Screening phage peptide library identifies peptide which disrupts MMP-2 gelatinolysis

Proteolysis by matrix metalloproteinases (MMPs) requires substrate binding via specific exodomains such as the collagen binding domain (CBD) in MMP-2. Objectives: We here tested our working hypothesis that disrupting CBD-mediated substrate interactions with binding site specific peptides can inhibit the catalytic activity of MMP-2. Methods: To identify CBD binding motifs, we screened a fUSE5/15mer phage displayed peptide library containing 2 x 10⁸ primary clones using recombinant CBD as bait and tested synthetic peptides corresponding to isolated phage sequences in MMP-2 activity assays. Results: The numbers of CBD-bound phages were ~8 x 10^-5 %, 0.02 %, and 0.06 % of input after the 1st, 2nd, and 3rd rounds of selection. Input phage and phage clones isolated in the 1st round screening had 100% random amino acid sequences. However, 50% and 100% of the clones isolated and sequenced (16) in the 2nd and 3rd rounds of selections, respectively, revealed one identical sequence. Phage capture assays with CBD testing 33 of the 3rd round clones demonstrated binding by 3.9 % of the total input phages. This was 44 fold higher than the binding of phages from 8 random control clones and showed that the selected phage clones bound CBD stronger than random phage. Importantly, binding of selected phage to CBD was competed by gelatin, a key CBD ligand, and gelatin released eluted phage bound to immobilized CBD. This indicated that the phage peptide and gelatin bound the same site(s) on CBD. A synthetic peptide designed from the isolated phage sequence inhibited MMP-2 cleavage of gelatin but not a collagen-like peptide. Conclusions: The results indicate that we have identified a peptide which inhibits MMP-2 cleavage of gelatin by disrupting CBD-substrate interactions independently of the catalytic site (Supported by NIH grants DE14236 and DE016312, and San Antonio Area Foundation).