title: Fluorescent substrates for ADAM15 useful for assaying and high throughput screening.
abstract: A disintegrin and metalloproteinase 15 (ADAM15), also known as metargidin, plays important roles in regulating inflammation, wound healing, neovascularization, and is an attractive drug target. Fluorescence resonance energy transfer (FRET)-based peptide substrates were tested to identify candidate reagents for high throughput screening and detection of ADAM15 in biological samples. ADAM15 exhibits a unique and diverse activity profile compared to other metalloproteinases. Two FRET substrates, Dabcyl-Gly-Pro-Leu-Gly-Met-Arg-Gly-Lys(FAM)-NH2 (PEPDAB011) and Dabcyl-Ala-Pro-Arg-Trp-Ile-Gln-Asp-Lys(FAM)-NH2 (PEPDAB017), which also detect activities of several matrix metalloproteinases (MMPs -2, -9, and -13), were efficiently cleaved by ADAM15 with specificity constants of 5800 M-1s-1and 4300 M-1s-1, respectively. Additionally, ADAM15 efficiently processed Dabcyl-Leu-Arg-Glu-Gln-Gln-Arg-Leu-Lys-Ser-Lys(FAM)-NH2 (PEPDAB022), which is based on a physiological CD23 cleavage site, with a specificity constant (kcat/Km) of 5200 M-1s-1. PEPDAB022 was used to screen the ability of known metalloproteinase inhibitors including TAPI-2, marimastat, GI-254023, and the Tissue Inhibitor of Metalloproteinases(TIMPs) 1 and 3 to block ADAM15 activity. Even though ADAM15 exhibits similar substrate preferences to other metalloproteinases, many broad spectrum inhibitors failed to block ADAM15 activity at concentrations as high as 50 μM. Thus, a clear need exists to develop potent and selective ADAM15 inhibitors, and the FRET substrates described herein should aid future research efforts towards this aim.
authors.affiliation: BioZyme Inc, 1513 Old White Oak Church Rd., Apex, NC 27523, USA. Electronic address: mossscientist0@gmail.com.
authors.forename: Marcia L
authors.initials: ML
authors.lastname: Moss
authors.affiliation: Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Miles A
authors.initials: MA
authors.lastname: Miller
authors.affiliation: University of Pittsburgh Cancer Institute, Departments of Pathology, VA Healthcare System, Pittsburgh, PA, USA.
authors.forename: Nikola
authors.initials: N
authors.lastname: Vujanovic
authors.affiliation: University of Pittsburgh Cancer Institute, Departments of Pathology, VA Healthcare System, Pittsburgh, PA, USA.
authors.forename: Toshie
authors.initials: T
authors.lastname: Yoneyama
authors.affiliation: BioZyme Inc, 1513 Old White Oak Church Rd., Apex, NC 27523, USA.
authors.forename: Fred H
authors.initials: FH
authors.lastname: Rasmussen
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D004151:Dipeptides / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000378:metabolism
chemical_list: C555398:3-(formylhydroxyamino)-2-(3-phenyl-1-propyl)butanoic acid (2,2-dimethyl-1-methylcarbamoyl-1-propyl)amide; D004151:Dipeptides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D006877:Hydroxamic Acids; D008565:Membrane Proteins; D010455:Peptides; C099410:TAPI-2; D051722:ADAM Proteins; C098660:ADAM15 protein, human
keywords: ADAM15; Disintegrin; FRET; Fluorescent substrate; Metalloproteinase; TIMP