Evaluation of ketoclomazone and its analogues as inhibitors of 1-deoxy-d-xylulose 5-phosphate synthases and other thiamine diphosphate (ThDP)-dependent enzymes.

Most pathogenic bacteria, apicomplexan parasites and plants rely on the methylerythritol phosphate (MEP) pathway to obtain precursors of isoprenoids. 1-Deoxy-d-xylulose 5-phosphate synthase (DXPS), a thiamine diphosphate (ThDP)-dependent enzyme, catalyses the first and rate-limiting step of the MEP pathway. Due to its absence in humans, DXPS is considered as an attractive target for the development of anti-infectious agents and herbicides. Ketoclomazone is one of the earliest reported inhibitors of DXPS and antibacterial and herbicidal activities have been documented. This study investigated the activity of ketoclomazone on DXPS from various species, as well as the broader ThDP-dependent enzyme family. To gain further insights into the inhibition, we have prepared analogues of ketoclomazone and evaluated their activity in biochemical and computational studies. Our findings support the potential of ketoclomazone as a selective antibacterial agent.

Discovery of antiplasmodial pyridine carboxamides and thiocarboxamides.

Malaria continues to be a significant burden, particularly in Africa, which accounts for 95% of malaria deaths worldwide. Despite advances in malaria treatments, malaria eradication is hampered by insecticide and antimalarial drug resistance. Consequently, the need to discover new antimalarial lead compounds remains urgent. To help address this need, we evaluated the antiplasmodial activity of twenty-two amides and thioamides with pyridine cores and their non-pyridine analogues. Twelve of these compounds showed in vitro anti-proliferative activity against the intraerythrocytic stage of Plasmodium falciparum, the most virulent species of Plasmodium infecting humans. Thiopicolinamide 13i was found to possess submicromolar activity (IC50 = 142 nM) and was >88-fold less active against a human cell line. The compound was equally effective against chloroquine-sensitive and -resistant parasites and did not inhibit ß-hematin formation, pH regulation or PfATP4. Compound 13i may therefore possess a novel mechanism of action.

Correction: A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

[This corrects the article DOI: 10.1371/journal.ppat.1011517.].

A screen of drug-like molecules identifies chemically diverse electron transport chain inhibitors in apicomplexan parasites.

Apicomplexans are widespread parasites of humans and other animals, and include the causative agents of malaria (Plasmodium species) and toxoplasmosis (Toxoplasma gondii). Existing anti-apicomplexan therapies are beset with issues around drug resistance and toxicity, and new treatment options are needed. The mitochondrial electron transport chain (ETC) is one of the few processes that has been validated as a drug target in apicomplexans. To identify new inhibitors of the apicomplexan ETC, we developed a Seahorse XFe96 flux analyzer approach to screen the 400 compounds contained within the Medicines for Malaria Venture 'Pathogen Box' for ETC inhibition. We identified six chemically diverse, on-target inhibitors of the ETC in T. gondii, at least four of which also target the ETC of Plasmodium falciparum. Two of the identified compounds (MMV024937 and MMV688853) represent novel ETC inhibitor chemotypes. MMV688853 belongs to a compound class, the aminopyrazole carboxamides, that were shown previously to target a kinase with a key role in parasite invasion of host cells. Our data therefore reveal that MMV688853 has dual targets in apicomplexans. We further developed our approach to pinpoint the molecular targets of these inhibitors, demonstrating that all target Complex III of the ETC, with MMV688853 targeting the ubiquinone reduction (Qi) site of the complex. Most of the compounds we identified remain effective inhibitors of parasites that are resistant to Complex III inhibitors that are in clinical use or development, indicating that they could be used in treating drug resistant parasites. In sum, we have developed a versatile, scalable approach to screen for compounds that target the ETC in apicomplexan parasites, and used this to identify and characterize novel inhibitors.

Inhibition of Thiamine Diphosphate-Dependent Enzymes by Triazole-Based Thiamine Analogues.

