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A substrate-optimized electrophoretic mobility shift assay for ADAM12.

Alexander Kotzsch1, Tine Skovgaard1, Uwe Buus1

  • 1Facility for Protein Purification and Function at the Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark.

Analytical Biochemistry
|February 19, 2014
PubMed
Summary
This summary is machine-generated.

Researchers developed a new assay for ADAM12, a protein involved in growth factor activation. This reliable method uses an optimized peptide substrate for better validation of potential drug inhibitors targeting ADAM metalloproteases.

Keywords:
ADAM12Fluorimetric assayMetalloproteaseMobility shift assayPeptide arraySmall-molecule inhibitor

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Area of Science:

  • Biochemistry
  • Enzymology
  • Drug Discovery

Background:

  • ADAM12 is a secreted metalloprotease within the ADAM family, known for activating growth factors like EGFR ligands and TNF-α.
  • ADAM proteases, particularly ADAM17, are pursued as drug targets, but challenges with inhibitor potency and side effects hinder clinical progress.
  • Developing selective ADAM inhibitors necessitates robust biochemical assays for validating screening-identified molecular probes.

Purpose of the Study:

  • To describe a novel and reliable assay for quantifying ADAM12 activity.
  • To introduce an optimized peptide substrate for enhanced assay performance and reproducibility.
  • To facilitate the screening and validation of new ADAM12 inhibitor candidates.

Main Methods:

  • Development of an electrophoretic mobility shift assay (EMSA) specifically designed for ADAM12.
  • Identification and optimization of a peptide substrate tailored for ADAM12 enzymatic activity.
  • Characterization of the assay's performance regarding sensitivity, specificity, and reproducibility.

Main Results:

  • An optimized peptide substrate was identified, demonstrating excellent performance for ADAM12.
  • The developed electrophoretic mobility shift assay provides a reliable method for measuring ADAM12 activity.
  • The assay exhibits high reproducibility, crucial for large-scale screening and inhibitor validation.

Conclusions:

  • The described EMSA represents a significant advancement for biochemical studies of ADAM12.
  • This assay enables more effective validation of potential therapeutic agents targeting ADAM12.
  • The optimized substrate and assay methodology contribute to overcoming challenges in ADAM protease inhibitor development.