Lysine Deacetylase Inhibitors in Parasites: Past, Present, and Future

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Perspective

Lysine Deacetylase Inhibitors in Parasites: Past, Present and Future Perspectives Gebremedhin Solomon Hailu, Dina Robaa, Mariantonietta Forgione, Wolfgang Sippl, Dante Rotili, and Antonello Mai J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01595 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 27, 2017

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Journal of Medicinal Chemistry

Lysine Deacetylase Inhibitors in Parasites: Past, Present and Future Perspectives Gebremedhin S. Hailu,1 Dina Robaa,2 Mariantonietta Forgione,1,3 Wolfgang Sippl,2* Dante Rotili,1* Antonello Mai,1,4*

1

Dipartimento di Chimica e Tecnologie del Farmaco “Sapienza” Università di Roma, 00185

Rome, Italy;

2

Institute of Pharmacy, Martin-Luther-Universitat Halle-Wittenberg, Halle,

Germany; 3Center for Life Nano Science@Sapienza, Italian Institute of Technology, Viale Regina Elena 291, 00161 Rome, Italy.

4

Istituto Pasteur, Fondazione Cenci-Bolognetti,

“Sapienza” Università di Roma, 00185 Rome, Italy.

1 ACS Paragon Plus Environment


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ABSTRACT Current therapies for human parasite infections rely on a few drugs, most of which have severe side effects, and their helpfulness is being seriously compromised by the drug resistance problem. Globally, this is pushing discovery research of anti-parasitic drugs towards new agents endowed with new mechanisms of action. By using a “drug repurposing” strategy, histone deacetylase inhibitors (HDACi), which are presently clinically approved for cancer use, are now under investigation for various parasite infections. Since parasitic Zn2+- and NAD+-dependent HDACs play crucial roles in the modulation of parasite gene expression, and many of them are pro-survival for several parasites under various conditions, they are now emerging as novel potential anti-parasitic targets. This Perspective summarizes the state of knowledge of HDACi (both class I/II HDACi and sirtuin inhibitors) targeted to the main human parasitic diseases (schistosomiasis, malaria, trypanosomiasis, leishmaniasis and toxoplasmosis) and provides visions into the main issues that challenge their development as anti-parasitic agents.


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INTRODUCTION Human parasitic diseases such as schistosomiasis, malaria, trypanosomiasis, leishmaniasis and toxoplasmosis present major health and economic problems, particularly in underdeveloped regions of the world.1 Schistosomiasis is one of the major neglected parasitic diseases which still represents a serious public health problem in many regions worldwide, especially in Africa, the Middle East, South America, and Asia.2,3 Human schistosomiasis can be caused by six species of Schistosoma trematodes, with S. mansoni and S. haematobium being the main species causing the disease.4 Schistosoma infection affects almost 259 million people worldwide5 causing an annual death toll of 280,0006 and millions of people suffering from long-term morbidity due to chronic schistosomiasis.7-9 Despite its introduction in the mid-1970s, praziquantel remains the only drug of choice for the treatment of schistosomiasis.9,10 Praziquantel is a low-cost and highly effective antischistosomial agent, which is active against all Schistosoma species, albeit its exact mechanism of action is still not fully understood.12,13 It is adminstered orally as a single dose, showing no notable side effects.14 The long-term use of praziquentel as a sole antischistosomal treatment alongside its implementation in mass drug administration campaigns has raised deep concerns over the potential for emergence of drug-resistance.12,13,15-17 Indeed, incidences of a reduced efficacy of praziquantel against some Schistosoma species18-19 and the induction of drugresistance in laboratrory strains21-24 have already been reported. Evidently, a diminished efficacy of praziquantel would have a serious impact on the ongoing efforts to combat the disease, highlighting the need to develop new antischistosomal agents. Malaria, a mosquito-borne infectious disease caused by six Plasmodium species, remains a devastating cause of death in tropical and subtropical regions with 40% of world population



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being at risk of infection. According to WHO 2014, malaria causes 600,000 deaths and there are approximately 200 million clinical cases of infection each year.25 Mosquito control and chemotherapy are the main strategies for the prevention and treatment of this disease, and only recently the vaccine RTS,S/AS01 (also known as Mosquirix) has been approved by European Medicines Agency for active immunization of children against malaria in highly affected areas.26 Regrettably, P. falciparum, the principal malarial protozoan parasite in humans, has become increasingly resistant to chloroquine (CQ), the best drug for the treatment of P. falciparum.27 Indeed, alarming signs of emerging resistance and decreased efficacy to artemisinin and its derivatives are now threatening the last line of defense against this devastating disease.28,29 This is pushing research efforts into development and discovery of new antimalarial drugs endowed with new mechanisms of action on novel targets in the parasite. Human African Trypanosomiasis (HAT) is another neglected disease in sub-Saharan Africa where its epidemics have been a significant public health problem in the past, but the disease is reasonably well-controlled at present, with fewer than 10,000 new cases reported annually in recent years.30 The causative parasite Trypanosoma brucei is transmitted to humans by Tsetse fly and causes African sleeping sickness.31 In mammals, the parasite survives free in the bloodstream and is able to evade the host immune response through antigenic variation.32 If the disease is not treated before the parasites reach the central nervous system, it can be lethal. In addition, various wild animals can serve as host for the parasites, adding difficulty to the eradication of the disease.33 Trypanosoma cruzi is another intracellular pathogen that is responsible for Chagas disease, a chronic infectious sickness affecting several million people. Chagas disease is endemic in Latin America; however, a significant increase in confirmed cases of this disease has recently been


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reported in developed regions as well, indicating that it is an emerging disease. It is estimated that 6-7 million people worldwide are infected by Chagas disease.34 Current therapies rely on a limited number of drugs (suramin, pentamidine, melarsoprol, eflornithine and nifurtimox for African sleeping sickness,30 while benznidazole and nifurtimox are used or the acute phase of Chagas disease).34 Most of these drugs are highly toxic, have numerous side effects, and suffer from emergence of drug resistance.30,34-36 Thus, for continual control and disease elimination efforts, development of new therapeutic agents with novel mechanisms of action and with improved safety and efficacy is crucial. Leishmaniasis is also a neglected vector-borne tropical infection, which is caused by the protozoan parasites of the genus Leishmania. There are three main types of leishmaniasis: visceral (kala-azar), cutaneous, and mucocutaneous, and the first type is fatal when untreated. It is estimated that 0.9-1.3 million new cases and 20,000-30,000 deaths occur annually, with approximately 350 million individuals at risk of infection.37,38 As in the case of other parasitic diseases, treatment of leishmaniasis depends on limited drug options such as amphotericin B, miltefosine, paromomycin, and antimony agents, including sodium stibogluconate and meglumine antimoniat. These drugs, however, require a long course of parenteral injection, and many suffer from unacceptable toxicity and poor efficacy.39,40 Opportunistic infection in immune-compromised patients, especially due to HIV infection,41 is also causing more problems with the currently available treatments. Likewise, toxoplasmosis is caused by the obligate intracellular protozoan Toxoplasma gondii. In healthy individuals, the symptoms of toxoplasmosis are self-limiting, but can cause more serious problems or, if women contract the disease while pregnant, be lethal to the fetus. Toxoplasmosis can also cause severe sickness in immune-compromised individuals or in people



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submitted to chemotherapy or organ transplants.42,43 Current treatment options for toxoplasmosis are limited to pyrimethamine combination (mostly with a sulfonamide) therapies and suffer from serious adverse effects and efficacy problems.44 With the only exception being RTS,S/AS01 for malaria,26 in the absence of approved vaccines for human parasitic diseases, research on novel anti-parasitic agents, along with dedicated public health measures, are still essential to address the growing health and economic worries caused by these sicknesses. Unfortunately, the actual drugs to treat such diseases (when available) are old, act with unknown mechanisms of action and show poor efficacy and/or disfavored safety profiles. Moreover, pandemic drug resistance, consequent to treatment of all main parasitic pathologies, put the currently-available drugs under an increasing threat of failure. Therefore, it is urgent to identify new drugs for parasitic sicknesses, and to overcome the rising problem of drug resistance. One extremely promising strategy to face these problems is represented by the “repurposing” strategy that, centering on targets and associated drugs already validated for other human diseases, try to apply them to new indications such as parasitic diseases. tThis strategy is fairly attractive, because it can accelerate progression of drug development due to lower costs, reduced risk and decreased time to market due to availability of preclinical data. However, selectivity for the parasite remains one of the main challenges to overcome in moving such compounds into clinical trials as potential novel anti-parasitic agents.44,45 Following this “repurposing” approach, histone deacetylase (HDAC) inhibitors (HDACi), which were originally developed against cancer, are now being investigated for treatment of parasitic diseases.45 This Perspective focuses on the current state of knowledge of HDACs and HDACi in the major human parasitic diseases: schistosomiasis, malaria, trypanosomiasis, leishmaniasis and toxoplasmosis.


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HUMAN HDACs BIOLOGY AND CLASSIFICATION HDAC enzymes catalyze the removal of the acetyl group from histone lysine residues and regulate cell chromatin structure, transcription, and gene expression.46,47 In addition to the regulation of the acetylation level of chromatin histones, HDACs have also various non-histone substrates, including many proteins involved in tumor progression, cell cycle control, apoptosis, angiogenesis, and cell invasion.46-48 HDACs are associated with many diseases in humans ranging from cancer to neurodegenerative, metabolic and immunological disorders; as a result they arose as a significant class of drug targets.46,47,48 To date, 18 HDACs have been identified in eukaryotes, and are grouped into four classes based on sequence similarity and cofactor dependence. Mammalian class-I HDACs, expressed almost ubiquitously, are represented by HDAC1, 2, 3 and 8. Class-I HDACs are nucleus-localized and have high homology with yeast RPD3. Class-II HDACs include HDAC4-7, HDAC9 and HDAC10, which shuttle between the nucleus and cytoplasm. They are tissue specific and share homology with yeast HDA1.48,50 The mammalian HDAC11, localized in both the nucleus and cytoplasm, is the only member of classIV HDACs identified so far, and is associated with the class-II enzyme HDAC6 rather than with multi-protein complexes of class-I HDACs.51 HDAC inhibitors (HDACi) typically target the “classical” class-I/II HDACs, which share a similar catalytic core that uses Zn2+ as cofactor, but differ in size and structural organization. On the other hand, class-III HDACs (also called sirtuins, SIRTs) are a set of seven NAD+-dependent enzymes (SIRT1-7) that share homology with yeast Sir2-related protein.52-54 The roles of SIRTs in many cellular functions range from the modulation of gene expression to apoptosis, from the DNA repair to the promotion of longevity.48,53-57 Hence, sirtuin inhibitors (SIRTi) and activators have been indicated as potential



