title: Synthesis and characterization of fluorescent antagonists and agonists for human oxytocin and vasopressin V(1)(a) receptors.
abstract: The fluoresceinyl (Flu) group has been linked by an amide bond to the side chain amino group at position 8 of (a) two oxytocin (OT) antagonists, to give d(CH(2))(5)[Tyr(Me)(2),Thr(4),Orn(8)(5/6C-Flu),Tyr-NH(2)(9)]VT (Orn(8)(5/6C-Flu)OTA) (1) and desGly-NH(2),d(CH(2))(5)[D- Tyr(2),Thr(4),Orn(8)(5/6C-Flu)]VT (2), and (b) eight Lys(8) and Orn(8) analogues of potent OT agonists, to give d[Lys(8)(5/6C-Flu)]VT (3), d[Thr(4),Lys(8)(5/6C-Flu)]VT (4), [HO(1)][Lys(8)(5/6C-Flu)]VT (5), [HO(1)][Thr(4),Lys(8)(5/6C-Flu)]VT (6), d[Orn(8)(5/6C-Flu)]VT (7), d[Thr(4),Orn(8)(5/6C-Flu)]VT (8), [HO(1)][Orn(8)(5/6C-Flu)]VT (9), and [HO(1)][Thr(4),Orn(8)(5/6C-Flu)]VT (10). The tetramethylrhodamyl (Rhm) group was attached to the precursor peptide of 9 to give [HO(1)][Orn(8)(5/6C-Rhm)]VT (11). All 11 fluorescent peptides were evaluated in human OT and vasopressin V(1a) (vasoconstrictor), V(1b) (pituitary), and V(2) (antidiuretic) receptor binding and functional assays. With K(d) = 6.24, 217, >10000, and >10000 nM for the OT, V(1a), V(1b), and V(2) receptors, peptide 1 is a potent and selective fluorescent OT antagonist and may be useful for specifically labeling OT receptors while peptide 2 exhibits low affinities for all the receptors. The fluorescent peptides 3-10 are all very potent agonists for the human OT receptor. They exhibit the following K(d) values (nM) for the human OT, V(1a), V(1b), and V(2) receptors, respectively: (3) 0.29, 57, 124, >10000; (4) 1.8, 25.5, 150, >10000; (5) 0.34, 13.7, 66, nd (not determined); (6) 0.32, 17.3, 53, >10000; (7) 0.25, 107, 393, >10000; (8) 0.40, 30, 282, >10000; (9) 0.18, 12.2, 126, nd; (10) 0.17, 11.8, 87, >1000; (11) 0.092, 7.36, nd, nd. Peptide 7 exhibits both a high affinity and a high selectivity for human OT receptors. Peptides 7 and 11 were utilized to study the internalization of the OT receptor-ligand complex. Preliminary studies indicate that this process is similar to that observed for the vasopressin V(1a) receptor and differs from that observed for vasopressin V(2) receptors. Some or all of the fluorescent OT antagonists and agonists reported here are very promising new fluorescent ligands for labeling cells which express the human OT receptor and are also useful tools to follow endocytosis of the receptor-ligand complex.
authors.affiliation: INSERM U 469, 141 rue de la Cardonille, 34094 Montpellier CEDEX 5, France.
authors.forename: Sonia
authors.initials: S
authors.lastname: Terrillon
authors.forename: Ling Ling
authors.initials: LL
authors.lastname: Cheng
authors.forename: Stoytcho
authors.initials: S
authors.lastname: Stoev
authors.forename: Bernard
authors.initials: B
authors.lastname: Mouillac
authors.forename: Claude
authors.initials: C
authors.lastname: Barberis
authors.forename: Maurice
authors.initials: M
authors.lastname: Manning
authors.forename: Thierry
authors.initials: T
authors.lastname: Durroux
mesh_terms: D000818:Animals; D065092:Antidiuretic Hormone Receptor Antagonists; D001667:Binding, Competitive; D016466:CHO Cells; D006224:Cricetinae; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D008024:Ligands; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D011869:Radioligand Assay; D018045:Receptors, Oxytocin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000187:drug effects*; D017483:Receptors, Vasopressin / Q000819:agonists / Q000187:drug effects*; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship
chemical_list: D065092:Antidiuretic Hormone Receptor Antagonists; D005456:Fluorescent Dyes; D007295:Inositol Phosphates; D008024:Ligands; D009842:Oligopeptides; D018045:Receptors, Oxytocin; D017483:Receptors, Vasopressin