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


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          ► We present crystal structures of 6 peptide aldehydes in complex with SARS-CoV M pro. ► The aldehydes form thiohemiacetals with the catalytic cysteine of the enzyme. ► Two different configurations of the thiohemiacetal can be observed for one aldehyde. ► Asp and Ser side-chains of the aldehydes bind to the hydrophobic S2 pocket. ► P2-Asp interacts with two methionines of the enzyme via O...S nonbonded interactions.


          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.

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          Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs.

          A novel coronavirus has been identified as the causative agent of severe acute respiratory syndrome (SARS). The viral main proteinase (Mpro, also called 3CLpro), which controls the activities of the coronavirus replication complex, is an attractive target for therapy. We determined crystal structures for human coronavirus (strain 229E) Mpro and for an inhibitor complex of porcine coronavirus [transmissible gastroenteritis virus (TGEV)] Mpro, and we constructed a homology model for SARS coronavirus (SARS-CoV) Mpro. The structures reveal a remarkable degree of conservation of the substrate-binding sites, which is further supported by recombinant SARS-CoV Mpro-mediated cleavage of a TGEV Mpro substrate. Molecular modeling suggests that available rhinovirus 3Cpro inhibitors may be modified to make them useful for treating SARS.
            • Record: found
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            A graphical user interface to the CCP4 program suite.

            CCP4i is a graphical user interface that makes running programs from the CCP4 suite simpler and quicker. It is particularly directed at inexperienced users and tightly linked to introductory and scientific documentation. It also provides a simple project-management system and visualization tools. The system is readily extensible and not specific to CCP4 software.
              • Record: found
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              Production of Authentic SARS-CoV M pro with Enhanced Activity: Application as a Novel Tag-cleavage Endopeptidase for Protein Overproduction

              The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (Mpro) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV Mpro was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV Mpro with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV Mpro with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of Mpro are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV Mpro and its complex with a Michael acceptor inhibitor were determined to 1.6 Å and 1.95 Å resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV Mpro could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV Mpro is more appropriate for mechanistic characterization and inhibitor design.

                Author and article information

                Antiviral Res
                Antiviral Res
                Antiviral Research
                Elsevier B.V.
                11 August 2011
                November 2011
                11 August 2011
                : 92
                : 2
                : 204-212
                [a ]Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
                [b ]Leibniz Institute for Molecular Pharmacology (FMP), Robert-Rössle-Strasse 10, 13125 Berlin, Germany
                [c ]German-Jordanian University, Chemical Pharmaceutical Engineering Department, P.O. Box 35247, Amman 11180, Jordan
                [d ]Institute of Pharmacy, Leipzig University, Brüderstraße 34, 04103 Leipzig, Germany
                [e ]Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China
                [f ]Laboratory for Structural Biology of Infection and Inflammation, c/o DESY, Building 22a, Notkestr. 85, 22603 Hamburg, Germany
                Author notes
                [* ]Corresponding author at: Institute of Biochemistry, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. Tel.: +49 451 500 4060; fax: +49 451 500 4068. hilgenfeld@ 123456biochem.uni-luebeck.de

                Present address: Aurigene Discovery Technologies Ltd., #39&40, KIADB Industrial Area, Electronic City Phase-2, Hosur Road, Bangalore 560100, India.


                Present address: University Chemical Laboratory, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, UK.

                Copyright © 2011 Elsevier B.V. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                : 1 July 2011
                : 3 August 2011

                Infectious disease & Microbiology
                sars-cov mpro, sars coronavirus main protease,cm-ff-h, cinnamoyl-phe-phe-h,boc, tert-butyl oxycarbonyl,ibx, 2-iodoxybenzoic acid,fret, fluorescence resonance energy transfer,peg, polyethylene glycol,mpd, 2-methyl-2,4-pentanediol,mes, 2-(n-morpholino)ethanesulfonic acid,dmso, dimethylsulfoxide,aldehyde inhibitor,antiviral drug design,cysteine protease,methionine–aspartic acid interaction,x-ray crystallography


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