The antimalarial activity of the pantothenamide α-PanAm is via inhibition of pantothenate phosphorylation

Abstract

The biosynthesis of the major acyl carrier Coenzyme A from pantothenic acid (PA) is critical for survival of Plasmodium falciparum within human erythrocytes. Accordingly, a PA analog α-PanAm showed potent activity against blood stage parasites in vitro; however, its efficacy in vivo and its mode of action remain unknown. We developed a new synthesis route for α-PanAm and showed that the compound is highly effective against blood stages of drug-sensitive and -resistant P. falciparum strains, inhibits development of P. berghei in hepatocytes, and at doses up to 100 mg/kg also inhibits blood stage development of P. chabaudi in mice. We used yeast and its pantothenate kinase Cab1 as models to characterize mode of action of α-PanAm and found that α-PanAm inhibits yeast growth in a PA-dependent manner, and its potency increases dramatically in a yeast mutant with defective pantothenate kinase activity. Biochemical analyses using 14C-PA as a substrate demonstrated that α-PanAm is a competitive inhibitor of Cab1. Interestingly, biochemical and mass spectrometry analyses also showed that the compound is phosphorylated by Cab1. Together, these data suggest that α-PanAm exerts its antimicrobial activity by direct competition with the natural substrate PA for phosphorylation by the pantothenate kinase.

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

“…The 1,2,3‐triazole ring is among the most commonly used scaffolds in drugs . The triazole derivatives 1 and 2 are stable in serum, show excellent antiplasmodial activity, are not toxic to human cells and have a new mode of action relative to clinical drugs, making them attractive hits for the development of novel antimalarials.…”

“…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 .…”

The yeast pantothenate kinase Cab1 is a master regulator of sterol metabolism and of susceptibility to ergosterol biosynthesis inhibitors

“…A mutant, cab1 ts , which produces a kinase enzyme with glycine 351 substituted to serine, was found to be viable at 30°C but is completely inviable at 37°C (8). Biochemical assays using purified WT and mutated Cab1p enzymes showed that the G351S substitution results in ϳ94% loss of enzyme activity (9). Recent studies showed that the pantothenate analog ␣-PanAm 2 inhibits yeast growth in a pantothenic acid dose-dependent manner (9).…”

“…Biochemical assays using purified WT and mutated Cab1p enzymes showed that the G351S substitution results in ϳ94% loss of enzyme activity (9). Recent studies showed that the pantothenate analog ␣-PanAm 2 inhibits yeast growth in a pantothenic acid dose-dependent manner (9). The MIC 50 of the compound was found to be ϳ4 times higher in medium supplemented with 1 M pantothenic acid (MIC 50 ϳ 7.6 g/ml) compared with medium with 100 nM pantothenic acid (MIC 50 ϳ 1.9 g/ml) (9).…”

“…Recent studies showed that the pantothenate analog ␣-PanAm 2 inhibits yeast growth in a pantothenic acid dose-dependent manner (9). The MIC 50 of the compound was found to be ϳ4 times higher in medium supplemented with 1 M pantothenic acid (MIC 50 ϳ 7.6 g/ml) compared with medium with 100 nM pantothenic acid (MIC 50 ϳ 1.9 g/ml) (9). In vitro pantothenate phosphorylation assays and MS analysis showed that ␣-PanAm is also phosphorylated by Cab1p and acts as a competitor of pantothenic acid at the enzyme catalytic site (9).…”