Discovering potent and selective reversible inhibitors of enzymes in complex proteomes
Discovering potent and selective reversible inhibitors of enzymes in complex proteomes
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DOI：
10.1038/nbt826
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发表时间：
2003-06-01
影响因子：
46.9
通讯作者：
Cravatt, BF
中科院分区：
文献类型：
--
作者：
Leung, D;Hardouin, C;Cravatt, BF
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选择性调节蛋白质活性的小分子。为此，组合合成的进步提供了前所未有的访问大型化合物库相当大的结构复杂性和多样性（1，2），转移药物发现的瓶颈，以开发有效的筛选蛋白质靶点（3）。可逆酶抑制剂的筛选通常需要大量的靶向特异性工作，包括蛋白质表达和纯化，以及特定底物测定的开发。在这里，我们报告了一种蛋白质组学方法，发现可逆酶抑制剂，避免这些步骤。我们表明，在具有基于活性的化学探针的复杂蛋白质组中的候选丝氨酸水解酶抑制剂文库的竞争性分析（4-6）同时鉴定了几种酶的纳摩尔可逆抑制剂，包括内源性大麻素降解酶脂肪酸酰胺水解酶（FAAH）（7）、三酰甘油水解酶（TGH）（8）和缺乏已知底物的未表征的膜相关水解酶。该策略平行测试抑制剂对多种酶的抑制作用，为每种药物分配效力和选择性因子。通过这种方式，混杂的抑制剂很容易被拒绝，而有利于对它们的靶点具有500倍或更高选择性的同等有效的化合物。
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(1,2), shifting the bottleneck in drug discovery to the development of efficient screens for protein targets(3). 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(4-6) identifies nanomolar reversible inhibitors of several enzymes simultaneously, including the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH)(7), triacylglycerol hydrolase (TGH)(8) 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.