Despite decades of dedicated research, there remains a dire need for new drugs against tuberculosis (TB). Current therapies are generations old and problematic. Resistance to these existing therapies results in an ever-increasing burden of patients with disease that is difficult or impossible to treat. Novel chemical entities with new mechanisms of action are therefore earnestly required. The biosynthesis of coenzyme A (CoA) has long been known to be essential in Mycobacterium tuberculosis (Mtb), the causative agent of TB. The pathway has been genetically validated by seminal studies in vitro and in vivo. In Mtb, the CoA biosynthetic pathway is comprised of nine enzymes: four to synthesize pantothenate (Pan) from L-aspartate and a-ketoisovalerate; five to synthesize CoA from Pan and pantetheine (PantSH). This review gathers literature reports on the structure/ mechanism, inhibitors, and vulnerability of each enzyme in the CoA pathway. In addition to traditional inhibition of a single enzyme, the CoA pathway offers an antimetabolite strategy as a promising alternative. In this review, we provide our assessment of what appear to be the best targets, and, thus, which CoA pathway enzymes present the best opportunities for antitubercular drug discovery moving forward.

Pantothenamides (PanAms) are analogues of pantothenate, the biosynthetic precursor of coenzyme A (CoA), and show potent antimicrobial activity against several bacteria and the malaria parasite in vitro. However, pantetheinase enzymes that normally degrade pantetheine in human serum also act on the PanAms, thereby reducing their potency. In this study, we designed analogues of the known antibacterial PanAm N-heptylpantothenamide (N7-Pan) to be resistant to pantetheinase by using three complementary structural modification strategies. We show that while two of these are effective in imparting resistance, the introduced modifications impact on the analogues’ interaction with pantothenate kinase (PanK, the first CoA biosynthetic enzyme), which act as metabolic activator and/or target of the PanAms. This, in turn, directly affects their mode of action. Importantly, we discover that the phosphorylated version of N7-Pan shows pantetheinase-resistance and antistaphylococcal activity, providing a lead for future studies in the ongoing search of PanAm analogues that show in vivo efficacy.

N-substituted pantothenamides (PanAms) are pantothenate analogues with up to nanomolar potency against blood-stage Plasmodium falciparum (the most virulent species responsible for malaria). Although these compounds are known to target coenzyme A (CoA) biosynthesis and/or utilization, their exact mode of action (MoA) is still unknown. Importantly, PanAms that retain the natural β-alanine moiety are more potent than other variants, consistent with the involvement of processes that are selective for pantothenate (the precursor of CoA) or its derivatives. The transport of pantothenate and its phosphorylation by P. falciparum pantothenate kinase (PfPanK, the first enzyme of CoA biosynthesis) are two such processes previously highlighted as potential targets for the PanAms' antiplasmodial action. In this study, we investigated the effect of PanAms on these processes using their radiolabeled versions (synthesized here for the first time), which made possible the direct measurement of PanAm uptake by isolated blood-stage parasites, and PanAm phosphorylation by PfPanK present in parasites lysates. We found that the MoA of PanAms does not involve interference with pantothenate transport, and that inhibition of PfPanK-mediated pantothenate phosphorylation does not correlate with PanAm antiplasmodial activity. Instead, PanAms that retain the β-alanine moiety were found to be metabolically activated by PfPanK in a selective manner, forming phosphorylated products that likely inhibit other steps in CoA biosynthesis, or are transformed into CoA antimetabolites that can interfere with CoA utilization. These findings provide direction for the ongoing development of CoA-targeted inhibitors as antiplasmodial agents with clinical potential.

We report a method for the simultaneous quantification of the essential metabolic cofactor coenzyme A (CoA) and its thiol-bearing precursors—including 4′-phosphopantetheine, which was recently shown to play a potentially important role as nexus metabolite in CoA biology—with pmol sensitivity. This sensitivity is gained by making use of an established thiol-derivatisation agent that produces a fluorophore upon labeling, which is subsequently separated by HPLC and quantified by fluorescence detection. While previous reports have made use of a similar strategy to quantify CoA, very few have extended the method to the CoA biosynthetic intermediates (some of which occur at levels much lower than CoA) or have critically evaluated its analytical performance. In this study we addressed these shortcomings, and also overcame the difficulty associated with the independent confirmation of the concentrations of the analytical standards used for quantification. The method's utility is showcased through time-course analyses of in vitro reconstituted enzyme reactions and by analysis of extracts from Escherichia coli and Staphylococcus aureus, demonstrating its potential in advancing studies of CoA biosynthesis and CoA-dependent biology in a wide range of systems.

The consensus has been that intracellular coenzyme A (CoA) is obtained exclusively by de novo biosynthesis via a universal, conserved five-step pathway in the cell cytosol. However, old and new evidence suggest that cells (and some microorganisms) have several strategies to obtain CoA, with 4′-phosphopantetheine (P-PantSH; the fourth intermediate in the canonical CoA biosynthetic pathway) serving as a 'nexus' metabolite. © 2016 Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.