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 general virus growth cycle includes virus entry, viral genome replication, and virus release. To further validate virus genome replication is the major target of DHODHi, we used the influenza-A-virus mini-replicon system to quantify viral genome replication. Brequinar, another potent inhibitor of DHODH was included as a positive control 30 , whereas, Osel targeting influenza NA protein served as a negative control. The results in Fig.4E showed no inhibition on viral genome replication in the Osel-treated group, but there were obvious inhibitions on viral genome replication in both S312-and S416-treated groups as well as Brequinar-treated group in dosedependent manners. Almost 90% of viral genome replication was suppressed by 10IC50 of S312 (24μM) and S416 (0.6μM). As DHODH catalyzes oxidation of dihydroorotate (DHO) to produce orotic acid (ORO) and finally forms UTP and CTP， we add four nucleotides (adenine nucleotide(A), guanine nucleotide(G), uracil nucleotide(U), and cytosine nucleotide(C)), DHO, ORO respectively to mini-replicon system to identify the target of S312 and S416. The results in Fig. 4F showed that the addition of 50μM either U or C could effectively rescue viral genome replication in S312-and S416-treated cells (as well as Brequinar-treated cells), whereas addition of neither A nor G changed the inhibitory effects. Moreover, supplement of DHODH substrate DHO cannot rescue viral genome replication (Fig. 4G) , but a supplement of DHODH product ORO can gradually reverse the inhibition effects of S312 and S416 (Fig. 4H) . The results further confirm that compounds S312 and S416 inhibit viral genome replication via targeting DHODH and interrupting the fourth step in de novo pyrimidine synthesis. The copyright holder for this preprint (which was not peer-reviewed) is the . S312 is advantageous over the DAA drug to treat advanced and late-phase disease with decreasing cytokine/chemokine storm.