Tsuboi Katsunori, Bachovchin Daniel A, Speers Anna E, Brown Steven J, Spicer Timothy, Fernandez-Vega Virneliz, Ferguson Jill, Cravatt Benjamin F, Hodder Peter, Rosen Hugh
The Scripps Research Institute, La Jolla CA
The Scripps Research Institute, Jupiter, FL
Glutathione transferases (GSTs) are a superfamily of enzymes that conjugate glutathione to a wide variety of both exogenous and endogenous compounds for biotransformation and/or removal. Using activity-based proteomic methods, glutathione S-tranferase omega (GSTO1) has been shown to be overexpressed in human cancer cell lines that show enhanced aggressiveness. Other studies have implicated GSTO1 in chemotherapeutic resistance. Cancer remains one of the most life-threatening diseases for which effective treatments and cures are lacking. Recently, targeted therapeutics (i.e., selective inhibitors that block individual enzymes) have shown great promise for the treatment of cancer. Inhibiting GSTO1 may thus offer a new therapeutic strategy for cancer. The Scripps Research Institute Molecular Screening Center (SRIMSC), part of the Molecular Libraries Probe Production Centers Network (MLPCN), identified a selective GSTO1 inhibitor probe, ML175, by high-throughput screening using fluorescence polarization-activity-based protein profiling (FluoPol-ABPP), and several rounds of gel-based competitive activity-based protein profiling (ABPP) secondary assays employing SAR analysis of selected compounds. ML175, a hindered alpha-chloroacetamide, was identified as a highly potent and selective inhibitor of the target enzyme GSTO1. ML175 has an IC of 28 nM, greater than 350-fold selectivity against potential anti-targets as assessed by competitive ABPP profiling, and has been shown to be active in situ, completely inhibiting GSTO1 at 250 nM compound concentration. As determined from LC-MS/MS analysis, ML175 is an activity-based inhibitor; it covalently labels the active site cysteine nucleophile of GSTO1. ML175 has favorable stability, solubility, and cytotoxicity profile, and improves on the current state of the art for GSTO1 inhibitors. Taken together, our findings suggest that it is very possible to develop potent and selective probes based on tempered electrophilic scaffolds, and that ML175 will be a highly successful probe for biochemical investigation of GSTO1.
谷胱甘肽转移酶（GSTs）是一类酶的超家族，可将谷胱甘肽与多种外源性和内源性化合物结合，以进行生物转化和/或清除。使用基于活性的蛋白质组学方法，已证明谷胱甘肽S-转移酶ω（GSTO1）在显示出更强侵袭性的人类癌细胞系中过表达。其他研究表明GSTO1与化疗耐药性有关。癌症仍然是最危及生命的疾病之一，缺乏有效的治疗方法和治愈手段。最近，靶向治疗药物（即阻断单个酶的选择性抑制剂）在癌症治疗方面显示出巨大的前景。因此，抑制GSTO1可能为癌症治疗提供一种新的治疗策略。斯克里普斯研究所分子筛选中心（SRIMSC）是分子文库探针生产中心网络（MLPCN）的一部分，通过基于荧光偏振活性的蛋白质谱分析（FluoPol-ABPP）的高通量筛选，以及使用所选化合物的SAR分析进行的几轮基于凝胶的竞争性活性蛋白质谱分析（ABPP）二级检测，鉴定出一种选择性GSTO1抑制剂探针ML175。ML175是一种受阻的α-氯乙酰胺，被鉴定为目标酶GSTO1的高效选择性抑制剂。ML175的IC50为28 nM，通过竞争性ABPP分析评估，对潜在抗靶点的选择性大于350倍，并且已证明在原位具有活性，在250 nM化合物浓度下可完全抑制GSTO1。通过LC-MS/MS分析确定，ML175是一种基于活性的抑制剂；它共价标记GSTO1的活性位点半胱氨酸亲核试剂。ML175具有良好的稳定性、溶解性和细胞毒性特征，并改进了目前GSTO1抑制剂的技术水平。综上所述，我们的研究结果表明，基于适度亲电支架开发强效和选择性探针是非常有可能的，并且ML175将成为用于GSTO1生化研究的非常成功的探针。