Thiamine is metabolized into the coenzyme thiamine diphosphate (ThDP). Interrupting thiamine utilization leads to disease states. Oxythiamine, a thiamine analogue, is metabolized into oxythiamine diphosphate (OxThDP), which inhibits ThDP-dependent enzymes. Oxythiamine has been used to validate thiamine utilization as an anti-malarial drug target. However, high oxythiamine doses are needed in vivo because of its rapid clearance, and its potency decreases dramatically with thiamine levels. We report herein cell-permeable thiamine analogues possessing a triazole ring and a hydroxamate tail replacing the thiazolium ring and diphosphate groups of ThDP. We characterize their broad-spectrum competitive inhibition of ThDP-dependent enzymes and of Plasmodium falciparum proliferation. We demonstrate how the cellular thiamine-utilization pathway can be probed by using our compounds and oxythiamine in parallel.

Pantothenate biosynthesis in Toxoplasma gondii tachyzoites is not a drug target.

Toxoplasma gondii is a pervasive apicomplexan parasite that can cause severe disease and death in immunocompromised individuals and the developing foetus. The treatment of toxoplasmosis often leads to serious side effects and novel drugs and drug targets are therefore actively sought. In 2014, Mageed and colleagues suggested that the T. gondii pantothenate synthetase, the enzyme responsible for the synthesis of the vitamin B5 (pantothenate), the precursor of the important cofactor, coenzyme A, is a good drug target. Their conclusion was based on the ability of potent inhibitors of the M. tuberculosis pantothenate synthetase to inhibit the proliferation of T. gondii tachyzoites. They also reported that the inhibitory effect of the compounds could be antagonised by supplementing the medium with pantothenate, supporting their conclusion that the compounds were acting on the intended target. Contrary to these observations, we find that compound SW314, one of the compounds used in the Mageed et al. study and previously shown to be active against M. tuberculosis pantothenate synthetase in vitro, is inactive against the T. gondii pantothenate synthetase and does not inhibit tachyzoite proliferation, despite gaining access into the parasite in situ. Furthermore, we validate the recent observation that the pantothenate synthetase gene in T. gondii can be disrupted without detrimental effect to the survival of the tachyzoite-stage parasite in the presence or absence of extracellular pantothenate. We conclude that the T. gondii pantothenate synthetase is not essential during the tachyzoite stage of the parasite and it is therefore not a target for drug discovery against T. gondii tachyzoites.

Correction: Thiamine analogues as inhibitors of pyruvate dehydrogenase and discovery of a thiamine analogue with non-thiamine related antiplasmodial activity.

[This corrects the article DOI: 10.1039/D2MD00085G.].

Roseoflavin, a Natural Riboflavin Analogue, Possesses In Vitro and In Vivo Antiplasmodial Activity.

The ability of the human malaria parasite Plasmodium falciparum to access and utilize vital nutrients is critical to its growth and proliferation. Molecules that interfere with these processes could potentially serve as antimalarials. We found that two riboflavin analogues, roseoflavin and 8-aminoriboflavin, inhibit malaria parasite proliferation by targeting riboflavin metabolism and/or the utilization of the riboflavin metabolites flavin mononucleotide and flavin adenine dinucleotide. An additional eight riboflavin analogues were evaluated, but none were found to be more potent than roseoflavin, nor was their activity on target. Focusing on roseoflavin, we tested its antimalarial activity in vivo against Plasmodium vinckei vinckei in mice. We found that roseoflavin decreased the parasitemia by 46-fold following a 4 day suppression test and, on average, increased the survival of mice by 4 to 5 days. Our data are consistent with riboflavin metabolism and/or the utilization of riboflavin-derived cofactors being viable drug targets for the development of new antimalarials and that roseoflavin could serve as a potential starting point.

Thiamine analogues as inhibitors of pyruvate dehydrogenase and discovery of a thiamine analogue with non-thiamine related antiplasmodial activity.

A series of derivatives of a triazole analogue of thiamine has been synthesised. When tested as inhibitors of porcine pyruvate dehydrogenase, the benzoyl ester derivatives proved to be potent thiamine pyrophosphate (TPP) competitive inhibitors, with the affinity of the most potent analogue (K i = 54 nM) almost matching the affinity of TPP itself. When tested as antiplasmodials, most of the derivatives showed modest activity (IC50 value >60 µM), except for a 4'-N-benzyl derivative, which has an IC50 value in the low micromolar range. This activity was not affected by increasing the extracellular concentration of thiamine in the culture medium for any of the compounds (except a modest increase in the IC50 for the unfunctionalized benzoyl ester), nor by overexpressing thiamine pyrophosphokinase in the parasite, making it unlikely to be due to an effect on thiamine transport or metabolism.

