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Background and Purpose: α- and β-melanocyte-stimulating hormones (MSH) are derived from pro-opiomelanocortin (POMC) and are the natural agonist ligands of the melanocortin 4 receptor, a key regulator of energy homeostasis. Recent rodent and human data have implicated the MAGEL2 gene, which may regulate activation of POMC neurons, as a significant contributor to the metabolic symptoms observed in Prader-Willi Syndrome (PWS). Firstly, patients with protein truncating mutations in MAGEL2 exhibit numerous clinical characteristics of PWS. Secondly, Magel2-null mice may not normally activate MC4 receptors, as they are defective in the activation of their POMC neurons and hence may fail to normally release the POMC-derived MC4 receptor agonist ligands α- and β-MSH. Magel2-null mice represent a tractable animal model for the metabolic and appetitive imbalance seen in patients with PWS., Experimental Approach: We tested a dose titration of the MC4 receptor agonist setmelanotide, in development for rare monogenic forms of obesity, in Magel2-null mice., Key Results: We show that Magel2-null mice are hypersensitive to the appetite suppressing and metabolic effects of setmelanotide., Conclusion and Implications: Setmelanotide may be a useful investigational hormone/neuropeptide replacement therapy for PWS and rare monogenic forms of obesity exhibiting impaired function of POMC neurons., (© 2016 The British Pharmacological Society.)

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain, where it controls energy balance through pathways including α-melanocyte-stimulating hormone (α-MSH)-dependent signaling. We have reported that the MC4R can exist in an active conformation that signals constitutively by increasing cAMP levels in the absence of receptor desensitization. We asked whether synthetic MC4R agonists differ in their ability to increase intracellular cAMP over time in Neuro2A cells expressing endogenous MC4R and exogenous, epitope-tagged hemagglutinin-MC4R-green fluorescent protein. By analyzing intracellular cAMP in a temporally resolved Förster resonance energy transfer assay, we show that withdrawal of α-MSH leads to a quick reversal of cAMP induction. By contrast, the synthetic agonist melanotan II (MTII) induces a cAMP signal that persists for at least 1 hour after removal of MTII from the medium and cannot be antagonized by agouti related protein. Similarly, in mHypoE-42 immortalized hypothalamic neurons, MTII, but not α-MSH, induced persistent AMP kinase signal, which occurs downstream of increased cAMP. By using a fluorescence recovery after photobleaching assay, it appears that the receptor exposed to MTII continues to signal after being internalized. Similar to MTII, the synthetic MC4R agonists, THIQ and BIM-22511, but not LY2112688, induced prolonged cAMP signaling after agonist withdrawal. However, agonist-exposed MC4R desensitized to the same extent, regardless of the ligand used and regardless of differences in receptor intracellular retention kinetics. In conclusion, α-MSH and LY2112688, when compared with MTII, THIQ, and BIM-22511, vary in the duration of the acute cAMP response, showing distinct temporal signaling selectivity, possibly linked to specific cell compartments from which cAMP signals may originate.

Context: Activation of the melanocortin-4 receptor (MC4R) with the synthetic agonist RM-493 decreases body weight and increases energy expenditure (EE) in nonhuman primates. The effects of MC4R agonists on EE in humans have not been examined to date., Objective, Design, and Setting: In a randomized, double-blind, placebo-controlled, crossover study, we examined the effects of the MC4R agonist RM-493 on resting energy expenditure (REE) in obese subjects in an inpatient setting., Study Participants and Methods: Twelve healthy adults (6 men and 6 women) with body mass index of 35.7 ± 2.9 kg/m(2) (mean ± SD) received RM-493 (1 mg/24 h) or placebo by continuous subcutaneous infusion over 72 hours, followed immediately by crossover to the alternate treatment. All subjects received a weight-maintenance diet (50% carbohydrate, 30% fat, and 20% protein) and performed 30 minutes of standardized exercise daily. Continuous EE was measured on the third treatment day in a room calorimeter, and REE in the fasting state was defined as the mean of 2 30-minute resting periods., Results: RM-493 increased REE vs placebo by 6.4% (95% confidence interval, 0.68-13.02%), on average by 111 kcal/24 h (95% confidence interval, 15-207 kcal, P = .03). Total daily EE trended higher, whereas the thermic effect of a test meal and exercise EE did not differ significantly. The 23-hour nonexercise respiratory quotient was lower during RM-493 treatment (0.833 ± 0.021 vs 0.848 ± 0.022, P = .02). No adverse effect on heart rate or blood pressure was observed., Conclusions: Short-term administration of the MC4R agonist RM-493 increases REE and shifts substrate oxidation to fat in obese individuals.

