistant tumour cells (MCF7/Mitox, lane 2) using a Fairbanks gel system. The parental drug sensitive cells (MCF7, lane 1) showed a much weaker signal for this 70-kDa polypeptide; while membrane extracts from several human erythrocytes (lanes 3-4) showed a significant expression of this 70-kDa protein.
These observations are consistent with the
expression of ABCG2 protein in breast tumour cells that have been selected for mitoxantrone resistance [23] from the drug-sensitive breast tumour cell lines MCF7. In addition, lanes 3 and 4 revealed a 140-kDa BXP-21 reactive protein that was not observed in membrane extracts from tumour cells (lanes 1 and 2).
The 140-kD
polypeptide has also been previously described as a homodimer of ABCG2 [13]. Hence, the assumption that the 70-kDa polypeptide is ABCG2 is consistent with: i) tumour cell lines selected for resistance to mitoxantrone express mostly ABCG2 [24]; ii) ABCG2 encodes 655 amino acid polypeptide migrates with a molecular mass of 72kDa on SDS PAGE [13, 15]; iii) BXP-21 is a well characterized monoclonal antibody raised against 126 amino acids polypeptide (271-396 a.a. of ABCG2) and binds to an intracellular epitope in ABCG2 [25]. Although equal amounts of membrane proteins were loaded in each of the lanes of Figure 1A, it was not possible to compare the loading between two membrane preparations derived from breast tumour cells (MCF7 and MCF7/Mitox) and mature erythrocytes. Consequently, a comparison was only possible between MCF7 and MCF7/Mitox or the two erythrocytes samples. Figure 1B shows the Western blot of the same resolved proteins as in Figure 1A probed with a monoclonal antibody specific to the alpha subunit of Na+/K+ ATPase (lanes 1 and 2 or 3 77
and 4, respectively).
The results of Figure 1B show 110-kDa polypeptide in
membranes from breast tumour cells
Figure 1- Detection of ABCG2 homodimer in mature erythrocytes. Plasma membranes from MCF7 parental and MCF7/Mitox drug resistant or erythrocyte ghosts (10-50 µg) were loaded onto Fairbanks gel system, and transferred onto nitrocellulose membranes. Panel 1A shows a Western blot probed with an ABCG2 specific monoclonal antibody, BXP-21. A 70-kDa reactive polypeptide is detected in MCF7/Mitox and to a much less extent in MCF7 parental cells (lanes 2 and 1, respectively). A 70-kDa reactive polypeptide is also detected in lanes 3 and 4 of Panel 1A which contain resolved membrane proteins from human erythrocyte (lane 3: South East Asian female, B+ blood type or lane 4: caucasian male, A+ blood type). Panel 1B shows the same Western blot as in 1A probed with a monoclonal antibody to Na+/K+-ATPase, hence confirming equal protein loading between lanes 1 and 2 or 3 and 4. Panel 1C shows a Coomassie blue stained gel of replicate samples as described in 1A. Densitometric 78
quantification of ABCG2 expression in mature erythrocyte membranes relative to MCF7/Mitox is shown in panel D and is represented by a histogram as mean ± S.D. of seven independent Western blot analyses. and mature erythrocytes, confirming equal loading between the parental and Mitox resistant tumour cells or the two different erythrocyte samples. Interestingly, membrane extracts from breast tumour cells expressed much higher levels of the Na+/K+ ATPase alpha subunit than from mature erythrocytes. To rule out the possibility that the BXP-21 reactive polypeptides (e.g., 70-kDa or 140kDa) is due to non-specific immuno-reactivity of BXP-21 mAb with abundant proteins in erythrocyte ghosts, total cell extracts from tumour cells and erythrocytes were resolved on SDS PAGE and stained with Coomassie blue. As expected, the results in Figure 1C show several highly expressed proteins, ankyrin and α-, β-spectrin, which migrates higher than the homodimer bands of ABCG2 (e.g., 210-kDa to 240-kDa versus 140-kDa) and band 3 which migrates significantly higher than ABCG2 monomer (e.g., 95-kDa versus 70-kDa). It should be mentioned that such cross-tissue comparison is rather difficult, especially where the above 4 proteins (α-, β-spectrin, ankyrin and band 3) account for the bulk of membrane proteins in erythrocytes. Hence, the expression of ABCG2 in erythrocytes is likely to be underestimated. Quantitative densitometry of ABCG2 immuno-reactive bands as determined by Western blots analyses of seven independent experiments revealed mature erythrocytes to express roughly 20% ABCG2 relative to MCF7/Mitox drug resistant cells (Figure 1D). The presence of the 140-kDa ABCG2 homodimer in erythrocytes, but not in tumour cells, under the Fairbanks denaturing conditions prompted us to examine other erythrocytes or blood donors, other than the two blood donors shown in Figure 1A. Figure 2 shows the results of a Western blot analysis of erythrocyte ghosts from five different subjects resolved on Fairbanks SDS PAGE and the Western blot probed with BXP-21 mAb (lanes 1-5, South Asian female, B+ blood type; caucasian male, O+ blood type; caucasian female, B+ blood type; caucasian female, B+ blood type; caucasian female, O+). The results in Figure 2 (lanes 1-5) demonstrate the presence of ABCG2 79
homodimer in all five erythrocyte samples. Hence, the presence of homodimer in erythrocytes appears to be independent of blood type or gender (Figure 2). Earlier studies have demonstrated gender-based differences in the expression of ABC proteins that result in gender-biased pharmacokinetics and ultimately contribute to variations seen in drug disposition and therapeutic response and drug toxicity (as reviewed by [26]).
Figure 2 – ABCG2 expression in erythrocytes from different ethnic groups and blood types. Briefly, erythrocyte ghosts (50 µg) were loaded onto Fairbanks gel system, and transferred onto nitrocellulose membranes. Blots were probed with anti-ABCG2 (e.g. BXP-21) mAb. Lanes 1-5 of the Western blot contain resolved proteins from South East Asian female, B+ blood type (lane 1); caucasian male, O+ blood type (lane 2); caucasian female, B+ blood type (lane 3); caucasian female, B+ blood type (lane 4); and caucasian female, O+ (lane 5). Anti-actin polyclonal antibody was used to estimate protein loading.
Panel B shows a histogram, showing the relative expression of
ABCG2 in different samples (lanes 1-5) relative to actin expression. 80
Our results in Figure 2 show no significant differences in ABCG2 expression levels based on the sample set tested (data not shown). In addition, we looked at different blood types to determine if any antigenic variability (or different blood types) different ethnic background could influence the expression levels of the ABCG2; again no significant differences were observed between donors of various racial groups (caucasian: n=8; South Asian: n=3; African: n=1). It has been previously shown that two ABCG2 monomers are required for active transport [10-12]. As such, analyses of ABCG2 on SDS PAGE under non-reducing conditions show a 140-kDa homodimer in membranes from drug resistant tumour cells. Indeed, a higher molecular weight band, in addition to that at 140-kDa of ABCG2 homodimer, has been shown to migrate with an apparent molecular mass of ~210-kDa (Figure 3) or a trimmer with speculation of even higher oligomerization [9]. The observation that while both tumour cells and mature erythrocytes expressed ABCG2 monomer, only erythrocyte membranes contained a homodimer, when membrane extracts were examined on Fairbanks SDS PAGE. ABCG2 contains 11 cysteine residues, including three extracellular cysteines, Cys-592, 603 and 608. Earlier reports have suggested that inter-disulfide bridge at Cys-603 in the 3rd extracellular domain between two monomers is important for oligomerization of ABCG2 [15]. However, given the role of disulfide bridges in ABCG2 oligomerization and functions, it was of interest to determine the effect of increasing concentrations of reducing agents on oligomerization of ABCG2.
To this effect, we examined the
increasing concentrations of DTT 50, and 100 mM on the mobility of ABCG2 on SDS PAGE, as a measure of ABCG2 oligomerization. The results of Figure 3 show that increasing concentrations of DTT causes the complete conversion of the dimer ABCG2 (e.g., 140-kDa polypeptide) into the monomer form (or a 70-kDa polypeptide). Taken together these results imply that the homodimer in erythrocytes is reduced under excessively high reducing conditions of 50 mM DTT or higher. The variation seen between MCF7/Mitox and erythrocytes has yet to further be explored; however, we speculate that the high oxidative conditions under which erythrocytes survive is likely to play a role in ABCG2 oligomerization, relative to ABCG2 in tumour cells. Indeed, 81
the results in Figure 3 show that in the absence of exogenously added reducing agent, both tumour and erythrocyte ABCG2 migrated mainly as homodimer. Consequently, one is left to speculate that in erythrocytes, ABCG2 homodimerization involves additional disulfides than those formed in ABCG2 extracted from tumour cells. The former ABCG2 (or erythrocyte ABCG2) homodimers are stable at low concentrations of DTT in Fairbanks gel system.
Figure 3 – Effects of increasing concentrations of DTT on homodimer/monomer pools of ABCG2 in MCF7/Mitox and erythrocyte membrane ghosts. Membrane extracts from MCF7/Mitox tumour cells and erythrocyte ghosts were incubated in the absence or presence of DTT (50 mM and 100 mM final). Membrane proteins (10-50 μg) were resolved on 7.5 % Laemmli gel and transferred to nitrocellulose membrane. Membranes were probed with anti-ABCG2 monoclonal antibody BXP-21. (RBC ghosts #1: caucasian female, B+ blood type; RBC ghosts #2: South East Asian female, B+ blood type). The arrows to the left of the figure show the mobility of the homodimer and monomer forms of ABCG2, migrating with apparent molecular masses of 140-kDa and 70-kDa, respectively. To determine if ABCG2 is active in erythrocytes, ABCG2-mediated transport in MCF7/Mitox resistant cells and whole fresh erythrocytes was analyzed by flow cytometry. Cells were incubated with PheA, a fluorescent substrate of ABCG2, in the 82
absence and presence of a specific inhibitor (FTC). The results in Figure 4 show that in the presence of FTC (up to 10 µM)), PheA retention is significantly (~60 %) increased in MCF7/Mitox cells.
Similar incubation of intact erythrocytes with PheA in the
presence of increasing concentrations of FTC shows a significant increase in PheA
Figure 4: Modulation of Pheophorbide A (PheA) transport. Drug sensitive and Mitox resistant MCF7 tumour cells and erythrocytes are incubated with 2 µM PheA for 30 min at 37 oC in the presence or absence of 1-10 µM fumitremorgin C (FTC). Cells were washed and incubated again for 60 min in PheA-free incubation medium, in the presence or absence of ABCG2 specific inhibitors. Drug accumulation was measured, by FACS analysis, as a function of total remaining fluorescence. Panels A-C show % change in mean fluorescence in MCF7, MCF7/Mitox, or erythrocytes in the presence of 83
increasing FTC relative to cells incubated with PheA alone. Values are means ± S.D. of at least one experiment done in triplicate. accumulation (~15 %), but to a lesser extent than in MCF7/Mitox and more than in MCF7 parental cells (Figure 4). It is interesting that our estimate of relative levels of ABCG2 expression in MCF7/Mitox and erythrocytes, roughly 1:4 folds, are consistent with the observed estimate of ABCG2 transport as measured by inhibition of ABCG2 transport with FTC and consequent increase in PheA retention (Figure 4). Taken together, our results demonstrate that ABCG2 in erythrocytes is functionally active and can mediate active transport. Future work will address the kinetics of ABCG2 transport in erythrocytes relative to tumour cells using inside out in vitro transport system. It is interesting to speculate in light of these results that although disulfide bridges appear important to the functions of ABCG2, the fine details of their role on ABCG2 functions remain elusive. For one thing, higher disulfide bonds between two homodimers, as predicted from these results in erythrocyte membranes, does not appear to inhibit ABCG2 function, nor does it cause hyper-activation of ABCG2 transport function. It could however, affect the turn over of the protein. Hence, it would of interest to determine the role of disulfide bridges on the relative stability or turn over of ABCG2 in different membranes from the same or different tissues.
ACKNOWLEDGMENTS: This work is supported by grant from the Natural Sciences and Engineering Research Council (NSERC) to EG. ML is a recipient of studentship from the Latvian Relief Society of Canada and the Latvian National Federation of Canada.
