title: Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease.
abstract: 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.
authors.affiliation: Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
authors.forename: Lili
authors.initials: L
authors.lastname: Zhu
authors.forename: Shyla
authors.initials: S
authors.lastname: George
authors.forename: Marco F
authors.initials: MF
authors.lastname: Schmidt
authors.forename: Samer I
authors.initials: SI
authors.lastname: Al-Gharabli
authors.forename: Jörg
authors.initials: J
authors.lastname: Rademann
authors.forename: Rolf
authors.initials: R
authors.lastname: Hilgenfeld
mesh_terms: D000447:Aldehydes / Q000494:pharmacology*; D000086782:Coronavirus 3C Proteases; D018360:Crystallography, X-Ray; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D045473:Severe acute respiratory syndrome-related coronavirus / Q000187:drug effects* / Q000201:enzymology*; D013379:Substrate Specificity / Q000187:drug effects; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000447:Aldehydes; D004791:Enzyme Inhibitors; D010455:Peptides; D014764:Viral Proteins; C000706207:3C-like protease, SARS coronavirus; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases