title: Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.
abstract: Melanocortin 4 (MC4) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC4receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC4receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ1/2=160min) and UTBC102 (τ1/2=7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ1/2=224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC4receptor specific ligands and could complement each other in these studies.
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14a, 50411, Tartu, Estonia.
authors.forename: Reet
authors.initials: R
authors.lastname: Link
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14a, 50411, Tartu, Estonia.
authors.forename: Santa
authors.initials: S
authors.lastname: Veiksina
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14a, 50411, Tartu, Estonia.
authors.forename: Ago
authors.initials: A
authors.lastname: Rinken
authors.affiliation: Institute of Chemistry, University of Tartu, Ravila 14a, 50411, Tartu, Estonia. Electronic address: sergei.kopanchuk@ut.ee.
authors.forename: Sergei
authors.initials: S
authors.lastname: Kopanchuk
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D015195:Drug Design; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008024:Ligands; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D044105:Receptor, Melanocortin, Type 4 / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D005456:Fluorescent Dyes; D008024:Ligands; D009842:Oligopeptides; D044105:Receptor, Melanocortin, Type 4
keywords: Budded baculoviruses; Fluorescence anisotropy; Fluorescent ligands; Melanocortin 4 receptors; UTBC101; UTBC102