Hydroxamate and thiosemicarbazone: Two highly promising scaffolds for the development of SARS-CoV-2 antivirals

Abstract

The emerging COVID-19 pandemic generated by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has severely threatened human health. The main protease (M^pro) of SARS-CoV-2 is promising target for antiviral drugs, which plays a vital role for viral duplication. Development of the inhibitor against M^pro is an ideal strategy to combat COVID-19. In this work, twenty-three hydroxamates 1a-i and thiosemicarbazones 2a-n were identified by FRET screening to be the potent inhibitors of M^pro, which exhibited more than 94% (except 1c) and more than 69% inhibition, and an IC₅₀ value in the range of 0.12-31.51 and 2.43-34.22 μM, respectively. 1a and 2b were found to be the most effective inhibitors in the hydroxamates and thiosemicarbazones, with an IC₅₀ of 0.12 and 2.43 μM, respectively. Enzyme kinetics, jump dilution and thermal shift assays revealed that 2b is a competitive inhibitor of M^pro, while 1a is a time-dependently inhibitor; 2b reversibly but 1a irreversibly bound to the target; the binding of 2b increased but 1a decreased stability of the target, and DTT assays indicate that 1a is the promiscuous cysteine protease inhibitor. Cytotoxicity assays showed that 1a has low, but 2b has certain cytotoxicity on the mouse fibroblast cells (L929). Docking studies revealed that the benzyloxycarbonyl carbon of 1a formed thioester with Cys145, while the phenolic hydroxyl oxygen of 2b formed H-bonds with Cys145 and Asn142. This work provided two promising scaffolds for the development of M^pro inhibitors to combat COVID-19.

Keywords: Hydroxamates; Inhibitor; Main protease; SARS-CoV-2; Thiosemicarbazones.

Graphical abstract

Fig. 1

The SDS-PAGE of the purified SARS-CoV-2 M^pro (a). Lane 1: protein molecular weight marker, lane 2: purified M^pro, lane 3: M^pro before cleavage with HRV 3C-protease. Activity of M^pro was confirmed by quantification crack of the fluorescent decapeptide Mca–AVLQSGFRK(Dnp)K as substrate (b).

Fig. 2

Structures of the tested hydroxamates (above) and thiosemicarbazones (below) against SARS-CoV-2 M^pro.

Fig. 3

Percent inhibition of hydroxamates 1a-i and thiosemicarbazones 2a-n (50 µM) against M^pro. 0.5% DMSO was used as negative control and ebselen was used as positive control.

Fig. 4

Time-dependent inhibition curve of hydroxamate 1a (1.25 µM) on M^pro (a). Progress curves of M^pro activity change in the presence of hydroxamate 1a and thiosemicarbazone 2b (b). 0.5% DMSO was used for the blank control.

Fig. 5

The hyperbolic plots of K_obs against concentrations of hydroxamate 1a (a). The Lineweaver-Burk plots of M^pro catalyzed hydrolysis of thiosemicarbazone 2b. The concentrations of inhibitors were 0 (●), 2.5 (○), 5(▾), 10 (▽) µM (b).

Fig. 6

The melting temperature (T_m) of M^pro in the absence and presence of 1a and 2b (a). Fluorescence based thermal shift assays of 2b interaction with M^pro as indicated by dF/dT (b). Dose-dependent melting temperature shift (c).

Fig. 7

Inhibition (a) and dose-dependent inhibition (b) of hydroxamate 1a on M^pro in the presence and absence of DTT.

Fig. 8

The lowest-energy conformations of the complex of inhibitors with M^pro. Interactions formed between hydroxamate 1a (a) and thiosemicarbazone 2b (b) and surrounding residues, the M^pro skeleton is exhibited as a green cartoon and the inhibitors and residues are exhibited as sticks colored by elements (N, blue; O, red; H, white; S, yellow; C, purple). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 9

The cytotoxicity assays of inhibitors (1–400 µM) hydroxamate 1a (a) and thiosemicarbazone 2b (b) on mouse fibroblast (L929) cells.