susceptibilities to ganaplacide and diversity in potential resistance mediators in Ugandan isolates.

Novel antimalarials are urgently needed to combat rising resistance to available drugs. The imidazolopiperazine ganaplacide is a promising drug candidate, but decreased susceptibility of laboratory strains has been linked to polymorphisms in the cyclic amine resistance locus (PfCARL), acetyl-CoA transporter (PfACT), and UDP-galactose transporter (PfUGT). To characterize parasites causing disease in Africa, we assessed drug susceptibilities to ganaplacide in 750 . isolates collected in Uganda from 2017 to 2023. Drug susceptibilities were assessed using a 72-hour SYBR Green growth inhibition assay. The median IC for ganaplacide was 13.8 nM, but some isolates had up to 31-fold higher ICs (31/750 with IC > 100 nM). To assess genotype-phenotype associations, we sequenced genes potentially mediating altered ganaplacide susceptibility in the isolates using molecular inversion probe and dideoxy sequencing methods. PfCARL was highly polymorphic, with eight mutations present in >5% of isolates. None of these eight mutations had previously been selected in laboratory strains with drug pressure and none were found to be significantly associated with decreased ganaplacide susceptibility. Mutations in PfACT and PfUGT were found in ≤5% of isolates, except for two frequent (>20%) mutations in PfACT; one mutation in PfACT (I140V) was associated with a modest decrease in susceptibility. Overall, Ugandan isolates were mostly highly susceptible to ganaplacide. Known resistance mediators were polymorphic, but mutations previously selected with drug pressure were not seen, and mutations identified in the Ugandan isolates were generally not associated with decreased ganaplacide susceptibility.