Structure-Based Discovery and Structural Basis of a Novel Broad-Spectrum Natural Product against the Main Protease of Coronavirus

Abstract

Over the past 20 years, the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome CoV (MERS-CoV), and SARS-CoV-2 emerged, causing severe human respiratory diseases throughout the globe. Developing broad-spectrum drugs would be invaluable in responding to new, emerging coronaviruses and to address unmet urgent clinical needs. Main protease (M^pro; also known as 3CL^pro) has a major role in the coronavirus life cycle and is one of the most important targets for anti-coronavirus agents. We show that a natural product, noncovalent inhibitor, shikonin, is a pan-main protease inhibitor of SARS-CoV-2, SARS-CoV, MERS-CoV, human coronavirus (HCoV)-HKU1, HCoV-NL63, and HCoV-229E with micromolar half maximal inhibitory concentration (IC₅₀) values. Structures of the main protease of different coronavirus genus, SARS-CoV from the betacoronavirus genus and HCoV-NL63 from the alphacoronavirus genus, were determined by X-ray crystallography and revealed that the inhibitor interacts with key active site residues in a unique mode. The structure of the main protease inhibitor complex presents an opportunity to discover a novel series of broad-spectrum inhibitors. These data provide substantial evidence that shikonin and its derivatives may be effective against most coronaviruses as well as emerging coronaviruses of the future. Given the importance of the main protease for coronavirus therapeutic indication, insights from these studies should accelerate the development and design of safer and more effective antiviral agents. IMPORTANCE The current pandemic has created an urgent need for broad-spectrum inhibitors of SARS-CoV-2. The main protease is relatively conservative compared to the spike protein and, thus, is one of the most promising targets in developing anti-coronavirus agents. We solved the crystal structures of the main protease of SARS-CoV and HCoV-NL63 that bound to shikonin. The structures provide important insights, have broad implications for understanding the structural basis underlying enzyme activity, and can facilitate rational design of broad-spectrum anti-coronavirus ligands as new therapeutic agents.

Keywords: broad-spectrum; coronavirus; inhibitor; main protease; nature product.

FIG 1

Structure-based compound discovery for the main protease of SARS-CoV-2. (A) N3 binding site of SARS-CoV-2-M^pro. Structure of SARS-CoV-2 M^pro is shown as gray sphere. N3 is presented as yellow sticks. (B) Close-up view of the N3 binding pocket. N3 recognition region is conserved between SARS-CoV M^pro and SARS-CoV-2 M^pro. Hydrogen bonds are shown as dashed lines. (C) Overlaid docking poses of 17 compounds selected for experimental testing. (D) Shikonin binding site of crystal structure of SARS-CoV-2 M^pro. Shikonin is shown as green sticks. Hydrogen bonds are shown as dashed lines.

FIG 2

Inhibition of shikonin on the recombinant M^pro of SARS-CoV-2 (A), SARS-CoV (B), MERS-CoV (C), HCoV-HKU1 (D), HCoV-NL63 (E), and HCoV-229E (F) against the inhibitor shikonin. IC₅₀ is given as mean ± standard deviation. The values are the mean ±standard deviation from three replicates. The main proteases from different coronaviruses were preincubated in the reaction buffer (50 mM Tris 7.3,150mM NaCl, 1 mM EDTA) with various concentrations of shikonin at room temperature for 30 min. The enzymatic reaction was initiated by adding the FRET substrate. The IC₅₀ of shikonin was evaluated using the a dose-response curve in GraphPad Prism as described in Materials and Methods.

FIG 3

Crystal structures in apo form of SARS-CoV M^pro and SARS-CoV-2 M^pro in complex with shikonin. (A) Comparison of overall structures in apo form of SARS-CoV M^pro- and SARS-CoV-2 M^pro-shikonin. Structure in apo form of SARS-CoV M^pro is shown in green. Structure of M^pro with shikonin is shown in yellow. The shikonin is shown in blue. Carbon atoms of shikonin are blue, and oxygen atoms are red. (B) A zoomed in view of shikonin binding pocket for SARS-CoV M^pro.

FIG 4

Comparison of the binding pocket of M^pro from different coronaviruses. (A) Surface of SARS-CoV-2 M^pro, SARS-CoV M^pro, and HCoV-NL63 M^pro. Shikonin is shown as cyan, yellow, and blue sticks in SARS-CoV-2 M^pro, SARS-CoV M^pro and HCoV-NL63 M^pro, respectively. Oxygen atoms are red. (B) Superposition of the binding pocket of SARS-CoV-2 M^pro, SARS-CoV M^pro, and HCoV-NL63 M^pro. (C) Sequence alignment of the binding pocket of seven human CoVs.

FIG 5

Cocrystal structures of SARS-CoV-2 M^pro, SARS-CoV M^pro, and HCoV-NL63 M^pro in complex with shikonin. (A to C) SARS-CoV-2 M^pro-shikonin complex. (A) Fo_Fc omit map contoured at 0.5 σ for shikonin. (B) Shikonin (red) in the S1, S2, and S3 positions of the active site of SARS-CoV-2 M^pro. (C) Hydrogen bonding (dashed lines) interactions between SARS-CoV-2 M^pro and shikonin. (D to F) SARS-CoV M^pr o-shikonin complex. (D) Fo_Fc omit map contoured at 0.5 σ for shikonin. (E) Shikonin (yellow) in the S1, S2, and S3 positions of the active site of SARS-CoV M^pro. (F) Hydrogen bonding (dashed lines) interactions between SARS-CoV M^pro and shikonin. (G to I) HCoV-NL63 M^pro-shikonin complex. (G) Fo_Fc omit map contoured at 0.5 σ for shikonin. (H) Shikonin (blue) in the S1, S2, and S3 positions of the active site of HCoV-NL63 M^pro. (I) Hydrogen bonding (dashed lines) interactions between HCoV-NL63 M^pro and shikonin.