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therapeutics for a range of diseases, including cancer, obesity, diabetes, cardiovascular diseases, inflammation, neuronal, metabolic and various ageing-related diseases.52,54 BIOLOGICAL ROLES OF HDACs IN THE MOST RELEVANT HUMAN PARASITES Schistosoma HDACs. Studies have shown that class-I HDACs (smHDAC1, 3 and 8) are expressed in all stages of the Schistosoma lifecycle, with smHDAC8 being the most abundant.58 Conversely, the human orthologue HDAC8 was reported to generally show less abundance than HDAC1 and HDAC3 in human cells, except in some tumor cells which show up-regulated levels of HDAC8.59 Initial studies investigating the effect of pan-HDACi demonstrated their ability to induce schistosome mortality; the exact mechanism however is not completely clarified yet.60,61 Incubation of schistosomula larvae with 1 (trichostatin A, TSA) or 2 (valproic acid, VPA) caused parasite mortality via an apoptotic mechanism. The results indicated that 1 induces a sustained hyperacetylation of H4 leading to an increased expression of CASP7, which is responsible of inducing apoptosis.61 Another study demonstrated that 1 arrests the transformation of Schistosoma miracidia to intramolluskal sporocysts.60 These experiments shed light on the potential mechanism of HDAC inhibitors-mediated mortality of schistosomes. Several studies have thus focused on smHDAC8 as a potential therapeutic target for the treatment of schistosomiasis.62-65 SmHDAC8 was found in in vivo studies to play an important role in the homeostasis of the parasite and to be essential for its pathogenicity. Mice infected with smHDAC8 knocked-down schistosomules showed, 35 days post incubation, a reduced number of recovered adult worms and lower egg burden compared to control mice. This indicated the importance of smHDAC8 for the survival and maturation of the parasite in its host.64


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Plasmodium HDACs. Five HDAC-encoding genes have been identified in the Plasmodium falciparum genome. Three of these genes encode proteins with homology to class I (PfHDAC1) or class-II (PfHDAC2 and 3) mammalian HDACs, while two genes are class-III HDAC homologs (PfSir2A and PfSir2B).45,66-69 PfHDAC1 is localized in the parasite nucleus, has almost 55% sequence identity to other eukaryotic class I HDACs, and is expressed/transcribed in different lifecycle stages of the protozoan (asexual intraerythrocytic parasites, gametocytes, and sporozoites).45,67,68,70,71 Although the functional roles of PfHDAC1 have not been fully characterized, the consequences of treatment of P. falciparum parasites with HDAC inhibitors have recently begun to be clarified and confirm that PfHDAC1 takes part in post-translational modification of histone and nonhistone Plasmodium proteins, and the consequent modulation of its gene expression, and seems important for the parasites survival.45,67,68,72-75 As shown by homology modeling analyses, the predicted active site tunnel of PfHDAC1 is highly conserved with that of human HDACs, but displays differences at its entrance. These could account for the higher in vitro growth inhibition of P. falciparum compared with mammalian cells that has been observed with several HDACi (Table 1 and discussion below),45,67,68 and could also be more efficiently exploited by novel HDACi tailored to be selective for PfHDAC1 over hHDACs.45,67,68,70-73 Both PfHDAC2 and PfHDAC3 are predicted to be high molecular weight proteins, that share less than 14 % amino acid identity to each other, and have limited sequence homology with other class II HDACs.66,69-71 Recently, by the mean of knock-down experiments, PfHDAC3 has been reported to be essential to the asexual-stage P. falciparum parasite growth and survival, and to play a role in P. falciparum transcriptional control.66



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PfSir2A and PfSir2B have been assigned as type III and IV sirtuins, since they have 30% and 38% sequence identity to an Archaeoglobus fulgidus class-III HDAC and group IV sirtuins, respectively.76,77 In addition to both histone deacetylase and ADP-ribosyltransferase activity,77,78 PfSir2A is also able to effectively remove medium and long chain fatty acyl groups from lysine residues.79 All these catalytic activities make PfSir2A a player in maintaining P. falciparum telomere length, in establishing heterochromatin in subtelomeric genomic regions, and in regulation of a subset of P. falciparum genes involved in virulence, antigenic variation and cytoadhesion/pathogenesis.74,76 Both PfSir2A and PfSir2B are involved in the mutually exclusive silencing (or expression) of different telomeric-associated var gene subsets, with distinct promoter types, that encode for the parasite-derived P. falciparum erythrocyte membrane protein 1 (PfEMP1) molecules that are displayed on the erythrocyte surface.74,76 The resulting antigenic variation of PfEMP1 accounts for the ability of P. falciparum to evade the host immune surveillance during infection.76 Moreover, PfSir2B has been also found to have a role in the telomeric end protection.76 Knock-out of the two P. falciparum Sir2 genes has shown that the absence of either one of them is not lethal to the parasite, and also established their functional redundancy in the parasite.76 However, to the best of our knowledge, the effect of a simultaneous knock-out of both PfSir2 genes has not been examined yet. Despite both PfSir2A and PfSir2B seem to be dispensable for the in vitro growth and development of P. falciparum,69,74 both of them, for their crucial role in regulating var genes expression, are thought to be essential for the persistence parasite survival in vivo (or inside a host), and have been proposed as potential targets for antimalarial therapies. In particular, these Pf sirtuins could disturb the cytoadhesion of infected erythrocytes to host cell receptors that mediate serious forms of the disease, or could hamper the Plasmodium evasion from the host innate immune system.74,76 In other Plasmodium


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species, even though no var genes have been identified, a role for these sirtuins (Sir2A and Sir2B) in the mutually exclusive silencing (or expression) of sub-telomeric genes able to undergo antigenic switching has been proposed.76 Trypanosoma HDACs. Trypanosoma brucei possesses four class-I/II HDAC orthologs (TbDAC1-4).45,80,81 Both TbDAC1 and 2 share similar sequence identity with mammalian class-I HDACs, while TbDAC3 and 4 are more closely related to class-II HDACs.80-83 Furthermore, it has been indicated that both TbDAC1 and 3 are nucleus-localized and are essential for the parasite survival,82 whereas TbDAC2 and 4 are predominantly localized to cytoplasm.83 In the bloodstream of the human host, T. brucei avoids immune destruction by periodic switching of the single variant surface glycoproteins (VSGs), which are encoded by a large repertoire of VSG genes, but with only one VSG expression site transcriptionally active at a time.82 In this regard, TbDAC3 has been shown to be required for VSG expression site silencing/cycling in both bloodstream and insect-stage cells, so allowing the parasite to evade the host immune defenses.83 By contrast, Trypanosoma brucei has three different sirtuin homologs (TbSir2rp1 to -3).45,84,85 TbSir2rp1 is a nuclear chromosome-associated protein and was found to be neither essential for in vitro parasite survival nor required for antigenic variation (VSG expression site silencing), despite its involvement in RNA polymerase I-mediated transcription repression of subtelomeric genes in both the insect and blood stages. However, under genotoxic stress, e.g. under DNA damaging conditions, TbSir2rp1 ought to be important for the parasite survival since the histone (especially H2A and H2B) modifications (ADP-ribosylation and deacetylation) catalyzed by this enzyme were shown to be involved in the DNA repair, and under- or over-expression of TbSir2rp1 was able to decrease or increase, respectively, the cellular resistance to DNA damage.84,85 In this regard, selective TbSir2rp1 inhibitors have been proposed as potentially



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promising adjuncts to be employed in combination with the conventional DNA-targeting chemotherapeutic agents to maximize the anti-parasitic efficacy.84,85 TbSir2rp2 and TbSir2rp3 are mitochondrial proteins, and their independent knockouts do not affect the proliferation and differentiation of bloodstream forms.84,85 Trypanosoma cruzi has only two genes coding for sirtuins, TcSir2rp1 and TcSir2rp3, which are cytosolic and mitochondrial proteins, respectively.86,87 Both sirtuins play important roles in the proliferation of T. cruzi replicative forms, in the host cell-parasite interplay, and in differentiation among different lifecycle stages, but each one is responsible of different outcomes in most of these processes.86,87 Indeed, the over-expression of TcSir2rp1 acts to impair parasite growth and differentiation, while the over-expression of wild-type TcSir2rp3 improves both.87 These recent findings, together with the evidence that the SIRTi 3 (salermide) was able to inhibit T. cruzi proliferation and differentiation in vitro and in vivo, and to fully revert the effects observed with overexpressed TcSir2rp3, likely through the inhibition of TcSir2rp3, highlighted for the first time the possibility that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy (see below in the specific section).87 Leishmania HDACs. The Leishmania genome also contains four genes putatively encoding class-I/II HDAC homologs, while three genes encoding for class-III HDAC (sirtuin) homologs.45 To date, none of the class-I/II HDACs of Leishmania species has been functionally characterized, but one homolog has been shown to be transiently up-regulated during promastigote-toamastigote differentiation in a host-free model system, indicating a possible role in chromatin structure modification and/or in transcriptional regulation.45,88 In contrast, several studies have investigated the biological roles of sirtuins of different Leishmania species, and, in particular, of the so called Sir2-related protein 1 (Sir2rp1). This


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protein has been found in cytoplasmic granules in different parasite developmental stages (promastigotes and amastigotes) of L. major, L. infantum and, more recently, L. amazonensis.45,89,90 Both LiSir2rp1 and LmSir2rp1 present NAD+-dependent deacetylase and ADP-ribosyltransferase activities unrelated to epigenetic silencing and, as indicated by overexpression and gene disruption studies, have been demonstrated to be essential for the in vitro and in vivo survival of the corresponding two Leishmania species.90-92 LiSir2rp1 has been also found associated with the cytoskeleton network where it deacetylates α-tubulin, resembling the function of human SIRT2 and HDAC6.91 Leishmania sirtuins also appear to possess immunomodulatory functions.45,93,94 Indeed, LmSir2rp1 elicits B-cell effector function and stimulates murine B-cell differentiation and in vivo production of specific antibodies.93 Similarly, LiSir2rp1 is capable of activating splenic B cells via a Toll-like receptor-2 (TLR2)-dependent mechanism, leading to up-regulation of major histocompatibility complex (MHC)-II and the co-stimulatory molecules CD40 and CD86, and secretion of the tumor necrosis factor in the mouse model employed.94 HDAC proteins, and especially the sirtuins, are crucial for both the in vitro and in vivo growth of the Leishmania parasite, and in the past few years they have been targeted by several inhibitors with the aim of identifying potential anti-leishmanial agents (see below in the specific section).95-97 Toxoplasma HDACs. The T. gondii genome contains five class-I/II HDAC homologs (TgHDAC1-5) as well as two homologs of the Sir2 (class-III HDAC) subtype, but with the exception of TgHDAC3, little is known about their biological functions.45 TgHDAC3 is localized in the nucleus, has more than 60% sequence identity to hHDAC1, and is part of a large multiprotein complex termed T. gondii co-repressor complex, which is similar to the human HDAC3-



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containing complex. T. gondii HDACs (together with acetylases) have also been associated with gene modulation in different forms of this parasite (tachyzoite to bradyzoite stages).98 In contrast to what observed with P. falciparum, because no subtelomeric gene family has been identified in T. gondii, it appears that its sirtuin proteins (TgSir2A and TgSir2B) are likely involved in processes other than antigenic variation.76 ANTIPARASITIC ACTIVITIES OF HDAC INHIBITORS Antischistosomal HDAC Inhibitors. To further investigate and confirm the role of smHDAC8 in schistosome biology, several investigations were dedicated to the development of smHDAC8 small molecule inhibitors and examine their effect in in vitro studies.62-65 A structurebased virtual screening campaign was successful in finding 25 hydroxamic acid derivatives with an in vitro inhibitory potency against smHDAC8. Nine compounds exhibited activity in the low micromolar range.63 Among the identified inhibitors, 4 (J1075, Figure 1) was found to induce apoptosis and mortality in schistosomula larvae.64 Heimburg et al. recently reported on a new proof of concept study, where several benzoylhydroxamic derivatives were designed as parasitespecific inhibitors. The developed inhibitors were evaluated for their inhibitory activity against schistosomal and human HDACs (smHDAC8, hHDAC1, hHDAC6 and hHDAC8). Twentyseven compounds exhibited an inhibitory activity in the nanomolar range in in vitro assays. Interestingly, many of the reported compounds showed a notable selectivity for smHDAC8 over the major human HDAC isoforms (HDAC1 and HDAC6), some of which also exhibited a preference for smHDAC8 over human HDAC8 [see for instance 5 (TH31) and 6 (TH65), Figures 1 and 2].62 The obtained crystal structures of smHDAC8 apo and inhibited forms as well as docking studies show that, besides the expected coordination of the catalytic zinc-ion by the hydroxamate group, there are active-site differences between the primary structures of


