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

Lili Zhu, Shyla George, Marco F. Schmidt, Samer I. Al-Gharabli, Jörg Rademann, Rolf Hilgenfeld*

*Corresponding author for this work
20 Citations (Scopus)


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±0.58μM. 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.

Original languageEnglish
JournalAntiviral Research
Issue number2
Pages (from-to)204-212
Number of pages9
Publication statusPublished - 01.11.2011

Research Areas and Centers

  • Academic Focus: Center for Infection and Inflammation Research (ZIEL)

Coronavirus related work

  • Research on SARS-CoV-2 / COVID-19


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  • HPLC/ESI Ion Trap Mass Spectrometer

    Hilgenfeld, R.


    Project: DFG ProjectsDFG Individual Projects

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