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Pantothenate and CoA biosynthesis in Apicomplexa and their promise as antiparasitic drug targets

['Laura E. De Vries', 'Department Of Medical Microbiology', 'Radboudumc Center For Infectious Diseases', 'Radboud Institute For Molecular Life Sciences', 'Radboud University Medical Center', 'Nijmegen', 'The Netherlands', 'Matteo Lunghi', 'Department Of Microbiology', 'Molecular Medicine']

Date: 2022-02 

The Apicomplexa phylum comprises thousands of distinct intracellular parasite species, including coccidians, haemosporidians, piroplasms, and cryptosporidia. These parasites are characterized by complex and divergent life cycles occupying a variety of host niches. Consequently, they exhibit distinct adaptations to the differences in nutritional availabilities, either relying on biosynthetic pathways or by salvaging metabolites from their host. Pantothenate (Pan, vitamin B5) is the precursor for the synthesis of an essential cofactor, coenzyme A (CoA), but among the apicomplexans, only the coccidian subgroup has the ability to synthesize Pan. While the pathway to synthesize CoA from Pan is largely conserved across all branches of life, there are differences in the redundancy of enzymes and possible alternative pathways to generate CoA from Pan. Impeding the scavenge of Pan and synthesis of Pan and CoA have been long recognized as potential targets for antimicrobial drug development, but in order to fully exploit these critical pathways, it is important to understand such differences. Recently, a potent class of pantothenamides (PanAms), Pan analogs, which target CoA-utilizing enzymes, has entered antimalarial preclinical development. The potential of PanAms to target multiple downstream pathways make them a promising compound class as broad antiparasitic drugs against other apicomplexans. In this review, we summarize the recent advances in understanding the Pan and CoA biosynthesis pathways, and the suitability of these pathways as drug targets in Apicomplexa, with a particular focus on the cyst-forming coccidian, Toxoplasma gondii, and the haemosporidian, Plasmodium falciparum.

The Apicomplexa phylum comprises thousands of parasitic species, among which the causative agents of malaria, toxoplasmosis and cryptosporidiosis. New parasiticidal compounds and drugs are urgently needed for treatment of these devastating diseases. As these parasites have adapted innovative pathways for nutrient acquisition, several studies have investigated vitamins and cofactor synthesis and salvage, with the aim of identifying unexplored drug targets. Coenzyme A (CoA) is an essential cofactor for cell biology and is synthesized from pantothenate (Pan, vitamin B5). The discovery of the druggability of CoA synthesis in Plasmodium falciparum has sparked intensive research toward lead compounds identification and preclinical development. Here, we review the current literature on the topic from biological and pharmacological perspectives. Focusing on Plasmodium species and Toxoplasma gondii, we describe recent findings on the importance of Pan synthesis, salvage, and metabolization to CoA in this phylum. In addition, we summarize recent promising advances in the exploration and exploitation of these pathways for lead compounds optimization and drug development.

Funding: LEdV was supported by a PhD fellowship from the Radboud Institute for Molecular Life Sciences, Radboudumc (RIMLS015-010) and TWAK by the Netherlands Organization for Scientific Research (NWO-VIDI 864.13.009). ML is supported by a PhD salary award granted by the Institute of Genetics and Genomics of Geneva (IGE3), AK and DSF are supported by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under grant agreement no. 695596. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Introduction