Purification of functional Plasmodium falciparum tubulin allows for the identification of parasite-specific microtubule inhibitors.

Cytoskeletal proteins are essential for parasite proliferation, growth, and transmission, and therefore have the potential to serve as drug targets.1-5 While microtubules and their molecular building block αß-tubulin are established drug targets in a variety of cancers,6,7 we still lack sufficient knowledge of the biochemistry of parasite tubulins to exploit the structural divergence between parasite and human tubulins. For example, it remains to be determined whether compounds of interest can specifically target parasite microtubules without affecting the host cell cytoskeleton. Such mechanistic insights have been limited by the lack of functional parasite tubulin. In this study, we report the purification and characterization of tubulin from Plasmodium falciparum, the causative agent of malaria. We show that the highly purified tubulin is fully functional, as it efficiently assembles into microtubules with specific parameters of dynamic instability. There is a high degree of amino-acid conservation between human and P. falciparum α- and ß-tubulin, sharing approximately 83.7% and 88.5% identity, respectively. However, Plasmodium tubulin is more similar to plant than to mammalian tubulin, raising the possibility of identifying compounds that would selectively disrupt parasite microtubules without affecting the host cell cytoskeleton. As a proof of principle, we describe two compounds that exhibit selective toxicity toward parasite tubulin. Thus, the ability to specifically disrupt protozoan microtubule growth without affecting human microtubules provides an exciting opportunity for the development of novel antimalarials.

A novel heteromeric pantothenate kinase complex in apicomplexan parasites.

Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that PfPanK1 and PfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf14-3-3I. Similarly, we demonstrate that TgPanK1 and TgPanK2 form a single complex that possesses PanK activity. Both TgPanK1 and TgPanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.

Exploring Heteroaromatic Rings as a Replacement for the Labile Amide of Antiplasmodial Pantothenamides.

Malaria-causing Plasmodium parasites are developing resistance to antimalarial drugs, providing the impetus for new antiplasmodials. Although pantothenamides show potent antiplasmodial activity, hydrolysis by pantetheinases/vanins present in blood rapidly inactivates them. We herein report the facile synthesis and biological activity of a small library of pantothenamide analogues in which the labile amide group is replaced with a heteroaromatic ring. Several of these analogues display nanomolar antiplasmodial activity against Plasmodium falciparum and/or Plasmodium knowlesi, and are stable in the presence of pantetheinase. Both a known triazole and a novel isoxazole derivative were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro. Although they fail to suppress Plasmodium berghei proliferation in vivo, the pharmacokinetic and contact time data presented provide a benchmark for the compound profile likely required to achieve antiplasmodial activity in mice and should facilitate lead optimization.

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.

Toward a Stable and Potent Coenzyme A-Targeting Antiplasmodial Agent: Structure-Activity Relationship Studies of N-Phenethyl-α-methyl-pantothenamide.

Pantothenamides (PanAms) are potent antiplasmodials with low human toxicity currently being investigated as antimalarials with a novel mode of action. These structural analogues of pantothenate, the vitamin precursor of the essential cofactor coenzyme A, are susceptible to degradation by pantetheinase enzymes present in serum. We previously discovered that α-methylation of the ß-alanine moiety of PanAms increases their stability in serum and identified N-phenethyl-α-methyl-pantothenamide as a pantetheinase-resistant PanAm with potent, on-target, and selective antiplasmodial activity. In this study, we performed structure-activity relationship investigations to establish whether stability and potency can be improved further through alternative modification of the scissile amide bond and through substitution/modification of the phenyl ring. Additionally, for the first time, the importance of the stereochemistry of the α-methyl group was evaluated in terms of stability versus potency. Our results demonstrate that α-methylation remains the superior choice for amide modification, and that while monofluoro-substitution of the phenyl ring (that often improves ADME properties) was tolerated, N-phenethyl-α-methyl-pantothenamide remains the most potent analogue. We show that the 2S,2'R-diastereomer is far more potent than the 2R,2'R-diastereomer and that this cannot be attributed to preferential metabolic activation by pantothenate kinase, the first enzyme of the coenzyme A biosynthesis pathway. Unexpectedly, the more potent 2S,2'R-diastereomer is also more prone to pantetheinase-mediated degradation. Finally, the results of in vitro studies to assess permeability and metabolic stability of the 2S,2'R-diastereomer suggested species-dependent degradation via amide hydrolysis. Our study provides important information for the continued development of PanAm-based antimalarials.

