Acoziborole resistance associated mutations in trypanosome CPSF3

Abstract

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 Asn232His edits conferred moderate resistance to acoziborole. Using a novel combinatorial oligo targeting method we edited multiple sites simultaneously, including sites that differ in human CPSF3, and found that an Asn232His, Tyr383Phe, Asn448Gln 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. Author SummaryDiagnosis and treatment options, previously limited for sleeping sickness, have been transformed in recent years. Acoziborole, for example, is a new, safe, single dose, oral therapy for the treatment of this deadly disease. This drug can also be used without the need for cumbersome disease-stage diagnosis. Additional boron-based drugs also show great promise against a whole range of other infectious diseases. Acoziborole targets an RNA processing enzyme in African trypanosomes, and triggers its degradation, but human cells express a similar enzyme, and alternative trypanosomal targets have also been suggested. Insights into how a drug interacts with its target can help to understand selective action against a pathogen, and to predict resistance, an ever-present threat for many drugs. We used a precision gene editing method to change the target protein in trypanosomes, editing single sites or multiple sites simultaneously. A novel triple-mutant was found to be both highly resistant to acoziborole and highly resistant to rapid degradation. Using computational models, we were able to explain how multiple mutations interfered with acoziborole binding to its target. The findings show how selective binding of a specific parasite enzyme makes acoziborole such a safe and effective drug.