Activity of alkoxyamide-based histone deacetylase inhibitors against Plasmodium falciparum malaria parasites.

There are more than 240 million cases of malaria and 600,000 associated deaths each year, most due to infection with Plasmodium falciparum parasites. While malaria treatment options exist, new drugs with novel modes of action are needed to address malaria parasite drug resistance. Protein lysine deacetylases (termed HDACs) are important epigenetic regulatory enzymes and prospective therapeutic targets for malaria. Here we report the antiplasmodial activity of a panel of 17 hydroxamate zinc binding group HDAC inhibitors with alkoxyamide linkers and different cap groups. The two most potent compounds (4a and 4b) were found to inhibit asexual P. falciparum growth with 50% inhibition concentrations (IC50's) of 0.07 µM and 0.09 µM, respectively, and demonstrated >200-fold more selectivity for P. falciparum parasites versus human neonatal foreskin fibroblasts (NFF). In situ hyperacetylation studies demonstrated that 4a, 4b and analogs caused P. falciparum histone H4 hyperacetylation, suggesting HDAC inhibition, with structure activity relationships providing information relevant to the design of new Plasmodium-specific aliphatic chain hydroxamate HDAC inhibitors.

Thymidine Kinase-Independent Click Chemistry DNADetect Probes for DNA Proliferation Assessment in Malaria Parasites.

Metabolic chemical probes are small-molecule reagents that utilize naturally occurring biosynthetic enzymes for in situ incorporation into biomolecules of interest. These reagents can be used to label, detect, and track important biological processes within living cells including protein synthesis, protein glycosylation, and nucleic acid proliferation. A limitation of current chemical probes, which have largely focused on mammalian cells, is that they often cannot be applied to other organisms due to metabolic differences. For example, the thymidine derivative 5-ethynyl-2'-deoxyuridine (EdU) is a gold standard metabolic chemical probe for assessing DNA proliferation in mammalian cells; however, it is unsuitable for the study of malaria parasites due to Plasmodium species lacking the thymidine kinase enzyme that is essential for metabolism of EdU. Herein, we report the design and synthesis of new thymidine-based probes that sidestep the requirement for a thymidine kinase enzyme in Plasmodium. Two of these DNADetect probes exhibit robust labeling of replicating asexual intraerythrocytic Plasmodium falciparum parasites, as determined by flow cytometry and fluorescence microscopy using copper-catalyzed azide-alkyne cycloaddition to a fluorescent azide. The DNADetect chemical probes are synthetically accessible and thus can be made widely available to researchers as tools to further understand the biology of different Plasmodium species, including laboratory lines and clinical isolates.

Isolation and In Vitro and In Vivo Activity of Secondary Metabolites from Clerodendrum polycephalum Baker against Plasmodium Malaria Parasites.

Chemical investigation of the antimalarial medicinal plant Clerodendrum polycephalum led to the isolation of five new diterpenoids, including ajugarins VII-X (1-4) and teuvincenone K (5), along with four known compounds, namely, 12,16-epoxy-6,11,14,17-tetrahydroxy-17(15 → 16)-abeo-5,8,11,13,15-abietapentaen-7-one (6), methyl pheophorbide A (7), loliolide (8), and acacetin (9). The chemical structures of the new compounds were elucidated using NMR spectroscopy, mass spectrometry, circular dichroism, as well as density functional theory calculations. All compounds were evaluated for in vitro activity against Plasmodium falciparum 3D7 malaria parasites with methyl pheophorbide A (7) showing the strongest activity (IC50 4.49 µM). Subsequent in vivo testing in a Plasmodium berghei chemosuppression model showed that compound 7 significantly attenuated peripheral blood parasitemia, leading to 79% and 87% chemosuppression following oral doses at 10 and 20 mg/kg, respectively.

QSAR Classification Models for Prediction of Hydroxamate Histone Deacetylase Inhibitor Activity against Malaria Parasites.

Malaria, caused by Plasmodium parasites, results in >400,000 deaths annually. There is no effective vaccine, and new drugs with novel modes of action are needed because of increasing parasite resistance to current antimalarials. Histone deacetylases (HDACs) are epigenetic regulatory enzymes that catalyze post-translational protein deacetylation and are promising malaria drug targets. Here, we describe quantitative structure-activity relationship models to predict the antiplasmodial activity of hydroxamate-based HDAC inhibitors. The models incorporate P. falciparum in vitro activity data for 385 compounds containing a hydroxamic acid and were subject to internal and external validation. When used to screen 22 new hydroxamate-based HDAC inhibitors for antiplasmodial activity, model A7 (external accuracy 91%) identified three hits that were subsequently verified as having potent in vitro activity against P. falciparum parasites (IC50 = 6, 71, and 84 nM), with 8 to 51-fold selectivity for P. falciparum versus human cells.