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REFERENCES [1] L.A. Doyle, W. Yang, L.V. Abruzzo, T. Krogmann, Y. Gao, A.K. Rishi, D.D. Ross, A multidrug resistance transporter from human MCF-7 breast cancer cells, Proc Natl Acad Sci U S A 95 (1998) 15665-15670. [2] E.M. Leslie, R.G. Deeley, S.P. Cole, Multidrug resistance proteins: role of Pglycoprotein, MRP1, MRP2, and BCRP (ABCG2) in tissue defense, Toxicol Appl Pharmacol 204 (2005) 216-237. [3] F. Staud, P. Pavek, Breast cancer resistance protein (BCRP/ABCG2), Int J Biochem Cell Biol 37 (2005) 720-725. [4] S. Zhou, J.D. Schuetz, K.D. Bunting, A.M. Colapietro, J. Sampath, J.J. Morris, I. Lagutina, G.C. Grosveld, M. Osawa, H. Nakauchi, B.P. Sorrentino, The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype, Nat Med 7 (2001) 1028-1034. [5] S. Zhou, J.J. Morris, Y. Barnes, L. Lan, J.D. Schuetz, B.P. Sorrentino, Bcrp1 gene expression is required for normal numbers of side population stem cells in mice, and confers relative protection to mitoxantrone in hematopoietic cells in vivo, Proc Natl Acad Sci U S A 99 (2002) 12339-12344. [6] S. Zhou, Y. Zong, P.A. Ney, G. Nair, C.F. Stewart, B.P. Sorrentino, Increased expression of the Abcg2 transporter during erythroid maturation plays a role in decreasing cellular protoporphyrin IX levels, Blood 105 (2005) 2571-2576. [7] J.W. Jonker, M. Buitelaar, E. Wagenaar, M.A. Van Der Valk, G.L. Scheffer, R.J. Scheper, T. Plosch, F. Kuipers, R.P. Elferink, H. Rosing, J.H. Beijnen, A.H. Schinkel, The breast cancer resistance protein protects against a major chlorophyll-derived dietary phototoxin and protoporphyria, Proc Natl Acad Sci U S A 99 (2002) 15649-15654. [8] P. Krishnamurthy, D.D. Ross, T. Nakanishi, K. Bailey-Dell, S. Zhou, K.E. Mercer, B. Sarkadi, B.P. Sorrentino, J.D. Schuetz, The stem cell marker Bcrp/ABCG2 enhances hypoxic cell survival through interactions with heme, J Biol Chem 279 (2004) 2421824225. [9] J. Xu, Y. Liu, Y. Yang, S. Bates, J.T. Zhang, Characterization of oligomeric human half-ABC transporter ATP-binding cassette G2, J Biol Chem 279 (2004) 19781-19789. 85
[10] C. Ozvegy, T. Litman, G. Szakacs, Z. Nagy, S. Bates, A. Varadi, B. Sarkadi, Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells, Biochem Biophys Res Commun 285 (2001) 111-117. [11] C. Ozvegy, A. Varadi, B. Sarkadi, Characterization of drug transport, ATP hydrolysis, and nucleotide trapping by the human ABCG2 multidrug transporter. Modulation of substrate specificity by a point mutation, J Biol Chem 277 (2002) 4798047990. [12] T. Janvilisri, H. Venter, S. Shahi, G. Reuter, L. Balakrishnan, H.W. van Veen, Sterol transport by the human breast cancer resistance protein (ABCG2) expressed in Lactococcus lactis, J Biol Chem 278 (2003) 20645-20651. [13] K. Kage, S. Tsukahara, T. Sugiyama, S. Asada, E. Ishikawa, T. Tsuruo, Y. Sugimoto, Dominant-negative inhibition of breast cancer resistance protein as drug efflux pump through the inhibition of S-S dependent homodimerization, Int J Cancer 97 (2002) 626-630. [14] T. Litman, U. Jensen, A. Hansen, K.M. Covitz, Z. Zhan, P. Fetsch, A. Abati, P.R. Hansen, T. Horn, T. Skovsgaard, S.E. Bates, Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2, Biochim Biophys Acta 1565 (2002) 6-16. [15] K. Kage, T. Fujita, Y. Sugimoto, Role of Cys-603 in dimer/oligomer formation of the breast cancer resistance protein BCRP/ABCG2, Cancer Sci 96 (2005) 866-872. [16] G.J. Poelarends, W.N. Konings, The transmembrane domains of the ABC multidrug transporter LmrA form a cytoplasmic exposed, aqueous chamber within the membrane, J Biol Chem 277 (2002) 42891-42898. [17] C. Kast, P. Gros, Topology mapping of the amino-terminal half of multidrug resistance-associated protein by epitope insertion and immunofluorescence, J Biol Chem 272 (1997) 26479-26487. [18] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the Folin phenol reagent, J Biol Chem 193 (1951) 265-275.
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[19] T.L. Steck, R.S. Weinstein, J.H. Straus, D.F. Wallach, Inside-out red cell membrane vesicles: preparation and purification, Science 168 (1970) 255-257. [20] G. Fairbanks, T.L. Steck, D.F. Wallach, Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane, Biochemistry 10 (1971) 2606-2617. [21] H. Towbin, T. Staehelin, J. Gordon, Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications, Proc Natl Acad Sci U S A 76 (1979) 4350-4354. [22] R.W. Robey, K. Steadman, O. Polgar, K. Morisaki, M. Blayney, P. Mistry, S.E. Bates, Pheophorbide a is a specific probe for ABCG2 function and inhibition, Cancer Res 64 (2004) 1242-1246. [23] O. Alqawi, S. Bates, E. Georges, Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding, Biochem J 382 (2004) 711-716. [24] K. Miyake, L. Mickley, T. Litman, Z. Zhan, R. Robey, B. Cristensen, M. Brangi, L. Greenberger, M. Dean, T. Fojo, S.E. Bates, Molecular cloning of cDNAs which are highly overexpressed in mitoxantrone-resistant cells: demonstration of homology to ABC transport genes, Cancer Res 59 (1999) 8-13. [25] M. Maliepaard, G.L. Scheffer, I.F. Faneyte, M.A. van Gastelen, A.C. Pijnenborg, A.H. Schinkel, M.J. van De Vijver, R.J. Scheper, J.H. Schellens, Subcellular localization and distribution of the breast cancer resistance protein transporter in normal human tissues, Cancer Res 61 (2001) 3458-3464. [26] M.E. Morris, H.J. Lee, L.M. Predko, Gender differences in the membrane transport of endogenous and exogenous compounds, Pharmacol Rev 55 (2003) 229-240.
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Connecting Statement 1
In order to understand drug-protein interactions, one must first characterize the protein. Some of the biochemical characteristics of ABCG2 in mature human erythrocytes were explored in the previous chapter. For other ABC multidrug transporters that have been well characterized, the challenge becomes to understand the complex biochemical nature of the multidrug binding site with its many substrates. ABCC2 is primarily expressed in the liver, and like other MDR transporters, it has multiple substrates. It was of interest to further characterize the protein and its interactions with a photoactive, radiolabelled compound IAALTC4. Based on transport assays, LTC4 is thought to interact with ABCC2, however in this chapter we show direct binding of the drug to ABCC2.
Additionally, we elucidate the binding competition profile of other
endogenous and exogenous substrates: GSH, Quercetin, and MK571.
Lastly, we
demonstrate a drug binding domain for the drug. Together, these results contribute to our understanding of the complex mutidrug binding site in ABCC2, a protein shown to contribute to both detoxification (ex:
patients lacking ABCC2 suffer from
hyperbilirubinemia), and chemoprotection.
88
Chapter 3 Characterization of LTC4 Binding to ABCC2 Membrane Transporter
Mara L. Leimanis, Joel Karwatsky, Elias Georges
Institute of Parasitology, McGill University, Quebec, Canada (In preparation)
89
ABSTRACT A photoreactive analog of LTC4 (IAALTC4) was used to elucidate the drug binding characteristics of MRP2 (ABCC2, cMOAT).
Binding studies revealed specific
photoaffinity labeling of IAALTC4 to MDCKII-ABCC2 transfected cells. This result was supported by probing the plasma membrane with an ATPase specific antibody, which was found to be negative for any cross-reactivity (with other membrane ATPase). Photoaffinity labeling was reduced up to 75 % using 50 µM of MK571, a well characterized ABCC2 inhibitor. Unmodified LTC4 enhanced the binding, whereas GSH and Quercetin only mildly inhibited the binding. Based on the trypsin digestion pattern of the photolabeled ABCC2 protein we conclude that there is a high affinity binding site for IAALTC4 in the C-terminal region of ABCC2.
Furthermore, MK571 and LTC4
share dissimilar binding sites. Together these results should aid in understanding the binding and transport characteristics of this ABC transporter.
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INTRODUCTION ABCC2 is a member of the ABC superfamily of membrane transporters thought to mediate the movement of endogenous metabolites and xenobiotics across the cell membrane. ABCC1, 2 and 3 (or MRP1-3) are shown to mediate the transport of a wide variety of organic anions and compounds that are conjugated with sulfate, glucuronate or glutathione (GSH) (reviewed in [1, 2]). ABCC1 was shown to cause the transport of leukotriene C4 (or LTC4) and has been implicated in inflammatory diseases [1, 3]. A related transporter, ABCC2 or MRP2, is associated with Dubin-Johnson syndrome [4]. In rats, ABCC2 is thought to mediate hepatobiliary excretion of numerous organic anions and has been implicated in hyperbilirubinemia [5-7], in addition to its role in multidrug resistance (MDR) to anti-cancer drugs [8]. However unlike ABCC1 which is expressed in most tissues, the tissue distribution of ABCC2 is limited to the apical surface of the epithelial cells of the hepatocytes, lungs, kidney, and colon [11-13]; consistent with a role in protecting normal tissues from toxic metabolites and xenobiotics (reviewed in [9, 10]). ABCC2 therefore, plays a key role in regulating liver detoxification from metabolites into the bile. Several reports have now demonstrated the efflux of LTC4, a potent mediator of smooth muscle contraction and an important mediator in inflammation [9-11]. In a study by Keppler et al. up to 77 % of [3H]LTC4 was excreted in the bile using a rat model, hence LTC4 is primarily metabolized by the liver and eliminated in the bile [12]. ABCC2, similar to ABCC1 and 3, encodes three hydrophobic
transmembrane
domains
(TM0,
TM1
and
TM2)
with
5+6+6
transmembrane helices and two hydrophilic domains each encoding an ATP binding cassette (or ABC) [2, 13-15]. In addition to this topologic homology with ABCC1, ABCC2 shares 48% sequence identity with ABCC1. Collectively, the sequence and structural homology between ABCC1 and 2 has been taken to imply functional homology and substrate specificity. LTC4, a normal cell metabolite is one of the highest affinity substrates for ABCC1 with Km of ~100 nM [16-18]. Similar to ABCC1, LTC4 is also a high-affinity substrate for ABCC2 with a Km of 1.0 μM [19], a roughly 10-fold higher Km [2]. However, unlike ABCC1, direct binding between LTC4 and ABCC2 has not been demonstrated. Earlier 91
photoaffinity labeling studies [20] using IAALTC4 had demonstrated its binding specificity to ABCC1. Moreover, IAALTC4 was shown to bind several sites in ABCC1 that have been previously shown to be important for drug binding and transport of LTC4 and other drugs [20-22]. To further characterize ABCC2 drug interactions we studied its interaction with LTC4 utilizing a photoreactive analogue, aryl azido leukotriene C4 (AALTC4).
The results of this study are the first demonstration of direct binding
between LTC4 and ABCC2.
92
MATERIALS AND METHODS Materials- Iodine-125 (100 mCi/ml) and Protein-A Sepharose were purchased from Amersham Biosciences (Baie D’Urfe, Montreal, Quebec).
NHS-ASA, and all
chemiluminescent substrates were purchased from Pierce (Rockford, IL). Trypsin was purchased from Gibco-BRL (Burlington, Ontario). Leukotreine C4 was purchased from Biomol (Plymouth Meeting, PA). The anti-ABCC2 (M2III-6) and anti-ABCC1 QCRL-1 mAb’s were purchased from Kamiya Biomedicals Company (Seattle, Washington). The ABCC1 pAb was generated against a C-terminal peptide as previously characterized earlier [23]. The Na+/K+ ATPase (α-subunit-Clone M7-PB-E9) Ab and the protease inhibitor cocktail were purchased from Sigma (St. Louis, MO).
All
chemicals used were of the highest grade available. Cell Culture, Plasma Membrane and Vesicle Preparation- HeLa, and HeLa-ABCC1 transfected cells and MDCKII (Madine Darby Canine Kidney cells) wild-type and with ABCC2 transfectant were grown in α-MEM media containing 10 % fetal calf serum (Bio Media, Canada).
