Varied Sensitivities to Novel MMV Pipeline Antimalarials of Ugandan Plasmodium Falciparum Isolates and Associations with Specific Genotypes

Among novel compounds under investigation as potential antimalarials are inhibitors of PfATP4, PfDHODH, PfEF2, PfCYTB, PfDHFR and compounds with resistance reportedly mediated by variation in PfCARL. We assessed ex vivo sensitivities to these compounds of 561 fresh Plasmodium falciparum isolates from Tororo and Busia districts in Uganda from 2016- 2019. We then characterized genotypes of parasites with varied sensitivity to these antimalarials. We observed varied degrees of sensitivities of Ugandan isolates to all tested compounds. For inhibitors of PfEF2 (DDD498) and PfDHODH (DSM265, DSM421, DSM632, DSM705, and MMV258), sequencing of target genes demonstrated no mutations clearly associated with altered sensitivities. For the PfCYTb inhibitor ELQ300, a A205V mutation located close to the cytochrome b inhibitor-binding site in a single isolate was associated with an unusually high IC50. For the PfDHFR inhibitor P218, the I164L mutation was associated with decreased sensitivity (mean IC50 1 nM for WT, 5 nM for mutant, p<0.05). Previous studies showed that occurrence of I164L mutation increased levels of resistance to other antifolates. For KAF156 the Y353C (mean IC50 36 nM for WT, 75 nM for mixed) and Q605L (mean IC50 27 nM for WT, 94 nM for mixed) mutations in the resistance mediator PfCARL were associated with decreased sensitivity. For the three PfATP4 inhibitors mean IC50s were 57 nM for SJ733, 9 nM for PA92, and 0.4 nM for KAE609. A G223S mutation was associated with decreased sensitivity to SJ733 (mean IC50 71 nM for WT, 93 nM for mutant), PA92 (IC50 10 nM for WT, 15 nM for mutant) and KAE609 (IC50 0.5 nM for WT, 0.7 nM for mutant); for all comparisons p<0.05. Interestingly, previous studies showed that incubation of a laboratory strain with an analog of KAE609 selected for resistant parasites with a mutation (G223R) at the same codon. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms associated with varied sensitivity to MMV pipeline compounds, although the clinical relevance of the differences in sensitivity of field isolates is uncertain.