Therapeutics developed and sold as racemates can exhibit a limited therapeutic index because of side effects resulting from the undesired enantiomer (distomer) and/or its metabolites, which at times, forces researchers to abandon valuable scaffolds. Therefore, most chiral drugs are developed as single enantiomers. Unfortunately, the development of some chirally pure drug molecules is hampered by rapid in vivo racemization. The class of compounds known as immunomodulatory drugs derived from thalidomide is developed and sold as racemates because of racemization at the chiral center of the 3-aminoglutarimide moiety. Herein, we show that replacement of the exchangeable hydrogen at the chiral center with deuterium allows the stabilization and testing of individual enantiomers for two thalidomide analogs, including CC-122, a compound currently in human clinical trials for hematological cancers and solid tumors. Using "deuterium-enabled chiral switching" (DECS), in vitro antiinflammatory differences of up to 20-fold are observed between the deuterium-stabilized enantiomers. In vivo, the exposure is dramatically increased for each enantiomer while they retain similar pharmacokinetics. Furthermore, the single deuterated enantiomers related to CC-122 exhibit profoundly different in vivo responses in an NCI-H929 myeloma xenograft model. The (-)-deuterated enantiomer is antitumorigenic, whereas the (+)-deuterated enantiomer has little to no effect on tumor growth. The ability to stabilize and differentiate enantiomers by DECS opens up a vast window of opportunity to characterize the class effects of thalidomide analogs and improve on the therapeutic promise of other racemic compounds, including the development of safer therapeutics and the discovery of new mechanisms and clinical applications for existing therapeutics.

Background: The gastrointestinal peptide hormone ghrelin was discovered in 1999 as the endogenous ligand of the growth hormone secretagogue receptor. Increasing evidence supports more complicated and nuanced roles for the hormone, which go beyond the regulation of systemic energy metabolism., Scope of Review: In this review, we discuss the diverse biological functions of ghrelin, the regulation of its secretion, and address questions that still remain 15 years after its discovery., Major Conclusions: In recent years, ghrelin has been found to have a plethora of central and peripheral actions in distinct areas including learning and memory, gut motility and gastric acid secretion, sleep/wake rhythm, reward seeking behavior, taste sensation and glucose metabolism.

We assessed the efficacy of simultaneous agonism at the glucagon-like peptide-1 receptor (GLP-1R) and the melanocortin-4 receptor (MC4R) for the treatment of obesity and diabetes in rodents. Diet-induced obese (DIO) mice were chronically treated with either the long-acting GLP-1R agonist liraglutide, the MC4R agonist RM-493 or a combination of RM-493 and liraglutide. Co-treatment of DIO mice with RM-493 and liraglutide improves body weight loss and enhances glycemic control and cholesterol metabolism beyond what can be achieved with either mono-therapy. The superior metabolic efficacy of this combination therapy is attributed to the anorectic and glycemic actions of both drugs, along with the ability of RM-493 to increase energy expenditure. Interestingly, compared to mice treated with liraglutide alone, hypothalamic Glp-1r expression was higher in mice treated with the combination therapy after both acute and chronic treatment. Further, RM-493 enhanced hypothalamic Mc4r expression. Hence, co-dosing with MC4R and GLP-1R agonists increases expression of each receptor, indicative of minimized receptor desensitization. Together, these findings suggest potential opportunities for employing combination treatments that comprise parallel MC4R and GLP-1R agonism for the treatment of obesity and diabetes., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

