ABSTRACT
A series of small molecules derived from MK-0677, a potent synthetic GHS, mimicking the N-terminal Gly-Ser-O-(n-octanoyl)-L-Ser-Phe segment of ghrelin was synthesized and tested in a binding and in a functional assay measuring intracellular calcium elevation in HEK-293 cells expressing hGHSR1a. Replacement of Phe in this tetrapeptide with a spiro(indoline-3,4'-piperidine) group, Gly-Ser with 2-aminoisobutyric acid, and O-(n-octanoyl)-L-Ser with O-benzyl-D-Ser provided synthetic GHS agonists with similar functional potency as ghrelin.
Subject(s)
Calcium/metabolism , Indoles/metabolism , Peptide Hormones , Peptides/metabolism , Piperidines/metabolism , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Spiro Compounds/metabolism , Binding Sites/physiology , Cells, Cultured , Ghrelin , Humans , Indoles/chemistry , Inhibitory Concentration 50 , Luminescence , Molecular Mimicry , Peptides/chemistry , Piperidines/chemical synthesis , Protein Binding/physiology , Receptors, Ghrelin , Spiro Compounds/chemistryABSTRACT
The superfamily of seven-transmembrane-domain G-protein-coupled receptors (GPCRs) is the largest and most diverse group of transmembrane proteins involved in signal transduction. Each of the approximately 1000 family members found in vertebrates responds to stimuli as diverse as hormones, neurotransmitters, odorants and light, which selectively activate intracellular signaling events mediated by heterotrimeric G proteins. Because GPCRs are centrally positioned in the plasma membrane to initiate a cascade of cellular responses by diverse extracellular mediators, it is not surprising that modulation of GPCR function has been successful in the development of many marketed therapeutic agents. It has become clear that GPCRs for which a natural activating ligand has not yet been identified (orphan GPCRs) might provide a path to discovering new cellular substances that are important in human physiology. The process of 'de-orphanizing' these novel proteins has accelerated significantly and opened up new avenues for research in human physiology and pharmacology.
Subject(s)
Receptors, Odorant/pharmacology , Receptors, Odorant/physiology , Animals , Humans , Ligands , Receptors, Odorant/isolation & purification , Signal TransductionABSTRACT
N-Substituted nipecotic and iso-nipecotic amides of beta-methylTrpLys tert-butyl ester were found to be novel, selective and potent agonists of the somatostatin subtype-2 receptor in vitro. For example iso-nipecotic amide 8a showed high hsst2 binding affinity (Ki = 0.5 nM) and good selectivity (h5/h2 = 832).
Subject(s)
Nipecotic Acids/metabolism , Receptors, Somatostatin/agonists , Animals , Combinatorial Chemistry Techniques , Humans , Isomerism , Nipecotic Acids/chemical synthesis , Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Protein Binding , Receptors, Somatostatin/metabolism , Structure-Activity RelationshipABSTRACT
Quinazolinone derivatives were synthesized and evaluated as non-peptidic growth hormone secretagogues. Modeling guided design of quinazolinone compound 21 led to a potency enhancement of greater than 200-fold compared to human growth hormone secretagogue affinity of a screening lead 4.
Subject(s)
Drug Design , Human Growth Hormone/metabolism , Quinazolines/chemical synthesis , Quinazolines/pharmacology , Receptors, Cell Surface/agonists , Receptors, G-Protein-Coupled , Animals , Binding Sites , Humans , Inhibitory Concentration 50 , Kinetics , Models, Molecular , Quinazolines/chemistry , Quinazolines/metabolism , Rats , Receptors, Cell Surface/metabolism , Receptors, Ghrelin , Secretory Rate/drug effects , Structure-Activity RelationshipABSTRACT
Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.
