title: Peptidyl diazomethyl ketones inhibit the human rhinovirus 3C protease: effect on virus yield by partial block of P3 polyprotein processing.
abstract: The efficacy of a series of diazomethyl ketones (DMKs) was measured in rhinovirus-infected cultures and against the HRV14 3C protease. Their specificity and potency were confirmed against purified recombinant enzyme expressed in a yeast secretion system. An internally quenched fluorescent peptide substrate was used to assess the potency against the enzyme, obtaining a 50% inhibitory concentration (IC50) of 1 microM for both Z-L-F-Q-CHN2 and Z-V-L-F-Q-CHN2, while a lower affinity was observed for Z-F-Q-CHN2. The tripeptide Z-L-F-Q-CHN2 blocked viral replication with an IC50 value of 30 microM as judged by the reduction in viral induced cytopathy of HeLa-H1 cells, as well as a marked reduction in viral plaque formation (50% effective concentration=20 microM). Western blot analysis of viral proteins from infected cells indicates that this inhibitor works specifically by blocking viral polyprotein maturation, displaying a reduction of detectable 3C protease and an accumulation of the 3CD polypeptide. These results indicate that DMK inhibitors of the 3C protease have antiviral potency. Furthermore, the pattern of viral protein processing observed suggests that reducing the concentration of mature HRV 3C protease even in the presence of increased 3CD protein is sufficient to block proper viral processing and significantly reduce virus yield.
authors.affiliation: Structural Genomix, San Diego, Calif, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: Murray
authors.forename: J W
authors.initials: JW
authors.lastname: Janc
authors.forename: S
authors.initials: S
authors.lastname: Venkatraman
authors.forename: L M
authors.initials: LM
authors.lastname: Babé
mesh_terms: D000086042:3C Viral Proteases; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003978:Diazomethane / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D015967:Gene Expression Regulation, Viral / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007659:Ketones / Q000737:chemistry / Q000494:pharmacology*; D015394:Molecular Structure; D020815:Polyproteins / Q000737:chemistry / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D012229:Rhinovirus / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism*; D013329:Structure-Activity Relationship; D010948:Viral Plaque Assay; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D007659:Ketones; D020815:Polyproteins; D014764:Viral Proteins; C021232:peptidyl diazomethyl ketones; D003978:Diazomethane; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases