松弛素家族多肽受体4的小分子配体发现和结构功能研究

Relaxin/insulin like family peptide receptor 4 (RXFP4) belongs to class A G protein-coupled receptor (GPCR) family with its cognate ligand named insulin-like peptide 5 (INSL5) which is a member of insulin superfamily. Various studies suggest that INSL5/RXFP4 is involved in glucose metabolism, appetite control and pancreatic islet function. Our recent results demonstrated that RXFP4 is highly expressed in islet β cells, and INSL5 is capable of promoting insulin release from β cells and enhancing glucagon-like peptide-1 (GLP-1) secretion from intestinal L cells. Subsequent in vivo investigations established its effects on glucose-stimulated insulin secretion (GSIS) and blood glucose regulation suggesting that the INSL5/RXFP4 system may be a potential therapeutic target for metabolic disorders such as type 2 diabetes. Further studies, however, are hampered by extremely limited access to native INSL5 (very difficult to produce an adequate amount) and the lack of small molecule ligands of RXFP4. To solve this problem, we developed a high-throughput screening (HTS) assay based on measurement of cAMP content and performed HTS campaigns which identified a few small molecule agonist-like compounds for RXFP4. In this project, we plan to conduct structural modification and optimization of these confirmed hits, study their structure-activity relationship (SAR) and molecular mechanism of action, and evaluate anti-diabetic their property in vitro and in vivo. The outcome of this project will help us better understand the signal transduction pathways of RXFP4 and discover one to two lead compounds for anti-diabetic drug development.

松弛素家族多肽受体4（RXFP4）属于A类GPCR，其天然配体为胰岛素样肽5（INSL5）。前期研究表明二者均与糖代谢、摄食以及胰岛功能等密切相关，故推测INSL5/RXFP4系统在胰岛β细胞功能维持及胰岛素分泌中发挥作用。申请人的最新研究成果显示RXFP4在胰岛β细胞上高表达，INSL5可促进胰岛细胞释放胰岛素并诱导肠L细胞分泌GLP-1，动物水平研究也证实INSL5可以显著改善小鼠的糖耐量，这表明RXFP4是糖尿病治疗的潜在靶点。然而，INSL5制备极其困难，为解决RXFP4配体匮乏的问题，我们建立了基于cAMP通路的高通量筛选方法并筛选发现了其小分子配体。本项目拟对小分子配体进行结构优化改造，研究受体配体的结构与功能相关性及其分子机制，并选取效果最优的活性化合物开展系统的体内外抗糖尿病药效评价。项目的成功实施可望阐释RXFP4信号转导机制，发现1-2个先导化合物用于抗糖尿病药用开发。

松弛素/胰岛素样肽受体4（RXFP4）属于A类G蛋白偶联受体（GPCR）家族成员，其内源性配体INSL5属于松弛素/胰岛素超家族。RXFP4主要分布于结直肠，参与众多生理功能的调控，包括能量代谢、血糖稳态、食欲调节、肠道蠕动、免疫生殖及肿瘤的发生和发展等。但目前针对RXFP4的研究较少，受限于INSL5结构复杂合成困难；已报道的RXFP4小分子激动剂（compound 1-4）特异性不佳（可激活同源受体RXFP3）。配体的缺乏限制了RXFP4的深入研究。本项目基于实验室前期以RXFP4为靶点经高通量筛选所获得的11个潜在活性化合物，通过cAMP水平检测，从中挑选了两个效果最优、结构不同的化合物进行后续研究。经细胞毒性分析、GPCR交叉反应检测以及非特异性PDE抑制剂排除实验，最终确定JK0621-D008化合物可特异性激活RXFP4下游信号。在此化合物的基础上进行结构优化改造，设计并合成了30个结构优化的小分子化合物。通过系统的下游信号通路功能检测，发现化合物14b具有相对较好的激动效果。对其进行对映异构体拆分，发现9-(S)-14b具有比原化合物JK0621-D008更优的效能和RXFP4靶点特异性。通过分子对接和突变验证，我们初步解释了该类化合物选择性结合RXFP4的原因。为更好地究INSL5与RXFP4的结合模式及小分子激动剂发挥拟肽作用的分子机制，我们利用冷冻电镜技术解析INSL5、小分子激动剂compound 4（RXFP3/RXFP4双激动剂）及DC591053（RXFP4单激动剂）与RXFP4的复合物三维结构，结合突变实验、SAR分析及分子动力学模拟，我们揭示了INSL5激活RXFP4的结构基础，明确了小分子激动剂发挥拟肽效应的关键位点，同时阐释了DC591053实现受体亚型选择性激活的分子机制。本研究对开发高效的RXFP4小分子激动剂，并进一步推动对该受体的生理功能研究和靶点可行性验证具有重要意义。