The Hedgehog (Hh) signaling pathway plays a critical role in controlling patterning, growth and cell migration during embryonic development. Aberrant activation of Hh signaling has been linked to tumorigenesis in various cancers, such as basal cell carcinoma (BCC) and medulloblastoma. As a key member of the Hh pathway, the Smoothened (Smo) receptor, a member of the G protein-coupled receptor (GPCR) family, has emerged as an attractive therapeutic target for the treatment and prevention of human cancers. The recent determination of several crystal structures of Smo in complex with different antagonists offers the possibility to perform structure-based virtual screening for discovering potent Smo antagonists with distinct chemical scaffolds. In this study, based on the two Smo crystal complexes with the best capacity to distinguish the known Smo antagonists from decoys, the molecular docking-based virtual screening was conducted to identify promising Smo antagonists from ChemDiv library. A total of 21 structurally novel and diverse compounds were selected for experimental testing, and six of them exhibited significant inhibitory activity against the Hh pathway activation (IC₅₀ < 10 μM) in a GRE (Gli-responsive element) reporter gene assay. Specifically, the most potent compound (compound 20: 47 nM) showed comparable Hh signaling inhibition to vismodegib (46 nM). Compound 20 was further confirmed to be a potent Smo antagonist in a fluorescence based competitive binding assay. Optimization using substructure searching method led to the discovery of 12 analogues of compound 20 with decent Hh pathway inhibition activity, including four compounds with IC₅₀ lower than 1 μM. The important residues uncovered by binding free energy calculation (MM/GBSA) and binding free energy decomposition were highlighted and discussed. These findings suggest that the novel scaffold afforded by compound 20 can be used as a good starting point for further modification/optimization and the clarified interaction patterns may also guide us to find more potent Smo antagonists.

刺猬（Hh）信号通路在控制胚胎发育过程中的模式，生长和细胞迁移中起着至关重要的作用。Hh信号的异常激活已与各种癌症（如基底细胞癌（BCC）和髓母细胞瘤）中的肿瘤发生有关。作为Hh通路的关键成员，Smoothened（Smo）受体是G蛋白偶联受体（GPCR）家族的成员，已成为治疗和预防人类癌症的有吸引力的治疗靶标。最近确定了具有不同拮抗剂的Smo的几种晶体结构，提供了进行基于结构的虚拟筛选的可能性，以发现具有不同化学骨架的有效Smo拮抗剂。在这项研究中，基于两种Smo晶体配合物，它们具有最佳能力将已知的Smo拮抗剂与诱饵区分开，进行了基于分子对接的虚拟筛选，从ChemDiv库中鉴定出有前途的Smo拮抗剂。总共选择了21种结构新颖多样的化合物进行实验测试，其中有6种化合物对Hh途径的活化具有显着的抑制活性（IC_50（  <10μM）在GRE（Gli响应元件）报告基因测定中。具体而言，最有效的化合物（化合物20：47纳米）显示出相当的Hh信号传导抑制到vismodegib（46纳米）。在基于荧光的竞争结合测定中，进一步证实化合物20是有效的Smo拮抗剂。使用亚结构搜索方法进行的优化导致发现了具有类似的Hh途径抑制活性的化合物20的12种类似物，包括四种IC ₅₀低于1μM的化合物。突出并讨论了通过结合自由能计算（MM / GBSA）和结合自由能分解发现的重要残基。这些发现表明，化合物提供的新型支架20可以用作进一步修饰/优化的良好起点，明确的相互作用模式也可以指导我们寻找更有效的Smo拮抗剂。