HDAC inhibitors in parasitic diseases - Nature

Immunology and Cell Biology (2012) 90, 66–77 & 2012 Australasian Society for Immunology Inc. All rights reserved 0818-9641/12 www.nature.com/icb

REVIEW

HDAC inhibitors in parasitic diseases Katherine T Andrews1,2, Ashraful Haque2 and Malcolm K Jones2,3 Parasitic diseases cause significant global morbidity and mortality, particularly in underdeveloped regions of the world. Malaria alone causes B800 000 deaths each year, with children and pregnant women being at highest risk. There is no licensed vaccine available for any human parasitic disease and drug resistance is compromising the efficacy of many available anti-parasitic drugs. This is driving drug discovery research on new agents with novel modes of action. Histone deacetylase (HDAC) inhibitors are being investigated as drugs for a range of diseases, including cancers and infectious diseases such as HIV/AIDS, and several parasitic diseases. This review focuses on the current state of knowledge of HDAC inhibitors targeted to the major human parasitic diseases malaria, schistosomiasis, trypanosomiasis, toxoplasmosis and leishmaniasis. Insights are provided into the unique challenges that will need to be considered if HDAC inhibitors are to be progressed towards clinical development as potential new anti-parasitic drugs. Immunology and Cell Biology (2012) 90, 66–77; doi:10.1038/icb.2011.97; published online 29 November 2011 Keywords: HDAC inhibitor; malaria; schistosomiasis; leishmaniasis; trypanosomiasis; toxoplasmosis

Infectious diseases are among the leading causes of death globally, collectively second only to cardiovascular diseases (Figure 1).1 In 2004, 16.2% of all deaths were attributed to infectious and parasitic diseases, with almost half of those due to three major causes—tuberculosis, HIV/AIDS and the parasitic disease malaria (Figure 1a). Other parasitic diseases, such as trypanosomiasis, schistosomiasis, leishmaniasis and lymphatic filariasis, also pose significant health and economic burdens, particularly in underdeveloped regions of the world. Together parasitic diseases, cause around 1 million deaths annually and a significant global burden of disease in terms of disabilityadjusted life years1 (Figure 1b). In the absence of a licensed vaccine targeted to any human parasitic disease, anti-parasitic drugs, together with appropriate public health measures, continue to be crucial to addressing the health and economic burdens caused by these diseases. Unfortunately, where drugs are available, they are under increasing threat of failure owing to drug-resistant parasites. Intense research efforts are therefore underway to discover and develop new drugs to treat neglected diseases and to address the growing problem of parasite resistance to existing agents for diseases such as malaria.2 One promising strategy is a ‘piggyback’ approach that focuses on drug targets that have been validated for other human diseases and for which chemical starting points are available. Using this approach, histone deacetylase (HDAC) inhibitors, drugs originally targeted for cancer use, are now being investigated to target a range of parasitic diseases. Here we review the status of HDAC inhibitors as new antiparasitic agents and critically examine the viability of progressing HDAC inhibitors towards clinical use for diseases that are a particular problem in underdeveloped regions of the world.

PARASITIC DISEASES OF HUMANS There are a number of parasitic pathogens that cause disease in humans (examples in Table 1), and for many disease severity is linked with the immune status of the host. For example, infection with Plasmodium falciparum, the parasite that causes most malaria-related deaths, may be asymptomatic or cause only mild symptoms in adults living in malaria-endemic regions that have acquired immunity to this pathogen. By contrast, children under the age of 5 in malaria-endemic regions are at most risk of severe malarial disease and death, primarily because of their underdeveloped immune systems.1 Women undergoing their first pregnancy are more susceptible to a severe form of malaria than their non-pregnant counterparts or women from the same regions who have had multiple pregnancies.3 Other groups at risk of severe malaria include immunologically naı¨ve people (for example, travellers to malaria endemic regions) and immunecompromised people such as those with HIV/AIDS. Likewise infection with Toxoplasma gondii, the causative agent of toxoplasmosis, is usually self-limiting but can pose more serious threat or be fatal to the foetus of women who first contract the disease while pregnant. Toxoplasma parasites can cause also cause serious opportunistic infections in immune-compromised people such as those with AIDS, or in people undergoing chemotherapy or organ transplants.4 Relationships between immune status and human diseases caused by helminths, such as schistosomiasis, are also complex and may be negatively associated with HIV infection.5 In addition to the influence of pre-existing immunity or immune status, interactions between parasites and their hosts are complex, confounded by many factors, including parasite virulence, age and

1Eskitis Institute for Cell and Molecular Therapies, Griffith University, Nathan, Queensland, Australia; 2Queensland Institute of Medical Research, Herston, Queensland, Australia and 3University of Queensland, School of Veterinary Sciences, Gatton, Queensland, Australia Correspondence: Dr KT Andrews, Tropical Parasitology Laboratory, Queensland Institute of Medical Research, PO Box Royal Brisbane Hospital, Herston, Queensland 4029, Australia. E-mail: [email protected] Received 19 September 2011; revised 28 September 2011; accepted 5 October 2011; published online 29 November 2011

HDAC inhibitors in parasitic infectious diseases KT Andrews et al 67

Figure 1 Percentage deaths and disability-adjusted life years in 2004 in WHO regions. The pie charts show (a) percentage of deaths and (b) disabilityadjusted life years (DALYs) owing to communicable diseases (grey), non-communicable diseases (black) and injury (white) in WHO regions in 2004. The pie charts were generated using data taken from reference 1.

nutritional status, and co-infection. These factors often combine in resource-poor settings and pose significant challenges in the fight against major human parasitic pathogens. Other challenges facing researchers engaged in anti-parasitic drug discovery include difficulty dissecting the molecular mechanisms underlying parasite resistance and identification and validation of new drug targets. Drug discovery is often hindered by the complex lifecycles of human-infecting parasites (more than one host, distinct morphological forms, and asexual and sexual replication), the genetic divergence of parasites, and the inability to easily genetically manipulate and biochemically characterize many species. However, in the past decade, sequencing and

annotation of the complete genomes of many of the major parasites that infect humans has begun to improve our understanding of the biology of these parasites. As a result we are now able to identify and functionally characterize homologues/orthologues of validated drug targets in other organisms, including histone-modifying proteins such as HDACs (Table 2), the focus of this review. HDACS AND GENE EXPRESSION Histone-modifying enzymes are crucial for modulating cell chromatin structure and gene expression in eukaryotic organisms. Acetylation of nucleosomal histone proteins H2A, H2B, H3 and H4, mediated by Immunology and Cell Biology


Table 1 Major human parasitic pathogens discussed in this review Disease

Transmission

Species

Features/pathophysiology

Malaria

Bite of infected female Anopheles mosquito

P. falciparum P. vivax

P. falciparum causes most morbidity and mortality. Simple fever-like symptoms to severe complications (severe malaria) ranging from renal failure, metabolic acidosis, loss of liver

P. ovale (two species) P. malariae

function, severe anaemia and coma; severe forms include cerebral malaria and pregnancy-associated malaria89

P. knowlesi T. gondii

Cysts in various tissues, including the CNS, eye, heart, skeletal muscle and placenta; ‘’resting-stage’

Toxoplasmosis

Ingestion of infected

bradyzoites in cysts can persist in host for life; tachyzoites can cross the maternal–foetal barrier, infect the foetus, and cause abortion or congenital abnormalities; serious opportunistic infections

meat/fomites; mother to foetus

in immunocompromised people (for example, AIDS) or people undergoing chemotherapy/organ transplants90 Trypanosomiasis

Bite of infected Tsetse fly/Assassin bug

T. brucei T. cruzi

T. brucei (African sleeping sickness): Phase-I—fever, headache, joint pain, itching and swelling of lymph nodes. Phase-II—neurological symptoms; fatal without treatment; T. cruzi (Chagas diseases) —mild acute-phase symptoms (local swelling of infection site) to chronic infection (asymptomatic in most, but life-threatening heart and digestive complications in 20–40% of cases)

Schistosomiasis

Intermediate hosts— freshwater snails;

