Federica Giordani 1 , Daniel Paape 1 , Isabel M. Vincent 1 , Andrew W. Pountain 1 , Fernando Fernández-Cortés 1 , Eva Rico 2 , Ning Zhang 2 , Liam J. Morrison 3 , Yvonne Freund 4 , Michael J. Witty 5 , Rosemary Peter 5 , Darren Y. Edwards 2 , Jonathan M. Wilkes 1 , Justin J. J. van der Hooft 6 , 7 , Clément Regnault 6 , Kevin D. Read 2 , David Horn 2 , Mark C. Field 2 , 8 , Michael P. Barrett 1 , 6 , *
3 November 2020
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.
AN11736 is a member of the benzoxaborole class identified as a development candidate for animal African trypanosomiasis, a deadly livestock disease with huge economic impact. As part of its early evaluation phase, we set to unravel the risk and mode of resistance to this new trypanocide. We discovered that AN11736 behaves as a prodrug that, once inside trypanosomes, is cleaved by the activity of specific serine carboxypeptidases. AN11736-resistant Trypanosoma brucei and T. congolense had deletions within the serine carboxypeptidase gene array, resulting in their being unable to efficiently process the parent drug. Other benzoxaboroles with a similar sub-structure are also substrates for the serine carboxypeptidases, hence our findings assume great importance in considering the future development and deployment of this class of compounds.