title: Molecular design of a highly selective and strong protein inhibitor against matrix metalloproteinase-2 (MMP-2).
abstract: Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (Ki(app) = 0.68 pm), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC50 > 1 μm). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t&frac12; = 30 min), APP-IP-TIMP-2 (t&frac12; ≫ 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.
authors: Department of Genome System Science, Graduate School of Nanobioscience, Yokohama City University, 641-12, Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan. shigashi@yokohama-cu.ac.jp
authors: Shouichi
authors: S
authors: Higashi
authors: Tomokazu
authors: T
authors: Hirose
authors: Tomoka
authors: T
authors: Takeuchi
authors: Kaoru
authors: K
authors: Miyazaki
mesh_terms: D000682:Amyloid / Q000737:chemistry; D001665:Binding Sites; D045744:Cell Line, Tumor; D024141:Collagen Type IV / Q000378:metabolism; D003208:Concanavalin A / Q000494:pharmacology; D015195:Drug Design; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D020128:Inhibitory Concentration 50; D007700:Kinetics; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D013997:Time Factors
chemical_list: D000682:Amyloid; D024141:Collagen Type IV; D004791:Enzyme Inhibitors; D061965:Matrix Metalloproteinase Inhibitors; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003208:Concanavalin A; D020778:Matrix Metalloproteinase 2; D016650:Fluorescein-5-isothiocyanate
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