| Summary: | <p>The dysregulation of proteinase activity is a hallmark of osteoarthritis (OA), a disease characterized by progressive degradation of articular cartilage by catabolic proteinases such as a disintegrin and metalloproteinase with thrombospondin type I motifs-5 (ADAMTS-5). The ability to detect such activity sensitively would aid disease diagnosis and the evaluation of targeted therapies. Förster resonance energy transfer (FRET) peptide substrates can detect and monitor disease-related proteinase activity. To date, FRET probes for detecting ADAMTS-5 activity are nonselective and relatively insensitive. We describe the development of rapidly cleaved and highly selective ADAMTS-5 FRET peptide substrates through <em>in silico</em> docking and combinatorial chemistry. The lead substrates <strong>3</strong> and <strong>26</strong> showed higher overall cleavage rates (∼3–4-fold) and catalytic efficiencies (∼1.5–2-fold) compared to the best current ADAMTS-5 substrate <em>ortho</em>-aminobenzoyl(Abz)-TESE↓SRGAIY-<em>N</em>-3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl(Dpa)-KK-NH<sub>2</sub>. They exhibited high selectivity for ADAMTS-5 over ADAMTS-4 (∼13–16-fold), MMP-2 (∼8–10-fold), and MMP-9 (∼548–2561-fold) and detected low nanomolar concentrations of ADAMTS-5.</p>
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