tapi-2 and apratastat

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.

Topics: ADAM Proteins; ADAM10 Protein; ADAM12 Protein; ADAM17 Protein; Amyloid Precursor Protein Secretases; Dipeptides; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Humans; Hydroxamic Acids; Kinetics; Membrane Proteins; Morpholines; Protease Inhibitors; Protein Binding