The Apicomplexa phylum encompasses a large and diverse group of parasites exhibiting distinct lifestyles within one or more cellular niches and hosts. In humans, these parasites can cause debilitating and deadly diseases such as malaria, toxoplasmosis, and cryptosporidiosis [1–3]. Arguably, the most successful zoonotic parasite is Toxoplasma gondii, with the highly proliferative form (tachyzoites) capable of infecting virtually all warm-blooded animals and replicating in most nucleated cell types. Upon encountering immune pressure from the intermediate host, the tachyzoites are rapidly eradicated, while few of them differentiate into the slow growing, cyst-forming bradyzoites that are responsible for chronic infection to ensure persistence and transmission [4,5]. Malaria is caused by parasites of the genus Plasmodium, which reside mainly intracellularly in the vertebrate host, while extracellularly in the mosquito host. First, the malaria parasite develops in liver cells in humans, and after release, parasites multiply via a continuous cycle of asexual replication in red blood cells. Some of these parasites enter a different developmental program and form sexual stages (gametocytes) that are required for successful colonization of a mosquito, the site of sexual reproduction [6,7]. Intracellular Plasmodium and Toxoplasma parasites have several ways to scavenge nutrients from the host, through the induction of new permeation pathways (NPPs) in Plasmodium-infected erythrocytes, the selectively permeable parasitophorous vacuole membrane, and by membrane transporter proteins [8–14]. Understanding the acquisition and de novo synthesis of metabolites and the characterization of metabolic pathways for the production of essential vitamins or cofactors is of significant therapeutic interest [15]. In this review, we focus on recent advances in our understanding of the biology of the pathways for the synthesis of pantothenate (Pan, vitamin B5) and coenzyme A (CoA) in the apicomplexan parasites T. gondii and P. falciparum, and their exploration as antiparasitic drug targets.

Pantothenate biosynthesis occurs only in the coccidian branch of the Apicomplexa Pan is the precursor for the biosynthesis of the essential cofactor CoA. Most bacteria, plants and fungi, can synthesize it de novo, while animals need to acquire this vitamin from their diet. Pan is synthesized from the branched-chain amino acid, L-valine, which undergoes 3 consecutive biotransformation reactions: (i) a hydroxymethyl transfer of ketoisovalerate, a metabolite of valine, to form α-ketopantoate; (ii) reduction of α-ketopantoate to pantoate; and (iii) ligation of pantoate with β-alanine (commonly derived from L-aspartate) to form Pan (Fig 1). These reactions are performed by the enzymes ketopantoate hydroxymethyl transferase (KPHMT), ketopantoate reductase (KPR), and pantoate-β-alanine ligase (PBAL, also called Pan synthetase), respectively. Whereas other apicomplexans lack this pathway altogether, the coccidians, including T. gondii, encode the enzymes catalyzing the 3 Pan synthesis reactions in only 2 genes, with the first 2 transformations of the pathway catalyzed by a bifunctional KPHMT-KPR enzyme (Fig 2). In non-apicomplexan alveolates, Pan synthesis genes are duplicated and organized in several ORFs. The photosynthetic symbionts Vitrella brassicaformis and Chromera velia contain KPHMT and KPR as single genes, as well as in 1 trifunctional ORF also comprising PBAL. The KPHMT-KPR fusion can also be observed in the oyster parasite Perkinsus marinus, in which PBAL activity is encoded by 2 genes instead [15]. Recent works in T. gondii on the localization of KPHMT-KPR and PBAL revealed that these enzymes localize to the mitochondrion and nucleus, respectively [16,17]. The branched-chain-aminotransferase (BCAT), which generates α-ketoisovalerate from L-valine, also localizes to the mitochondrion [18], possibly pointing toward the initiation of Pan synthesis within this organelle. The localization of PBAL in the nucleus is surprising but is confirmed by a proteome-wide fractionation study (localization of organelle proteins by isotope tagging (LOPIT) [17]), which reports localization for only this enzyme in the pathway. However, the precursors and product of the enzyme are expected to diffuse freely between the cytosol and nucleus. Previously, a pharmacological study described the Pan synthesis pathway in T. gondii and proposed its druggability by testing the efficacy of compounds designed to target Mycobacterium tuberculosis Pan synthetase [19]. Consistent with whole-genome fitness score study performed via CRISPR-Cas9 editing in T. gondii [20], and in sharp contrast to the pharmacological evidence [19], KPHMT-KPR and PBAL were found to be fully dispensable in in vitro cultured tachyzoites, as well as in the mouse model of acute infection [16]. Moreover, while PBAL has been shown to be a functional enzyme in vitro and in vivo, stable isotope labeling experiments demonstrated that Pan synthesis is not occurring in in vitro standard cultured T. gondii tachyzoites and that parasites are dependent on Pan salvage from the host cell. Remarkably, given the dispensability and inactivity of the synthesis pathway in tachyzoites, individual deletion of either KPHMT-KPR or PBAL gene in the cyst-forming type II (ME49) strain of T. gondii resulted in the dramatic reduction in the number of cysts in the mouse brain, clearly supporting the utilization of Pan synthesis in bradyzoites [16]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Pan and CoA biosynthesis pathways and the predicted essentiality in T. gondii, P. berghei, and P. falciparum. Phenotypic data for T. gondii tachyzoites and P. berghei and P. falciparum asexual blood stages are derived from Sidik and colleagues [20], Bushell and colleagues [21], and Zhang and colleagues [22], respectively. Since the essentiality data for each of the species (T. gondii, P. berghei, and P. falciparum) were obtained using different genetic approaches, the most representative forms are displayed. The FS for T. gondii (circles) is an experimentally observed measure (ranging from −6.9 to +3 for the fitness cost associated with the disruption of a given gene for parasite survival. Fitness-conferring genes display a lower FS and dispensable genes display a higher FS [20]. Phenotypic data for P. falciparum genes based on their MIS, which estimates the potential mutability of a gene, are represented as diamonds [22]. For P. berghei, a gene is indicated as dispensable or essential based on the relative growth rate observed upon genetic disruption within the asexual blood stage (squares) [21]. Dashed lines represent putative routes. Pantothenamides and pantetheine can further be hydrolyzed into Pan via the action of host-encoded pantetheinases (vanins) [23]. BCAT, branched-chain amino acid transaminase; CoA, coenzyme A; DPCK, dephospho-CoA kinase; FS, fitness score; KPHMT, ketopantoate hydroxymethyltransferase; KPR, α-ketopantoate reductase; MIS, mutagenesis index score; PBAL, pantoate-β-alanine ligase; Pan, pantothenate; PANK, pantothenate kinase; PPAT, phosphopantetheine adenylyltransferase; PPCDC, phosphopantothenoylcysteine decarboxylase; PPCS, phosphopantothenoylcysteine synthetase. https://doi.org/10.1371/journal.ppat.1010124.g001 PPT PowerPoint slide

