Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs. Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs

African trypanosomes cause disease in both humans and animals. The livestock disease primarily caused by Trypanosoma congolense, T. vivax or T. brucei, in cattle is known as nagana and, is responsible for billions of dollars in lost annual food production. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious sub-class of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance emerges readily, due to mutations in an array of CBP genes. A T. brucei line with three tandem copies of the CBP gene readily developed resistance by partial deletion within the locus. A T. congolense line with an array of related CBP genes also developed resistance through deletion within the locus. A genome-scale genetic screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance, CRISPR-cas9 editing confirmed that partial deletion within the locus was sufficient to confer resistance and CBP re-expression in either resistant T. brucei or T. congolense lines restored drug-susceptibility. The peptidases themselves cleave the compounds to carboxylic acid derivatives. Thus, the current veterinary trypanocidal benzoxaboroles are CBP-activated prodrugs. Drug-resistance due to CBP loss-of-function may impact their efficacy.