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Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease

Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease RESEARCH ARTICLES Cite as: W. Dai et al., Science 10.1126/science.abb4489 (2020). Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease 1,2 3 4 1 1,5 3 1 Wenhao Dai *, Bing Zhang *, Xia-Ming Jiang *, Haixia Su *, Jian Li , Yao Zhao , Xiong Xie , 3 1 3 1 6 3 3 Zhenming Jin , Jingjing Peng , Fengjiang Liu , Chunpu Li , You Li , Fang Bai , Haofeng Wang , 1 6 1 3 1 7 4 Xi Cheng , Xiaobo Cen , Shulei Hu , Xiuna Yang , Jiang Wang , Xiang Liu , Gengfu Xiao , 1,2,3 3 4 1 3 1,2,5 Hualiang Jiang , Zihe Rao , Lei-Ke Zhang †, Yechun Xu †, Haitao Yang †, Hong Liu † State Key Laboratory of Drug Research, CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 2 3 201203, China. School of Pharmacy, China Pharmaceutical University, Nanjing 210009, Jiangsu, China. Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, China. College of Pharmacy, Nanjing University of Chinese Medicine, Qixia District, Nanjing 210023, China. National Chengdu Center for Safety Evaluation of Drugs, Westchina Hospital of Sichuan Unverisity, High-Tech Development Zone, Chengdu, Sichuan 610041, China. State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for Cell Response, College of Life Sciences, College of Pharmacy, Nankai University, Tianjin 300353, China. *These authors contributed equally to this work. †Corresponding author. Email: [email protected] (H.L.); [email protected] (H.Y.); [email protected] (L.-K.Z.); [email protected] (Y.X.) SARS-CoV-2 is the etiological agent responsible for the global COVID-19 outbreak. The main protease pro (M ) of SARS-CoV-2 is a key enzyme that plays a pivotal role in mediating viral replication and pro transcription. We designed and synthesized two lead compounds (11a and 11b) targeting M . Both exhibited excellent inhibitory activity and potent anti-SARS-CoV-2 infection activity. The X-ray crystal pro structures of SARS-CoV-2 M in complex with 11a or 11b, both determined at 1.5 Å resolution, showed that pro the aldehyde groups of 11a and 11b are covalently bound to Cys145 of M . Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity, suggesting that these compounds are promising drug candidates. In late December 2019, a cluster of pneumonia cases caused the treatment of COVID-19, but more data are needed to by a novel coronavirus (CoV) was reported in Wuhan, China prove its efficacy (10–12). Specific anti-SARS-CoV-2 drugs (1–3). Genomic sequencing showed that this pathogenic with efficiency and safety are urgently needed. coronavirus is 96.2% identical to a bat coronavirus and A maximum likelihood tree based on the genomic se- shares 79.5% sequence identify to SARS-CoV (4–6). This quence showed that the virus falls within the subgenus Sar- novel coronavirus was named severe acute respiratory syn- becovirus of the genus Betacoronavirus (6). Coronaviruses drome coronavirus 2 (SARS-CoV-2) by the International are enveloped, positive-sense, single-stranded RNA viruses. Committee on Taxonomy of Viruses, and the pneumonia The genomic RNA of CoVs is approximately 30 k nt in was designated as COVID-19 by the World Health Organiza- length with a 5′-cap structure and 3′-poly-A tail, and con- tion (WHO) on February 11, 2020 (7). The epidemic spread tains at least 6 open reading frames (ORFs) (13, 14). The rapidly to more than 212 countries and was announced as a first ORF (ORF 1a/b), about two-third of genome length, global health emergency by WHO (8). No clinically effective directly translates two polyproteins: pp1a and pp1ab, be- vaccines or specific antiviral drugs are currently available cause there is an a-1 frameshift between ORF1a and ORF1b. pro for the prevention and treatment of COVID-19 infections. ), These polyproteins are processed by a main protease (M pro also known as the 3C-like protease (3CL ), and one or two The combination of α-interferon and the anti-HIV drugs papain-like proteases (PLPs), into 16 non-structural proteins Lopinavir/Ritonavir (Kaletra®) has been used, but the cura- (nsps). These nsps engage in the production of subgenomic tive effect remains very limited and there can be toxic side RNAs that encode four main structural proteins (envelope effects (9). Remdesivir, a broad-spectrum antiviral drug de- (E), membrane (M), spike (S), and nucleocapsid (N) pro- veloped by Gilead Sciences, Inc., is also being explored for First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 1 pro teins) and other accessory proteins (15, 16). Therefore, these Recombinant SARS-CoV-2 M was expressed and puri- pro proteases, especially M , play a vital role in the life cycle of fied from Escherichia coli (E. coli) (18, 25). A fluorescently coronavirus. labeled substrate, MCA-AVLQ↓SGFR-Lys (Dnp)-Lys-NH , pro M is a three-domain (domains I to III) cysteine prote- derived from the N-terminal auto-cleavage sequence from ase involved in most maturation cleavage events within the the viral protease was designed and synthesized for the en- pro precursor polyprotein (17–19). Active M is a homodimer zymatic assay. pro pro containing