title: Development of High Affinity and High Specificity Inhibitors of Matrix Metalloproteinase 14 through Computational Design and Directed Evolution.
abstract: Degradation of the extracellular matrices in the human body is controlled by matrix metalloproteinases (MMPs), a family of more than 20 homologous enzymes. Imbalance in MMP activity can result in many diseases, such as arthritis, cardiovascular diseases, neurological disorders, fibrosis, and cancers. Thus, MMPs present attractive targets for drug design and have been a focus for inhibitor design for as long as 3 decades. Yet, to date, all MMP inhibitors have failed in clinical trials because of their broad activity against numerous MMP family members and the serious side effects of the proposed treatment. In this study, we integrated a computational method and a yeast surface display technique to obtain highly specific inhibitors of MMP-14 by modifying the natural non-specific broad MMP inhibitor protein N-TIMP2 to interact optimally with MMP-14. We identified an N-TIMP2 mutant, with five mutations in its interface, that has an MMP-14 inhibition constant (Ki) of 0.9 pm, the strongest MMP-14 inhibitor reported so far. Compared with wild-type N-TIMP2, this variant displays ∼900-fold improved affinity toward MMP-14 and up to 16,000-fold greater specificity toward MMP-14 relative to other MMPs. In anin vitroand cell-based model of MMP-dependent breast cancer cellular invasiveness, this N-TIMP2 mutant acted as a functional inhibitor. Thus, our study demonstrates the enormous potential of a combined computational/directed evolution approach to protein engineering. Furthermore, it offers fundamental clues into the molecular basis of MMP regulation by N-TIMP2 and identifies a promising MMP-14 inhibitor as a starting point for the development of protein-based anticancer therapeutics.
authors.affiliation: Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel.
authors.forename: Valeria
authors.initials: V
authors.lastname: Arkadash
authors.affiliation: Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel.
authors.forename: Gal
authors.initials: G
authors.lastname: Yosef
authors.affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
authors.forename: Jason
authors.initials: J
authors.lastname: Shirian
authors.affiliation: Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel.
authors.forename: Itay
authors.initials: I
authors.lastname: Cohen
authors.affiliation: Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel.
authors.forename: Yuval
authors.initials: Y
authors.lastname: Horev
authors.affiliation: Department of Biological Regulation, Weizmann Institute of Science, 76100 Rehovot, Israel.
authors.forename: Moran
authors.initials: M
authors.lastname: Grossman
authors.affiliation: Department of Biological Regulation, Weizmann Institute of Science, 76100 Rehovot, Israel.
authors.forename: Irit
authors.initials: I
authors.lastname: Sagi
authors.affiliation: Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Jacksonville, Florida 32224.
authors.forename: Evette S
authors.initials: ES
authors.lastname: Radisky
authors.affiliation: Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Electronic address: jshifman@mail.huji.ac.il.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Shifman
authors.affiliation: Department of Biotechnology Engineering and the National Institute of Biotechnology in the Negev, Ben-Gurion University of the Negev, 84105 Beer-Sheva, Israel. Electronic address: papo@bgu.ac.il.
authors.forename: Niv
authors.initials: N
authors.lastname: Papo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution; D015195:Drug Design; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000737:chemistry / Q000378:metabolism*; D061965:Matrix Metalloproteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D062105:Molecular Docking Simulation; D009154:Mutation; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D019716:Tissue Inhibitor of Metalloproteinase-2; D053511:Matrix Metalloproteinase 14
keywords: binding affinity; computational protein design; directed evolution; matrix metalloproteinases; metastasis; protease inhibitor; protein-protein interaction; proteolysis; yeast surface display