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smHDAC8 and hHDAC8.64 First, two H-bond interactions are formed between the amide linker and the side chains of the protein residues smHis292 (corresponding to hMet274 in the human structure) and smLys20.64 More importantly, the smPhe151 side chain is turned away from the catalytic pocket (flipped-out conformation), whereas the side chain of its human counterpart, hPhe152, is turned towards the active site adopting a flipped-in conformation similarly to all the other known inhibitor- and substrate-bound hHDAC8 structures. Interestingly, hPhe152 cannot adopt a flipped-out conformation in the human HDAC8 structure for the steric hindrance due to the hLeu31 side chain, present in the active site. The corresponding residue in the smHDAC8 structure is replaced by the smaller serine (smSer18) side chain, that enlarges the pocket allowing the flipped-out conformation for the smPhe151 side chain.64 This could partly account for the selectivity of the compounds over HDAC1 and HDAC6 (Figure 1). Further phenotypic assays demonstrated that two of the reported compounds caused significant dose-dependent reduction of cultured schistosomula larvae,62 as opposed to praziquantel which is known to be less effective against larval developing stages of the parasite.99 Moreover, 6 caused a noticeable separation of female and male worm pairs and an impairment of egg-laying by adult worms.62 Further hits from other chemical series, partially including a thiol group as warheads to chelate the catalytic zinc-ion, were also reported to possess in vitro inhibitory activity against schistosomula of S. mansoni.65 Another study was dedicated to investigating schistosomal NAD+-dependent histone deacetylases (sirtuins).100 Altogether, five sirtuins were found to be encoded in S. mansoni genome, which are orthologues of the human sirtuins SIRT1, SIRT2, SIRT5, SIRT6 and SIRT7. The encoded sirtuins are expressed at all stages of S. mansoni life cycle; however, the different classes of SIRTs show a distinct expression pattern in the various parasite lifecycle stages. With



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the aim of assessing the potential of schistosomal sirtuins as targets for anti-parasitic drug development, several reported mammalian SIRTi were tested in in vitro assays to investigate their effects on cultured schistosomula and adult worms. Compound 7 (sirtinol), 3 and the thiobarbiturate derivative 8 (MS3) (Figure 1) significantly reduced the viability of schistosomula through induction of apoptosis. Moreover, incubation of the aforementioned sirtuin inhibitors with adult worms caused separation of female and male worms and reduction in egg laying. Interestingly, 3 also caused a remarkable change in the morphology of ovaries and testes.100


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Figure 1. Most relevant antischistosomal HDAC and sirtuin inhibitors: chemical structures and biological data. Color code: biological data for parasite in green; IC50s for HDAC inhibition in orange; IC50s for antiproliferative effects in mammalian cells in brown.



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Figure 2. (A) X-ray structure of the inhibitor 5 in complex with SmHDAC8 (PDB ID 5FUE). (B) Docking pose of the inhibitor 6 in SmHDAC8. Compounds 5 and 6 are shown in yellow, protein residues involved in the interaction in white, zinc-ion as orange spheres, and H-bond as well as interactions with the metal ion as yellow dashed lines.

ANTIMALARIAL HDAC INHIBITORS The utility of HDACi as antimalarial agents was first reported two decades ago when the cyclic tetrapeptide 9 (apicidin) was described to potentially target Plasmodium and other Apicomplexan parasites.101 Since then, a growing number of HDACi belonging to various structural classes have been reported to exert antimalarial activity. Cyclic Tetrapeptide HDAC Inhibitors. In a pioneering study in 1996, Darkin-Ratray et al. disclosed that the natural cyclic tetrapeptide 9 (Table 1) is endowed with a potent in vitro activity against P. falciparum (IC50 = 200 nM) and a panel of Apicomplexan parasites (T. gondii, Cr. parvum, N. caninum, B. jellisoni, E. tenella, etc.).101 Subsequent studies showed that 9 is able to induce substantial changes to the Plasmodium intra-erythrocytic developmental cycle transcriptional cascade, with ~ 30-60% of the genes showing altered expression,102,103 so


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resembling the effects of many HDACi, including 6 itself, on higher eukaryotic cells.104,105 Indeed, 9 causes in situ parasite histone hyperacetylation101 and inhibits the PfHDAC1 activity in the low nanomolar range (IC50 ∼ 1 nM).75 Despite being orally active (2-20 mg/kg for 3 days) in a lethal P. berghei mouse model of malaria,106 due to its poor bioavailability and a substantial lack of selectivity for P. falciparum-infected erythrocytes versus mammalian cell lines (Table 1) 9 has never been considered suitable for clinical applications. While the structural similarities between one of the side chains of 9 called Aoda (2-amino-8-oxodecanoic acid) and the acetylated histone lysines enable the chelation of the zinc ion at the bottom of the HDAC catalytic tunnel, the other components of the cyclic peptide presumably do not distinguish among the flanking amino acid residues that characterize the different entrances to the catalytic active site of the different HDAC isoforms, so providing a possible explanation for its inability to selectively inhibit P. falciparum over human HDACs.101,106 To address the selectivity issue, many analogues of 9 were prepared over the years by Merck Research Laboratories by modifying either its Aoda side chain or its tryptophan moiety,106-109 and it was found that the replacement of tryptophan with quinolone/quinoline nuclei led to analogues of 9 showing 50- to >230-fold parasite selectivity over mammalian cells (e.g. compound 10, Table 1).106-108 Together with a series of synthetic analogues, 9 was also comparatively investigated for inhibitory activity against two Trypanosoma species (T. cruzi and T. brucei), P. falciparum and L. donovani. Both 9 and its analogues showed potent and nonselective activity toward T. brucei, similarly to P. falciparum, but revealed to be toxic against T. cruzi and L. donovani.109 Recently, the cyclic depsipeptide 11 (romidepsin, also called FK228 in Table 1), together with the other three HDACi approved as anticancer drugs (see below in the text), was assessed for its in vitro activity against drug sensitive (3D7) and drug resistant (Dd2) asexual P. falciparum parasites and the bloodstream



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forms of T. b brucei. parasites.110 Compound 11 displayed activity in the low nanomolar range against all parasite lines, caused hyperacetylation of both histone and non-histone proteins in P. falciparum, and inhibited deacetylase activity of parasite nuclear extracts and recombinant PfHDAC1, but unfortunately exhibited no selectivity over mammalian cells (NFF and HEK293) for both parasites (SI < 1).110 Short-chain fatty acid HDAC Inhibitors. Short-chain fatty acid-type HDACi, such as 2 (Table 1), sodium butyrate, 4-phenylbutyrate and related derivatives, despite being weak inhibitors of mammalian HDACs, show low toxicity. Inhibitor 2 is already approved for some therapeutic indications (epilepsy and mood disorders), is now undergoing several trials for HDAC-related diseases, and could be intriguing for repurposing in parasitic diseases.111 These compounds are active in vitro in the (sub)millimolar range against different parasites such as P. falciparum, T. gondii and S. mansoni and suffer from poor selectivity for parasites (especially P. falciparum) over mammalian cells.60,112,113 Hydroxamate-Based HDAC Inhibitors. As in other human diseases such as cancer, HDACi bearing a hydroxamate warhead as zinc-binding group (ZBG) are the best studied anti-parasitic HDACi and have shown promising in vitro activity profiles against P. falciparum and other parasites. Many natural and synthetic class-I/II HDACi such as 1,114 12 (suberoylanilide hydroxamic acid or SAHA),110 13 (suberic bishydroxamate or SBHA),114 14 (MW2796)114 and aroyl-pyrrolylhydroxyamides (APHAs, e.g. 15),115 have been studied for their antimalarial potential (Table 1).45,67,68,75,114-116 These compounds showed inhibitory activities against P. falciparum in the range of low/sub micromolar (12, 14, 15) to nanomolar (1) concentrations, but almost all suffered, despite to a different extent, from poor selectivity versus host cells, with the only exception of 13, which displayed somewhat better selectivity for the parasite based on


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mammalian cytotoxicity data (Table 1).45,67,68,75,114-116 The extremely potent compound 1, despite being unsuitable for clinical progression due to its lack of selectivity, has been useful to clarify how HDAC inhibition affects the malarial parasite’s growth, development and gene transcription.75,102,114 Indeed, 1 inhibited PfHDAC1 activity in the subnanomolar range (IC50 ∼ 0.6 nM),75 and, like 9, caused hyperacetylation of parasite histones and genome-wide transcriptional changes in the protozoan.102 Compound 12 (vorinostat), the first HDACi approved for the clinical treatment of cutaneous T-cell lymphoma in humans in 2006, is less potent than 1 against P. falciparum parasites in vitro (IC50 values in the submicromolar range), but displays a somewhat improved parasite selectivity (up to ∼ 200-fold, Table 1).45,67,116 Despite its clinical utility for cancer, the in vivo efficacy of 12 against Plasmodium parasites in mouse models of malaria has still to be evaluated. In contrast, compound 13, less potent but more parasite-selective in vitro than 12 (Table 1), has been examined in vivo in P. berghei-infected BALB/c mice (200 mg/kg, twice daily intra-peritoneally for 3 days) where it showed a cytostatic effect by significantly inhibiting peripheral parasitemia.114 Although no mouse efficacy was observed, these early data about compound 13 suggested that hydroxamate-based HDACi deserved further investigation as potential antimalarial agents.45,67,114 Inspired by the promising initial results, a number of HDACi with better in vitro potency against P. falciparum parasites than 12 or 13, and with varying improvements on selectivity, were identified in the subsequent years by screening compounds that, keeping intact the hydroxamic acid as the ZBG, showed variations into the three other structural motifs of the general pharmacophoric model for HDAC inhibition: the CAP group that, acting as an enzyme surface recognition moiety, interacts at the entrance of the catalytic tunnel; the linker region that connecting the CAP with the ZBG fits the narrow, hydrophobic, tubular enzyme cavity; and the



