Inhibition of cysteine proteases by 6,6′‐dihydroxythiobinupharidine (Dtbn) from nuphar lutea

The specificity of inhibition by 6,6′‐dihydroxythiobinupharidine (DTBN) on cysteine pro-teases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural‐steric and biochemical differences. Cathepsin S (IC50 = 3.2 μM) was most sen-sitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 μM respectively). DTBN is inactive for the inhibition of M^pro of SARS‐CoV‐2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close prox-imity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins‐DTBN complexes, respectively. The lack of reactivity of DTBN against SARS‐CoV‐2 M^pro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of M^pro. Thus, grada-tions in reactivity among the tested Cathepsins may be conducive for a mechanism‐based search for derivatives of nupharidine against COVID‐19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.