title: A simple solid phase, peptide-based fluorescent assay for the efficient and universal screening of HRV 3C protease inhibitors.
abstract: With over a 100 different serotypes, the human rhinovirus (HRV) is the major aetiological agent for the common cold, for which only symptomatic treatment is available. HRV maturation and replication is entirely dependent on the activity of a virally encoded 3C protease that represents an attractive target for the development of therapeutics to treat the common cold. Although a variety of small molecules and peptidomimetics have been found to inhibit HRV 3C protease, no universally compatible assay exists to reliably quantify the activity of the enzyme in vitro. Herein we report the development of a universal and robust solid phase peptide assay that utilizes the full HRV-14 3C protease recognition sequence and the release of 5(6)-carboxyfluorescein to sensitively quantify protease activity. This novel assay overcomes several limitations of existing assays allowing for the simple and efficient analysis of HRV-14 3C protease activity facilitating both high-throughput screening and the accurate kinetic study of HRV-14 3C protease inhibitors.
authors: School of Chemical Sciences, The University of Auckland, Auckland, New Zealand.
authors: Katrin
authors: K
authors: Schünemann
authors: Stephen
authors: S
authors: Connelly
authors: Renata
authors: R
authors: Kowalczyk
authors: Jonathan
authors: J
authors: Sperry
authors: Ian A
authors: IA
authors: Wilson
authors: John D
authors: JD
authors: Fraser
authors: Margaret A
authors: MA
authors: Brimble
mesh_terms: D000086042:3C Viral Proteases; D003546:Cysteine Endopeptidases / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D057075:Enzyme Assays; D005452:Fluoresceins / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D012229:Rhinovirus / Q000201:enzymology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D015853:Cysteine Proteinase Inhibitors; D005452:Fluoresceins; D010455:Peptides; D014764:Viral Proteins; C024098:6-carboxyfluorescein; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases