key: cord-297044-tpp40j0g authors: Jin, Zhenming; Zhao, Yao; Sun, Yuan; Zhang, Bing; Wang, Haofeng; Wu, Yan; Zhu, Yan; Zhu, Chen; Hu, Tianyu; Du, Xiaoyu; Duan, Yinkai; Yu, Jing; Yang, Xiaobao; Yang, Xiuna; Yang, Kailin; Liu, Xiang; Guddat, Luke W.; Xiao, Gengfu; Zhang, Leike; Yang, Haitao; Rao, Zihe title: Structural basis for the inhibition of SARS-CoV-2 main protease by antineoplastic drug Carmofur date: 2020-04-28 journal: bioRxiv DOI: 10.1101/2020.04.09.033233 sha: doc_id: 297044 cord_uid: tpp40j0g The antineoplastic drug Carmofur was shown to inhibit SARS-CoV-2 main protease (Mpro). Here the X-ray crystal structure of Mpro in complex with Carmofur reveals that the carbonyl reactive group of Carmofur is covalently bound to catalytic Cys145, whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral replication in cells (EC50 = 24.30 μM) and it is a promising lead compound to develop new antiviral treatment for COVID-19. The antineoplastic drug Carmofur was shown to inhibit SARS-CoV-2 main protease 24 (M pro ). Here the X-ray crystal structure of M pro in complex with Carmofur reveals that 25 the carbonyl reactive group of Carmofur is covalently bound to catalytic Cys145, 26 whereas its fatty acid tail occupies the hydrophobic S2 subsite. Carmofur inhibits viral 27 replication in cells (EC50 = 24.30 μM) and it is a promising lead compound to develop 28 new antiviral treatment for COVID-19. 29 30 COVID-19, a highly infectious viral disease, has spread since its appearance in 31 December 2019, causing an unprecedented pandemic. The number of confirmed cases 32 worldwide continues to grow at a rapid rate, but there are no specific drugs or vaccines 33 available to control symptoms or the spread of this disease at this time. 34 The etiological agent of the disease is the coronavirus SARS-CoV-2. This virus has 35 a ~30,000 nt RNA genome. The N-terminus of the viral genome encodes two 36 translational products, polyproteins 1a and 1ab (pp1a and pp1ab) 1,2 , which are 37 processed into mature non-structural proteins, by the main protease (M pro ) and a papain-38 like protease 3 . M pro has been proposed as a therapeutic target for anti-coronavirus (CoV) 39 drug development 4-6 . We previously screened over 10,000 compounds and identified 40 Carmofur as compound that can inhibit M pro in vitro, with an IC50 of 1.82 μM 7 . Carmofur (1-hexylcarbamoyl-5-fluorouracil) is a derivative of 5-fluorouracil (5-42 FU) (Fig. 1a) and an approved antineoplastic agent. Carmofur has been used to treat 43 colorectal cancer since 1980s 8 , and has shown clinical benefits on breast, gastric, and 44 bladder cancers 9-11 . The target for Carmofur is believed to be thymidylate synthase 12,13 , 45 but it has also been shown to inhibit human acid ceramidase (AC) 14 , through covalent 46 modification of its catalytic cysteine 15 . The molecular details for how Carmofur inhibits M pro activity were unresolved. Here, we present the 1.6 Å X-ray crystal structure of SARS-CoV-2 M pro in complex 49 with Carmofur (Fig. 1b, domain II feature the catalytic dyad residues Cys145 and His41 (Fig. 1c, d) . The 57 substrate-binding pocket is divided into a series of subsites (including S1, S2, S4, and 58 S1′), each accommodating a single but consecutive amino acid residue in the substrate. The first residue serine of one protomer interacts with residue Phe140 and Glu166 of 60 the other protomer to stabilize the S1 subsite (Extended Data Fig. 1c) , and this structural 61 feature is essential for catalysis 7 . The electron density map unambiguously shows that the fatty acid moiety . Another difference is that Carmofur only occupies the S2 subsite (Fig. 1d) , whereas 86 N3 occupies four subsites (S1, S2, S4 and S1′, see Extended Data Fig. 3b , c). The lactam 87 ring of N3 is located in the S1 subsite, which is filled by a DMSO molecule in the M pro - 88 Carmofur structure (Extended Data Fig. 3b, c) . These observations demonstrate the 89 potential for structural elaboration of Carmofur and will be useful to design more potent 90 derivatives against the M pro of SARS-CoV-2. 91 We previously showed that treatment with 10 μM Ebselen (EC50 = 4.67 μM) 92 inhibited infection of Vero cells with SARS-CoV-2 whereas Carmofur did not showed 93 detectable antiviral activity at this concentration 7 . Here we determined the inhibitory 94 effect of Carmofur against SARS-CoV-2 infection on Vero E6 cells, as previously 95 described 20 (Fig. 2) . By measuring viral RNA in supernatant, we determined the EC50 96 for Carmofur as 24.30 μM (Fig. 2a) . To verify this result, we fixed infected cells and 97 stained them using anti-sera against viral nucleocapsid protein (NP) and observed a decrease in NP levels after Carmofur treatment (Fig. 2b) . We also performed 99 cytotoxicity assays for Carmofur in Vero E6 cells and determined the CC50 value of 100 133.4 μM (Fig. 2c) . Thus, Carmofur has a favorable selectivity index (SI) of 5.36, but 101 further optimization will be required to develop into an effective drug. In conclusion, the crystal structure of M pro in complex with Carmofur shows that 103 the compound directly modifies the catalytic Cys145 of SARS-CoV-2 M pro . Our study 104 also provides a basis for rational design of Carmofur analogs with enhanced inhibitory 105 efficacy to treat COVID-19. Since M pro is highly conserved among all CoV M pro s, 106 Carmofur and its analogs may be effective against a broader spectrum of coronaviruses. The best crystals were grown using a well buffer containing 0. to immunofluorescence to monitor intracellular NP level as described previously 20 . For 227 cytotoxicity assays, Vero E6 cells were suspended in growth medium in 96-well plates. The next day, appropriate concentrations of Carmofur were added to the medium. After Ceramidase in Glioblastoma: A Review of Its Role, Potential Treatment, and 145 Challenges Molecular Mechanism of Inhibition of Acid Ceramidase by 147 Structures of two coronavirus main proteases: implications for 149 substrate binding and antiviral drug design The newly emerged SARS-like coronavirus HCoV-EMC also has 151 an "Achilles' heel": current effective inhibitor targeting a 3C-like protease Structure of Main Protease 154 from Human Coronavirus NL63: Insights for Wide Spectrum Anti-Coronavirus Design of wide-spectrum inhibitors targeting coronavirus main References Phaser crystallographic software Macromolecular structure determination using X-rays, 244 neutrons and electrons: recent developments in Phenix Features and development 247 of Coot Towards automated crystallographic structure refinement 249 with phenix.refine We are grateful to the staff at the BL17U1, BL18U1 and BL19U1 at Shanghai 252 This work 253 was supported by grants from National Key R&D Program of China (grants No. 254 Department of Science and Technology of Guangxi Zhuang Autonomous Region 2020AB40007), and the Natural Science Foundation of China (grant No conceived the project performed qRT-266 PCR and cytotoxicity assay analysis 270 The authors declare no competing interests.