Identification of Vitamin K3 and its analogues as covalent inhibitors of SARS-CoV-2 3CL(pro)

Wang, Ruyu; Hu, Qing; Wang, Haonan; Zhu, Guanghao; Wang, Mengge; Zhang, Qian; Zhao, Yishu; Li, Chunyu; Zhang, Yani; Ge, Guangbo; Chen, Hongzhuan; Chen, Lili

After the emergence of the pandemic, repurposed drugs have been considered as a quicker way of finding potential antiviral agents. SARS-CoV-2 3CL(pro) is essential for processing the viral polyproteins into mature non-structural proteins, making it an attractive target for developing antiviral agents. Here we show that Vitamin K3 screened from the FDA-Approved Drug Library containing an array of 1,018 compounds has potent inhibitory activity against SARS-CoV-2 3CL(pro) with the IC(50) value of 4.78 ± 1.03 μM, rather than Vitamin K1, K2 and K4. Next, the time-dependent inhibitory experiment was carried out to confirm that Vitamin K3 could form the covalent bond with SARS-CoV-2 3CL(pro). Then we analyzed the structure-activity relationship of Vitamin K3 analogues and identified 5,8-dihydroxy-1,4-naphthoquinone with 9.8 times higher inhibitory activity than Vitamin K3. Further mass spectrometric analysis and molecular docking study verified the covalent binding between Vitamin K3 or 5,8-dihydroxy-1,4-naphthoquinone and SARS-CoV-2 3CL(pro). Thus, our findings provide valuable information for further optimization and design of novel inhibitors based on Vitamin K3 and its analogues, which may have the potential to fight against SARS-CoV-2.