The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei, the proteins that make up the kinetochore are highly divergent; the inner kinetochore comprises at least 20 distinct and essential proteins (KKT1–20) that include four protein kinases—CLK1 (also known as KKT10), CLK2 (also known as KKT19), KKT2 and KKT3. Here, we report the identification and characterization of the amidobenzimidazoles (AB) protein kinase inhibitors that show nanomolar potency against T. brucei bloodstream forms, Leishmania and Trypanosoma cruzi. We performed target deconvolution analysis using a selection of 29 T. brucei mutants that overexpress known essential protein kinases, and identified CLK1 as a primary target. Biochemical studies and the co-crystal structure of CLK1 in complex with AB1 show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with Cys 215 in the ATP-binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell-cycle progression, leading to cell death. This research highlights a unique drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

线粒体是在染色体的着丝粒上组装的大分子结构，并在有丝分裂期间为纺锤体微管提供了主要附着点。在布鲁氏锥虫中，组成动球体的蛋白质高度不同。内部的线粒体至少包含20种不同的必需蛋白（K​​KT1–20），其中包括四种蛋白激酶-CLK1（也称为KKT10），CLK2（也称为KKT19），KKT2和KKT3。在这里，我们报告的酰胺基苯并咪唑类（AB）蛋白激酶抑制剂的鉴定和表征，其对T. brucei血流形式，利什曼原虫和克鲁斯锥虫表现出纳摩尔效价。我们使用29种选择进行了目标反卷积分析T. brucei突变体过表达已知的必需蛋白激酶，并确定CLK1为主要靶标。生化研究以及与AB1结合的CLK1的共晶体结构表明，对CLK1的不可逆竞争性抑制取决于与ATP结合口袋中Cys 215形成不可逆键的迈克尔受体，该残基在人体中不存在CLK1，从而提供了选择性。CLK1的化学抑制作用会破坏内部的线粒体募集并损害细胞周期进程，从而导致细胞死亡。这项研究突出了锥虫原虫寄生虫的独特药物靶标，以及研究其多样化动植物功能的新化学工具。