title: 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.
authors.affiliation: Department of Chemistry and Biochemistry, Florida Atlantic University, Boca Raton, FL 33431, USA.
authors.forename: Janelle L
authors.initials: JL
authors.lastname: Lauer-Fields
authors.forename: Timothy P
authors.initials: TP
authors.lastname: Spicer
authors.forename: Peter S
authors.initials: PS
authors.lastname: Chase
authors.forename: Mare
authors.initials: M
authors.lastname: Cudic
authors.forename: Gayle D
authors.initials: GD
authors.lastname: Burstein
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.forename: Peter
authors.initials: P
authors.lastname: Hodder
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Fields
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors*; D000071121:ADAMTS4 Protein; D003094:Collagen / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D011348:Procollagen N-Endopeptidase / Q000037:antagonists & inhibitors*; D011480:Protease Inhibitors / Q000494:pharmacology
chemical_list: D011480:Protease Inhibitors; D003094:Collagen; D051722:ADAM Proteins; D011348:Procollagen N-Endopeptidase; D000071121:ADAMTS4 Protein