Overcoming synthetic challenges in targeting coenzyme A biosynthesis with the antimicrobial natural product CJ-15,801.

The biosynthesis of the essential metabolic cofactor coenzyme A (CoA) has been receiving increasing attention as a new target that shows potential to counter the rising resistance to established antimicrobials. In particular, phosphopantothenoylcysteine synthetase (PPCS)-the second CoA biosynthesis enzyme that is found as part of the bifunctional CoaBC protein in bacteria, but is monofunctional in eukaryotes-has been validated as a target through extensive genetic knockdown studies in Mycobacterium tuberculosis. Moreover, it has been identified as the molecular target of the fungal natural product CJ-15,801 that shows selective activity against Staphylococcus aureus and the malaria parasite Plasmodium falciparum. As such, CJ-15,801 and 4'-phospho-CJ-15,801 (its metabolically active form) are excellent tool compounds for use in the development of new antimicrobial PPCS inhibitors. Unfortunately, further study and analysis of CJ-15,801 is currently being hampered by several unique challenges posed by its synthesis. In this study we describe how these challenges were overcome by using a robust palladium-catalyzed coupling to form the key N-acyl vinylogous carbamate moiety with retention of stereochemistry, and by extensive investigation of protecting groups suited to the labile functional group combinations contained in this molecule. We also demonstrate that using TBAF for deprotection causes undesired off-target effects related to the presence of residual tertiary ammonium salts. Finally, we provide a new method for the chemoenzymatic preparation of 4'-phospho-CJ-15,801 on multi-milligram scale, after showing that chemical synthesis of the molecule is not practical. Taken together, the results of this study advances our pursuit to discover new antimicrobials that specifically target CoA biosynthesis and/or utilization.

Structure-Activity Relationships of Antiplasmodial Pantothenamide Analogues Reveal a New Way by Which Triazoles Mimic Amide Bonds.

Pantothenamides are potent growth inhibitors of the malaria parasite Plasmodium falciparum. Their clinical use is, however, hindered due to the ubiquitous presence of pantetheinases in human serum, which rapidly degrade pantothenamides into pantothenate and the corresponding amine. We previously reported that replacement of the labile amide bond with a triazole ring not only imparts stability toward pantetheinases, but also improves activity against P. falciparum. A small library of new triazole derivatives was synthesized, and their use in establishing structure-activity relationships relevant to antiplasmodial activity of this family of compounds is discussed herein. Overall it was observed that 1,4-substitution on the triazole ring and use of an unbranched, one-carbon linker between the pantoyl group and the triazole are optimal for inhibition of intraerythrocytic P. falciparum growth. Our results imply that the triazole ring may mimic the amide bond with an orientation different from what was previously suggested for this amide bioisostere.

Mutations in the pantothenate kinase of Plasmodium falciparum confer diverse sensitivity profiles to antiplasmodial pantothenate analogues.

The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase (PfPanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes (Pfpank1) in each resistant line. These mutations significantly alter PfPanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive. We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.

Structure-activity analysis of CJ-15,801 analogues that interact with Plasmodium falciparum pantothenate kinase and inhibit parasite proliferation.

Survival of the human malaria parasite Plasmodium falciparum is dependent on pantothenate (vitamin B5), a precursor of the fundamental enzyme cofactor coenzyme A. CJ-15,801, an enamide analogue of pantothenate isolated from the fungus Seimatosporium sp. CL28611, was previously shown to inhibit P. falciparum proliferation in vitro by targeting pantothenate utilization. To inform the design of next generation analogues, we set out to synthesize and test a series of synthetic enamide-bearing pantothenate analogues. We demonstrate that conservation of the R-pantoyl moiety and the trans-substituted double bond of CJ-15,801 is important for the selective, on-target antiplasmodial effect, while replacement of the carboxyl group is permitted, and, in one case, favored. Additionally, we show that the antiplasmodial potency of CJ-15,801 analogues that retain the R-pantoyl and trans-substituted enamide moieties correlates with inhibition of P. falciparum pantothenate kinase (PfPanK)-catalyzed pantothenate phosphorylation, implicating the interaction with PfPanK as a key determinant of antiplasmodial activity.

