Purification of active matrix metalloproteinase catalytic domains and its use for screening of specific stromelysin-3 inhibitors.
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.
Kannan
R
R
Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, Illkirch Cedex, 67404, France.
Ruff
M
M
Kochins
J G
JG
Manly
S P
SP
Stoll
I
I
El Fahime
M
M
Noël
A
A
Foidart
J M
JM
Rio
M C
MC
Dive
V
V
Basset
P
P
D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000302:isolation & purification; D001483:Base Sequence; D016466:CHO Cells; D020134:Catalytic Domain / Q000235:genetics; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D053506:Matrix Metalloproteinase 10; D053507:Matrix Metalloproteinase 11; D020783:Matrix Metalloproteinase 7; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology
D000970:Antineoplastic Agents; D017931:DNA Primers; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D053507:Matrix Metalloproteinase 11; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D053506:Matrix Metalloproteinase 10; D020783:Matrix Metalloproteinase 7