Subject(s)
Fishes/genetics , Receptors, Cell Surface/chemistry , Receptors, G-Protein-Coupled , Amino Acid Sequence , Animals , Blotting, Southern , Cell Line , Cloning, Molecular , Conserved Sequence , Genomic Library , Humans , Ligands , Models, Genetic , Molecular Sequence Data , Protein Structure, Tertiary , Receptors, Cell Surface/genetics , Receptors, Ghrelin , Sequence Alignment , Sequence Homology, Amino Acid , TransfectionABSTRACT
A series of structurally diverse growth hormone (GH) releasing substances have been synthesized that are distinct from the naturally occurring GH releasing hormone (GHRH). These synthetic molecules range from the family of GH releasing peptides and mimetics such as MK-0677. The physiological importance of these molecules and their receptor is exemplified by studies in the elderly. For example, when MK-0677 was administered chronically to 70- to 90-year-old subjects, once daily, the age-related reduced amplitude of GH pulses was reversed to that of the physiological profile typical of young adults. In 1996, the synthesis of (35)S-MK-0677 was reported and used as a ligand to characterize a common receptor (GH secretagogue receptor [GHS-R]) for the GH releasing substances. The GHS-R is distinct from the GHRH receptor. Subsequently, the GHS-R gene was cloned and shown to encode a unique G-protein coupled receptor with a deduced protein sequence that was 96% identical in human and rat. Because of the physiological importance of the GHS-R, a search for family members (FMs) was initiated and its molecular evolution investigated. Three FMs GPR38, GPR39 and FM3 were isolated from human genomic libraries. To accelerate the identification of other FMs, a vertebrate organism with a compact genome distant in evolutionary terms from humans was exploited. The pufferfish (Spheroides nephelus) genome provides an ideal model for the discovery of human genes. Three distinct full-length clones encoding proteins of significant sequence identity to the human GHS-R were cloned from the pufferfish. Remarkably, the pufferfish gene with highest sequence homology to the human receptor was activated by the hexapeptide and non-peptide ligands. These intriguing results show that the structure and function of the ligand binding pocket of the human GHS-R has been highly conserved in evolution ( approximately 400 million years) and strongly suggests that an endogenous natural ligand has been conserved. This new information is consistent with a natural ligand for the GHS-R playing a fundamentally important and conserved role in physiology.
Subject(s)
Growth Hormone-Releasing Hormone , Receptors, Cell Surface/physiology , Receptors, G-Protein-Coupled , Aged , Aged, 80 and over , Amino Acid Sequence , Animals , Binding Sites , Human Growth Hormone/metabolism , Humans , Indoles/pharmacology , Molecular Sequence Data , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Receptors, Ghrelin , Receptors, Neuropeptide , Receptors, Pituitary Hormone-Regulating Hormone , Spiro Compounds/pharmacologyABSTRACT
Replacement of the phenyl in 3 with a 2-pyridyl or 4-thiazolyl group resulted in increased potency in the rat pituitary cell GH release assay and in beagles.
Subject(s)
Growth Hormone/chemical synthesis , Growth Hormone/metabolism , Thiazoles/chemical synthesis , Animals , Biological Availability , Dogs , Models, Chemical , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thiazoles/administration & dosage , Thiazoles/pharmacokinetics , Time FactorsABSTRACT
Motilin is a 22-amino acid peptide hormone expressed throughout the gastrointestinal (GI) tract of humans and other species. It affects gastric motility by stimulating interdigestive antrum and duodenal contractions. A heterotrimeric guanosine triphosphate-binding protein (G protein)-coupled receptor for motilin was isolated from human stomach, and its amino acid sequence was found to be 52 percent identical to the human receptor for growth hormone secretagogues. The macrolide antibiotic erythromycin also interacted with the cloned motilin receptor, providing a molecular basis for its effects on the human GI tract. The motilin receptor is expressed in enteric neurons of the human duodenum and colon. Development of motilin receptor agonists and antagonists may be useful in the treatment of multiple disorders of GI motility.
Subject(s)
Colon/metabolism , Gastric Mucosa/metabolism , Intestine, Small/metabolism , Motilin/metabolism , Receptors, Gastrointestinal Hormone/chemistry , Receptors, Gastrointestinal Hormone/genetics , Receptors, Neuropeptide/chemistry , Receptors, Neuropeptide/genetics , Alternative Splicing , Amino Acid Sequence , Base Sequence , Binding Sites , Calcium/metabolism , Cell Line , Chromosome Mapping , Chromosomes, Human, Pair 13 , Cloning, Molecular , Erythromycin/metabolism , GTP-Binding Proteins/metabolism , Humans , In Situ Hybridization , Ligands , Molecular Sequence Data , Motilin/analogs & derivatives , Receptors, Gastrointestinal Hormone/metabolism , Receptors, Neuropeptide/metabolism , Thyroid Gland/metabolism , TransfectionABSTRACT
The synthesis and biological activities of a series of spiroheterocyclic growth hormone secretagogues are reported. Modification of the spiroindane part-structure of the prototypal secretagogue L-162,752 revealed that the spiroindane could be replaced with spirobenzodihydrothiophen derivatives to enhance not only in vitro potency but also oral activity. In this study non-aromatic D-2-amino-4-cyclohexylbutanoic analogs (8a-8d) were also identified to be active secretagogues.
