Pentafluoro‐3‐hydroxy‐pent‐2‐en‐1‐ones Potently Inhibit FNT‐Type Lactate Transporters from all Five Human‐Pathogenic Plasmodium Species

Abstract

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.

Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors

“…We circumvented the PfFNT G107S resistance by a strictly linear vinylogous acid-type compound with a nitrogen atom in the aromatic ring for hydrogen bonding with the serine-hydroxyl, i.e., BH267.meta (Figure 1) [12]. The compound further blocks FNT-type lactate transporters of the remaining four human-pathogenic plasmodia at sub-micromolar concentrations [13]. Sub-lethal doses of MMV007839 selected resistant parasites that carried a single PfFNT G107S point mutation shifting the MMV007839 efficacy by two orders of magnitude (IC 50 21 µM, EC 50 35 µM) [4].…”

“…We circumvented the PfFNT G107S resistance by a strictly linear vinylogous acid-type compound with a nitrogen atom in the aromatic ring for hydrogen bonding with the serine-hydroxyl, i.e., BH267.meta ( Figure 1 ) [ 12 ]. The compound further blocks FNT-type lactate transporters of the remaining four human-pathogenic plasmodia at sub-micromolar concentrations [ 13 ].…”

Structural characterization of the Plasmodium falciparum lactate transporter PfFNT alone and in complex with antimalarial compound MMV007839 reveals its inhibition mechanism

“…falciparum parasites, BH267.meta prevented the formation of drug resistance even when the parasite was treated with it for a long period [ 18 ]. Moreover, a recent study revealed that BH267.meta inhibits a broad spectrum of FNTs, including all causal agents of human malaria, indicating its potential application in further chemotherapeutic development [ 27 ].…”

Discovery and Development of Inhibitors of the Plasmodial FNT-Type Lactate Transporter as Novel Antimalarials

“…Sequence comparison of the FNT proteins from the five Plasmodium species that infect humans (P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi) showed > 84% overall similarity with the internal substrate transport path, with the isoforms being virtually identical [24]. The vital functionality and lack of similarity with the human MCTs appeared to be beneficial for the design of PfFNT-specific antimalarials.…”

“…Eventually, with BH267.meta, a compound was generated exhibiting nanomolar efficacy on the PfFNT wildtype and G107S mutant protein, as well as in 3D7 P. falciparum parasite in vitro cultures. Furthermore, BH267.meta showed very low cytotoxicity towards human cells [24] and minimal off-target potency on the human lactate transporter, MCT1 [24,37]. Cell viability as determined by ATP quantity and resazurin reduction and cell proliferation measured by nuclei count was tested using human kidney (HEK293) and liver (HepG2) cell lines.…”