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.

The Key Glycolytic Enzyme Phosphofructokinase Is Involved in Resistance to Antiplasmodial Glycosides.

Plasmodium parasites rely heavily on glycolysis for ATP production and for precursors for essential anabolic pathways, such as the methylerythritol phosphate (MEP) pathway. Here, we show that mutations in the Plasmodium falciparum glycolytic enzyme, phosphofructokinase (PfPFK9), are associated with in vitro resistance to a primary sulfonamide glycoside (PS-3). Flux through the upper glycolysis pathway was significantly reduced in PS-3-resistant parasites, which was associated with reduced ATP levels but increased flux into the pentose phosphate pathway. PS-3 may directly or indirectly target enzymes in these pathways, as PS-3-treated parasites had elevated levels of glycolytic and tricarboxylic acid (TCA) cycle intermediates. PS-3 resistance also led to reduced MEP pathway intermediates, and PS-3-resistant parasites were hypersensitive to the MEP pathway inhibitor, fosmidomycin. Overall, this study suggests that PS-3 disrupts core pathways in central carbon metabolism, which is compensated for by mutations in PfPFK9, highlighting a novel metabolic drug resistance mechanism in P. falciparumIMPORTANCE Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue, causing 405,000 deaths and 228 million cases in 2018. Understanding key metabolic processes in malaria parasites is critical to the development of new drugs to combat this major infectious disease. The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways. Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Our work addresses this unmet need, providing new mechanistic insights into this key pathway.

An ELISA method to assess HDAC inhibitor-induced alterations to P. falciparum histone lysine acetylation.

The prevention and treatment of malaria requires a multi-pronged approach, including the development of drugs that have novel modes of action. Histone deacetylases (HDACs), enzymes involved in post-translational protein modification, are potential new drug targets for malaria. However, the lack of recombinant P. falciparum HDACs and suitable activity assays, has made the investigation of compounds designed to target these enzymes challenging. Current approaches are indirect and include assessing total deacetylase activity and protein hyperacetylation via Western blot. These approaches either do not allow differential compound effects to be determined or suffer from low throughput. Here we investigated dot blot and ELISA methods as new, higher throughput assays to detect histone lysine acetylation changes in P. falciparum parasites. As the ELISA method was found to be superior to the dot blot assay using the control HDAC inhibitor vorinostat, it was used to evaluate the histone H3 and H4 lysine acetylation changes mediated by a panel of six HDAC inhibitors that were shown to inhibit P. falciparum deacetylase activity. Vorinostat, panobinostat, trichostatin A, romidepsin and entinostat all caused an ~3-fold increase in histone H4 acetylation using a tetra-acetyl lysine antibody. Tubastatin A, the only human HDAC6-specific inhibitor tested, also caused H4 hyperacetylation, but to a lesser extent than the other compounds. Further investigation revealed that all compounds, except tubastatin A, caused hyperacetylation of the individual N-terminal H4 lysines 5, 8, 12 and 16. These data indicate that tubastatin A impacts P. falciparum H4 acetylation differently to the other HDAC inhibitors tested. In contrast, all compounds caused hyperacetylation of histone H3. In summary, the ELISA developed in this study provides a higher throughput approach to assessing differential effects of antiplasmodial compounds on histone acetylation levels and is therefore a useful new tool in the investigation of HDAC inhibitors for malaria.

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.

Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

As a result of limited classes of anthelmintics and an over-reliance on chemical control, there is a great need to discover new compounds to combat drug resistance in parasitic nematodes. Here, we show that deguelin, a plant-derived rotenoid, selectively and potently inhibits the motility and development of nematodes, which supports its potential as a lead candidate for drug development. Furthermore, we demonstrate that deguelin treatment significantly increases gene transcription that is associated with energy metabolism, particularly oxidative phosphorylation and mitoribosomal protein production before inhibiting motility. Mitochondrial tracking confirmed enhanced oxidative phosphorylation. In accordance, real-time measurements of oxidative phosphorylation in response to deguelin treatment demonstrated an immediate decrease in oxygen consumption in both parasitic (Haemonchus contortus) and free-living (Caenorhabditis elegans) nematodes. Consequently, we hypothesize that deguelin is exerting its toxic effect on nematodes as a modulator of oxidative phosphorylation. This study highlights the dynamic biologic response of multicellular organisms to deguelin perturbation.-Preston, S., Korhonen, P. K., Mouchiroud, L., Cornaglia, M., McGee, S. L., Young, N. D., Davis, R. A., Crawford, S., Nowell, C., Ansell, B. R. E., Fisher, G. M., Andrews, K. T., Chang, B. C. H., Gijs, M. A. M., Sternberg, P. W., Auwerx, J., Baell, J., Hofmann, A., Jabbar, A., Gasser, R. B. Deguelin exerts potent nematocidal activity via the mitochondrial respiratory chain.

Assessing the anthelmintic activity of pyrazole-5-carboxamide derivatives against Haemonchus contortus.

BACKGROUND: In this study, we tested five series of pyrazole-5-carboxamide compounds (n = 55) for activity against parasitic stages of the nematode Haemonchus contortus (barber's pole worm), one of the most pathogenic parasites of ruminants. METHODS: In an optimised, whole-organism screening assay, using exsheathed third-stage (xL3) and fourth-stage (L4) larvae, we measured the inhibition of larval motility and development of H. contortus. RESULTS: Amongst the 55 compounds, we identified two compounds (designated a-15 and a-17) that reproducibly inhibit xL3 motility as well as L4 motility and development, with IC50 values ranging between ~3.4 and 55.6 µM. We studied the effect of these two 'hit' compounds on mitochondrial function by measuring oxygen consumption. This assessment showed that xL3s exposed to each of these compounds consumed significantly less oxygen and had less mitochondrial activity than untreated xL3s, which was consistent with specific inhibition of complex I of the respiratory electron transport chain in arthropods. CONCLUSIONS: The present findings provide a sound basis for future work, aimed at identifying the targets of compounds a-15 and a-17 and establishing the modes of action of these chemicals in H. contortus.

Investigating the antiplasmodial activity of primary sulfonamide compounds identified in open source malaria data.

In the past decade there has been a significant reduction in deaths due to malaria, in part due to the success of the gold standard antimalarial treatment - artemisinin combination therapies (ACTs). However the potential threat of ACT failure and the lack of a broadly effective malaria vaccine are driving efforts to discover new chemical entities (NCEs) to target this disease. The primary sulfonamide (PS) moiety is a component of several clinical drugs, including those for treatment of kidney disease, glaucoma and epilepsy, however this chemotype has not yet been exploited for malaria. In this study 31 PS compounds sourced from the GlaxoSmithKline (GSK) Tres Cantos antimalarial set (TCAMS) were investigated for their ability to selectively inhibit the in vitro growth of Plasmodium falciparum asexual stage malaria parasites. Of these, 14 compounds were found to have submicromolar activity (IC50 0.16-0.89 µM) and a modest selectivity index (SI) for the parasite versus human cells (SI > 12 to >43). As the PS moiety is known to inhibit carbonic anhydrase (CA) enzymes from many organisms, the PS compounds were assessed for recombinant P. falciparum CA (PfCA) mediated inhibition of CO2 hydration. The PfCA inhibition activity did not correlate with antiplasmodial potency. Furthermore, no significant difference in IC50 was observed for P. falciparum versus P. knowlesi (P > 0.05), a Plasmodium species that is not known to contain an annotated PfCA gene. Together these data suggest that the asexual intraerythrocytic stage antiplasmodial activity of the PS compounds examined in this study is likely unrelated to PfCA inhibition.

Adaptation of the [3H]Hypoxanthine Uptake Assay for In Vitro-Cultured Plasmodium knowlesi Malaria Parasites.

The zoonotic malaria parasite Plasmodium knowlesi has recently been established in continuous in vitro culture. Here, the Plasmodium falciparum [(3)H]hypoxanthine uptake assay was adapted for P. knowlesi and used to determine the sensitivity of this parasite to chloroquine, cycloguanil, and clindamycin. The data demonstrate that P. knowlesi is sensitive to all drugs, with 50% inhibitory concentrations (IC50s) consistent with those obtained with P. falciparum This assay provides a platform to use P. knowlesi in vitro for drug discovery.

Defining the targets of antiparasitic compounds.