Subject(s)
Growth Hormone/chemical synthesis , Growth Hormone/metabolism , Indoles/chemical synthesis , Spiro Compounds/chemical synthesis , Animals , Dogs , Growth Hormone/pharmacology , Humans , Indoles/administration & dosage , Indoles/pharmacology , Kinetics , Models, Chemical , Rats , Spiro Compounds/administration & dosage , Spiro Compounds/pharmacologyABSTRACT
A series of carbohydroxamido-oxazolidine inhibitors of UDP-3-O-[R-3-hydroxymyristoyl]-GlcNAc deacetylase, the enzyme responsible for the second step in lipid A biosynthesis, was identified. The most potent analog L-161,240 showed an IC50 = 30 nM in the DEACET assay and displayed an MIC of 1-3 microg/mL against wild-type E. coli.
Subject(s)
Anti-Bacterial Agents/pharmacology , Hydroxamic Acids/pharmacology , Lipid A/biosynthesis , Oxazoles/pharmacology , Amidohydrolases/antagonists & inhibitors , Anti-Bacterial Agents/chemistry , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Escherichia coli/drug effects , Hydroxamic Acids/chemistry , Microbial Sensitivity Tests , Oxazoles/chemistryABSTRACT
Backbone cyclization of urea-based somatostatin agonists resulted in novel, orally bioavailable agonists. Binding assays confirmed that the resulting conformationally constrained cyclic ureas retained the potency of their acyclic counterparts. SAR studies subsequently led to highly potent analogs, selective for receptor subtype 2, and having good oral bioavailability.
Subject(s)
Somatostatin/agonists , Somatostatin/pharmacology , Urea/chemistry , Administration, Oral , Animals , Benzimidazoles , Biological Availability , Dogs , Indoles , Somatostatin/chemistry , Somatostatin/pharmacokinetics , Structure-Activity RelationshipABSTRACT
Nonpeptide agonists of each of the five somatostatin receptors were identified in combinatorial libraries constructed on the basis of molecular modeling of known peptide agonists. In vitro experiments using these selective compounds demonstrated the role of the somatostatin subtype-2 receptor in inhibition of glucagon release from mouse pancreatic alpha cells and the somatostatin subtype-5 receptor as a mediator of insulin secretion from pancreatic beta cells. Both receptors regulated growth hormone release from the rat anterior pituitary gland. The availability of high-affinity, subtype-selective agonists for each of the somatostatin receptors provides a direct approach to defining their physiological functions.
Subject(s)
Amides/pharmacology , Receptors, Somatostatin/agonists , Amides/metabolism , Amino Acid Sequence , Animals , Cell Line , Cells, Cultured , Cricetinae , Drug Design , Glucagon/metabolism , Growth Hormone/metabolism , Insulin/metabolism , Insulin Secretion , Islets of Langerhans/drug effects , Islets of Langerhans/metabolism , Ligands , Membrane Proteins , Mice , Models, Chemical , Molecular Sequence Data , Pituitary Gland, Anterior/drug effects , Pituitary Gland, Anterior/metabolism , Rats , Receptors, Somatostatin/physiologyABSTRACT
A series of nonpeptide somatostatin agonists which bind selectively and with high affinity to somatostatin receptor subtype 2 (sst2) have been synthesized. One of these compounds, L-054,522, binds to human sst2 with an apparent dissociation constant of 0.01 nM and at least 3,000-fold selectivity when evaluated against the other somatostatin receptors. L-054,522 is a full agonist based on its inhibition of forskolin-stimulated adenylate cyclase activity in Chinese hamster ovary-K1 cells stably expressing sst2. L-054,522 has a potent inhibitory effect on growth hormone release from rat primary pituitary cells and glucagon release from isolated mouse pancreatic islets. Intravenous infusion of L-054,522 to rats at 50 microgram/kg per hr causes a rapid and sustained reduction in growth hormone to basal levels. The high potency and selectivity of L-054, 522 for sst2 will make it a useful tool to further characterize the physiological functions of this receptor subtype.