two protomers. The CoV M features a non- Both 11a and 11b exhibited high SARS-CoV-2 M inhi- canonical Cys-His dyad located in the cleft between domains bition activity, which reached 100% for 11a and 96% for 11b pro I and II (17–19). M is conserved among CoVs and several at 1 µM, respectively. We used a fluorescence resonance en- pro common features are shared among the substrates of M in ergy transfer (FRET)-based cleavage assay to determine the different CoVs. The amino acids in substrates from the N IC values. The results revealed excellent inhibitory potency terminus to C terminus are numbered as fellows (-P4-P3-P2- with IC values of 0.053 ± 0.005 µM and 0.040 ± 0.002 µM, P1↓P1′-P2′-P3′-), and the cleavage site is between the P1 for 11a and 11b respectively (Fig. 2). and P1′. In particular, a Gln residue is almost always re- In order to elucidate the mechanism of inhibition of pro quired in the P1 position of the substrates. There is no hu- SARS-CoV-2 M by 11a, we determined the high-resolution pro man homolog of M which makes it an ideal antiviral crystal structure of this complex at 1.5-Å resolution (table pro target (20–22). S1). The crystal of M -11a belong to the space group C2 and pro The active sites of M are highly conserved among all an asymmetric unit contains only one molecule (table S1). pro CoV’s M s and are usually composed of four sites (S1′, S1, Two molecules (designated protomer A and protomer B) S2 and S4) (22). By analyzing the substrate-binding pocket associate into a homodimer around a crystallographic 2-fold pro of SARS-CoV M (PDB ID: 2H2Z), novel inhibitors target- symmetry axis (fig. S2). The structure of each protomer con- pro ing the SARS-CoV-2 M were designed and synthesized tains three domains with the substrate-binding site located (Fig. 1). The thiol of a cysteine residue in the S1′ sites an- in the cleft between domain I and II. At the active site of pro chors inhibitors by a covalent linkage that is important for SARS-CoV-2 M , Cys145 and His41 (Cys-His) form a catalyt- the inhibitors to maintain antiviral activity. In our design of ic dyad (fig. S2). new inhibitors, an aldehyde was selected as a new warhead The electron density map clearly showed compound 11a pro in P1 in order to form a covalent bond with cysteine. The in the substrate binding pocket of SARS-CoV-2 M in an pro reported SARS-CoV M inhibitors often have an (S)-γ- extended conformation (Fig. 3A and fig. S3, A and B). De- pro lactam ring that occupies the S1 site of M , and this ring tails of the interaction are shown in Fig. 3, B and C. The was expected to be a good choice in P1 (23). Furthermore, electron density shows that the C of the aldehyde group of pro pro the S2 site of coronavirus M is usually large enough to 11a and the catalytic site Cys145 of SARS-CoV-2 M form a accommodate the bigger P2 fragment. To test the im- standard 1.8-Å C–S covalent bond. The oxygen atom of the portance of different ring systems, a cyclohexyl or 3- aldehyde group also plays a crucial role in stabilizing the fluorophenyl were introduced in P2, with the fluorine ex- conformations of the inhibitor by forming a 2.9-Å hydrogen pected to enhance activity. An indole group was introduced bond with the backbone of residues Cys145 in the S1′ site. into P3 in order to form new hydrogen bonds with S4 and The (S)-γ-lactam ring of 11a at P1 fits well into the S1 site. improve drug-like properties. The oxygen of the (S)-γ-lactam group forms a 2.7-Å hydrogen The synthetic route and chemical structures of the bond with the side chain of His163. The main chain of compounds (11a and 11b) are shown in scheme S1. The Phe140 and side chain of Glu166 also participate in stabiliz- starting material (N-Boc-L-glutamic acid dimethyl ester 1) ing the (S)-γ-lactam ring by forming 3.2-Å and 3.0-Å hydro- was obtained from commercial suppliers and used without gen bonds with its NH group, respectively. In addition, the further purification to synthesize the key intermediate 3 amide bonds on the chain of 11a are hydrogen-bonded with according to the literature (24). The intermediates 6a and the main chains of His164 (3.2 Å) and Glu166 (2.8 Å), re- 6b were synthesized from 4 and acids 5a, 5b. Removal of spectively. The cyclohexyl moiety of 11a at P2 deeply inserts the t-butoxycarbonyl group from 6a and 6b yielded 7a and into the S2 site, stacking with the imidazole ring of His41. 7b. Coupling 7a and 7b with the acid 8 yielded the esters The cyclohexyl group is also surrounded by the side chains 9a and 9b. The peptidomimetic aldehydes 11a and 11b were of Met49, Tyr54, Met165, Asp187 and Arg188, producing ex- approached through a two-step route in which the ester de- tensive hydrophobic interactions. The indole group of 11a at rivatives 9 were first reduced with NaBH to generate the P3 is exposed to solvent (S4 site) and is stabilized by Glu166 primary alcohols 10a and 10b, which were subsequently through a 2.6-Å hydrogen bond. The side chains of residues oxidized into aldehydes 11a and 11b with Dess-Martin Peri- Pro168 and Gln189 interact with the indole group of 11a odinane (DMP). through hydrophobic interactions. Interestingly, multiple First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 2 water molecules (named W1-W6) play an important role in 87.8% were observed when the compound 11a was given binding 11a. W1 interacts with the amide bonds of 11a intraperitoneally. Metabolic stability of 11a in mice was also through a 2.9-Å hydrogen bond, whereas W2-6 form a num- good (Clearance (CL) = 17.4 mL/min/mg). When adminis- ber of hydrogen bonds with the aldehyde group of 11a and tered intraperitoneally (20 mg/kg), subcutaneously (5 the residues of Asn142, Gly143, Thr26, Thr25, His41 and mg/kg) and intravenously (5 mg/kg), compound 11b also Cys44, which contributes to stabilizing 11a in the binding showed good PK properties (the bioavailability of intraperi- pocket. toneally and subcutaneously are more than 80%, and a pro The crystal structure of SARS-CoV-2 M in complex longer T of 5.21 hours when 11b was given intraperitone- 1/2 with 11b is very similar to that of the 11a complex and ally). Considering the danger of COVID-19, we selected the shows a similar inhibitor binding mode (Fig. 3D and figs. intravenous drip administration to further study for the S3, C and D, and S4A). The difference in binding mode is reason that value of the area under the curve (AUC) is high most probably due to the 3-fluorophenyl group of 11b at P2. and the effect is rapid. Compared with 11a administrated Compared with the cyclohexyl group in 11a, the 3- intravenously, the T (1.65h) of 11b is shorter and the 1/2 fluorophenyl group undergoes a significant downward rota- clearance rate is faster (CL = 20.6 mL/min/mg). Compound tion (Fig. 3D). The side chains of residues His41, Met49, 11a was selected for further investigation with intravenous Met165, Val186, Asp187 and Arg188 interact with this aryl drip dosing in Sprague-Dawley (SD) rats and Beagle dogs. group through hydrophobic interactions and the side chain The results showed (table S3) that 11a exhibited long T 1/2 of Gln189 stabilizes the 3-fluorophenyl group with an addi- (SD rat, 7.6 hours and Beagle dog, 5.5h), low clearance rate tional 3.0-Å hydrogen bond (Fig. 3, E and F). In short, these (rat, 4.01 mL/min/kg and dog, 5.8 mL/min/kg) and high two crystal structures reveal a similar inhibitory mechanism AUC value (rat, 41500 hours*ng/mL and dog, 14900 in which both compounds occupy the substrate-binding hours*ng/mL)). Those above PK results indicate that com- pro pocket and block the enzyme activity of SARS-CoV-2 M . pound 11a is worth to warrant further study. Compared with those of N1, N3 and N9 in SARS-CoV An in vivo toxicity study (table S4) of 11a has been car- pro M complex structures reported previously, the binding ried out on SD rats and Beagle dogs. The acute toxicity of pro modes of 11a and 11b in SARS-CoV-2 M complex struc- 11a was measured on SD rats. No SD rats died after receiv- tures are similar and the differences among these overall ing 40 mg/kg by intravenous drip administration. When the structures are small (Fig. 4 and fig. S4, B to F) (22). The dif- dosage was raised to 60 mg/kg, one of four SD rats died. ferences mainly lie in the interactions at S1′, S2 and S4 sub- The dose range toxicity study of 11a was conducted for sev- sites, possibly due to various sizes of functional groups at en days at dosing levels of 2, 6, and 18 mg/kg on SD rats and corresponding P1′, P2 and P4 sites in the inhibitors (Fig. 4, at 10-40 mg/kg on Beagle dogs. All animals received once A and C). daily dosing (QD), by intravenous drip, and all animals were To further substantiate the enzyme inhibition results, clinically observed at least once a day. No obvious toxicity we evaluated the ability of these compounds to inhibit was observed in either group. These above data indicated SARS-CoV-2 in vitro (Fig. 5 and fig. S5). 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Trans. 39, 1371–1375 (2011). doi:10.1042/BST0391371 Medline 16. Z. Ren, L. Yan, N. Zhang, Y. Guo, C. Yang, Z. Lou, Z. Rao, The newly emerged First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 4 ACKNOWLEDGMENTS We thank Prof. James Halpert and LetPub (www.letpub.com) for linguistic assistance during the preparation of this manuscript. We also thank the staff from beamlines BL17U1, BL18U1 and BL19U1 at Shanghai Synchrotron Radiation Facility (SSRF) for assistance during data collection. Funding: We are grateful to the National Natural Science Foundation of China (Nos. 21632008, 21672231, 21877118, 31970165, 91953000 and 81620108027), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12040107 and XDA12040201) and Chinese Academy of Engineering and Ma Yun Foundation (No. 2020-CMKYGG-05) and Science and Technology Commission of Shanghai Municipality (Nos. 20431900100), and National Key R&D Program of China (Nos. 2017YFC0840300 and 2020YFA0707500 to Z.R.), and Science and Technology Commission of Shanghai Municipality (No. 20431900200), and Department of Science and Technology of Guangxi Zhuang Autonomous Region (No. 2020AB40007), and Frontier Biotechnologies Inc. for financial support. Author contributions: H. Y. and H. L. conceived the project. Y. X., L. Z., H. Y., and H. L. designed the experiments; W. D. and J. L. designed and synthesized the compounds; X. J. and H. S tested the inhibitory activities; X. X., J. P., C. L., S. H., J. W., performed the chemical experiments and collected the data. B. Z., Y. Z., Z. J., F. L., F. B., H. W., X. C., X. L., and X. Y. collected the diffraction data and solved the crystal structure; Y. L. and X. C. performed the toxicity experiments. G. X., H. J., Z. R., L, Z., Y. X., H. Y. and H. L., analyzed and discussed the data. L. Z., Y. X., H. Y., and H. L., wrote the manuscript. Competing interests: The Shanghai Institute of Materia Medica has applied for PCT and Chinese patents which cover 11a, 11b and related peptidomimetic aldehyde compounds. Data and materials availability: All data are available in the main text or the supplementary materials. The PDB accession No. for the coordinates of SARS- CoV-2 Mpro in complex with 11a is 6LZE, and the PDB accession No. for the pro coordinates of SARS-CoV-2 M in complex with 11b is 6M0K. The plasmid encoding pro the SARS-CoV-2 M will be freely available. Compounds 11a and 11b are available from H. L under a material transfer agreement with Shanghai Institute of Materia Medica. There is currently an international effort to join forces to design better pro inhibitors of SARS-CoV-2 M as described in the following website: https://covid.postera.ai/covid. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material. SUPPLEMENTARY MATERIALS science.sciencemag.org/cgi/content/full/science.abb4489/DC1 Materials and Methods Scheme S1 Figs. S1 to S5 Tables S1 to S4 References (26–29) 18 March 2020; accepted 20 April 2020 Published online 22 April 2020 10.1126/science.abb4489 First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 5 Fig. 1. Design strategy of novel SARS-CoV-2 main protease inhibitors and the chemical structures of 11a and 11b. pro Fig. 2. Inhibitory activity profiles of compounds 11a (A) and 11b (B) against SARS-CoV-2 M . First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 6 pro Fig. 3. M -inhibitor binding modes for 11a and 11b. (A) Cartoon representation of the crystal structure of SARS-CoV-2 pro M in complex with 11a. The compound 11a is shown as magenta sticks; water molecules shown as red spheres. (B) Close-up view of the 11a binding pocket. Four subsites, S1′, S1, S2 and S4, are labeled. The residues involved in inhibitor binding are shown as wheat sticks. 11a and water molecules are shown as magenta sticks and red spheres, respectively. pro Hydrogen bonds are indicated as dashed lines. (C) Schematic diagram of SARS-CoV-2 M -11a interactions shown in (B). pro (D) Comparison of the binding modes between 11a and 11b for SARS-CoV-2 M . The major differences between 11a and 11b are marked with dashed circles. The compounds of 11a and 11b are shown as magenta and yellow sticks, respectively. (E) Close-up view of the 11b binding pocket. Hydrogen bonds are indicated as dashed lines. (F) Schematic diagram of pro SARS-CoV-2 M -11b interactions shown in (E). First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 7 pro Fig. 4. Comparison of the inhibitor binding modes in SARS-CoV and SARS-CoV-2 M s. (A) Comparison of pro pro pro binding modes of 11a in SARS-CoV-2 M with those of N1, N3 and N9 in SARS-CoV M . SARS-CoV-2 M -11a pro pro (wheat, PDB code: 6LZE), SARS-CoV M -N1 (sky blue, PDB code:1WOF), SARS-CoV M -N3 (gray, PDB code: pro 2AMQ) and SARS-CoV M -N9 (olive, PDB code: 2AMD).11a, N1, N3 and N9 are shown in magenta, cyan, dirty pro violet and salt, respectively. (B) Comparison of the 11a and N3 binding pockets. Residues in M -11a structure and pro M -N3 structure are colored in wheat and gray, respectively. 11a and N3 are shown as sticks colored in magenta pro and dirty violet, respectively. (C) Comparison of binding modes of 11b in SARS-CoV-2 M with those of N1, N3 and pro pro N9 in SARS-CoV M . SARS-CoV-2 M -11b (pale cyan, PDB code: 6M0K). 11b, N1, N3 and N9 are shown in yellow, pro cyan, dirty violet and salt, respectively. (D) Comparison of the 11b and N9 binding pockets. Residues in M -11b pro structure and M -N9 structure are colored in pale cyan and olive, respectively. 11b and N9 are shown as sticks colored in yellow and salt, respectively. First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 8 Fig. 5. In vitro inhibition of viral main protease inhibitors against SARS-CoV-2. (A and B) Vero E6 cells were treated with a series concentration of indicated compounds 11a and 11b and infected with SARS-CoV-2 at an MOI of 0.05. At 24 hours post infection, viral yield in the cell supernatant was quantified by plaque assay. The cytotoxicity of these compounds in Vero E6 cells was also determined by using CCK8 assays. The left and right Y-axis of the graphs represent mean % inhibition of virus yield and mean % cytotoxicity of the drugs, respectively. (C and D) Viral RNA copy numbers in the cell supernatants were quantified by qRT-PCR. Data are mean ± SD, n = 3 biological replicates. First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 9 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Science (New York, N.y.) Pubmed Central

Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease

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RESEARCH ARTICLES Cite as: W. Dai et al., Science 10.1126/science.abb4489 (2020). Structure-based design of antiviral drug candidates targeting the SARS-CoV-2 main protease 1,2 3 4 1 1,5 3 1 Wenhao Dai *, Bing Zhang *, Xia-Ming Jiang *, Haixia Su *, Jian Li , Yao Zhao , Xiong Xie , 3 1 3 1 6 3 3 Zhenming Jin , Jingjing Peng , Fengjiang Liu , Chunpu Li , You Li , Fang Bai , Haofeng Wang , 1 6 1 3 1 7 4 Xi Cheng , Xiaobo Cen , Shulei Hu , Xiuna Yang , Jiang Wang , Xiang Liu , Gengfu Xiao , 1,2,3 3 4 1 3 1,2,5 Hualiang Jiang , Zihe Rao , Lei-Ke Zhang †, Yechun Xu †, Haitao Yang †, Hong Liu † State Key Laboratory of Drug Research, CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 2 3 201203, China. School of Pharmacy, China Pharmaceutical University, Nanjing 210009, Jiangsu, China. Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China. State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, China. College of Pharmacy, Nanjing University of Chinese Medicine, Qixia District, Nanjing 210023, China. National Chengdu Center for Safety Evaluation of Drugs, Westchina Hospital of Sichuan Unverisity, High-Tech Development Zone, Chengdu, Sichuan 610041, China. State Key Laboratory of Medicinal Chemical Biology, Frontiers Science Center for Cell Response, College of Life Sciences, College of Pharmacy, Nankai University, Tianjin 300353, China. *These authors contributed equally to this work. †Corresponding author. Email: [email protected] (H.L.); [email protected] (H.Y.); [email protected] (L.-K.Z.); [email protected] (Y.X.) SARS-CoV-2 is the etiological agent responsible for the global COVID-19 outbreak. The main protease pro (M ) of SARS-CoV-2 is a key enzyme that plays a pivotal role in mediating viral replication and pro transcription. We designed and synthesized two lead compounds (11a and 11b) targeting M . Both exhibited excellent inhibitory activity and potent anti-SARS-CoV-2 infection activity. The X-ray crystal pro structures of SARS-CoV-2 M in complex with 11a or 11b, both determined at 1.5 Å resolution, showed that pro the aldehyde groups of 11a and 11b are covalently bound to Cys145 of M . Both compounds showed good PK properties in vivo, and 11a also exhibited low toxicity, suggesting that these compounds are promising drug candidates. In late December 2019, a cluster of pneumonia cases caused the treatment of COVID-19, but more data are needed to by a novel coronavirus (CoV) was reported in Wuhan, China prove its efficacy (10–12). Specific anti-SARS-CoV-2 drugs (1–3). Genomic sequencing showed that this pathogenic with efficiency and safety are urgently needed. coronavirus is 96.2% identical to a bat coronavirus and A maximum likelihood tree based on the genomic se- shares 79.5% sequence identify to SARS-CoV (4–6). This quence showed that the virus falls within the subgenus Sar- novel coronavirus was named severe acute respiratory syn- becovirus of the genus Betacoronavirus (6). Coronaviruses drome coronavirus 2 (SARS-CoV-2) by the International are enveloped, positive-sense, single-stranded RNA viruses. Committee on Taxonomy of Viruses, and the pneumonia The genomic RNA of CoVs is approximately 30 k nt in was designated as COVID-19 by the World Health Organiza- length with a 5′-cap structure and 3′-poly-A tail, and con- tion (WHO) on February 11, 2020 (7). The epidemic spread tains at least 6 open reading frames (ORFs) (13, 14). The rapidly to more than 212 countries and was announced as a first ORF (ORF 1a/b), about two-third of genome length, global health emergency by WHO (8). No clinically effective directly translates two polyproteins: pp1a and pp1ab, be- vaccines or specific antiviral drugs are currently available cause there is an a-1 frameshift between ORF1a and ORF1b. pro for the prevention and treatment of COVID-19 infections. ), These polyproteins are processed by a main protease (M pro also known as the 3C-like protease (3CL ), and one or two The combination of α-interferon and the anti-HIV drugs papain-like proteases (PLPs), into 16 non-structural proteins Lopinavir/Ritonavir (Kaletra®) has been used, but the cura- (nsps). These nsps engage in the production of subgenomic tive effect remains very limited and there can be toxic side RNAs that encode four main structural proteins (envelope effects (9). Remdesivir, a broad-spectrum antiviral drug de- (E), membrane (M), spike (S), and nucleocapsid (N) pro- veloped by Gilead Sciences, Inc., is also being explored for First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 1 pro teins) and other accessory proteins (15, 16). Therefore, these Recombinant SARS-CoV-2 M was expressed and puri- pro proteases, especially M , play a vital role in the life cycle of fied from Escherichia coli (E. coli) (18, 25). A fluorescently coronavirus. labeled substrate, MCA-AVLQ↓SGFR-Lys (Dnp)-Lys-NH , pro M is a three-domain (domains I to III) cysteine prote- derived from the N-terminal auto-cleavage sequence from ase involved in most maturation cleavage events within the the viral protease was designed and synthesized for the en- pro precursor polyprotein (17–19). Active M is a homodimer zymatic assay. pro pro containing two protomers. The CoV M features a non- Both 11a and 11b exhibited high SARS-CoV-2 M inhi- canonical Cys-His dyad located in the cleft between domains bition activity, which reached 100% for 11a and 96% for 11b pro I and II (17–19). M is conserved among CoVs and several at 1 µM, respectively. We used a fluorescence resonance en- pro common features are shared among the substrates of M in ergy transfer (FRET)-based cleavage assay to determine the different CoVs. The amino acids in substrates from the N IC values. The results revealed excellent inhibitory potency terminus to C terminus are numbered as fellows (-P4-P3-P2- with IC values of 0.053 ± 0.005 µM and 0.040 ± 0.002 µM, P1↓P1′-P2′-P3′-), and the cleavage site is between the P1 for 11a and 11b respectively (Fig. 2). and P1′. In particular, a Gln residue is almost always re- In order to elucidate the mechanism of inhibition of pro quired in the P1 position of the substrates. There is no hu- SARS-CoV-2 M by 11a, we determined the high-resolution pro man homolog of M which makes it an ideal antiviral crystal structure of this complex at 1.5-Å resolution (table pro target (20–22). S1). The crystal of M -11a belong to the space group C2 and pro The active sites of M are highly conserved among all an asymmetric unit contains only one molecule (table S1). pro CoV’s M s and are usually composed of four sites (S1′, S1, Two molecules (designated protomer A and protomer B) S2 and S4) (22). By analyzing the substrate-binding pocket associate into a homodimer around a crystallographic 2-fold pro of SARS-CoV M (PDB ID: 2H2Z), novel inhibitors target- symmetry axis (fig. S2). The structure of each protomer con- pro ing the SARS-CoV-2 M were designed and synthesized tains three domains with the substrate-binding site located (Fig. 1). The thiol of a cysteine residue in the S1′ sites an- in the cleft between domain I and II. At the active site of pro chors inhibitors by a covalent linkage that is important for SARS-CoV-2 M , Cys145 and His41 (Cys-His) form a catalyt- the inhibitors to maintain antiviral activity. In our design of ic dyad (fig. S2). new inhibitors, an aldehyde was selected as a new warhead The electron density map clearly showed compound 11a pro in P1 in order to form a covalent bond with cysteine. The in the substrate binding pocket of SARS-CoV-2 M in an pro reported SARS-CoV M inhibitors often have an (S)-γ- extended conformation (Fig. 3A and fig. S3, A and B). De- pro lactam ring that occupies the S1 site of M , and this ring tails of the interaction are shown in Fig. 3, B and C. The was expected to be a good choice in P1 (23). Furthermore, electron density shows that the C of the aldehyde group of pro pro the S2 site of coronavirus M is usually large enough to 11a and the catalytic site Cys145 of SARS-CoV-2 M form a accommodate the bigger P2 fragment. To test the im- standard 1.8-Å C–S covalent bond. The oxygen atom of the portance of different ring systems, a cyclohexyl or 3- aldehyde group also plays a crucial role in stabilizing the fluorophenyl were introduced in P2, with the fluorine ex- conformations of the inhibitor by forming a 2.9-Å hydrogen pected to enhance activity. An indole group was introduced bond with the backbone of residues Cys145 in the S1′ site. into P3 in order to form new hydrogen bonds with S4 and The (S)-γ-lactam ring of 11a at P1 fits well into the S1 site. improve drug-like properties. The oxygen of the (S)-γ-lactam group forms a 2.7-Å hydrogen The synthetic route and chemical structures of the bond with the side chain of His163. The main chain of compounds (11a and 11b) are shown in scheme S1. The Phe140 and side chain of Glu166 also participate in stabiliz- starting material (N-Boc-L-glutamic acid dimethyl ester 1) ing the (S)-γ-lactam ring by forming 3.2-Å and 3.0-Å hydro- was obtained from commercial suppliers and used without gen bonds with its NH group, respectively. In addition, the further purification to synthesize the key intermediate 3 amide bonds on the chain of 11a are hydrogen-bonded with according to the literature (24). The intermediates 6a and the main chains of His164 (3.2 Å) and Glu166 (2.8 Å), re- 6b were synthesized from 4 and acids 5a, 5b. Removal of spectively. The cyclohexyl moiety of 11a at P2 deeply inserts the t-butoxycarbonyl group from 6a and 6b yielded 7a and into the S2 site, stacking with the imidazole ring of His41. 