Histone deacetylase inhibitor AR-42 and achiral analogues kill malaria parasites in vitro and in mice.

Malaria is caused by infection with Plasmodium parasites and results in significant health and economic impacts. Malaria eradication is hampered by parasite resistance to current drugs and the lack of a widely effective vaccine. Compounds that target epigenetic regulatory proteins, such as histone deacetylases (HDACs), may lead to new therapeutic agents with a different mechanism of action, thereby avoiding resistance mechanisms to current antimalarial drugs. The anticancer HDAC inhibitor AR-42, as its racemate (rac-AR-42), and 36 analogues were investigated for in vitro activity against P. falciparum. Rac-AR-42 and selected compounds were assessed for cytotoxicity against human cells, histone hyperacetylation, human HDAC1 inhibition and oral activity in a murine malaria model. Rac-AR-42 was tested for ex vivo asexual and in vitro exoerythrocytic stage activity against P. berghei murine malaria parasites. Rac-AR-42 and 13 achiral analogues were potent inhibitors of asexual intraerythrocytic stage P. falciparum 3D7 growth in vitro (IC50 5-50 nM), with four of these compounds having >50-fold selectivity for P. falciparum versus human cells (selectivity index 56-118). Rac-AR-42 induced in situ hyperacetylation of P. falciparum histone H4, consistent with PfHDAC(s) inhibition. Furthermore, rac-AR-42 potently inhibited P. berghei infected erythrocyte growth ex vivo (IC50 40 nM) and P. berghei exoerythrocytic forms in hepatocytes (IC50 1 nM). Oral administration of rac-AR-42 and two achiral analogues inhibited P. berghei growth in mice, with rac-AR-42 (50 mg/kg/day single dose for four days) curing all infections. These findings demonstrate curative properties for HDAC inhibitors in the oral treatment of experimental mouse malaria.

Drug resistance in Giardia: Mechanisms and alternative treatments for Giardiasis.

The use of chemotherapeutic drugs is the main resource against clinical giardiasis due to the lack of approved vaccines. Resistance of G. duodenalis to the most used drugs to treat giardiasis, metronidazole and albendazole, is a clinical issue of growing concern and yet unknown impact, respectively. In the search of new drugs, the completion of the Giardia genome project and the use of biochemical, molecular and bioinformatics tools allowed the identification of ligands/inhibitors for about one tenth of ≈150 potential drug targets in this parasite. Further, the synthesis of second generation nitroimidazoles and benzimidazoles along with high-throughput technologies have allowed not only to define overall mechanisms of resistance to metronidazole but to screen libraries of repurposed drugs and new pharmacophores, thereby increasing the known arsenal of anti-giardial compounds to some hundreds, with most demonstrating activity against metronidazole or albendazole-resistant Giardia. In particular, cysteine-modifying agents which include omeprazole, disulfiram, allicin and auranofin outstand due to their pleiotropic activity based on the extensive repertoire of thiol-containing proteins and the microaerophilic metabolism of this parasite. Other promising agents derived from higher organisms including phytochemicals, lactoferrin and propolis as well as probiotic bacteria/fungi have also demonstrated significant potential for therapeutic and prophylactic purposes in giardiasis. In this context the present chapter offers a comprehensive review of the current knowledge, including commonly prescribed drugs, causes of therapeutic failures, drug resistance mechanisms, strategies for the discovery of new agents and alternative drug therapies.

Cyclization-blocked proguanil as a strategy to improve the antimalarial activity of atovaquone.

Atovaquone-proguanil (Malarone®) is used for malaria prophylaxis and treatment. While the cytochrome bc1-inhibitor atovaquone has potent activity, proguanil's action is attributed to its cyclization-metabolite, cycloguanil. Evidence suggests that proguanil has limited intrinsic activity, associated with mitochondrial-function. Here we demonstrate that proguanil, and cyclization-blocked analogue tBuPG, have potent, but slow-acting, in vitro anti-plasmodial activity. Activity is folate-metabolism and isoprenoid biosynthesis-independent. In yeast dihydroorotate dehydrogenase-expressing parasites, proguanil and tBuPG slow-action remains, while bc1-inhibitor activity switches from comparatively fast to slow-acting. Like proguanil, tBuPG has activity against P. berghei liver-stage parasites. Both analogues act synergistically with bc1-inhibitors against blood-stages in vitro, however cycloguanil antagonizes activity. Together, these data suggest that proguanil is a potent slow-acting anti-plasmodial agent, that bc1 is essential to parasite survival independent of dihydroorotate dehydrogenase-activity, that Malarone® is a triple-drug combination that includes antagonistic partners and that a cyclization-blocked proguanil may be a superior combination partner for bc1-inhibitors in vivo.

