以 DNA 编码分子库为工具的活细胞表面 G-蛋白偶联受体的动态修饰以及小分子拮抗剂与激动剂的研究

G protein-coupled-receptor (GPCR) is a superfamily of membrane proteins on cell surface. GPCRs have participated in nearly every aspects of the biological processes in cells and play important roles in a myriad of signaling transduction pathways. Naturally, GPCRs are implicated in the pathobiology of numerous human diseases and are the most extensively studied drug targets. However, similar to other membrane proteins, it is highly difficult to maintain the native structure of GPCRs in the purified form; and more importantly, purified proteins cannot recapitulate the interactions of GPCR with the endogenous binding partners and native post-translational modifications. These issues have become one of the major obstacles in GPCR research. Here we propose a novel method based on the DNA-encoded chemical library technology, aiming to realize the dynamic modifications of GPCR proteins directly on the surface of live cells and to realize target-specific discovery of GPCR antagonists and agonists with large-scale DNA-encoded libraries. This project will provide a powerful and effective tool for the high throughput screening against GPCRs as well as other membrane proteins on live cells, thereby facilitating the corresponding development of novel molecular probes and drug candidates. We will first conduct the methodology development, and then use β2-adrenergic receptor (β2AR), glucagon-like peptide-1 receptor (GLP-1R), and an orphan receptor GPR84 as representative examples to validate the proposed research method and to develop novel functional ligands for these GPCRs.

G蛋白偶联受体(G-protein-coupled receptor; GPCR)是一类重要的膜蛋白，其种类繁多，参与众多的细胞生命过程，具有重要的生物功能。GPCR与许多人类重大疾病密切相关，是研究最为广泛的药物靶点类型。然而和许多膜蛋白类似，纯化后单独的GPCR较难保持其天然蛋白结构，失去了与内源配体的相互作用以及翻译后修饰，难以反映其天然的生物功能，成为GPCR研究中亟待解决的重要科学问题。本项目拟发展一种基于DNA编码分子库的新技术方法，实现对活细胞表面GPCR蛋白的动态化学修饰，并实现GPCR不同活性状态下拮抗剂和激动剂的筛选和发现，为推动基于GPCR这一重要靶点蛋白的分子探针发展和创新药物研究，提供一种新颖而有效的化学生物学工具方法。本项目首先进行方法学的发展，再以β2-肾上腺素受体、 胰高血糖素样肽-1受体、孤儿受体GPR84三个重要GPCR蛋白进行针对性的研究。

G蛋白偶联受体 (G-protein-coupled receptor; GPCR) 是一类重要的膜蛋白，其种类繁多，参与众多的细胞生命过程，具有重要的生物功能。GPCR与许多人类重大疾病密切相关，是研究最为广泛的药物靶点类型。然而和许多膜蛋白类似，纯化后单独的GPCR 蛋白较难保持其天然结构，失去了与内源配体的相互作用以及翻译后修饰，难以反映其天然的生物功能，成为GPCR研究中亟待解决的重要科学问题。本项目成功发展了一系列分子库的新高通量筛选技术方法，实现对活细胞表面包膜蛋白的动态化学修饰，并实现膜蛋白在不同活性状态下拮抗剂和激动剂的筛选和发现，为推动基于包括GPCR的膜蛋白这一重要靶点蛋白的分子探针发展和创新药物研究，提供了一种新颖而有效的化学生物学工具方法。本项目研究中，我们首先进行方法学的发展，着重发展了基于DNA编码分子库对活细胞表面膜蛋白的高通量筛选方法，进一步发展了对膜蛋白进行常规、非DNA编码分子库的天然产物分子库的筛选方法；项目最后进行了对整个活细胞，“无靶点”的分子库筛选方法。此外，我们利用这些方法学，对对 一系列重要膜蛋白，以及多种肿瘤细胞进行了大规模的高通量筛选，获得了多种新颖的配体分子，为下游的新药研发提供了新的探索途径。