人源羧酸酯酶1亚型选择性荧光探针设计、合成与生物学应用

Human carboxylesterase 1(hCE1), an important subtype of carboxylesterase in human, play an important role in the metabolism of endogenous and exogenous esters compounds, as well as multiple physiological and pathological processes. It is important to detect the hCE1 content in biological systems accurately for the real catalytic activity evaluation of hCE1 in tissues or cell lysate, formulating individualized dosage regimen by predicting the metabolism rate of drugs mediated by hCE1, screening specific inhibitors, early warning of potential risks of drug-drug interactions, and further exploring its role in disease process. At present, a selective and rapid quantitation of hCE1 in biological samples has not been realized. In this project, a series of fluorescence substrate for hCE1 will be designed, synthesized and well-characterized, based on our previous understanding in structure and subtype-selectivity of hCE1. The aim of this project is to develop practical fluorescent probe with high subtype specificity, sensitivity. The probe will be used for measuring the activity of hCE1 in different biological system, high-throughput screening inhibitors and inducers of hCE1. Ultimately, we will construct an optimized development and application system of fluorescence probe for hCE1 subtype.

人源羧酸酯酶1（hCE1）作为人体内一种重要的羧酸酯酶亚型，不仅能催化多种内源性和外源性酯类分子的代谢，并且在机体多种生理和病理过程中发挥重要作用。生物体系中hCE1含量的精确测定对于观察组织或细胞制备液中hCE1的真实催化活性，预测其介导的药物代谢速率来制定个性化给药方案，筛选特异性抑制剂，对潜在的药-药相互作用风险进行预警，以及探究其在疾病过程中所扮演的角色都十分重要。目前，通过荧光技术对生物样品中hCE1进行快速选择性定量一直未能实现。本项目拟在前期工作基础上，基于hCE1 偏好底物的结构特点，系统开展hCE1 亚型选择性荧光探针的设计、合成、荧光及生物学属性表征等研究，旨在研发具有亚型选择性、高灵敏度、抗环境干扰能力强的实用型荧光探针，用于不同生物体系中hCE1含量的测定，以及hCE1抑制剂/诱导剂的高通量筛选。最终构建一整套hCE1亚型选择性荧光探针的研发及应用技术体系。

基于羧酸酯酶1偏好底物的结构特点，系统开展hCE1亚型选择性荧光探针的设计、合成、荧光及生物学属性表征等研究，研发了三个具有亚型选择性、高灵敏度、抗环境干扰能力强的实用型荧光探针。针对不同生物体系和研究目的，开发相应的检测流程和操作规范，并将其用于组织微粒体、细胞、组织、斑马鱼以及小鼠等体系中羧酸酯酶1的快速表征，及建立了利用人肝微粒体为酶源的hCE1抑制剂高通量筛选方法，构建了一整套hCE1亚型选择性荧光探针底物的研发及应用技术体系。这些探针可作为有效工具分子用于CE1抑制剂的高通量筛选，对于相关药物的开发以及预警由CE1介导的药物相互作用方面具有指导意义。此外，探针应用于生物内CE1的实时原位检测，实现多种生物体系下CE1活性的可视化，对其酶活性及其在生理病理过程中的功能研究具有重要意义。