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.

Figure 1

Domain structures of ADAMTS-4 and ADAMTS-5. The catalytic domain contains a typical reprolysin-type zinc-binding motif, and is followed by a disintegrin-like domain, a single thrombospondin type I module, and a Cys-rich domain. The C-terminal portion of the proteins consists of a spacer domain, which shows little similarity between ADAMTS family members or other protein domain structures [49]. ADAMTS-5 has an additional thrombospondin type I repeat after the spacer domain [49].

Figure 2

Inhibition of ADAMTS-4-2 by MMP inhibitor III. The change in relative fluorescence units (ΔRFU) for 10 nM ADAMTS-4-2 hydrolysis of 10 μM fSSPa was monitored over an MMP inhibitor III concentration range of 1 nM to 10μM as described in Materials and Methods. Assays were performed in triplicate; bars indicate standard deviations.

Figure 3

Results of the 384-well screen for inhibitors of ADAMTS-4-2. Displayed are the results of test compounds (n = 960; yellow arrow) as well as positive (100% inhibition; green arrow) and negative (0% inhibition; blue arrow) controls. Circled are the inhibition results for the five active compounds (“hits”) identified in the screen.

Figure 4

Structures of (a) piceatannol [(E)-4-[2-(3,5-dihydroxyphenyl)ethenyl] 1,2-benzenediol; trans-3,3′,4,5′-tetrahydroxystilbene]; (b) (R,R)-cis-diethyltetrahydro-2,8-chrysenediol; (c) (S)-(+)-camptothecin (4-ethyl-4-hydroxy-1H-pyrano[3′,4′:6,7] indolizino[1,2-b] quinoline-3,14(4H,12H)dione); (d) N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a] indole-11-yl)ethanamine [IIK7]; and (e) 8-(p-sulfophenyl)theophylline.

Figure 5

Inhibition of ADAMTS-4-2 by piceatannol (closed triangles) or (S)-(+)-camptothecin (closed circles). The change in relative fluorescence units (ΔRFU) for 10 nM ADAMTS-4-2 hydrolysis of 10 μM fSSPa was monitored over an inhibitor concentration range of 1 nM to 10 μM as described in Materials and Methods. IC₅₀ = 1.0 and 4.0 μM for piceatannol and (S)-(+)-camptothecin, respectively. Assays were performed in triplicate; bars indicate standard deviations.

Figure 6

RP-HPLC elution profiles of ADAMTS-4-2 hydrolysis of fSSPa in the absence (blue) and presence (magenta) of picetannol. ADAMTS-4-2 (10 nM) hydrolysis of 10 μM fSSPa was examined after 24 h following addition of 0 or 10 μM piceatannol as described in Materials and Methods. The intact substrate eluted at 14.667 min, while cleavage products were observed at 12.069, 12.361, and 13.410 min. The identities of the cleavage products have been described previously [19].

Figure 7

Inhibition of ADAMTS-4-2 by piceatannol, as monitored by RP-HPLC and fluorescence. The change in RP-HPLC peak areas (closed circles) or relative fluorescence units (ΔRFU) (closed triangles) for 10 nM ADAMTS-4-2 hydrolysis of 10 μM fSSPa was monitored over a piceatannol concentration range of 0.1 to 10 μM as described in Materials and Methods. Assays were performed in duplicate.

Figure 8

Inhibition of ADAMTS-4-2 by piceatannol (open circles), (R,R)-cis-diethyltetrahydro-2,8-chrysenediol (closed squares), (S)-(+)-camptothecin (closed diamonds), N-butanoyl-2-(2-methoxy-6H-isoindolo[2,1-a] indole-11-yl)ethanamine (closed circles), and 8-(p-sulfophenyl)theophylline (closed triangles), as monitored by RP-HPLC. The change in RP-HPLC peak areas for 10 nM ADAMTS-4-3 hydrolysis of 10 μM fSSPa was monitored over an inhibitor concentration range of 0.1 to 10 μM as described in Materials and Methods. Assays were performed in duplicate.

Figure 9

Hydrolysis of fSSPa as a function of ADAMTS-4-2 concentration in different assay volumes. The change in relative fluorescence units (ΔRFU) for ADAMTS-4-2 hydrolysis of 10 μM fSSPa was monitored using total reaction volumes of 10 (closed circles), 20 (closed triangles), 40 (closed squares), or 80 (closed diamonds) μL as described in Materials and Methods. Assays were performed in triplicate; bars indicate standard deviations.