The biosynthesis of coenzyme A (CoA) from pantothenate and the utilization of CoA in essential biochemical pathways represent promising antimalarial drug targets. Pantothenamides, amide derivatives of pantothenate, have potential as antimalarials, but a serum enzyme called pantetheinase degrades pantothenamides, rendering them inactive in vivo. In this study, we characterize a series of 19 compounds that mimic pantothenamides with a stable triazole group instead of the labile amide. Two of these pantothenamides are active against the intraerythrocytic stage parasite with 50% inhibitory concentrations (IC 50 s) of ϳ50 nM, and three others have submicromolar IC 50 s. We show that the compounds target CoA biosynthesis and/or utilization. We investigated one of the compounds for its ability to interact with the Plasmodium falciparum pantothenate kinase, the first enzyme involved in the conversion of pantothenate to CoA, and show that the compound inhibits the phosphorylation of [ 14 C]pantothenate by the P. falciparum pantothenate kinase, but the inhibition does not correlate with antiplasmodial activity. Furthermore, the compounds are not toxic to human cells and, importantly, are not degraded by pantetheinase. Due to drug resistance by Plasmodium falciparum, the parasite responsible for malaria, it is vital to identify new chemotherapeutics targeting novel parasite pathways. P. falciparum does not survive its asexual red blood cell (RBC) stage without access to exogenous pantothenate (vitamin B 5 ) (1-3). Pantothenate is metabolized by five enzymes into coenzyme A (CoA), a cofactor estimated to be required by 9% of all enzymes (4). The first enzyme in the CoA biosynthetic pathway is pantothenate kinase (PanK), which catalyzes the phosphorylation of pantothenate into phosphopantothenate. Several pantothenate analogues have been shown to inhibit the growth of P. falciparum in vitro, including pantothenol (5), 801 (6), and various other analogues (7,8). Pantothenol (Fig. 1) has also been shown to possess antiplasmodial activity in vivo in a mouse model of malaria (5). Recently, pantothenamides, amides of pantothenate initially investigated for antibacterial activity (9-11), have been shown to possess potent antiplasmodial activity (12). Unfortunately, the effectiveness of pantothenamides as antiplasmodials is attenuated by pantetheinase (12), an enzyme found in human serum (13). The endogenous substrate of pantetheinase is pantetheine, which is broken down by pantetheinase into pantothenate and cysteamine (14). Pantetheinase also breaks down pantothenamides (12), including the prototypical pantothenamide, N-pentylpantothenamide (N5-Pan [ Fig. 1]). The breakdown of pantothenamides is an obstacle that needs to be overcome if these compounds are to be of any use as antimicrobial agents.There are two obvious ways by which the breakdown of pantothenamides by pantetheinase can be prevented: (i) development of a pantetheinase inhibitor that can be coadministered with the pantothenamide, a strategy recently demonstrated to b...

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.

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.

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.

SummaryPantothenamides are known for their in vitro antimicrobial activity. Our group has previously reported a new stereoselective route to access derivatives modified at the geminal dimethyl moiety. This route however fails in the addition of large substituents. Here we report a new synthetic route that exploits the known allyl derivative, allowing for the installation of larger groups via cross-metathesis. The method was applied in the synthesis of a new pantothenamide with improved stability in human blood.