Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

Anacor Pharmaceuticals Inc Palo Alto California United States of America

Current address Bioinformatics Group Wageningen University Wageningen the Netherlands

Glasgow Polyomics Institute of Infection Immunity and Inflammation College of Medical Veterinary and Life Sciences University of Glasgow Glasgow United Kingdom

Global Alliance for Livestock and Veterinary Medicine Pentlands Science Park Penicuik Edinburgh United Kingdom

Institute of Parasitology Biology Centre of the Czech Academy of Sciences České Budějovice Czech Republic

Roslin Institute Royal School of Veterinary Studies University of Edinburgh Midlothian United Kingdom

Wellcome Centre for Anti Infectives Research School of Life Sciences University of Dundee Dundee United Kingdom

Wellcome Centre for Integrative Parasitology Institute of Infection Immunity and Inflammation University of Glasgow Glasgow United Kingdom

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