Acoziborole resistance associated mutations in Trypanosoma brucei CPSF3

Acoziborole is a safe, single dose, oral therapy, for treatment of both early and late-stage sleeping sickness, a deadly disease caused by African trypanosomes. Other benzoxaboroles show efficacy against other trypanosomatids, apicomplexans, fungi, bacteria, and viruses. Acoziborole targets the trypanosome pre-mRNA processing endonuclease, cleavage and polyadenylation specificity factor 3 (CPSF3), and triggers CPSF3 degradation, but it remains unclear whether additional mechanisms contribute to efficacy. We used oligo targeting for site saturation mutagenesis of the native CPSF3 gene. Among >1,500 edits around the putative drug binding site, only Asn 232 His edits conferred moderate resistance to acoziborole. Using a combinatorial oligo targeting method we edited multiple sites simultaneously, including sites that differ in human CPSF3, and found that an Asn 232 His, Tyr 383 Phe, Asn 448 Gln triple-mutant strain was > 40-fold resistant to acoziborole. We used gene tagging to show that all three edits were on the same allele, and to show that triple-mutant CPSF3 was highly resistant to rapid acoziborole and proteasome-dependent degradation. Computational modelling revealed how the combinatorial mutations can disrupt acoziborole – CPSF3 interactions by introducing steric clash and by disrupting hydrophobic and water-mediated interactions. We conclude that acoziborole safety and efficacy can be explained by selective affinity for, and rapid turnover of, trypanosome CPSF3.