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TIFF original image Download: Fig 2. Genes encoding Pan and CoA biosynthesis enzymes. Phenotypic data of T. gondii tachyzoites and P. berghei and P. falciparum asexual blood stages are derived from Sidik and colleagues [20], Bushell and colleagues [21], and Zhang and colleagues [22], respectively. Since the essentiality data for each of the species (T. gondii, P. falciparum, and P. berghei) were obtained using different genetic approaches, the scores are not comparable. The FS for T. gondii is an experimentally observed measure (ranging from −6.9 to +3) for the fitness cost associated with the disruption of a given gene for parasite survival. Fitness-conferring genes display a low FS and dispensable genes display a high FS [20]. The presented fitness score for P. falciparum is based on the MFS, which estimates the relative growth fitness cost for mutating a gene based on its normalized quantitative insertion-site sequencing reads distribution. The scores range from −4.1 to 2.8, with lower scores indicating nonmutability of a gene [22]. Experimental localization data (in black) were obtained from Oppenheim and colleagues [34], Lunghi and colleagues [16], and Barylyuk and colleagues (only PBAL is identified in this study) [17] for T. gondii, Tjhin and colleagues [35] and Swift and colleagues [36] for P. falciparum. Predicted localization is shown in gray. Variability in conservation is shown with different colors: light gray when only 1 copy of the gene is present, gray when 2 ORFs are present in a single gene, and dark gray when 3 ORFs exist in a single gene. Light orange is when more than 1 gene is identified in the whole group for the indicated catalytic activity, orange when only some species within the group encode more than 1 gene, and dark orange when the gene can be identified but has poor sequence similarity to the genes found in other species. BCAT, branched-chain amino acid transaminase; CoA, coenzyme A; DPCK, dephospho-CoA kinase; FS, fitness score; KPHMT, ketopantoate hydroxymethyltransferase; KPR, α-ketopantoate reductase; LOPIT, localization of organelle proteins by isotope tagging; MFS, mutagenesis fitness score; Pan, pantothenate; PANK, pantothenate kinase; PBAL, pantoate-β-alanine ligase; PPAT, phosphopantetheine adenylyltransferase; PPCDC, phosphopantothenoylcysteine decarboxylase; PPCS, phosphopantothenoylcysteine synthetase. https://doi.org/10.1371/journal.ppat.1010124.g002 The retention of the Pan synthesis pathway exclusively in coccidians and the critical role of the Pan synthesis pathway for establishment of chronic infection by T. gondii are intriguing. Perhaps, the preferential niches occupied by bradyzoites have limited Pan levels, leaving scarce amounts for scavenge by the parasite. The most studied route of synthesis of β-alanine in bacteria is from aspartate by aspartate decarboxylase [24] and in yeast by degradation of spermine [25]. In mammalian organisms, β-alanine can be synthesized by a variety of routes, such as a 3-enzymatic step from uracil [26], from purine catabolism via the β-alanine synthetase [27], and from glutamate by glutamic acid decarboxylase–like 1 enzyme [28]. While β-alanine accumulates in brain tissues [29] and plays a role as neurotransmitter [30], it is also necessary for synthesis of the histidine-containing dipeptides carnosine and anserine [31], both of which have physiological roles as an antioxidant, pH buffer, and neurotransmitter, and accumulate in brain and muscle tissues as well [32]. Mining of the Toxoplasma genome failed to identify homologous enzymes capable of synthesizing β-alanine via the various routes known in mammalian, bacterial, or yeast systems. Concordantly, T. gondii tachyzoites are dependent on exogenous β-alanine to produce Pan and PBAL activity could only be identified upon supplementation with both pantoate and β-alanine [16]. Furthermore, uptake of labeled β-alanine and formation of Pan was only observed in parasites expressing PBAL [16]. The presence of a highly divergent pathway, active in a different life cycle stage of the parasite where β-alanine supply is limited, cannot be ruled out. However, given that Toxoplasma cysts reside in β-alanine and carnosine-rich tissues [30], it is plausible that a selective pressure has led to the retention of an active Pan synthesis and acquisition of its precursors from the host. Pan is a small metabolite with a MW of 219 Da that presumably diffuses freely through the molecular sieve of the parasitophorous vacuole membrane [9,33]. Accessibility of host-derived nutrients to encysted bradyzoites is currently unknown, and reduced permeability across the cyst wall and limited diffusion through large cysts could explain the necessity for Pan biosynthesis. The precise transport mechanism and molecular entity mediating Pan transport to the intracellular environment, as well as the molecular basis for Toxoplasma tissue preference for cyst formation, remain unanswered.

