1 Salinomycin and other ionophores as new class of antimalarial ...

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Jun 8, 2015 - 3. Introduction. 40. Malaria caused 627.000 deaths and 207 million .... naturally-clotted heat-inactivated 0+ human serum (Interstate Blood Bank, Inc.), ... final hematocrit 1%) and incubated for 72 h at 37°C. Chloroquine (CQ).
AAC Accepted Manuscript Posted Online 8 June 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.04332-14 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Salinomycin and other ionophores as new class of antimalarial drugs with transmission blocking

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activity

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Sarah D’Alessandroa, Yolanda Corbetta, Denise Ilboudoa, Paola Misianoa, Nisha Dahiyab, Solomon

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M. Abayb, Annette Habluetzelb, Romualdo Grandec, Maria R. Gismondoc, Koen Decheringd, Karin

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Koolend, Robert W. Sauerweine, Donatella Taramellia, Nicoletta Basilicof#, Silvia Parapinia

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Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, Milan,

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Italy a; Scuola di Scienze del Farmaco e dei Prodotti della Salute, Università di Camerino,

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Camerino, MC, Italyb; L.Sacco University Hospital, Milan, Italyc; TropIQ Health Sciences,

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Nijmegen 6525 GA, The Netherlandsd; Department of Medical Microbiology, Radboud University

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Nijmegen Medical Center, Nijmegen, The Netherlands, 6525 GAe; Dipartimento di Scienze

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Biomediche, Chirurgiche e Odontoiatriche, Università degli Studi di Milano, Milan, Italyf

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Running Head: Ionophores as antimalarials

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#Address correspondence to Nicoletta Basilico, [email protected]

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Abstract

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The drug target profile proposed by Medicine for Malaria Venture for the malaria

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elimination/eradication policy focuses on molecules active on both asexual and sexual stages of

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Plasmodium, thus with both curative and transmission blocking activity. The aim of the present

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work was to investigate whether the class of monovalent ionophores, which includes drugs used in

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veterinary medicine and recently proposed as human anticancer agents, meets these requirements.

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Here, the activity of salinomycin, monensin and nigericin on Plasmodium falciparum (Pf) asexual

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and sexual erythrocytic stages and on the development of the P. berghei (Pb) and Pf mosquito

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stages is reported.

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Gametocytogenesis of the 3D7 Pf strain was induced in vitro, gametocytes at stage II-III or stage

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IV-V of development were treated for different lengths of time with the ionophores and their

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viability measured with the pLDH assay. The monovalent ionophores efficiently killed both asexual

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parasites and gametocytes with nanomolar IC50. Salinomycin showed a fast speed of kill compared

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to standard drugs and the potency was higher on stage IV-V than on stage II and III gametocytes.

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The ionophores inhibited ookinete development and subsequent oocyst formation in the mosquito

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midgut, confirming a transmission blocking activity. Potential toxicity due to hemolysis was

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excluded since only infected and not normal erythrocytes were damaged by ionophores.

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Our data strongly support downstream exploration of monovalent ionophores for repositioning as

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new antimalarial and transmission blocking leads.

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Introduction

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Malaria caused 627.000 deaths and 207 million reported cases in 2012 (1). Among the five species

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that infect humans, Plasmodium falciparum (Pf) is responsible for the majority of deaths and severe

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cases. The recommended malaria control measures include drug treatment, in particular with

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artemisinin-based combination therapy (ACT), and protection from the vectors with insecticide

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treated bed-nets and indoor residual spraying (1). However, the effectiveness of control tools is

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seriously threatened by the emergence and spread of drug and insecticide resistance (2). Even for

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artemisinins, which up to now were safe and effective, resistance is a growing issue in Asia,

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particularly in the Cambodia-Thailand border that is the cradle of antimalarial resistance (3, 4). New

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drugs and new lead compounds for antimalarial drug development are highly needed (5).

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In the past few years, the international strategy against malaria has changed towards malaria

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elimination, and ultimately eradication. The Medicines for Malaria Venture (MMV) has defined

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four Target Candidate Profiles (TCPs) that describe the requirements for novel tools for control and

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elimination of malaria (6). In particular, new multi-stage antimalarial drugs able to kill the liver or

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sexual stages of the parasite and/or capable of preventing the parasite development in the mosquito

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are needed (5, 6). Gametocytes are the sexual stage of Plasmodium, which appear in the circulation

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concomitantly or after the asexual intraerythrocytic stage. They undergo five stages of maturation,

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from I to V. Stage V gametocytes, when taken up by Anopheles mosquitoes during a blood meal,

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become gametes and fuse to form a zygote (7). Subsequently, the zygote transforms into a motile

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ookinete, and becomes an oocyst, which divides to produce sporozoites, ready to restart the cycle.

