Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease

Antiviral Res. 2011 Nov;92(2):204-12. doi: 10.1016/j.antiviral.2011.08.001. Epub 2011 Aug 11.


SARS coronavirus main protease (SARS-CoV M(pro)) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M(pro). Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M(pro) requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M(pro) in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M(pro), with K(i) values in the μM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2=Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M(pro) in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M(pro), with K(i)=2.24±0.58 μM. These results show that the stringent substrate specificity of the SARS-CoV M(pro) with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehydes / pharmacology*
  • Coronavirus 3C Proteases
  • Crystallography, X-Ray
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / metabolism*
  • Enzyme Inhibitors / pharmacology*
  • Kinetics
  • Models, Molecular
  • Peptides / metabolism
  • Protein Conformation
  • SARS Virus / drug effects*
  • SARS Virus / enzymology*
  • Substrate Specificity / drug effects
  • Viral Proteins / antagonists & inhibitors*
  • Viral Proteins / chemistry
  • Viral Proteins / metabolism*


  • Aldehydes
  • Enzyme Inhibitors
  • Peptides
  • Viral Proteins
  • 3C-like protease, SARS coronavirus
  • Cysteine Endopeptidases
  • Coronavirus 3C Proteases