Structural basis for the inhibition of the SARS-CoV-2 main protease by the anti-HCV drug narlaprevir

Dear Editor, The second wave of the coronavirus disease (COVID-19) pandemic has recently appeared in Europe. Most European countries, such as France, Germany, and Italy, have announced the implementation of a new round of epidemic prevention and control measures. However, no clinical drug or vaccine has been approved for the treatment of COVID-19. The interim results of the solidarity therapy trial coordinated by the World Health Organization (WHO) indicated that remdesivir, hydroxychloroquine, lopinavir/ritonavir, and interferon appear to have little or no effect on the 28-day mortality of hospitalized patients or the hospitalization process of new COVID-19 patients. Therefore, there is an urgent need to develop new drugs against COVID-19. Many viral protease inhibitors, such as telaprevir, asunaprevir, grazoprevir, simeprevir, and darunavir, have been successfully approved for the treatment of HCV and HIV. For coronavirus, the main protease (M, 3CL) and papain-like protease (PL) are responsible for the digestion of viral polyproteins 1a and 1ab to produce 16 active viral nonstructural proteins. These nonstructural proteins are critical for viral replication and transcription. In particular, M cleaves 11 substrate sites of viral polyprotein 1ab and 7 substrate sites of viral polyprotein 1a. Therefore, M is recognized as an attractive drug target. The structures of the covalent inhibitors 13b and N3 when complexed with M have been determined at first. Based on the complex structure, structure-based design of the covalent inhibitors 11a and 11b targeting M has led to better antiviral activities. Compared with these preclinical drugs, repurposing approved drugs is a feasible method for emergent treatment of COVID-19 patients. The antineoplastic drug carmofur was screened and it exhibited M inhibitory activity. The crystal structure, when complexed with M, revealed that the carbonyl reactive group of carmofur can covalently bind to catalytic Cys145. We also found that the antiHCV drug boceprevir can effectively inhibit SARS-CoV-2 in Vero cells by targeting M with an EC50 of 15.57 μM. Further, structural analysis revealed that boceprevir can occupy the substratebinding pocket of M and form a covalent bond with the catalytic Cys145. Narlaprevir is a potent second-generation inhibitor of the HCV NS3 protease based on boceprevir and now is in phase III clinical trials. Unlike boceprevir, narlaprevir is a single isoform and shows an improved pharmacokinetic profile and physicochemical characteristics. Using an enzyme activity inhibition assay, we found that narlaprevir (Fig. 1a) showed moderate inhibitory activity against SARS-CoV-2 M, with an IC50 value of 16.11 μM (Fig. 1a). To validate the binding of narlaprevir with SARS-CoV-2 M and exclude any false-positive results of the enzyme activity inhibition test, we performed isothermal titration calorimetry (ITC) to measure the binding affinity between narlaprevir and SARS-CoV2 M. The Kd value of narlaprevir binding with SARS-CoV-2 M is 82 μM. In contrast, boceprevir and GC376 have Kd values of 21 μM and 0.46 μM, respectively (Supplementary Fig. S1). These results were consistent with the enzyme activity inhibition assay. Narlaprevir showed an antiviral effect against SARS-CoV-2 with an EC50 value of 7.23 μM (Fig. 1b). As a positive control, remdesivir and boceprevir inhibited SARS-CoV-2 replication with EC50 values of 0.58 μM and 14.13 μM, respectively. Additionally, narlaprevir exhibited no cytotoxicity in Vero cells at different concentrations up to 200 μM (Supplementary Fig. S2). Treatment with narlaprevir infection demonstrated a dose-dependent inhibitory effect on SARS-CoV-2 plaque formation (Fig. 1c). The plaques were completely inhibited in the presence of 50 μM narlaprevir. The crystal structure of the M-narlaprevir complex was determined at 1.78 Å resolution (Supplementary Table S1). The M molecule contains three domains and narlaprevir binds to the substrate-binding site located in the cavity between domains I and II of M in an extended conformation (Fig. 1d). The unambiguous electron density map shows that narlaprevir binds to the active site of M through a C–S covalent bond interaction with catalytic C145 (Fig. 1e and Supplementary Fig. S3). In the Mnarlaprevir complex, residues H41, N142, G143, and H164 form four hydrogen-bonds with the amide backbone of narlaprevir on one side, and residue E166 forms three hydrogen-bonds with narlaprevir on the other side (Fig. 1f). According to the Berger and Schechter nomenclature, narlaprevir can be divided into five moieties, P1–P4 and P1’, as shown in Figs. 1a and 1f. The S1 subsite of M was found to be a polarity pocket composed of Phe140, Tyr161, His162, Glu166, and His172. The norleucine moiety at P1 of narlaprevir can fit the S1 pocket shape well (Fig. 1g). The rigid P2 dimethyl-cyclopropyl proline (DMCP) residue lies in the S2 hydrophobic pocket, which is composed of His41, Met49, Met165, Phe181, and Asp187. The hydrophobic P3 tert-butyl (tBu) residue is exposed to solvents in the S3 subsite. The cyclohexyl moiety at P4 is buried deep in the S4 pocket. However, the appended tBu sulfone group is exposed to solvents. In addition, the cyclopropyl moiety at P1’ can also be tolerated by the S1’ pocket due to its small size (Fig. 1g). Compared with the HCV NS3/4A-narlaprevir complex (Fig. 1h), narlaprevir undergoes a large conformational change to fit the M substrate-binding pocket (Fig. 1g). This is similar to boceprevir binding (Supplementary Fig. S4a and S4b). However, narlaprevir has a weaker protease inhibitory activity than boceprevir. The tBu sulfone tail of narlaprevir, which does not appear to favor the S4 pocket of M, may contribute to the reduction in enzyme potency. In contrast, the tBu sulfone tail and cyclopropyl moiety at P1’ of narlaprevir can increase its biological activity across the cell membrane. This leads to the improved antiviral activity of narlaprevir over boceprevir against SARS-CoV-2. We also compared the structures of the newly identified compounds complexed with SARS-CoV-2 M and found that all target the active site of SARS-CoV-2 M. These compounds were covalently bound to the catalytic residue Cys145 (Supplementary Fig. S4c–f).

1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 1.78 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based. The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density. The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor fit to the electron density. The numeric value is given above the bar.

Mol Chain Length
Quality of chain 1 A 302 2 Entry composition i ○ There are 2 unique types of molecules in this entry. The entry contains 2512 atoms, of which 0 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
• Molecule 1 is a protein. 3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The first graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor fit to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.

Mol Chain Residues
• Molecule 1: Xtriage's analysis on translational NCS is as follows: The largest off-origin peak in the Patterson function is 6.91% of the height of the origin peak. No significant pseudotranslation is detected.
5 Model quality i ○

Standard geometry i ○
Bond lengths and bond angles in the following residue types are not validated in this section: LIG The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no chirality outliers.

Mol Chain
There are no planarity outliers.

Too-close contacts i ○
In the following The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 3.
All (16) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude. There are no symmetry-related clashes.

Protein backbone i ○
In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues. In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.

Mol Chain
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.

RNA i ○
There are no RNA molecules in this entry.

5.4
Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligand geometry i ○
There are no ligands in this entry.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry. 6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled 'Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9. 6.2 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligands i ○
There are no ligands in this entry.

Other polymers i ○
There are no such residues in this entry.