title: Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening.
abstract: In this paper we describe novel fluorescent substrates for the human ADAM family members ADAM17, ADAM10, ADAM8, and ADAM12 that have good specificity constants and are useful for high-throughput screening of inhibitors. The fluorescence resonance energy transfer substrates contain a 4-(4-dimethylaminophenylazo)benzoyl and 5-carboxyfluorescein (Dabcyl/Fam) pair and are based on known cleavage sequences in precursor tumor necrosis factor-alpha (TNF-alpha) and CD23. The precursor TNF-alpha-based substrate, Dabcyl-Leu-Ala-Gln-Ala-Homophe-Arg-Ser-Lys(Fam)-NH2, is a good substrate for all the ADAMs tested, including ADAM12 for which there is no reported fluorescent substrate. The CD23-based substrate, Dabcyl-His-Gly-Asp-Gln-Met-Ala-Gln-Lys-Ser-Lys(Fam)-NH2, is more selective, being hydrolyzed efficiently only by ADAM8 and ADAM10. The substrates were used to obtain inhibition constants for four inhibitors that are commonly used in shedding assays: TMI-1, GM6001, GW9471, and TAPI-2. The Wyeth Aerst compound, TMI-1, is a potent inhibitor against all of the ADAMs tested and is slow binding against ADAM17.
authors.affiliation: BioZyme, Inc., Apex, NC 27523-9299, USA. mmoss@biozyme-inc.com
authors.forename: Marcia L
authors.initials: ML
authors.lastname: Moss
authors.forename: Fred H
authors.initials: FH
authors.lastname: Rasmussen
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000072197:ADAM10 Protein; D000072199:ADAM12 Protein; D000072198:ADAM17 Protein; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009025:Morpholines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding
chemical_list: D004151:Dipeptides; D005456:Fluorescent Dyes; D006877:Hydroxamic Acids; D008565:Membrane Proteins; D009025:Morpholines; C078131:N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide; D011480:Protease Inhibitors; C099410:TAPI-2; C484302:apratastat; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; D000072199:ADAM12 Protein; C000606480:ADAM12 protein, human; C068490:ADAM8 protein, human; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human