Selective covalent targeting of SARS-CoV-2 main protease by enantiopure chlorofluoroacetamide

Abstract

The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The main protease (M^pro) is a promising target for COVID-19 treatment. Here, we report an irreversible SARS-CoV-2 M^pro inhibitor possessing chlorofluoroacetamide (CFA) as a warhead for the covalent modification of M^pro. Ugi multicomponent reaction using chlorofluoroacetic acid enabled the rapid synthesis of dipeptidic CFA derivatives that identified 18 as a potent inhibitor of SARS-CoV-2 M^pro. Among the four stereoisomers, (R,R)-18 exhibited a markedly higher inhibitory activity against M^pro than the other isomers. Reaction kinetics and computational docking studies suggest that the R configuration of the CFA warhead is crucial for the rapid covalent inhibition of M^pro. Our findings highlight the prominent influence of the CFA chirality on the covalent modification of proteinous cysteines and provide the basis for improving the potency and selectivity of CFA-based covalent inhibitors.

Fig. 1. Chemical structures of SARS-CoV-2 M^pro inhibitors. (A) Structure of nirmatrelvir (PF-07321332) and its binding mode in the M^pro active site (the conformation was retrieved from the cocrystal structure, PDB code 7RFW). (B) The structures of dipeptidic M^pro inhibitors generated by Ugi multicomponent reaction and the binding mode of Jun8-76-3A in the M^pro active site (the conformation was retrieved from the cocrystal structure, PDB code 7KX5).

Fig. 2. (A) Preparation of the dipeptidic inhibitor 3via Ugi multicomponent reaction (MCR). (B) Enzyme inhibitory activities of the dipeptidic inhibitors against SARS-CoV-2 M^pro. Compounds 3A and 3B are the less and more polar diastereomers, respectively. The reported IC₅₀ values for Jun8-76-3A, 1, and 2 are 0.31, 2.95, and 2.72 μM, respectively.

Fig. 3. Structure–activity relationship of chlorofluoroacetamide (CFA) derivatives. (A) Screening of the CFA derivatives with the different R¹, R², and R³ substituents. ND, not determined. A and B indicate the less and more polar diastereomers separated by silica gel column chromatography. (B) The structures and inhibitory activities of the CFA dipeptides 18 and 19. (C) The relative configuration of 18B determined by X-ray crystallography (see Fig. S1†).

Scheme 1. The optical resolution of chlorofluoroacetic acid using (R)-(−)-2-phenylglycinol as a chiral resolving agent. See ESI for the detail.

Fig. 4. Inhibition properties of the four stereoisomers of 18 against SARS-CoV-2 M^pro. (A) The absolute configurations of the stereoisomers of 18 (R = (3-fluorophenyl)ethyl). The R and S configurations at the CFA unit are highlighted in green and magenta, respectively. (B) The inhibitory activity of the stereoisomers against SARS-CoV-2 M^pro. Each plot represents the mean of triplicate experiments ± standard deviation. (C) Time-course plot of the M^pro-catalyzed hydrolysis of the fluorogenic substrate in the presence of various concentrations of (R,R)-18. [P] represents the concentration of the hydrolyzed product of the fluorogenic peptide substrate determined by the fluorescence intensity. Each plot represents the mean of triplicate experiments ± standard error of the mean. (D) Summary of IC₅₀ values and kinetic parameters of the chiral stereoisomers of 18. ND, not determined. (E) and (F) The computationally predicted, most stable conformations of (R,R)- and (R,S)-18 in the M^pro active site. See ESI for a detailed computational method.

Fig. 5. Selectivity profiles of the CFA inhibitors toward SARS-CoV-2 M^pro. (A) Inhibitory activity of (R,R)-18 and Jun8-76-3A toward the different cysteine proteases. The data are represented as the average of two independent experiments. (B) In-gel fluorescence analysis of the labeling of the recombinant M^pro with CFA probe 22 (37 °C, 1 h). (C) Reactivity profiles of (R,R)-22 toward the M^pro spiked into A431 cell lysate (37 °C, 1 h). Left: concentration-dependent labeling with (R,R)-22. Right: competition labeling by PF-00835231, (R,R)-18, and Jun8-76-3A. The red arrow indicates the fluorescence band of SARS-CoV-2 M^pro. (D) Proteome-wide reactivity profile of 22 and acrylamide probe 23 in live A431 cells (37 °C, 1 h).