Ghrelin, a stomach-derived orexigenic hormone, has stimulated great interest as a potential target for obesity control. Pharmacological evidence indicates that ghrelin’s effects on food intake are ...mediated by neuropeptide Y (NPY) and agouti-related protein (AgRP) in the central nervous system. These include intracerebroventricular application of antibodies to neutralize NPY and AgRP, and the application of an NPY Y1 receptor antagonist, which blocks some of the orexigenic effects of ghrelin. Here we describe treatment of Agrp−/−;Npy−/− and Mc3r−/−;Mc4r−/− double knockout mice as well as Npy−/− and Agrp−/− single knockout mice with either ghrelin or an orally active nonpeptide ghrelin agonist. The data demonstrate that NPY and AgRP are required for the orexigenic effects of ghrelin, as well as the involvement of the melanocortin pathway in ghrelin signaling. Our results outline a functional interaction between the NPY and AgRP pathways. Although deletion of either NPY or AgRP caused only a modest or nondetectable effect, ablation of both ligands completely abolished the orexigenic action of ghrelin. Our results establish an in vivo orexigenic function for NPY and AgRP, mediating the effect of ghrelin.
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 ∼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.
I. Introduction
II. Identification of Peptidomimetic GH Secretagogues
A. Mechanism of action of GHRH, GHRP-6, and somatostatin
B. In vitro assays
III. Molecular Design by Medicinal Chemistry
A. ...Benzolactams and L-692,429
B. Spiroindanes and MK-0677
C. Isonipecotic acid peptidomimetics
IV. Characterization of the MK-0677 Receptor
A. Pituitary gland
B. Hypothalamus
V. Signal Transduction Pathway
VI. Cloning the GH Secretagogue Receptor
A. Chromosomal localization
VII. Action of the Peptidomimetic GH Secretagogues in the Central
Nervous System
VIII. Peptidomimetic GH Secretagogues in Vivo
A. Animal models
B. Clinical studies in humans
IX. Regulation of Pulsatile GH Release
A. GHRH and somatostatin
B. The role of GHS-R
X. Concluding Comments
A potent, orally active growth hormone (GH) secretagogue L-163,191 belonging to a recently synthesized structural class has been characterized. L-163,191 releases GH from rat pituitary cells in ...culture with EC50= 1.3 ± 0.09 nM and is mechanistically indistinguishable from the GH-releasing peptide GHRP-6 and the prototypical nonpeptide GH secretagogue L-692,429 but clearly distinguishable from the natural GH secretagogue, GH-releasing hormone. L-163,191 elevates GH in dogs after oral doses as low as 0.125 mg/kg and was shown to be specific in its release of GH without significant effect on plasma levels of aldosterone, luteinizing hormone, thyroxine, and prolactin after oral administration of 1 mg/kg. Only modest increases in cortisol were observed. Based on these properties, L-163,191 has been selected for clinical studies.
MB243 (a 1,3-disubstituted piperazine) is a new, potent, and selective melanocortin receptor subtype-4 agonist with potential application in the treatment of obesity and/or erectile dysfunction. ...MB243 was observed to covalently bind extensively to liver microsomal proteins from rats and humans. In the presence of glutathione, two thioether adducts were detected in liver microsomal incubations by radiochromatography and LC/MS/MS analysis. These adducts were also formed when bile duct-cannulated rats were dosed with MB243. The two adducts were isolated, and their structures were determined by accurate mass MS/MS and NMR analyses. The proposed structures resulted from a novel contraction of the piperazine ring to yield a substituted imidazoline. A mechanism is proposed, which involves an initial six electron oxidation of the piperazine ring to form a reactive intermediate, which is trapped by glutathione. Hydrolysis of the glutamic acid residue followed by internal aminolysis by the cysteine amino group resulted in opening of the piperazine ring, which is followed by ring closure to an imidazoline. The resulting cysteinyl−glycine conjugate underwent subsequent hydrolysis of the glycine residue. Understanding of the mechanism of bioactivation led to the design of MB243 analogues that exhibited reduced covalent protein binding.
