PAPO LAB
SERINE PROTEASE INHIBITORS
Engineered protein therapeutics offer advantages, including strong target affinity, selectivity and low toxicity, but like natural proteins can be susceptible to proteolytic degradation, thereby limiting their effectiveness. A compelling therapeutic target is mesotrypsin, a protease up-regulated with tumour progression, associated with poor prognosis, and implicated in tumour growth and progression of many cancers. However, with its unique capability for cleavage and inactivation of proteinaceous inhibitors, mesotrypsin presents a formidable challenge to the development of biological inhibitors. We are using a powerful yeast display platform for directed evolution, employing novel multi-modal library screening strategies, to engineer the human amyloid precursor protein Kunitz protease inhibitor domain (APPI) simultaneously for increased proteolytic stability, stronger binding affinity and improved selectivity for mesotrypsin inhibition.
Molecular dynamics simulations reveal suppressed dynamics in Kunitz domains with engineered disulfide bonds. (A) Global dynamics of unbound APPIM17G/I18F/F34V and APPIM17C/I18F/F34C, compared according to backbone atom (NCαCO) average RMSD from MD simulations, show only modest differences in amplitude. (B) Magnitudes of positional fluctuations per residue of APPI variants over the length of the MD simulation are plotted as Cα average RMSF. (C) Positional RMSF amplitudes from (B) are heat-mapped onto the protein structures in the blue-white-red spectrum (scale bar shown). Note the peak of elevated mobility spanning binding loop residues 15-18 of APPIM17G/I18F/F34V that is absent in APPIM17C/I18F/F34C. (D) Global dynamics of unbound KD1TFPI1K15R vs KD1TFPI1K15R/I17C/I34C, compared according to backbone atom (NCαCO) average RMSD from MD simulations, show lower amplitudes of backbone atom deviation from average positions in the engineered disulfide variant. (E) Magnitudes of positional fluctuations per residue of KD1TFPI variants over the length of the MD simulation are plotted as Cα average RMSF. (F) Positional RMSF amplitudes from (E) are heat-mapped onto the protein structures in the blue-white-red spectrum (scale bar shown). Note that several peaks of elevated mobility in KD1TFPI1K15R, including binding loop residues 16-19, are reduced in KD1TFPI1K15R/I17C/I34C.
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