Plasma membrane preparations were prepared as described
previously [24]. Briefly, cells were grown to 90 % confluency and harvested in cold phosphate buffered saline pH 7.4 (PBS). The cells were washed three times for five minutes in ice cold PBS at 230 x g. The cell pellet was re-suspended in hypotonic buffer [10 mM KCl, 1.5 mM MgCl2 and 10 mM Tris-HCl buffer (pH 7.4)] supplemented with protease inhibitor cocktail (dilution of 1:500) and left on ice for 30 min. The cell pellet was homogenized (Dounce homogenizer, 150 strokes) and then the cell sample was centrifuged at 10,000 x g for five minutes at 4 oC to remove any unlysed cells and nuclei. Supernatant fraction was spun at 100,000 x g for 60 min to obtain the crude membrane fraction. Final membranes were re-suspended in labeling buffer (5 mM Tris-HCl, pH 7.4, 250 mM sucrose). For vesicle preparation, plasma membrane pellet was passed through a 27-gauge needle 30 times.
Protein
concentrations were determined by the Lowry method [25], plasma membranes and vesicles were stored at -70 oC.
93
IAA-LTC4 Synthesis, Iodination and Photoaffinity labelling of Plasma MembraneThe synthesis of a photoreactive analogue of LTC4, aryl azido- LTC4 (AALTC4), was done as previously described [24, 26] with some modifications. Purified AALTC4 was characterized by MALDI-mass spectrometry, dried and stored at -70 oC.
For
iodination, AALTC4 was re-suspended in 10 µl of 100 % methanol to which 100 µl of 13.18 mM Chloramine T was added along with 2-5 mCi (100 mCi/ml) of carrier-free [125Na]. The reaction was allowed to proceed for 5 min at room temperature. The reaction was arrested with the addition of 100 µl of 263 mM sodium metabisulfate. in 0.01 M Na-PO4, pH 8.5.
Reaction mixture was collected and loaded onto pre-
equilibrated and washed C-18 Sep-Pak cartridge as previously described [20, 32]. The iodinated-AALTC4 (IAALTC4) was eluted with 100 % methanol and dried. IAALTC4 was re-suspended in a final volume of 100-200 µl LTC4 buffer (35 % ammonium acetate buffer, 65 % methanol, pH 5.6). Plasma membrane aliquots expressing ABCC1 or 2 proteins were photoaffinity labelled with IAALTC4, (1.0 µM), in the absence or presence of increasing concentrations of various anticancer drugs as previously described [21, 24, 27]. SDS PAGE and Immunoblotting- Photoaffinity labelled plasma membranes were solubilized in Fairbanks buffers I and II (BI: 10 mM Tris HCl, pH 8.0 containing 40 mM dithiolthreitol (DTT), 1 mM EDTA, 2 % SDS and BII: 2 x BI and 10 M urea). The photolabelled proteins were then resolved on SDS-PAGE using the Fairbanks gel system [28] and gels were either stained with Coomassie blue or transferred to nitrocellulose membrane for Western blotting according to the method of Towbin et al. [29]. Immuno-detection of ABCC1 or 2 was achieved by probing the nitrocellulose membranes with ABCC1 or 2 specific antibodies (ABCC1 pAb [23] recognized the last 15 amino acids in the.a. C-terminal, ABCC1 mAb QCRL-1 recognizes amino acids 918-924 [30] region, and clone M2III-6 recognizes the 202-C-terminal amino acid sequence of ABCC2) at recommended dilutions of 1:5000 (v/v) for ABCC1 pAb, and 1:125 (v/v) for M2III-6. Membranes were reincubated with goat anti-mouse or antirabbit antibody conjugated to horseradish peroxidase. Immuno-reactive proteins were 94
visualized by chemiluminescence using Pico or Femto SuperSignal Substrate from Pierce (Rockford, IL). Proteolytic Digestion and Immunoprecipitation- To determine the trypsin digestion sites in the ABCC2, plasma membranes with or without photoaffinity labeling with IAALTC4 were incubated with increasing concentrations of sequence grade trypsin at 37 oC for 40 min. Samples were placed on ice to terminate digestion and protease inhibitor cocktail was added (1:20). Photolabelled plasma membrane were trypsin digested and immunoprecipitated using the ABCC1, 2 or Na+/K+ ATPase Ab, as previously described [31]. The immunoprecipitated proteins are then resolved on SDSPAGE using the Fairbanks gel system [28]. Gels with radiolabelled proteins were fixed (ddH20/methanol/glacial acetic acid (50:40:10)), dried and exposed to Kodak BIOMAX MS films at -70 oC.
95
RESULTS To characterize the drug binding of ABCC2, we made use of a photoactive, iodinated LTC4 drug analog (e.g., IAALTC4; Figure 1) previously used to study ABCC1 drug binding [20]. Earlier studies [19] had shown that ABCC2 mediates the transport of LTC4, however direct evidence for ABCC2-LTC4 interaction is lacking. The results in
Figure 1: Chemical structure of iodo-aryl-azido-leucotriene C4 (IAALTC4) Figure 2 show the photoaffinity labeling of plasma membranes from un-transfected control and ABCC2 transfected MDCKII cells (or MDCKIIABCC2) which have been previously described [32]. Figure 2A shows that IAALTC4 bound directly to a 190-kDa protein (lane 2) in MDCKIIABCC2 plasma membrane that was immunoprecipitated specifically with ABCC2-specific monoclonal antibody (e.g., M2III-6 mAb, directed to the C-terminal 202 amino acid; [33]). Photoaffinity labeling of plasma membrane from MDCKII cells with IAALTC4, and subsequent immuunoprecipitation with ABCC2specific mAb did not reveal a 190-kDa photolabeled protein (lane 1, Figure 2A). The absence of 190-kDa photoaffinity labeled protein in membranes from MDCKII cells (lane 1, Figure 2A), together with the apparent molecular mass of the photoaffinity labeled protein from MDCKIIABCC2 cells are consistent with the identity of the 190-kDa as ABCC2 (lane 2, Figure 2A). To rule out the possibility that plasma membranes from MDCKIIABCC2 cells expressed large amounts of a 190-kDa protein that was non96
specifically photoaffinity labeled with IAALTC4, Figure 2B shows a Coomassie Blue
Figure 2:
Direct photolabeling of IAALTC4 to MDCKII and MDCKII/ABCC2.
IAALTC4 photolabeling is detected by immunoprecipitation in lanes 1 and 2 using MDCKII and MDCKII/ABCC2 plasma membranes (20 µg) using an MRP2-specific mAb M2III-6, followed by Coomassie blue staining in lanes 3 and 4 of Figure 2A. In Figure 2B a saturation curve is demonstrated showing the saturable photoaffinity labeling of IAALTC4 to ABCC2. Where not otherwise specified ~1 µL of IAALTC4 was used. staining of proteins from MDCKII and MDCKIIABCC2 cells (lanes 3 and 4, respectively). The results in Figure 2B show similar levels of protein expression (as revealed by Coomassie blue staining) in MDCKII and MDCKIIABCC2 cells; consistent with a specific photoaffinity labeling of ABCC2 by IAALTC4. The presence of a photoaffinity labeled protein migrating higher than the 190-kDa protein has been previously observed and is due potentially to differential conformation of 97
transmembrane protein. Indeed, such apparent differential mobility of ABC proteins on SDS PAGE appears to be enhanced in the presence of the IAALTC4 and is not entirely clear.
To confirm the specificity of ABCC2 photoaffinity labeling, fixed
amounts of plasma membranes from MDCKIIABCC2 ABCC2-transfected MDCKII cells were photoaffinity labeled with increasing concentrations of IAALTC4 (1 to 8 µM). Figure 2C shows saturable photoaffinity labeling of a 190-kDa with increasing concentrations of IAALTC4. We have previously used the same photoractive analogue of LTC4 (IAALTC4) to show a direct binding to ABCC1 in plasma membranes expressing ABCC1 [20]. Hence, the specificity of IAALTC4 towards ABCC1 in HelaABCC1 cells was used as a positive control [21, 27]. The results of Figure 3B (lanes 1 and 2) show the photoaffinity labeling of plasma membranes from HeLa and HeLaABCC1cells, showing a direct and specific photolabeling of ABCC1.
Interestingly, however, IAALTC4 photoaffinity
labeled ABCC1 more intensely or with higher affinity, than ABCC2 (lane 2 versus lane 6, respectively). This dramatic difference in photoaffinity labeling of ABCC1 and ABCC2 by IAALTC4 is consistent with earlier LTC4 binding studies [19] demonstrating ABCC1 to have a higher affinity than ABCC2 for LTC4 (e.g., Km of ~100 nM versus 1.0 μM, respectively). Given this differential affinity of ABCC1 and ABCC2 towards LTC4, it was important to show that photoaffinity labeling of ABCC2 was due to specific protein-drug interactions and not due to the mere presence in the lipid bilayer.
To rule out the latter possibility, it was of interest to determine if
IAALTC4 photoaffinity labeled a ubiquitously expressed transmembrane protein, for example the Na+/K+ ATPase subunit in both membrane preparations. The results in Figure 3A (lanes 3-4 and 7-8) do not show the photoaffinity labeling of the Na+/K+ ATPase subunit large subunit, consistent with specific, but low affinity, of ABCC2 with IAALTC4. Figure 3B shows the expression of ABCC1 and ABCC2 in HelaABCC1 and MDCKIIABCC2 cells and Na+/K+ ATPase subunit (lanes 1-8, respectively). Differences in the apparent expression of ABCC1 and ABCC2 in HelaABCC1 and MDCKIIABCC2 cells, as determined by Western blotting with their respective antibodies (ABCC1QCRL-1, ABCC2- M2III-6) is not readily explainable as differences in the apparent 98
signal of these two proteins are likely due to differences in the affinities of antibodies to each protein rather than quantitative differences in protein expression (Figure 3A).
Figure 3: Specificity of IAALTC4 binding to ABCC2 as detected by western blot and immunoprecipitation. In lanes 1 and 2 HeLa, HeLa/ABCC1 plasma membrane are probed the ABCC1 polypeptide Ab, and same membrane re-probed with the Na+/K+ ATPase mAb, as shown in lanes 3 and 4. Lanes 5 and 6 of Figure 3A are MDCKII and MDCKII/ABCC2 plasma membranes respectively, as probed with the ABCC2-specific mAb, M2III-6. ABCC2 is detected at the 190-kDa range. This same blot was re-probed with a Na+/K+ ATPase mAb and similarly lanes 3, 4 (HeLa, HeLa/ABCC1) and lanes 7, 8 (MDCKII and MDCKII/ABCC2) the Na+/K+ ATPase is detected in the 110-kDa range. In Figure 3B an immunoprecipitation is demonstrated using several antibodies. In lanes 1 and 2 HeLa, HeLa/ABCC1 plasma membrane is used with the ABCC1 polypeptide Ab. In lanes 3 and 4 we have the same membrane as probed with the Na+/K+ ATPase mAb.
Lanes 5 and 6 MDCKII and MDCKII/ABCC2 plasma
membranes are probed with the ABCC2-specific mAb M2III-6. Lanes 7 and 8 are MDCKII and MDCKII/ABCC2 probed with the Na+/K+ ATPase mAb. A figure legend is shown for further visual clarification. 99
To determine if IAALTC4 photoaffinity labels ABCC2 at a physiologically relevant site, plasma membranes from MDCKIIABCC2 cells were photoaffinity labeled with IAALTC4 (1 uM) in the presence of increasing concentrations of normal cell metabolites and drugs. The results of Figure 4A show photoaffinity labeling of ABCC2 in the presence of increasing concentrations of unmodified LTC4 (2.5-fold and 25-fold molar excess). Interestingly, and by contrast to the photoaffinity labeling of ABCC1
Figure 4: IAALTC4 competition experiment using substrates of ABCC2. Using both endogenous and exogenous compounds as competitive substrates as seen in Figure 4A, LTC4 (endogenous), MK571 (exogenous), GSH (endogenous), and Quercetin (exogenous) we see variation of inhibiting potential. LTC4 was competed at 2.5 and 25 molar excess, MK571 was competed at 25 and 50 molar excess, and both GSH and Quercetin were competed at 25 and 250 molar excess. MDCKII and MDCKII/ABCC2 plasma membranes are immunoprecipitated with the ABCC2-specific mAb M2III-6. Competition experiments are summarized as the relative intensity of the bands (measured as % relative to control) as seen in Figure 4B. At least 2 experiments done in duplicate are represented in Figure 4B. 100
with IAALTC4 [20], the increasing concentrations of LTC4 caused a significant increase in the photolabeling of ABCC2 by IAALTC4. Although not entirely clear, it is likely that the observed increase in ABCC2 photoaffinity labelling in the presence of molar excess of LTC4 increases the affinity of the protein to LTC4, including IAALTC4 and consequently this translates into higher photoaffinity labeling (~200 % relative increase; Figure 4B). These results are in contrast with those obtained with ABCC1 using the same photoreative analogue of LTC4, where increasing concentrations of LTC4 inhibited or caused a marked reduction in the photoaffinity labeling of the protein [20]. Photoaffinity labeling in the presence of molar excess of MK571 (25-fold and 50-fold) shows a significant decrease in ABCC2 photoaffinity labeling (~75 % relative decrease, Figure 4B). Together, these observations suggest for the first time different binding domains for LTC4 and MK571 on ABCC2. Indeed, it is not entirely clear if the same is true for ABCC1 as both LTC4 and MK571 are thought to inhibit ABCC1 binding and transport to normal cell metabolites and drugs. Of considerable interest are the findings that 25-250 fold molar excess of GSH and Quercetin, the most abundant dietary flavanoid, have no significant effect on ABCC2 photoaffinity labeling by IAALTC4 (Figure 4). Earlier studies using photoreactive drugs and mutational analysis have lead to the identification of specific domains in ABC proteins as sites for protein-drug interactions [34-36]).
