title: Development of a high-throughput screening assay for inhibitors of aggrecan cleavage using luminescent oxygen channeling (AlphaScreen ).
abstract: Aggrecan is one of the most important structural components of joint cartilage, and members of the metalloprotease (MMP) and ADAM (a disintegrin and metalloproteinase) protease families have been shown to degrade aggrecan in vivo. A robust assay for aggrecan-degrading activity suitable for high-throughput screening (HTS) was set up and measured using AlphaScreen. In this technology, beads brought into proximity through cross-linking and stimulated with laser light generate a signal through luminescent oxygen tunneling, the outcome of which is a time-resolved fluorescent signal. Specific antibodies to the carbohydrate side chains of aggrecan were harnessed to create a scaffold whereby aggrecan could form a cross-link between donor and acceptor AlphaScreen detector beads. Digested aggrecan, which failed to form a cross-link, generated no signal, so that inhibitors of the digestion could be detected as a restoration of signal. The development of this assay and its validation for HTS are described in this report.
authors.affiliation: Aventis Pharmaceuticals, Mailstop: JR1-303C, P.O. Box 6800, Route 202-206, Bridgewater, NJ 08807, USA. jane.peppard@aventis.com
authors.forename: Jane
authors.initials: J
authors.lastname: Peppard
authors.forename: Fraser
authors.initials: F
authors.lastname: Glickman
authors.forename: Yang
authors.initials: Y
authors.lastname: He
authors.forename: Shou-Ih
authors.initials: SI
authors.lastname: Hu
authors.forename: John
authors.initials: J
authors.lastname: Doughty
authors.forename: Ronald
authors.initials: R
authors.lastname: Goldberg
mesh_terms: D053674:Aggrecans; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D016326:Extracellular Matrix Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000737:chemistry / Q000378:metabolism; D037181:Lectins, C-Type; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D011509:Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results
chemical_list: D053674:Aggrecans; D000906:Antibodies; D004791:Enzyme Inhibitors; D016326:Extracellular Matrix Proteins; D007202:Indicators and Reagents; D037181:Lectins, C-Type; D061965:Matrix Metalloproteinase Inhibitors; D011509:Proteoglycans; D010450:Endopeptidases; D020782:Matrix Metalloproteinases; C083641:aggrecanase; D010100:Oxygen