Novel and potent inhibitors targeting DHODH, a rate-limiting enzyme in de novo pyrimidine biosynthesis, are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2

Rui Xiong; Leike Zhang; Shiliang Li; Yuan Sun; Minyi Ding; Yong Wang; Yongliang Zhao; Yan Wu; Weijuan Shang; Xiaming Jiang; Jiwei Shan; Zihao Shen; Yi Tong; Liuxin Xu; Chen Yu; Yingle Liu; Gang Zou; Dimitri Lavillete; Zhenjiang Zhao; Rui Wang; Lili Zhu; Gengfu Xiao; Ke Lan; Honglin Li; Ke Xu

To examine the antiviral activities of these DHODHi, we use the influenza A virus as a model virus. A labor stain of A/WSN/33(H1N1, WSN) with 20 TCID50 was applied to infect MDCK cells, and serial dilutions of drugs (DMSO as controls) were added at the same time when cells were infected. Drug efficacies were evaluated by quantification of cell viability in both infected and non-infected cells, and the halfmaximal effective concentration (EC50) and the half-cytotoxic concentration (CC50) of the indicated drug were obtained accordingly. The selectivity index (SI) was calculated by CC50/EC50. As shown in Fig. 1D , the antiviral effect of Leflunomide is hardly detectable at the cell culture level (EC50>25 μ M). However, Teriflunomide, the active metabolite of Leflunomide, exhibited a clear antiviral effect against the WSN virus (EC50=29.33μM, CC50=178.50μM，SI=6.08). As compared to Teriflunomide, the potent DHODHi S312 is ~12-fold stronger (EC50=2.36μM) and S416 is ~480-fold stronger (EC50=0.061μM) than Teriflunomide in their antiviral efficacies. We also tested different influenza A virus subtypes of H3N2 and H9N2. The antiviral efficacies of DHODHi followed the same pattern as they were against H1N1, which is S416>S312>Teriflunomide>Leflunomide in viral inhibitory efficacies (summarized in Table 1 ). The drug effective curve of S416 to H3N2 (EC50=0.013μM) and H9N2 (EC50=0.020μM) is shown in Fig. 1E and 1F . When we compared the drug efficacy by virus plaque assay, the results in Fig. 1G showed that the positive control DAA drug Oseltamivir (Osel) could reduce the plaque size to needlepoint size. However, the virus plaque in equivalent S312-treatment was not observable at all indicating that S312 is more efficient in inhibiting virus replication than Osel.