Insights into the crystal structure of BRD2-BD2 – phenanthridinone complex and theoretical studies on phenanthridinone analogs

Bromodomain and extra-terminal family proteins recognize the acetylated histone code on chromatin and participate in downstream processes like DNA replication, modification, and repair. As part of epigenetic approaches, BRD2 and BRD4 were identified as putative targets, for the management of chronic diseases. We have recently reported the discovery of a new scaffold of the phenanthridinone-based inhibitor (L10) of the second bromodomain of BRD2 (BRD2-BD2). Here, we present the crystal structure of the BRD2-BD2, refined to 1.4 Å resolution, in complex with β-mercaptoethanol (a component of the protein buffer). The β-mercaptoethanol covalently links to C425 of BD2 in the acetyl-lysine binding pocket, to form a modified cysteine mercaptoethanol (CME). The CME modification significantly hinders the entry of ligands into the BD2 binding pocket, suggesting that β-mercaptoethanol should be removed during protein production process. Next, to confirm whether phenanthridionone scaffold is a new inhibitor family of BRD2-BD2, we have determined the crystal structure of BD2 in complex with 6(5H)-Phenanthridinone (a core moiety of L10), refined to 1.28 Å resolution. It confirmed that the phenanthridinone molecule, unambiguously, binds to BD2. Moreover, we performed molecular docking and molecular dynamic studies on selected phenanthridinone analogs. The predicted L10 analogs are stable with essential hydrophobic and hydrophilic interactions with BD2 during molecular dynamic simulations. We propose that the predicted phenanthridinone analogs may be potential molecules for inhibiting the BD2 function of acetylated histone recognition.

溴结构域与额外末端结构域（Bromodomain and extra-terminal, BET）家族蛋白可识别染色质上的乙酰化组蛋白密码，并参与DNA复制、修饰与修复等下游生物学过程。作为表观遗传学治疗策略的组成部分，BRD2与BRD4已被鉴定为慢性疾病治疗的潜在靶点。我们近期报道了一类靶向BRD2第二个溴结构域（BRD2-BD2）的菲啶酮类抑制剂（L10）的全新母核结构。本研究解析了分辨率达1.4 Å的BRD2-BD2晶体结构，该蛋白与β-巯基乙醇（β-mercaptoethanol，蛋白缓冲液组分）形成复合物。β-巯基乙醇会在乙酰赖氨酸结合口袋中与BD2的C425位点共价结合，形成修饰后的半胱氨酸巯基乙醇（Cysteine Mercaptoethanol, CME）。CME修饰会显著阻碍配体进入BD2结合口袋，这提示在蛋白制备过程中应去除β-巯基乙醇。为验证菲啶酮母核是否为BRD2-BD2的新型抑制剂家族，我们解析了分辨率达1.28 Å的BD2与6(5H)-菲啶酮（6(5H)-Phenanthridinone，L10的核心基团）复合物的晶体结构。该结构明确证实菲啶酮分子可结合至BD2。此外，我们对筛选得到的菲啶酮类似物开展了分子对接与分子动力学研究。在分子动力学模拟过程中，预测得到的L10类似物可与BD2形成关键的疏水与亲水相互作用，保持结构稳定。我们据此提出，所预测的菲啶酮类似物有望成为抑制乙酰化组蛋白识别相关BD2功能的潜在候选分子。