cPantothenamides inhibit blood-stage Plasmodium falciparum with potencies (50% inhibitory concentration [IC 50 ], ϳ20 nM) similar to that of chloroquine. They target processes dependent on pantothenate, a precursor of the essential metabolic cofactor coenzyme A. However, their antiplasmodial activity is reduced due to degradation by serum pantetheinase. Minor modification of the pantothenamide structure led to the identification of ␣-methyl-N-phenethyl-pantothenamide, a pantothenamide resistant to degradation, with excellent antiplasmodial activity (IC 50 , 52 ؎ 6 nM), target specificity, and low toxicity.O ne-half of the world's population (ϳ3.4 billion people) is at risk of contracting malaria, with pregnant women and children Ͻ5 years of age being especially vulnerable. In 2013, the WHO estimated that malaria caused ϳ584,000 deaths globally, with the majority occurring in Africa (1). Although efforts to control and to eliminate malaria in the past 15 years have saved an estimated 3.3 million lives (1), drug-resistant parasites continue to emerge (2). This places the progress in the fight against the disease under pressure, especially since there is no effective vaccine against malaria (3). Several new drug targets have been identified in recent years (4); however, these targets now need to be exploited through the development of directed treatments.We are interested in targeting the biosynthesis of the essential cofactor coenzyme A (CoA) from the water-soluble vitamin B 5 (pantothenate, compound 1 in Fig. 1) for antimalarial drug development (5, 6). It has been shown that extracellular pantothenate is essential for intracellular malaria parasites (7), which indicates that Plasmodium falciparum does not utilize exogenous CoA but must synthesize CoA de novo (8).Pantothenate analogues interfere with the ability of P. falciparum to utilize the vitamin, with many analogues being characterized as growth inhibitors of the blood-stage parasites (9-11). Furthermore, a recent study showed that CoA biosynthesis can be targeted by a chemically diverse set of inhibitors that do not resemble pantothenate, the most potent of which had a 50% inhibitory concentration (IC 50 ; the concentration that inhibits parasite proliferation by 50%) of 120 nM against blood-stage parasites (12). These studies support pantothenate utilization (and therefore CoA biosynthesis and CoA-dependent processes) as an antiplasmodial target.Recently we showed that N-substituted pantothenamides (PanAms), a specific class of pantothenate analogues, have excellent antiplasmodial activity. Among these, N-phenethyl-pantothenamide (N-PE-PanAm) (compound 2 in Fig. 1) exhibited an IC 50 of 20 nM (13); this potency is comparable to that of chloroquine (14, 15). In practice, however, the antiplasmodial activity of the PanAms is decreased since they are degraded by pantetheinase (13), a ubiquitous enzyme of the Vanin protein family that is present in serum (16,17). Pantetheinase normally catalyzes the hydrolysis of pantetheine (a CoA-derived metabolite) to for...

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 (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 β-alanine moiety of PanAms increases their stability in serum and identified N-phenethyl-α-methylpantothenamide 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.

Intracellular parasites, such as the apicomplexan Toxoplasma gondii, are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. TgApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T. gondii. Here, we show that the abundance of TgApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite’s external environment, increasing in response to decreased [Arg]. Using a luciferase-based ‘biosensor’ strain of T. gondii, we demonstrate that the expression of TgApiAT1 varies between different organs within the host, indicating that parasites are able to modulate TgApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo. Finally, we show that Arg-dependent regulation of TgApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the TgApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.