Structure-Activity and Structure-Toxicity Relationships of Peptoid-Based Histone Deacetylase Inhibitors with Dual-Stage Antiplasmodial Activity.

Novel malaria intervention strategies are of great importance, given the development of drug resistance in malaria-endemic countries. In this regard, histone deacetylases (HDACs) have emerged as new and promising malaria drug targets. In this work, we present the design, synthesis, and biological evaluation of 20 novel HDAC inhibitors with antiplasmodial activity. Based on a previously discovered peptoid-based hit compound, we modified all regions of the peptoid scaffold by using a one-pot multicomponent pathway and submonomer routes to gain a deeper understanding of the structure-activity and structure-toxicity relationships. Most compounds displayed potent activity against asexual blood-stage P. falciparum parasites, with IC50 values in the range of 0.0052-0.25 µm and promising selectivity over mammalian cells (SIPf3D7/HepG2 : 170-1483). In addition, several compounds showed encouraging sub-micromolar activity against P. berghei exo-erythrocytic forms (PbEEF). Our study led to the discovery of the hit compound N-(2-(benzylamino)-2-oxoethyl)-N-(4-(hydroxycarbamoyl)benzyl)-4-isopropylbenzamide (2 h) as a potent and parasite-specific dual-stage antiplasmodial HDAC inhibitor (IC50 Pf3D7=0.0052 µm, IC50 PbEEF=0.016 µm).

Effect of clinically approved HDAC inhibitors on Plasmodium, Leishmania and Schistosoma parasite growth.

Malaria, schistosomiasis and leishmaniases are among the most prevalent tropical parasitic diseases and each requires new innovative treatments. Targeting essential parasite pathways, such as those that regulate gene expression and cell cycle progression, is a key strategy for discovering new drug leads. In this study, four clinically approved anti-cancer drugs (Vorinostat, Belinostat, Panobinostat and Romidepsin) that target histone/lysine deacetylase enzymes were examined for in vitro activity against Plasmodium knowlesi, Schistosoma mansoni, Leishmania amazonensis and L. donovani parasites and two for in vivo activity in a mouse malaria model. All four compounds were potent inhibitors of P. knowlesi malaria parasites (IC50 9-370 nM), with belinostat, panobinostat and vorinostat having 8-45 fold selectivity for the parasite over human neonatal foreskin fibroblast (NFF) or human embryonic kidney (HEK 293) cells, while romidepsin was not selective. Each of the HDAC inhibitor drugs caused hyperacetylation of P. knowlesi histone H4. None of the drugs was active against Leishmania amastigote or promastigote parasites (IC50 > 20 µM) or S. mansoni schistosomula (IC50 > 10 µM), however romidepsin inhibited S. mansoni adult worm parings and egg production (IC50 ∼10 µM). Modest in vivo activity was observed in P. berghei infected mice dosed orally with vorinostat or panobinostat (25 mg/kg twice daily for four days), with a significant reduction in parasitemia observed on days 4-7 and 4-10 after infection (P < 0.05), respectively.

Profiling the anti-protozoal activity of anti-cancer HDAC inhibitors against Plasmodium and Trypanosoma parasites.

Histone deacetylase (HDAC) enzymes work together with histone acetyltransferases (HATs) to reversibly acetylate both histone and non-histone proteins. As a result, these enzymes are involved in regulating chromatin structure and gene expression as well as other important cellular processes. HDACs are validated drug targets for some types of cancer, with four HDAC inhibitors clinically approved. However, they are also showing promise as novel drug targets for other indications, including malaria and other parasitic diseases. In this study the in vitro activity of four anti-cancer HDAC inhibitors was examined against parasites that cause malaria and trypanosomiasis. Three of these inhibitors, suberoylanilide hydroxamic acid (SAHA; vorinostat(®)), romidepsin (Istodax(®)) and belinostat (Beleodaq(®)), are clinically approved for the treatment of T-cell lymphoma, while the fourth, panobinostat, has recently been approved for combination therapy use in certain patients with multiple myeloma. All HDAC inhibitors were found to inhibit the growth of asexual-stage Plasmodium falciparum malaria parasites in the nanomolar range (IC50 10-200 nM), while only romidepsin was active at sub-µM concentrations against bloodstream form Trypanosoma brucei brucei parasites (IC50 35 nM). The compounds were found to have some selectivity for malaria parasites compared with mammalian cells, but were not selective for trypanosome parasites versus mammalian cells. All compounds caused hyperacetylation of histone and non-histone proteins in P. falciparum asexual stage parasites and inhibited deacetylase activity in P. falciparum nuclear extracts in addition to recombinant PfHDAC1 activity. P. falciparum histone hyperacetylation data indicate that HDAC inhibitors may differentially affect the acetylation profiles of histone H3 and H4.

Lysine acetylation in sexual stage malaria parasites is a target for antimalarial small molecules.

Therapies to prevent transmission of malaria parasites to the mosquito vector are a vital part of the global malaria elimination agenda. Primaquine is currently the only drug with such activity; however, its use is limited by side effects. The development of transmission-blocking strategies requires an understanding of sexual stage malaria parasite (gametocyte) biology and the identification of new drug leads. Lysine acetylation is an important posttranslational modification involved in regulating eukaryotic gene expression and other essential processes. Interfering with this process with histone deacetylase (HDAC) inhibitors is a validated strategy for cancer and other diseases, including asexual stage malaria parasites. Here we confirm the expression of at least one HDAC protein in Plasmodium falciparum gametocytes and show that histone and nonhistone protein acetylation occurs in this life cycle stage. The activity of the canonical HDAC inhibitors trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA; Vorinostat) and a panel of novel HDAC inhibitors on early/late-stage gametocytes and on gamete formation was examined. Several compounds displayed early/late-stage gametocytocidal activity, with TSA being the most potent (50% inhibitory concentration, 70 to 90 nM). In contrast, no inhibitory activity was observed in P. falciparum gametocyte exflagellation experiments. Gametocytocidal HDAC inhibitors caused hyperacetylation of gametocyte histones, consistent with a mode of action targeting HDAC activity. Our data identify HDAC inhibitors as being among a limited number of compounds that target both asexual and sexual stage malaria parasites, making them a potential new starting point for gametocytocidal drug leads and valuable tools for dissecting gametocyte biology.

Identification of Potent and Selective Inhibitors of the Plasmodium falciparum M18 Aspartyl Aminopeptidase (PfM18AAP) of Human Malaria via High-Throughput Screening.

The target of this study, the PfM18 aspartyl aminopeptidase (PfM18AAP), is the only AAP present in the genome of the malaria parasite Plasmodium falciparum. PfM18AAP is a metallo-exopeptidase that exclusively cleaves N-terminal acidic amino acids glutamate and aspartate. It is expressed in parasite cytoplasm and may function in concert with other aminopeptidases in protein degradation, of, for example, hemoglobin. Previous antisense knockdown experiments identified a lethal phenotype associated with PfM18AAP, suggesting that it is a valid target for new antimalaria therapies. To identify inhibitors of PfM18AAP function, a fluorescence enzymatic assay was developed using recombinant PfM18AAP enzyme and a fluorogenic peptide substrate (H-Glu-NHMec). This was screened against the Molecular Libraries Probe Production Centers Network collection of ~292,000 compounds (the Molecular Libraries Small Molecule Repository). A cathepsin L1 (CTSL1) enzyme-based assay was developed and used as a counter screen to identify compounds with nonspecific activity. Enzymology and phenotypic assays were used to determine mechanism of action and efficacy of selective and potent compounds identified from high-throughput screening. Two structurally related compounds, CID 6852389 and CID 23724194, yielded micromolar potency and were inactive in CTSL1 titration experiments (IC50>59.6 µM). As measured by the K(i) assay, both compounds demonstrated micromolar noncompetitive inhibition in the PfM18AAP enzyme assay. Both CID 6852389 and CID 23724194 demonstrated potency in malaria growth assays (IC504 µM and 1.3 µM, respectively).

Antimalarial activity of compounds comprising a primary benzene sulfonamide fragment.

Despite the urgent need for effective antimalarial drugs with novel modes of action no new chemical class of antimalarial drug has been approved for use since 1996. To address this, we have used a rational approach to investigate compounds comprising the primary benzene sulfonamide fragment as a potential new antimalarial chemotype. We report the in vitro activity against Plasmodium falciparum drug sensitive (3D7) and resistant (Dd2) parasites for a panel of fourteen primary benzene sulfonamide compounds. Our findings provide a platform to support the further evaluation of primary benzene sulfonamides as a new antimalarial chemotype, including the identification of the target of these compounds in the parasite.

Antimalarial activity of the anticancer histone deacetylase inhibitor SB939.