Antiplasmodial Mode of Action of Pantothenamides: Pantothenate Kinase Serves as a Metabolic Activator Not as a Target.

N-Substituted pantothenamides (PanAms) are pantothenate analogues with up to nanomolar potency against blood-stage Plasmodium falciparum (the most virulent species responsible for malaria). Although these compounds are known to target coenzyme A (CoA) biosynthesis and/or utilization, their exact mode of action (MoA) is still unknown. Importantly, PanAms that retain the natural ß-alanine moiety are more potent than other variants, consistent with the involvement of processes that are selective for pantothenate (the precursor of CoA) or its derivatives. The transport of pantothenate and its phosphorylation by P. falciparum pantothenate kinase (PfPanK, the first enzyme of CoA biosynthesis) are two such processes previously highlighted as potential targets for the PanAms' antiplasmodial action. In this study, we investigated the effect of PanAms on these processes using their radiolabeled versions (synthesized here for the first time), which made possible the direct measurement of PanAm uptake by isolated blood-stage parasites and PanAm phosphorylation by PfPanK present in parasite lysates. We found that the MoA of PanAms does not involve interference with pantothenate transport and that inhibition of PfPanK-mediated pantothenate phosphorylation does not correlate with PanAm antiplasmodial activity. Instead, PanAms that retain the ß-alanine moiety were found to be metabolically activated by PfPanK in a selective manner, forming phosphorylated products that likely inhibit other steps in CoA biosynthesis or are transformed into CoA antimetabolites that can interfere with CoA utilization. These findings provide direction for the ongoing development of CoA-targeted inhibitors as antiplasmodial agents with clinical potential.

Biological characterization of chemically diverse compounds targeting the Plasmodium falciparum coenzyme A synthesis pathway.

BACKGROUND: In the fight against malaria, the discovery of chemical compounds with a novel mode of action and/or chemistry distinct from currently used drugs is vital to counteract the parasite's known ability to develop drug resistance. Another desirable aspect is efficacy against gametocytes, the sexual developmental stage of the parasite which enables the transmission through Anopheles vectors. Using a chemical rescue approach, we previously identified compounds targeting Plasmodium falciparum coenzyme A (CoA) synthesis or utilization, a promising target that has not yet been exploited in anti-malarial drug development. RESULTS: We report on the outcomes of a series of biological tests that help to define the species- and stage-specificity, as well as the potential targets of these chemically diverse compounds. Compound activity against P. falciparum gametocytes was determined to assess stage-specificity and transmission-reducing potential. Against early stage gametocytes IC50 values ranging between 60 nM and 7.5 µM were obtained. With the exception of two compounds with sub-micromolar potencies across all intra-erythrocytic stages, activity against late stage gametocytes was lower. None of the compounds were specific pantothenate kinase inhibitors. Chemical rescue profiling with CoA pathway intermediates demonstrated that most compounds acted on either of the two final P. falciparum CoA synthesis enzymes, phosphopantetheine adenylyltransferase (PPAT) or dephospho CoA kinase (DPCK). The most active compound targeted either phosphopantothenoylcysteine synthetase (PPCS) or phosphopantothenoylcysteine decarboxylase (PPCDC). Species-specificity was evaluated against Trypanosoma cruzi and Trypanosoma brucei brucei. No specific activity against T. cruzi amastigotes was observed; however three compounds inhibited the viability of trypomastigotes with sub-micromolar potencies and were confirmed to act on T. b. brucei CoA synthesis. CONCLUSIONS: Utilizing the compounds we previously identified as effective against asexual P. falciparum, we demonstrate for the first time that gametocytes, like the asexual stages, depend on CoA, with two compounds exhibiting sub-micromolar potencies across asexual forms and all gametocytes stages tested. Furthermore, three compounds inhibited the viability of T. cruzi and T. b. brucei trypomastigotes with sub-micromolar potencies and were confirmed to act on T. b. brucei CoA synthesis, indicating that the CoA synthesis pathway might represent a valuable new drug target in these parasite species.