title: Improving the Selectivity of Engineered Protease Inhibitors: Optimizing the P2 Prime Residue Using a Versatile Cyclic Peptide Library.
abstract: Standard mechanism inhibitors are attractive design templates for engineering reversible serine protease inhibitors. When optimizing interactions between the inhibitor and target protease, many studies focus on the nonprimed segment of the inhibitor's binding loop (encompassing the contact β-strand). However, there are currently few methods for screening residues on the primed segment. Here, we designed a synthetic inhibitor library (based on sunflower trypsin inhibitor-1) for characterizing the P2' specificity of various serine proteases. Screening the library against 13 different proteases revealed unique P2' preferences for trypsin, chymotrypsin, matriptase, plasmin, thrombin, four kallikrein-related peptidases, and several clotting factors. Using this information to modify existing engineered inhibitors yielded new variants that showed considerably improved selectivity, reaching up to 7000-fold selectivity over certain off-target proteases. Our study demonstrates the importance of the P2' residue in standard mechanism inhibition and unveils a new approach for screening P2' substitutions that will benefit future inhibitor engineering studies.
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology , Brisbane, Queensland QLD 4059, Australia.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: de Veer
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , 306 Carmody Road, Building 80, Queensland Bioscience Presinct, Brisbane, Queensland, QLD 4072, Australia.
authors.forename: Conan K
authors.initials: CK
authors.lastname: Wang
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology , Brisbane, Queensland QLD 4059, Australia.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Harris
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , 306 Carmody Road, Building 80, Queensland Bioscience Presinct, Brisbane, Queensland, QLD 4072, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland , 306 Carmody Road, Building 80, Queensland Bioscience Presinct, Brisbane, Queensland, QLD 4072, Australia.
authors.forename: Joakim E
authors.initials: JE
authors.lastname: Swedberg
mesh_terms: D000595:Amino Acid Sequence; D001777:Blood Coagulation / Q000187:drug effects; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D005342:Fibrinolysis / Q000187:drug effects; D007610:Kallikreins / Q000037:antagonists & inhibitors / Q000737:chemistry; D008958:Models, Molecular; D010456:Peptides, Cyclic / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D015202:Protein Engineering; D015842:Serine Proteinase Inhibitors / Q000138:chemical synthesis* / Q000494:pharmacology*
chemical_list: D010456:Peptides, Cyclic; C448501:SFTI-1 peptide, sunflower; D015842:Serine Proteinase Inhibitors; D007610:Kallikreins