COMBINATORIAL ENGINEERING OF PROTEOLYTICALLY RESISTANT APPI VARIANTS THAT INHIBIT HUMAN MESOTRYPSIN FOR CANCER THERAPY

Among the human serine proteases involved in pathological conditions, mesotrypsin is considered an unusual, distinct, enigmatic and perhaps the most challenging form in terms of elucidating its mechanism of action. Although its specific pathological roles are yet to be revealed, the dysregulation and overexpression of mesotrypsin correlate with poor prognosis in many human tumors and pancreatitis, thereby making it an attractive target for therapeutic intervention. Nevertheless, developing selective inhibitors for mesotrypsin presents special challenges, as mesotrypsin shares high sequence homology and structural similarity with various trypsins, and is resistant to inhibition by many polypeptide inhibitors. Among these inhibitors, the amyloid β-protein precursor inhibitor domain (APPI), is unique by its high affinity to mesotrypsin, but is cleaved very rapidly.

Herein, we used directed evolution to generate novel APPI mutants with improved affinity, specificity and high proteolytic resistance to mesotrypsin. For that, we developed unique strategy that involved high-throughput screening of an all-gene random mutagenesis APPI library against mesotrypsin with evolutionary stimulus that allowed the rapid isolation of a triple APPI mutant (APPI_3M). Remarkably, this mutant was shown to have 1459-fold higher affinity, up to 350000-fold higher specificity and 83-fold higher proteolytic stability vis-à-vis APPI_WT. Herein, we demonstrate that APPI_3M acts as a functional inhibitor against mesotrypsin in cell-based assays, for which high binding affinities and stabilities are crucial. In addition, by solving the crystal structure of the APPI_3M/mesotrypsin complex, we were able to draw insights for the newly formed interactions and how these interactions contribute to the increased binding and resistance towards mesotrypsin. Our study thus establishes mesotrypsin as a target for cancer therapy and justifies further testing APPI_3M in additional pre-clinical studies.