The treatment of major human parasitic infections is dependent on drugs that are plagued by issues of drug resistance. New chemotherapeutics with novel mechanisms of action (MOA) are desperately needed to combat multi-drug-resistant parasites. Although widespread screening strategies are identifying potential new hits for development against most major human parasitic diseases, in many cases such efforts are hindered by limited MOA data. Although MOA data are not essential for drug development, they can facilitate compound triage and provide a mechanism to combat drug resistance. Here we describe and discuss methods currently used to identify the targets of antiparasitic compounds, which could circumvent this bottleneck and facilitate the development of new antiparasitic drugs.

Sulfonamide inhibition studies of the η-class carbonic anhydrase from the malaria pathogen Plasmodium falciparum.

The η-carbonic anhydrases (CAs, EC 4.2.1.1) were recently discovered as the sixth genetic class of this metalloenzyme superfamily, and are so far known only in protozoa, including various Plasmodium species, the causative agents of malaria. We report here an inhibition study of the η-CA from Plasmodium falciparum (PfCA) against a panel of sulfonamides and one sulfamate compound, some of which are clinically used. The strongest inhibitors identified were ethoxzolamide and sulthiame, with KIs of 131-132 nM, followed by acetazolamide, methazolamide and hydrochlorothiazide (KIs of 153-198 nM). Brinzolamide, topiramate, zonisamide, indisulam, valdecoxib and celecoxib also showed significant inhibitory action against PfCA, with KIs ranging from 217 to 308 nM. An interesting observation was that the more efficient PfCA inhibitors are representative of several scaffolds and chemical classes, including benzene sulfonamides, monocyclic/bicyclic heterocyclic sulfonamides and compounds with a more complex scaffold (i.e., the sugar sulfamate derivative, topiramate, and the coxibs, celecoxib and valdecoxib). A comprehensive inhibition study of small molecules for η-CAs is needed as a first step towards assessing PfCA as a druggable target. The present work identifies the first known η-CA inhibitors and provides a platform for the development of next generation novel PfCA inhibitors.

Discovery of a new family of carbonic anhydrases in the malaria pathogen Plasmodium falciparum--the η-carbonic anhydrases.

The genome of the protozoan parasite Plasmodium falciparum, the causative agent of the most lethal type of human malaria, contains a single gene annotated as encoding a carbonic anhydrase (CAs, EC 4.2.1.1) thought to belong to the α-class, PfCA. Here we demonstrate the kinetic properties of PfCA for the CO2 hydration reaction, as well as an inhibition study of this enzyme with inorganic and complex anions and other molecules known to interact with zinc proteins, including sulfamide, sulfamic acid, and phenylboronic/arsonic acids, detecting several low micromolar inhibitors. A closer examination of the sequence of this and the CAs from other Plasmodium spp., as well as a phylogenetic analysis, revealed that these protozoa encode for a yet undisclosed, new genetic family of CAs termed the η-CA class. The main features of the η-CAs are described in this report.

Synthesis and evaluation of antimalarial properties of novel 4-aminoquinoline hybrid compounds.

Pharmacophore hybridization has recently been employed in the search for antimalarial lead compounds. This approach chemically links two pharmacophores, each with their own antimalarial activity and ideally with different modes of action, into a single hybrid molecule with the goal to improve therapeutic properties. In this paper, we report the synthesis of novel 7-chloro-4-aminoquinoline/primary sulfonamide hybrid compounds. The chlorinated 4-aminoquinoline scaffold is the core structure of chloroquine, an established antimalarial drug, while the primary sulfonamide functional group has a proven track record of efficacy and safety in many clinically used drugs and was recently shown to exhibit some antimalarial activity. The activity of the hybrid compounds was determined against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum strains. While the hybrid compounds had lower antimalarial activity when compared to chloroquine, they demonstrated a number of interesting structure-activity relationship (SAR) trends including the potential to overcome the resistance profile of chloroquine.

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.

Synthesis and antimalarial evaluation of novel benzopyrano[4,3-b]benzopyran derivatives.

7-Methoxyflavenes and 5,7,8-trimethoxyflavenes were found to undergo stereoselective acid-catalyzed rearrangement to generate the benzopyrano[4,3-b]benzopyran ring system present in the natural product, dependensin. Dependensin and its analogs were subjected to antimalarial growth inhibition assays against Plasmodium falciparum and found to have IC(50) values ranging between 1.9 and 3.9 µM.