Protein lysine methyltransferase G9a is widely considered as an appealing antineoplastic target. Herein we present an integrated workflow combining shape-based virtual screening and structure-based molecular modification for the identification of novel G9a inhibitors. The shape-based similarity screening through ROCS overlay on the basis of the structure of UNC0638 was performed to identify CPUY074001 contained a 6H-anthra[1,9-cd]isoxazol-6-one scaffold as a hit. Analysis of the binding mode of CPUY074001 with G9a and 3D-QSAR results, two series compounds were designed and synthesized. The derivatives were confirmed to be active by in vitro assay and the SAR was explored by docking stimulations. Besides, several analogues showed acceptable anti-proliferative effects against several cancer cell lines. Among them, CPUY074020 displayed potent dual G9a inhibitory activity and anti-proliferative activity. Furthermore, CPUY074020 induced cell apoptosis in a dose-dependent manner and displayed a significant decrease in dimethylation of H3K9. Simultaneously, CPUY074020 showed reasonable in vivo PK properties. Altogether, our workflow supplied a high efficient strategy in the identification of novel G9a inhibitors. Compounds reported here can serve as promising leads for further study.

蛋白质赖氨酸甲基转移酶G9a被广泛认为是有吸引力的抗肿瘤靶标。在这里，我们提出了一个集成的工作流程，结合了基于形状的虚拟筛选和基于结构的分子修饰，用于鉴定新型G9a抑制剂。通过基于UNC0638的结构通过ROCS覆盖进行基于形状的相似性筛选，以鉴定CPUY074001包含6 H-蒽[1,9 - cd ]异恶唑-6-一个骨架作为命中。CPUY074001的绑定方式分析根据G9a和3D-QSAR结果，设计并合成了两个系列化合物。通过体外测定证实了该衍生物是有活性的，并且通过对接刺激探索了SAR。此外，几种类似物对几种癌细胞系显示出可接受的抗增殖作用。其中，CPUY074020显示出强大的双重G9a抑制活性和抗增殖活性。此外，CPUY074020以剂量依赖性方式诱导细胞凋亡，并显示H3K9的二甲基化显着降低。同时，CPUY074020显示出合理的体内PK特性。总之，我们的工作流程为鉴定新型G9a抑制剂提供了高效的策略。本文报道的化合物可作为有希望的进一步研究的线索。