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Combinatorial Protein Engineering of Proteolytically Resistant Mesotrypsin Inhibitors for Cancer Therapy



Pharmaceutical protease inhibitors targeting the HIV or HCV viruses were already approved and their market is significantly expanding. The same rationale in cancer is more challenging as it requires protease inhibition which is solely tumor specific. Such a therapeutic target enzyme is the human Mesotrypsin, a novel serine protease associated with the metastatic progression of many cancers including; Prostate, Breast, Pancreatic and Melanoma but has no activity in naïve healthy cells. 


The Technology


We used our powerful yeast display platform for directed evolution, employing a novel multi-modal library screening strategy to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity, and improved selectivity of reversible blocking of the human Mesotrypsin.   We identified a triple mutant with a mesotrypsin inhibition constant (Ki) of 89 pM, as the strongest mesotrypsin inhibitor yet reported; this variant displays 1459-fold improved affinity, up to 350,000-fold greater specificity, and 83-fold improved proteolytic stability vs wild-type APPI. We demonstrated that this mutant acts as a functional inhibitor in cell-based models of mesotrypsin-dependent prostate cancer cellular invasiveness. Finally, initial preclinical evaluation of the lead candidate was highly encouraging both as a targeting agent for tumor imaging and as an anti-cancer therapeutic. 




  • Silencing of Mesotrypsin expression was shown to inhibit cancer growth, invasion and metastasis both in-vitro and invivo, implicating this is a putative therapeutic target
  • Unlike past inhibitor the current one is proteolytically resistant, highly specific in pico molar concentrations only to Mesotrypsin and not to any other variants.
  • We solved the crystal structure of the APPI triple mutant/mesotrypsin complex and obtained new insights into the structural and mechanistic basis for improved binding and proteolytic resistance.
  • An in-vivo proof of concept in mice was already demonstrated.


Patent Status


International Patent Application No. PCT/IL2016/051318


Principal Investigator


Prof. Niv Papo, NIBN and the Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Israel

Collaborator: Professor Evette S. Radisky, Department of Cancer Biology, Mayo Clinic Cancer Center, Jacksonville, USA