title: Bicyclic peptide inhibitor of urokinase-type plasminogen activator: mode of action.
abstract: The development of protease inhibitors for pharmacological intervention has taken a new turn with the use of peptide-based inhibitors. Here, we report the rational design of bicyclic peptide inhibitors of the serine protease urokinase-type plasminogen activator (uPA), based on the established monocyclic peptide, upain-2. It was successfully converted to a bicyclic peptide, without loss of inhibitory properties. The aim was to produce a peptide cyclised by an amide bond with an additional stabilising across-the-ring covalent bond. We expected this bicyclic peptide to exhibit a lower entropic burden upon binding. Two bicyclic peptides were synthesised with affinities similar to that of upain-2, and their binding energetics were evaluated by isothermal titration calorimetry. Indeed, compared to upain-2, the bicyclic peptides showed reduced loss of entropy upon binding to uPA. We also investigated the solution structures of the bicyclic peptide by NMR spectroscopy to map possible conformations. An X-ray structure of the bicyclic-peptide-uPA complex confirmed an interaction similar to that for the previous upain-1/upain-2-uPA complexes. These physical studies of the peptide-protease interactions will aid future designs of bicyclic peptide protease inhibitors.
authors.affiliation: Danish-Chinese Centre for Proteases and Cancer, Aarhus University, 10C Gustav Wieds Vej, 8000 Aarhus C (Denmark); Department of Chemistry, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C (Denmark).
authors.forename: Renée
authors.initials: R
authors.lastname: Roodbeen
authors.forename: Berit
authors.initials: B
authors.lastname: Paaske
authors.forename: Longguang
authors.initials: L
authors.lastname: Jiang
authors.forename: Jan K
authors.initials: JK
authors.lastname: Jensen
authors.forename: Anni
authors.initials: A
authors.lastname: Christensen
authors.forename: Jakob T
authors.initials: JT
authors.lastname: Nielsen
authors.forename: Mingdong
authors.initials: M
authors.lastname: Huang
authors.forename: Frans A A
authors.initials: FA
authors.lastname: Mulder
authors.forename: Niels Chr
authors.initials: NC
authors.lastname: Nielsen
authors.forename: Peter A
authors.initials: PA
authors.lastname: Andreasen
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D015195:Drug Design; D006801:Humans; D007700:Kinetics; D010456:Peptides, Cyclic / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D010456:Peptides, Cyclic; D011480:Protease Inhibitors; C000592736:upain-2 peptide; D014568:Urokinase-Type Plasminogen Activator
keywords: NMR spectroscopy; X-ray structures; cancer; isothermal titration calorimetry; peptide-protease interactions