Design and characterization of a metalloproteinase inhibitor-tethered resin for the detection of active MMPs in biological samples.
Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases, are implicated in tumor progression. We describe herein the development of a resin-immobilized, broad-spectrum synthetic MMP inhibitor for selective binding of the active forms of MMPs from different experimental samples. We confirmed the activity-based binding of MMPs to the inhibitor-tethered resin with purified human recombinant MMP-2, -9, and -14, samples of cultured cells, and tissue extracts. Our results show that only the free active MMPs, and not the zymogens or MMP/TIMP (enzyme-protein inhibitor) complexes, bound specifically to the resin. In our comparison of benign and carcinoma tissue extracts, we detected active MMP-2 and MMP-14 forms only in the cancerous tissue samples, indicating that a pool of the tumor MMPs is free of endogenous inhibitors (TIMPs), and is thus likely to contribute to proteolytic events that precipitate tumor metastasis.
Hesek
Dusan
D
Department of Chemistry and Biochemistry and The Walther Cancer Research Center, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Toth
Marta
M
Meroueh
Samy O
SO
Brown
Stephen
S
Zhao
Huiren
H
Sakr
Wael
W
Fridman
Rafael
R
Mobashery
Shahriar
S
D001943:Breast Neoplasms / Q000201:enzymology; D015195:Drug Design; D005260:Female; D018093:Gelatinases / Q000032:analysis / Q000037:antagonists & inhibitors; D006801:Humans; D066298:In Vitro Techniques; D007822:Laryngeal Neoplasms / Q000201:enzymology; D061965:Matrix Metalloproteinase Inhibitors; D020782:Matrix Metalloproteinases / Q000032:analysis*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008958:Models, Molecular; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000032:analysis / Q000037:antagonists & inhibitors; D012117:Resins, Synthetic / Q000138:chemical synthesis / Q000737:chemistry
D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D012117:Resins, Synthetic; D018093:Gelatinases; D020782:Matrix Metalloproteinases; D053504:Matrix Metalloproteinases, Membrane-Associated