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.

非洲锥虫（African trypanosomes）可引发人类与动物共患疾病。其中在牛群中主要由刚果锥虫（Trypanosoma congolense）、伊氏锥虫（T. vivax）或布氏锥虫（T. brucei）引发的家畜疾病被称为那加那病（nagana），每年造成的粮食生产损失高达数十亿美元，因此亟需开发新型治疗药物。令人鼓舞的是，极具应用前景的抗锥虫苯并氧硼杂环类化合物（benzoxaboroles）正处于兽药研发阶段。本研究证实，这类化合物中疗效最优的亚类属于前体药物，其活化依赖于锥虫丝氨酸羧肽酶（serine carboxypeptidases, CBPs）。由于一系列CBP基因发生突变，此类药物极易产生耐药性。携带三串联拷贝CBP基因的布氏锥虫（T. brucei）菌株可通过基因座内部分缺失快速获得耐药性；而携带相关CBP基因阵列的刚果锥虫（T. congolense）菌株同样可通过基因座内缺失产生耐药性。布氏锥虫的全基因组遗传筛选证实，CBP功能丧失是耐药性产生的核心机制；CRISPR-Cas9基因编辑实验进一步确认，仅需在基因座内发生部分缺失即可赋予耐药性；而在耐药的布氏锥虫或刚果锥虫菌株中重新表达CBP，均可恢复其药物敏感性。此类肽酶可将该类化合物切割为羧酸衍生物，因此当前的兽用杀锥虫苯并氧硼杂环类化合物实为CBP活化型前药。由CBP功能丧失介导的耐药性，或将影响此类药物的临床疗效。