Using photoreactive analogues, we have previously identified several
domains in ABCC1 as possible drug binding sites [20-22]. In this study and in an attempt to elucidate IAALTC4 binding domain(s), ABCC2 was photoaffinity labeled with IAALTC4 and subjected to limited trypsin digestion prior to immunoprecipitation. The digested and photoaffinity labeled polypeptides were immunoprecipitated with ABCC2 mAb (M2III-6) which recognizes the C-terminal 202 amino acids of ABCC2 [33]. Figure 5A, shows an immunoblot of ABCC2 digested with increasing amounts of trypsin and probed with M2III-6 mAb. The results in Figure 5A show three major and four minor tryptic polypeptides of ABCC2 that contain M2III-6 epitope, migrating with apparent molecular masses of 72-kDa, 50-kDa, 25-kDa and 115-kDa, 45-kDa, 40-kDa, 101
and 37-kDa
(see topological representation).
Figure 5B shows the results of
immunoprecipitation of ABCC2 photoaffinity labeled protein following tryptic digestion. Two IAALTC4 photoaffinity labeled tryptic peptides with, apparent molecular masses of 70-kDa and 40-kDa, are immunoprecipitated with M2III-6 mAb.
Figure 5:
Digestion experiments are demonstrated using the MDCKII and
MDCKII/ABCC2 plasma membranes as probed with the ABCC2-specific mAb M2III6. In Figure 5A we show a western blot of ABCC2 peptides as generated by limited trypsin proteolysis. Similarly in Figure 5B and C ABCC2 peptides are generated by trypsin digest and are detected using immunoprecipitation. A topological representation of the digested peptides is represented in the diagram whereby the size and the location of the peptides are shown.
102
The 70-kDa peptide, one of three major tryptic polypeptides, is intensely photolabelled (Figure 5B) and encodes the C-terminal ATP binding domain and the entire TMD2 (Figure 5C). The 70-kDa peptide represents a sensitive tryptic site in the charged linker sequence (L1) between TMD1-NBD1 and TMD2 (Figure 5C). A similar protease sensitive site has been previously observed in ABCC1 and ABCB1 [21, 26, 27]. This protease sensitivity in the L1 linker domain is thought to be due to the presence of R and K amino acids, in addition to being accessible or exposed sequences. The second major photolabeled peptide (e.g., 40-kDa) encodes the C-terminal ATP binding domain and two transmembrane helices (eg., TM16-17) of TMD2 (Figure 5C; [20-22]. This peptide appears to be very labile relative to the 70-kDa peptide and several nonphotoaffinity labeled peptides. With respect to the latter, a major tryptic peptide of ABCC2 is a 25-kDa peptide which encodes the C-terminal ATP-binding domain, minus TM16-17).
103
DISCUSSION It is well established that ABCC2 plays a role in maintaining homeostasis in cells of the liver by hepatobiliary excretion of numerous organic anions. In addition, in cases where ABCC2 expression is low or absent serious clinical conditions may ensue such as hyperbilirubinemia. Earlier studies have shown ABCC2 mediates the transport of LTC4 [24], however it was not known if LTC4 binds to ABCC2 and how various other normal metabolites modulate LTC4 binding. We have previously synthesized a photoreactive analogue of LTC4 (IAALTC4) and used it to characterize its binding kinetics and sites in ABCC1 [20]. Using this previously characterized photoreactive analogue of LTC4, we now demonstrate for the first time a direct and specific binding of LTC4 to ABCC2. However, as expected IAALTC4 appears to photolabel ABCC2 with lesser intensity than ABCC1. The reduced photoaffinity labeling of ABCC2 relative to ABCC1 is consistent with earlier results showing roughly a 10-fold lower affinity of ABCC2 to LTC4 [2], however this result may also reflect the affinity of the Ab’s for their respective epitopes. Also consistent with ABCC2 having lower affinity towards LTC4, the addition of cold unmodified LTC4 increases the photoaffinity labeling of ABCC2. It is not clear what function ABCC2 transport of LTC4 serves in light of a much higher affinity transporter, ABCC1. Using both endogenous and exogenous compounds, the binding was competed, demonstrating that our drug is also binding to a similar binding site to a known inhibitor of ABCC2, MK571. Surprisingly, non-modified LTC4 did not compete IAALTC4 binding but rather enhanced the binding. This might imply that our non-modified LTC4 is not binding to the same binding site, but rather that the binding is in a site that enhanced the binding of IAALTC4 to another site in ABCC2. Based on work by Zelcer et al., two distinct binding sites in human ABCC2 were found. In their work, they proposed a “S” and “M” working model for substrate binding, whereby the “S” site transports the substrate, and the “M” site is able to modulate this transport [37]. Stated differently, they proposed that ABCC2 contains an allosteric binding site that modulates the transport from a neighboring site, when examining the interaction of drugs, bile acids with organic anions with three different substrates of ABCC2 (estradiol-17-β-D-glucuronide, methotrexate, glutathione-S-dinitrophenol). 104
Similar results were found when exploring the transport of E217βG and summarized in a review by Borst et al. where a two-site mechanistic model was suggested [9]. Using this template of a two-site model LTC4 binds to modulate the affinity of IAALTC4 for ABCC2, and therefore likely binds to the “M” site, where IAALTC4 binds to the “S” site. With increasing concentrations of LTC4 we see an increase in the binding signal, the significance of this result has yet to be elucidated. In contrast to this we see that MK571 reduces significantly the binding of IAALTC4 to ABCC2. Therefore, MK571 shares the same binding site as IAALTC4 in ABCC2. Lastly, we were able to determine a potential binding domain for ABCC2 using tryptic digestion studies. It was clear based on the availability of an antibody that recognizes the C-terminal of ABCC2 that this was the only domain that we would be able to characterize, however based on previous studies it was not surprising that we did in fact see binding in this region.
From the immuno-detection of digested ABCC2, 3
fragments were detected: a band in the 115 kDa range, the 72 kDa range and in the lower molecular weight range 40 kDa in size. The same experiment performed on immunoprecipitated and photolabelled ABCC2 showed peptides with the same molecular weight. Our results correspond with previous findings from other groups that have implicated certain TM regions in ABCC2 substrate specificity [38, 39]. Specifically, in a site-mutagenesis study it was shown that replacing the tryptophan residue at position 1254 of ABCC2 resulted in a dramatic change in the affinity for both [3H]LTC4 and [3H]Methotrexate (MTX). When there was a conservative substitution made (tryptophan to tyrosine), [3H]LTC4 transport was preserved. However a nonconservative substitution for either an alanine or a cysteine abolished uptake. In the case of [3H]MTX, the results were even more absolute since any substitution yielded a complete void of uptake.
Taken together, the results from this previous study
demonstrate that a substitution in TM 17 position 1254 dramatically alters the substrate specificity of certain drugs, and that likely [3H]LTC4 transport requires an aromatic amino acid to preserve function. In this report we demonstrate initial observations and more work will be done to further elucidate the binding site.
105
ACKNOWLEDGMENT:
This work is supported by a grant from the Natural
Sciences and Engineering Research Council (NSERC) to EG. ML is a recipient of studentship from Latvian National Federation of Canada.
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[12] Keppler D, Huber M, Weckbecker G et al. Leukotriene C4 metabolism by hepatoma cells and liver. Adv Enzyme Regul 1987; 26:211-24. [13] Borst P, Evers, R., Kool, M. and J. Wijnholds. The multidrug resistance protein family. Biochem Biophys Acta 1999; 1461:347-357. [14] Buchler M, Konig J, Brom M et al. cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J Biol Chem 1996; 271:15091-8. [15] Hipfner DR, Almquist, K.C., Leslie, E.M., Gerlach, J.H., Grant, C.E., Deeley, R.G., and S.P.C. Cole. Membrane topology of the multidrug resistance protein (MRP). J Biol Chem 1997; 272:23623-23630. [16] Leier I, Jedlitschky G, Buchholz U et al. The MRP gene encodes an ATPdependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem 1994; 269:27807-10. [17] Loe DW, Almquist KC, Deeley RG, Cole SP. Multidrug resistance protein (MRP)mediated transport of leukotriene C4 and chemotherapeutic agents in membrane vesicles. Demonstration of glutathione-dependent vincristine transport. J Biol Chem 1996; 271:9675-82. [18] Stride BD, Grant CE, Loe DW et al. Pharmacological characterization of the murine and human orthologs of multidrug-resistance protein in transfected human embryonic kidney cells. Mol Pharmacol 1997; 52:344-53. [19] Cui Y, Konig J, Buchholz JK et al. Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells. Mol Pharmacol 1999; 55:929-37. [20] Karwatsky J, Leimanis M, Cai J et al. The leucotriene C4 binding sites in multidrug resistance protein 1 (ABCC1) include the first membrane multiple spanning domain. Biochemistry 2005; 44:340-51. [21] Daoud R, Julien M, Gros P, Georges E. Major photoaffinity drug binding sites in multidrug resistance protein 1 (MRP1) are within transmembrane domains 10-11 and 16-17. J Biol Chem 2001; 276:12324-30.
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[22] Karwatsky J, Daoud R, Cai J et al. Binding of a photoaffinity analogue of glutathione to MRP1 (ABCC1) within two cytoplasmic regions (L0 and L1) as well as transmembrane domains 10-11 and 16-17. Biochemistry 2003; 42:3286-94. [23] Laberge RM, Karwatsky J, Lincoln MC et al. Modulation of GSH levels in ABCC1 expressing tumour cells triggers apoptosis through oxidative stress. Biochem Pharmacol 2007; 73:1727-37. [24] Karwatsky J, Leimanis M, Cai J et al. The Leucotriene C(4) Binding Sites in Multidrug Resistance Protein 1 (ABCC1) Include the First Membrane Multiple Spanning Domain. Biochemistry 2005; 44:340-51. [25] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193:265-75. [26] Nare B, Prichard RK, Georges E. Characterization of rhodamine 123 binding to Pglycoprotein in human multidrug-resistant cells. Mol Pharmacol 1994; 45:1145-52. [27] Daoud R, Kast C, Gros P, Georges E. Rhodamine 123 binds to multiple sites in the multidrug resistance protein (MRP1). Biochemistry 2000; 39:15344-52. [28] Fairbanks G, Steck TL, Wallach DF. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 1971; 10:2606-17. [29] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979; 76:4350-4. [30] Hipfner DR, Almquist KC, Stride BD et al. Location of a protease-hypersensitive region in the multidrug resistance protein (MRP) by mapping of the epitope of MRPspecific monoclonal antibody QCRL-1. Cancer Res 1996; 56:3307-14. [31] Georges E, Zhang JT, Ling V. Modulation of ATP and drug binding by monoclonal antibodies against P-glycoprotein. J Cell Physiol 1991; 148:479-84. [32] Evers R, Kool M, van Deemter L et al. Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA. J Clin Invest 1998; 101:1310-9.
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[33] Scheffer GL, Kool M, Heijn M et al. Specific detection of multidrug resistance proteins MRP1, MRP2, MRP3, MRP5, and MDR3 P-glycoprotein with a panel of monoclonal antibodies. Cancer Res 2000; 60:5269-77. [34] Greenberger LM. Major photoaffinity drug labeling sites for iodoaryl azidoprazosin in P-glycoprotein are within, or immediately C-terminal to, transmembrane domains 6 and 12. J Biol Chem 1993; 268:11417-25. [35] Karwatsky JM, Georges E. Drug binding domains of MRP1 (ABCC1) as revealed by photoaffinity labeling. Curr Med Chem Anticancer Agents 2004; 4:19-30. [36] Zhang X, Collins KI, Greenberger LM. Functional evidence that transmembrane 12 and the loop between transmembrane 11 and 12 form part of the drug-binding domain in P-glycoprotein encoded by MDR1. J Biol Chem 1995; 270:5441-8. [37] Zelcer N, Huisman MT, Reid G et al. Evidence for two interacting ligand binding sites in human multidrug resistance protein 2 (ATP binding cassette C2). J Biol Chem 2003; 278:23538-44. [38] Ryu S, Kawabe T, Nada S, Yamaguchi A. Identification of basic residues involved in drug export function of human multidrug resistance-associated protein 2. J Biol Chem 2000; 275:39617-24. [39] Ito K, Oleschuk CJ, Westlake C et al. Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity. J Biol Chem 2001; 276:38108-14.
