title: Synthesis and evaluation of bivalent ligands for binding to the human melanocortin-4 receptor.
abstract: Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25±10Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH2 was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-c[Asp-His-2'-D-Nal-Arg-Trp-Lys]-NH2 and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)3 units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA.
authors.forename: Steve M
authors.initials: SM
authors.lastname: Fernandes
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA. Electronic address: yeon@email.arizona.edu.
authors.forename: Yeon Sun
authors.initials: YS
authors.lastname: Lee
authors.affiliation: H. Lee Moffitt Cancer Center and Research Institute, Department of Cancer Imaging and Metabolism, Tampa, FL 33612, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Gillies
authors.affiliation: Department of Chemistry and Biochemistry, University of Arizona, Tucson, AZ 85721, USA. Electronic address: hruby@email.arizona.edu.
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Hruby
mesh_terms: D000595:Amino Acid Sequence; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D028581:Lanthanoid Series Elements / Q000737:chemistry; D008024:Ligands; D009074:Melanocyte-Stimulating Hormones / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D044105:Receptor, Melanocortin, Type 4 / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D028581:Lanthanoid Series Elements; D008024:Ligands; C470072:MC4R protein, human; D009842:Oligopeptides; D044105:Receptor, Melanocortin, Type 4; C103272:SHU 9119; D009074:Melanocyte-Stimulating Hormones
keywords: Bivalent ligands; Eu-binding assay; Linkers; Melanocortin receptors; Solid phase synthesis