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.
MeSH terms
-
ADAM Proteins / antagonists & inhibitors
-
ADAM Proteins / metabolism*
-
ADAM10 Protein
-
ADAM12 Protein
-
ADAM17 Protein
-
Amyloid Precursor Protein Secretases / antagonists & inhibitors
-
Amyloid Precursor Protein Secretases / metabolism
-
Dipeptides / chemistry
-
Dipeptides / metabolism
-
Dipeptides / pharmacology
-
Fluorescence Resonance Energy Transfer
-
Fluorescent Dyes / chemistry
-
Fluorescent Dyes / metabolism*
-
Humans
-
Hydroxamic Acids / chemistry
-
Hydroxamic Acids / metabolism
-
Hydroxamic Acids / pharmacology
-
Kinetics
-
Membrane Proteins / antagonists & inhibitors
-
Membrane Proteins / metabolism*
-
Morpholines / chemistry
-
Morpholines / metabolism
-
Morpholines / pharmacology
-
Protease Inhibitors / chemistry
-
Protease Inhibitors / metabolism*
-
Protease Inhibitors / pharmacology
-
Protein Binding
Substances
-
Dipeptides
-
Fluorescent Dyes
-
Hydroxamic Acids
-
Membrane Proteins
-
Morpholines
-
N-(2(R)-2-(hydroxamidocarbonylmethyl)-4-methylpentanoyl)-L-tryptophan methylamide
-
Protease Inhibitors
-
TAPI-2
-
apratastat
-
Amyloid Precursor Protein Secretases
-
ADAM Proteins
-
ADAM12 Protein
-
ADAM12 protein, human
-
ADAM8 protein, human
-
ADAM10 Protein
-
ADAM10 protein, human
-
ADAM17 Protein
-
ADAM17 protein, human