Current drugs to treat African sleeping sickness are inadequate and new therapies are urgently required. As part of a medicinal chemistry programme based upon the simplification of acetogenin-type ether scaffolds, we previously reported the promising trypanocidal activity of compound 1, a bis-tetrahydropyran 1,4-triazole (B-THP-T) inhibitor. This study aims to identify the protein target(s) of this class of compound in Trypanosoma brucei to understand its mode of action and aid further structural optimisation. We used compound 3, a diazirine- and alkyne-containing bi-functional photoaffinity probe analogue of our lead B-THP-T, compound 1, to identify potential targets of our lead compound in the procyclic form T. brucei. Bi-functional compound 3 was UV cross-linked to its target(s) in vivo and biotin affinity or Cy5.5 reporter tags were subsequently appended by Cu(II)-catalysed azide-alkyne cycloaddition. The biotinylated protein adducts were isolated with streptavidin affinity beads and subsequent LC-MSMS identified the F_oF₁-ATP synthase (mitochondrial complex V) as a potential target. This target identification was confirmed using various different approaches. We show that (i) compound 1 decreases cellular ATP levels (ii) by inhibiting oxidative phosphorylation (iii) at the F_oF₁-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the F_oF₁-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The F_oF₁-ATP synthase is a target of our simplified acetogenin-type analogues. This mitochondrial complex is essential in both procyclic and bloodstream forms of T. brucei and its identification as our target will enable further inhibitor optimisation towards future drug discovery. Furthermore, the photo-affinity labeling technique described here can be readily applied to other drugs of unknown targets to identify their modes of action and facilitate more broadly therapeutic drug design in any pathogen or disease model.

当前用于治疗非洲昏睡病的药物不足，迫切需要新的疗法。作为基于简化的产乙酸素类醚支架的药物化学计划的一部分，我们先前报道了化合物1（一种双四氢吡喃1,4-三唑（B-THP-T）抑制剂）的有希望的锥虫杀灭活性。这项研究旨在确定布鲁氏锥虫中此类化合物的蛋白质靶标，以了解其作用方式并有助于进一步的结构优化。我们使用化合物3（我们的先导B-THP-T化合物1的含重氮基和炔烃的双功能光亲和探针类似物）来鉴定前环形式T的我们的先导化合物的潜在靶标。布鲁西。将双功能化合物3在体内进行UV交联，然后通过Cu（II）催化的叠氮化物-炔烃环加成反应附加生物素亲和力或Cy5.5报告分子标签。用链霉亲和素亲和珠分离生物素化的蛋白质加合物，随后的LC-MSMS将F _o F ₁ -ATP合酶（线粒体复合物V）鉴定为潜在的靶标。使用多种不同方法确认了目标识别。我们表明（i）化合物1通过抑制F _o F _1的氧化磷酸化（iii）降低细胞ATP水平（ii）-ATP合酶。此外，使用F _o F ₁ -ATP合酶的GFP-PTP标记亚基表明我们的化合物与ATP合酶的α-和β-亚基特异性结合。F _o F ₁ -ATP合酶是我们简化的acetogenin型类似物的目标。这种线粒体复合体在T的前循环和血液形式中都是必不可少的。布鲁西并将其确定为我们的目标将使进一步的抑制剂优化成为未来药物开发的目标。此外，此处描述的光亲和标记技术可轻松应用于未知靶标的其他药物，以识别其作用方式并促进在任何病原体或疾病模型中进行更广泛的治疗性药物设计。