Mutations within and outside the PfFNT transport pathway drive MMV007839 resistance in Plasmodium falciparum

Plasmodium falciparum, the parasite responsible for the majority of malaria cases and deaths worldwide, poses a major global health challenge due to its ability to rapidly develop resistance to antimalarial drugs. To circumvent existing resistance pathways, it is of interest to identify both new inhibitors and new therapeutic targets within the parasite. One such target is the Plasmodium falciparum Formate-Nitrite Transporter (PfFNT), which mediates essential lactate and proton (H+) export from the parasite cytosol during the intra-erythrocytic stage. Several inhibitors of PfFNT have been identified that block lactate/H+ transport and/or kill parasites in vitro, including MMV007839, which binds within the substrate transport pathway as revealed by cryo-EM structures. However, three resistance-conferring mutations have previously been identified following prolonged in vitro exposure of parasites to PfFNT inhibitors. Here, we performed further in vitro evolution experiments with MMV007839 in P. falciparum and identified two additional mutations in PfFNT, V200L and L198M, the latter of which is located outside the substrate transport pathway. These mutations are associated with 6-fold and 13-fold changes in the IC50 of MMV007839. Using molecular dynamics simulations, and thermodynamic integration, we show that these mutations, together with previously identified resistance mutations, destabilise inhibitor binding within the transport pathway; and we suggest the molecular mechanisms underlying this destabilisation. Finally, using both molecular docking and additional simulations, we suggest new sites where additional mutations that confer resistance to PfFNT inhibitors may arise.