Fluorescent substrates for the proteinases ADAM17, ADAM10, ADAM8, and ADAM12 useful for high-throughput inhibitor screening

Anal Biochem. 2007 Jul 15;366(2):144-8. doi: 10.1016/j.ab.2007.04.043. Epub 2007 May 3.

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