Subject(s)
Caudate Nucleus/metabolism , Dioxanes/metabolism , Indoles/metabolism , Piperidines/metabolism , Receptors, Serotonin/metabolism , Animals , Cattle , Cell Membrane , Humans , Indoles/pharmacology , Iodine Radioisotopes , Radioligand Assay , Receptors, Serotonin, 5-HT4 , Serotonin Antagonists/metabolism , Serotonin Receptor Agonists/metabolism , Serotonin Receptor Agonists/pharmacologyABSTRACT
To identify determinants that form nonapeptide hormone binding domains of the white sucker Catostomus commersoni [Arg8]vasotocin receptor, chimeric constructs encoding parts of the vasotocin receptor and parts of the isotocin receptor have been analyzed by [(3,5-3H)Tyr2, Arg8]vasotocin binding to membranes of human embryonic kidney cells previously transfected with the different cDNA constructs and by functional expression studies in Xenopus laevis oocytes injected with mutant cRNAs. The results indicate that the N terminus and a region spanning the second extracellular loop and its flanking transmembrane segments, which contains a number of amino acid residues that are conserved throughout the nonapeptide receptor family, contribute to the affinity of the receptor for its ligand. Nonapeptide selectivity, however, is mainly defined by transmembrane region VI and the third extracellular loop. These results are complemented by a molecular model of the vasotocin receptor obtained by aligning its sequence with those of other G-protein coupled receptors as well as that of bacteriorhodopsin. The model indicates that amino acid residues of transmembrane regions II-VII that are located close to the extracellular surface also contribute to the binding of vasotocin.
Subject(s)
Protein Structure, Secondary , Receptors, Vasopressin/chemistry , Receptors, Vasopressin/metabolism , Vasotocin/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , Cell Membrane/metabolism , DNA Mutational Analysis , Female , Fishes , Humans , Kidney , Kinetics , Models, Molecular , Models, Structural , Molecular Sequence Data , Mutagenesis, Site-Directed , Oocytes/physiology , Polymerase Chain Reaction , Rats , Receptors, Vasopressin/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Transfection , Tritium/metabolism , Xenopus laevisABSTRACT
The design and synthesis of a new class of potent and selective 5-HT4 receptor agonists containing an indole nucleus linked to a carbazimidamide are presented. A conformational study of the 5-HT4 receptor agonists serotonin and zacopride led to the identification of an initial pharmacophore and to the definition of a three-dimensional map of the 5-HT4 agonist recognition site. 1, a representative member of our new class of 5-HT4 receptor agonists, incorporates all reference structural features and matched perfectly with these models. 1 is a highly potent, full agonist at 5-HT4 receptors present in the isolated electrically stimulated guinea pig ileum preparation, with a pD2 value of 8.8, displaying selectivity (ranging from 40- to over 10,000-fold) versus other members of the serotonin receptor family.
Subject(s)
Receptors, Serotonin/metabolism , Serotonin Receptor Agonists/chemical synthesis , Animals , Binding Sites , Drug Design , Guinea Pigs , Ileum/drug effects , In Vitro Techniques , Molecular Conformation , Serotonin Receptor Agonists/metabolism , Serotonin Receptor Agonists/pharmacologyABSTRACT
A number of substituted indole carbazimidamides were prepared and evaluated as 5-HT4 receptor agonists by using the isolated field-stimulated guinea pig ileum preparation. Their selectivity for the 5-HT4 receptor was established by examining their affinity for other 5-HT receptors using radioligand-binding techniques. Several selective and highly potent full as well as partial agonists emerged from this study. For example, 1b,d were found to be the most potent, full 5-HT4 receptor agonist described so far (EC50 = 0.5 and 0.8 nM, respectively), being 6 and 4 times more potent than serotonin itself. On the other hand, 5b and 1h appeared as partial 5-HT4 receptor agonists in the nonstimulated guinea pig ileum preparation with potencies, evaluated against serotonin action, respectively similar (5b, Ki = 12 nM) to and 300-fold higher (1h, Ki = 0.04 nM) than serotonin.
