title: Engineered protease inhibitors based on sunflower trypsin inhibitor-1 (SFTI-1) provide insights into the role of sequence and conformation in Laskowski mechanism inhibition.
abstract: Laskowski inhibitors regulate serine proteases by an intriguing mode of action that involves deceiving the protease into synthesizing a peptide bond. Studies exploring naturally occurring Laskowski inhibitors have uncovered several structural features that convey the inhibitor's resistance to hydrolysis and exceptional binding affinity. However, in the context of Laskowski inhibitor engineering, the way that various modifications intended to fine-tune an inhibitor's potency and selectivity impact on its association and dissociation rates remains unclear. This information is important as Laskowski inhibitors are becoming increasingly used as design templates to develop new protease inhibitors for pharmaceutical applications. In this study, we used the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1), as a model system to explore how the inhibitor's sequence and structure relate to its binding kinetics and function. Using enzyme assays, MD simulations and NMR spectroscopy to study SFTI variants with diverse sequence and backbone modifications, we show that the geometry of the binding loop mainly influences the inhibitor's potency by modulating the association rate, such that variants lacking a favourable conformation show dramatic losses in activity. Additionally, we show that the inhibitor's sequence (including both the binding loop and its scaffolding) influences its potency and selectivity by modulating both the association and the dissociation rates. These findings provide new insights into protease inhibitor function and design that we apply by engineering novel inhibitors for classical serine proteases, trypsin and chymotrypsin and two kallikrein-related peptidases (KLK5 and KLK14) that are implicated in various cancers and skin diseases.
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059, Australia.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: de Veer
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Joakim E
authors.initials: JE
authors.lastname: Swedberg
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: Muharrem
authors.initials: M
authors.lastname: Akcan
authors.affiliation: School of Biomedical Sciences, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: K Johan
authors.initials: KJ
authors.lastname: Rosengren
authors.affiliation: Department of Medical Biosciences, Umeå University, 901 87 Umeå, Sweden.
authors.forename: Maria
authors.initials: M
authors.lastname: Brattsand
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Craik
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD 4059, Australia j2.harris@qut.edu.au.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Harris
mesh_terms: D006801:Humans; D007610:Kallikreins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D010456:Peptides, Cyclic; D010940:Plant Proteins; D011994:Recombinant Proteins; C448501:SFTI-1 peptide, sunflower; C425336:KLK14 protein, human; C400147:KLK5 protein, human; D007610:Kallikreins
keywords: Laskowski mechanism; cyclic peptides; drug design; kallikrein-related peptidase; protease inhibitors