![]() (28) (C) Representative Western blot and Coomassie stain of purified TS5-5. Domains and cutoffs are defined according to Gendron et al. (B) Schematic representation of the domain organization of full-length ADAMTS-5 and the TS5-5 domain deletion mutant used here. (A) Proteinase activity can be measured by the increase in signal intensity resulting from cleavage of FRET peptide substrates. The current ADAMTS-5 FRET peptide substrate lacks selectivity. (23−26) Developing a selective ADAMTS-5 FRET substrate would enable earlier diagnostic imaging of osteoarthritic processes and provide more effective tracking of disease progression. Furthermore, current FRET substrates for ADAMTSs are limited by low rates of cleavage and/or poor target selectivity. (20−22) However, these FRET substrates were limited by only being able to distinguish between osteoarthritic joints and healthy joints once damage was already clearly visible by histology. (19) Selective in vivo FRET substrates have been developed for MMP-13 to monitor disease progression and response to therapy in rodent models of OA. Upon substrate cleavage, an increase in fluorescence is observed due to an increase in separation between donor and acceptor (>70 Å), resulting in disruption of the FRET process. (18) Composed of a peptide sequence with a blue fluorophore (BlueF) and quencher (Q) on either side of the scissile bond, (19) the FRET probe does not fluoresce prior to cleavage as the excitation of the BlueF (the donor) is tunneled to the quencher (the acceptor) over distances between 0 and 70 Å ( Figure 1A). (11,12)įörster resonance energy transfer (FRET) substrates are a useful tool for measuring proteinase activity longitudinally due to their capacity to amplify signal intensity through continuous substrate cleavage and potential for high target selectivity. (8−10) Additionally, in both murine and human osteoarthritic cartilage explants, loss of aggrecan staining is observed earlier than loss of type II collagen staining. (8−15) The temporal nature of specific proteinase activity in cartilage degradation is supported by in vitro experiments with bovine cartilage explants treated with interleukin-1, in which aggrecan degradation occurs in the first week of culture and type II collagen breakdown thereafter. (5−7) Aggrecan degradation is likely followed by the breakdown of type II collagen by members of the matrix metalloproteinase family (MMP-1, MMP-8, and MMP-13). More specifically, ADAMTS-5 has been identified as the main aggrecanase in surgical mouse models of osteoarthritis, (3,4) whereas both ADAMTS-4 and ADAMTS-5 have been implicated in human disease progression. OA is thought to be characterized by the initial degradation of aggrecan by proteinases of the a disintegrin and metalloproteinase with thrombospondin type I motifs (ADAMTS) family. (1,2) The extracellular matrix of cartilage consists of two major structural components: type II collagen and aggrecan. The degradation of articular cartilage is a major pathological feature of osteoarthritis (OA), a joint disease affecting more than 300 million people worldwide. 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. 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- l-2,3-diaminopropionyl(Dpa)-KK-NH 2. We describe the development of rapidly cleaved and highly selective ADAMTS-5 FRET peptide substrates through in silico docking and combinatorial chemistry. To date, FRET probes for detecting ADAMTS-5 activity are nonselective and relatively insensitive. Förster resonance energy transfer (FRET) peptide substrates can detect and monitor disease-related proteinase activity. ![]() ![]() The ability to detect such activity sensitively would aid disease diagnosis and the evaluation of targeted therapies. 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).
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