Department of Pharmacology, University of Michigan School of Medicine, Ann Arbor, Michigan (A.L.V., M.Y., G.G.T.); and.
Departments of Molecular and Cellular Physiology (M.J.R., G.S.) and Structural Biology (G.S.), Stanford University School of Medicine, Stanford, California.
Mol Pharmacol. 2023 Jul;104(1):28-41. doi: 10.1124/molpharm.123.000688. Epub 2023 Jun 8.
GPR56 is a widely expressed adhesion GPCR (AGPCR) that has pleotropic roles in brain development, platelet function, cancer, and more. Nearly all AGPCRs possess extracellular regions that bind protein ligands and conceal a cryptic tethered peptide agonist. AGPCR reception of mechanical or shear force is thought to release the tethered agonist permitting its binding to the AGPCR orthosteric site for consequent activation of G protein signaling. This multistep mechanism of AGPCR activation is difficult to target, emphasizing the need for tool compounds and potential therapeutics that modulate AGPCRs directly. We expanded our cell-based pilot screen for GPR56 small molecule activators to screen >200,000 compounds and identified two promising agonists: 2-(furan-2-yl)-1-[(4-phenylphenyl)carbonyl]pyrrolidine, or compound 4, and propan-2-yl-4-(2-bromophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate, or compound 36. Both compounds activated GPR56 receptors enginered to have impaired tethered agonists and/or be cleavage deficient. Compound 4 activated a subset of group VIII AGPCRs while compound 36 had exclusive specificity for GPR56 among the GPCRs tested. Compound 36 SAR analysis identified an analog with the isopropyl R group replaced with a cyclopentyl ring and the electrophilic bromine replaced with a CF group. Analog 36.40 had 40% increased potency over compound 36 and was 20-fold more potent than synthetic peptidomimetics designed from the GPR56 tethered agonist. The new GPCR56 tool compounds discovered in this screen may be used to further advance understanding of GPR56 function and aid development of AGPCR-targeted therapeutics. SIGNIFICANCE STATEMENT: Adhesion G protein coupled receptors (AGPCRs) are a large, clinically relevant class of GPCRs with no available therapeutics, in part due to their unique mechanism of activation. GPR56 is a widely expressed model AGPCR involved in cancer metastasis, hemostasis, and neuron myelination. In the present study, we identified novel small molecule agonists for GPR56. These molecules are among the most potent identified thus far and may become useful leads in the development of a GPR56-targeted therapeutic.
GPR56 是一种广泛表达的黏附 G 蛋白偶联受体（AGPCR），在大脑发育、血小板功能、癌症等方面具有多种作用。几乎所有的 AGPCR 都具有结合蛋白配体并隐藏隐蔽的连接肽激动剂的细胞外区域。AGPCR 接收机械或剪切力被认为会释放连接的激动剂，使其能够与 AGPCR 变构位点结合，从而激活 G 蛋白信号。这种多步骤的 AGPCR 激活机制难以靶向，这强调了需要具有工具化合物和潜在治疗剂的方法，以直接调节 AGPCR。我们扩展了我们基于细胞的 GPR56 小分子激活剂的初步筛选，以筛选超过 200,000 种化合物，并确定了两种有前途的激动剂：2-（呋喃-2-基）-1-（（4-苯基苯基）羰基）吡咯烷或化合物 4，和丙烷-2-基-4-（2-溴苯基）-2,7,7-三甲基-5-氧代-1,4,5,6,7,8-六氢喹啉-3-羧酸酯，或化合物 36。这两种化合物都激活了具有受损连接激动剂和/或缺乏切割的工程 GPR56 受体。化合物 4 激活了一组 VIII 型 AGPCR，而化合物 36 在测试的 GPCR 中是 GPR56 的特异性配体。化合物 36 的 SAR 分析确定了一种类似物，其异丙基 R 基团被环戊基取代，亲电溴被 CF 取代。类似物 36.40 的效力比化合物 36 增加了 40%，比根据 GPR56 连接激动剂设计的合成肽模拟物强 20 倍。在该筛选中发现的新型 GPCR56 工具化合物可能用于进一步推进对 GPR56 功能的理解，并有助于开发针对 AGPCR 的治疗方法。意义声明：黏附 G 蛋白偶联受体（AGPCR）是一大类具有临床相关性的 GPCR，目前尚无可用的治疗方法，部分原因是其独特的激活机制。GPR56 是一种广泛表达的模型 AGPCR，参与癌症转移、止血和神经元髓鞘形成。在本研究中，我们鉴定了 GPR56 的新型小分子激动剂。这些分子是迄今为止最有效的分子之一，可能成为开发 GPR56 靶向治疗方法的有用先导物。
