A cyclic peptide inhibitor of the SARS-CoV-2 main protease

Abstract

This paper presents the design and study of a first-in-class cyclic peptide inhibitor against the SARS-CoV-2 main protease (M^pro). The cyclic peptide inhibitor is designed to mimic the conformation of a substrate at a C-terminal autolytic cleavage site of M^pro. The cyclic peptide contains a [4-(2-aminoethyl)phenyl]-acetic acid (AEPA) linker that is designed to enforce a conformation that mimics a peptide substrate of M^pro. In vitro evaluation of the cyclic peptide inhibitor reveals that the inhibitor exhibits modest activity against M^pro and does not appear to be cleaved by the enzyme. Conformational searching predicts that the cyclic peptide inhibitor is fairly rigid, adopting a favorable conformation for binding to the active site of M^pro. Computational docking to the SARS-CoV-2 M^pro suggests that the cyclic peptide inhibitor can bind the active site of M^pro in the predicted manner. Molecular dynamics simulations provide further insights into how the cyclic peptide inhibitor may bind the active site of M^pro. Although the activity of the cyclic peptide inhibitor is modest, its design and study lays the groundwork for the development of additional cyclic peptide inhibitors against M^pro with improved activities.

Keywords: COVID-19; Cyclic peptide inhibitor; Cyclophane; Main protease; SARS-CoV-2.

Graphical abstract

Fig. 1

(A) Chemical structure of a general cyclic peptide inhibitor illustrating the arrangement of the P2, P1, P1′, and P2′ positions and [4-(2-aminoethyl)phenyl]-acetic acid (AEPA) and the envisioned binding interactions with the S2, S1, S1′, S2′, and S3′ pockets in the M^pro active site. (B) Chemical structure of UCI-1.

Fig. 2

Crystal structure of M^pro₃₁₆ showing two M^pro₃₁₆ dimers in two adjacent asymmetric units (PDB 5B6O). One dimer is shown in grey surface view; the other dimer is shown in green cartoons. The inset shows a detailed view of residues 301–310 of the C-terminal autolytic cleavage site of one M^pro₃₁₆ molecule in the active site of another M^pro₃₁₆ molecule.

Fig. 3

Design process for creating the cyclic peptide inhibitor UCI-1 from the C-terminal autolytic substrate in the active site of M^pro: (1) Delete residues 301–304 and 310 as well as the carbonyl of Phe309. (2) Build a CH₂CO group on the para position of the phenyl group on Phe309. (3) Create a bond between the carbonyl carbon of the newly created CH₂CO group on Phe309 and the amino group of Phe305 and minimize (clean) the structure. (4) Mutate Gly307 to serine.

Scheme 1

Synthesis of UCI-1.

Fig. 4

(A) Lowest energy conformer of UCI-1. (B) Superposition of the 20 lowest energy conformers from conformational searching. The difference in energy between the lowest and highest energy conformers among these 20 is 7.0 kJ/mol (C) UCI-1 in complex with the SARS-CoV-2 M^pro active site generated in Autodock Vina. The SARS-CoV-2 M^pro crystal structure with PDB accession number 6YB7 was used in the docking study.

Fig. 5

Enzyme inhibition assay of UCI-1 (A) and control peptides peptide-1a (B) and peptide-1b (C). The activity of M^pro was measured in the presence of increasing concentrations of UCI-1, peptide-1a, or peptide-1b. A dose-response curve was determined by non-linear regression and used to estimate the IC₅₀. All data are shown as the mean of three technical replicates, with error bars representing the standard deviation. Panels D and E show the chemical structures of peptide-1a and peptide-1b.

Fig. 6

Global minimum energy conformations of UCI-1 (A) and tri-Ala-UCI-1 (B).

Fig. 7

Global minimum energy conformations of homologous AEPA macrocycles derived from UCI-1 that contain two (A), three (B) and five (C) amino acids spanned across the AEPA linker.