title: Purification and characterization of catalytic domains of gelatinase A with or without fibronectin insert for high-throughput inhibitor screening.
abstract: Gelatinase A represents an attractive therapeutic target for cancer invasion and metastasis. In order to screen for gelatinase A inhibitors, we have cloned, overexpressed in a bacterial system, and purified the catalytic domain of human gelatinase A with (GaCDfn) or without (GaCD) fibronectin-like insert. GaCDfn and GaCD were purified to homogeneity and refolded in vitro. GaCDfn was refolded to a stable and active form in the presence of calcium and zinc ions. GaCD was refolded through direct dialysis against Tris-HCl (pH 7.5) buffer without calcium and zinc ions. GaCD is unstable in the presence of calcium and zinc ions. The enzymatic activities of GaCDfn and GaCD require calcium and zinc ions, but high concentration of zinc and calcium ions inhibited the activities. The GaCDfn and GaCD cleaved several synthetic substrates including a chromogenic thiopeptolide (TPL) and fluorogenic peptides with optimal activity around pH 7.5. Moreover, GaCDfn and GaCD cleave gelatin and collagen VII and display similar cleavage patterns on the gel, but the digestion rate of these protein substrates by GaCD is apparently slower than GaCDfn. EDTA, 1,10-phenanthroline, and reference inhibitors potently blocked GaCDfn and GaCD enzymatic activities. A set of 3596 compounds from our center collection were screened by using GaCDfn and GaCD to cleave TPL. Further analysis by using MMP inhibitors indicated there is a correlation between IC(50) values on GaCDfn and GaCD. A few compounds with selectivity toward gelatinase A catalytic domain were identified for structure modification.
authors.affiliation: National Center for Drug Screening, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
authors.forename: Donghang
authors.initials: D
authors.lastname: Cheng
authors.forename: Qiang
authors.initials: Q
authors.lastname: Shen
authors.forename: Fajun
authors.initials: F
authors.lastname: Nan
authors.forename: Zhen
authors.initials: Z
authors.lastname: Qian
authors.forename: Qi-Zhuang
authors.initials: QZ
authors.lastname: Ye
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D020134:Catalytic Domain; D002614:Chelating Agents / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D005353:Fibronectins / Q000737:chemistry*; D006801:Humans; D020128:Inhibitory Concentration 50; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D000077423:Polidocanol; D011092:Polyethylene Glycols / Q000494:pharmacology; D020673:Protein Renaturation / Q000187:drug effects; D015032:Zinc / Q000494:pharmacology
chemical_list: D002614:Chelating Agents; D004791:Enzyme Inhibitors; D005353:Fibronectins; D061965:Matrix Metalloproteinase Inhibitors; D000077423:Polidocanol; D011092:Polyethylene Glycols; D020778:Matrix Metalloproteinase 2; D015032:Zinc; D002118:Calcium