Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Eur Rev Med Pharmacol Sci. 2024 Sep;28(18):4313-4325. doi: 10.26355/eurrev_202409_36791.
The coronavirus disease (COVID-19) pandemic, resulting from human-to-human transmission of a novel severe acute respiratory syndrome coronavirus (SARS-CoV-2), has caused a global health emergency. The lack of a specific drug or treatment strategy against this disease makes it devastating. Given that the main protease (Mpro) of SARS-CoV-2 plays an indispensable role in viral polyprotein processing, its successful inhibition prevents viral replication and constrains virus spread. Therefore, developing an effective SARS-CoV-2 Mpro inhibitor to treat COVID-19 is imperative.
We employed a high-throughput screening (HTS) method based on fluorescence polarization (FP) assay and further confirmed by the fluorescence resonance energy transfer (FRET) method for the discovery of Mpro inhibitors. Then multiple approaches were taken to investigate the inhibition profiles of the hit compounds against Mpro, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) proliferation assay, surface plasmon resonance analysis (SPR), high-performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS), cytopathic effect (CPE) assay, molecule docking, and the drug-likeness analysis.
In this study, four Mpro inhibitors with low toxicity were selected from HTS. According to SPR, all the hit compounds had medium binding affinities toward SARS-CoV-2 Mpro. HPLC-Q-TOF-MS results revealed the non-covalent linkage of each compound with SARS-CoV-2 Mpro. Molecule docking simulated the molecule interactions between each compound and the substrate binding pocket of SARS-CoV-2 Mpro. CPE assay was used to detect their inhibitory activities against coronaviruses HCoV-OC43 and HCoV-229E. In particular, the IMB63-8G compound demonstrated the highest antiviral potency [50% effective concentration (IC50) value of 1.71 μg/mL] and selectivity against HCoV-OC43 (SI = 39), which was more than 4-fold higher than that of ribavirin (RBV). Besides, the IMB63-8G compound possessed favorable drug-likeness characteristics.
Our results will highlight the therapeutic potential of these compounds for the treatment of SARS-CoV-2 infection.
由新型严重急性呼吸系统综合征冠状病毒（SARS-CoV-2）人际传播引起的冠状病毒病（COVID-19）大流行，已造成全球卫生紧急状态。针对该疾病缺乏特定药物或治疗策略，导致其破坏性极大。鉴于 SARS-CoV-2 的主要蛋白酶（Mpro）在病毒多蛋白加工中发挥不可或缺的作用，成功抑制 Mpro 可阻止病毒复制并限制病毒传播。因此，开发有效的 SARS-CoV-2 Mpro 抑制剂以治疗 COVID-19 势在必行。
我们采用基于荧光偏振（FP）测定法的高通量筛选（HTS）方法，并进一步通过荧光共振能量转移（FRET）方法进行确认，以发现 Mpro 抑制剂。然后采用多种方法研究了候选化合物对 Mpro 的抑制特性，包括 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴化物（MTT）增殖测定法、表面等离子体共振分析（SPR）、高效液相色谱-四极杆飞行时间质谱法（HPLC-Q-TOF-MS）、细胞病变效应（CPE）测定法、分子对接和药物相似性分析。
在本研究中，我们从 HTS 中筛选出四种低毒性的 Mpro 抑制剂。根据 SPR 分析，所有候选化合物与 SARS-CoV-2 Mpro 均具有中等结合亲和力。HPLC-Q-TOF-MS 结果表明，每种化合物均与 SARS-CoV-2 Mpro 发生非共价结合。分子对接模拟了各化合物与 SARS-CoV-2 Mpro 底物结合口袋之间的分子相互作用。CPE 测定法用于检测各化合物对冠状病毒 HCoV-OC43 和 HCoV-229E 的抑制活性。特别是，IMB63-8G 化合物表现出最高的抗病毒活性[半数有效浓度（IC50）值为 1.71μg/mL]和对 HCoV-OC43 的选择性（SI = 39），比利巴韦林（RBV）高 4 倍以上。此外，IMB63-8G 化合物具有良好的药物相似性特征。
我们的研究结果将突出这些化合物在治疗 SARS-CoV-2 感染方面的治疗潜力。
