Heteroaromatic ester inhibitors of hepatitis A virus 3C proteinase: Evaluation of mode of action

The related 3C and 3C-like proteinase (3C(pro) and 3CL(pro)) of picornaviruses and coronaviruses, respectively, are good drug targets. As part of an effort to generate broad-spectrum inhibitors of these enzymes, we screened a library of inhibitors based on a halopyridinyl ester from a previous study of the severe acute respiratory syndrome (SARS) 3CL proteinase against Hepatitis A virus (HAV) 3C(pro). Three of the compounds, which also had furan rings, inhibited the cleavage activity of HAV 3C(pro) with K(ic)s of 120–240 nM. HPLC-based assays revealed that the inhibitors were slowly hydrolyzed by both HAV 3C(pro) and SARS 3CL(pro), confirming the identity of the expected products. Mass spectrometric analyses indicated that this hydrolysis proceeded via an acyl-enzyme intermediate. Modeling studies indicated that the halopyridinyl moiety of the inhibitor fits tightly into the S1-binding pocket, consistent with the lack of tolerance of the inhibitors to modification in this portion of the molecule. These compounds are among the most potent non-peptidic inhibitors reported to date against a 3C(pro).