Ex vivo susceptibilities to ganaplacide and diversity in potential resistance mediators in Ugandan Plasmodium falciparum isolates

Abstract

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 Plasmodium falciparum cyclic amine resistance locus (PfCARL), acetyl-CoA transporter (PfACT), and UDP-galactose transporter (PfUGT). To characterize parasites causing disease in Africa, we assessed ex vivo drug susceptibilities to ganaplacide in 750 P. falciparum isolates collected in Uganda from 2017 to 2023. Drug susceptibilities were assessed using a 72-hour SYBR Green growth inhibition assay. The median IC₅₀ for ganaplacide was 13.8 nM, but some isolates had up to 31-fold higher IC₅₀s (31/750 with IC₅₀ > 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 in vitro 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 P. falciparum isolates were mostly highly susceptible to ganaplacide. Known resistance mediators were polymorphic, but mutations previously selected with in vitro drug pressure were not seen, and mutations identified in the Ugandan isolates were generally not associated with decreased ganaplacide susceptibility.

Keywords: PfACT; PfCARL; PfUGT; Plasmodium falciparum; antimalarial drugs; drug susceptibility; ganaplacide; malaria.

Fig 1

Susceptibilities to ganaplacide and mutations in resistance mediators of Ugandan parasites. (A) IC₅₀s of individual isolates (gray dots). The horizontal line shows the median and whiskers the interquartile range. Mean IC₅₀s of control parasites 3D7 (red) and Dd2 (blue) are depicted as colored dots. Mutations in PfCARL (B) and PfACT (C) were observed in >5% of Ugandan P. falciparum isolates. Depicted are mutation frequencies of mixed (WT and mutant; blue) and mutant (red) isolates.

Fig 2

Spearman rank correlation between susceptibilities of Ugandan parasites to lumefantrine and ganaplacide. Blue dots depict results for single isolates. The black line demonstrates the linear regression.

Fig 3

Ganaplacide susceptibilities of Ugandan P. falciparum isolates with validated K13 mutations. Dots depict single isolates and bar graphs the median IC₅₀s.