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dispensable polar connection unit (CU) between the CAP and the linker.45,47,67,68 Among them, supported by in silico molecular modeling of PfHDAC1 and docking studies, the Fairlie group reported some L-cysteine-based (thioether in the linker region, e.g. 16) and 2-aminosuberic acid (2-ASA)-based (only methylene groups in the linker region) hydroxamates that showed similar in vitro anti-parasitic potency (IC50s in the low nanomolar range) against both CQ-sensitive (3D7) and CQ-resistant (Dd2) P. falciparum strains, with the better selectivity generally observed for the 2-ASA compounds (Table 1). Among the latter, 17 (2-ASA-9) and 18 (2-ASA-14), in addition to causing a marked hyperacetylation of P. falciparum histones, showed the interesting capability to inhibit the P. falciparum growth in erythrocytes at both early and late stages of the parasite’s life cycle.73 In one study, 1, 12 and 17 were profiled for their effects on gene expression in P. falciparum parasites. Each compound caused genome-wide transcriptional changes (up to 2-21%), consistent with inhibition of HDAC activity in the parasite. Though the three inhibitors had very different overall effects on gene expression profiles, α-tubulin II was found to be one of the small set of genes up-regulated by all three HDACi and its identification as transcriptional marker induced by structurally different HDACi in P. falciparum has been proposed as an important finding since this marker might be utilized for developing HDACi as “specific” antimalarial agents.117 The ASA compounds 17 and 18, as well as 12, were also the first HDACi to be tested against P. vivax, that is the second most important human infecting malaria parasite because, despite not generally responsible of death, is the cause of significant malaria-related morbidity and relapses due to parasite stages that can remain dormant in the liver.67 All three hydroxamates were able to inhibit the ex vivo growth of multi-drug resistant P. falciparum and P. vivax isolates obtained directly from infected patients.118 The similar activity profiles obtained for 12, 17 and 18 against both P. falciparum (IC50 310, 533, and 266 nM,


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respectively) and P. vivax (IC50 170, 503, and 278 nM, respectively), despite somewhat higher than those reported in vitro against laboratory strains, provided the first examples of HDACi targeting multiple human-infecting malaria parasite species, which is nowadays thought to be highly beneficial for clinical applications. Several series of 2-ASA compounds, such as the one containing typical non-steroidal anti-inflammatory (NSAID) components in the CAP region (e.g. 19, Table 1) were tested over the years for their inhibitory activity against P. falciparum. But although many compounds displayed an extremely potent activity against P. falciparum (IC50s in the low nanomolar range), significant improvements in parasite selectivity in comparison to 17 were not obtained (Table 1).119 In 2009, Patel et al. screened in a high-throughput viability assay the antimalarial efficacy of a library of ∼ 2000 HDACi characterized by an acyl hydrazone moiety as CU and with chemical diversity in the recognition CAP, the ZBG, and the hydrophobic linker length (4-6 methylene units).75 Although many compounds potently inhibited P. falciparum parasite growth and recombinant PfHDAC1 activity, only 17 derivatives demonstrated anti-parasitic activity in the low nanomolar range, coupled with minimum perturbation of mammalian cell (human myeloma MM.1S cells) histone acetylation, that was used as an indicator of selectivity.75 Within this series, the selective inhibition of P. falciparum proliferation was highly favored by the presence of ortho-substituents (mainly bromine and hydroxyl) in the CAP aromatic group, of a hydroxamic acid as metal chelator, and of a linker of five methylene units (e.g 20, Table 1).75 In the same period, Kozikowski and collaborators reported two series of suberoylamide hydroxamates bearing substituted triazolylphenyl120 and phenylthiazolyl (WR compounds)116 moieties as CAP groups. Among the triazolylphenyl-based HDACi, compound 21, endowed with an activity in the low nanomolar range (Table 1), was 10-fold more potent than other congeners,



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and more active than mefloquine and chloroquine against the multiple drug-resistant P. falciparum strains C235 and C2A. However, in the best case 21 was only ∼ 23-fold more selective for C235 over mammalian cells.120 Interestingly, in a panel of 50 phenylthiazolyl hydroxamate-based HDACi, three very potent compounds (IC50 < 3 nM) were identified that had more than 600-fold selectivity toward P. falciparum compared to human cells. The most promising HDACi from this set resulted the derivative 22 (WR301801), that exhibited IC50 values in the (sub)nanomolar range against several drug-resistant strains (D6, W2, C235 and C2A) of P. falciparum (Table 1), with a significant inhibition of HDAC activity in P. falciparum nuclear extracts (IC50 ∼ 10 nM), and a strong hyperacetylation of parasite histones in situ.116 In one study, 22 caused a significant suppression of parasitemia but did not cure P. berghei-infected mice when administered orally as monotherapy at doses of up to 640 mg/kg, while some, but not all, mice were cured when it was combined with sub-curative doses of CQ (52 mg/kg of 22 plus 64 mg/kg of CQ).116 Likewise, oral administration of 22 (32 mg/kg/day for 3 days) to P. falciparum-infected Aotus monkeys resulted in parasite suppression but not eradication.116 In another study, 22 improved survival and completely and irreversibly suppressed parasitemia in P. berghei-infected mice when given by intra-peritoneal injection at a dose of 50 mg/kg/day for 4 days, with an experimental follow up period of 6 weeks.121 Optimization of the pharmacokinetic properties of 22 appears desirable, since it is rapidly hydrolyzed both in vitro and in vivo to the corresponding inactive carboxylic acid.116 Nevertheless, these findings clearly demonstrate the potential of HDACi in mono- and/or combination therapy for the treatment of malaria.45,67,116,121 In 2010, a series of aryltriazolyl hydroxamate-based HDACi was tested by Oyelere’s team for their inhibitory activity against promastigote stages of L. donovani and asexual P. falciparum blood-stage parasites.122 Under the tested conditions, several compounds achieved better


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inhibitory activity (IC50s in the nanomolar range) and selectivity than 12 against P. falciparum growth (D6 and W2 strains). Despite less active than against P. falciparum, some compounds possessed also modest inhibitory potency versus L. donovani, with IC50 values from 2- to 4-fold better than those of 12 and comparable to miltefosine, the standard oral drug for the treatment of visceral leishmaniasis. Notably, the anti-parasitic activity was dependent on the length of polymethylene linker and the nature of the CAP group. For any given CAP moiety, the activity against both parasites was maximal in analogues with 5 or 6 methylene units in the spacer region between the CAP and the ZBG. Indeed, compounds 23 and 24 (Table 1), characterized by a 3’biphenyltriazolyl moiety as CAP and by a 6 and 5 methylene units linker, respectively, displayed the best activity and selectivity against P. falciparum, with 24 resulting also the most active against L. donovani (IC50 ∼ 32 µM).122 Oyelere and collaborators also investigated the antimalarial and antileishmanial activity of five tricyclic ketolide-based phenyltriazolyl HDACi.123 Under the tested conditions, the optimal length of the linker between the CAP and the hydroxamic acid was of 6 methylene units for the best antimalarial activity that also mirrored the most potent PfHDAC1 inhibition (25a), while of 9 methylene units for the best antileishmanial activity that did not correlate with the PfHDAC1 inhibition (25b).123 More in detail, compound 25a showed IC50 values against both CQ-sensitive (D6) and CQ-resistant (W2) P. falciparum strains from 7- to 10-fold lower than those of 12, resulting up to 10-fold more selective over mammalian cells (Vero) compared to it (Table 1), and was devoid of antileishmanial activity, while compound 25b showed an activity against L. donovani (IC50 ∼ 5 µM) 16-fold stronger than 12 (IC50 ∼ 81 µM) together with modest antimalarial effects (Table 1).123 The same group also reported the antimalarial and antileishmanial activities of HDACi characterized by nonpeptide macrocyclic skeletons derived from 14- and 15-membered macrolides linked to a phenyltriazolyl



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moiety as large recognition CAP groups. All compounds inhibited the proliferation of both CQsensitive (D6 clone) and CQ-resistant (W2 clone) strains of P. falciparum with IC50 values in the (sub)micromolar range.124 For both macrolide skeletons, the maximum activity and selectivity against P. falciparum was achieved with 6 methylene units in the linker group separating the triazole ring of the CAP group from the active-site zinc binding hydroxamate, accordingly to previous reports.122,123 The best among these compounds resulted the derivative 26 (Table 1), that is characterized by a 15-membered macrolide skeleton, showed the highest anti PfHDAC1 activity (IC50 = 29 nM) and exhibited up to 11-fold more potent antiplasmodial activity and up to 14-fold increased selectivity over mammalian cells (Vero) in comparison to 12. Interestingly, for both macrolide skeletons, compounds with 5 to 7 methylene units at the linker group were devoid of activity against the promastigote stage of L. donovani, while maximum activity was obtained for those compounds having either 8 or 9 methylene units,124 as in the case of ketolide-based HDACi,123 but differently from SAR observed in aryltriazolyl hydroxamates.122 In particular, compound 27 (Table 1), that contains a 14-membered macrolide skeleton and a 9 methylene units linker, was up to 25-fold more active than 12 (IC50 ∼ 81 µM), displaying the maximum antileishmanial activity with IC50 values of 3.2 and 4.7 µM against the promastigote and amastigote stages of the parasite, respectively.124 In 2012, Andrews and coworkers reported the in vitro and in vivo antiplasmodial activities of the orally active anticancer HDACi 28 (pracinostat, also indicated as SB939).125 Compound 28 potently inhibited the growth of P. falciparum asexual-stage parasites in human erythrocytes in vitro (IC50s in the nanomolar range), causing hyperacetylation of parasite histone and non-histone proteins, and showing selectivity indexes over mammalian cells ranging from 4 to > 1250, depending on the different tested cell lines (Table 1).125 In addition to a promising additive effect


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in vitro in combination with the antimalarial protease inhibitor lopinavir, 28 offered the first evidence of HDACi as liver-stage antimalarial drug leads. Indeed, 28, as well as 12, displayed a potent inhibition of the in vitro growth of exo-erythrocytic-stage P. berghei ANKA mouse malaria parasites within HepG2 human hepatocytes (IC50 ∼ 150 nM).125 Orally administrated 28 reduced peripheral parasitemia and total parasite burden at doses of up to 100mg/kg/day in the P. berghei ANKA mouse model of malaria. Administartion of 28 to mice prevented the development of experimental cerebral malaria-like symptoms, and the animals did not get hyperparasitemia until 2-3 weeks after the interruption of the treatment.125 Subsequently, a panel of 21 HDACi characterized by a pentyloxyamide connection unit/linker moiety and by a substituted benzene ring as CAP were tested by the same group in vitro for their effects against different Plasmodium life cycle stages: asexual blood stage of P. falciparum (3D7 line); tissue schizontocidal stage of P. berghei (exo-erythrocytic forms cultured in HepG2 human liver cells); and late stage (IV and V) P. falciparum gametocytes. All compounds displayed activity (IC50 values in the range of 0.09-1.12 µM) against the asexual form of P. falciparum, with the potency and selectivity increasing along with the bulkiness of the alkyl/alkoxy substituents at the para position of the phenyl ring, as exemplified by the most active compound 29a (Table 1). Three derivatives revealed nanomolar activity against all three life cycle stages (e.g. 29b, Table 1), and several compounds showed an improved parasite selectivity compared to 12 for at least the asexual and exo-erythrocytic life cycle stages (e.g. 29c, Table 1).126 The same team also reported the antimalarial activity and a SAR investigation of a series of related alkoxyurea-based HDACi.127,128 Several compounds were active at (sub)micromolar level against the 3D7 line of P. falciparum,127 and some of them also displayed early/late stage gametocytocidal activity in the single digit micromolar range (IC50 = 1.68-6.65 µM).127,128 The



