The P 2 ' residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity
Abstract
PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P2' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P1 and P2' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant Ki of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of themore »
- Authors:
- Publication Date:
- Research Org.:
- Brookhaven National Lab. (BNL), Upton, NY (United States)
- Sponsoring Org.:
- USDOE SC OFFICE OF SCIENCE (SC)
- OSTI Identifier:
- 1069432
- Report Number(s):
- BNL-100004-2013-JA
Journal ID: ISSN 0264-6021
- DOE Contract Number:
- DE-AC02-98CH10886
- Resource Type:
- Journal Article
- Journal Name:
- Biochemical Journal
- Additional Journal Information:
- Journal Volume: 440; Journal Issue: 1; Journal ID: ISSN 0264-6021
- Publisher:
- Biochemical Society
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 42 ENGINEERING
Citation Formats
Salameh, Moh'd A., Soares, Alexei S., Hockla, Alexandra, Radisky, Derek C., and Radisky, Evette S. The P 2 ' residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity. United States: N. p., 2011.
Web. doi:10.1042/BJ20110788.
Salameh, Moh'd A., Soares, Alexei S., Hockla, Alexandra, Radisky, Derek C., & Radisky, Evette S. The P 2 ' residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity. United States. https://doi.org/10.1042/BJ20110788
Salameh, Moh'd A., Soares, Alexei S., Hockla, Alexandra, Radisky, Derek C., and Radisky, Evette S. 2011.
"The P 2 ' residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity". United States. https://doi.org/10.1042/BJ20110788.
@article{osti_1069432,
title = {The P 2 ' residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity},
author = {Salameh, Moh'd A. and Soares, Alexei S. and Hockla, Alexandra and Radisky, Derek C. and Radisky, Evette S.},
abstractNote = {PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumour progression. Mesotrypsin inhibitors could potentially provide valuable research tools and novel therapeutics, but small-molecule trypsin inhibitors have low affinity and little selectivity, whereas protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. In the present study, we use mutagenesis of a mesotrypsin substrate, APPI (amyloid precursor protein Kunitz protease inhibitor domain), and of a poor mesotrypsin inhibitor, BPTI (bovine pancreatic trypsin inhibitor), to dissect mesotrypsin specificity at the key P2' position. We find that bulky and charged residues strongly disfavour binding, whereas acidic residues facilitate catalysis. Crystal structures of mesotrypsin complexes with BPTI variants provide structural insights into mesotrypsin specificity and inhibition. Through optimization of the P1 and P2' residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant Ki of 5.9 nM, a >2000-fold improvement in affinity over native BPTI. Using this engineered inhibitor, we demonstrate the efficacy of pharmacological inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. Although further improvements in inhibitor selectivity will be important before clinical potential can be realized, the results of the present study support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.},
doi = {10.1042/BJ20110788},
url = {https://www.osti.gov/biblio/1069432},
journal = {Biochemical Journal},
issn = {0264-6021},
number = 1,
volume = 440,
place = {United States},
year = {Tue Nov 15 00:00:00 EST 2011},
month = {Tue Nov 15 00:00:00 EST 2011}
}