Chang Jae Won, Cognetta Armand B, Niphakis Micah J, Cravatt Benjamin F
The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute , La Jolla, California, The United States of America.
ACS Chem Biol. 2013 Jul 19;8(7):1590-9. doi: 10.1021/cb400261h. Epub 2013 May 23.
Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo, using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and α-β hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome.
丝氨酸水解酶是自然界中最大且最多样化的酶类之一。丝氨酸水解酶抑制剂被用于治疗多种疾病，包括肥胖症、糖尿病、认知性痴呆以及细菌和病毒感染。尽管如此，哺乳动物体内200多种丝氨酸水解酶中的大多数仍然缺乏用于其功能表征的选择性抑制剂。我们和其他人已经表明，活化氨基甲酸酯通过与丝氨酸水解酶保守的丝氨酸亲核试剂发生共价反应，可以作为该酶家族成员的有效抑制剂。然而，氨基甲酸酯与其他蛋白质类别的交叉反应程度大多仍未被探索。在这里，我们通过结合竞争性和点击化学（CC）-基于活性的蛋白质谱分析（ABPP），在体外和体内研究多种活化氨基甲酸酯的全蛋白质组反应性，来解决这个问题。我们鉴定出多种类型的氨基甲酸酯，包括O-芳基、O-六氟异丙基（HFIP）和O-N-羟基琥珀酰亚胺基（NHS）氨基甲酸酯，它们在体内能与整个小鼠组织蛋白质组中的丝氨酸水解酶选择性反应。我们利用HFIP氨基甲酸酯的全蛋白质组特异性来创建用于内源性大麻素水解酶单酰甘油脂肪酶（MAGL）和α-β水解酶-6（ABHD6）的原位成像探针。综上所述，这些发现表明氨基甲酸酯是丝氨酸水解酶的一个特殊反应基团，它可以容纳多种结构修饰，从而产生在哺乳动物蛋白质组中显示出卓越效力和选择性的抑制剂。
