The spreading of malaria parasites, Plasmodium falciparum, with resistance to all known drugs calls for novel classes of inhibitors with new modes of action. Recently, we discovered and validated the plasmodial L-lactate transporter, PfFNT, as a novel antimalarial drug target. However, treatment of parasites with a screening hit from the malaria box compound collection, MMV007839, gave rise to a PfFNT Gly107Ser resistance mutation decreasing inhibitor affinity by 2 orders of magnitude. Here, we show that newly introduced nitrogen atoms into the inhibitor scaffold can act as hydrogen bond acceptor sites to the serine hydroxyl. The gain in affinity led to almost equal inhibition of wildtype PfFNT and the Gly107Ser mutation. The most potent inhibitor of this work, BH267.meta, killed cultured P. falciparum parasites with nanomolar efficacy and did not give rise to new resistance formation in vitro. Its deduced pharmacokinetic properties appear suitable for further drug development.

The protozoan parasite Plasmodium falciparum causes the most severe and prevailing form of malaria in sub-Saharan Africa. Previously, we identified the plasmodial lactate transporter, PfFNT, a member of the microbial formate-nitrite transporter family, as a novel antimalarial drug target. With the pentafluoro-3-hydroxy-pent-2-en-1-ones, we discovered PfFNT inhibitors that potently kill P. falciparum parasites in vitro. Four additional human-pathogenic Plasmodium species require attention, that is, P. vivax, most prevalent outside of Africa, and the regional P. malariae, P. ovale and P. knowlesi. Herein, we show that the plasmodial FNT variants are highly similar in terms of protein sequence and functionality. The FNTs from all human-pathogenic plasmodia and the rodent malaria parasite were efficiently inhibited by pentafluoro-3-hydroxy-pent-2-en-1-ones. We further established a phenotypic yeast-based FNT inhibitor screen, and found very low compound cytotoxicity and monocarboxylate transporter 1 off-target activity on human cells, particularly of the most potent FNT inhibitor BH267.meta, allowing these compounds to proceed towards animal model malaria studies.

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Malaria parasites in the blood stage express a single transmembrane transport protein for the release of the glycolytic end product l -lactate/H + from the cell. This transporter is a member of the strictly microbial formate-nitrite transporter (FNT) family and a novel putative drug target.