Antiplasmodial Mode of Action of Pantothenamides: Pantothenate Kinase Serves as a Metabolic Activator Not as a Target

Abstract

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 parasite 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.

Structure–Activity Relationships of Antiplasmodial Pantothenamide Analogues Reveal a New Way by Which Triazoles Mimic Amide Bonds

“…It can be difficult to draw SARs with pantothenamides or their analogues, because their antimicrobial activity is affected by multiple factors such as the participation of more than one enzyme during bioactivation, the possible involvement of multiple different targets in the mode of action (as expected for pantothenate antimetabolites), and of course, cell permeability. Our results with triazole derivatives mimicking pantothenamides clearly imply that the 1,4‐substitution pattern on the triazole ring, with a simple methylene linker between the triazole and the pantoyl moieties, can overcome all the mechanistic barriers and display antiplasmodial activity at low nanomolar concentrations.…”

“…It can be difficult to draw SARs with pantothenamides or their analogues, [5,8,10,16,18,24,25,30] because their antimicrobiala ctivity is affected by multiple factors such as the participation of more than one enzyme during bioactivation, the possible involvement of multiple differentt argets in the mode of action (as expected forp antothenatea ntimetabolites), and of course,c ell Table 1. Effect of compounds 6a-b and 8a-i on the proliferation of P. falciparum in the presence of pantetheinases (unlesso therwise noted), reported as the 50 %i nhibitory concentration( IC 50 ), and predicted physicochemical parameters of compounds 1, 2, 6a-b,a nd 8a-i,i ncluding lipophilicity (LogP), molarr efractivity (MR), topological polar surface area (tPSA) or aqueous solubility (LogS).…”

“…Pantothenamides were later found to be metabolically activated by enzymes of the coenzyme A (CoA) biosynthetic pathway in P . falciparum . This may not only affect CoA biosynthesis but the resulting metabolites are also inferred to inhibit downstream CoA‐utilizing pathways, leading to lethal effects on P .…”

“…falciparum. [9][10][11] This may not only affect CoA biosynthesis but the resulting metabolites are also inferred to inhibit downstream CoA-utilizing pathways, leading to lethal effects on P. falciparum, [9,10] as suggested for bacteria. [12][13][14][15][16][17][18][19][20] Based on their synthetic accessibility,h igh potency and low cytotoxicity,pantothenamides would be excellent candidatesf or antimalarial drug development.…”

Genetic Characterization of Coenzyme A Biosynthesis Reveals Essential Distinctive Functions during Malaria Parasite Development in Blood and Mosquito

“…Earlier studies in bird malaria parasites and recent studies in the human malaria parasite P. falciparum have shown that pantothenate analogs and pantothenamides inhibit intra-erythrocytic development of P. falciparum (Spry et al, 2005, 2010, 2013; de Villiers et al, 2017), and exert their antimalarial activity by possibly inhibiting pantothenate phosphorylation or the phosphorylation of alternative substrates (e.g., pantetheine) and thus blocking CoA biosynthesis. The cellular machinery for the biosynthesis of CoA from exogenous pantothenate involves a putative pantothenate transporter (PAT) and five enzymes, PanK (Pantothenate Kinase), PPCS (Phosphopantothenylcysteine Synthase), PPCDC (Phosphopantothenylcysteine Decarboxylase), PPAT (Phosphopantetheine Adenylyltransferase), and DPCK (Dephospho-CoA Kinase) (Leonardi et al, 2005; Spry et al, 2008).…”