The P2’ residue is a key determinant of mesotrypsin specificity: Engineering a high-affinity inhibitor with anticancer activity
PRSS3/mesotrypsin is an atypical isoform of trypsin, the up-regulation of which has been implicated in promoting tumor 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 P'{sub 2} position. We find that bulky and charged residues strongly disfavor 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 P{sub 1} and P'{sub 2} residues of BPTI, we generate a stable high-affinity mesotrypsin inhibitor with an equilibrium binding constant K{sub i} 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.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States)
- Sponsoring Organization:
- DOE-OFFICE OF BIOLOGICAL & ENVIRONMENTAL RESEARCH
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 1033586
- Report Number(s):
- BNL-96572-2011-JA; BIJOAK; R&D Project: BO-070; KP1605010; TRN: US201203%%17
- Journal Information:
- Biochemical Journal, Vol. 440, Issue 1; ISSN 0006-2936
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
60 APPLIED LIFE SCIENCES
AFFINITY
CATALYSIS
CATTLE
CRYSTAL STRUCTURE
ENZYMES
INHIBITION
MAMMARY GLANDS
MUTAGENESIS
NEOPLASMS
OPTIMIZATION
PRECURSOR
PROTEIN ENGINEERING
PROTEINS
RESIDUES
SPECIFICITY
TRYPSIN
crystal structure
drug discovery
endopeptidase
enzyme inhibition
mesotrypsin
protease inhibitor
protein engineering