S. mansoni S. japonicum

Chronic pathology arises from host reaction to eggs that become entrapped in tissues; site of egg entrapment is linked to site of infection by adult parasites—disease may be hepatosplenic

cercariae (larval stages) penetrate skin

S. haemotobium S. intercalatum

or genitourinary91

S. mekongi Leishmaniasis

Bite of infected sand flies

L. donovani L. infantatum

Depending on species—skin lesions, fever, liver and spleen damage, anaemia; visceral leishmaniasis—potentially fatal if untreated92,93

histone acetyltransferases and HDACs, is one of the best-studied post-translational modifications of histones.6 In general, increased acetylation by histone acetyltransferase proteins causes an openingup of chromatin to facilitate gene transcription, whereas the opposing action of HDACs results in a more ‘closed’ chromatin state and transcriptional repression. In addition to their action on histones, an increasing number of non-histone proteins have also been shown to be HDAC targets in eukaryotic cells, including structural proteins, chaperones, transcription factors, chromatinremodelling proteins, signalling mediators and nuclear import proteins.7 It is therefore becoming increasingly evident that the biological function of HDACs is complex, influencing numerous important functions, including protein stability, protein–protein interactions, protein localization, and alteration of the DNA-binding properties of proteins. HDACs are grouped into four classes based on sequence similarity and cofactor dependence (reviewed in reference 7). In human cells, class-I HDACs comprise HDAC1–3, and HDAC8; class-II HDACs comprise HDAC4–7, HDAC9 and HDAC10; and class-III (Silent Information Regulator-2 (Sir2)-related proteIN (sirtuin)) HDACs comprise seven members (SIRT1–7) that share homology with yeast SIR2. HDAC11, which has some similarity with class-I HDACs, has been assigned as the only member of class-IV. Class-I and II HDAC proteins share a similar catalytic core that uses zinc as a cofactor, but differ in size and structural organization. Class-I HDACs are primarily localized in the nucleus and are expressed almost ubiquitously. By contrast, class-III HDACs have more tissue specificity, can be located in the nucleus and cytoplasm, and use nicotinamide adenine dinucleotide (NAD+) as cofactor. Recent work has highlighted a role for class-III sirtuins in many cellular functions, including gene repression, apoptosis, DNA repair and promotion of longevity.8,9 FUNCTIONAL ROLES OF HDACS IN MAJOR HUMAN PARASITES HDACs have been identified in all the major human parasitic pathogens (summarized in Table 2). This section will focus on key Immunology and Cell Biology

functional findings available for characterized or partially characterized HDACs. Plasmodium HDACs Only one of the three identified class-I/II HDAC homologues has been investigated in any detail for Plasmodium falciparum (Table 2). PfHDAC1 has up to B55% amino-acid identity to other eukaryotic class-I HDACs,10 is nucleus-localized11 and expressed/transcribed across multiple lifecycle stages of the parasite.11–14 In silico homology modelling of PfHDAC1 predicts a high level of conservation of the active-site tunnel, but differences at the entrance to the active-site tunnel compared with human HDACs.15,16 These differences may explain the better in vitro growth inhibition activity of some HDAC inhibitors for P. falciparum compared with mammalian cells (Table 3 and below). The functional role(s) of PfHDAC1 in P. falciparum have not yet been directly demonstrated; however, as discussed below, the consequence of HDAC inhibitor treatment of P. falciparum parasites is beginning to be elucidated and it is likely PfHDAC1 is involved in the post-translational modification of histones and therefore control of gene expression. Whereas little is known about the other class-I/II P. falciparum HDACs, the two class-III homologues have been better studied (Table 2). PfSir2A, which has both histone deacetylase and ADP-ribosyltransferase activity, and PfSir2B are classified as type-III and type-IV sirtuins, respectively.17–19 PfSir2A has a role in maintaining P. falciparum telomere length, heterochromatin establishment in sub-telomeric regions, and the regulation of a subset of P. falciparum virulence genes involved in antigenic variation and cytoadhesion/ pathogenesis.18,20,21 PfSir2B also silences a subset of these P. falciparum genes, however, a set controlled by a different type of promoter than those regulated by PfSir2A.18 While neither PfSir2 is essential for P. falciparum growth in vitro,18,20 their role in regulating virulence gene expression makes them potential targets for anti-disease therapies, for example by interfering with infected erythrocyte cytoadhesion to host cell receptors that mediate severe forms of the diseases and/or by blocking malarial parasite immune evasion.


Table 2 HDAC homologues from human-infecting parasites Name

Gene IDa

HDAC

MW

class

(BkDa)

Characteristics/function

P. falciparum PfHDAC1

PFI1260c

I

51

Expressed in asexual and gametocyte stages; nuclear localization (asexual stage); inhibited by apicidin,

PfHDAC2

PF14_0690

II

269

Expressed in asexual and gametocyte stages; nuclear localization signal; putative interaction with

PfHDAC3

PF10_0078

II

282

Expressed in asexual and gametocyte stages; putative interaction with knob-associated histidine-rich

PfSir2A

PF13_0152

III

30

TSA and SAHA11,13,14,41,94 S-adenosyl-L-homocysteine hydrolase (PFE1050w)13,14,94,95 protein (KAHRP; PFB0100c)13,14,94,95 Type-III sirtuin; transcribed in asexual and asexual stages; nuclear pole/punctate perinuclear localization; colocalization with heterochromatin and telomeric clusters; HDAC and ADP-ribosyltransferase activity; inhibited by nicotinamide; not essential in vitro; associated with silencing of some var and rif genes; role in maintaining telomere length13,14,19–21,94 PfSir2B

PF14_0489

III

155

Type-IV sirtuin; silences some var genes (different promoters to PfSir2A); not essential in vitro13,14,18,94

T. gondii TgHDAC2

TGME49_049620

I

79

B55% amino-acid identity to hHDAC196

TgHDAC3

TGME49_027290

I

50

460% amino-acid identity to hHDAC1; associated with bradyzoite-specific promoters; absent from tachyzoite-specific promoters; nuclear localized (not nucleolus); forms complex with actin, TgHSP70a and TgHDP70b; inhibited by FR235222 in tachyzoites; amino-acid T99A and T99I mutations affect basal activity22,50,96

TgHDAC1

TGME49_081420

II

250

Conserved domains: HDAC, AcuC, IPK, PRK

TgHDAC4

TGME49_057790

I/II

30

TgHDAC5

TGME49_002230

II

154

TgSir2

TGME49_027020

III

40

Conserved domains: SIR2 superfamily

Sir2-related

TGME49_067360

III

187

Conserved domains: SIR2 superfamily

Conserved domains: HDAC superfamily (AcuC domain); homology to hHDAC11 Conserved domains: ANK, AcuC, HDAC

T. brucei TbDAC1

Tb927.10.1680

I

43

Essential in vitro in the bloodstream form; nuclear localized; antagonizes telomeric VSG silencing in

TbDAC2

Tb11.01.7240

I

61

Predominantly localized to cytoplasm26

TbDAC3

Tb927.2.2190

II

75

Essential in vitro in the bloodstream form; localized to nucleus; mutation of H316 abolishes HDAC activity23,26

TbDAC4

Tb927.5.2900

II

64

Predominantly localized to cytoplasm26

TbSIR2rp1

Tb927.7.1690

III

38

Nucleus-localized; required for basal telomeric silencing but not VSG expression site silencing; not essential27

TbSIR2rp4

Tb927.8.3140

III

34

Mitochondrial localization; not essential in T. brucei; not present in the T. cruzi genome27,32

TbSIR2

Tb927.4.2520

III

27

Mitochondrial localization; not essential27

SmHDAC1

Smp_005210

I

52

Mammalian HDAC1 orthologue; transcribed in all lifecycle stages; putative transcriptional repressor28

SmHDAC3

Smp_093280

I

48

Mammalian HDAC3 orthologue; transcribed in all lifecycle stages28

SmHDAC8

Smp_091990.1/

I

48/50

Mammalian HDAC8 orthologue; transcribed in all lifecycle stages28

bloodstream form parasites23,26

S. mansoni

2mp_091990.2

9 128 > >Putative orthologues of mammalian HDACs 2, 4, 5, 6/10 and 929 > > = 23 33 > > > > ; 31

–

Smp_138770

II

–

Smp_138780

II

–

Smp_177250

II

–

Smp_191310

II

Smp_138640

III

68

Putative chromatin-regulatory protein sir2

Smp_134630

III

185


Smp_024670

III

60


Smp_055090

III

34


Smp_084140.1,

III

40/38/34


3, and 4 L. major –

LmjF.21.0680

I

47

Histone deacetylase, putative; transiently upregulated during promastigote-to-amastigote differentiation

–

LmjF.24.1370

I

58

Histone deacetylase, putative

–

LmjF.08.1090

II

57


–

LmjF.21.1870

II

67


LmSIR2RP1

LmjF.26.0210

III

41

Cytoplasmic localization; excreted-secreted antigen; interacts with HSP83; L. infantum homologue

in a host-free system33

(LiSIR2RP1) expressed in the cytoplasm of promastigotes and amastigotes; LmSIR2RP1 and LiSIR2RP1 have a potential role in parasite survival; overexpression causes increased parasite survival; immunogenic during human and canine infections; potential immunomodulatory role34,35,38,39,97 Sir2-like

LmjF.34.2140

III

27

Putative NAD-dependent deacetylase/nicotinic acid mononucleotide-5,6-dimethylbenzimidazole (Cobb) protein35

Abbreviation: HDAC, histone deacetylase. aReferences for database gene ID numbers are as follows: P. falciparum: 12; T. gondii: 96; S. mansoni: 98; and from GeneDB (www.genedb.org and http://tritrypdb.org/tritrypdb/) for L. major and T. brucei, respectively.

