KAF156 Resistant Lines Conferring Mutations in Acetyl-CoA Transporter. Plasmodium falciparum strain:3D7 and Dd2

The growing emergence of resistance to anti-infectives is a global problem, threatening to derail disease control programs. Malaria drug resistance is no exception. As new classes of drugs are poised to enter the antimalarial pipeline, understanding the mechanisms of resistance (MoR) becomes critical for disease control in the broader context of proper combination therapies, phenotypic and/or genotypic diagnostics, and targeted treatment in the clinic. Here, we report the establishment of a platform for the in vitro selection of spontaneous Plasmodium falciparum drug-resistant mutants. The unique advantages of this approach include the rapid selection and elucidation of resistance-associated genes with minimal genetic drift, identification of low-fitness mutants as a result of early clonal dilutions, and prediction of the parasite mutation rate for a given drug. The success of this forward chemical genetics approach was exemplified in our selection for in vitro spontaneous mutants resistant to KAF156 and KAF179, imidazolopiperazine analogs with activity in both liver- and blood-stage parasites. Two new mechanisms of resistance, comprising a UDP-galactose transporter (pfugt) and acetyl-CoA transporter (pfact) were identified, in addition to the previously identified cyclic amine resistance locus (pfcarl), for this compound series. Mutations in pfugt and pfact resulted in elevated levels of resistance, extensive morphological changes and a noticeable loss of fitness, in contrast to pfcarl mutants which displayed minimal fitness loss. The new mechanisms of resistance were validated by reverse genetics using the CRISPR/Cas9 system and through an in vivo selection of resistant P. berghei mutants. Our establishment of this platform to rapidly select new spontaneous mutants will help facilitate the deconvolution of resistance mechanisms and the identification of potential new targets for the design of new antimalarial therapies.