In search of a pantothenate transporter in Apicomplexa All apicomplexans are expected to rely on salvage of Pan from the host. This has been demonstrated in T. gondii by mass isotope labeling experiments [16], and a seminal study also clearly demonstrated that P. falciparum requires exogenous Pan for growth [37]. Pan transport is increased drastically in infected compared to uninfected red blood cells and is mediated by the NPP [38]. The existence of a Na+-independent, pH-dependent Pan transporter in P. falciparum has been formally demonstrated [39], and, therefore, inhibiting the Pan transporter presents a promising drug-based intervention strategy. However, the transporter remains unidentified. In model organisms, highly divergent Pan transporters have been described, including the Na+ /Pan symporter PanF in Escherichia coli [40], a major facilitator superfamily transporter Fen2 in yeast [41] and a Na+-dependent multivitamin transporter SLC5A6 in mammals [42]. Surprisingly, PanF, Fen2, and SLC5A6 share no sequence similarity, and, therefore, it is plausible that apicomplexans harbor yet another divergent Pan transporter. An attempt to identify the P. falciparum Pan transporter by homology with Fen2 led to the characterization of a putative transporter, PfPAT, although with a relatively poor homology [43]. While the scavenging of exogenous Pan is essential for P. falciparum growth in erythrocytes, PAT is dispensable for blood-stage growth of rodent malaria parasites and only essential for mosquito transmission [44,45]. Later studies revealed a major role of PbPAT (and its homolog TFP1 in T. gondii) in secretion of osmiophilic bodies and microneme maturation, respectively [46,47], rendering its role as a Pan transporter highly unlikely. A complementary strategy to the homology search for the identification of the Pan transporter could take advantage of the currently available genome-wide datasets, the LOPIT dataset for T. gondii [17] and large-scale genetic screens [20–22]. The candidate list would be limited to candidates with the following: (i) plasma membrane predicted localization; (ii) essentiality for both T. gondii and Plasmodium growth; and (iii) presence of predicted transmembrane domains. Taking a comparable approach, a recent review by Martin proposed novel, highly divergent candidate transporters for Pan in P. falciparum [10].

The CoA biosynthesis pathway is conserved and essential in Apicomplexa Pan is the precursor for the synthesis of CoA, which is an essential cofactor for a broad range of functions within all cells. CoA provides activated acyl groups and the prosthetic 4′-phosphopantetheine group for gene regulation, posttranslational modification of proteins via acetylation, and various metabolic functions, such as the tricarboxylic acid cycle (TCA cycle) and fatty acid synthesis (FAS) [48]. While differences are present in Archaea, the canonical pathway for CoA synthesis is present in most bacteria and eukaryotes [49]. The first step is the phosphorylation of Pan to 4′-phosphopantothenate, catalyzed by Pan kinase (PanK). This reaction is followed by the sequential formation of 4′-phosphopantothenoylcysteine by phosphopantothenoylcysteine synthetase (PPCS), 4′-phosphopantetheine by phosphopantothenoylcysteine decarboxylase (PPCDC), dephospho-CoA by phosphopantetheine adenylyltransferase (PPAT), and, finally, CoA by dephospho-CoA kinase (DPCK). Although this essential CoA pathway is highly conserved, there are differences between phyla and even between apicomplexan parasites (Fig 2) [20–22,36,50,51]. Most bacteria and Saccharomyces cerevisiae contain a single PanK [52]. Conversely, mammals harbor 3 PanK genes and 1 pseudo-PanK [53], which are expressed in different compartments and tissues [54,55] and have different regulatory properties [56]. The apicomplexans possess 2 distinct PanK genes, which have been thoroughly studied in Plasmodium species for their potential druggability. In P. falciparum, PanK1 and PanK2 are part of a macromolecular complex as heterodimers, a feature that is also conserved in T. gondii and unique to the Apicomplexa phylum [16,57]. PfPanK1 is essential and the mutant PfPanK2 generated by transposon insertional mutagenesis is predicted to have a reduced fitness [22,35]. Both PanKs are essential in T. gondii [16,57]. In these species, the heterodimerization is likely essential for the phosphorylation of Pan [57], whereas in Plasmodium yoelii, both PanKs are individually dispensable [51], indicative of a possible redundant function. Contrastingly and unexpectedly, the simultaneous knockout of both PanK1 and PanK2 appears to be viable in blood-stage P. berghei parasites [58]. Other unexpected cases that need further elucidation are the presence of 2 PPCS genes in P. falciparum, which are predicted to be dispensable, and the dispensability of PfPPAT [22]. For the latter, a nicotinamide nucleotidyltransferase (PF3D7_1327600) has been suggested as an alternative PPAT-encoding gene with an adenylyltransferase activity comparable to that of PPAT [59,60], possibly explaining the dispensability of PPAT. Studies in rodent malaria parasites have highlighted that single-copy PPCS and PPCDC are also dispensable [21,50]. The ability of PanK to phosphorylate pantetheine [61] points toward a possible salvage of pantetheine for CoA synthesis (Fig 1), similar to observations in some bacteria [62], potentially rendering PPCS and PPCDC dispensable. Nevertheless, the presence of pantetheine-hydrolyzing enzymes, pantetheinases (vanins), in the serum possibly limits the amount of freely available pantetheine. At this stage, the mode of a possible pantetheine uptake, much like that of Pan, remains elusive. This may occur through a conserved Pan transporter, or through diffusion, as has been suggested for 4′-phosphopantetheine [63]. Salvage of 4′-phosphopantetheine would make PanK activity also dispensable, possibly explaining the viability of the PanK double knockout in P. berghei. Interestingly, the presence of a single transporter for ketopantoate, pantoate, and CoA intermediates has recently been proposed in Salmonella enterica [64]; however, no homolog could be identified in Apicomplexa. While all these CoA synthesis enzymes, including TgDPCK, are likely cytoplasmic, the last step performed by PfDPCK has recently been localized to the apicoplast. This enzyme is essential for intra-erythrocytic parasite survival and remains active in the vesicles that accumulate after apicoplast disruption [36].

