title: Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).
abstract: Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 μM. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.
authors.affiliation: Department of Basic Science, Charles E. Schmidt College of Medicine, Florida Atlantic University, Boca Raton, Florida 33431, USA.
authors.forename: Harinath
authors.initials: H
authors.lastname: Bahudhanapati
authors.forename: Yingnan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
authors.forename: Keith
authors.initials: K
authors.lastname: Brew
mesh_terms: D019943:Amino Acid Substitution; D017364:Collagenases; D014644:Genetic Variation; D006801:Humans; D061965:Matrix Metalloproteinase Inhibitors; D045726:Metalloproteases / Q000037:antagonists & inhibitors; D019151:Peptide Library; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism*
chemical_list: D061965:Matrix Metalloproteinase Inhibitors; D019151:Peptide Library; C560823:TIMP2 protein, human; D019716:Tissue Inhibitor of Metalloproteinase-2; D045726:Metalloproteases; D017364:Collagenases; C417249:collagenase 1