Subject(s)
Amides/chemistry , Amides/pharmacology , Indoles/chemistry , Indoles/pharmacology , Serotonin Receptor Agonists/pharmacology , Animals , Guinea Pigs , Ileum/drug effects , In Vitro Techniques , Spectrum Analysis , Structure-Activity RelationshipABSTRACT
Human somatostatin receptor subtypes (SSTR1-5) bind their natural ligands SRIF-14 and SRIF-28 with high affinity. By contrast, short synthetic SRIF analogues such as SMS 201-995, a peptide agonist used for the treatment of various endocrine and malignant disorders, display sub-nanomolar affinity only for the receptor subtype SSTR2. To understand the molecular nature of selective peptide agonist binding to somatostatin receptors we have now, by site-directed mutagenesis, identified amino acids mediating SMS 201-995 specificity for SSTR2. Sequentially, amino acids in SSTR1, a receptor subtype exhibiting low affinity for SMS 201-995, were exchanged for the corresponding SSTR2 residues. After three consecutive steps, in which eight amino acids were exchanged, a SSTR1 mutant receptor with high affinity for SMS 201-995 was obtained. Receptor mutants with different combinations of these eight amino acids were then constructed. A single Ser305 to Phe mutation in TM VII increased the affinity of SSTR1 for SMS 201-995 nearly 100-fold. When this mutation was combined with an exchange of Gln291 to Asn in TM VI, almost full susceptibility to SMS 201-995 was obtained. Thus, it is concluded that the specificity of SMS 201-995 for SSTR2 is mainly defined by these two amino acids in transmembrane domains VI and VII. Using the conjugate gradient method we have, by analogy to the well established structure of bacteriorhodopsin, built a model for SRIF receptor-ligand interactions that explains the importance of Gln291 and Ser305 for the selectivity of agonists.
Subject(s)
Octreotide/pharmacology , Receptors, Somatostatin/drug effects , Amino Acid Sequence , Bacteriorhodopsins/chemistry , Base Sequence , Humans , In Vitro Techniques , Membrane Proteins/chemistry , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligonucleotide Probes , Protein Structure, Tertiary , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
The enantiomers of beta,gamma-dimethyl- and beta-methyl-alpha-methylene-gamma-butyrolactones have been synthesized stereospecifically from glutamic acid and beta-hydroxy isobutyric acid, respectively. Guinea pigs have been sensitized (Freund complete adjuvant technique) and tested to them. Both enantiomers of beta-methyl lactone as well as (+)-beta,gamma-dimethyl lactone induced enantiospecific allergic contact dermatitis (ACD); in turn, (-)-beta,gamma-dimethyl lactone showed no specificity. An interpretation is proposed.
Subject(s)
4-Butyrolactone/immunology , Allergens , Dermatitis, Contact/etiology , Furans/immunology , Animals , Female , Guinea Pigs , Skin Tests , Stereoisomerism , Structure-Activity RelationshipABSTRACT
In order to develop new theories for desensitization, a potential skin sensitizer, 1, with two different haptenic ends, a pyrocatechol group and an alpha-methylene-gamma-butyrolactone moiety, separated by a straight nine-carbon-atom chain has been prepared and used to sensitize guinea pigs. A "monohapten" 8 containing an electrophylic alpha-methylene-gamma-butyrolactone, connected to a dimethoxybenzene group, has also been prepared. Both the "bihapten" 1 and the "monohapten" 8 were shown to be sensitizers. Bihapten 1 sensitized animals recognized only bihapten 1 (and not the lactone 8) while monohapten 8 sensitized guinea pigs reacted to both 8 and 1. Bihapten 1 treated animals were further sensitized to monohapten 8 and challenged to the latter: the skin intensity reaction was significantly lower than the test to 8 in 8-sensitized animals. Bihapten 1 seems therefore to "tolerize" against the alpha-methylene-gamma-butyrolactone end.