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SAR studies revealed that the hydroxamic acid as ZBG and a linker of 5 methylene units were crucial for the antiplasmodial activity. Indeed, alternative ZBGs such as N-methylhydroxamic acid, o-hydroxyanilide and o-aminoanilide were inactive, and chain-shortened analogues (less than 5 methylene units at the linker) exhibited lower potency.127 Also in this series, bulky alkyl/alkoxy substituents at the 4-position of the phenyl CAP group, and its replacement with bulky aromatic rings led to the most potent and selective compounds against both asexual and gametocyte P. falciparum forms, as exemplified by the 4-tert-butyl derivative 30 and the 1naphthyl derivative 31, that anyway both resulted not better than 12 in terms of potency while, under the tested conditions, 31 was more parasite-selective than 12 in its asexual stage and latestage gametocyte killing activity (Table 1).128 In 2015, Giannini et al. assessed the potential anti-parasitic (P. falciparum, L. donovani, T. cruzi, T. brucei, G. lamblia) efficacy and SAR of a few 12 analogues characterized by the substitution with β/γ lactam-carboxamides at the position α of the anilide CU and with (trifluoro)methyl groups in the meta position of the phenyl ring CAP, as well as by an hydroxamic acid or a thiol function as ZBG.129 Remarkably, all hydroxamates showed a potency comparable with CQ (3-23 versus 6 nM) and slightly better than 12 (3-23 versus 25 nM) against P. falciparum, the most sensitive parasite, and were highly selective for P. falciparum over mammalian L6 cells (SIs = 205-3953), while the thio derivatives, both the free and the pro-drug, exhibited poor anti-parasitic activity. It can be pointed out that a hydroxamate as ZBG, a γlactam carboxamide in α to the anilide CU, and a methyl or trifluoromethyl substituent in the meta phenyl ring position associate with an enhanced anti-parasitic activity (at least toward P. falciparum and, to a lesser extent, T. cruzi and brucei species), though the phenyl modifications are also responsible of an increased cytotoxicity and reduced selectivity over mammalian cells.129


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Indeed, in a preliminary test in vivo the best results in a P. berghei mouse model of malaria were obtained with the unsubstituted phenyl derivative 32, that was the most selective in vitro (Table 1) and that inhibited ∼ 88% of the Plasmodium development versus 99.8% of dihydroartemisinine.129 In 2015, as already above mentioned, Andrews and coworkers reported the in vitro activity against P. falciparum (Table 1) and T. b. brucei parasites of all four HDACi clinically approved for the treatment of cancer [11, 12, 33 (belinostat), and 34 (panobinostat)] up to now.110 All compounds inhibited the growth of asexual-stage P. falciparum parasites with nanomolar potencies (Table 1), while only 11 was active at nanomolar level against the bloodstream form T. b. brucei (IC50 = 35 nM), despite being devoid of any selectivity over mammalian cells. The four HDACi also inhibited the deacetylase activity of P. falciparum nuclear extracts and of recombinant PfHDAC1 and caused hyperacetylation of (non)histone proteins, differentially affecting the acetylation profiles of histones H3 and H4, but no one, with the exception of 12 and, to a lesser extent, of 33, displayed some selectivity for malaria parasites over mammalian cells (NFF and HEK293).110 Thiol-based HDAC Inhibitors. The thiol-based HDAC6 (class IIb) selective inhibitor 35,130 when tested against CQ-sensitive (3D7) and CQ-resistant (Dd2) P. falciparum strains, was only poorly active (IC50s in the micromolar range) in inhibiting the proliferation of the parasite (Table 1), so providing a further evidence of the necessity to focus on hydroxamate-based and likely on pan-HDACi as potential antimalarial agents.73 Ortho-Aminoanilides HDAC Inhibitors. The same conclusions can be drawn by considering the series of HDACi containing an ortho-aminoanilide moiety as ZBG. The prototype of these HDACi is the class-I HDAC selective inhibitor 36 (MS275, entinostat),47,131,132 that in multiple



1 2 3 screening over the years has always resulted a modest inhibitor of both PfHDAC1 and 4 5 proliferation with IC50 values of 0.94 µM and ∼ 8 µM, respectively.45,67,68,73,75 6 7 8 9 10 11 Table 1. In vitro Antimalarial Activity of Selected Class-I/II HDAC Inhibitors P. 12 Nuclear extract Mammalia falciparu a 13 Class/ Inhibitor [rPfHDAC1] Structure n cells m IC50 14 IC50 (nM) IC50 (µM) (µM) 15 Cyclic Tetrapeptides 16 17 O 18 N NH 19 O O 20 H 21 9 O NA 22 (apicidin) 0.200 0.05-0.1 HN NH [1±0.1] 23 O 24 O N 25 26 27 28 29 30 31 32 33 34 NA 0.063 >15 10 35 36 37 38 39 40 41 42 43 44 45 0.9±0.8 11 46 0.001[48±39% 0.09-0.13 47 (romidepsin, 100 1350 56 (valproic acid, VPA) 57 HO O 58 59 60 ACS Paragon Plus Environment

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parasite

SIb

Referen ces

0.2-0.5

101108

>238

106-109

20

5-17

115

NA

0.048

0.6

12

73

NA

0.0150.039

1.24

30-83

73, 117, 118

O HOHN NHOH O

H N O

O N H

N

NHOH O



1 2 3 4 5 6 7 18 8 (2-ASA-14) 9 10 11 12 13 14 15 16 17 19 18 19 20 21 22 23 20 24 25 26 27 28 29 21 30 31 32 33 34 35 22 36 (WR301801) 37 38 39 40 41 42 23 43 44 45 46 47 48 24 49 50 51 52 53 54 25a 55 (n=6) 56 57 58 59 60

Br

O N

O

N H

NHOH

N N

NA

0.0130.033

0.34

10-26

73, 118

NA

0.013

0.26

20

119

NA [37]

0.015

NA

NA

75

NA

0.0170.035

0.02-0.8

0.6-22.8

120

1-10 [NA]

0.00060.0016

0.6

4001016

116, 121

NA

0.069

NC

>190.4

122

NA

0.0740.107

NC

>127 (>183)

122

NA [10±0.5]

0.1440.148

>5.2

O

H N

N

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5 NHOH O

O

H N

N S

5 NHOH O

H2N

>36 (>35)


123

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1 2 3 4 25b 5 (n=9) 6 7 8 9 10 11 26 12 13 14 15 16 17 18 19 20 21 22 27 23 24 25 26 27 28 29 30 31 28 32 33 (pracinostat, SB939) 34 35 36 37 38 39 40 29 41 42 43 44 45 46 30 47 48 49 50 51 31 52 53 54 55 56 57 58 59 60


NA [304±17]

0.93-1.24

>5

>5.4 (>4)

123

NA [29±0.9]

0.0940.226

NC

>20.7 (>47.6)

124

NA [401±19.7]

0.76-1.32

NC

>3.4 (>6)

124

NA

0.08-0.15

0.8->100

4->1250

125

O N

NHOH

N N

O R

O N H

O 5

a. R = 4-t-Bu-Ph NHOH b. R = 3,4-CH3-Ph

c. R = 4-BuO-Ph

[68.6±1.2% @1µM] [82.6±4.2% @1µM] [72.1±5% @1µM]

0.09

12.47

139

126

0.12

3.24

27

126

0.17

>50

>294

126

NA

0.16 (3.45)c

4.9

31 (2)c

127

NA

0.25-0.32 (2.122.25)c

19.5

36-78 (9)c

128



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1 2 3 4 5 6 NA 0.019 75.1 >3950 129 32 7 8 9 10 11 O 12 O 214.7±15.3 O 33 13 S [78.5±4.7% 0.06-0.13 1.4-2.4 11-40 110 N NHOH 14 (belinostat) @1µM] H 15 16 HN 17 18 3.3±0.7 19 N 34 H [100±0% 0.01-0.03 0.07-0.18 2-18 110 20 (panobinostat ) NHOH @1µM] 21 22 O 23 24 Thiols 25 O 26 27 HN O H NA 15.2-19.9 NA NA 73 35 28 N SH 29 30 O 31 Ortho-Aminoanilides 32 O 33 34 36 O N NH2 NA H 35 H 7.8-8.3 >20 73,75 (MS275, ∼2.5 N [940±90] 36 entinostat) N 37 O 38 a b 39 Deacetylase activity tested using either P. falciparum nuclear lysates (no brackets) or c recombinant PfHDAC1[brackets]; SI: Selectivity Index - fold difference between mammalian cell and P. falciparum IC50 values (IC50 mammalian cells/IC50 P. falciparum); Relative to gametocytocidal activity; NA: data not available; NC: no cytotoxicity at 40 the maximum tested concentration. 41 42 43 Other Amides as HDAC Inhibitors. Very recently, Ontoria et al., cast doubt on PfHDAC1 44 45 inhibition data reported in the literature so far. They described an innovative screening approach 46 47 48 that led to the identification of a novel series of P. falciparum growth inhibitors claimed as 49 50 selective for PfHDACi and characterized by a secondary amide as the ZBG, a hexamethylene 51 52 chain as linker and two groups such as an (hetero)aryl substituted imidazole ring and a 253 54 55 thiazolylcarboxamide as surface contact CAP moieties.133 Some derivatives in the series showed 56 57 submicromolar inhibition of P. falciparum proliferation (EC50 < 500 nM) with only a modest 58 59 34 60 ACS Paragon Plus Environment

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inhibition of human class I HDACs in HeLa cells (IC50 values in the micromolar range), used as an indicator of selectivity.133 Both activity and selectivity were strongly influenced by the nature of the (hetero)aryl substituent attached to the imidazole core. Although clear SAR trends were difficult to identify, compounds based on phenyl-/ biphenyl-/ benzimidazole- or benzotriazolesubstituted imidazoles were either weak or poorly selective inhibitors of parasite growth. Conversely, analogues bearing indole-, indazole-, isoquinoline- and mainly pyrazolylphenyl- (37 and 38) or quinoline-substituted (39) imidazoles showed the best activity and selectivity for the parasite (Table 2).133 Support for selective PfHDACs inhibition as the mechanism of action of these compounds was provided by the evidence that compound 37, one the most potent and selective (SI = 37) of the series, induced the hyperacetylation of the histone H4K8 in parasite cells at a concentration (EC50 = 350 nM) quite close to the potency measured in the parasite growth inhibition assay (EC50 = 450 nM) and quite far from the concentration necessary to inhibit recombinant (EC50 > 5 µM) and cellular (EC50 = 16.7 µM) hHDACs and to promote the same extent of

histone H4 hyperacetylation in human HeLa cells (EC50 > 25 µM).133 In

summary, the screening strategy proposed by Ontoria et al. for evaluating the parasite selectivity of potential antimalarial HDACi consists of two steps: (i) comparison of parasite growth inhibition in erythrocytes with hHDAC inhibition in HeLa cells as preliminary readout of selectivity; (ii) conclusive validation of the parasite selectivity of the most promising compounds by measurements of nuclear histone hyperacetylation in both human and parasite treated cells. This approach seems quite attractive because it avoids the problems of the common PfHDAC preparations that are likely to be inactive without endogenous cofactors, are endowed with only low in vitro activities due to contamination by copurified host HDACs arising from the cellular expression vectors, and therefore are not reliable for inhibition studies.



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Table 2. In vitro Antimalarial Activity of N-Methyl Carboxamides as HDAC Inhibitors Nuclear extract Class/ P. falciparum Structure [rPfHDAC Inhibitor EC50 (µM) a 1] IC50 (nM)

Class I hHDACs in HeLa cells IC50 (µM)

SIb

References

37

NA

0.45

16.6

37

133

38

NA

0.90

20.5

23

133

39

NA

0.5

>25

54

133

a Deacetylase activity tested using either P. falciparum nuclear lysates (no brackets) or recombinant PfHDAC1[brackets]; b SI: Selectivity Index [HeLa class I HDACs IC50 (µM) / Pfgrowth EC50 (µM)]; NA: data not available.

