Varied susceptibility to proteasome inhibitors and associations with genetic polymorphisms in Plasmodium falciparum isolates from Uganda

The proteasome is a multi-protein complex responsible for protein degradation in eukaryotes. Proteasome components are promising targets for antimalarial chemotherapy. In considering these targets, it is important to characterize naturally occurring variation in sensitivity of P. falciparum from malaria-endemic regions to proteasome inhibitors under study. We assessed ex vivo susceptibilities of fresh clinical isolates of P. falciparum collected in Tororo and Busia districts in Uganda from 2017-2020 to nine asparagine ethylenediamine and three macrocyclic peptide proteasome inhibitors. IC50 values were determined using a 72-h microplate growth inhibition assay with SYBR Green. We observed a range of proteasome inhibitor potencies, with median IC50 s from 1.1 - 1942 nM (28 - 170 isolates tested per compound), with 10 of the compounds having median IC50 s < 100 nM. TDI8304, a lead compound with drug-like properties, had a median IC50 of 16 nM (range 3.0 - 50 nM). Positive associations (r = 0.36 - 0.94) were seen between IC50 values for all asparagine ethylenediamine compounds. We sequenced the β5 (catalytic) proteasome subunit, which is the target of these inhibitors, and the β6 (structural) proteasome subunit, in Ugandan isolates. We observed two mutations in the β5 subunit, A142S and D150E, in two isolates. We observed no mutations in the β6 subunit. Considering the β2 (catalytic) subunit, we observed two mutations, I204T and S214F, in three isolates. Mutations in the β2 and β5 subunits were not associated with decreased susceptibility to the twelve proteasome inhibitors tested. In vitro screening of susceptibilities to the peptide proteasome inhibitors WLW and WLL, which target the β2 subunit and β2 and β5 subunits, respectively, demonstrated low nM activity, and did not show decreased susceptibilities to β2 S214F mutant parasites. Our results showed that a number of proteasome inhibitors had potent activity against P. falciparum isolates circulating in Uganda. Genetic variation in presumed proteasome targets was uncommon, and this variation was not clearly associated with changes in inhibitor susceptibility.