Targeting the pantothenate and CoA synthesis pathways for drug development While mammalian cells lack the ability to synthesize Pan, bradyzoites rely on this ability, making this pathway a plausible target for intervention against the chronic stages of T. gondii [16,65]. Interestingly, one of the proposed mechanisms of action of the current standard drug against M. tuberculosis, pyrazinamide, is the inhibition of Pan synthesis through the degradation of aspartate decarboxylase [66–69]. Unfortunately, the lack of an obvious aspartate decarboxylase homolog in T. gondii renders repurposing pyrazinamide for treatment of chronic T. gondii infections unlikely. Other targets for the inhibition of Pan synthesis in T. gondii may be KPHMT-KPR or PBAL [70]. While no inhibitors of the first enzyme have been identified, a panel of 13 Pan synthetase inhibitors that were designed against M. tuberculosis inhibited T. gondii growth with varying potencies, including SW413 (IC 50 of 20 nM) (Fig 3) [19]. As discussed above, T. gondii tachyzoites do not rely on Pan synthesis in vitro [20], indicating that the activity of the Pan synthetase inhibitors may rather be a consequence of off-target effects. As parasiticidal activity of these compounds could be rescued by addition of excess Pan in the culture media, the off-target effect is potentially related to Pan uptake or CoA synthesis. Nevertheless, specific PBAL inhibitors may be efficacious against bradyzoites, though it should be noted that the limited permeability of the blood brain barrier and the cyst wall present an additional challenge to the design of drugs targeted to kill this stage of the parasite. Plasmodium species cannot synthesize Pan and therefore rely entirely on Pan uptake. The presumed divergence of the human and Plasmodium Pan transporter could be exploited to develop parasite-specific inhibitors, although this would require the identification of the parasite transporter in the first place [39,71]. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Pan and CoA synthesis targeting compounds. All IC 50 s are determined in P. falciparum, unless stated otherwise. CoA, coenzyme A; Pan, pantothenate. https://doi.org/10.1371/journal.ppat.1010124.g003 Even though there are differences in the CoA biosynthesis pathway between apicomplexans, the majority of enzymes involved are essential for parasite survival (Fig 2), indicating their potential as antiparasitic drug targets. However, antimicrobials that target the Pan-fueled CoA biosynthesis pathway could have deleterious consequences in the host if its activity is not specific for the metabolism of the parasite. Defects in the CoA biosynthesis pathway in humans caused by mutations in PanK, PPCS, or CoA synthase (CoASY, a bifunctional PPAT/DPCK enzyme) lead to a variety of diseases including neurodegeneration and dilated cardiomyopathy [72–75]. In addition, mice fed on a Pan-free diet and treated with hopantenate, a Pan analog that inhibits PPCS [76], died within 5 to 15 days, even though no toxicity had been observed when concentrations of up to 800 μM were tested on a variety of cell lines [77]. The effects of hopantenate highlight the importance of thorough safety testing in animal models and developing compounds that are very specific and ideally target parasite enzymes only. The dispensability of PPCS in Plasmodium and PPCDC in rodent malaria parasites precludes these 2 enzymes as ideal drug targets [21,22,50]. In contrast, the bifunctional CoaBC enzyme (with both PPCS and PPCDC function) in M. tuberculosis is a potential drug target based on the bactericidal effects upon knockdown [70,78]. More promising targets in P. falciparum are PanK1, DPCK, and, possibly, PPAT. This is especially the case for PfPPAT, if essential, which exhibits weak homology to the human bifunctional PPAT/DPCK enzyme CoASY [79]. PanK1 and DPCK have higher homology to the human equivalents, and this would require the development of parasite-specific compounds. The