Purification of active matrix metalloproteinase catalytic domains and its use for screening of specific stromelysin-3 inhibitors

Protein Expr Purif. 1999 Jun;16(1):76-83. doi: 10.1006/prep.1999.1068.

Abstract

The matrix metalloproteinase (MMP) stromelysin-3 (ST3) has been shown to be involved in malignant tumor progression and therefore represents an attractive therapeutical target. In order to screen for ST3 synthetic inhibitors, we have produced and purified the catalytic domain of ST3, matrilysin, stromelysin-2, and membrane type-1 MMP from inclusion bodies in a bacterial system. Our strategy allowed the purification of MMPs directly in the active form, thereby avoiding in vitro activation. A total of 140,000 synthetic compounds from the Bristol-Myers Pharmaceutical Research Institute chemical deck were tested, using a substrate-based colorimetric enzymatic assay, in which ST3 activity was evaluated through its ability to cleave and inactivate alpha-1 proteinase inhibitor. One ST3 inhibitor belonging to the cephalosporin family of antibiotics was thereby identified.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antineoplastic Agents / chemistry
  • Antineoplastic Agents / isolation & purification
  • Base Sequence
  • CHO Cells
  • Catalytic Domain / genetics
  • Cricetinae
  • DNA Primers / genetics
  • Drug Evaluation, Preclinical
  • Escherichia coli / genetics
  • Humans
  • In Vitro Techniques
  • Kinetics
  • Matrix Metalloproteinase 10
  • Matrix Metalloproteinase 11
  • Matrix Metalloproteinase 7
  • Matrix Metalloproteinases, Membrane-Associated
  • Metalloendopeptidases / antagonists & inhibitors*
  • Metalloendopeptidases / chemistry*
  • Metalloendopeptidases / genetics
  • Mice
  • Protease Inhibitors / chemistry
  • Protease Inhibitors / isolation & purification
  • Protease Inhibitors / pharmacology*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / pharmacology

Substances

  • Antineoplastic Agents
  • DNA Primers
  • Protease Inhibitors
  • Recombinant Proteins
  • Matrix Metalloproteinase 11
  • Matrix Metalloproteinases, Membrane-Associated
  • Metalloendopeptidases
  • Matrix Metalloproteinase 10
  • Matrix Metalloproteinase 7