Immunology and Cell Biology


Table 3 In vitro anti-parasitic activity of selected HDAC inhibitors Class/inhibitor

Structure

Mammalian cell

Parasite IC50 (mM) (selectivity index)a

IC50 (mM) P. falciparum Cyclic tetrapeptide

0.001–0.1

Apicidinb

B0.200 (o1)

T. gondii 0.013–0.015

S. mansoni Leishmania sp. ND

Toxic

B5–50e

ND

(o8)

O HN

N

HN

NH

O O O

N O

O

Short-chain fatty acid Valproic acidc

433–1350

4100d (o4–13)

31–1600 (1–43)

(B8–270)

HO

O

Hydroxamic acid TSAf O

0.008–0.120 (2–38) 0.041 (5–7)

o2–200g

5–20

0.1–0.3 (17–200)

0.083 (60–241)

410 (o2) 112.4 (o1)

50–300

0.8–2.3 (22–375)

0.213 (235–1408) ND

ND

45

0.15 (433)

ND

ND

443

45

1.23 (44)

ND

ND

4.96 (41)

0.2–0.3

O N H

4.5 (o1)

OH

N

SAHAh

O

OH N H

O HN

SBHAi

OH HN

O

O Tricyclic ketolide 15bj (n¼2)

O

N N N

N

O N

O HO

O O

OH N H

O

N

O

n( )

O

O

NH HO

Tricyclic ketolide 15ej (n¼5)

Abbreviations: HDAC, histone deacetylase; ND, not determined. aSelectivity index: IC 50 mammalian cells/IC50 parasite species. bReferences 10,48–50,99. cReferences 53,100,101. dOur unpublished data. eB80% reduction in miracidia development with 50 mM (4 h).54 B30% % inhibition of schistosomula cell viability with 5 mM (7 days).29 fReferences 52,56,65,102. g67–98% reduction in miracidia development with 200 mM (4 h).54 100% inhibition of schistosomula cell viability with 2 mM (7days).29 hReferences 52,65,77,102,103. iReferences 52,56,104. jReference 66.



Toxoplasma HDACs The T. gondii genome contains five class-I/II HDAC homologues (Table 2). TgHDAC3 has been shown to localize in the nucleus22 and to be part of a large multi-protein complex termed the T. gondii co-repressor complex. The T. gondii co-repressor complex includes TgHDAC3, T. gondii transducin b-like protein-1 (TgTBL1), actin, two heat-shock protein-70 (HSP70)-like proteins and subunits of a T. gondii TCP-1 ring complex (TRiC).22 Homologues of the TgHDAC3 complex members are present in human HDAC3-containing complexes.22 Deacetylase activity in the affinity-purified T. gondii co-repressor complex has been shown to be sensitive to HDAC inhibitors (for example, B75% deacetylase activity inhibition with butyrate (2 mM) and trichostatin-A (TSA; 0.02 mM)).22 T. gondii HDACs (and histone acetyltransferase enzymes) have also been shown to function in stage-specific gene regulation in different forms of this parasite (tachyzoite to bradyzoite forms).22 Trypanosoma HDACs There are four class-I/II HDACs in Trypanosoma brucei (Table 2). Two of these proteins (TbDAC1 and TbDAC3) are nucleus-localized and essential to parasite survival.23 In the bloodstream of the human host, the T. brucei parasite surface is coated with GPI-anchored variant surface glycoproteins (VSGs). VSG members are encoded in one of B15 polycistronic transcribed telomeric expression sites that can be altered to help the parasite evade host immune defences.24 Although there are hundreds of VSG genes in the T. brucei genome, only one VSG expression site is transcriptionally active at a time.25 TbDAC3 is required for VSG expression site silencing in bloodstream and insect stage cells.26 By contrast, a T. brucei SIR2 homologue (TbSIR2rp1) has been shown to be required for basal telomeric silencing but not VSG expression site silencing.27 T. brucei DAC1 antagonizes SIR2rp1dependent telomeric silencing in bloodstream form parasites, which is indicative of a role in the control of silent chromatin domains. A mutation in the active site of TbDAC3 (H316) abolishes HDAC activity.26

have investigated the functional roles of class-III HDACs of Leishmania species. A Leishmania major protein with extensive homology to yeast SIR2p (L. major SIR2-related protein-1 (LmSIR2RP1)) has been identified,34 and homologues of this protein are expressed in the cytoplasm of different Leishmania species and parasite developmental stages (promastigotes and amastigotes).34,35 The L. infantum SIR2related protein-1 (LiSIR2RP1) has been found to have NAD+-dependent deacetylase activity and possible ADP-ribosyltransferase activity.36 LiSIR2RP1 deacetylates a-tubulin and is partially associated with the microtubule network.36 Overexpression (LiSIR2RP1) and gene disruption studies (LmSIR2RP1 and LiSIR2RP1) indicate a role for Leishmania SIR2RP1 proteins in parasite survival.35,37 Studies of soluble fractions of parasites overexpressing LmSIR2RP1 showed that this HDAC co-immunoprecipitates with HSP83, the Leishmania orthologue of mammalian HSP90.38 However, the amount of intracellular LiSIR2RP1 did not alter the acetylation state of LiHSP83, so it is not yet clear if LiHSP83 is a substrate for this HDAC. Recombinant LmSIR2RP1 expressed and purified from Escherichia coli is able to trigger B-cell effector function, suggesting an immune modulation role for this HDAC during infection.39 Likewise, LiSIR2RP1 activates splenic B cells through a Toll-like receptor-2 (TLR2)-dependent mechanism, resulting in upregulation of major histocompatibility complex (MHC)-II and the co-stimulatory molecules CD40 and CD86, and secretion of tumour necrosis factor in the mouse model used.40 The TLR-signalling pathway is involved in initial recognition of Leishmania parasites by the host innate immune system, and activation of this pathway is thought to lead to resistance to infection and to aid in the development of acquired immunity. However, despite the observation of TLR2-dependent activation of B cells, TLR2-deficient mice were still able to secrete antibodies to LiSIR2RP1 after immunization, suggesting that LiSIR2RP1 may also interact with other cell types in the absence of TLR2.40 The Leishmania SIR2RP1-mediated effects discussed above did not appear to be due to endotoxin contaminants. In both studies, recombinant SIR2RP1 protein was passed through an EndoTrapred column (Profos, Regensburg, Germany), and in one, polymyxin-B, which was able to significantly abrogate lipopolysaccharide-induced activity, did not affect LmSIR2-induced changes.39 However, given that the literature on pathogen and host products signalling through TLR2 and TLR4 remains unclear, it is important that future studies give appropriate consideration to possible effects of contaminating bacterial products. The immunomodulatory role of Leishmania SIR2RP1 does, however, raise the possibility that this protein might act as a possible adjuvant in a multi-component vaccine.40

Schistosoma HDACs Three HDACs from Schistosoma mansoni have been characterized as orthologues of mammalian class-I HDACs (Table 2).28 Several putative class-II HDAC orthologues have also been identified in S. mansoni, however none has been characterized functionally.29 The class-I homologue SmHDAC1 transcriptionally represses the transcription factor Gal4 or a Gal4-NF-kB fusion protein in a co-transfection system in mammalian cells.28 This has been shown to be HDAC-dependent as repression was partially restored by treatment with the HDAC inhibitor TSA. When the two histidine residues in the active site of the SmHDAC1 protein (H176/177A) were modified, repression of Gal4-NF-kB-dependent transcriptional activity in the co-transfection system was abrogated,30 indicating a key role for these residues in the activity of the protein. No class-III HDACs have been characterized for schistosomes, although expressed sequence tags (ESTs) encoding multiple putative members are present in the S. mansoni genomic data sets (Table 2).31

ANTI-PARASITIC ACTIVITY OF HDAC INHIBITORS The potential of HDAC inhibitors as anti-parasitic agents was first realized over a decade ago when the cyclic tetrapeptide apicidin was found to have broad-spectrum anti-parasitic activity.10 Since then a growing number of studies have focused on the anti-parasitic activity of HDAC inhibitors of various structural classes, highlighting the potential of these chemotypes for anti-parasitic intervention.

Leishmania HDACs The Leishmania genome also contains multiple genes encoding putative HDACs (Table 2).32 While four class-I/II HDAC homologues have been annotated, none has been functionally characterized. One (Table 2, LmjF21.0680) has, however, been shown to be transiently upregulated during promastigote-to-amastigote differentiation in a model host-free system, suggesting a possible role in alteration of chromatin structure and transcription.33 By contrast, several studies

Cyclic tetrapeptide HDAC inhibitors Darkin-Ratray et al.10 showed that apicidin has potent in vitro activity against P. falciparum, T. gondii (Table 3) and other protozoan parasites. Apicidin causes hyper-acetylation of P. falciparum histones10 and inhibits the activity of recombinant PfHDAC1 enzyme (IC50 B1 nM).41 This compound has also been shown recently to cause significant alterations to the P. falciparum intra-erythrocytic developmental cycle transcriptional cascade, with B30–60% of the Immunology and Cell Biology


genes showing altered expression.42,43 These findings are similar to the effect of HDAC inhibitors on higher eukaryotic cells.44,45 Orally administered apicidin (2-20 mg kg1 for 3 days) was found to be effective against Plasmodium berghei in mice;10 however, the poor selectivity of this compound for parasites versus mammalian cells (Table 3), and its poor bioavailability, means that it is not considered clinically suitable.46 The inability of apicidin to selectively inhibit P. falciparum growth as compared with mammalian cell growth has been suggested to be due to structural similarities with one of the side chains (2-amino-8-oxodecanonic acid) and acetylated histone lysines.10,46 To try to address the issue of selectivity, apicidin analogues with indole modifications, and tryptophan– or quinolone –replacements, have been tested against P. falciparum parasites. Some quinolone derivatives, but not N-substituted indole derivatives, were found to have increased selectivity (up to B200-fold) for P. falciparum versus mammalian cells at the whole-cell level.46–48 The activity of apicidin and a series of synthetic analogues have been compared for two Trypanosoma species (Trypanosoma cruzi and T. brucei), P. falciparum and Leishmania donovani.49 As reported previously10 apicidin had potent, but non-selective, activity against P. falciparum and T. brucei, but was found to be toxic to L. donovani and T. cruzi parasites and host cells.49 The species-specific effect observed for Trypanosoma species was also seen for the three synthetic analogues, with 50% inhibitory (IC50) values of B2–13 mg ml1 achieved for T. brucei parasite growth, whereas the IC50 for intracellular T. cruzi was 430 mg ml1 for all three compounds. The three synthetic analogues also showed poor activity against P. falciparum (IC50 41 mg ml1) and L. donovani (IC50 430 mg ml1).49 More recently, the cyclic tetrapeptide FR235222, isolated from fermentation broth of Acremonium species, was shown to have potent activity against T. gondii tachyzoites, and drug-sensitive and resistant P. falciparum-infected erythrocytes (IC50 B10 nM).50 This compound caused hyper-acetylation of T. gondii histone H4, indicating that it acts, at least in part, through HDAC inhibition. In addition, T. gondii lines with mutations in the TgHDAC3 gene (T99A and T99I) were more resistant to the compound (IC50 B50 nM versus wild-type IC50 B10 nM). There did, however, appear to be a fitness cost to the resistant parasites, and alternative modes of action were also considered a possibility based on the levels of resistance observed.50 In an effort to investigate genes that might be controlled by TgHDAC3, chromatin immunoprecipitation (ChIP) and microarray (ChIP-onchip assay) were used to investigate patterns of DNA-bound acetylated histone H4 after treatment of T. gondii parasites with FR235222 as compared with untreated parasites. FR235222-mediated hyper-acetylation effects were observed across all 14 chromosomes of the parasite, with B5% of genes having increased H4 acetylation levels. FR235222 treatment induced differentiation of the replicative tachyzoite stage parasites into the non-replicative bradyzoite stage. These data provide evidence for TgHDAC3 as a regulator of gene expression and stage-specific conversion in T. gondii.50 Short-chain fatty acid HDAC inhibitors Short-chain fatty acid-type HDAC inhibitors include valproic acid, sodium butyrate and its derivatives. This class of HDAC inhibitors have weak HDAC inhibitory activity against mammalian HDACs, but also have other targets, making their effects difficult to attribute only to HDAC inhibition. Some of these compounds have, however, shown clinical efficacy. For example valproic acid, which has been widely used as an antiepileptic drug and mood stabilizer, is undergoing several clinical trials for HDAC-relevant diseases.51 Valproic acid, sodium butyrate and a derivative (4-phenylbutyrate) all have relatively poor Immunology and Cell Biology

in vitro activity against T. gondii tachyzoites (IC50 B1.0, 1.6 and 5.4 mM, respectively)52,53 and poor selectivity for the parasite versus HS69 mammalian cells. Likewise, valproic acid has only modest activity against P. falciparum and S. mansoni (Table 3). For S. mansoni, an B80 reduction in miracidia development was achieved with 50 mM valproic acid after 4 h;54 however only an B30% loss of viability of schistosomula was observed after 7-day continued exposure to the compound at 5 mM.29 To our knowledge, valproic acid has yet to be evaluated in vivo in animal models for any of these parasitic diseases. This is despite the in vitro activity of valproic acid against some parasites being within the therapeutic range for this compound for its primary use as a moodstabilizing drug for schizophrenia or bipolar disorder.52,53 Hydroxamate HDAC inhibitors Hydroxamate-based HDAC inhibitors include natural product derivatives such as TSA, and synthetic compounds including suberoylanilidie hydroxamic acid (SAHA, Vorinostat; Merck & Co., Whitehouse Station, NJ, USA), which was approved for clinical use for treatment of cutaneous T-cell lymphoma in 2006.55 As has been the case for HDAC inhibitors for other human diseases, such as cancers, hydroxamates are also the best studied anti-parasitic HDAC inhibitors. Several studies have examined the effect of hydroxamate HDAC inhibitors on malarial parasite growth. TSA is a highly potent inhibitor of in vitro growth of P. falciparum (IC50 8–12 nM);56 however this compound kills mammalian cells at similar concentrations as it does the parasite (Table 3), so it is not considered suitable for clinical progression. Despite this, TSA has proved to be a highly useful tool to help understand how HDAC inhibition effects malarial parasite growth, development and transcriptional control. TSA inhibits recombinant PfHDAC1 enzyme activity (IC50 B0.6 nM),41 causes in situ hyper-acetylation of P. falciparum histones10 and, like apicidin, causes large-scale genome-wide transcriptional changes in the parasite.42 TSA has also been used as a molecular tool to identify HSP90 (PfHSP90) as the first potential non-histone target of HDACs in P. falciparum.57 This is an important finding as identification of nonhistone targets of malarial parasite HDACs may lead to new insights into the regulation of essential molecular pathways in the parasite and the identification of novel drug targets. In contrast to TSA, SAHA has less potent activity against P. falciparum laboratory lines in vitro (IC50 B100–300 nM),58 but somewhat improved parasite-specific selectivity58 (Table 3, up to B200-fold). SAHA, and two other anti-malarial hydroxamates based on a 2-aminosuberic acid scaffold,15 have also been shown to inhibit ex vivo growth of P. falciparum (SAHA IC50 120–484 nM) and Plasmodium vivax (SAHA IC50 67–281 nM) isolates obtained directly from infected people.59 The activity obtained against P. vivax is very promising. Although infection by this malarial parasite species does not generally result in death, it is the cause of significant malaria-related morbidity, and drugs that potentially target multiple Plasmodium species are desirable. Despite its clinical use for cancer, the in vivo efficacy of SAHA against Plasmodium parasites in murine malaria models has not yet been reported. By contrast, the hydroxamate suberic bishydroxamate (SBHA) has been studied in vivo in a mouse model of malaria. SBHA has lower in vitro potency than SAHA against P. falciparum, but, depending on the cell line examined, better selectivity for the parasite versus mammalian cells (Table 3). In P. berghei-infected BALB/c mice, SBHA (200 mg kg1, twice daily (intra-peritoneally) for 3 days) significantly inhibited peripheral parasitemia, however mice were not cured. The compound was therefore suggested to have a cytostatic effect.56 More recently, several hydroxamic acid-based HDAC inhibitor


analogues have been described with better potency against P. falciparum parasites in vitro than SAHA or SBHA, and in some cases much better selectivity. HDAC inhibitor analogues screened in those studies included compounds based on L-cysteine, 2-aminosuberic acid,15 triazolylphenyl,60 compounds with cinnamate or non-steroidal antiinflammatory components,61 and a panel of 50 phenyl-thiazolylhydroxamate-based HDAC inhibitor analogues.58 The latter study identified three compounds with P. falciparum IC50 values o3 nM and selectivity indices of 4600.58 The lead compound from this panel (WR301801) was found to hyper acetylate P. falciparum histones and inhibit P. falciparum deacetylase activity in nuclear extracts (IC50 B10 nM). WR301801 caused a significant suppression of parasitemia in P. berghei-infected mice after oral administration as monotherapy (50 mg kg1 day1 for 4 days).58 However, orally administered WR301801 was not able to cure mice unless combined with sub-curative doses of the anti-malarial drug chloroquine (52 mg kg1 WR301801 and 64 mg kg1 chloroquine). These findings, together with another study that showed that this same compound could cure mice when administered through the intra peritoneal route,62 do however demonstrate the potential of hydroxamate-based HDAC inhibitors for malaria therapy, provided the pharmacokinetics of these compounds can be improved. It should be noted however, that no information is currently available on whether in vivo efficacy is due to a direct anti-parasitic effect or immune modulation effects on the host. Several hydroxamates have been tested for activity against T. gondii tachyzoite-stage parasites.52 TSA (IC50 41 nM), SAHA (IC50 83 nM) and scriptaid (IC50 39 nM) all have similar in vitro potency against T. gondii tachyzoite-stage parasite, whereas SBHA is B2- to 5-fold less potent than the other three compounds (IC50 213 nM; Table 3).53 As for P. falciparum, TSA has a low level of selective activity for T. gondii parasites versus mammalian cells, whereas SAHA and scriptaid show better selectivity.52 Interestingly, low concentrations of TSA, SAHA and scriptaid (1–50 nM) had an apparently stimulatory effect on T. gondii tachyzoite proliferation and/or survival, but a suppressive effect on tachyzoite infectivity.52 In Schistosoma parasites, TSA has been found to partially inhibit total deacetylase activity of protein lysates across five different lifecycle stages of S. mansoni, the miracidia, sporocysts, cercariae, schistosomula and adult males.29 Incomplete inhibition was suggested to be due to differences in substrate specificity for the S. mansoni HDACs versus mammalian HDACs, or residual class-III (sirtuin) activity in the parasites.29 TSA and SAHA inhibited total deacetylase activity in protein lysates by 70–90% in schistosomula and adult male worms,29 and TSA blocked the in vitro development of miracidia into sporocysts.54 Longer-term exposure (7 days) of schistosomula to TSA (1 and 2 mM) is toxic to the parasite,29 in contrast to SAHA, which was not toxic to in vitro cultured schistosomula at concentrations up to 10 mM. The authors suggested that this might be due to inability of SAHA to penetrate the double apical tegument membrane of the larvae; however it should be noted that TSA and SAHA have similar cLogP values (1.9 and 1.0, respectively). There was some evidence that the anti-parasitic activity of TSA was due to apoptotic mechanisms, although high levels of this compound were required to achieve significant increases in apoptosis indicator assays (caspase-3/7 activity and TUNEL assays).29 TSA causes an increase in acetylated histone H4 in S. mansoni miracidia, schistosomula and adult worms,29,54 and, to a lesser extent, histone H3 in schistosomula and adult worms.29 Using ChIP assays TSA has been shown to increase histone H4 acetylation in the proximal promoter region of the caspase-7 gene, which is transcriptionally upregulated by this compound.29 Together, these

data provide evidence that hydroxamate HDAC inhibitors target deacetylase activity in Schistosoma parasites. Very little information is available on the activity of hydroxamate or other class-I/II HDAC inhibitors against typanosomes or Leishmania species. Some studies have attempted to use TSA to understand HDAC-related regulatory mechanisms in typanosomes. One study reported that TSA ‘suppressed the growth’ of the bloodstream form of T. brucei at B7 mM and that this concentration had no effect on the derepression of a silent VSG expression site.63 In another study TSA was found to inhibit 450% of activity of recombinant TgDAC1 and TgDAC3 at 5 mM;26 however TSA (0.3 mM) did not alter histone acetylation in T. cruzi parasites.64 The activity of a series of aryltriazolyl hydroxamate-based HDAC inhibitors was investigated for their activity against the promastigote stages of L. donovani as well as asexual P. falciparum blood-stage parasites.65 Several compounds showed nM activity against P. falciparum growth, which was better than the activity obtained for the control HDAC inhibitor SAHA. Although the compounds were overall less active against L. donovani than P. falciparum, a series of six analogues had L. donovani IC50 values 2- to 4-fold better than SAHA and comparable to miltefosine, a clinically approved oral drug for visceral leishmaniasis. Structure– activity relationship analysis of the anti-parasitic effect for P. falciparum and L. donovani showed that activity was maximal in analogues with 5 or 6 methylenes in the spacer region between the compounds’ active site group and capping group.65 The same group also investigated five tricyclic ketolide-based HDAC inhibitor derivatives for activity against L. donovani and P. falciparum parasite growth. The most active compound against P. falciparum has an IC50 similar to SAHA (Table 2, 15b). All five compounds were also tested against recombinant PfHDAC1 enzyme, with IC50 values between 10 and 303 nM (Table 3, compound 15b and 15e, respectively) being achieved.66 However, these values were similar to those obtained for HeLa nuclear extracts (1 and 208 nM, respectively), indicating poor selectivity. The activity of the five analogues against L. donovani was modest for one (Table 3, 15e (IC50 4.96 mM)) but poor for the other four (Table 2, for example, 15b (IC50 443 mM)). The better activity of one of these analogues against L. donovani may be due to this compound having the longest methylene linker region. Class-III HDAC inhibitors A number of Sir2 inhibitors have been tested for anti-proliferative activity against P. falciparum-infected erythrocytes and Leishmania parasites. Overall the activity of Sir2 inhibitors against P. falciparum parasite growth is modest. This is not surprising given the low homology between the parasite and other eukaryotic Sir2 proteins, and that PfSir2A and PfSir2B can be genetically disrupted in P. falciparum parasites in vitro18,20 Double knockout of the two PfSir2 paralogues has not yet been reported. The functions of these proteins are also not yet fully known and, although PfSir2A and PfSir2B only share 26% amino-acid identity,18 it may be possible that a level of functional redundancy exists. Sir2 inhibitors that have been examined for growth inhibition activity against P. falciparum include sirtinol (IC50 B9–13 mM), surfactin (IC50B9 mM), splitomycin (IC50 410 mM) and hyperforin (IC50 1.5–2.1 mM)15,17,41,67 Nicotinamide, which is a product of the Sir2-catalysed reaction, and a non-competitive inhibitor of both acetylated peptide and NAD+, is much less active at the whole-cell level (IC50B10 mM), with a delayed parasite growth effect observed.68 Nicotinic acid, also a product of Sir2dependent enzymatic pathways, did not show this effect.68 Recombinant PfSir2A, but not PfSir2B, is available and variations in inhibitory activity have been observed. For example, surfactin and nicotinamide Immunology and Cell Biology


have similar activity against PfSir2A (IC50 35 and 51 mM, respectively); however splitomycin and sirtinol are less active (IC50 4400 and 450 mM, respectively).17,19 In a recent study, lysine-based tripeptide analogues were synthesized to try to target PfSir2 through competition with the peptide binding pocket.69 Three of four analogues examined had similar or better activity (IC50 23–34 mM) against PfSir2A compared with surfactin and nicotinamide.69 However, as was the case for all the Sir2 inhibitors discussed above, none of these compounds were selective for the parasite Sir2 as compared with human SIRT1.69 The most active of the compounds was tested in vitro against P. falciparum and had similar activity to other Sir2 inhibitors (IC50 B10 mM).69 The Sir2 activators isonicotinic acid and resveratrol (IC50 B60 mM70) have also been examined against recombinant PfSir2A, however activity was not affected by these compounds using the assays conditions reported.19 Sirtinol has been shown to have activity against L. infantum parasites, with activity observed for axenic amastigotes (IC50 B30 mM) but not promastigotes (IC504B60 mM).71 This stage-specific activity is supported by targeted gene disruption studies where deletion of a single LiSIR2 gene did not affect promastigotes whereas amastigotes had reduced capacity to multiply in vitro and in vivo.37 Parasites overexpressing L. major SIR2 have also been shown to be resistant to DNA fragmentation caused by sirtinol treatment.71 Nicotinamide has been shown to inhibit L. major growth; however overexpression of LmSIR2 protein did not protect the parasites from growth inhibition by this compound, indicating alternative targets may be involved.72 Enzyme studies using LiSIR2RP1 have shown that sirtinol, nicotinamide and suramin all have inhibitory activity (IC50 B194, 40 and 7 mM, respectively); however similar levels of activity were observed for human SIRT1, indicating lack of selectivity.73 In silico homology modelling studies have, however, shown that there are some differences between the LmSIR2 and human SIRT2 proteins, which may be able to be exploited for design of selective inhibitors/ activators.74 In a recent structure–activity relationship study of bisnaphthalimidopropyl derivatives, 12 compounds were shown to have activity against amastigotes (IC50 B1–10 mM).73 Although some of these showed moderate selectivity for the LiSIR2RP1 versus human SIRT1 enzyme (up to 17-fold), this did not correlate with antiparasitic activity.73 Little information is available on the effect of class-III HDAC inhibitors on Schistosoma, Trypanosoma or Toxoplasma parasite growth, with the exception that nicotinamide has been shown to be inactive against T. gondii tachyzoites (IC50 450 mM).52 Suramin, a compound reported to be an inhibitor of human sirtuins (reviewed in reference 9), is used to treat trypanosomiasis. However this compound has been shown recently to competitively inhibit pyruvate kinases from humans (Ki¼1.1–17 mM), T. cruzi (Ki¼108 mM) and Leishmania mexicana (Ki¼116 mM), all of which have similar active sites.75 These data indicate likely promiscuous activity for this compound against multiple targets. TOWARDS CLINICAL USE OF HDAC INHIBITORS FOR PARASITIC DISEASES While there is increasing interest in the anti-parasitic effects of HDAC inhibitors, there are still many critical factors that need to be considered if these compounds are to be used clinically. Chemoprevention and therapy of parasitic infections represent a very different target treatment group than for other diseases. Importantly, most of the morbidity and mortality associated with these diseases occurs in the underdeveloped regions of the world and people most at risk are generally children, pregnant women or those who are in some Immunology and Cell Biology

way immune-compromised. Co-infection with different infectious agents is also common and needs to be considered in terms of potential drug–drug interactions. Important considerations in the development of new anti-parasitic drugs therefore include: (i) compounds with high potency and selectivity for the parasite; (ii) a high safety profile, including for use in children and pregnant women; (iii) low cost in order to potentially treat hundreds of millions of people; (iv) activity against drug-resistant organisms; and (v) complementary pharmacokinetics with potential partner drugs. Some of these considerations are discussed in more detail below using individual examples; however most are equally applicable to different parasitic pathogens. To progress HDAC inhibitors towards clinical trials as drugs for parasitic diseases a high level of potency and selectivity for parasites versus host cells is essential. In this context it is promising that some isoform-selective HDAC inhibitors have now been developed to target human HDACs.76 This paves the way for medicinal chemistry programs to specifically target parasitic HDAC isoforms. Such approaches are still challenging, however, as they require cloning and expression of recombinant parasitic HDAC isoforms for enzyme assays and crystallization studies, as well as information on the molecular function of these enzymes. Next-generation anti-parasite HDAC inhibitors will also require improved pharmacokinetic profiles to accommodate the unique challenges facing the application of drugs for developing-world diseases, such as oral efficacy and complementary profiles with combination drugs, for single infections (as in the case of malaria), or poly-parasitism and polymicrobial infections (for example, HIV and parasite co-infection). As with any new anti-parasitic drug, activity against drug-resistant organisms is essential. It is promising that several anti-malarial HDAC inhibitors, for example, have shown similar in vitro activities against P. falciparum laboratory lines that have differing sensitivities to drugs such as chloroquine and mefloquine.56,58,77 Three hydroxamatebased HDAC inhibitors, including SAHA and two compounds based on 2-aminosuberic acid, also showed nM potency against multi-drugresistant clinical isolates of P. falciparum and P. vivax.59 Interestingly, susceptibility to SAHA was correlated with mefloquine in P. falciparum but not P. vivax, and susceptibility to the 2-aminosuberic acid compounds was correlated with mefloquine in P. vivax but not P. falciparum. While these findings may be due to the small numbers of clinical isolates examined (n¼4–9), it may be that there are differences in susceptibility profiles for different Plasmodium species. Further studies will be required to investigate this in more detail. An approach that has proved useful in assessing potential for resistance to new drugs is the in vitro generation of resistant laboratory lines, either by selection or mutagenesis, for molecular studies on resistance mechanisms and activity screening.78 While such lines have not yet been reported for anti-malarial HDAC inhibitors, as discussed above, T. gondii parasites resistant to the HDAC inhibitor FR235222 have been generated. Amino-acid residues (T99A and T99I) in the TgHDAC3 gene (a homologue of PfHDAC1) have been identified as being involved in reduced sensitivity to FR235222,50 and one (T99) is part of a two amino-acid-motif present in apicomplexan HDACs (A75 and T76 in PfHDAC1) but not in class-I HDAC homologues from other organisms, including human HDAC1 and HDAC8. In silico structural homology modelling of PfHDAC1 with bound HDAC inhibitors has found that amino acid T76 (as well as several other residues) are predicted to be key binding site residues in PfHDAC1.15,16 Future studies of the role of these amino acids on HDAC inhibitor activity, as well as the in vitro selection of HDAC inhibitor tolerant/resistant parasite lines, will provide