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Being the stage responsible for transmission, the gametocytes are an essential target for malaria

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elimination/eradication and the identification of gametocytocidal compounds has become an

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absolute priority.

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The strategy of drug repositioning (the usage of existing drugs for new therapeutic indications)

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allows to significantly reduce the development costs, the time-to-market and the risks of failure.

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This is particularly important for diseases of developing countries, for which research funds are 3

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limited (8). Here, we provide evidence in support of the repurposing of salinomycin and/or other

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ionophores as antimalarial and transmission blocking agents. Salinomycin is a polyether antibiotic

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isolated from Streptomyces spp. and a monovalent ionophore for alkali ions with relative K+

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selectivity, able thus of interfering with mitochondrial functions (9). Salinomycin was patented as

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an anticoccidial agent in 1974 and has been used in poultry and other livestock since then. More

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recently, salinomycin and other ionophores such as monensin and nigericin (sodium and potassium

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antiporters, respectively) were found to inhibit cancer stem cell growth by modulating the Wnt

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pathway (10-12). This finding has prompted the usage of salinomycin for compassionate use in a

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few cancer patients with promising results (13).

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Salinomycin and other ionophores (gramicidin, lasalocid and monensin) have already been reported

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to be active against Pf parasites (14-16), but the potency of this class of compounds as transmission

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blocking agents has not been fully investigated, yet. The aim of the present work was to evaluate

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the antimalarial activity of salinomycin, monensin and nigericin on both asexual and transmission

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stages of Pf.

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Materials and methods

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Plasmodium falciparum cultures

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P. falciparum (Pf) cultures were carried out according to Trager and Jensen (17) with minor

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modifications. The strains used in this study are either CQ-sensitive (D10, 3D7 and Pf Ghana

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isolate) or CQ-resistant (W2 and Pf Burkina Faso isolate). The resistance profile of W2 is well

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documented in the literature (19), whereas the Pf Burkina Faso isolate is considered resistant since

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its IC50 to chloroquine is higher than 100 nM, as commonly accepted as threshold for resistance

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(18). All the strains were maintained at 5% hematocrit (human type A-positive red blood cells for

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D10, W2 and Pf isolates; 0-positive red blood cells for 3D7) in RPMI 1640 medium containing 24

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mM sodium bicarbonate (EuroClone, Celbio) with the addition of 0.01 % hypoxantine, 20 mM

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Hepes, 2 mM glutamine. All the parasites were cultured in the presence of 1 % AlbuMaxII (lipid4

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rich bovine serum albumin), except the 3D7 strain that was cultured in the presence of 10% (v/v)

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naturally-clotted heat-inactivated 0+ human serum (Interstate Blood Bank, Inc.), which ensures

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constant and high gametocyte production. All the cultures were maintained at 37 °C in a standard

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gas mixture consisting of 1 % O2, 5 % CO2, 94 % N2. The Pf Ghana and Burkina Faso isolates

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derived from two different patients with malaria admitted to the Ospedale Sacco (Milano, Italy)

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returning to Milan from Ghana or Burkina Faso, respectively. The parasites were adapted to grow in

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culture and are not clonal strains.

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Gametocytogenesis was triggered as previously described (20). Briefly, Pf 3D7 asexual parasites

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cultures were diluted to 0.5% parasitaemia and medium was changed daily without of the addition

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of fresh red blood cells (RBC). When a parasitaemia higher than 5% was obtained and the parasites

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were stressed by nutrient deprivation, the cultures were treated for 48-72h with N-

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acetylglucosamine (NAG) (Sigma-Aldrich) to clear residual asexual parasites. Stage II-III

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gametocytes were obtained and used for the experiments after 4 days from the addition of NAG to

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the culture, while stage IV-V after 7-8 days. Gametocytes stages were routinely checked in Giemsa

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stained smears.

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In vitro Plasmodium falciparum drug susceptibility assay

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Salinomycin, monensin and nigericin sodium salts (Sigma Aldrich, Figure 1) were dissolved in

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either DMSO (salinomycin and nigericin) or ethanol (monensin) and then diluted with medium to

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achieve the required concentrations (final DMSO or ethanol concentration 500

14.3 ± 8.7

IV-V

>500

156.3 ± 78.3

II-III

>100

12.1 ± 4.5

IV-V

>100

6.7 ± 1.9

SI* 72h# 72+72h##

11.4

25.0

27.8

29.8

9.3

96.0

NA

19.8

NA

1.7

HMEC-1 IC50 (nM) 157.5 ± 45.0

169.6 ± 48.0

86.4 ± 31.0

3094.8 ± 316.5

11.7 ± 3.1

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