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

The authors declare no competing interests. The copyright holder for this preprint (which was not peer-reviewed) is the . concentrations of Teriflunomide, Brequinar, S312 and S416 respectively. Inhibitory effects of these compounds (EC50) to EBOV mini-genome replication were determined using Bright-Glo Luciferase Assay (left-hand scale, red curve). CC50 of compounds were determined by analyzing BSR-T7/5 cell viability using CellTiterGlo Assay (righthand scale, green curve). The results are presented as a mean of at least two replicates ± SD. (B) Anti-Zika virus efficacy. Huh7 cells were infected with Zika virus (MOI=0.05) for 4 hours and then treated with increasing concentrations of compounds Teriflunomide, Brequinar, S312 and S416 respectively. The viral yields in cell supernatants were then quantified by qRT-PCR to reflect the replication efficiency of Zika virus. (C) Anti-SARS-CoV-2 virus efficacy. Aliquots of Vero E6 cells were seeded in 96-well plates and then infected with Beta CoV/Wuhan/WIV04/2019 at MOI of 0.03. At the same time, different concentrations of the compounds were added for co-culture. Cell supernatants were harvested 48 h.p.i. and RNA was extracted and quantified by qRT-PCR to determine the numbers of viral RNA copies. (D) Immuno-fluorescence assay of SARS-CoV-2-infected cells. Vero E6 cells were infected with SARS-CoV-2 under the same procedure of C. Cells were fixed and permeabilized for staining with anti-viral NP antibody, followed by staining with Alexa 488-labeled secondary antibody. Green represents infected cells. Nuclei were stained by DAPI, and the merge of NP and nuclei were shown. Scale bar, 400uM. The results (B, C) are presented as a mean of at least three replicates ± SD. Statistical analysis, One-way ANOVA for (B). NS, p >0.05; *, p <0.05; **, p <0.01; ***, p <0.001. (2.5, 5, 10mg/kg), Oseltamivir (20mg/kg) and S312+Oseltamivir (10mg/kg+20mg/kg) once per day from D1-D14 respectively. The body weight and survival were monitored for 14 days or until body weight reduced to 75% (n = 4 mice per group). (C) Mice were inoculated intranasally with 600 PFU of A/SC/09 (H1N1) and then i.p. with S312 (10mg/kg), Oseltamivir (20mg/kg) and S312+Oseltamivir (10mg/kg+20mg/kg) once per day from D1 to D14. The body weight and survival were monitored until 14 days post-infection or when the bodyweight reduced to 75%. The dotted line indicates endpoint for mortality (75% of initial weight). The body weights are present as the mean percentage of the initial weight ±SD of 4-5 mice per group and survival curve were shown.