important insights into the molecular mechanisms of HDAC inhibitor resistance. HDAC inhibitor resistance in mammalian cells is not yet well characterized. Depsipeptide (FK-228) resistance in leukemia cells has been shown to be mediated by both P-glycoprotein-dependent and independent mechanisms.79,80 Adenocarcinoma cell lines that are tolerant to SAHA and valproic acid have phenotypes that are independent of multi-drug resistance. As both P-glycoprotein and/or multi-drug resistance-mediated drug resistance mechanisms have been demonstrated for several parasites, it will be important to determine whether these also have a role in potential HDAC inhibitor resistance in parasites. In addition, increased reactive oxygen species expression may contribute to clinical resistance to the HDAC inhibitor SAHA (Vorinostat) in leukemia patients.81 This is worth considering for all parasite species, including malarial parasites, which are exposed to high fluxes of toxic reactive oxygen species due to the different intracellular and extracellular environments they survive in during their lifecycle; their high metabolic rates; and the reactive oxygen species that are produced by the host’s immune response.82 Finally, for HDAC inhibitors to be developed clinically as antiparasitic drugs, their potential role as immune modulators must also be taken into account. Different HDAC enzymes have been shown to have both positive and negative roles on innate and adaptive immunity. An increasing body of evidence has also demonstrated that HDAC inhibitors can potently effect many cellular components of the immune system, although the mechanism behind this are not well understood (for a recent review see reference Shakespear et al.83). Antiviral CD8+ T-cell function can be compromised in vitro,84 and immunosuppressive CD4+ Foxp3+ regulatory T-cell numbers increase in vivo after HDAC inhibitor treatment.85 The production of proinflammatory cytokines by macrophages and dendritic cells can be strongly suppressed by HDAC inhibitors.86,87 In the case of dendritic cells, which have a pivotal role in the generation of adaptive T- and Bcell responses, their ability to express the co-stimulatory molecules CD40, CD86 and CD80 is reduced, and production of the immunosuppressive molecule indoleamine-2,3-dioxygenase is increased after HDAC inhibitor treatment.87 Various reports have also suggested that HDAC inhibitor treatment leads to increased susceptibility to viral, bacterial and fungal pathogens.84,86 Furthermore, HDAC inhibitors protect mice against symptoms of lupus, septic shock and graftversus-host-disease, all of which are immune-mediated diseases.85–87 Given that T cells, dendritic cells and macrophages are known to have crucial roles in immunity to human-infecting parasites, it will be of considerable importance to assess the relative in vivo effects of various HDAC inhibitors (used at different doses and routes of administration) on host immune responses versus direct anti-parasitic effects. The potential use of HDAC inhibitors as adjunctive therapy, for example against cerebral malaria,88 is also intriguing and warrants further scrutiny in potentially targeting severe forms of parasitic diseases. ACKNOWLEDGEMENTS We thank the Australian Research Council (ARC Future Fellowship to KTA) and the National Health and Medical Research Council (NHMRC) of Australia for funding support.

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World Health Organization. Global burden of disease: 2004 update. WHO Press: Geneva, 2008. Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med 2009; 361: 455–467.

33

Brabin BJ. An analysis of malaria in pregnancy in Africa. Bull World Health Organ 1983; 61: 1005–1016. Porter SB, Sande MA. Toxoplasmosis of the central nervous system in the acquired immunodeficiency syndrome. N Engl J Med 1992; 327: 1643–1648. van Riet E, Hartgers FC, Yazdanbakhsh M. Chronic helminth infections induce immunomodulation: consequences and mechanisms. Immunobiology 2007; 212: 475–490. Cairns BR. Emerging roles for chromatin remodeling in cancer biology. Trends Cell Biol 2001; 11: S15–S21. Beumer JH, Tawbi H. Role of histone deacetylases and their inhibitors in cancer biology and treatment. Curr Clin Pharmacol 2010; 5: 196–208. Alcain FJ, Villalba JM. Sirtuin activators. Expert Opin Ther Pat 2009; 19: 403–414. Alcain FJ, Villalba JM. Sirtuin inhibitors. Expert Opin Ther Pat 2009; 19: 283–294. Darkin-Rattray SJ, Gurnett AM, Myers RW, Dulski PM, Crumley TM, Allocco JJ et al. Apicidin: a novel antiprotozoal agent that inhibits parasite histone deacetylase. Proc Natl Acad Sci USA 1996; 93: 13143–13147. Joshi MB, Lin DT, Chiang PH, Goldman ND, Fujioka H, Aikawa M et al. Molecular cloning and nuclear localization of a histone deacetylase homologue in Plasmodium falciparum. Mol Biochem Parasitol 1999; 99: 11–19. Aurrecoechea C, Brestelli J, Brunk BP, Dommer J, Fischer S, Gajria B et al. PlasmoDB: a functional genomic database for malaria parasites. Nucleic Acids Res 2009; 37: D539–D543. Le Roch KG, Zhou Y, Blair PL, Grainger M, Moch JK, Haynes JD et al. Discovery of gene function by expression profiling of the malaria parasite life cycle. Science 2003; 301: 1503–1508. Bozdech Z, Llinas M, Pulliam BL, Wong ED, Zhu J, DeRisi JL. The transcriptome of the intraerythrocytic developmental cycle of Plasmodium falciparum. PLoS Biol 2003; 1: E5. Andrews KT, Tran TN, Lucke AJ, Kahnberg P, Le GT, Boyle GM et al. Potent antimalarial activity of histone deacetylase inhibitor analogues. Antimicrob Agents Chemother 2008; 52: 1454–1461. Mukherjee P, Pradhan A, Shah F, Tekwani BL, Avery MA. Structural insights into the Plasmodium falciparum histone deacetylase 1 (PfHDAC-1): a novel target for the development of antimalarial therapy. Bioorg Med Chem 2008; 16: 5254–5265. Chakrabarty SP, Saikumari YK, Bopanna MP, Balaram H. Biochemical characterization of Plasmodium falciparum Sir2, a NAD(+)-dependent deacetylase. Mol Biochem Parasitol 2008; 158: 139. Tonkin CJ, Carret CK, Duraisingh MT, Voss TS, Ralph SA, Hommel M et al. Sir2 paralogues cooperate to regulate virulence genes and antigenic variation in Plasmodium falciparum. PLoS Biol 2009; 7: e84. Merrick CJ, Duraisingh MT. Plasmodium falciparum Sir2: an unusual sirtuin with dual histone deacetylase and ADP-ribosyltransferase activity. Eukaryot Cell 2007; 6: 2081–2091. Duraisingh MT, Voss TS, Marty AJ, Duffy MF, Good RT, Thompson JK et al. Heterochromatin silencing and locus repositioning linked to regulation of virulence genes in Plasmodium falciparum. Cell 2005; 121: 13–24. Freitas-Junior LH, Hernandez-Rivas R, Ralph SA, Montiel-Condado D, RuvalcabaSalazar OK, Rojas-Meza AP et al. Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell 2005; 121: 25–36. Saksouk N, Bhatti MM, Kieffer S, Smith AT, Musset K, Garin J et al. Histonemodifying complexes regulate gene expression pertinent to the differentiation of the protozoan parasite Toxoplasma gondii. Mol Cell Biol 2005; 25: 10301–10314. Ingram AK, Horn D. Histone deacetylases in Trypanosoma brucei: two are essential and another is required for normal cell cycle progression. Mol Microbiol 2002; 45: 89–97. Pays E. Regulation of antigen gene expression in Trypanosoma brucei. Trends Parasitol 2005; 21: 517–520. Hertz-Fowler C, Figueiredo LM, Quail MA, Becker M, Jackson A, Bason N et al. Telomeric expression sites are highly conserved in Trypanosoma brucei. PLoS One 2008; 3: e3527. Wang QP, Kawahara T, Horn D. Histone deacetylases play distinct roles in telomeric VSG expression site silencing in African trypanosomes. Mol Microbiol 2010; 77: 1237–1245. Alsford S, Kawahara T, Isamah C, Horn D. A sirtuin in the African trypanosome is involved in both DNA repair and telomeric gene silencing but is not required for antigenic variation. Mol Microbiol 2007; 63: 724–736. Oger F, Dubois F, Caby S, Noel C, Cornette J, Bertin B et al. The class I histone deacetylases of the platyhelminth parasite Schistosoma mansoni. Biochem Biophys Res Commun 2008; 377: 1079–1084. Dubois F, Caby S, Oger F, Cosseau C, Capron M, Grunau C et al. Histone deacetylase inhibitors induce apoptosis, histone hyperacetylation and upregulation of gene transcription in Schistosoma mansoni. Mol Biochem Parasitol 2009; 168: 7–15. Theurkauf WE, Baum H, Bo J, Wensink PC. Tissue-specific and constitutive alphatubulin genes of Drosophila melanogaster code for structurally distinct proteins. Proc Natl Acad Sci USA 1986; 83: 8477–8481. Berriman M, Haas BJ, LoVerde PT, Wilson RA, Dillon GP, Cerqueira GC et al. The genome of the blood fluke Schistosoma mansoni. Nature 2009; 460: 352–358. Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M et al. The genome of the kinetoplastid parasite, Leishmania major. Science 2005; 309: 436–442. Saxena A, Lahav T, Holland N, Aggarwal G, Anupama A, Huang Y et al. Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and



