Leung Donmienne, Hardouin Christophe, Boger Dale L, Cravatt Benjamin F
The Skaggs Institute for Chemical Biology and the Department of Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, California 92037, USA.
Nat Biotechnol. 2003 Jun;21(6):687-91. doi: 10.1038/nbt826. Epub 2003 May 12.
To realize the promise of genomics-based therapeutics, new methods are needed to accelerate the discovery of small molecules that selectively modulate protein activity. Toward this end, advances in combinatorial synthesis have provided unprecedented access to large compound libraries of considerable structural complexity and diversity, shifting the bottleneck in drug discovery to the development of efficient screens for protein targets. Screening for reversible enzyme inhibitors typically requires extensive target-specific work, including protein expression and purification, as well as the development of specific substrate assays. Here we report a proteomic method for the discovery of reversible enzyme inhibitors that avoids these steps. We show that competitive profiling of a library of candidate serine hydrolase inhibitors in complex proteomes with activity-based chemical probes identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH), triacylglycerol hydrolase (TGH) and an uncharacterized membrane-associated hydrolase that lacks known substrates. The strategy tests inhibitors against numerous enzymes in parallel, assigning both potency and selectivity factors to each agent. In this way, promiscuous inhibitors were readily rejected in favor of equally potent compounds with 500-fold or greater selectivity for their targets.
为了实现基于基因组学的治疗方法的前景，需要新的方法来加速发现能够选择性调节蛋白质活性的小分子。为此，组合合成技术的进步使人们能够前所未有的方式获取具有相当结构复杂性和多样性的大型化合物库，从而将药物发现的瓶颈转移到针对蛋白质靶点的高效筛选方法的开发上。筛选可逆性酶抑制剂通常需要大量针对特定靶点的工作，包括蛋白质表达和纯化，以及特定底物检测方法的开发。在此，我们报告一种用于发现可逆性酶抑制剂的蛋白质组学方法，该方法可避免这些步骤。我们表明，使用基于活性的化学探针在复杂蛋白质组中对候选丝氨酸水解酶抑制剂库进行竞争性分析，可同时鉴定出几种酶的纳摩尔级可逆抑制剂，包括内源性大麻素降解酶脂肪酸酰胺水解酶（FAAH）、三酰甘油水解酶（TGH）以及一种缺乏已知底物的未表征膜相关水解酶。该策略可并行测试针对多种酶的抑制剂，并为每种试剂确定效力和选择性因子。通过这种方式，混杂的抑制剂很容易被排除，而更倾向于对其靶点具有500倍或更高选择性的同等效力的化合物。
