Targeting Thiamine (Vitamin B1) Metabolism and Utilisation in Plasmodium

Abstract

Despite having the ability to synthesize thiamine, Plasmodium falciparum still needs to obtain it from external sources to meet its requirements. Thiamine is converted into thiamine pyrophosphate (TPP), by the enzyme thiamine pyrophosphokinase (TPK). TPP serves as a cofactor for several TPP-dependent enzymes. This study explored thiamine metabolism in Plasmodium as a potential target for antimalarial drugs using several approaches.

Oxythiamine, a thiamine analogue, is metabolized by TPK into the antimetabolite oxythiamine pyrophosphate (OxyPP), which is toxic to Plasmodium parasites. To discover a more potent antiplasmodial thiamine analog, various commercial and synthesized analogs were tested against P. falciparum, P. knowlesi and P. berghei. From a series of 19 triazole derivatives synthesised by our colleagues, the 4'-N-benzyl derivative exhibited low micromolar antiplasmodial activity, whilst a hydroxamate derivative showed improvements in the antiplasmodial activity compared to the oxythiamine. Among the commercially available thiamine analogs, N3-pyridyl thiamine (N3PT) was found to suppress P. falciparum proliferation with an IC50 value that is 10 times lower than that of oxythiamine. It was also active against P. knowlesi and less toxic to human fibroblast cells compared to oxythiamine. The antiplasmodial activity of N3PT was reduced when the thiamine concentration in the culture medium was increased, consistent with the compound acting on the thiamine metabolism/utilisation pathway. Transgenic P. falciparum parasites overexpressing TPK were highly sensitive to N3PT, consistent with the compound being metabolised into an inactive TPP analog. N3PT decreased [3H]thiamine accumulation (a combination of transport and metabolism) in isolated parasites. Furthermore, N3PT reduced the parasitemia in P. berghei-infected mice, extended the time to display malaria symptoms, and appeared to be non-toxic.

To deepen our understanding of the antiplasmodial mechanism of action of oxythiamine, in vitro evolution was used to generate oxythiamine-resistant parasites. Whole-genome sequencing identified a mutation in PfTPK at position 284. Oxythiamine-resistant parasites accumulated five times less [3H]thiamine/[3H]TPP than wild-type parasites, and the activity of oxythiamine against parasites expressing a GFP-tagged version of mutated or wild-type PfTPK was altered. Overall, the data are consistent with the mutated PfTPK having reduced activity, providing an explanation for the oxythiamine resistance phenotype.

To investigate the role of TPK throughout the parasite's life cycle, P. berghei parasites lacking TPK (PbTPK-KO) were generated. No defect was observed in PbTPK-KO during the intraerythrocytic stage, but they were resistant (5-fold) to oxythiamine in vivo, confirming a role for TPK in the antiplasmodial activity of oxythiamine. Although PbTPK-KO produce a similar number of oocysts compared to wild-type parasites, PbTPK-KO oocyst failed to fully mature. Transmission electron microscopy (TEM) of mosquito midguts infected with PbTPK-KO parasites revealed small oocysts containing large empty spaces and no sporozoites. Mosquito dissection and mouse infection via mosquito bite confirmed the absence of sporozoites in PbTPK-KO parasites.

Transketolase (TK) is a TPP-dependent enzyme involved in the non-oxidative branch of the pentose phosphate pathway. Transgenic P. falciparum parasites were generated that express a GFP-tagged PfTK (PfTK-GFP) in addition to the endogenous PfTK. PfTK-GFP localized primarily to the parasite cytosol, in contrast to a previous study. Using a fluorometric TK assay, immunoprecipitated PfTK-GFP was found to be active and could be inhibited by phosphorylated oxythiamine. Lysates prepared from PfTK-GFP-expressing parasites have 1.7-times higher TK activity compared to control parasite lysates. Surprisingly, parasites expressing PfTK-GFP did not have an altered sensitivity to oxythiamine. Show more