The gastrointestinal peptide hormone ghrelin is the endogenous ligand of the growth hormone secretagogue receptor (a.k.a. ghrelin receptor, GHR). Currently, ghrelin is the only circulating peripheral hormone with the ability to promote a positive energy balance by stimulating food intake while decreasing energy expenditure and body fat utilization, as defined in rodents. Based on these and additional, beneficial effects on metabolism, the endogenous ghrelin system is considered an attractive target to treat diverse pathological conditions including those associated with eating/wasting disorders and cachexia. As the pharmacological potential of ghrelin is hampered by its relatively short half-life, ghrelin analogs with enhanced pharmacokinetics offer the potential to sustainably improve metabolism. One of these ghrelin analogs is the pentapeptide RM-131, which promotes food intake and adiposity with higher potency as compared to native ghrelin in rodents. Whereas, the effect of RM-131 on energy metabolism is solidly confirmed in rodents, it remains elusive whether RM-131 exerts its effect solely via the ghrelin receptor. Accordingly, we assessed the receptor specificity of RM-131 to promote food intake and adiposity in mice lacking the GHR. Our data show that in wildtype mice RM-131 potently promotes weight gain and adiposity through stimulation of food intake. However, RM-131 fails to affect food intake and body weight in mice lacking the GHR, underlining that the anabolic effects of RM-131 are mediated via the ghrelin receptor in mice.

Design, syntheses and structure-activity relationships of N-acetylated piperazine privileged structures containing MC4R agonist compounds were described. The most potent derivatives were low nM MC4R selective full agonists. Several compounds from the series had modest pharmacokinetic properties., (Copyright 2010 Elsevier Ltd. All rights reserved.)

NOTCH signaling is deregulated in the majority of T-cell acute lymphoblastic leukemias (T-ALL) as a result of activating mutations in NOTCH1. Gamma secretase inhibitors (GSI) block proteolytic activation of NOTCH receptors and may provide a targeted therapy for T-ALL. We have investigated the mechanisms of GSI sensitivity across a panel of T-ALL cell lines, yielding an approach for patient stratification based on pathway activity and also providing a rational combination strategy for enhanced response to GSI. Whereas the NOTCH1 mutation status does not serve as a predictor of GSI sensitivity, a gene expression signature of NOTCH pathway activity does correlate with response, and may be useful in the selection of patients more likely to respond to GSI. Furthermore, inhibition of the NOTCH pathway activity signature correlates with the induction of the cyclin-dependent kinase inhibitors CDKN2D (p19(INK4d)) and CDKN1B (p27(Kip1)), leading to derepression of RB and subsequent exit from the cell cycle. Consistent with this evidence of cell cycle exit, short-term exposure of GSI resulted in sustained molecular and phenotypic effects after withdrawal of the compound. Combination treatment with GSI and a small molecule inhibitor of CDK4 produced synergistic growth inhibition, providing evidence that GSI engagement of the CDK4/RB pathway is an important mechanism of GSI action and supports further investigation of this combination for improved efficacy in treating T-ALL.

Objective: To further address the function of the Y5 receptor in energy homeostasis, we investigated the effects of a novel spironolactone Y5 antagonist in diet-induced obese (DIO) mice., Methods and Procedures: Male C57BL/6 or Npy5r(-/-) mice were adapted to high-fat (HF) diet for 6-10 months and were submitted to three experimental treatments. First, the Y5 antagonist at a dose of 10 or 30 mg/kg was administered for 1 month to DIO C57BL/6 or Npy5r(-/-) mice. Second, the Y5 antagonist at 30 mg/kg was administered for 1.5 months to DIO C57BL/6 mice, and insulin sensitivity was evaluated using an insulin tolerance test. After a recovery period, nuclear magnetic resonance measurement was performed to evaluate body composition. Third, DIO mice were treated with the Y5 antagonist alone, or in combination with 10% food restriction, or with another anorectic agent, sibutramine at 10 mg/kg, for 1.5 months. Plasma glucose, insulin, and leptin levels, and adipose tissue weights were quantified., Results: The spironolactone Y5 antagonist significantly reduced body weight in C57BL DIO mice, but not in Npy5r(-/-) DIO mice. The Y5 antagonist produced a fat-selective loss of body weight, and ameliorated obesity-associated insulin resistance in DIO mice. In addition, the Y5 antagonist combined with either food restriction or sibutramine tended to produce greater body weight loss, as compared with single treatment., Discussion: These findings demonstrate that the Y5 receptor is an important mediator of energy homeostasis in rodents.

Discovery of a series of tert-butyl pyrrolidine derived, potent and orally bioavailable melanocortin receptor subtype-4 (MC4R) selective modulators is disclosed.

We document in vitro and in vivo effects of a novel, selective cannabinoid CB(1) receptor inverse agonist, Imidazole 24b (5-(4-chlorophenyl)-N-cyclohexyl-4-(2,4-dichlorophenyl)-1-methyl-imidazole-2-carboxamide). The in vitro binding affinity of Imidazole 24b for recombinant human and rat CB(1) receptor is 4 and 10 nM, respectively. Imidazole 24b binds to human cannabinoid CB(2) receptor with an affinity of 297 nM; in vitro, it is a receptor inverse agonist at both cannabinoid CB(1) and CB(2) receptors as it causes a further increase of forskolin-induced cAMP increase. Oral administration of Imidazole 24b blocked CP-55940-induced hypothermia, demonstrating cannabinoid CB(1) receptor antagonist efficacy in vivo. Using ex vivo autoradiography, Imidazole 24b resulted in dose-dependent increases in brain cannabinoid CB(1) receptor occupancy (RO) at 2h post-dosing in rats, indicating that approximately 50% receptor occupancy is sufficient for attenuation of receptor agonist-induced hypothermia. Imidazole 24b administered to C57Bl/6 mice and to dietary-induced obese (DIO) Sprague-Dawley rats attenuated overnight food intake with a minimal effective dose of 10 mg/kg, p.o. Administration had no effect in cannabinoid CB(1) receptor-deficient mice. DIO rats were dosed orally with vehicle, Imidazole 24b (1, 3 or 10 mg/kg), or dexfenfluramine (3 mg/kg) for 2 weeks. At 3 mg/kg, Imidazole 24b reduced cumulative food intake, leading to a non-significant decrease in weight gain. Imidazole 24b at 10 mg/kg and dexfenfluramine treatment inhibited food intake and attenuated weight gain. These findings suggest that selective cannabinoid CB(1) receptor inverse agonists such as Imidazole 24b have potential for the treatment of obesity.

SAR about the piperidine core in a series of MC4R agonists is described. A number of alkyl substituents that furnish compounds with good affinity and functional potency are reported.

The cannabinoid-1 receptor (CB1R) has been implicated in the control of energy balance. To explore the pharmacological utility of CB1R inhibition for the treatment of obesity, we evaluated the efficacy of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-[[5-(trifluoromethyl)pyridin-2-yl]oxy]propanamide (MK-0364) and determined the relationship between efficacy and brain CB1R occupancy in rodents. MK-0364 was shown to be a highly potent CB1R inverse agonist that inhibited the binding and functional activity of various agonists with a binding K(i) of 0.13 nM for the human CB1R in vitro. MK-0364 dose-dependently inhibited food intake and weight gain, with an acute minimum effective dose of 1 mg/kg in diet-induced obese (DIO) rats. CB1R mechanism-based effect was demonstrated for MK-0364 by its lack of efficacy in CB1R-deficient mice. Chronic treatment of DIO rats with MK-0364 dose-dependently led to significant weight loss with a minimum effective dose of 0.3 mg/kg (p.o.), or a plasma C(max) of 87 nM. Weight loss was accompanied by the loss of fat mass. Partial occupancy (30-40%) of brain CB1R by MK-0364 was sufficient to reduce body weight. The magnitude of weight loss was correlated with brain CB1R occupancy. The partial receptor occupancy requirement for efficacy was also consistent with the reduced food intake of the heterozygous mice carrying one disrupted allele of CB1R gene compared with the wild-type mice. These studies demonstrated that MK-0364 is a highly potent and selective CB1R inverse agonist and that it is orally active in rodent models of obesity.

Optimization of the biological activity for 5,6-diarylpyridines as CB1 receptor inverse agonists is described. Food intake and pharmacokinetic evaluation of 3f and 15c indicate that these compounds are effective orally active modulators of CB1.

Neuropeptide Y (NPY) is thought to have a significant role in the physiological control of energy homeostasis. We recently reported that an NPY Y5 antagonist inhibits body weight gain in diet-induced obese (DIO) mice, with a moderate reduction in food intake. To clarify the mechanism of the antiobesity effects of the Y5 antagonist, we conducted a pair-feeding study in DIO mice. The Y5 antagonist at 100 mg/kg produced a moderate feeding suppression leading to an 18% decrease in body weight, without altering body temperature. In contrast, the pair-fed group showed only a transient weight reduction and a reduced body temperature, thus indicating that the Y5 antagonist stimulates thermogenesis. The Y5 antagonist-treated mice showed an up-regulation of uncoupling protein mRNA in brown adipose tissue (BAT) and white adipose tissue (WAT), suggesting that both BAT and WAT contribute to energy expenditure. Thus, the Y5 antagonist induces its antiobesity effects by acting on both energy intake and expenditure.

The discovery of novel acyclic amide cannabinoid-1 receptor inverse agonists is described. They are potent, selective, orally bioavailable, and active in rodent models of food intake and body weight reduction. A major focus of the optimization process was to increase in vivo efficacy and to reduce the potential for formation of reactive metabolites. These efforts led to the identification of compound 48 for development as a clinical candidate for the treatment of obesity.

Structure-activity relationships of a 4-aminoquinoline MCH1R antagonist lead series were explored by synthesis of analogs with modifications at the 2-, 4-, and 6-positions of the original HTS hit. Improvements to the original screening lead included lipophilic groups at the 2-position and biphenyl, cyclohexyl phenyl, and hydrocinnamyl carboxamides at the 6-position. Modifications of the 4-amino group were not well tolerated.

A series of 2-aminoquinoline compounds was prepared and evaluated in MCH1R binding and functional antagonist assays. Small dialkyl, methylalkyl, methylcycloalkyl, and cyclic amines were tolerated at the quinoline 2-position. The in vivo efficacy of compound 12 was explored and compared to that of a related inactive analog to determine their effects on food intake and body weight in rodents.

Neuropeptide Y (NPY) is thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, the NPY Y5 receptor (Y5R) is a prime candidate to mediate some of the effects of NPY on energy homeostasis, although its role in physiologically relevant rodent obesity models remains poorly defined. We examined the effect of a potent and highly selective Y5R antagonist in rodent obesity and dietary models. The Y5R antagonist selectively ameliorated diet-induced obesity (DIO) in rodents by suppressing body weight gain and adiposity while improving the DIO-associated hyperinsulinemia. The compound did not affect the body weight of lean mice fed a regular diet or genetically obese leptin receptor-deficient mice or rats, despite similarly high brain Y5R receptor occupancy. The Y5R antagonist acts in a mechanism-based manner, as the compound did not affect DIO of Y5R-deficient mice. These results indicate that Y5R is involved in the regulation and development of DIO and suggest utility for Y5R antagonists in the treatment of obesity.

Design and synthesis of potent MC4 selective agonists based on cyclohexylpiperidine derived cyclic urea, oxazolidinones, and sulfonamide based privileged structures are disclosed.

Synthesis, SAR, and binding affinities are described for a new class of 1,8-naphthyridinone CB1 receptor specific inverse agonists. Food intake, knockout mouse, and pharmacokinetic evaluation of 14 indicate that this compound is an effective orally active modulator of CB1.

