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@ARTICLE{Cohen:306286,
      author       = {I. Cohen and S. Naftaly and E. Ben-Zeev and A. Hockla and
                      E. S. Radisky and N. Papo},
      title        = {{P}re-equilibrium competitive library screening for tuning
                      inhibitor association rate and specificity toward serine
                      proteases.},
      journal      = {Biochemical journal},
      volume       = {475},
      number       = {7},
      issn         = {0006-2936},
      address      = {London},
      publisher    = {Portland Press},
      reportid     = {DKFZ-2025-02512},
      pages        = {1335 - 1352},
      year         = {2018},
      note         = {#DKFZ-MOST-GR-2495#},
      abstract     = {High structural and sequence similarity within protein
                      families can pose significant challenges to the development
                      of selective inhibitors, especially toward proteolytic
                      enzymes. Such enzymes usually belong to large families of
                      closely similar proteases and may also hydrolyze, with
                      different rates, protein- or peptide-based inhibitors. To
                      address this challenge, we employed a combinatorial yeast
                      surface display library approach complemented with a novel
                      pre-equilibrium, competitive screening strategy for facile
                      assessment of the effects of multiple mutations on inhibitor
                      association rates and binding specificity. As a proof of
                      principle for this combined approach, we utilized this
                      strategy to alter inhibitor/protease association rates and
                      to tailor the selectivity of the amyloid β-protein
                      precursor Kunitz protease inhibitor domain (APPI) for
                      inhibition of the oncogenic protease mesotrypsin, in the
                      presence of three competing serine proteases, anionic
                      trypsin, cationic trypsin and kallikrein-6. We generated a
                      variant, designated APPIP13W/M17G/I18F/F34V, with up to
                      30-fold greater specificity relative to the parental
                      APPIM17G/I18F/F34V protein, and 6500- to 230 000-fold
                      improved specificity relative to the wild-type APPI protein
                      in the presence of the other proteases tested. A series of
                      molecular docking simulations suggested a mechanism of
                      interaction that supported the biochemical results. These
                      simulations predicted that the selectivity and specificity
                      are affected by the interaction of the mutated APPI residues
                      with nonconserved enzyme residues located in or near the
                      binding site. Our strategy will facilitate a better
                      understanding of the binding landscape of multispecific
                      proteins and will pave the way for design of new drugs and
                      diagnostic tools targeting proteases and other proteins.},
      keywords     = {Amyloid beta-Protein Precursor: chemistry / Amyloid
                      beta-Protein Precursor: genetics / Amyloid beta-Protein
                      Precursor: metabolism / Binding, Competitive / Humans /
                      Models, Molecular / Molecular Docking Simulation / Peptide
                      Library / Protease Inhibitors: chemistry / Protease
                      Inhibitors: metabolism / Substrate Specificity / Trypsin:
                      genetics / Trypsin: metabolism / directed evolution (Other)
                      / protease inhibitor (Other) / protein engineering (Other) /
                      protein–protein interactions (PPIs) (Other) / serine
                      proteases (Other) / APP protein, human (NLM Chemicals) /
                      Amyloid beta-Protein Precursor (NLM Chemicals) / Peptide
                      Library (NLM Chemicals) / Protease Inhibitors (NLM
                      Chemicals) / Trypsin (NLM Chemicals)},
      ddc          = {540},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:29535275},
      pmc          = {pmc:PMC5929103},
      doi          = {10.1042/BCJ20180070},
      url          = {https://inrepo02.dkfz.de/record/306286},
}