小分子抑制剂（SMI）能够靶向Oncostatin M (OSM)信号通路，是应对癌症、炎症性肠病和CNS疾病的新型治疗方法。最近，报道了一种名为SMI-10B的首例SMI，它能够靶向OSM并阻止其与受体（OSMR）的相互作用。然而，SMI-10B在OSM上的结合口袋和相互作用模式仍不为人们所了解，这阻碍了靶向OSM的SMIs的合理设计。本研究利用SMI-10B作为探针，通过无偏分子动力学（MD）模拟广泛探索了OSM上与小分子结合的多个口袋。随后，通过分子力学广义Born表面积（MM/GBSA）结合能漏斗鉴定了该复合物的近原生态结构。此外，通过额外的独立MD运行和绝对自由能扰动（FEP）计算验证了近原生态和非原生态构象下的蛋白质-配体复合物的结合稳定性。总之，本研究所表征的SMI-10B自发结合于OSM的独特特征不仅为理解SMI-10B与OSM结合的分子机制提供了详细的信息，还将促进新型更强效的SMI阻断OSM信号通路的合理设计。版权所有 ©2023 Elsevier Ltd.发表。

Small molecule inhibitors (SMIs) targeting oncostatin M (OSM) signaling pathway represent new therapeutics to combat cancer, inflammatory bowel disease (IBD) and CNS disease. Recently, the first-in-class SMI named SMI-10B that target OSM and block its interaction with receptor (OSMR) were reported. However, the binding pocket and interaction mode of the compound on OSM remain poorly understood, which hampering the rational design of SMIs that target OSM. Here, using SMI-10B as a probe, the multiple pockets on OSM for small molecules binding were extensively explored by unbiased molecular dynamics (MD) simulations. Then, the near-native structure of the complex was identified by molecular mechanics generalized Born surface area (MM/GBSA) binding energy funnel. Moreover, the binding stabilities of the protein-ligand complexes in near- and non-native conformations were verified by additional independent MD runs and absolute free energy perturbation (FEP) calculation. In summary, the unique feature of SMI-10B spontaneously binds to OSM characterized here not only provide detailed information for understanding the molecular mechanism of SMI-10B binding to OSM, but also will facilitate the rational design of novel and more potent SMIs to block OSM signaling.Copyright © 2023. Published by Elsevier Ltd.