title: Luminometric method for screening retroviral protease inhibitors.
abstract: We have developed a sensitive luminometric assay for determining the activity of retroviral proteases that uses proteolytic cleavage of polypeptide substrate immobilized on Ni-NTA HisSorb Strips microplates. The protease substrate derived from the Gag precursor protein of Mason-Pfizer monkey virus (M-PMV) was conjugated with horseradish peroxidase (HRP), which catalyzes oxidation of luminol in the assay. The cleavage of the substrate was monitored as a decrease in luminescent signal caused by the release of the cleavage product conjugated to HRP. Testing of a set of M-PMV protease inhibitors confirmed that this method is sufficiently sensitive and specific for high-throughput screening of retroviral protease inhibitors.
authors.affiliation: Department of Biochemistry and Microbiology, Institute of Chemical Technology, Technická 3, 166 28 Prague, Czech Republic.
authors.forename: Dana
authors.initials: D
authors.lastname: Horáková
authors.forename: Michaela
authors.initials: M
authors.lastname: Rumlová
authors.forename: Iva
authors.initials: I
authors.lastname: Pichová
authors.forename: Tomás
authors.initials: T
authors.lastname: Ruml
mesh_terms: D004353:Drug Evaluation, Preclinical / Q000379:methods; D010450:Endopeptidases / Q000737:chemistry*; D008163:Luminescent Measurements / Q000379:methods; D016093:Mason-Pfizer monkey virus / Q000201:enzymology*; D011480:Protease Inhibitors / Q000032:analysis*; D013379:Substrate Specificity
chemical_list: D011480:Protease Inhibitors; D010450:Endopeptidases; C445563:Mason-Pfizer monkey virus protease