Histone deacetylase (HDAC) enzymes posttranslationally modify lysines on histone and nonhistone proteins and play crucial roles in epigenetic regulation and other important cellular processes. HDAC inhibitors (e.g., suberoylanilide hydroxamic acid [SAHA; also known as vorinostat]) are used clinically to treat some cancers and are under investigation for use against many other diseases. Development of new HDAC inhibitors for noncancer indications has the potential to be accelerated by piggybacking onto cancer studies, as several HDAC inhibitors have undergone or are undergoing clinical trials. One such compound, SB939, is a new orally active hydroxamate-based HDAC inhibitor with an improved pharmacokinetic profile compared to that of SAHA. In this study, the in vitro and in vivo antiplasmodial activities of SB939 were investigated. SB939 was found to be a potent inhibitor of the growth of Plasmodium falciparum asexual-stage parasites in vitro (50% inhibitory concentration [IC(50)], 100 to 200 nM), causing hyperacetylation of parasite histone and nonhistone proteins. In combination with the aspartic protease inhibitor lopinavir, SB939 displayed additive activity. SB939 also potently inhibited the in vitro growth of exoerythrocytic-stage Plasmodium parasites in liver cells (IC(50), ~150 nM), suggesting that inhibitor targeting to multiple malaria parasite life cycle stages may be possible. In an experimental in vivo murine model of cerebral malaria, orally administered SB939 significantly inhibited P. berghei ANKA parasite growth, preventing development of cerebral malaria-like symptoms. These results identify SB939 as a potent new antimalarial HDAC inhibitor and underscore the potential of investigating next-generation anticancer HDAC inhibitors as prospective new drug leads for treatment of malaria.

Inhibition of hypoxanthine-guanine phosphoribosyltransferase by acyclic nucleoside phosphonates: a new class of antimalarial therapeutics.

The purine salvage enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is essential for purine nucleotide and hence nucleic acid synthesis in the malaria parasite, Plasmodium falciparum. Acyclic nucleoside phosphonates (ANPs) are analogues of the nucleotide product of the reaction, comprising a purine base joined by a linker to a phosphonate moiety. K(i) values for 19 ANPs were determined for Pf HGXPRT and the corresponding human enzyme, HGPRT. Values for Pf HGXPRT were as low as 100 nM, with selectivity for the parasite enzyme of up to 58. Structures of human HGPRT in complex with three ANPs are reported. On binding, a large mobile loop in the free enzyme moves to partly cover the active site. For three ANPs, the IC(50) values for Pf grown in cell culture were 1, 14, and 46 microM, while the cytotoxic concentration for the first compound was 489 microM. These results provide a basis for the design of potent and selective ANP inhibitors of Pf HGXPRT as antimalarial drug leads.

The M18 aspartyl aminopeptidase of the human malaria parasite Plasmodium falciparum.

A member of the M18 family of aspartyl aminopeptidases is expressed by all intra-erythrocytic stages of the human malaria parasite Plasmodium falciparum (PfM18AAP), with highest expression levels in rings. Functionally active recombinant enzyme, rPfM18AAP, and native enzyme in cytosolic extracts of malaria parasites are 560-kDa octomers that exhibit optimal activity at neutral pH and require the presence of metal ions to maintain enzymatic activity and stability. Like the human aspartyl aminopeptidase, the exopeptidase activity of PfM18AAP is exclusive to N-terminal acidic amino acids, glutamate and aspartate, making this enzyme of particular interest and suggesting that it may function alongside the malaria cytosolic neutral aminopeptidases in the release of amino acids from host hemoglobin-derived peptides. Whereas immunocytochemical studies using transgenic P. falciparum parasites show that PfM18AAP is expressed in the cytosol, immunoblotting experiments revealed that the enzyme is also trafficked out of the parasite into the surrounding parasitophorous vacuole. Antisense-mediated knockdown of PfM18AAP results in a lethal phenotype as a result of significant intracellular damage and validates this enzyme as a target at which novel antimalarial drugs could be directed. Novel phosphinic derivatives of aspartate and glutamate showed modest inhibition of rPfM18AAP but did not inhibit malaria growth in culture. However, we were able to draw valuable observations concerning the structure-activity relationship of these inhibitors that can be employed in future inhibitor optimization studies.

CLAG 9 is located in the rhoptries of Plasmodium falciparum.

Clag 9, a gene located on chromosome 9 of Plasmodium falciparum has previously been associated with the cytoadherence of parasitized erythrocytes to CD36. This gene is part of a multi-gene family found in all Plasmodium species studied to date. Using data from the Malaria Genome Sequencing Project, peptides specific for clag 9 were designed, synthesized and used to immunize mice. This antisera was used in Western blotting and immunofluorescence experiments to determine the cellular localization of CLAG 9 in the parasitized erythrocyte. Co-localization using immunofluorescence of wildtype and knockout parasites unequivocally shows that CLAG 9 is localized to the rhoptry organelles of P. falciparum.