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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 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.

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.

Malaria kills more than half a million people each year. There is no vaccine, and recent reports suggest that resistance is developing to the antimalarial regimes currently recommended by the World Health Organization. New drugs are therefore needed to ensure malaria treatment options continue to be available. The intra-erythrocytic stage of the malaria parasite's life cycle is dependent on an extracellular supply of pantothenate (vitamin B5), the precursor of CoA (coenzyme A). It has been known for many years that proliferation of the parasite during this stage of its life cycle can be inhibited with pantothenate analogues. We have shown recently that pantothenamides, a class of pantothenate analogueswith antibacterial activity, inhibit parasite proliferation at submicromolar concentrations and do so competitively with pantothenate. These compounds, however, are degraded, and therefore rendered inactive, by the enzyme pantetheinase (vanin), which is present in serum. In the present mini-review, we discuss the two strategies that have been put forward to overcome pantetheinase-mediated degradation of pantothenamides. The strategies effectively provide an opportunity for pantothenamides to be tested in vivo. We also put forward our 'blueprint' for the further development of pantothenamides (and other pantothenate analogues) as potential antimalarials.

We describe here a simple, miniaturized radiation-based phosphorylation assay that can be used to monitor phosphorylation of a diverse range of small molecule substrates in the presence of purified and crude enzyme preparations. Ba(OH)2 and ZnSO4 are used to terminate phosphoryl transfer and to precipitate selectively the phosphorylated reaction product in a single step; non-phosphorylated substrate is removed by filtration prior to quantification. The key advantages over alternative radiation-based assays are that: (i) high-energy/short-lived radioactive emitters are not required; (ii) high-quality data can be obtained without the need for high radioactivity concentrations; and (iii) the assay is compatible with high-throughput applications.

Pantothenamides are secondary or tertiary amides of pantothenic acid, the vitamin precursor of the essential cofactor and universal acyl carrier coenzyme A. A recent study has demonstrated that pantothenamides inhibit the growth of blood-stage Plasmodium

In the absence of an effective vaccine against malaria suitable for widespread deployment, the control of this lethal infectious disease relies heavily on antimalarial chemotherapies. The most virulent of the parasites that cause malaria (Plasmodium falciparum) has, however, developed resistance to all antimalarial agents in clinical use, and there is a desperate need for new antimalarial agents that target previously unexploited parasite processes. P. falciparum requires an extracellular supply of pantothenate to support its proliferation during the erythrocytic stage of its development in humans. This requirement highlights the mechanisms involved in the utilization (uptake and metabolism) of pantothenate as potential targets for chemotherapeutic attack. Here we review the evidence demonstrating pantothenate to be an essential nutrient for P. falciparum and data from studies investigating whether this parasite has the capacity to utilize exogenous supplies of the cofactor (coenzyme A; CoA) for which pantothenate serves as a precursor. The results of recent studies aimed at characterizing the mechanisms by which pantothenate is taken up by the P. falciparum-infected erythrocyte and intracellular parasite, and metabolized to CoA, are described. The unique properties that may be exploited to develop selective inhibitors of pantothenate utilization by P. falciparum-infected erythrocytes are highlighted. The molecular identities of P. falciparum pantothenate transporter(s) and CoA biosynthesis enzymes remain unconfirmed. We consider the possible identities, and emphasize the importance of generating these proteins in pure, functionally-active form. The tools currently available for identifying inhibitors of pantothenate utilization that may be potent antiplasmodial agents are also discussed.

Pantothenic acid, a precursor of coenzyme A (CoA), is essential for the growth of pathogenic microorganisms. Since the structure of pantothenic acid was determined, many analogues of this essential metabolite have been prepared. Several have been demonstrated to exert an antimicrobial effect against a range of microorganisms by inhibiting the utilization of pantothenic acid, validating pantothenic acid utilization as a potential novel antimicrobial drug target. This review commences with an overview of the mechanisms by which various microorganisms acquire the pantothenic acid they require for growth, and the universal CoA biosynthesis pathway by which pantothenic acid is converted into CoA. A detailed survey of studies that have investigated the inhibitory activity of analogues of pantothenic acid and other precursors of CoA follows. The potential of inhibitors of both pantothenic acid utilization and biosynthesis as novel antibacterial, antifungal and antimalarial agents is discussed, focusing on inhibitors and substrates of pantothenate kinase, the enzyme catalysing the rate-limiting step of CoA biosynthesis in many organisms. The best strategies are considered for identifying inhibitors of pantothenic acid utilization and biosynthesis that are potent and selective inhibitors of microbial growth and that may be suitable for use as chemotherapeutic agents in humans.