Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 422, New York, New York 10065, USA.
J Am Chem Soc. 2010 Feb 17;132(6):1748-9. doi: 10.1021/ja9088549.
Semisynthetic, mechanism-based protein inhibitors of ubiquitin (Ub) and ubiquitin-like modifier (Ubl) activating enzymes (E1s) have been developed to target E1-catalyzed adenylation and thioesterification of the Ub/Ubl C-terminus during the processes of protein SUMOylation and ubiquitination. The inhibitors were generated by intein-mediated expressed protein ligation using a truncated Ub/Ubl protein (SUMO residues 1-94; Ub residues 1-71) with a C-terminal thioester and synthetic tripeptides having a C-terminal adenosine analogue and an N-terminal cysteine residue. SUMO-AMSN (4a) and Ub-AMSN (4b) contain a sulfamide group as a nonhydrolyzable mimic of the phosphate group in the cognate Ub/Ubl-AMP adenylate intermediate in the first half-reaction, and these constructs selectively inhibit SUMO E1 and Ub E1, respectively, in a dose-dependent manner. SUMO-AVSN (5a) and Ub-AVSN (5b) contain an electrophilic vinyl sulfonamide designed to trap the incoming E1 cysteine nucleophile (Uba2 Cys173 in SUMO E1; Uba1 Cys593 in Ub E1) in the second half-reaction, and these constructs selectively, covalently, and stably cross-link to SUMO E1 and Ub E1, respectively, in a cysteine nucleophile-dependent manner. These inhibitors are powerful tools to probe outstanding mechanistic questions in E1 function and can also be used to study the biological functions of E1 enzymes.
半合成、基于机制的泛素（Ub）和泛素样修饰物（Ubl）激活酶（E1s）的蛋白质抑制剂已被开发出来，以针对 E1 催化的蛋白质 SUMOylation 和 ubiquitination 过程中 Ub/Ubl C 末端的腺苷酸化和硫酯化。抑制剂是通过使用具有 C 末端硫酯和合成三肽的内含子介导的表达蛋白连接产生的，该三肽具有 C 末端腺苷类似物和 N 末端半胱氨酸残基。SUMO-AMSN（4a）和 Ub-AMSN（4b）含有磺酰胺基团，作为第一半反应中相应 Ub/Ubl-AMP 腺苷酸中间产物中磷酸基团的不可水解类似物，这些构建体分别以剂量依赖的方式选择性抑制 SUMO E1 和 Ub E1。SUMO-AVSN（5a）和 Ub-AVSN（5b）含有设计用于捕获进入的 E1 半胱氨酸亲核试剂（SUMO E1 中的 Uba2 Cys173；Ub E1 中的 Uba1 Cys593）的亲电乙烯基磺酰胺，这些构建体分别以半胱氨酸亲核试剂依赖的方式选择性、共价和稳定地交联到 SUMO E1 和 Ub E1。这些抑制剂是研究 E1 功能中突出的机制问题的有力工具，也可用于研究 E1 酶的生物学功能。
