Tulloch Lindsay B, Menzies Stefanie K, Fraser Andrew L, Gould Eoin R, King Elizabeth F, Zacharova Marija K, Florence Gordon J, Smith Terry K
EaStChem School of Chemistry and School of Biology, Biomedical Science Research Complex, University of St Andrews, St Andrews, Fife, United Kingdom.
PLoS Negl Trop Dis. 2017 Sep 5;11(9):e0005886. doi: 10.1371/journal.pntd.0005886. eCollection 2017 Sep.
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.
目前用于治疗非洲昏睡病的药物并不理想，迫切需要新的治疗方法。作为基于简化产乙酸素型醚支架的药物化学项目的一部分，我们之前报道了化合物1（一种双四氢吡喃1,4-三唑（B-THP-T）抑制剂）具有良好的杀锥虫活性。本研究旨在确定这类化合物在布氏锥虫中的蛋白质靶点，以了解其作用模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的一种含重氮丙啶和炔烃的双功能光亲和探针类似物）来确定我们的先导化合物在布氏锥虫前循环形式中的潜在靶点。双功能化合物3在体内与它的靶点进行紫外光交联，随后通过铜（II）催化的叠氮化物-炔烃环加成反应连接生物素亲和或Cy5.5报告标签。用链霉亲和素亲和珠分离生物素化的蛋白质加合物，随后的液相色谱-串联质谱鉴定出F₀F₁-ATP合酶（线粒体复合物V）为潜在靶点。使用各种不同方法证实了这一靶点鉴定。我们表明：（i）化合物1降低细胞ATP水平；（ii）通过抑制氧化磷酸化；（iii）在F₀F₁-ATP合酶处。此外，使用F₀F₁-ATP合酶的绿色荧光蛋白-PTP标记亚基表明，我们的化合物特异性结合ATP合酶的α亚基和β亚基。F₀F₁-ATP合酶是我们简化的产乙酸素型类似物的一个靶点。这种线粒体复合物在布氏锥虫的前循环和血流形式中都至关重要，将其鉴定为我们的靶点将有助于进一步优化抑制剂，以用于未来的药物发现。此外，这里描述的光亲和标记技术可以很容易地应用于其他靶点未知的药物，以确定它们的作用模式，并更广泛地促进在任何病原体或疾病模型中的治疗药物设计。