Antimalarial Class-III HDAC (Sirtuin) Inhibitors. Although less studied than class I/II/IV HDACi, several SIRTi were tested in vitro for growth arrest against erythrocytes infected by P. falciparum and for inhibition of the recombinant PfSir2A protein.45,67 Known natural and synthetic SIRTi that have been examined over the years for P. falciparum growth inhibition comprise 7 (IC50 ∼ 9-13 µM),77,78 40 (splitomycin, IC50 > 10 µM),75,77,78 41 (surfactin, IC50 ∼ 9 µM),77 and 42 (hyperforin, IC50 ∼ 1.5-2 µM)134,135 (Figure 3). Compound 43 (nicotinamide), the


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physiological product of the Sir2-catalyzed NAD+-dependent deacylation reaction, was found to be significantly less active at whole cell level (IC50 ∼ 9.9 mM) (Figure 3).77,78,136 The depsipeptide 41 and, to a lesser extent, 43 are more potent inhibitors of PfSir2A (IC50 = 35 and 51 µM, respectively) than hSIRT1 (IC50 > 600 and = 88-250 µM, respectively), while 7 and 40 are less potent than them versus PfSir2A (IC50 > 50 and > 400 µM, respectively). In 2009, Chakrabarty et al. applied a click chemistry approach to synthesize lysine-based tripeptide analogues designed to hit PfSir2A by competing with the peptide binding pocket.137 Although three out of four tested analogues had similar or higher potency (IC50s = 23-34 µM) against PfSir2A compared with 41 and 43, all of them were devoid of selectivity for the parasite sirtuin over human SIRT1. The most potent compound 44 versus PfSir2A was also tested in vitro against P. falciparum-infected erythrocytes where it was as potent as 7 and 41 in inhibiting the growth of the parasite (IC50 = 9.8 µM).137 The natural hSIRT1 activator resveratrol,52 able to modestly inhibit in vitro the growth of P. falciparum (IC50 ∼ 60 µM), was also tested against recombinant PfSir2A, but no enzyme activation neither inhibition was detected.78 Overall, the in vitro activity of the SIRTi tested so far against P. falciparum growth is modest, but this is quite expected because PfSir2A and PfSir2B appear to be not essential for the in vitro parasite growth and development, and share low sequence homology with the other eukaryotic Sir2 proteins.76,138 Moreover, the somewhat low consistency between the PfSir2A inhibitory potencies and the in vitro antiproliferative effects promoted by some compounds (e.g. 7, 40, 43 and, to a lesser extent, 41) and the pleiotropic nature of some of them (e.g. 7 and 43) do not exclude additional mechanisms involved in their P. falciparum growth inhibitory activity. For these reasons, it is presently highly desirable to identify and develop inhibitors with significantly increased potency against PfSir2 enzymes and selectivity over human sirtuins, that could be 37 ACS Paragon Plus Environment


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useful as tools for studying the biology of P. falciparum sirtuins and potentially for their pharmacological validation as targets of drugs exerting a direct or an indirect (by blocking the parasite evasion from the host innate immune surveillance) anti-parasitic activity (see above in text).76,138

Figure 3. Antimalarial class-III HDAC (SIRT) inhibitors: chemical structures and biological data. Color code: IC50s for parasite growth inhibition in green; IC50s for SIRT inhibition in orange.


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Antitrypanosomal HDAC Inhibitors. Unlike the case for malaria, only a few HDACi were tested for the treatment of Trypanosoma species infections (see also above in the antimalarial section).45,110,129 In one study, 1 was reported to suppress the growth of the bloodstream form of T. brucei at ~ 7 µM, but without de-repression of a silent VSG expression site.139 In other studies, 1 inhibited > 50% of the activity of recombinant TbDAC1 and TbDAC3 at 5 µM,83 while it was not able to alter the parasite histone acetylation at 0.3 µM concentration.140 As above mentioned, Engel et al. tested the antitrypanosomal and antimalarial activity of the four HDACi (11, 12, 33, and 34) clinically approved for the treatment of cancer so far.110 All inhibitors demonstrated activity in the range of submicromolar to single-digit micromolar concentrations against T. brucei brucei, with the depsipeptide 11 (Figure 4) exhibiting the highest potency (IC50 = 35 nM), but all of them were also cytotoxic to human cells (NFF and HEK 293 lines) at lower concentrations (SI < 1).110 In 2012, many hydroxamates belonging to 4 series of human HDACi patented as anticancer agents (sulphonamides, sulphonylpiperazines, long chain amides, and heterocycle-containing acrylamides) were screened for their inhibitory effect against cultured bloodstream form Trypanosoma brucei.141 All tested classes, with the only exception of the long chain amides (IC50s > 10 µM), revealed a significant parasite growth inhibitory activity in the (sub)micromolar range (IC50s = 0.034-1.54 µM). Sulphonylpiperazines resulted the most potent compounds, expecially if containing an heteroaryl ring linked to the piperazine moiety, such as the derivative 45 (Figure 4) that showed an IC50 of 34 nM and was able to induce the death of the parasite within 4 h after treatment when used at 2 µg/mL.141 The evidence that, despite all 4 series were



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potent inhibitors of the hHDAC activity of HeLa cell lysates (IC50s = 0.010-0.212 µM), only 3 were active as antitrypanosomal agents, and among them no correlation was observed between hHDAC inhibitory potency and anti-parasitic activity, suggested that TbDACs likely possess unique structural features that may be exploited for the development of new more potent and parasite-selective HDACi.141 Recently, a panel of known hydroxamate-based HDACi (including two HDACi recently approved and four in clinical trials as anticancer agents) were tested for their ability to inhibit the proliferation of cultured bloodstream T. brucei brucei. All reported compounds displayed moderate potency against the parasites, with EC50 ranging from 0.029 µM for the most potent 46 (quisinostat, Figure 4) to 11 µM for givinostat.142 The anticancer drugs 33 and 34 were promising because significantly effective at the concentrations reached with their tolerated doses in humans, but when tested after a single dose administration, both of them were unable to kill cultured parasites. In addition, 34, the most slowly cleared HDACi, did not show synergistic effect when co-administered with the standard current treatment drugs (pentamidine, suramin, melarsoprol, and nifurtimox) of HAT.142 Collectively, these evidences led the authors to state that any direct repurposing of these two HDACi for the HAT treatment as single agents or in combination is precluded.142 Finally, in the light of the absence of correlations between the potency against any hHDAC isoform and the inhibition of T. brucei proliferation, also in this case, the authors suggested that trypanosome histone deacetylases might possess a unique specificity that could be exploited to develop potent HDACi selective for parasite over human HDACs.142 Antitrypanosomal Class-III HDAC (Sirtuin) Inhibitors. In 2012, 43 has been reported as a putative inhibitor of the T. cruzi sirtuin TcSir2rp1 as well as of the growth of T. cruzi at both the epimastigote and trypomastigote forms, displaying a significant reduction in the number of


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amastigotes in the T. cruzi-infected macrophages.143 Although 43, beyond its SIRT inhibitory activity, also holds a pleiotropic nature (see also above in the antimalarial section), this was the first report highlighting the potential of sirtuin inhibition in T. cruzi infection.143 In 2015, Moretti et al., in addition to the characterization of the two T. cruzi sirtuins (TcSir2rp1 and TcSir2rp3), reported the effects of 3 (Figure 4), a known SIRTi endowed with a promising antitumor potential in different cancer models,52,144,145 against T. cruzi growth and differentiation.87 Both in vitro and in vivo assays indicated that 3 is able to impair the epimastigote proliferation (EC50 = 10.6 µM) and differentiation to infective forms at concentrations that did not affect host cell viability. Furthermore, it inhibited the activity of the recombinant TcSir2rp3, with an IC50 ∼1 µM (lower than those measured with hSIRT1 and hSIRT2), suggesting that the observed effects in the parasite were accomplished through inhibition of TcSir2rp3 activity. This finding was also supported by the experimental evidence that all phenotypic effects observed with the over-expression of TcSir2rp3 were fully reverted by adding 3.87 Importantly, the parasitemia reduction without side effects in a mouse (BALB/c) model of the T. cruzi infection was observed at doses lower than those reported to be effective for eliminating cancer cells in vivo. However, 3 was not able to prevent the mice mortality with the employed doses, so convincing the authors about the necessity to develop more potent and parasite isoform (TcSir2rp3) selective SIRTi as agents potentially useful in the management of T. cruzi infection.87 Moving toward this direction, molecular docking investigations of pan-SIRTi (7 and 43) and specific class-III SIRTi (thiobarbiturate derivatives) have recently revealed that TcSir2rp3 shows strong analogies with the mitochondrial human SIRT5 in terms of binding mode and interaction strength.146 However, the comparison of the catalytic pocket of TcSir2rp3 with the homologous



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human protein SIRT5 and in silico structural and surface analysis of trypanosomal and human SIRTs have allowed the identification of minor but significant structural differences in the corresponding inhibitor catalytic domains, that suggest the possibility to developing selective TcSir2rp3 inhibitors that could lead to specific anti T. cruzi agents.146,147

Figure 4. Most relevant antitrypanosomal class I/II HDAC and sirtuin inhibitors: chemical 42 ACS Paragon Plus Environment

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structures and biological data. Color code: IC50s or EC50s for parasite growth inhibition in green; IC50s for antiproliferative effects in mammalian cells in brown; IC50s for HDAC/SIRT inhibition in orange.

Antileishmanial HDAC Inhibitors. Although the identity and biological characterization of the specific Leishmania HDAC isoforms which confer antileishmanial activity to HDACi is currently unknown, Leishmania parasites respond to HDACi, implying that specific HDACs could be crucial for their survival.45,95,122-124 Some hydroxamate-based class-I/II HDACi (24, 25b and 27 in Figure 5A) endowed with both antimalarial and antileishmanial activities have been described in the last years by the Oyelere’s group as above discussed (see the antimalarial section).122-124 In 2014, the same team in order to dissect the role of class I and II HDACs into the antileishmanial activity of HDACi, determined the effects of a series of 3-hydroxypyridine-2thiones (3HPTs), previously reported as hHDAC6/hHDAC8 inhibitors, on the viability of the L. donovani amastigote and promastigote forms.95 In addition to the pan-HDACi 12, they also included in the test for comparison purposes the hydroxamate-based HDACi 47 (tubastatin A) and PCI-34051 as reference compounds for hHDAC6 and hHDAC8 selective inhibition, respectively.95 In the tests, the 3-HPT-derived HDACi resulted cytotoxic to both extracellular and intracellular stages of L. donovani with IC50 values ranging for the most potent compounds (48 and 49 in Figure 5) from 0.1 to 6.5 µg/mL, depending on the specific parasite stage. Since the same antileishmanial activity against both forms of L. donovani was also obtained with the hHDAC6 selective inhibitor 47, but not with the hHDAC8 selective inhibitor PCI-34051 and the pan HDACi 12, the authors proposed that the observed antileishmanial activity was due to the



