title: A microplate assay for the screening of ADAMTS-4 inhibitors.
abstract: Aggrecanase plays a major role in cartilage proteoglycan degradation in rheumatic diseases such as osteoarthritis and rheumatoid arthritis. The search of new inhibitors of aggrecanase activity necessitates a robust assays in order to be able to screen large numbers of compounds. We present in this paper an assay based on the cleavage of His-tagged aggrecan interglobular domain by N- and C- terminus truncated, active aggrecanase-1/ADAMTS-4, with formation of the aggrecanase-specific ARGSV neoepitope. This is detected by anti-ARGSV antibody, in turn recognized by a fluorescent anti-IgG. Furthermore, the formation of the reaction products was confirmed by high-pressure capillary electrophoresis. This assay allows the rapid screening of aggrecanase inhibitors in a 96-well plate format, allowing an immediate transposition to high-throughput scale up.
authors.affiliation: Division of Rheumatology, Institut de Recherches Servier, IdRS, 11 rue des Moulineaux, 92150 Suresnes, France.
authors.forename: Marie
authors.initials: M
authors.lastname: Thomas
authors.forename: Massimo
authors.initials: M
authors.lastname: Sabatini
authors.forename: Fabrice
authors.initials: F
authors.lastname: Bensaude
authors.forename: Benoit
authors.initials: B
authors.lastname: Mignard
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Ortuno
authors.forename: Isabelle
authors.initials: I
authors.lastname: Caron
authors.forename: Jean A
authors.initials: JA
authors.lastname: Boutin
authors.forename: Gilles
authors.initials: G
authors.lastname: Ferry
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors*; D000071121:ADAMTS4 Protein; D053674:Aggrecans / Q000378:metabolism; D000906:Antibodies; D000918:Antibody Specificity; D001681:Biological Assay / Q000295:instrumentation* / Q000379:methods*; D006801:Humans; D011348:Procollagen N-Endopeptidase / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000032:analysis*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D053674:Aggrecans; D000906:Antibodies; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein; C000603918:ADAMTS4 protein, human