7b. Coupling 7a and 7b with the acid 8 yielded the esters The cyclohexyl group is also surrounded by the side chains 9a and 9b. The peptidomimetic aldehydes 11a and 11b were of Met49, Tyr54, Met165, Asp187 and Arg188, producing ex- approached through a two-step route in which the ester de- tensive hydrophobic interactions. The indole group of 11a at rivatives 9 were first reduced with NaBH to generate the P3 is exposed to solvent (S4 site) and is stabilized by Glu166 primary alcohols 10a and 10b, which were subsequently through a 2.6-Å hydrogen bond. The side chains of residues oxidized into aldehydes 11a and 11b with Dess-Martin Peri- Pro168 and Gln189 interact with the indole group of 11a odinane (DMP). through hydrophobic interactions. Interestingly, multiple First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 2 water molecules (named W1-W6) play an important role in 87.8% were observed when the compound 11a was given binding 11a. W1 interacts with the amide bonds of 11a intraperitoneally. Metabolic stability of 11a in mice was also through a 2.9-Å hydrogen bond, whereas W2-6 form a num- good (Clearance (CL) = 17.4 mL/min/mg). When adminis- ber of hydrogen bonds with the aldehyde group of 11a and tered intraperitoneally (20 mg/kg), subcutaneously (5 the residues of Asn142, Gly143, Thr26, Thr25, His41 and mg/kg) and intravenously (5 mg/kg), compound 11b also Cys44, which contributes to stabilizing 11a in the binding showed good PK properties (the bioavailability of intraperi- pocket. toneally and subcutaneously are more than 80%, and a pro The crystal structure of SARS-CoV-2 M in complex longer T of 5.21 hours when 11b was given intraperitone- 1/2 with 11b is very similar to that of the 11a complex and ally). Considering the danger of COVID-19, we selected the shows a similar inhibitor binding mode (Fig. 3D and figs. intravenous drip administration to further study for the S3, C and D, and S4A). The difference in binding mode is reason that value of the area under the curve (AUC) is high most probably due to the 3-fluorophenyl group of 11b at P2. and the effect is rapid. Compared with 11a administrated Compared with the cyclohexyl group in 11a, the 3- intravenously, the T (1.65h) of 11b is shorter and the 1/2 fluorophenyl group undergoes a significant downward rota- clearance rate is faster (CL = 20.6 mL/min/mg). Compound tion (Fig. 3D). The side chains of residues His41, Met49, 11a was selected for further investigation with intravenous Met165, Val186, Asp187 and Arg188 interact with this aryl drip dosing in Sprague-Dawley (SD) rats and Beagle dogs. group through hydrophobic interactions and the side chain The results showed (table S3) that 11a exhibited long T 1/2 of Gln189 stabilizes the 3-fluorophenyl group with an addi- (SD rat, 7.6 hours and Beagle dog, 5.5h), low clearance rate tional 3.0-Å hydrogen bond (Fig. 3, E and F). In short, these (rat, 4.01 mL/min/kg and dog, 5.8 mL/min/kg) and high two crystal structures reveal a similar inhibitory mechanism AUC value (rat, 41500 hours*ng/mL and dog, 14900 in which both compounds occupy the substrate-binding hours*ng/mL)). Those above PK results indicate that com- pro pocket and block the enzyme activity of SARS-CoV-2 M . pound 11a is worth to warrant further study. Compared with those of N1, N3 and N9 in SARS-CoV An in vivo toxicity study (table S4) of 11a has been car- pro M complex structures reported previously, the binding ried out on SD rats and Beagle dogs. The acute toxicity of pro modes of 11a and 11b in SARS-CoV-2 M complex struc- 11a was measured on SD rats. No SD rats died after receiv- tures are similar and the differences among these overall ing 40 mg/kg by intravenous drip administration. When the structures are small (Fig. 4 and fig. S4, B to F) (22). The dif- dosage was raised to 60 mg/kg, one of four SD rats died. ferences mainly lie in the interactions at S1′, S2 and S4 sub- The dose range toxicity study of 11a was conducted for sev- sites, possibly due to various sizes of functional groups at en days at dosing levels of 2, 6, and 18 mg/kg on SD rats and corresponding P1′, P2 and P4 sites in the inhibitors (Fig. 4, at 10-40 mg/kg on Beagle dogs. All animals received once A and C). daily dosing (QD), by intravenous drip, and all animals were To further substantiate the enzyme inhibition results, clinically observed at least once a day. No obvious toxicity we evaluated the ability of these compounds to inhibit was observed in either group. These above data indicated SARS-CoV-2 in vitro (Fig. 5 and fig. S5). 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Trans. 39, 1371–1375 (2011). doi:10.1042/BST0391371 Medline 16. Z. Ren, L. Yan, N. Zhang, Y. Guo, C. Yang, Z. Lou, Z. Rao, The newly emerged First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 4 ACKNOWLEDGMENTS We thank Prof. James Halpert and LetPub (www.letpub.com) for linguistic assistance during the preparation of this manuscript. We also thank the staff from beamlines BL17U1, BL18U1 and BL19U1 at Shanghai Synchrotron Radiation Facility (SSRF) for assistance during data collection. Funding: We are grateful to the National Natural Science Foundation of China (Nos. 21632008, 21672231, 21877118, 31970165, 91953000 and 81620108027), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA12040107 and XDA12040201) and Chinese Academy of Engineering and Ma Yun Foundation (No. 2020-CMKYGG-05) and Science and Technology Commission of Shanghai Municipality (Nos. 20431900100), and National Key R&D Program of China (Nos. 2017YFC0840300 and 2020YFA0707500 to Z.R.), and Science and Technology Commission of Shanghai Municipality (No. 20431900200), and Department of Science and Technology of Guangxi Zhuang Autonomous Region (No. 2020AB40007), and Frontier Biotechnologies Inc. for financial support. Author contributions: H. Y. and H. L. conceived the project. Y. X., L. Z., H. Y., and H. L. designed the experiments; W. D. and J. L. designed