A microplate assay specific for the enzyme aggrecanase |
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Authors: | Miller Jeffery A Liu Rui-Qin Davis Gary L Pratta Michael A Trzaskos James M Copeland Robert A |
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Institution: | The Bristol-Myers Squibb Company Pharmaceutical Research Institute, Experimental Station, Route 141 and Henry Clay Road, Wilmington, DE 19880, USA. |
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Abstract: | We have identified a 41-residue peptide, bracketing the aggrecanase cleavage site of aggrecan, that serves as a specific substrate for this enzyme family. Biotinylation of the peptide allowed its immobilization onto streptavidin-coated plates. Aggrecanase-mediated hydrolysis resulted in an immobilized product that reveals an N-terminal neoepitope, recognized by the specific antibody BC-3. This assay is highly specific for aggrecanases; MMPs were inactive in this assay. Reduction of the peptide size below 30 amino acids resulted in a significant diminution of activity. Using the immobilized 41-residue peptide as a substrate, we have developed a 96-well microplate-based assay that can be conveniently used for high-throughput screening of samples for aggrecanase activity and for discovery of inhibitors of aggrecanase activity. |
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Keywords: | Aggrecanase Metalloprotease Arthritis Inflammatory joint disease High-throughput screening Assay design Drug discovery |
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