Photo-affinity labelling and biochemical analyses identify the target of trypanocidal simplified natural product analogues

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 photo-affinity 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 FoF1-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 FoF1-ATP synthase. Furthermore, the use of GFP-PTP-tagged subunits of the FoF1-ATP synthase, shows that our compounds bind specifically to both the α- and β-subunits of the ATP synthase. The FoF1-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（African sleeping sickness）的药物疗效欠佳，亟需开发全新的治疗方案。作为基于简化番荔枝内酯型醚骨架的药物化学研究项目的一部分，我们此前曾报道了化合物1——一种双四氢吡喃1,4-三唑（bis-tetrahydropyran 1,4-triazole，B-THP-T）抑制剂——具有颇具前景的锥虫杀伤活性。本研究旨在鉴定布氏锥虫（Trypanosoma brucei）中该类化合物的蛋白质靶点，以阐明其作用机制并助力后续的结构优化。我们使用化合物3——一种兼具双功能的重氮阿嗪（diazirine）与炔基修饰的光亲和探针类似物，对应先导化合物B-THP-T类的化合物1——来鉴定布氏锥虫前循环型中先导化合物的潜在靶点。将双功能化合物3在体内通过紫外交联与其靶点结合，随后通过铜(II)催化的叠氮-炔环加成反应添加生物素亲和标签或Cy5.5报告标签。利用链霉亲和素亲和磁珠分离得到生物素标记的蛋白质加合物，后续通过液相色谱-串联质谱（LC-MSMS）鉴定出FoF1-ATP合酶（FoF1-ATP synthase，线粒体复合物V）为潜在靶点。该靶点鉴定结果通过多种不同方法得到了验证。我们证实：(i) 化合物1会降低细胞内ATP水平；(ii) 其通过抑制氧化磷酸化（oxidative phosphorylation）发挥作用；(iii) 作用位点为FoF1-ATP合酶。此外，通过使用GFP-PTP标记的FoF1-ATP合酶亚基，我们证实本研究中的化合物可特异性结合ATP合酶的α与β亚基。FoF1-ATP合酶是我们简化的番荔枝内酯型类似物的靶点。该线粒体复合物在布氏锥虫的前循环型与血流型中均为必需组分，将其鉴定为靶点将推动后续抑制剂优化，助力未来的药物开发。此外，本文所述的光亲和标记技术可轻松应用于其他未知靶点的药物，以阐明其作用机制，并促进各类病原体或疾病模型中的治疗性药物设计。