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羧酸酯酶1(carboxylesterase 1,CE1)是哺乳动物体内分布的一种重要的丝氨酸水解酶,广泛参与多种内外源性酯类化合物(包括胆固醇酯等内源物,以及酯类药物及杀虫剂等外源物)的水解代谢。本研究基于人羧酸酯酶1(hCE1)偏好底物的结构特征,选取8-羧酸-BODIPY为荧光母核,设计合成了4种BODIPY-8-羧酸酯衍生物,进而通过单酶筛选和酶抑制实验考察了hCE1催化4种酯类底物的特异性。研究发现,BODIPY-8-羧酸酯的醇基部分越小,hCE1对底物的选择性越高,BODIPY-8-羧酸甲酯(BCM)和BODIPY-8-羧酸乙酯(BCE)均可以作为hCE1的特异性荧光探针底物。在此基础上,选择了水解速率更快的BCM为hCE1探针底物,进一步考察了BCM在人肝微粒体(HLM)和hCE1单酶中的水解动力学,并借助该底物开展了hCE1抑制剂的高效表征研究。研究发现BCM在HLM和hCE1中的酶动力学行为及K_m值非常接近,表明hCE1是HLM中参与BCM水解的主要代谢酶。此外,抑制剂表征实验表明BCM可用于hCE1抑制剂的高效筛选与评价,且可用HLM代替hCE1单酶进行酶抑制剂的筛选与评价。
Carboxylesterase 1 (CE1) is an important serine hydrolase distributed in mammals. It is widely involved in many kinds of endogenous and exogenous ester compounds (including endogenous substances such as cholesterol ester, Pests and other foreign matter) of the hydrolysis and metabolism. In this study, based on the structural characteristics of the human carboxylesterase 1 (hCE1) preferred substrate, eight BODIPY-8-carboxylate derivatives were designed and synthesized using 8-carboxylic acid-BODIPY as the fluorescent parent core, Screening and enzyme inhibition experiments examined hCE1 catalytic four ester substrate specificity. It was found that the smaller the alcohol group of BODIPY-8-carboxylate, the higher the selectivity of hCE1 to the substrate. BODIPY-8-carboxylate (BCM) and BODIPY-8-carboxylate (BCE) Can be used as hCE1 specific fluorescent probe substrate. On the basis of this, BCM with faster hydrolysis rate was chosen as the substrate of hCE1 probe. The kinetics of hydrolysis of BCM in human liver microsomes (HLM) and hCE1 single enzyme was further investigated. With this substrate, hCE1 Highly efficient characterization of inhibitors. It was found that the enzymatic kinetics and Km values of BCM in HLM and hCE1 are very close, indicating that hCEl is the major metabolic enzyme involved in BCM hydrolysis in HLM. In addition, inhibitor characterization experiments showed that BCM can be used for efficient screening and evaluation of hCE1 inhibitors, and screening and evaluation of enzyme inhibitors can be performed using HLM instead of hCE1 single enzyme.