Background: Ubiquitin-specific protease 7 (USP7) is one of the most widely studied Deubiquitinating enzymes (DUBs). The protein level of USP7 is highly expressed in a variety of malignant cancers, which suggests that it is a prognostic marker of cancers and a potential drug target for oncotherapy. Objective: The aim of this study was to identify natural and effective USP7 inhibitors in order to understand the activation of the USP7/p53 pathway by natural inhibitors. Methods: In this study, the USP7 enzyme activity screening system and p53 luciferase reporter system were applied for the discovery of natural USP7 inhibitors targeting the catalytic active site. Molecular docking and molecular dynamics (MD) simulation revealed the combined mechanism between USP7 with gardenin B. Results: The gardenin B was screened from our home-lab natural products (160 flavonoids) and had cytotoxicity in HCT116 cells (IC50 = 46.28 ± 2.16μM). Preliminary in vitro studies disclosed its antiproliferative activity and activated p53 signaling pathway in HCT116 cells. We found that the complex formed by gardenin B and 5vsk (ledock score = -6.8645, MM/GBSA score = -53.349) had the optimal binding conformation. Moreover, the MD simulation showed that the π-π interactions between gardenin B with His461 and Phe409 and the hydrogen bonds interaction between gardenin B with Leu406 and Phe409 played an important role in maintaining the close binding of the complexes. Conclusion: In conclusion, gardenin B could be used as a natural product inhibitor of USP7 for further optimized design and development potential.

中文翻译：

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Gardenin B，USP7 的天然抑制剂：体外评估和计算机鉴定

背景：泛素特异性蛋白酶 7 (USP7) 是研究最广泛的去泛素化酶 (DUB) 之一。USP7的蛋白水平在多种恶性肿瘤中呈高表达，这表明它是肿瘤的预后标志物和肿瘤治疗的潜在药物靶点。目的：本研究的目的是鉴定天然有效的 USP7 抑制剂，以了解天然抑制剂对 USP7/p53 通路的激活。方法：在本研究中，USP7 酶活性筛选系统和 p53 荧光素酶报告系统被应用于发现针对催化活性位点的天然 USP7 抑制剂。分子对接和分子动力学 (MD) 模拟揭示了 USP7 与 gardenin B 之间的联合机制。结果：gardenin B 是从我们的家庭实验室天然产物（160 种类黄酮）中筛选出来的，对 HCT116 细胞具有细胞毒性（IC50 = 46.28 ± 2.16μM）。初步体外研究揭示了其在 HCT116 细胞中的抗增殖活性和激活的 p53 信号通路。我们发现由 gardenin B 和 5vsk（ledock 分数 = -6.8645，MM/GBSA 分数 = -53.349）形成的复合物具有最佳结合构象。此外，MD模拟表明，gardenin B与His461和Phe409之间的π-π相互作用以及gardenin B与Leu406和Phe409之间的氢键相互作用在维持复合物的紧密结合方面发挥了重要作用。结论：综上所述，gardenin B可作为USP7的天然产物抑制剂，具有进一步优化设计和开发的潜力。