Screening of potential a disintegrin and metalloproteinase with thrombospondin motifs-4 inhibitors using a collagen model fluorescence resonance energy transfer substrate

Abstract

The major components of the cartilage extracellular matrix are type II collagen and aggrecan. Type II collagen provides cartilage with its tensile strength, whereas the water-binding capacity of aggrecan provides compressibility and elasticity. Aggrecan breakdown leads to an increase in proteolytic susceptibility of articular collagen; hence, aggrecan may also have a protective effect on type II collagen. Given their role in aggrecan degradation and differing substrate specificity profiles, the pursuit of inhibitors for both aggrecanase 1 (a disintegrin and metalloproteinase with thrombospondin motifs-4 [ADAMTS-4]) and aggrecanase 2 (ADAMTS-5) is desirable. We previously described collagen model fluorescence resonance energy transfer (FRET) substrates for aggrecan-degrading members of the ADAMTS family. These FRET substrate assays are also fully compatible with multiwell formats. In the current study, a collagen model FRET substrate was examined for inhibitor screening of ADAMTS-4. ADAMTS-4 was screened against a small compound library (n=960) with known pharmacological activity. Five compounds that inhibited ADAMTS-4>60% at a concentration of 1muM were identified. A secondary screen using reversed-phase high-performance liquid chromatography (RP-HPLC) was developed and performed for verification of the five potential inhibitors. Ultimately, piceatannol was confirmed as a novel inhibitor of ADAMTS-4, with an IC(50) value of 1muM. Because the collagen model FRET substrates have distinct conformational features that may interact with protease secondary substrate sites (exosites), nonactive site-binding inhibitors can be identified via this approach. Selective inhibitors for ADAMTS-4 would allow a more definitive evaluation of this protease in osteoarthritis and also represent a potential next generation in metalloproteinase therapeutics.

Regulated Proteolytic Processing of Reelin through Interplay of Tissue Plasminogen Activator (tPA), ADAMTS-4, ADAMTS-5, and Their Modulators

“…1F ). Thus, adding piceatannol (PIC), a selective inhibitor of ADAMTS-4 and -5 [41] , to the cell cultures induced a strong inhibition of Reelin cleavage at both, the N- and C-terminal site ( Fig. 2A and Fig.…”

“…Since it was shown that ADAMTS-4 and ADAMTS-5 (aggrecanase-2) have a number of common substrates and overlapping physiological roles [46] , and that both ADAMTS-4 and -5 are inhibited by α2M and piceatannol [41] , [43] , we tested if ADAMTS-5, that was also identified in our protease screen ( Table S1 ), could cleave Reelin. Although shRNA-mediated knock-down of ADAMTS-5 in HEK293 cells did not affect Reelin cleavage in the medium (data not shown), incubation of FL-Reelin with recombinant active ADAMTS-5 led to formation of Reelin proteolytic fragments, a process that could be fully inhibited by the addition of TIMP-3 ( Fig.…”

A high-throughput FRET-based assay for determination of Atg4 activity

“…Atg4A or Atg4B were incubated with FRET-LC3B or FRET-GATE-16 at different concentrations in Buffer B in a total volume of 200 µl at 37°C for the designated times. The kinetics were determined as previously described 32 with modifications. In brief, the initial velocity of the cleavage reaction was defined as V = [S] t /t, in which [S] t is the substrate concentration ([S]) at a given time point (t).…”

Calcium pentosan polysulfate is a multifaceted exosite inhibitor of aggrecanases

“…Among them, ADAMTS‐5 (aggrecanase 2) appears to be a major aggrecanase in mice (6, 7), but both ADAMTS‐4 (aggrecanase 1) and ADAMTS‐5 are considered to play a role in human disease (8). Thus interest has been growing in the development of inhibitors for ADAMTS‐4 and ‐5 (9–11), mirroring the development of MMP inhibitors for the treatment of diseases associated with elevated MMP activity. Numerous MMP inhibitors have been developed, and several were clinically tested in patients with cancer (12) and rheumatoid arthritis (13), but they failed to show efficacy and exhibited side effects such as musculoskeletal pain and mild thrombocytopenia (12).…”