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Connecting Statement 2
The biochemical characterization of a protein is a vital step in understanding both the basic biology as well as determining the potential of the protein as a drug target. As demonstrated in the previous chapter, characterization of drug binding provides some of the clues to help build on our current understanding.
In malaria research ABC
transporters as well as other transporters have long since been considered as potential mediators or modulators of drug resistance.
Consequently, the search for novel
antimalarials, which is an ongoing effort in malaria biology, looks to both target and evade parasite drug resistance mechanisms. The proteins of interest include those previously elucidated to be involved with drug resistance (namely PfCRT, Pgh1). This work has been largely done in the last two decades with various strains of Plasmodium falciparum (both New and Old World isolates), as well as transfected parasites. An alternative to novel drug development is to explore the antimalarial drug activity of drugs previously used for other diseases. An example of such a drug is MK571, which was previously synthesized as an anti-asthmatic, and has been shown to be an ABCC1 inhibitor. In this study the antimalarial potency of MK571 against CQS and CQR parasites strains was explored. In addition a potential site of action of MK571 was elucidated using site-specific mutagenesis in transfected parasites.
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Chapter 4 Chloroquine-resistant Plasmodium falciparum Are Hypersensitive to MK571, a leukotriene LTD4 receptor antagonist
Mara L. Leimanis and Elias Georges
Institute of Parasitology, McGill University, Quebec, Canada (In preparation)
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ABSTRACT The spread of drug resistant Plasmodium falciparum (P. falciparum) is a major concern for effective treatment programs of malaria in developing countries.
Molecular
characterization of drug resistant laboratory strains and field isolates of P. falciparum has led to the identification of two membrane transport proteins (e.g., PfCRT and Pgh1) as modulators of resistance to several antimalarial drugs, including the most widely used quinoline-based drug, chloroquine. Earlier studies have demonstrated the ability of verapamil, a calcium channel blocker and well-established reversing agent of Pglycoprotein 1 (or ABCB1)-mediated drug resistance in tumour cells, to reverse both pfcrt and pfmdr1-modulated drug resistance.
In this report, we have examined the
effects of an ABCC1-specific inhibitor, MK571 (a leukotriene LTD4 receptor antagonist), on the survival of chloroquine-sensitive and -resistant P. falciparum. MK571 was found to be more toxic to chloroquine-resistant- than -sensitive strain (e.g., FCR-3 and 3D7, respectively). The correlation between chloroquine-resistance and hypersensitivity to MK571 was observed with two other independent chloroquineresistant P. falciparum strains, the 7G8 and W2. Furthermore, transfection of the chloroquine resistant strains 7G8 with a mutant pfmdr1 encoding the chloroquine sensitive D10 genotype (e.g. Pgh1 with Ser1034, Asn1042, Asp1246 substitutions) partially abolished the MK571 increased sensitivity of the transfectant 7G8 strain.
Taken
together, the results in this report demonstrate for the first time a correlation between pfmdr1-modulated chloroquine resistance and increased sensitivity to MK571. Given the safety profile of MK571 in humans, the possibility of using MK571 as an antimalarial compound to treat chloroquine resistant malaria is being suggested.
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INTRODUCTION Malaria remains a major cause of global death in many parts of the world, with 1-2 million deaths annually [1, 2]. It is believed that the number of clinical cases of malaria may increase due to changes in climate [3, 4]. The most critical lapse in the global efforts at controlling malaria has been the re-emergence of the disease due to drug resistance and the lack of developing new antimalarials [5]. The mechanisms of chloroquine resistance have been widely explored, and largely attributed to the involvement of two membrane transporters expressed on the food vacuole membrane of the parasite [6-8]. These transporters are thought to decrease the effective chloroquine levels in the parasite food vacuole the site of action of the drug [9], although this has only been more conclusively determined for PfCRT [10]. Chloroquine drug resistance is therefore, due in part to a reduced accumulation of the drug [11, 12]. Similar drug transport mechanisms have been extensively studied in tumour cells and have been shown to be causative of drug resistance to chemotherapy [13, 14]. One such ATP Binding Cassette (or ABC) membrane transporter, the pfmdr1 or Pgh1, has been characterized in P. falciparum [6, 15, 16] and has been attributed to both gene amplification and point mutations; both of which have arisen from several independent events in different geographical locations [15, 17, 18]. Specifically, point mutations have been associated with changes in drug sensitivity of P. falciparum to mefloquine, chloroquine, quinine and artemisinin [19, 20], while changes in gene amplification have been shown to affect P. falciparum drug susceptibility to mefloquine, quinine, and artemisinin, as well as halofantrine and lumefantrine [21]. In one study by Foote et al. [17] four codon polymorphisms were found to be strongly associated with chloroquineresistance. A later study by Reed et al. [22] showed the effects of altering specific residues in pfdmr1 (1034Ser-Cys, 1042Asn-Asp, 1246Asp-Tyr) in 7G8 leads to decreased resistance to chloroquine. These initial results are in stark contrast to a later study by Sidhu et al [23] where the same three mutations were not found to contribute to chloroquine resistance. It was therefore anticipated that Pgh1 alone could not fully resolve the drug resistance phenotype in P. falciparum. Indeed, later studies identified another mediator of chloroquine resistance, the PfCRT, a membrane protein located on 114
the food vacuole [7] and a member of the drug-metabolite transporter superfamily [24, 25]. Specifically, a K76T mutation in PfCRT first transmembrane domain was shown to be causative of chloroquine resistance, facilitating the passive release or active transport of chloroquine from the food vacuole of resistant parasites [7, 26] The initial finding that verapamil, a well studied calcium channel blocker and a class I inhibitor of ABCB1 in tumour cells, reverses chloroquine drug resistance [27] was surprising but instrumental in initially linking chloroquine resistance to the function(s) of pfmdr1 in P. falciparum. Since that initial report, verapamil has largely been shown to reverse pfcrt-mediated drug resistance in independent isolates of chloroquine resistant P. falciparum [10, 28]. Although reversal of chloroquine drug resistance by verapamil in P. falciparum is not well understood, it is thought to be mediated by direct binding of verapamil to PfCRT (see review [29]). Of interest are the findings that verapamil also modulates the activities of two ABC transporters in tumour cells (e.g., ABCB1 and ABCC1). Indeed, verapamil alone has been shown to be highly toxic to drug resistant cells at non-toxic concentrations to drug sensitive cells [30-33]. Moreover, this hypersensitivity of drug resistant tumour cells to verapamil was recently shown to be mediated directly by ABCB1 function [34]. Although, it remains to be determined if verapamil effects in P. falciparum are due to direct reversal or hypersensitivity of chloroquine resistant parasites expressing Pgh1 or PfCRT membrane transporters; it is interesting in this respect that agents that modulate resistance in tumour cells function similarly in drug resistant parasites. Consistent with the latter, we have previously demonstrated that ABCC1 can mediate the transport (through direct binding), and confers resistance to several quinoline-based drugs, including chloroquine [35-37]. Consequently, in this report it was of interest to examine the effects of a well established inhibitor of ABCC1, MK571 (an LTD4 receptor-antagonist and a quinolinebased analogue) on the growth of drug sensitive and resistant P. falciparum, including the 7G8 strain transfected with the D10-chloroquine sensitive pfmdr1 genotype. The findings in this study show a clear increased sensitivity to MK571 in three independent isolates of chloroquine resistant P.falciparum. Interestingly, hypersensitivity to MK571 in chloroquine resistant malaria may be mediated through pfmdr1. 115
MATERIALS AND METHODS Materials- MK571 was purchased from Interscience (Markham, ON), chloroquine and artimisinin
was
purchased
from
Sigma-Aldrich.
[3Η]-Hypoxanthine
monohydrochloride was purchased from Amersham Pharmacia (Baie d’Urfe, QC). All other chemicals were of the highest grade available. Parasite culture- Plasmodium falciparum strains 3D7, 7G8 and FCR-3 (kindly provided by Dr. E. Schurr at the Centre for the Study of Host Resistance, McGill University), W2, D10-mdrD10 and 7G8-mdrD10 were obtained from MR4 (MRA-157, MRA-563, MRA-566 respectively, ATCC, Manassas, VA, USA). All cultures were grown in continuous culture as previously described by Trager and Jensen [38]. Washed human erythrocytes (type A+, or B+) from freshly drawn blood were suspended in culture medium (RPMI-1640 from Gibco supplemented with 0.32 mM hypoxanthine, 2 mM L-glutamine, 25 mM HEPES, 24 mM sodium bicarbonate, 11 mM glucose) with either 0.5 % Albumax II or 10 % human A+ pooled de-activated human sera at 5 % hematocrit and inoculated with infected erythrocytes. The flasks were incubated in twenty milliliters of parasite suspension at 37 oC in a T-75 tissue culture flask by candle jar method [38], FCR-3 was grown in 5 % CO2 incubator, all cultures underwent daily changes of medium. The percentage of infected cells (parasitemia) was determined microscopically in thin, Giemsa-stained smears. Assay for antimalarial activity - To determine the growth of chloroquine-sensitive and –resistant strains of P. falciparum (3D7 and FCR-3, respectively), the Desjardins radioisotope method was adopted [39]. Parasite cultures were washed twice in 10 ml of culture medium (RPMI-1640 from Gibco supplemented with 0.5 % Albumax II, 16 µM hypoxanthine, 2 mM L-glutamine, 25 mM HEPES, 24 mM sodium bicarbonate, 11 mM glucose), and diluted to 2 % parasitemia in 4% hematocrit (type B+) in 100 μl of the culture medium and added to each well of 96-well plates. After a brief incubation (30 minutes) at 37 oC allowing the cultures to settle, 100 μl of media containing increasing 116
concentrations of various drugs were added (final dilution; parasitemia 1%, hematocrit 2 %) and plates were incubated for 24 hrs at 37 oC. On the second day, 20 µl (25 µCi/ml) [3Η]-Hypoxanthine monohydrochloride was added to each well of the 96-well plates and incubated for an additional 18-24 hrs. The parasitized RBCs were harvested on glass fiber filters (Wallac Printed Filtermat A) using the Packard Cell Harvester (FilterMate 96-well), with distilled water as a wash medium and 30 µl of scintillation fluid (Betaplate) was added to each filter well. [3H]-Hypoxanthine monohydrochloride accumulation was determined by fluorometry using the Perkin Elmer 1450 Microbeta counter. The 50 % inhibitory concentration (IC50) values were determined using Prism software (version 4.02) using a non-linear regression sigmoidal dose-response curve. To determine significant changes between test samples relative to control an ANOVA test was performed with post-hoc analysis (Bonferroni posttest). Each concentration of the test substance was tested 2-3 independent times in quadruplicate. The maximum final concentrations of solvents (e.g., ethanol or methanol) were kept under 2 %, and reference wells contained solvent in the concentration of 0.5 %.
117
RESULTS AND DISCUSSION Membrane transport proteins have been shown to modulate drug responses in tumour cells and infectious organisms, including antimalarial drug resistance in P. falciparum (see reviews [14, 19, 40]). To better understand these drug resistance mechanisms, efforts have been focused on the use of inhibitors of such membrane transport mechanisms that appear to cross species boundaries. The calcium channel blocker, verapamil, has been one such example and has been shown to be an effective inhibitor of ABC transporters in tumour cells and a membrane transporter in malaria parasites [10, 28, 30-33]. In this study it was of interest to examine the effects of another ABC transporter inhibitor, the MK571 (previously shown to be an effective MRP1 or ABCC1 inhibitor [41-43]). Indeed, our interest in examining the effect of an ABCC1 inhibitor (MK571) on chloroquine resistant P. falciparum is a continuation of our earlier work showing ABCC1 to bind to, and transport chloroquine in tumour cells [36]. In this study, we examined the effects MK571 (Figure 1) on the growth of sensitive and chloroquine resistant isolates of P. falciparum, namely the 3D7 and W2 or FCR-3. The results in
Figure 1: Organic structure of MK571 Figure 2 show the survival curves for each of the three strains of P. falciparum 3D7 (CQS), W2 (CQR) and FCR-3 (CQR) grown in the presence of increasing concentrations of chloroquine (Figure 2A). As expected, the results in Figure 2A show choroquine to be very effective against 3D7 strain (IC50 = 8.7 nM), but much less against W2 strain (IC50 = 30.3 nM) and FCR-3 strain (IC50 = 60.6 nM). By contrast, no difference in survival was observed between 3D7 and FCR-3 when grown in the
118
presence of increasing concentrations of artemisinin (Figure 2B). The latter results are consistent with earlier studies [44-46] demonstrating differences in the mechanism of
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Figure 2: Effects of increasing concentrations of drugs on the in vitro growth of chloroquine-sensitive (3D7) and -resistant (W2, FCR-3) strains of Plasmodium falciparum. Parasitized red blood cells were exposed for 48 h to increasing concentrations of chloroquine diphosphate (3.9-500 nM) (A), artemisinin (7.8-500 nM) (B) and MK571 (0.78-200 µM) (C). The incorporation of [3H]-hypoxanthine was used to measure the effects of each drug on survival of 3D7 ( ), W2 (O) and FCR-3 ( ) strains. Results are expressed as % survival as compared to control, in the absence of added drugs. Each graph represents the means of three independent experiments done in quadruplicate. * When compared with 3D7 changes in drug concentration correlates with a significant difference as assessed by the post hoc test (P < 0.05).
resistance between that of chloroquine (including other quinoline drugs) and artemisinins. Interestingly, the presence of increasing concentrations of MK571 alone was more toxic to the chloroquine-resistant strains. Figure 2C shows the IC50 values for each of the two chloroquine-resistant strains, FCR-3 (IC50 = 36 µM), and W2 (IC50 = 30 µM), relative to the drug-sensitive 3D7 (IC50 = 60 µM). Moreover, there appears to be a correlation between the levels of resistance to chloroquine and the degree of hypersensitivity to MK571. This latter correlation is consistent with a parasite-related mechanism rather than host-cell related one, as ABCC1 expression has been demonstrated in normal red blood cells [47]. Also consistent with the effect of MK571 on the parasite rather than the host ABCC1, is a lack of effect on parasite growth in the presence of increasing concentrations of LTC4 (up to 1 µM), a very high affinity ligand and normal substrate of ABCC1 [42] (data not shown). MK571 is a potent and competitive LTD4/E4 antagonist both in humans and in animals [48-50]; where leukotrienes (LTC4, D4, and E4) have shown to play a role in the pathology of asthma [51]. Moreover, MK571 is well tolerated by humans where the drug was used to inhibit leukotriene synthesis [52, 53]. One clinical study revealed the plasma drug profiles and tolerability of MK571 in patients.
From this study we
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Figure 3: Effects of increasing concentrations of drugs on the in vitro growth of chloroquine-sensitive (D10(D10), 7G8(D10)) and -resistant (7G8) strains of Plasmodium falciparum.
Parasitized red blood cells were exposed for 48 h to increasing
concentrations of chloroquine diphosphate (3.9-500 nM) (A) and MK571 (0.78-200 µM) (B). The incorporation of [3H]-hypoxanthine was used to measure the effects of each drug on survival of 7G8 (♦), 7G8(D10) (◊) and D10(D10) (□) strains. Results are expressed as % survival as compared to control, in the absence of added drugs. Each graph represents the means of three independent experiments done in quadruplicate. * When compared with D10(D10)
changes in drug concentration correlates with a
significant difference as assessed by the post hoc test (P < 0.001).
121
determined that the IC50’s of MK571 for the CQ-resistant strains tested are in range (or below) the Cmax (the maximum drug level) found in humans after clinical dosing [53]. Thus, it would be of interest to further elucidate the mechanism of action of MK571, through the identification of target molecules which in turn could lead to development of more effective lead compounds and ultimately novel antimalarial drugs. To further confirm the increased sensitivity of MK571 on chloroquine-resistant P. falciparum, we examined the effect of this drug on another chloroquine-resistant isolate 7G8 (CQR) and D10-mdrD10 (CQS) and 7G8-mdrD10 (CQS) [22]. The D10-mdrD10 preserves the CQS phenotype, and 7G8-mdrD10 is a CQR-partial revertant (i.e. with a lower IC50 to CQ than the parental 7G8). The D10 strain alone was not included as D10 shares both the same drug sensitivity profiles and shares the same amino acid sequence as D10mdrD10 [22]. Both transfectants preserve the CQS phenotype (Ser1034, Asn1042, Asp1246), in pfmdr1 encoding the Pgh1 transport protein located on the food vacuole membrane of P. falciparum. The results in Figure 3A confirm earlier results [22] showing clearly that choroquine is very effective against D10-mdrD10 and 7G8-mdrD10 parasite lines (IC50 = 19 nM and IC50 = 18 nM), but is less active against 7G8 strain (IC50 = 50 nM). Surprisingly, the results in Figure 3B show MK571 was more toxic to the chloroquineresistant strain 7G8 (IC50 = 34 µM) than to the chloroquine-sensitive stains D10-mdrD10 (IC50 = 122 µM). The transfer pfmdr1 encoding an increased chloroquine-sensitive genotype into 7G8 P.falciparum strain (i.e., 7G8-mdrD10 CQR-partial revertant) shows decreased sensitivity to MK571, with an IC50 = 70 µM versus that seen in untransfected 7G8 with IC50 = 34 µM (Figure 3B). With the exception of Figure 2B, ANOVA test results demonstrate a significant difference between drug concentration and the various strains (P < 0.001), with additional post hoc analysis revealing significant differences at various drug concentrations (relative to control) as indicated on the graphs. These results suggest that MK571 hypersensitivity may be linked to pfmdr1 activity. A similar result was found by Hayward et al. [54] using the same transfectants where the 7G8-mdrD10 (CQR-partial revertant), was found to share the same tolerance of D10-
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Table 1: In-vitro antimalarial activity of chloroquine, artemsinin and MK571 against chloroquine-sensitive and chloroquine-resistant strains of P. falciparum
mdrD10 to verapamil, as compared to the 7G8-mdr7G8. This study supports our findings that Pgh1 may alter drug sensitivity. It should be noted that addition of fixed molar concentrations of chloroquine to the growth media containing increasing concentrations of MK571 did not show a synergistic or additive effects (results not shown). Hence, the two drugs appear to interact with different target or their effects are mediated by different mechanisms. Although it is difficult to rule out at this point if both drugs share the same target (Pgh1) and if so why no additive or synergistic effect on the growth of chloroquine-sensitive and –resistant parasites is observed; it is unlikely that MK571 which contains a quinoline-moiety, acts as a novel quinoline drug and specifically kills P. falciparum through its action on chloroquine target site or protein. Consistent with the possibility that MK571 is not just another quinoline-like drug is its preferential effect on chloroquine-resistant, relative to chloroquine-sensitive parasites. It is interesting in this respect that several structurally similar drugs to chloroquine namely, the 8-aminoquinolines (primaquine and pamaquine) were observed to be more potent against chloroquine-resistant than -sensitive P. falciparum strains [55].
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However, in those studies it was suggested that such an observed effect is likely due to enhanced drug activity against a chloroquine drug target. Similar hypersensitivity effect have been observed in mammalian tumour cells with verapamil and BSO in drug resistant cells overexpressing ABCB1 and ABCC1, respectively [34, 56].
It is
interesting that in both drug resistant model systems, the enhanced toxicity effect towards resistant tumour cells was due to the interactions of verapamil or BSO with ABCB1 or ABCC1, but the mechanism of hypersensitivity was different. In both cases, the enhanced cell killing was due to oxidative stress, albeit due to different initiator mechanisms. Work is in progress to fully characterize the molecular mechanism of MK571 hypersensitivity and its accumulation in parasitized erythrocytes.
ACKOWLEDGEMENTS: We thank MR4 for providing us W2 as contributed by Dr. D. Kyle and D10-mdrD10 and 7G8-mdrD10 parasites were contributed by Dr. A.F. Cowman. This work is supported by a grant from the Natural Sciences and Engineering Research Council (NSERC) to EG. ML is a recipient of a studentship from the Latvian National Federation of Canada, and the CHPI Bridge Funds.
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[13] Higgins CF. Multiple molecular mechanisms for multidrug resistance transporters. Nature 2007; 446:749-57. [14] Szakacs G, Paterson JK, Ludwig JA et al. Targeting multidrug resistance in cancer. Nat Rev Drug Discov 2006; 5:219-34. [15] Foote SJ, Thompson JK, Cowman AF, Kemp DJ. Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum. Cell 1989; 57:921-30. [16] Wilson CM, Serrano AE, Wasley A et al. Amplification of a gene related to mammalian mdr genes in drug-resistant Plasmodium falciparum. Science 1989; 244:1184-6. [17] Foote SJ, Kyle DE, Martin RK et al. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature 1990; 345:255-8. [18] Triglia T, Foote SJ, Kemp DJ, Cowman AF. Amplification of the multidrug resistance gene pfmdr1 in Plasmodium falciparum has arisen as multiple independent events. Mol Cell Biol 1991; 11:5244-50. [19] Duraisingh MT, Cowman AF. Contribution of the pfmdr1 gene to antimalarial drug-resistance. Acta Trop 2005; 94:181-90. [20] Uhlemann AC, Krishna S. Antimalarial multidrug resistance in Asia: mechanisms and assessment. Curr Top Microbiol Immunol 2005; 295:39-53. [21] Sidhu AB, Uhlemann AC, Valderramos SG et al. Decreasing pfmdr1 copy number in plasmodium falciparum malaria heightens susceptibility to mefloquine, lumefantrine, halofantrine, quinine, and artemisinin. J Infect Dis 2006; 194:528-35. [22] Reed MB, Saliba KJ, Caruana SR et al. Pgh1 modulates sensitivity and resistance to multiple antimalarials in Plasmodium falciparum. Nature 2000; 403:906-9. [23] Sidhu AB, Valderramos SG, Fidock DA. pfmdr1 mutations contribute to quinine resistance and enhance mefloquine and artemisinin sensitivity in Plasmodium falciparum. Mol Microbiol 2005; 57:913-26.
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[24] Martin RE, Kirk K. The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Mol Biol Evol 2004; 21:193849. [25] Tran CV, Saier MH, Jr. The principal chloroquine resistance protein of Plasmodium falciparum is a member of the drug/metabolite transporter superfamily. Microbiology 2004; 150:1-3. [26] Cooper RA, Ferdig MT, Su XZ et al. Alternative mutations at position 76 of the vacuolar transmembrane protein PfCRT are associated with chloroquine resistance and unique stereospecific quinine and quinidine responses in Plasmodium falciparum. Mol Pharmacol 2002; 61:35-42. [27] Martin SK, Oduola AM, Milhous WK. Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 1987; 235:899-901. [28] Lakshmanan V, Bray PG, Verdier-Pinard D et al. A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance. Embo J 2005; 24:2294-305. [29] Bray PG, Martin RE, Tilley L et al. Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance. Mol Microbiol 2005; 56:323-33. [30] Tsuruo T, Iida H, Tsukagoshi S, Sakurai Y. Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil. Cancer Res 1981; 41:1967-72. [31] Fojo A, Akiyama S, Gottesman MM, Pastan I. Reduced drug accumulation in multiply drug-resistant human KB carcinoma cell lines. Cancer Res 1985; 45:3002-7. [32] Rogan AM, Hamilton TC, Young RC et al. Reversal of adriamycin resistance by verapamil in human ovarian cancer. Science 1984; 224:994-6. [33] Slater LM, Murray SL, Wetzel MW et al. Verapamil restoration of daunorubicin responsiveness in daunorubicin-resistant Ehrlich ascites carcinoma. J Clin Invest 1982; 70:1131-4. [34] Karwatsky J, Lincoln MC, Georges E. A mechanism for P-glycoprotein-mediated apoptosis as revealed by verapamil hypersensitivity. Biochemistry 2003; 42:12163-73.
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[35] Vezmar M, Deady LW, Tilley L, Georges E. The quinoline-based drug, N-[4-[1hydroxy-2-(dibutylamino)ethyl] quinolin-8-yl]-4-azidosalicylamide, photoaffinity labels the multidrug resistance protein (MRP) at a biologically relevant site. Biochem Biophys Res Commun 1997; 241:104-11. [36] Vezmar M, Georges E. Direct binding of chloroquine to the multidrug resistance protein (MRP): possible role for MRP in chloroquine drug transport and resistance in tumour cells. Biochem Pharmacol 1998; 56:733-42. [37] Vezmar M, and E. Georges. Reversal of MRP-mediated doxorubicin resistance with quinoline-based drugs. Biochem Pharmacol 2000; 59:1245-1252. [38] Trager W, and J.B. Jenson. Cultivation of malarial parasites. Nature 1978; 273:621-622. [39] Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Chemother 1979; 16:710-8. [40] Wellems TE, Plowe CV. Chloroquine-resistant malaria. J Infect Dis 2001; 184:770-6. [41] Gekeler V, Ise W, Sanders KH et al. The leukotriene LTD4 receptor antagonist MK571 specifically modulates MRP associated multidrug resistance. Biochem Biophys Res Commun 1995; 208:345-52. [42] Leier I, Jedlitschky G, Buchholz U et al. The MRP gene encodes an ATPdependent export pump for leukotriene C4 and structurally related conjugates. J Biol Chem 1994; 269:27807-10. [43] Leier I, Jedlitschky G, Buchholz U, Keppler D. Characterization of the ATPdependent leukotriene C4 export carrier in mastocytoma cells. Eur J Biochem 1994; 220:599-606. [44] Eckstein-Ludwig U, Webb RJ, Van Goethem ID et al. Artemisinins target the SERCA of Plasmodium falciparum. Nature 2003; 424:957-61. [45] Robert A, Coppel Y, Meunier B. Alkylation of heme by the antimalarial drug artemisinin. Chem Commun (Camb) 2002:414-5.