and synthesized the compounds; X. J. and H. S tested the inhibitory activities; X. X., J. P., C. L., S. H., J. W., performed the chemical experiments and collected the data. B. Z., Y. Z., Z. J., F. L., F. B., H. W., X. C., X. L., and X. Y. collected the diffraction data and solved the crystal structure; Y. L. and X. C. performed the toxicity experiments. G. X., H. J., Z. R., L, Z., Y. X., H. Y. and H. L., analyzed and discussed the data. L. Z., Y. X., H. Y., and H. L., wrote the manuscript. Competing interests: The Shanghai Institute of Materia Medica has applied for PCT and Chinese patents which cover 11a, 11b and related peptidomimetic aldehyde compounds. Data and materials availability: All data are available in the main text or the supplementary materials. The PDB accession No. for the coordinates of SARS- CoV-2 Mpro in complex with 11a is 6LZE, and the PDB accession No. for the pro coordinates of SARS-CoV-2 M in complex with 11b is 6M0K. The plasmid encoding pro the SARS-CoV-2 M will be freely available. Compounds 11a and 11b are available from H. L under a material transfer agreement with Shanghai Institute of Materia Medica. There is currently an international effort to join forces to design better pro inhibitors of SARS-CoV-2 M as described in the following website: https://covid.postera.ai/covid. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material. SUPPLEMENTARY MATERIALS science.sciencemag.org/cgi/content/full/science.abb4489/DC1 Materials and Methods Scheme S1 Figs. S1 to S5 Tables S1 to S4 References (26–29) 18 March 2020; accepted 20 April 2020 Published online 22 April 2020 10.1126/science.abb4489 First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 5 Fig. 1. Design strategy of novel SARS-CoV-2 main protease inhibitors and the chemical structures of 11a and 11b. pro Fig. 2. Inhibitory activity profiles of compounds 11a (A) and 11b (B) against SARS-CoV-2 M . First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 6 pro Fig. 3. M -inhibitor binding modes for 11a and 11b. (A) Cartoon representation of the crystal structure of SARS-CoV-2 pro M in complex with 11a. The compound 11a is shown as magenta sticks; water molecules shown as red spheres. (B) Close-up view of the 11a binding pocket. Four subsites, S1′, S1, S2 and S4, are labeled. The residues involved in inhibitor binding are shown as wheat sticks. 11a and water molecules are shown as magenta sticks and red spheres, respectively. pro Hydrogen bonds are indicated as dashed lines. (C) Schematic diagram of SARS-CoV-2 M -11a interactions shown in (B). pro (D) Comparison of the binding modes between 11a and 11b for SARS-CoV-2 M . The major differences between 11a and 11b are marked with dashed circles. The compounds of 11a and 11b are shown as magenta and yellow sticks, respectively. (E) Close-up view of the 11b binding pocket. Hydrogen bonds are indicated as dashed lines. (F) Schematic diagram of pro SARS-CoV-2 M -11b interactions shown in (E). First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 7 pro Fig. 4. Comparison of the inhibitor binding modes in SARS-CoV and SARS-CoV-2 M s. (A) Comparison of pro pro pro binding modes of 11a in SARS-CoV-2 M with those of N1, N3 and N9 in SARS-CoV M . SARS-CoV-2 M -11a pro pro (wheat, PDB code: 6LZE), SARS-CoV M -N1 (sky blue, PDB code:1WOF), SARS-CoV M -N3 (gray, PDB code: pro 2AMQ) and SARS-CoV M -N9 (olive, PDB code: 2AMD).11a, N1, N3 and N9 are shown in magenta, cyan, dirty pro violet and salt, respectively. (B) Comparison of the 11a and N3 binding pockets. Residues in M -11a structure and pro M -N3 structure are colored in wheat and gray, respectively. 11a and N3 are shown as sticks colored in magenta pro and dirty violet, respectively. (C) Comparison of binding modes of 11b in SARS-CoV-2 M with those of N1, N3 and pro pro N9 in SARS-CoV M . SARS-CoV-2 M -11b (pale cyan, PDB code: 6M0K). 11b, N1, N3 and N9 are shown in yellow, pro cyan, dirty violet and salt, respectively. (D) Comparison of the 11b and N9 binding pockets. Residues in M -11b pro structure and M -N9 structure are colored in pale cyan and olive, respectively. 11b and N9 are shown as sticks colored in yellow and salt, respectively. First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 8 Fig. 5. In vitro inhibition of viral main protease inhibitors against SARS-CoV-2. (A and B) Vero E6 cells were treated with a series concentration of indicated compounds 11a and 11b and infected with SARS-CoV-2 at an MOI of 0.05. At 24 hours post infection, viral yield in the cell supernatant was quantified by plaque assay. The cytotoxicity of these compounds in Vero E6 cells was also determined by using CCK8 assays. The left and right Y-axis of the graphs represent mean % inhibition of virus yield and mean % cytotoxicity of the drugs, respectively. (C and D) Viral RNA copy numbers in the cell supernatants were quantified by qRT-PCR. Data are mean ± SD, n = 3 biological replicates. First release: 22 April 2020 www.sciencemag.org (Page numbers not final at time of first release) 9

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Published: Apr 22, 2020

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