34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51 52

53

54 55 56

57

58

59

60

permanent changes in gene expression during differentiation. Mol Biochem Parasitol 2007; 152: 53–65. Yahiaoui B, Taibi A, Ouaissi A. A Leishmania major protein with extensive homology to silent information regulator 2 of Saccharomyces cerevisiae. Gene 1996; 169: 115–118. Vergnes B, Sereno D, Madjidian-Sereno N, Lemesre JL, Ouaissi A. Cytoplasmic SIR2 homologue overexpression promotes survival of Leishmania parasites by preventing programmed cell death. Gene 2002; 296: 139–150. Tavares J, Ouaissi A, Santarem N, Sereno D, Vergnes B, Sampaio P et al. The Leishmania infantum cytosolic SIR2-related protein 1 (LiSIR2RP1) is an NAD+ dependent deacetylase and ADP-ribosyltransferase. Biochem J 2008; 415: 377–386. Vergnes B, Sereno D, Tavares J, Cordeiro-da-Silva A, Vanhille L, Madjidian-Sereno N et al. Targeted disruption of cytosolic SIR2 deacetylase discloses its essential role in Leishmania survival and proliferation. Gene 2005; 363: 85–96. Adriano MA, Vergnes B, Poncet J, Mathieu-Daude F, da Silva AC, Ouaissi A et al. Proof of interaction between Leishmania SIR2RP1 deacetylase and chaperone HSP83. Parasitol Res 2007; 100: 811–818. Silvestre R, Cordeiro-da-Silva A, Tavares J, Sereno D, Ouaissi A. Leishmania cytosolic silent information regulatory protein 2 deacetylase induces murine B-cell differentiation and in vivo production of specific antibodies. Immunology 2006; 119: 529–540. Silvestre R, Silva AM, Cordeiro-da-Silva A, Ouaissi A. The contribution of Toll-like receptor 2 to the innate recognition of a Leishmania infantum silent information regulator 2 protein. Immunology 2009; 128: 484–499. Patel V, Mazitschek R, Coleman B, Nguyen C, Urgaonkar S, Cortese J et al. Identification and characterization of small molecule inhibitors of a class I histone deacetylase from Plasmodium falciparum. J Med Chem 2009; 52: 2185–2187. Hu G, Cabrera A, Kono M, Mok S, Chaal BK, Haase S et al. Transcriptional profiling of growth perturbations of the human malaria parasite Plasmodium falciparum. Nat Biotechnol 2010; 28: 91–98. Chaal BK, Gupta AP, Wastuwidyaningtyas BD, Luah YH, Bozdech Z. Histone deacetylases play a major role in the transcriptional regulation of the Plasmodium falciparum life cycle. PLoS Pathog 2010; 6: e1000737. Glaser KB, Staver MJ, Waring JF, Stender J, Ulrich RG, Davidsen SK. Gene expression profiling of multiple histone deacetylase (HDAC) inhibitors: defining a common gene set produced by HDAC inhibition in T24 and MDA carcinoma cell lines. Mol Cancer Ther 2003; 2: 151–163. Peart MJ, Smyth GK, van Laar RK, Bowtell DD, Richon VM, Marks PA et al. Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors. Proc Natl Acad Sci USA 2005; 102: 3697–3702. Meinke PT, Colletti SL, Doss G, Myers RW, Gurnett AM, Dulski PM et al. Synthesis of apicidin-derived quinolone derivatives: parasite-selective histone deacetylase inhibitors and antiproliferative agents. J Med Chem 2000; 43: 4919–4922. Colletti SL, Myers RW, Darkin-Rattray SJ, Gurnett AM, Dulski PM, Galuska S et al. Broad spectrum antiprotozoal agents that inhibit histone deacetylase: structure– activity relationships of apicidin. Part 2. Bioorg Med Chem Lett 2001; 11: 113–117. Colletti SL, Myers RW, Darkin-Rattray SJ, Gurnett AM, Dulski PM, Galuska S et al. Broad spectrum antiprotozoal agents that inhibit histone deacetylase: structure– activity relationships of apicidin. Part 1. Bioorg Med Chem Lett 2001; 11: 107–111. Murray PJ, Kranz M, Ladlow M, Taylor S, Berst F, Holmes AB et al. The synthesis of cyclic tetrapeptoid analogues of the antiprotozoal natural product apicidin. Bioorg Med Chem Lett 2001; 11: 773–776. Bougdour A, Maubon D, Baldacci P, Ortet P, Bastien O, Bouillon A et al. Drug inhibition of HDAC3 and epigenetic control of differentiation in Apicomplexa parasites. J Exp Med 2009; 206: 953–966. Tan J, Cang S, Ma Y, Petrillo RL, Liu D. Novel histone deacetylase inhibitors in clinical trials as anticancer agents. J Hematol Oncol 2010; 3: 5. Strobl JS, Cassell M, Mitchell SM, Reilly CM, Lindsay DS. Scriptaid and suberoylanilide hydroxamic acid are histone deacetylase inhibitors with potent anti-Toxoplasma gondii activity in vitro. J Parasitol 2007; 93: 694–700. Jones-Brando L, Torrey EF, Yolken R. Drugs used in the treatment of schizophrenia and bipolar disorder inhibit the replication of Toxoplasma gondii. Schizophr Res 2003; 62: 237–244. Azzi A, Cosseau C, Grunau C. Schistosoma mansoni: developmental arrest of miracidia treated with histone deacetylase inhibitors. Exp Parasitol 2009; 121: 288–291. Grant S, Easley C, Kirkpatrick P. Vorinostat. Nat Rev Drug Discov 2007; 6: 21–22. Andrews KT, Walduck A, Kelso MJ, Fairlie DP, Saul A, Parsons PG. Anti-malarial effect of histone deacetylation inhibitors and mammalian tumour cytodifferentiating agents. Int J Parasitol 2000; 30: 761–768. Pallavi R, Roy N, Nageshan RK, Talukdar P, Pavithra SR, Reddy R et al. Heat shock protein 90 as a drug target against protozoan infections: biochemical characterization of HSP90 from Plasmodium falciparum and Trypanosoma evansi and evaluation of its inhibitor as a candidate drug. J Biol Chem 2010; 285: 37964–37975. Dow GS, Chen Y, Andrews KT, Caridha D, Gerena L, Gettayacamin M et al. Antimalarial activity of phenylthiazolyl-bearing hydroxamate-based histone deacetylase inhibitors. Antimicrob Agents Chemother 2008; 52: 3467–3477. Marfurt J, Chalfein F, Prayoga P, Wabiser F, Kenangalem E, Piera KA et al. Ex vivo activity of histone deacetylase inhibitors against multidrug-resistant clinical isolates of Plasmodium falciparum and P. vivax. Antimicrob Agents Chemother 2011; 55: 961–966. Chen Y, Lopez-Sanchez M, Savoy DN, Billadeau DD, Dow GS, Kozikowski AP. A series of potent and selective, triazolylphenyl-based histone deacetylases inhibitors with activity against pancreatic cancer cells and Plasmodium falciparum. J Med Chem 2008; 51: 3437–3448.


