Severe acute respiratory syndrome-coronavirus-1/2 (SARS-CoV-1/2) macrodomain 3 (Mac3) is critical for replication and transcription of the viral genome and is therefore a potential therapeutic target. Here, we solved the crystal structure of SARS-CoV-2 Mac3, which reveals a small-molecule binding pocket. Two low-molecular-weight drugs, oxaprozin and meclomen, induced different patterns of nuclear magnetic resonance (NMR) chemical shift perturbations (CSPs). Meclomen binds to site I of SARS-CoV-2 Mac3 with binding pose determined by NMR CSP and transferred paramagnetic relaxation enhancement, while oxaprozin binds to site II as revealed by the crystal structure. Interestingly, oxaprozin and meclomen both perturb residues in site I of SARS-CoV Mac3. Fluorescence polarization experiments further demonstrated that oxaprozin and meclomen inhibited the binding of DNA-G4s to SARS-CoV-2 Mac3. Our work identified two adjacent ligand-binding sites of SARS-CoV-2 Mac3 that shall facilitate structure-guided fragment linking of these compounds for more potent inhibitors.

中文翻译：

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Oxaprozin 和 Meclomen 探测的 SARS-CoV-2 宏结构域 3 的两个结合位点

严重急性呼吸综合征-冠状病毒-1/2 (SARS-CoV-1/2) 宏结构域 3 (Mac3) 对于病毒基因组的复制和转录至关重要，因此是一个潜在的治疗靶点。在这里，我们解析了 SARS-CoV-2 Mac3 的晶体结构，它揭示了一个小分子结合口袋。两种低分子量药物奥沙普秦和美可洛明诱导了不同模式的核磁共振 (NMR) 化学位移扰动 (CSP)。Meclomen 与 SARS-CoV-2 Mac3 的位点 I 结合，其结合位姿由 NMR CSP 确定并转移顺磁弛豫增强，而奥沙普秦与晶体结构显示的位点 II 结合。有趣的是，oxaprozin 和 meclomen 都扰乱了 SARS-CoV Mac3 位点 I 的残基。荧光偏振实验进一步证明，奥沙普秦和美克洛明抑制了 DNA-G4s 与 SARS-CoV-2 Mac3 的结合。我们的工作确定了 SARS-CoV-2 Mac3 的两个相邻配体结合位点，它们将促进这些化合物的结构导向片段连接以获得更有效的抑制剂。