Para Amelia:
M pro is a three-domain (domains I to III) cysteine protease involved in most maturation cleavage events within the precursor polyprotein (17–19). Active Mpro is a homodimer containing two protomers. The CoV Mpro features a non-canonical Cys-His dyad located in the cleft between domains I and II (17–19). Mpro is conserved among CoVs and several common features are shared among the substrates of Mpro in different CoVs. The amino acids in substrates from the N terminus to C terminus are numbered as fellows (-P4-P3-P2-P1↓P1′-P2′-P3′-), and the cleavage site is between the P1 and P1′. In particular, a Gln residue is almost always required in the P1 position of the substrates. There is no human homolog of Mpro which makes it an ideal antiviral target (20–22).
The active sites of Mpro are highly conserved among all CoV’s Mpros and are usually composed of four sites (S1′, S1, S2 and S4) (22). By analyzing the substrate-binding pocket of SARS-CoV Mpro (PDB ID: 2H2Z), novel inhibitors targeting the SARS-CoV-2 Mpro were designed and synthesized (Fig. 1). The thiol of a cysteine residue in the S1′ sites anchors inhibitors by a covalent linkage that is important for the inhibitors to maintain antiviral activity. In our design of new inhibitors, an aldehyde was selected as a new warhead in P1 in order to form a covalent bond with cysteine. The reported SARS-CoV Mpro inhibitors often have an (S)-γ-lactam ring that occupies the S1 site of Mpro, and this ring was expected to be a good choice in P1 (23). Furthermore, the S2 site of coronavirus Mpro is usually large enough to accommodate the bigger P2 fragment. To test the importance of different ring systems, a cyclohexyl or 3-fluorophenyl were introduced in P2, with the fluorine expected to enhance activity. An indole group was introduced into P3 in order to form new hydrogen bonds with S4 and improve drug-like properties.
he synthetic route and chemical structures of the compounds (11a and 11b) are shown in scheme S1. The starting material (N-Boc-L-glutamic acid dimethyl ester 1) was obtained from commercial suppliers and used without further purification to synthesize the key intermediate 3 according to the literature (24). The intermediates 6a and 6b were synthesized from 4 and acids 5a, 5b. Removal of the t-butoxycarbonyl group from 6a and 6b yielded 7a and 7b. Coupling 7a and 7b with the acid 8 yielded the esters 9a and 9b. The peptidomimetic aldehydes 11a and 11b were approached through a two-step route in which the ester derivatives 9 were first reduced with NaBH4 to generate the primary alcohols 10a and 10b, which were subsequently oxidized into aldehydes 11a and 11b with Dess-Martin Periodinane (DMP)
.
Recombinant SARS-CoV-2 Mpro was expressed and purified from Escherichia coli (E. coli) (18, 25). A fluorescently labeled substrate, MCA-AVLQ↓SGFR-Lys (Dnp)-Lys-NH2, derived from the N-terminal auto-cleavage sequence from the viral protease was designed and synthesized for the enzymatic assay.
Both 11a and 11b exhibited high SARS-CoV-2 Mpro inhibition activity, which reached 100% for 11a and 96% for 11b at 1 μM, respectively. We used a fluorescence resonance energy transfer (FRET)-based cleavage assay to determine the IC50 values. The results revealed excellent inhibitory potency with IC50 values of 0.053 ± 0.005 μM and 0.040 ± 0.002 μM, for 11a and 11b respectively (Fig. 2).
In order to elucidate the mechanism of inhibition of SARS-CoV-2 Mpro by 11a, we determined the high-resolution crystal structure of this complex at 1.5-Å resolution (table S1). The crystal of Mpro-11a belong to the space group C2 and an asymmetric unit contains only one molecule (table S1). Two molecules (designated protomer A and protomer B) associate into a homodimer around a crystallographic 2-fold symmetry axis (fig. S2). The structure of each protomer contains three domains with the substrate-binding site located in the cleft between domain I and II. At the active site of SARS-CoV-2 Mpro, Cys145 and His41 (Cys-His) form a catalytic dyad (fig. S2).
