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 falciparum with submicromolar potency by exerting an effect on pantothenic acid utilization, but only when the pantetheinase present in the growth medium has been inactivated. Here, we demonstrate that small modifications of the pantothenamide core structure are sufficient to counteract pantetheinase-mediated degradation and that the resulting pantothenamide analogues still inhibit the in vitro proliferation of P. falciparum by targeting a pantothenic acid-dependent process (or processes). Finally, we investigated the toxicity of the most potent analogues to human cells and show that the selectivity ratio exceeds 100 in one case. Taken together, these results provide further support for pantothenic acid utilization being a viable target for antimalarial drug discovery.
The redesign of enzymes to produce catalysts for a predefined transformation remains a major challenge in protein engineering. Here, we describe the structure-based engineering of methylaspartate ammonia lyase (which in nature catalyses the conversion of 3-methylaspartate to ammonia and 2-methylfumarate) to accept a variety of substituted amines and fumarates and catalyse the asymmetric synthesis of aspartic acid derivatives. We obtained two single-active-site mutants, one exhibiting a wide nucleophile scope including structurally diverse linear and cyclic alkylamines and one with broad electrophile scope including fumarate derivatives with alkyl, aryl, alkoxy, aryloxy, alkylthio and arylthio substituents at the C2 position. Both mutants have an enlarged active site that accommodates the new substrates while retaining the high stereo- and regioselectivity of the wild-type enzyme. As an example, we demonstrate a highly enantio- and diastereoselective synthesis of threo-3-benzyloxyaspartate (an important inhibitor of neuronal excitatory glutamate transporters in the brain).
N-substituted pantothenamides are analogues of pantothenic acid, the vitamin precursor of CoA, and constitute a class of well-studied bacterial growth inhibitors that show potential as new antibacterial agents. Previous studies have highlighted the importance of pantothenate kinase (PanK; EC 2.7.1.33) (the first enzyme of CoA biosynthesis) in mediating pantothenamide-induced growth inhibition by one of two proposed mechanisms: first, by acting on the pantothenamides as alternate substrates (allowing their conversion into CoA antimetabolites, with subsequent effects on CoA-and acyl carrier protein-dependent processes) or, second, by being directly inhibited by them (causing a reduction in CoA biosynthesis). In the present study we used structurally modified pantothenamides to probe whether PanKs interact with these compounds in the same manner. We show that the three distinct types of eubacterial PanKs that are known to exist (PanK I , PanK II and PanK III ) respond very differently and, consequently, are responsible for determining the pantothenamide mode of action in each case: although the promiscuous PanK I enzymes accept them as substrates, the highly selective PanK III s are resistant to their inhibitory effects. Most unexpectedly, Staphylococcus aureus PanK (the only known example of a bacterial PanK II ) experiences uncompetitive inhibition in a manner that is described for the first time. In addition, we show that pantetheine, a CoA degradation product that closely resembles the pantothenamides, causes the same effect. This suggests that, in S. aureus, pantothenamides may act by usurping a previously unknown role of pantetheine in the regulation of CoA biosynthesis, and validates its PanK as a target for the development of new antistaphylococcal agents.
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 biosynthesis and utilization of CoA (coenzyme A), the ubiquitous and essential acyl carrier in all organisms, have long been regarded as excellent targets for the development of new antimicrobial drugs. Moreover, bioinformatics and biochemical studies have highlighted significant differences between several of the bacterial enzyme targets and their human counterparts, indicating that selective inhibition of the former should be possible. Over the past decade, a large amount of structural and mechanistic data has been gathered on CoA metabolism and the CoA biosynthetic enzymes, and this has facilitated the discovery and development of several promising candidate antimicrobial agents. These compounds include both target-specific inhibitors, as well as CoA antimetabolite precursors that can reduce CoA levels and interfere with processes that are dependent on this cofactor. In the present mini-review we provide an overview of the most recent of these studies that, taken together, have also provided chemical validation of CoA biosynthesis and utilization as viable targets for antimicrobial drug development.
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