sequence homologies of the CoA synthesis enzymes and the potential to develop Plasmodium-specific compounds is extensively reviewed in Spry and colleagues’ paper [80]. Already since the 1940s, the CoA synthesis pathway has been considered and explored as a drug target [81]. Following the discovery of the structure of Pan [82], many Pan analogs have been generated that all act as antimicrobials [81]. Interestingly, some of these also showed weak activity against avian, nonhuman primate and human malaria parasites [83–85]. In 2005, 2 Pan analogs, pantothenol (provitamin B5) and CJ-15,801 (a natural fungal product) [86], were shown to have micromolar activity against P. falciparum asexual blood stages (Fig 3) [87–89]. More promising results were obtained with pantothenamides (PanAms), a class of Pan analogs that first showed micromolar activity, but potency was improved to the low nanomolar range by inhibiting or inactivating the pantetheinase enzymes present in the human serum [23,90–92]. These enzymes hydrolyze pantetheine to Pan and cysteamine and can also degrade PanAms (Fig 1) [91,93,94]. Different strategies have been used to generate PanAms that are resistant to degradation, including (i) addition of a methyl group adjacent to the distal amide [95,96]; (ii/iii) displacement of the distal amide bond in the pantothenamide structure by removal or addition of a methylene group in its β-alanine moiety producing α-PanAms and HoPanAms, respectively [23]; (iv) replacement of the pantetheinase-susceptible amide bond by a triazole isostere to make the triazole-substituted PanAms [97,98]; and (v) inversion of the labile amide bond, leading to the inverted-amide PanAms (iPanAms) [99,100]. This led to very promising drug candidates, including N-PE-αMe-nPanAm [96], N5-trz-C1-Pan [97], and MMV693183 with IC 50 s of 23 nM, 56 nM, and 2.5 nM, respectively, against asexual blood-stage P. falciparum (Fig 3). Although these strategies also led to stable and active compounds against gram-positive bacteria, they only showed micromolar activity in these organisms [101–103]. The lack of activity of an N7-PanAm and, possibly, other derivatives, against the gram-negative bacteria, E. coli, is partially due to the efflux of the drug through TolC-dependent pumps [104]. PanAms have also shown potent activity against Plasmodium knowlesi [105], Plasmodium vivax [100], P. falciparum sexual blood stages in vitro, and asexual blood stages in an in vivo humanized mouse model [99,100]. In addition, the iPanAm MMV688558 inhibits T. gondii growth in vitro with an IC 50 of 0.95 μM [106], showing its potential to target other apicomplexan parasites. The potent antimalarial activities of PanAms show that the Pan and CoA synthesis pathways are indeed plausible drug targets. Drug screenings have been performed to find compounds that are chemically different to Pan analogs and could target CoA synthesis. Recently, a library of compounds was tested to inhibit PfPanK1 activity and P. falciparum growth, and 4 inhibitors were identified with micromolar activity that may serve as new scaffolds [61]. Furthermore, a chemically diverse set of inhibitors against P. falciparum was suggested to target the CoA synthesis pathway, based on their reduced activity upon supplementation with metabolites from the CoA pathway [107,108]. One of the putative CoA biosynthesis targeting drugs, Amb180780, is part of the dihetarylthioether class of drugs that has been further developed into the promising new candidate, KuWei173 (Fig 3) [109]. A structurally related compound, compound 33 from Edlin and colleagues, reduced parasitemia by 34% in a rodent malaria model [110]. Activity of Amb180780 on Trypanosoma brucei brucei [107] and the potent dihetarylthioethers make these compounds an exciting basis for new drugs that may target the CoA pathway in diverse protozoan parasites belonging to the kinetoplastids and apicomplexans.

[END]

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