The electron density map clearly showed compound 11a in the substrate binding pocket of SARS-CoV-2 Mpro in an extended conformation (Fig. 3A and fig. S3, A and B). Details of the interaction are shown in Fig. 3, B and C. The electron density shows that the C of the aldehyde group of 11a and the catalytic site Cys145 of SARS-CoV-2 Mpro form a standard 1.8-Å C–S covalent bond. The oxygen atom of the aldehyde group also plays a crucial role in stabilizing the conformations of the inhibitor by forming a 2.9-Å hydrogen bond with the backbone of residues Cys145 in the S1′ site. The (S)-γ-lactam ring of 11a at P1 fits well into the S1 site. The oxygen of the (S)-γ-lactam group forms a 2.7-Å hydrogen bond with the side chain of His163. The main chain of Phe140 and side chain of Glu166 also participate in stabilizing the (S)-γ-lactam ring by forming 3.2-Å and 3.0-Å hydrogen bonds with its NH group, respectively. In addition, the amide bonds on the chain of 11a are hydrogen-bonded with the main chains of His164 (3.2 Å) and Glu166 (2.8 Å), respectively. The cyclohexyl moiety of 11a at P2 deeply inserts into the S2 site, stacking with the imidazole ring of His41. The cyclohexyl group is also surrounded by the side chains of Met49, Tyr54, Met165, Asp187 and Arg188, producing extensive hydrophobic interactions. The indole group of 11a at P3 is exposed to solvent (S4 site) and is stabilized by Glu166 through a 2.6-Å hydrogen bond. The side chains of residues Pro168 and Gln189 interact with the indole group of 11a through hydrophobic interactions. Interestingly, multiple water molecules (named W1-W6) play an important role in binding 11a. W1 interacts with the amide bonds of 11a through a 2.9-Å hydrogen bond, whereas W2-6 form a number of hydrogen bonds with the aldehyde group of 11a and the residues of Asn142, Gly143, Thr26, Thr25, His41 and Cys44, which contributes to stabilizing 11a in the binding pocket.
(A) Cartoon representation of the crystal structure of SARS-CoV-2 Mpro 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. Hydrogen bonds are indicated as dashed lines. (C) Schematic diagram of SARS-CoV-2 Mpro-11a interactions shown in (B). (D) Comparison of the binding modes between 11a and 11b for SARS-CoV-2 Mpro. 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 SARS-CoV-2 Mpro-11b interactions shown in (E).
The crystal structure of SARS-CoV-2 Mpro in complex with 11b is very similar to that of the 11a complex and shows a similar inhibitor binding mode (Fig. 3D and figs. S3, C and D, and S4A). The difference in binding mode is most probably due to the 3-fluorophenyl group of 11b at P2. Compared with the cyclohexyl group in 11a, the 3-fluorophenyl group undergoes a significant downward rotation (Fig. 3D). The side chains of residues His41, Met49, Met165, Val186, Asp187 and Arg188 interact with this aryl group through hydrophobic interactions and the side chain of Gln189 stabilizes the 3-fluorophenyl group with an additional 3.0-Å hydrogen bond (Fig. 3, E and F). In short, these two crystal structures reveal a similar inhibitory mechanism in which both compounds occupy the substrate-binding pocket and block the enzyme activity of SARS-CoV-2 Mpro.
Compared with those of N1, N3 and N9 in SARS-CoV Mpro complex structures reported previously, the binding modes of 11a and 11b in SARS-CoV-2 Mpro complex structures are similar and the differences among these overall structures are small (Fig. 4 and fig. S4, B to F) (22). The differences mainly lie in the interactions at S1′, S2 and S4 subsites, possibly due to various sizes of functional groups at corresponding P1′, P2 and P4 sites in the inhibitors (Fig. 4, A and C).
References and Notes
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 coordinates of SARS-CoV-2 Mpro in complex with 11b is 6M0K. The plasmid encoding the SARS-CoV-2 Mpro 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 inhibitors of SARS-CoV-2 Mpro 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.
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