3p95

Publication Abstract from PubMed

PRSS3/mesotrypsin is an atypical isoform of trypsin, the upregulation 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, while protein trypsin inhibitors bind poorly and are rapidly degraded by mesotrypsin. Here, we use mutagenesis of a mesotrypsin substrate, the Kunitz domain of the amyloid precursor protein (APPI), and of a poor mesotrypsin inhibitor, bovine pancreatic trypsin inhibitor (BPTI), to dissect mesotrypsin specificity at the key P2' position. We find that bulky and charged residues strongly disfavor binding, while 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 pharmacologic inhibition of mesotrypsin in assays of breast cancer cell malignant growth and pancreatic cancer cell invasion. While further improvements in inhibitor selectivity will be important before clinical potential can be realized, our studies support the feasibility of engineering protein protease inhibitors of mesotrypsin and highlight their therapeutic potential.

The P2' residue is a key determinant of mesotrypsin specificity: Engineering a high affinity inhibitor with anticancer activity.,Salameh MA, Soares AS, Hockla A, Radisky DC, Radisky ES Biochem J. 2011 Aug 2. PMID:21806544^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.