Chemical and genetic validation of thiamine utilization as an antimalarial drug target

Abstract

Thiamine is metabolized into an essential cofactor for several enzymes. Here we show that oxythiamine, a thiamine analog, inhibits proliferation of the malaria parasite Plasmodium falciparum in vitro via a thiamine-related pathway and significantly reduces parasite growth in a mouse malaria model. Overexpression of thiamine pyrophosphokinase (the enzyme that converts thiamine into its active form, thiamine pyrophosphate) hypersensitizes parasites to oxythiamine by up to 1,700-fold, consistent with oxythiamine being a substrate for thiamine pyrophosphokinase and its conversion into an antimetabolite. We show that parasites overexpressing the thiamine pyrophosphate-dependent enzymes oxoglutarate dehydrogenase and pyruvate dehydrogenase are up to 15-fold more resistant to oxythiamine, consistent with the antimetabolite inactivating thiamine pyrophosphate-dependent enzymes. Our studies therefore validate thiamine utilization as an antimalarial drug target and demonstrate that a single antimalarial can simultaneously target several enzymes located within distinct organelles.

BCKDH: The Missing Link in Apicomplexan Mitochondrial Metabolism Is Required for Full Virulence of Toxoplasma gondii and Plasmodium berghei

“…Apicomplexa have a canonical PDH, but this complex is targeted to the apicoplast, suggesting that other enzymes fulfil the function of the PDH in the mitochondrion [13]–[15]. Recent studies have raised the possibility that mitochondrial acetyl-CoA could be generated from glycolytic pyruvate via one of the other TPP-dependent mitochondrial dehydrogenases [22], [33]. However, direct evidence for involvement of either BCKDH or α-KDH, or another uncharacterized dehydrogenase has not been obtained.…”

“…The possibility that other mitochondrial dehydrogenases may individually or collectively fill this missing link was raised by the finding that the P. falciparum α-ketoglutarate dehydrogenase (α-KDH) can catalyze the conversion of pyruvate to acetyl-CoA in vitro , although slightly less efficiently than PDH [33]. On the other hand, Cobbold et al, proposed that the P. falciparum branched chain ketoacid dehydrogenase (BCKDH), the only enzyme implicated in BCAA degradation retained in the Plasmodium spp., may substitute for PDH based on the finding that catabolism of glucose in the TCA cycle in a P. falciparum PDH mutant was inhibited by oxythiamine, an inhibitor of thiamine pyrophosphate (TPP)-dependent dehydrogenases [22].…”

Multiphoton fluorescence lifetime imaging microscopy reveals free-to-bound NADH ratio changes associated with metabolic inhibition

“…A dramatic decrease in tumor cell proliferation after the administration of OT has been observed in several in vitro and in vivo tumor models, which has been interpreted as due to direct inhibition via TK of PPP reactions, which causes a G1 cell cycle arrest without interference with cell energy production. [30][31][32] However, a large variety of enzymes catalyzing many different classes of reactions require the coenzyme TPP, [33][34][35] and because of that, OT is expected to exhibit cell-type-specific confounding pleiotropic effects.…”

Exploiting the coenzyme A biosynthesis pathway for the identification of new antimalarial agents: the case for pantothenamides

“…[13]. Several nutrient acquisition, synthesis and utilization pathways have been validated as antiplasmodial drug targets [14,15] and some serve as targets of established antimalarials (e.g. the antifolate combination sulfadoxine/pyrimethamine [16]).…”

“…Knowledge of the precise mechanism will facilitate the design of further analogues. Targeted overexpression of specific genes [15] and generation of drug-resistant parasites coupled to whole-genome analysis (reviewed in [57]) have both proven to be effective strategies for identifying the mechanism of action of antiplasmodial agents.…”