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[46] Uhlemann AC, Cameron A, Eckstein-Ludwig U et al. A single amino acid residue can determine the sensitivity of SERCAs to artemisinins. Nat Struct Mol Biol 2005; 12:628-9. [47] Pulaski L, Jedlitschky G, Leier I et al. Identification of the multidrug-resistance protein (MRP) as the glutathione-S-conjugate export pump of erythrocytes. Eur J Biochem 1996; 241:644-8. [48] Jones TR, Zamboni R, Belley M et al. Pharmacology of L-660,711 (MK-571): a novel potent and selective leukotriene D4 receptor antagonist. Can J Physiol Pharmacol 1989; 67:17-28. [49] Krell RD. The emergence of potent and selective peptide leukotriene receptor antagonists. Pulm Pharmacol 1989; 2:27-31. [50] Mason PaJT. Effects of timolol, indomethacin and MK-571 on bronchoconstriction to infused leukotriene D4 in guinea pigs. . Can J Physiol Pharmacol 1990; 68:783-90. [51] Taylor GW, Taylor I, Black P et al. Urinary leukotriene E4 after antigen challenge and in acute asthma and allergic rhinitis. Lancet 1989; 1:584-8. [52] Amirav I, Pawlowski N. Inhibition of exercise-induced bronchoconstriction by MK-571, a potent leukotriene D4-receptor antagonist. N Engl J Med 1991; 324:1288. [53] Depre M, Margolskee DJ, Hsieh JY et al. Plasma drug profiles and tolerability of MK-571 (L-660,711), a leukotriene D4 receptor antagonist, in man. Eur J Clin Pharmacol 1992; 43:427-30. [54] Hayward R, Saliba KJ, Kirk K. Mutations in pfmdr1 modulate the sensitivity of Plasmodium falciparum to the intrinsic antiplasmodial activity of verapamil. Antimicrob Agents Chemother 2005; 49:840-2. [55] Geary TG, Divo AA, Jensen JB. Activity of quinoline-containing antimalarials against chloroquine-sensitive and -resistant strains of Plasmodium falciparum in vitro. Trans R Soc Trop Med Hyg 1987; 81:499-503. [56] Laberge RM, Karwatsky J, Lincoln MC et al. Modulation of GSH levels in ABCC1 expressing tumour cells triggers apoptosis through oxidative stress. Biochem Pharmacol 2007; 73:1727-37.
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Connecting Statement 3 Targeting parasite proteins to combat CQR is a well explored area that has exploited differences in parasite metabolism and homeostasis as compared to the human host. One such protein that has been previously described to modulate CQR is Pgh1. Pgh1 expression in the parasite could be targeted using MK571, resulting in increased sensitivity to the drug in CQ-resistant parasites as described in the previous chapter. Additional drugs may be used to target other parasite specific proteins and/or metabolic pathways. One of the most hopeful areas of drug development has been with the trioxolanes.
This particular class of drugs utilizes an alternative mechanism as
compared to chloroquine, and includes all artemisinins. These drugs are believed to interact with heme causing oxidative stress to the parasite. From our work we were able to determine the low level activity (~3 µM) of novel trioxolanes that were easily synthesized for further investigation.
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Chapter 5 Preliminary Synthesis and In Vitro Characterization of Novel Ozonides (trioxolanes) as Antimalarial Drugs
Mara L. Leimanisα, Sabine Thielgesβ, Oleg Shirobokovβ, George Justβ, Nicolas Moitessierβ, Elias Georgesα
α
Institute of Parasitology, and βDepartment of Chemistry, McGill University (unpublished to date)
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ABSTRACT The emergence of multidrug resistant malaria, especially to quinoline-based drugs, has led to the rapid deployment of artimisinin and its synthetic derivatives. Artimisinin is among the earliest natural product antimalarials and is a rapidly metabolized endoperoxide that is activated in the presence of free or heme-conjugated Fe(II). However, both artemisinin and its derivatives suffer from low extraction yields and synthesis scale-up; in addition to short in-vivo half-life.
Recent reports have
demonstrated the synthesis of several endoperoxides or trioxolanes as highly effective antimalarial compounds. In this study, we describe the synthesis and characterization of 11 novel trioxolanes that are easily scalable. The antimalarial activities of all 11 trioxolanes were tested in vitro using both chloroquine-sensitive (3D7) and -resistant (FCR-3) strains of Plasmodium falciparum. Interestingly, four trioxolane compounds (8, 9a, 9b, 10) had antimalarial activities in the low micromolar range (3µM – 15uM). One of the most active compounds (9b), contained one phenyl ring and a CF3 moiety fused to a 1,2,4 trioxolane group. Taken together, the results of this study show for the first time that modifications of the 1,2,4-trioxolane with a hydrophobic group and an electron-withdrawing group produces moderate antimalarial activity. Work is in progress to develop other novel trioxolanes with greater antimalarial activity based on our current active compounds.
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INTRODUCTION The spread of drug resistant malaria has made the development of novel antimalarial drugs more urgent. In a recent report released by the World Health Organization, all artemisinin-based monotherapies have been halted in the treatment of malaria [1]. This comes on the heels of the findings that the mainstay drug chloroquine has largely lost its efficacy due to the spread of chloroquine- and other quinoline-resistant Plasmodium falciparum (P. falciparum). The use of artemisinin alone and in combination with other antimalarial drugs in the treatment of chloroquine-sensitive and -resistant P. falciparum is currently one of the last drugs in the arsenal for treating P. falciparum [2]. Artemisinin, a natural product extract from the Chinese herbal sweet wormwood Artemisia annua (A. annua).
A major drawback of Artemisinin and its synthetic
derivatives (artesunate, artemether, and arteether) is their short half-lives (3-5 hrs). This necessitates multiple treatments over the course of a few days (see review [3]). This drawback has fueled the search for second-generation endoperoxides (trioxanes, tetraoxanes) [4-6] that have increased stability, and are highly potent against chloroquine resistant and multidrug resistant P.falciparum. In one study [7], it was shown that an adamatane ring fused onto the basic ozonide ring produced a novel class of antimalarial drugs with high efficacy in vitro and in vivo. Of the second generation trioxolanes, OZ277 was selected as a lead compound based on efficacy, bioavailability, and lack of side-effects and is currently in clinical trials. The mechanism of action of artemisinin has been attributed to the 1,2,4 trioxane pharmacophore containing the endoperoxide bridge [8].
Consistent with this
mechanism of action, the antimalarial activity was abolished when the artemisinin derivatives devoid of the endoperoxide bridge were tested for activity [9]. Once inside the parasite, the activity of these drugs is abolished due to chemical decomposition in the presences of parasite metabolite (the presence of free heme-Fe(II) resulting from the parasite digestion of hemoglobin) creating carbon-centered free radicals [5, 10, 11]. This irreversible redox reaction is believed to lead to the alkylation of heme [12] and several enzymes, including the P. falciparum sarco-endoplasmic calcium-transporter ATPase PfATP6 [13]. The inhibition of PfATP6 activity by artemisinin and other
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synthetic endoperoxides is thought to lead to the death of the parasite. More recently Uhlemann et al., [14] have demonstrated that a single amino acid residue in transmembrane segment 3 of PfATP6 abolishes susceptibility to artemisinins. This suggests a possible mechanism of acquired artemisinin resistance involving a single amino acid change in PfATP6 in vivo. In an effort to expand the list of novel endoperoxides as antimalarial drugs with improved efficacy, half-life (and possibly by-passing PfATP6-mutation mediated drug resistance), we synthesized several trioxolanes, whereby the adamatane ring was replaced by other hydrophobic moieties fused to the ozonide ring.
Some of the
objectives behind our efforts to synthesize novel trioxolanes are to lower costs and to increase the feasibility of scale up for these compounds. In this study, a total of 11 trioxolanes were synthesized and their antimalarial activity against P. falciparum was evaluated in vitro.
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MATERIALS AND METHODS Materials - [3Η]-Hypoxanthine monohydrochloride was purchased from Amersham Pharmacia (Baie d’Urfe, Quebec, Canada). All other chemicals were of the highest grade available. Parasite culture - Plasmodium falciparum strains 3D7 and FCR-3 were grown in continuous culture as previously described by Trager and Jensen [15]. Washed human erythrocytes (type B+) from freshly drawn blood were suspended in culture medium (RPMI-1640 from Gibco supplemented with 0.5 % Albumax II, 0.32 mM hypoxanthine, 2 mM L-glutamine, 25 mM HEPES, 24 mM sodium bicarbonate, 11 mM glucose) at 5 % hematocrit and inoculated with infected erythrocytes. The flasks were incubated in 10 ml of parasite suspension at 37 oC in a T-25 tissue culture flask by candle jar method [15], FCR-3 was grown in a 5% CO2 incubator. Both cultures underwent daily changes of medium. The percentage of infected cells (parasitemia) was determined microscopically in thin, Giemsa-stained smears. Assay for antimalarial activity - To determine the growth of chloroquine-sensitive and –resistant strains of P. falciparum (3D7 and FCR-3, respectively), the Desjardins radioisotope method was adopted [16]. Parasite cultures were washed twice in 10 ml of culture medium (RPMI-1640 from Gibco supplemented with 0.5 % Albumax II, 16 µM hypoxanthine, 2 mM L-glutamine, 25 mM HEPES, 24 mM sodium bicarbonate, 11 mM glucose), and diluted to 2 % parasitemia in 4 % hematocrit (type B+) in 100 μl of the culture medium and added to each well of 96-well plates. After a brief incubation (30 min) at 37 oC allowing the cultures to settle, 100 μl of media containing increasing concentrations of various drugs were added (final dilution; parasitemia 1 %, hematocrit 2 %) and plates were incubated for 24 hrs at 37 oC. On the second day, 20 µl (25 µCi/ml) [3Η]-Hypoxanthine monohydrochloride was added to each well of the 96-well plates and incubated for an additional 18-24 hrs. The parasitized RBCs were harvested on glass fiber filters (Wallac Printed Filtermat A) using the Packard Cell Harvester (FilterMate 96-well), with distilled water as a wash medium and 30 µl of scintillation
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fluid (Betaplate) was added to each filter well. [3H]-Hypoxanthine monohydrochloride accumulation was determined by fluorometry using the Perkin Elmer 1450 Microbeta counter. The 50 % inhibitory concentration (IC50) values were determined using Prism software (version 4.02) using a non-linear regression sigmoidal dose-response curve. Each concentration of the test substance was tested independently 2-3 times in quadruplicate. The concentration of solvents (ethanol and methanol) did not exceed 2 %.
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RESULTS AND DISCUSSION Earlier work by Vennerstrom [7] on the antimalarial endoperoxides focused on the derivatization of the spiro-adamantane-trioxolane core. Given the high cost of the adamantane functionality and the short half-life of the latter adamantine-trioxolanes, it was of interest to investigate the effects of replacing the adamantane core on antimalarial activity of trioxolanes. Figure 1 shows the synthesis scheme for all 11 compounds synthesized in this study. The first series of trioxolanes (5-7) were obtained in 50-65 % yields. The trioxolane core was formed by using a Griesbaum crossozonolysis reaction [17] in which an O-alkyl oxime was ozonized in the presence of a ketone. Other trioxolanes were synthesized by coupling two ketones (hexachloro-2propanone and the diethylketomalonate) to two O-alkyl oximes (2-3). Unfortunately, the malonate derivatives were not stable enough and could not be isolated. The hexachloro-ozonides 5 and 6 were found to be more stable and were obtained after HPLC purification. A second series of trioxolanes (8-11) were also prepared by the same type of cross-ozonolysis with four different ketones. Figure 2 shows the benzophenone and fluorenone (two hydrophobic cores) that were chosen as they are less costly than the previously described adamantanone core, while hexafluoro-2-propanone was used in order to improve the stability of the oxolane ring by increasing the electronegativity (Figure 2). The last ketone used was the 2,2,2-trifluoroacetophenone which combined a hydrophobic portion (phenyl ring) and an electron-withdrawing group (CF3). In the first series of trioxolanes, it was determined that the O-alkyl oxime partner was not stable for storage purposes and it often decomposed during ozonolysis, leading to the formation of several side-products. In order to improve this last step, we decided to replace the O-alkyl oxime 3 with a methoxyolefin, which has been shown to have the same reactivity as the oxime ether previously used [18]. The new partner 4 was prepared from the same ketone 1 by a Wittig olefination. The cross-ozonolysis was successful using this olefin. For each ozonolysis, we observed a total conversion of the starting material and the purity, as measured by HPLC before further purification, was superior to 90 % with the formation of only one diastereoisomer. A last series of trioxolanes (12 and 13) were prepared by saponification of the methyl ester, again with
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the rationale of increasing the stability of the trioxolanes and decreasing cost of synthesis (Figure 2).