Subject(s)
Benzimidazoles/chemical synthesis , Benzimidazoles/pharmacology , Indoles/chemical synthesis , Indoles/pharmacology , Molecular Mimicry , Receptors, Somatostatin/agonists , Animals , CHO Cells , Cricetinae , Glucagon/antagonists & inhibitors , Glucagon/metabolism , Growth Hormone/metabolism , Humans , Insulin/metabolism , Insulin Antagonists/pharmacology , Male , Mice , Mice, Inbred C57BL , RatsSubject(s)
Growth Hormone-Releasing Hormone/chemical synthesis , Growth Hormone-Releasing Hormone/physiology , Hormones/chemical synthesis , Human Growth Hormone/metabolism , Oligopeptides/chemical synthesis , Receptors, Somatotropin/physiology , Amino Acid Sequence , Animals , Drug Design , Growth Hormone-Releasing Hormone/chemistry , Growth Hormone-Releasing Hormone/pharmacology , Hormones/chemistry , Hormones/pharmacology , Human Growth Hormone/therapeutic use , Humans , Hypothalamo-Hypophyseal System/physiology , Molecular Sequence Data , Oligopeptides/chemistry , Oligopeptides/pharmacology , Protein Conformation , Receptors, Somatotropin/chemistrySubject(s)
Esters/chemical synthesis , Growth Hormone/metabolism , Piperidines/chemical synthesis , Administration, Oral , Animals , Biological Availability , Crystallography, X-Ray , Dogs , Esters/administration & dosage , Esters/pharmacokinetics , Esters/pharmacology , Growth Hormone/blood , In Vitro Techniques , Male , Piperidines/administration & dosage , Piperidines/pharmacokinetics , Piperidines/pharmacology , Pituitary Gland/cytology , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Rats , Rats, Sprague-Dawley , StereoisomerismSubject(s)
Indoles , Piperidines , Receptors, Somatostatin/agonists , Animals , CHO Cells , Cell Line , Cricetinae , Cyclic AMP/agonists , Growth Hormone/metabolism , Humans , In Vitro Techniques , Indoles/chemical synthesis , Indoles/chemistry , Indoles/metabolism , Indoles/pharmacology , Ligands , Mice , Models, Molecular , Piperidines/chemical synthesis , Piperidines/chemistry , Piperidines/metabolism , Piperidines/pharmacology , Pituitary Gland/cytology , Pituitary Gland/drug effects , Pituitary Gland/metabolism , Rats , Receptors, Somatostatin/metabolism , Structure-Activity RelationshipABSTRACT
Antibodies raised against an intracellular and extracellular domain of the GH secretagogue receptor (GHS-R) confirmed that its topological orientation in the lipid bilayer is as predicted for G protein-coupled receptors with seven transmembrane domains. A strategy for mapping the agonist-binding site of the human GHS-R was conceived based on our understanding of ligand binding in biogenic amine and peptide hormone G protein-coupled receptors. Using site-directed mutagenesis and molecular modeling, we classified GHS peptide and nonpeptide agonist binding in the context of its receptor environment. All peptide and nonpeptide ligand classes shared a common binding domain in transmembrane (TM) region 3 of the GHS-R. This finding was based on TM-3 mutation E124Q, which eliminated the counter-ion to the shared basic N+ group of all GHSs and resulted in a nonfunctional receptor. Restoration of function for the E124Q mutant was achieved by a complementary change in the MK-0677 ligand through modification of its amine side-chain to the corresponding alcohol. Contacts in other TM domains [TM-2 (D99N), TM-5 (M213K, S117A), TM-6 (H280F), and extracellular loop 1 (C116A)] of the receptor revealed specificity for the different peptide, benzolactam, and spiroindolane GHSs. GHS-R agonism, therefore, does not require identical disposition of all agonist classes at the ligand-binding site. Our results support the hypothesis that the ligand-binding pocket in the GHS-R is spatially disposed similarly to the well characterized catechol-binding site in the beta2-adrenergic receptor.
Subject(s)
GTP-Binding Proteins/chemistry , GTP-Binding Proteins/metabolism , Human Growth Hormone/metabolism , Peptides/metabolism , Peptides/physiology , Receptors, Cell Surface/chemistry , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled , Amino Acid Sequence , Animals , GTP-Binding Proteins/genetics , GTP-Binding Proteins/physiology , Human Growth Hormone/chemistry , Human Growth Hormone/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Peptides/chemistry , Peptides/genetics , Rats , Receptors, Cell Surface/genetics , Receptors, Ghrelin , SwineABSTRACT
Systematic SAR studies of the different regioisomers and homologues of the spiro(indane-1,4-piperidine) moiety in the growth hormone secretagogue L-162,752 are presented. Among them, spiro(3H-1-benzopyran-2,3-piperidine) was found to afford secretagogues with low nanomolar in vitro activity.