Abstract
Growth of the virulent human malaria parasite Plasmodium falciparum is dependent on an extracellular supply of pantothenate (vitamin B5) and is susceptible to inhibition by pantothenate analogues that hinder pantothenate utilization. In this study, on the hunt for pantothenate analogues with increased potency relative to those reported previously, we screened a series of pantothenamides (amide analogues of pantothenate) against P. falciparum and show for the first time that analogues of this type possess antiplasmodial activity. Although the active pantothenamides in this series exhibit only modest potency under standard in vitro culture conditions, we show that the potency of pantothenamides is selectively enhanced when the parasite culture medium is pre-incubated at 37°C for a prolonged period. We present evidence that this finding is linked to the presence in Albumax II (a serum-substitute routinely used for in vitro cultivation of P. falciparum) of pantetheinase activity: the activity of an enzyme that hydrolyzes the pantothenate metabolite pantetheine, for which pantothenamides also serve as substrates. Pantetheinase activity, and thereby pantothenamide degradation, is reduced following incubation of Albumax II-containing culture medium for a prolonged period at 37°C, revealing the true, sub-micromolar potency of pantothenamides. Importantly we show that the potent antiplasmodial effect of pantothenamides is attenuated with pantothenate, consistent with the compounds inhibiting parasite proliferation specifically by inhibiting pantothenate and/or CoA utilization. Additionally, we show that the pantothenamides interact with P. falciparum pantothenate kinase, the first enzyme involved in converting pantothenate to coenzyme A. This is the first demonstration of on-target antiplasmodial pantothenate analogues with sub-micromolar potency, and highlights the potential of pantetheinase-resistant pantothenamides as antimalarial agents.
Highlights
Every day approximately half of the world’s population is at risk of contracting malaria, a lethal infectious disease estimated to have claimed 655 000 lives [1] in 2010
We show, using an in vitro primary amine detection assay, that a pantothenamide selected from the series tested here is hydrolyzed in the presence of Albumax II, demonstrating Albumax II to be a source of pantetheinase activity
A series of 22 previously published [18] pantothenamides was tested in vitro for antiplasmodial activity against erythrocytic stage P. falciparum parasites in 96 h growth assays initiated with parasites predominantly in the ring stage
Summary
Every day approximately half of the world’s population is at risk of contracting malaria, a lethal infectious disease estimated to have claimed 655 000 lives [1] (if not more [2]) in 2010. Plasmodium falciparum has an absolute requirement for exogenous pantothenate (vitamin B5; Figure 1) [4,5,6], a precursor of the essential enzyme cofactor coenzyme A (CoA). The resultant 4’-phosphopantothenamides are further metabolized by the CoA biosynthesis pathway of bacteria to yield analogues of CoA [17]. Such CoA analogues have been shown to be incorporated by, and inhibit the function of, acyl carrier protein [16,19], a protein involved in fatty acid biosynthesis that requires the 49-phosphopantetheine moiety of CoA for activation. Whether the mechanism that results in bacteriostasis is inhibition of CoA biosynthesis [20], fatty acid biosynthesis [19] or another CoA-utilizing process, or a combination of the above, remains to be resolved
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