Development of Selective ADAMTS‑5 Peptide Substrates to Monitor Proteinase Activity

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 in silico docking and combinatorial chemistry.
The lead substrates 3 and 26 showed higher
overall cleavage rates (∼3–4-fold) and catalytic efficiencies
(∼1.5–2-fold) compared to the best current ADAMTS-5
substrate ortho-aminobenzoyl(Abz)-TESE↓SRGAIY-N-3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl(Dpa)-KK-NH2. 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.