Specific interactions of human melanocortin-4 receptor (hMC4R) with its nonpeptide and peptide agonists were studied using alanine-scanning mutagenesis. The binding affinities and potencies of two ...synthetic, small-molecule agonists (THIQ, MB243) were strongly affected by substitutions in transmembrane α-helices (TM) 2, 3, 6, and 7 (residues Glu100, Asp122, Asp126, Phe261, His264, Leu265, and Leu288). In addition, a I129A mutation primarily affected the binding and potency of THIQ, while F262A, W258A, Y268A mutations impaired interactions with MB243. By contrast, binding affinity and potency of the linear peptide agonist NDP-MSH were substantially reduced only in D126A and H264A mutants. Three-dimensional models of receptor−ligand complexes with their agonists were generated by distance−geometry using the experimental, homology-based, and other structural constraints, including interhelical H-bonds and two disulfide bridges (Cys40−Cys279, Cys271−Cys277) of hMC4R. In the models, all pharmacophore elements of small-molecule agonists are spatially overlapped with the corresponding key residues (His6, d-Phe7, Arg8, and Trp9) of the linear peptide: their charged amine groups interact with acidic residues from TM2 and TM3, similar to His6 and Arg6 of NDP-MSH; their substituted piperidines mimic Trp9 of the peptide and interact with TM5 and TM6, while the d-Phe aromatic rings of all three agonists contact with Leu133, Trp258, and Phe261 residues.
The potential application of small molecules in GH therapy has recently become a topic of increasing interest. The spiroindoline MK-0677, the benzolactam L-692,429, and the peptides, GHRP-6 and ...hexarelin, have been shown to possess potent and selective GH-secretory activity in several species including human. Moreover, these synthetic GH secretagogues act on a signal transduction pathway distinct from that of GHRH. A specific high affinity binding site in porcine and rat anterior pituitary membranes that mediates the activity of these secretagogues has now been identified. The binding affinity of these structurally diverse secretagogues is tightly correlated with GH-secretory activity. The binding is Mg(2+)-dependent, is inhibited by GTP-gamma-S, and is not displaced by GHRH and somatostatin. The receptor is distinct from that for GHRH and has the properties of a new G-protein-coupled receptor. It is speculated that these GH secretagogues mimic an unidentified natural hormone that regulates GH secretion in concert with GHRH and somatostatin.
1-(2R)-2-({(1S,2S)-1-amino-1,2,3,4-tetrahydronaphthalen-2-ylcarbonyl}amino)-3-(4-chlorophenyl)propanoyl-N-(tert-butyl)-4-cyclohexylpiperidine-4-carboxamide (1) is a potent melanocortin-4 receptor ...agonist that exhibited time-dependent inhibition of cytochrome P450 (P450) 3A in incubations with human liver microsomes. In incubations fortified with potassium cyanide, a cyano adduct was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis as a cyanonitrosotetrahydronaphthalenyl derivative. The detection of this adduct suggested that a nitroso species was involved in the formation of a metabolite intermediate (MI) complex that led to the observed P450 inactivation. Further evidence supporting this hypothesis derived from incubations of 1 with recombinant P450 3A4, which exhibited a max at approximately 450 nm. The species responsible for this absorbance required the presence of -nicotinamide adenine dinucleotide phosphate reduced form (NADPH), increased with increasing incubation time and decreased following the addition of potassium ferricyanide to the incubation mixture, suggestive of an MI complex. Similar results were obtained with rat liver microsomes and with recombinant P450 3A1. When rats were dosed with indinavir as a P450 3A probe substrate, plasma exposure to indinavir increased three-fold following pretreatment with 1, consistent with drug-drug interaction projections based on the kinact and KI parameters for 1 in rat liver microsomes. A similar approach was used to predict the magnitude of the corresponding drug-drug interaction potential in humans dosed with a drug metabolized predominantly by P450 3A, and the forecast area under the curve (AUC) increase ranged from four- to ten-fold. These data prompted a decision to terminate further evaluation of 1 as a development candidate, and led to the synthesis of the methyl analogue 2. Methyl substitution to the amino group in 2 was designed to reduce the propensity for formation of a nitroso intermediate and, indeed, 2 failed to exhibit time-dependent inhibition of P450 3A in human liver microsomal incubations. This case study highlights the importance of mechanistic studies in support of drug-discovery and decision-making processes.
The design and synthesis of potent MC4 agonists
19b and
27 are reported.
Design and synthesis of potent MC4 selective agonists based on cyclohexylpiperidine derived cyclic urea, oxazolidinones, and ...sulfonamide based privileged structures are disclosed.
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