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inhibition of the HDAC6-like activity of L. donovani and that targeting HDAC6-like activity of this protozoan with isoform selective inhibitors could represent a more focused therapeutic strategy for leishmaniasis.95 Antileishmanial Class-III HDAC (Sirtuin) Inhibitors. To date, a few SIRTi have also been reported as endowed with antileishmanial activity (Figure 5B).45 In 2005, 7 was reported to exhibit a stage-specific antiproliferative activity against L. infantum parasites.148 It inhibited the axenic amastigotes multiplication (IC50 ∼ 30 µM) by promoting an apoptosis-like cell death associated with DNA fragmentation, but substantially did not affect the in vitro growth of parasite promastigotes (IC50 > 60 µM).148 Interestingly, parasites overexpressing LmSir2rp1 resulted less susceptible to the DNA fragmentation caused by 7 treatment.148 In the same year, 43 has been shown to inhibit L. major growth, but, since the over-expression of LmSir2rp1 resulted unable to protect the parasites from the in vitro inhibitory effects of this compound, alternative mechanisms of action cannot be excluded.149 In 2008, an ensemble of in silico and biochemical studies, in addition to reveal the existence of a few subtle but potentially crucial differences between the LmSir2rp1 and human SIRTs catalytic domains, tried to exploit them for identifying selective LmSir2rp1 inhibitors.96 Although the study did not identify a truly potent and selective lead compound, among the four compounds resulted effective in inhibiting the L. infantum axenic amastigote growth in vitro, there was at least one, the nicotinamide derivative 50 (Figure 5B), that likely works via LmSir2rp1 inhibition.96 Afterwards, enzymes tests showed that 7, 43 and suramin were all able to inhibit recombinant LiSir2rp1 (IC50 ~ 194, 40 and 7 µM, respectively); however, these inhibitory potencies were


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similar as those against the human enzyme SIRT1, so denoting a substantial lack of parasite selectivity.97 In the same paper, Tavares et al. reported the in vitro antileishmanial activity and a SAR study of 12 bisnaphthalimidopropyl (BNIP) derivatives that differed in the length of the central alkyl chains, with 2, 3, or 4 nitrogen atoms, linking the two naphthalimidopropyl moieties.97 All BNIP derivatives were able to inhibit LiSir2rp1 with IC50 values in the range of 5.7-54.7 µM, and resulted in some cases selective over hSIRT1. Both LiSir2rp1 inhibitory activity and selectivity of these analogues appeared to depend on the length and net charge of the linker group. BNIP diamine derivatives with 4-7 methylene units in the linker were actually less active and less selective than those containing 8-12 methylene units, while the introduction of additional positively charged amino functions in the linker did not improve neither potency nor selectivity. The most potent compound 51 (BNIP9 in Figure 5B), with a linker group of nine methylene units, had an IC50 value of 5.7 µM against LiSir2rp1, with a 17-fold selectivity over hSIRT1.97 Moreover, BNIP derivatives were able to inhibit the intracellular development of L. infantum amastigotes (IC50 values ~ 1-10 µM). Despite the lack of a straight correlation between the observed LiSir2rp1 inhibitory activity and the amastigote antiproliferative effects, some of these derivatives confirmed their promising antileishmanial effects also in vivo (BALB/c mice chronically infected with L. infantum).150



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Figure 5. Most relevant antileishmanial class-I/II HDAC and sirtuin inhibitors: chemical structures and biological data. Color code: IC50s for parasite growth inhibition in green; IC50s for HDAC/SIRT inhibition in orange.

Antitoxoplasma HDAC Inhibitors. Several class-I/II HDACi have shown the capability to inhibit the growth of Toxoplasma gondii with the cyclic tetrapeptides that result the most effective so far. In addition to 9 that is extremely potent (IC50 = 13-15 nM) but not selective over mammalian cells,101 the cyclic tetrapeptide 52 (FR235222, Figure 6), isolated from the fermentation broth of Acremonium species, is endowed with a powerful activity against T. gondii tachyzoites (IC50 ~10 nM) and a 13-fold selectivity over human foreskin fibroblasts (HFF).151 Active also against drug-sensitive and -resistant P. falciparum-infected erythrocytes, 52 caused T. gondii histone H4 hyper-acetylation through specific inhibition of TgHDAC3. Indeed, parasite lines with decreased sensitivity to 52 or 9, and showing single-point mutations within TgHDAC3 (T99A and T99I) have been isolated, thus providing genetic evidence that TgHDAC3 is the target of these compounds. Interestingly, the inhibitory activity of 52 appears to depend on a tworesidue (T99 and A98) insertion within the catalytic site of TgHDAC3, exclusively conserved in the Apicomplexa HDAC3 family and lacking in any other eukaryotic HDACs characterized so far.151 Moreover, TgHDAC3 displays a regulatory role of in the modulation of gene expression and stage-specific conversion in T. gondii, because treatment with 52 induced differentiation of the parasites from their replicative tachyzoite into the non-replicative bradyzoite stage.151 In a subsequent study, 52 was able to affect also in vitro-converted cysts and bradyzoites, and was active on ex vivo T.gondii cysts. In particular, 52 impaired the capability of the bradyzoites to convert to tachyzoites without damage the cyst wall, so that free bradyzoites isolated after the



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lysis of the cell wall were not able to proliferate in vitro. Finally, in vivo inoculation of cysts previously treated with 52 failed to infect mice. In the same study were reported two analogues (W363 and W399) of 52 that resulted highly active against tachyzoites and endowed with a better selectivity (SI = 48-62) over HFF cells, and for this reason more suitable for future in vivo studies, that anyway at the moment are still missing.152 Short-chain fatty acids such as 2, sodium butyrate, and 4-phenylbutyrate showed relatively poor activity against T. gondii tachyzoites (IC50 values in the single digit millimolar range) and low selectivity versus HS69 mammalian cells.112 In the past years, also the activity of some hydroxamate-based HDACi has been evaluated against T. gondii tachyzoite-stage parasites.113 Among these compounds, 1, 12 and 53 (scriptaid) (Figure 6) inhibited T. gondii tachyzoites with IC50 values of 41, 83 and 39 nM, respectively, whereas 13 was 2- to 5-fold less potent than the others (IC50 = 213 nM).113 Similarly to what seen with P. falciparum, 12 and 53 showed better parasite-selectivity than 1 versus mammalian cells.113 Interestingly, low concentrations of 1, 12 and 53 (1-50 nM) had a suppressive effect on T. gondii tachyzoite infectivity, joined to an apparent stimulation of proliferation and survival.113 Very little information is available on the activity of class-III HDACi against Toxoplasma species, with only one report indicating 43 as substantially inactive against T. gondii tachyzoites (IC50 = 50 mM).113


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Figure 6. Most relevant antitoxoplasmal class-I/II/IV HDAC inhibitors: chemical structures and biological data (IC50s for parasite growth inhibition in green).

DISCUSSION AND CONCLUSIONS Histone deacetylase inhibitors are a promising new class of potential anti-parasitic agents, with several of them displaying significant in vitro, ex vivo and, in a few cases, in vivo activity against specific parasites. Despite the growing interest in their anti-parasitic effects, many critical points still need to be addressed before moving HDACi/SIRTi towards clinical trials as antiparasitic compounds.45,67 Indeed, the clinical treatment of parasitic infections is quite different from that of other diseases, such as cancer, since most morbidity and mortality associated with parasitic diseases occur in resource-constrained regions of the world, with children, pregnant women and immunecompromised people being at highest risk. Moreover, the co-infection of people in these regions 49 ACS Paragon Plus Environment


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with different infective agents (e.g. HIV) is quite common and needs to be taken into account due to potential drug-drug interactions. Consequently, the main features that a HDACi, as any other new anti-parasitic drug, should have before being considered for clinical use include: (1) high potency and selectivity in vivo for parasite over normal host cells; (2) activity against organisms resistant to currently employed drug(s); (3) an elevated degree of safety for use also in children and pregnant women; (4) low cost, to potentially treat hundreds of millions of deprived people; (5) oral bioavailability and effective pharmacokinetic profiles that allow single daily doses; and, (6) hopefully, pharmacokinetics compatible with those of potential partner drugs, to prevent or limit the development of drug resistance. To progress class I/II HDACi towards clinical trials as potential anti-parasitic drugs, a high level of potency and selectivity for parasites versus host cells is essential. From a medicinal chemistry point of view, there is opportunity to optimize the chemical structures of the currently available class I/II HDACi for therapeutic use against parasites by modifying each one of the four portions (CAP, CU, linker, and ZBG) of the general pharmacophoric model for class I/II HDAC inhibition. The zinc-coordinating residues in HDACs and the tubular cavity between the zinc and the surface of HDAC enzymes are relatively well-conserved between human and parasite enzymes,72,73 and the linker moiety of HDACi is not able to be varied a great deal, being restricted by the size and shape of the active site tunnel. Nevertheless, this region can still be successfully exploited to develop selective parasite HDACi, as exemplified by the selective smHDAC8 inhibitors recently reported by Heimburg et al. that show an (hetero)aromatic ring as linker group.62 Moreover, the length of the linker group has been found to be a major structural determinant for the antimalarial and antileishmanial activities of two classes of non-peptide macrocyclic HDACi, that displayed the maximal antileishmanial activity in analogues with


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linkers comprising eight or nine methylene units,123,124 whereas the best antimalarial potency was observed for analogues with five or six methylene unit spacers.123,124 The CAP group involved in the interaction with the rim at the entrance of the catalytic tunnel has more scope for variation and exploitation, and it is to be expected that most attempts to improve the activity and/or selectivity of the future anti-parasitic HDACi will focus on this. Substitutions on the connection unit might also be introduced to increase potency and/or selectivity, as exemplified by the introduction of a γ-lactam carboxamide in α to the anilide CU of the 12-like HDACi reported by Giannini et al..129 While the zinc-binding hydroxamate, present in some of the most effective HDACi, confers affinity and potency for HDAC enzymes, it is not an ideal ZBG. Indeed, the hydroxamate moiety is a potential site of metabolic instability,116,153 and it is not selective in coordinating the zinc ion in just HDACs, but can bind to other zinc-containing proteins or other metal ions in vivo leading to potential toxicity issues, such as those observed for hydroxamate containing matrix metalloprotease inhibitors.154 Although variations of the ZBG in most cases were not successful, Ontoria et al. were recently able to replace the hydroxamate group with a secondary amide moiety and reported selective P. falciparum HDACi (37-39 in Table 2), suggesting a way to develop new generation HDACi with improved selectivity and metabolic stability.133 In order to limit the potential for toxicity, future studies should also focus on discovery of compounds with selectivity for parasitic HDACs/SIRTs, and preferably with some isoform specificity. In this context it is promising that recently some isoform-selective HDACi have started to be developed to target hHDACs,155 and very recently have been reported the first examples of parasite selective HDACi (e.g. smHDAC8 selective inhibitors).62 The crystal structures as well as docking studies of smHDAC8 in complex with different HDACi of the 3-