61 Wheatley NC, Andrews KT, Tran TL, Lucke AJ, Reid RC, Fairlie DP. Antimalarial histone deacetylase inhibitors containing cinnamate or NSAID components. Bioorg Med Chem Lett 2010; 20: 7080–7084. 62 Agbor-Enoh S, Seudieu C, Davidson E, Dritschilo A, Jung M. Novel inhibitor of Plasmodium histone deacetylase that cures P. berghei-infected mice. Antimicrob Agents Chemother 2009; 53: 1727–1734. 63 Sheader K, te Vruchte D, Rudenko G. Bloodstream form-specific upregulation of silent vsg expression sites and procyclin in Trypanosoma brucei after inhibition of DNA synthesis or DNA damage. J Biol Chem 2004; 279: 13363–13374. 64 Respuela P, Ferella M, Rada-Iglesias A, Aslund L. Histone acetylation and methylation at sites initiating divergent polycistronic transcription in Trypanosoma cruzi. J Biol Chem 2008; 283: 15884–15892. 65 Patil V, Guerrant W, Chen PC, Gryder B, Benicewicz DB, Khan SI et al. Antimalarial and antileishmanial activities of histone deacetylase inhibitors with triazole-linked cap group. Bioorg Med Chem 2010; 18: 415–425. 66 Mwakwari SC, Guerrant W, Patil V, Khan SI, Tekwani BL, Gurard-Levin ZA et al. Nonpeptide macrocyclic histone deacetylase inhibitors derived from tricyclic ketolide skeleton. J Med Chem 2010; 53: 6100–6111. 67 Verotta L, Appendino G, Bombardelli E, Brun R. In vitro antimalarial activity of hyperforin, a prenylated acylphloroglucinol. A structure–activity study. Bioorg Med Chem Lett 2007; 17: 1544–1548. 68 Prusty D, Mehra P, Srivastava S, Shivange AV, Gupta A, Roy N et al. Nicotinamide inhibits Plasmodium falciparum Sir2 activity in vitro and parasite growth. FEMS Microbiol Lett 2008; 282: 266–272. 69 Chakrabarty SP, Ramapanicker R, Mishra R, Chandrasekaran S, Balaram H. Development and characterization of lysine based tripeptide analogues as inhibitors of Sir2 activity. Bioorg Med Chem 2009; 17: 8060–8072. 70 Bajsa J, Singh K, Nanayakkara D, Duke SO, Rimando AM, Evidente A et al. A survey of synthetic and natural phytotoxic compounds and phytoalexins as potential antimalarial compounds. Biol Pharm Bull 2007; 30: 1740–1744. 71 Vergnes B, Vanhille L, Ouaissi A, Sereno D. Stage-specific antileishmanial activity of an inhibitor of SIR2 histone deacetylase. Acta Trop 2005; 94: 107–115. 72 Sereno D, Alegre AM, Silvestre R, Vergnes B, Ouaissi A. In vitro antileishmanial activity of nicotinamide. Antimicrob Agents Chemother 2005; 49: 808–812. 73 Tavares J, Ouaissi A, Kong Thoo Lin P, Loureiro I, Kaur S, Roy N et al. Bisnaphthalimidopropyl derivatives as inhibitors of Leishmania SIR2 related protein 1. ChemMedChem 2010; 5: 140–147. 74 Kadam RU, Kiran VM, Roy N. Comparative protein modeling and surface analysis of Leishmania sirtuin: a potential target for antileishmanial drug discovery. Bioorg Med Chem Lett 2006; 16: 6013–6018. 75 Morgan HP, McNae IW, Nowicki MW, Zhong W, Michels PA, Auld DS et al. The trypanocidal drug suramin and other trypan blue mimetics are inhibitors of pyruvate kinases and bind to the adenosine site. J Biol Chem 2011; 286: 31232–31240. 76 Bieliauskas AV, Pflum MK. Isoform-selective histone deacetylase inhibitors. Chem Soc Rev 2008; 37: 1402–1413. 77 Chen Y, Lopez-Sanchez M, Savoy DN, Billadeau DD, Dow GS, Kozikowski AP. A series of potent and selective, triazolylphenyl-based histone deacetylases inhibitors with activity against pancreatic cancer cells and Plasmodium falciparum. J Med Chem 2008; 51: 3437–3448. 78 Witkowski B, Berry A, Benoit-Vical F. Resistance to antimalarial compounds: methods and applications. Drug Resist Updat 2009; 12: 42–50. 79 Glaser KB. Defining the role of gene regulation in resistance to HDAC inhibitors— mechanisms beyond P-glycoprotein. Leuk Res 2006; 30: 651–652. 80 Yamada H, Arakawa Y, Saito S, Agawa M, Kano Y, Horiguchi-Yamada J. Depsipeptide-resistant KU812 cells show reversible P-glycoprotein expression, hyper-acetylated histones, and modulated gene expression profile. Leuk Res 2006; 30: 723–734. 81 Garcia-Manero G, Yang H, Bueso-Ramos C, Ferrajoli A, Cortes J, Wierda WG et al. Phase 1 study of the histone deacetylase inhibitor vorinostat (suberoylanilide hydroxamic acid [SAHA]) in patients with advanced leukemias and myelodysplastic syndromes. Blood 2008; 111: 1060–1066. 82 Becker K, Tilley L, Vennerstrom JL, Roberts D, Rogerson S, Ginsburg H. Oxidative stress in malaria parasite-infected erythrocytes: host–parasite interactions. Int J Parasitol 2004; 34: 163–189. 83 Shakespear MR, Halili MA, Irvine KM, Fairlie DP, Sweet MJ. Histone deacetylases as regulators of inflammation and immunity. Trends Immunol 2011; 32: 335–343. 84 Mosley AJ, Meekings KN, McCarthy C, Shepherd D, Cerundolo V, Mazitschek R et al. Histone deacetylase inhibitors increase virus gene expression but decrease CD8+ cell antiviral function in HTLV-1 infection. Blood 2006; 108: 3801–3807. 85 Reilly CM, Thomas M, Gogal Jr R, Olgun S, Santo A, Sodhi R et al. The histone deacetylase inhibitor trichostatin A upregulates regulatory T cells and modulates autoimmunity in NZB/W F1 mice. J Autoimmun 2008; 31: 123–130. 86 Roger T, Lugrin J, Le Roy D, Goy G, Mombelli M, Koessler T et al. Histone deacetylase inhibitors impair innate immune responses to Toll-like receptor agonists and to infection. Blood 2011; 117: 1205–1217. 87 Reddy P, Sun Y, Toubai T, Duran-Struuck R, Clouthier SG, Weisiger E et al. Histone deacetylase inhibition modulates indoleamine 2,3-dioxygenase-dependent DC functions and regulates experimental graft-versus-host disease in mice. J Clin Invest 2008; 118: 2562–2573.

HDAC inhibitors in parasitic infectious diseases KT Andrews et al 77 88 John CC, Kutamba E, Mugarura K, Opoka RO. Adjunctive therapy for cerebral malaria and other severe forms of Plasmodium falciparum malaria. Expert Rev Anti Infect Ther 2010; 8: 997–1008. 89 Greenwood BM, Bojang K, Whitty CJ, Targett GA. Malaria. Lancet 2005; 365: 1487–1498. 90 Montoya JG, Liesenfeld O. Toxoplasmosis. Lancet 2004; 363: 1965–1976. 91 Fenwick A, Rollinson D, Southgate V. Implementation of human schistosomiasis control: challenges and prospects. Adv Parasitol 2006; 61: 567–622. 92 Reithinger R, Dujardin JC, Louzir H, Pirmez C, Alexander B, Brooker S. Cutaneous leishmaniasis. Lancet Infect Dis 2007; 7: 581–596. 93 Chappuis F, Sundar S, Hailu A, Ghalib H, Rijal S, Peeling RW et al. Visceral leishmaniasis: what are the needs for diagnosis, treatment and control? Nat Rev Microbiol 2007; 5: 873–882. 94 Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD et al. A proteomic view of the Plasmodium falciparum life cycle. Nature 2002; 419: 520–526. 95 LaCount DJ, Vignali M, Chettier R, Phansalkar A, Bell R, Hesselberth JR et al. A protein interaction network of the malaria parasite Plasmodium falciparum. Nature 2005; 438: 103–107. 96 Kissinger JC, Gajria B, Li L, Paulsen IT, Roos DS. ToxoDB: accessing the Toxoplasma gondii genome. Nucleic Acids Res 2003; 31: 234–236. 97 Zemzoumi K, Sereno D, Francois C, Guilvard E, Lemesre JL, Ouaissi A. Leishmania major: cell type dependent distribution of a 43 kDa antigen related to silent information regulatory-2 protein family. Biol Cell 1998; 90: 239–245.

98 Zerlotini A, Heiges M, Wang H, Moraes RL, Dominitini AJ, Ruiz JC et al. SchistoDB: a Schistosoma mansoni genome resource. Nucleic Acids Res 2009; 37: D579–D582. 99 Singh SB, Zink DL, Liesch JM, Mosley RT, Dombrowski AW, Bills GF et al. Structure and chemistry of apicidins, a class of novel cyclic tetrapeptides without a terminal alpha-keto epoxide as inhibitors of histone deacetylase with potent antiprotozoal activities. J Org Chem 2002; 67: 815–825. 100 Dokmanovic M, Clarke C, Marks PA. Histone deacetylase inhibitors: overview and perspectives. Mol Cancer Res 2007; 5: 981–989. 101 Rasheed WK, Johnstone RW, Prince HM. Histone deacetylase inhibitors in cancer therapy. Expert Opin Investig Drugs 2007; 16: 659–678. 102 Mai A, Cerbara I, Valente S, Massa S, Walker LA, Tekwani BL. Antimalarial and antileishmanial activities of aroyl-pyrrolyl-hydroxyamides, a new class of histone deacetylase inhibitors. Antimicrob Agents Chemother 2004; 48: 1435–1436. 103 Ungerstedt JS, Sowa Y, Xu WS, Shao Y, Dokmanovic M, Perez G et al. Role of thioredoxin in the response of normal and transformed cells to histone deacetylase inhibitors. Proc Natl Acad Sci USA 2005; 102: 673–678. 104 Brinkmann H, Dahler AL, Popa C, Serewko MM, Parsons PG, Gabrielli BG et al. Histone hyperacetylation induced by histone deacetylase inhibitors is not sufficient to cause growth inhibition in human dermal fibroblasts. J Biol Chem 2001; 276: 22491–22499.
