title: A cyclic peptidic serine protease inhibitor: increasing affinity by increasing peptide flexibility.
abstract: Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China.
authors.forename: Baoyu
authors.initials: B
authors.lastname: Zhao
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Peng
authors.initials: P
authors.lastname: Xu
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China.
authors.forename: Longguang
authors.initials: L
authors.lastname: Jiang
authors.affiliation: Nanoscience Center and Department of Chemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Berit
authors.initials: B
authors.lastname: Paaske
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Tobias
authors.initials: T
authors.lastname: Kromann-Hansen
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Hans Peter
authors.initials: HP
authors.lastname: Sørensen
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Zhuo
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Nanoscience Center and Department of Chemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Jakob T
authors.initials: JT
authors.lastname: Nielsen
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Anni
authors.initials: A
authors.lastname: Christensen
authors.affiliation: Department of Chemistry, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Masood
authors.initials: M
authors.lastname: Hosseini
authors.affiliation: Department of Chemistry, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Kasper K
authors.initials: KK
authors.lastname: Sørensen
authors.affiliation: Nanoscience Center and Department of Chemistry, University of Aarhus, Aarhus, Denmark.
authors.forename: Niels Christian
authors.initials: NC
authors.lastname: Nielsen
authors.affiliation: Department of Chemistry, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China.
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China; Danish-Chinese Centre for Proteases and Cancer, Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark.
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D018360:Crystallography, X-Ray; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry; D010456:Peptides, Cyclic / Q000737:chemistry / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D020349:Surface Plasmon Resonance
chemical_list: D050505:Mutant Proteins; D010456:Peptides, Cyclic; D015842:Serine Proteinase Inhibitors; C530075:mupain-1