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amidobenzohydroxamate series have revealed the existence of a schistosome-specific clamp formed by the two residues Lys20 and His292, that, distinctively interacting by hydrogen bonds with the CONH amide group of these inhibitors, suggests the molecular basis for improving activity versus smHDAC8, and selectivity at least over hHDAC1 and hHDAC6. Nevertheless, the search for selective parasite HDACi remains challenging because it requires the cloning and expression of each recombinant parasite histone deacetylase isoform, joined to information on their molecular function(s). To gain insights for selective HDACi design, three-dimensional (3D) models have been created for parasitic HDACs along with their human homologs, via various homology modeling approaches.62,63,72,96,143,146,147 In silico homology modeling studies of PfHDAC1 have shown that the predicted active site tunnel of PfHDAC1 is highly homologous to that of hHDACs, but displays differences at its entrance that could be exploited for the development of selective inhibitors.72 Recently, Melesina et al. reported homology models of relevant parasitic (Trypanosoma, Leishmania and Plasmodium species) HDACs and compared them to human and S. mansoni HDACs, where docking and molecular dynamics studies indicated the presence of several differences in the topology of the binding cavity as well as the amino acid residues in the conserved parts of the catalytic pocket that could guide the structurebased design of parasites selective HDACi.156 We are currently still at the stage of infancy in the functional characterization of most sirtuins from human parasites. Even though no 3-D structure has been experimentally solved for any of the parasitic SIRT enzymes yet, several 3-D structural models have been proposed for TbSir2rp1, TcSir2rp1, LmSir2rp1, and LiSir2rp1, along with the hSIRT1 (or SIRT2) via a homology modeling approach with the hSIRT2 crystal structure as the template.96,143,147 All these structural studies focused on the NAD+ binding pocket of the SIRT active site, and revealed subtle but significant differences in particular at the 43 binding site


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between parasite and human SIRTs that may be exploited to develop inhibitors selective towards parasitic sirtuins.96,146,147,157,158 Moreover, the aforementioned selective LiSir2rp1 inhibitor 51 was also shown to interact differently with the NAD+ binding site of the LiSir2rp1 when compared to that of hSIRT1, so confirming that the NAD+ binding pocket is exploitable to develop inhibitors selective for parasitic SIRTs over human enzymes.97 Since the activity against drug-resistant strains, as for any new anti-parasitic agent, is essential, the selection of a lead HDACi for a potential use in vivo must be coupled with early detection and avoidance of cross resistance through screening multiple parasite strains with different sensitivity to currently approved drugs. Encouragingly, several antimalarial HDACi have shown similar in vitro activities against P. falciparum strains that are sensitive or resistant to classic drugs such as chloroquine and mefloquine.73,110,114,116,118,120,125,128 Interestingly, the study that showed 12 and two 2-ASA HDACi to have nanomolar potency against multidrug resistant clinical isolates of P. falciparum and P. vivax, also revealed that the susceptibility to 12 was related to mefloquine in P. falciparum but not P. vivax. Susceptibility to the 2-ASA compounds was related to the same classical antimalarial drug in P. vivax but not P. falciparum, suggesting the possibility of dissimilarities in the susceptibility profiles of different Plasmodium species.118 A useful approach to assess the potential for resistance to new anti-parasitic drugs is the in vitro generation, either by selection or mutagenesis, of resistant laboratory strains. While such resistant or tolerant lines have not been reported for antimalarial HDACi yet, as discussed above, T. gondii parasites resistant to the HDACi 52 have been generated.151 T. gondii lines with mutations to amino-acid residues in the TgHDAC3 gene displayed reduced sensitivity to 52. Homology modeling of PfHDAC1 with bound HDACi has found that amino acid T76 is predicted to be one of the key binding site residues in PfHDAC1.72,73 Further studies of the role



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of these amino acids on HDAC inhibitor activity, as well as the in vitro selection of HDACi tolerant/resistant parasite lines, are needed to provide critical insights into the molecular mechanisms of HDACi resistance. In mammalian cells, mechanisms of HDACi resistance have recently started to be characterized.159,160 In particular, resistance to 11 in leukemia has been linked to both P-glycoprotein-dependent and independent mechanisms, and tolerance to 2 and 12 in adenocarcinoma cells seems to be independent by multi-drug resistance (MDR) expression.159,160 Since these two drug resistance mechanisms have been also shown in various parasites, it is possible that they could play a role also for HDACi resistance in parasites.45,67 To date, limited studies have been carried out to identify possible partner drugs for HDACi.45,67 Nevertheless, the experiments, in a few cases, have shown promising additive effects, as in the combination between chloroquine and 22 in P. berghei infected mice,116 and in the in vitro combination between 28 and lopinavir.125 In some cases, there are preliminary investigational proofs that suggest the potential utility of HDACi in the combination therapy of parasitic diseases.45,67, 157 For example, the experimental evidence that the Trypanosoma brucei sirtuin TbSir2rp1 is important for the parasite DNA repair and survival in response to genotoxic insults has suggested that specific TbSir2rp1 inhibitors could be used in co-treatment with conventional

DNA-targeting

chemotherapeutic

agents

to

improve

their

anti-parasitic

efficacy.84,85,157 Further studies, including extensive in vivo interaction studies, are however presently needed to evaluate the potential utility of HDACi and SIRTi in combination therapies. In this regard, the potential role of HDACi and SIRTi as immune modulators should be also taken into account. Different HDAC and SIRT enzymes have been shown to play important roles in the parasite antigenic variation and evasion from the host immune surveillance and on innate and adaptive immunity (see above in the text), and HDACi/SIRTi can potently affect many


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cellular components (such as T-cells,

dendritic cells, macrophages, etc.) of the immune

system.45-47,50,52,54 In particular, since T cells, dendritic cells and macrophages play crucial roles in immunity to human-infecting parasites, the effecs of in vivo treatment with HDACi and SIRTi on host immune responses and direct anti-parasitic effects should be taken into account together. The eventual use of Zn2+- and NAD+-dependent HDACi as a coadjutant therapy against the worse forms of parasitic diseases161 could open new opportunities, and should also be examined in depth.45,67,157

AUTHOR INFORMATION Corresponding Authors W.S.: phone, +49 345 5525040; fax, +49 345 5527355; E-mail, [email protected] D.R.: phone, +39 06 49913891; fax, +39 06 49693268; E-mail, [email protected] A.M.: phone, +39 06 49913392; fax, +39 06 49693268; E-mail, [email protected]. Author Contribution The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. Biographies Gebremedhin Solomon Hailu earned both his B. Pharm and M.Sc. from Addis Ababa University, Ethiopia. He worked at Mekelle University, Ethiopia. At the end of 2014, he started his Ph.D. at the Department of Chemistry and Pharmaceutical Technology, Sapienza University



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of Rome, in the research group of Prof. Antonello Mai. His research focuses on the development of small molecule epigenetic modulators targeting parasitic diseases. Dina Robaa studied Pharmacy at the University of Alexandria in Egypt. She obtained her Ph.D. in Pharmaceutical Chemistry at the University of Jena in the group of Jochen Lehmann. Since 2011, she has been working as a postdoctoral fellow in the research group of Wolfgang Sippl. Her research focuses on structure-based drug design of several epigenetic modulators. Mariantonietta Forgione studied Pharmacy at “Sapienza” University of Rome and got her degree in 2013. In the same year she started her Ph.D. in Pharmaceutical Sciences in the research group of Prof. Antonello Mai. Her research activity is focused on the development of small molecule epigenetic modulators as histone deacetylases, methyltransferases and demethylases, and inhibitors of the enzyme GST (gluthatione-S-transferase). Wolfgang Sippl is Professor for Medicinal Chemistry and Director of the Institute of Pharmacy at the Martin-Luther-University of Halle-Wittenberg (Germany). He obtained a Ph.D. in Pharmaceutical Chemistry at the University of Düsseldorf in the group of Hans-Dieter Höltje and was a postdoctoral fellow at the Université Louis-Pasteur in Strasbourg (France) where he worked with Camille G. Wermuth. Since 2003, he has been Full Professor at the Institute of Pharmacy in Halle. His main interests are focused on computational chemistry and structurebased drug design of novel epigenetic modulators for the therapy of cancer and parasitic diseases. Dante Rotili graduated in Medicinal Chemistry at the University of Rome ‘‘La Sapienza’’ (Italy) in 2003. He received his Ph.D. in Pharmaceutical Sciences at the same University in 2007. In 2009/2010 he was research associate at the Department of Chemistry of the University of Oxford, where he worked in collaboration with Prof. C. Schofield in the development of chemoproteomic probes for the characterization of 2-oxoglutarate-dependent enzymes. Since


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2011 he has been a tenured Assistant Professor of Medicinal Chemistry at the University of Rome “La Sapienza”. Since 2014 he has got the Italian National Habilitation to Associate Professor of Medicinal Chemistry. His research activity has been focusing mainly on the development of modulators of epigenetic enzymes with potential applications in cancer, neurodegenerative, metabolic, and infectious diseases. Antonello Mai graduated in Pharmacy at the University of Rome ‘‘La Sapienza,’’ Italy, in 1984. He received his Ph.D. in 1992 in Pharmaceutical Sciences, with a thesis entitled ‘‘Researches on new polycyclic benzodiazepines active on central nervous system,’’ advisor Prof. M. Artico. In 1998, he was appointed Associate Professor of Medicinal Chemistry at the same University. In 2011, Prof. Mai was appointed Full Professor of Medicinal Chemistry at the Faculty of Pharmacy and Medicine, Sapienza University of Rome. He published more than 200 papers on peer-review high-impact factor journals. His research interests include the synthesis and biological evaluation of new bio-active compounds, in particular small molecule modulators of epigenetic targets. In addition, he is working in the field of antibacterial/antimycobacterial , antiviral and CNS agents.

ACKNOWLEDGMENTS This work was supported by EU: COST-Action: EPICHEMBIO (CM1406); FP7: BLUEPRINT (contract n° 282510), A-PARADDISE (contract n° 602080); PRIN 2012 (Prot. 2012CTAYSY), PRIN 2016 (Prot. 20152TE5PK), AIRC Fondazione Cariplo TRIDEO Id. 17515.

ABBREVIATIONS USED Aoda, 2-amino-8-oxodecanoic acid; APHAs, aroyl-pyrrolylhydroxyamides; 2-ASA, 2aminosuberic acid; BNIP, bisnaphthalimidopropyl; CASP7, apoptosis-related cysteine peptidase; CQ, chloroquine; CU, connection unit; HAT, human African trypanosomiasis; HDACi, histone 57 ACS Paragon Plus Environment


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deacetylase inhibitors; 3HPT, 3-hydroxypyridine-2-thione; hSIRT, human sirtuins; LiSir2rp1, Leishmania infatum Sir2-related protein 1; LmSir2rp1, Leishmania major infantum Sir2-related protein 1; NFF, neonatal foreskin fibroblast; PfEMP1, Plasmodium falciparum erythrocyte membrane protein 1; PfHDAC, Plasmodium falciparum histone deacetylase; PfSir2A/B, Plasmodium falciparum sirtuins; SBHA, suberic bishydroxamate; SI, Selectivity Index; SIRTi, sirtuin inhibitors; SmHDAC, Schistosoma mansoni histone deacetylase; rPfHDAC1, recombinant Plasmodium falciparum histone deacetylase; TbDAC, Trypanosoma brucei histone deacetylase; TbSir2rp1-3, Trypanosoma brucei Sir2-related protein 1/3; TcSir2rp1/3, Trypanosoma cruzi Sir2-related protein 1/3; TgHDAC, Toxoplasma gondii histone deacetylase; TgSir2A/B, Toxoplasma gondii sirtuins; TSA, trichostatin A; VPA, valproic acid; VSGs, variant surface glycoproteins; WHO, World Health Organization; ZBG, zinc-binding group.

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“Table of Contents Graphic” S. mansoni T. gondii

P. falciparum HDACi SIRTi

L. donovani L. infantum

T. brucei T. cruzi


Lysine Deacetylase Inhibitors in Parasites: Past, Present, and Future

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