Figure 1: Reactions at the 1,4-dioxane yielding compounds 1-13. Percent (%) yields for each compound are as indicated. To investigate the antimalarial activity of the above synthesized trioxolanes, in vitro cytotoxicity assays were performed using both 3D7 and FCR-3 as P. falciparum test strains as summarized in Table 1. Initially the growth of chloroquine-sensitive (3D7) and – resistant (FCR-3) P.falciparum were tested in the presence of increasing molar concentrations of chloroquine and artemisinin. The results show clearly that unlike choroquine which was very effective against 3D7 strain (IC50 = 11.4 nM), the drug was
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much less against FCR-3 strain (IC50 = 106.1 nM). Artemisinin was found to be equally effective against choroquine-sensitive and –resistant P.falciparum (IC50 = 13.7 nM and 11.2 nM).
Figure 2: Organic structures of synthesized compounds Given these results, it was of interest to examine the effects of all 11 novel compounds against the two strains of P.falciparum. Only four of the 11 tested compounds had an effect on the growth of 3D7 and FCR-3 isolates of P.falciparum. Compounds 8, 9a, 9b and 10 display antimalarial activities in the low micromolar concentration, with IC50 values for 3D7 and FCR-3 of 11 µM and 6.3 µM (compound 8); 16.4 µM and 14.3 µM (compound 9a), 3.4 µM and 3.0 µM (compound 9b), and 21µM and 9.7 µM (compound 10). The remaining compounds were much less active in the low micromolar range, as summarized in Table 1.
These results suggest that some of these synthesized
trioxolanes did in fact have in vitro antimalarial activity and that the drugs were effective against both chloroquine drug resistant and sensitive parasites. In the above results compound 9b is shown to be the most active antimalarial trioxolane. The structure contains a hydrophobic phenyl ring together with an electronwithdrawing group (CF3) fused to the peroxide bridge.
Interestingly, its
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Table 1: In vitro antimalarial activity of compounds artemisinin, chloroquine and compounds 5-15 against the chloroquine-sensitive 3D7 and chloroquine-resistant FCR3 strains of Plasmodium falciparum diastereoisomer (9a) shows much lower antimalarial activity (e.g. ~3.4 µM versus ~16.4 µM, respectively). The fusion of two hydrophobic moieties (e.g. two phenyl rings or two methyl groups) or two electron-withdrawing moieties (e.g. CF3 or CCl3) to the peroxide bridge did not enhance the trioxolanes antimalarial activity. These results are consistent with earlier observations that iron potentiation of the peroxide bridge in artimisinin and synthetic trioxolanes is critical for activity. In that study, Dong et al. [19] demonstrates a loss of antimalarial activity when the trioxolane peroxide was too exposed and thereby metabolically unstable. Similarly, loss in activity was seen in compounds where the Fe(II) was sterically inaccessible. Consequently, it was proposed that optimal drug candidates have both a sterically hindered side and another side that allows for interaction of Fe(II) with a sterically unhindered peroxide oxygen atom.
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Taking together, our results are consistent with their findings whereby the best antimalarial activity was found in compounds with at least one hydrophobic group (810).
Work is ongoing to explore alternative side groups (with and without the
adamatane core) that may improve both antimalarial activity while maintaining desirable physiochemical and biopharmaceutical properties [20, 21].
ACKNOWLEDGMENTS:
This work is supported by grants from the Natural
Sciences and Engineering Research Council (NSERC) to EG and from the FQRNT Center for Host Parasite Interactions (CHPI) to EG and GJ. ML is a recipient of studentship from the Latvian National Federation of Canada.
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Concluding Statement
“Nothing in biology makes sense except in the light of evolution.”
THEODOSIUS DOBZHANSKY
In and effort to explore the biochemical characterization of several ABC transporters one must first determine the overall significance and questions posed.
Do ABC
transporters have great clinical significance? Are ABC transporter good drug targets? Is targeting MDR the answer to reversing clinically resistant cancers? In order to address these questions we must first summarize over 20 years of data. Largely this leads to two main camps in this area of research, those that believe that ABC transporters are good targets and those that do not. The subject is complicated in part to the following information: 1) structural data is lacking, 2) drug-binding sites are large and flexible, therefore transport of substrates in varied, and 3) multiple transporters may be over-expressed, complicating the interpretation of data. There is additional speculation to state that given the normal function of ABC transporters to protect an organism against cytotoxins, that any modulation in expression levels may simply be a stress response. As, this is seen both in antibiotic-resistance in bacteria, as well as in MDR in cancer patients [1]. In general, it appears as though there is data to suggest the contribution of ABC transporters to the MDR phenomenon. This thesis however, is exploring the positive side of the story. That is to say that the results in this thesis contribute to the greater knowledge of ABC transporters, in hopes
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that MDR will be circumvented in the future. Specifically, in the first part of the thesis (chapters two and three) we explored the fundamental biochemical characterization of two ABC transporters ABCG2 and ABCC2. The ABCG2 expression in whole mature RBC’s was characterized and found to be predominantly expressed as a homodimer in the presence of mild denaturing conditions. It has yet to be determined whether higher oligomerization of ABCG2 in RBC’s exists, however there is evidence to suggest the formation of tetramer or multiple dimers [2]. The expression of ABCG2 was present in multiple samples collected from various blood donors and was also found to be active. Future work in this area may serve to better understand the role of ABCG2 and its role in hypoxia. This may include an epidemiological survey of expression levels and a further understanding of the dimerization process. In addition, further understanding of drug binding and transport function in RBCs will further the understanding to those studying blood biology and heme transport mechanisms. This body of work contributes to the growing discoveries in ABCG2 research, with recent reviews suggesting that ABCG2 may have great potential clinically [3]. In the third chapter we explored the drug binding characteristics of a photoreactive azido-anoalogue of LTC4 (AALTC4), an endogenous substrate of ABCC2. There has been a lack of any information regarding the direct and specific binding of drug to ABCC2 and therefore for the first time, this work elucidates and supports previous finding that demonstrate LTC4 transport by ABCC2. Of greater interest was the finding that a known inhibitor of ABCC2, MK571 was able to compete for the same binding site as LTC4. This result supports similar transport assays, whereby MK571 was able to inhibit drug transport [4, 5]. Drug binding studies with ABCB1 and ABCC1, have aided to clarify and strengthen the fundamental science of these clinically relevant transporters and aids to provide a clearer understanding on how drug-protein and drugdrug interactions may be occurring.
Furthermore once drug binding studies are
explored there may be a better framework for future clinical work using protein-specific inhibitors. Further work is required to provide a more detailed map of drug binding to ABCC2.
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MDR researchers remain optimistic that significant contributions may still be made in this field, however what remains to be a greater question still, is whether we should be focusing on reversing the drug resistance or rather attempting to avoid it in the first place.
This requires a greater understanding and better informed alternatives for
treatment and disease prevention. Together, the studies in these chapters add to the fundamental research and the biochemical characterization of clinically relevant ABC transport proteins. In the second part of the thesis (chapters four and five) we turn our attention to exploring the drug development field in malaria research. This is of utmost importance to the field given that there is limited drug development for many tropical diseases, including malaria. Major efforts are underway to screen drugs currently used to treat other diseases, as well as synthesizing novel molecules based on the molecular structure of known antimalarials (namely the artemisinins). This effort is largely in response to the global urgency for antimalarial drug development due to widespread CQ drug resistance in P. falciparum. As compared to ABC transporters in mammalian cells perhaps the picture is a little clearer in P. faciparum. Pgh1 has been shown to be involved in modulating drug resistance. In addition, it has been clearly demonstrated that PfCRT, albeit not an ABC transporter (but a membrane transporter) is key in its involvement with CQR [6]. The findings presented in chapter four describe an unexpected increased sensitivity of MK571 against CQR parasites. Future work may include looking to determine in greater detail the involvement of additional transporters in the hypersensitivity to MK571, and to screen similarly structured drugs. Likewise, the studies presented in chapter five include preliminary synthesis and in vitro characterization of trioxolanes that may be further optimized. Future work will likely include drug synthesis with additional modifications to drugs found to be active, primarily with the addition of the adamatane core. Together, this work builds on the existing knowledge about structureactivity relationship of drugs (SAR), the mechanisms of drug action and subsequent drug resistance mechanisms. Work is ongoing to address these issues as well at to characterize novel ABC transporters in the malaria parasite as potential drug targets.
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This exploration may include transporters such as that described in chapter two, as it is unknown whether ABCG2 may play a role in host-parasite interactions of P. falciparum infected erythrocytes. In summary, it is clear that the attention and help of the entire global community is necessary to control malaria. In Canada, it is known that the largest amount of malaria is due to travelers from endemic areas, as it has been reported that since 1990 an average of 538 cases of malaria occur annually resulting in an average of one death per year [7]. To address this issue, ongoing surveying methods are necessary, to determine the causative species and to monitor the spread of drug resistance. Additionally, fastacting policy changes are required to adapt to findings of drug resistance in the field, and to implement strict drug monitoring to avoid the spread of counterfeit drugs. It is with great optimism that we anticipate the following decade, whereby we will look to the malaria vaccine initiatives and the development millennium goals to fulfill their mandate.
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References [1] Higgins CF. Multiple molecular mechanisms for multidrug resistance transporters. Nature 2007; 446:749-57. [2] Xu J, Liu Y, Yang Y et al. Characterization of oligomeric human half-ABC transporter ATP-binding cassette G2. J Biol Chem 2004; 279:19781-9. [3] Robey RW, Polgar O, Deeken J et al. ABCG2: determining its relevance in clinical drug resistance. Cancer Metastasis Rev 2007; 26:39-57. [4] Chen ZS, Kawabe T, Ono M et al. Effect of multidrug resistance-reversing agents on transporting activity of human canalicular multispecific organic anion transporter. Mol Pharmacol 1999; 56:1219-28. [5] Payen L, Courtois A, Campion JP et al. Characterization and inhibition by a wide range of xenobiotics of organic anion excretion by primary human hepatocytes. Biochem Pharmacol 2000; 60:1967-75. [6] Bray PG, Martin RE, Tilley L et al. Defining the role of PfCRT in Plasmodium falciparum chloroquine resistance. Mol Microbiol 2005; 56:323-33. [7] MacLean JD, Demers AM, Ndao M et al. Malaria epidemics and surveillance systems in Canada. Emerg Infect Dis 2004; 10:1195-201.
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Appendix I
Chapter 5 Supplemental Figure 1: Effects of increasing concentrations of drugs on the in vitro growth of chloroquine-sensitive (3D7) and -resistant (FCR-3) strains of Plasmodium falciparum. Parasitized red blood cells were exposed for 48 h to increasing concentrations of chloroquine diphosphate (3.9-500 nM) (A) and artemisinin (7.8-500 nM) (B). The incorporation of [3H]-hypoxanthine was used to measure the effects of each drug on the survival of 3D7 ( ) and FCR-3 ( ) strains. Results are expressed as % survival compared to control in the absence of added drugs. Points represent mean values of experiments done 2-3 times with each drug concentration in quadruplicate ± S.E of data.
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Chapter 5 Supplemental Figure 2: Effects of increasing concentrations of novel trioxolanes on the growth of chloroquine-sensitive (3D7) and -resistant (FCR-3) strains of Plasmodium falciparum. Parasitized red blood cells were exposed for 48 h to increasing concentrations of compounds 8, 9a, 9b, and 10 (12.2 nM-100 µM). The incorporation of [3H]-hypoxanthine was used to measure the effects of each drug on survival of 3D7 ( ) and FCR-3 ( ) strains. Results are expressed as % survival compared to control in the absence of added drugs. Each graph represents the means of experiments done 2-3 times with each drug concentration done in quadruplicate.
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Appendix II
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