Over the past decade, hypothalamic circuits have been described that impact energy homeostasis in rodents and humans. Our drug development efforts for the treatment of obesity and the metabolic syndrome have largely focused on selected genetic and/or pharmacologically validated pathways. The translation of these pathways into therapeutics for the treatment of obesity will find its first clinical successes over the coming decade. Initial efforts have focused on gaining a better understanding of the relevance of rodent pharmacological and genetic observations for the development of therapeutics for the treatment of human obesity. We pursue pathways defined by the expression of the ghrelin receptor, melanin-concentrating hormone receptors, melanocortin receptors, cannabinoid receptors and neuropeptide Y1 and Y5 receptors. In this review, we will discuss drug development efforts for the treatment of obesity, focused on selective melanocortin 4 receptor agonists and neuropeptide Y1 and Y5 receptor antagonists. These drug development efforts required an in-depth understanding of cell-based observations which drive the development of compound structure-activity relationships. These include understanding of receptor function in selected cell-based backgrounds and early evaluation and validation of ex vivo observations in appropriate in vivo models. In order to develop selective and safe anti-obesity drugs, diverse approaches are needed to increase the likelihood of clinical success, including: (i) developing a detailed understanding of the predictive value of rodent pathways for treatment of human disease; (ii) knowledge of the exact location of targeted receptor subtypes for the clinical indication under study in order to derive a suitable compound profile; (iii) predictive measures of in vivo and/or ex vivo receptor occupancy required to bring about a desired physiological effect; (iv) predictive parameters that outline that the drug-derived effects are safe and mechanism-based; and (v) the refinement of selected compound classes, aimed at their clinical use.

The melanocortin subtype-4 receptor (MC4R) has been implicated in the control of feeding behavior and body weight regulation. A series of tetrapeptides, based on Tic-DPhe-Arg-Trp-NH2-a mimic of the putative message sequence "His-Phe-Arg-Trp" and modified at the DPhe position, were prepared and pharmacologically characterized for potency and selectivity. Substitution of His with Tic gave peptides with significant increases in selectivity. The effects of the substitution pattern of DPhe were investigated and it has significant influences on potency and the level of the maximum cAMP accumulation. Intracerebroventricular administration of peptide 10 induced significant inhibition of cumulative overnight food intake and feeding duration in rats.

A novel isoquinuclidine containing selective melanocortin subtype-4 receptor small molecule agonist, 3 (RY764), is reported. Its in vivo characterization revealed mechanism-based food intake reduction and erectile activity augmentation in rodents.

The discovery of 1-amino-1,2,3,4-tetrahydronaphthalene-2-carboxylic acid analogs as potent human melanocortin-4 selective agonists is described.

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. The 5- and 6-alkylated piperazine compounds exhibit low bioactivation potential as measured by covalent binding in microsome preparations.

Melanin-concentrating hormone (MCH) exerts a positive regulation on appetite and binds to the G protein-coupled receptors, MCH1R and MCH2R. In rodents, MCH is produced by neurons in the lateral hypothalamus with projections to various hypothalamic and other brain sites. In the present study, MCH1R was shown, by immunocytochemistry, to be present in the human infundibular nucleus/median eminence, paraventricular nucleus, lateral hypothalamic area, and perifornical area, although in the latter two regions, only a few MCH1R-containing cells were found. In addition, MCH1R staining was found in nerve fibers in the periventricular nucleus, dorsomedial and ventromedial nucleus, suprachiasmatic nucleus, and tuberomammillary nucleus. A significant 1.6 times increase in the number of MCH1R cell body staining was found in the infundibular nucleus in postmortem brain material of cachectic patients, compared with matched controls, supporting a role for this receptor in energy homeostasis in the human.

Structure-activity relationship studies directed toward the optimization of 4,5-diarylimidazole-2-carboxamide analogs as human CB1 receptor inverse agonists resulted in the discovery of the two amide derivatives 24a and b (hCB1 IC50 = 6.1 and 4.0 nM) which also demonstrated efficacy in overnight feeding studies in the rat for reduction in both food intake and overall body weight.

Structure-activity relationship studies for two series of 2-benzyloxy-5-(4-chlorophenyl)-6-(2,4-dichlorophenyl)pyridines having either a 3-cyano or 3-carboxamide moiety resulted in the preparation of the 2-(3,4-difluorobenzyloxy)-3-nitrile analog 10d and the 2-(3,4-difluorobenzyloxy)-3-(N-propylcarboxamide) analog 16c, (hCB1 IC(50)=1.3 and 1.7 nM, respectively) as potent and selective hCB1 inverse agonists. Their synthesis and biological activities are described herein.

We report the discovery and optimization of substituted 2-piperazinecarboxamides as potent and selective agonists of the melanocortin subtype-4 receptor. Further in vivo development of lead agonist, MB243, is disclosed.

A homogeneous time-resolved fluorescence immunoassay for detection of beta-amyloid (A beta) peptides has been adapted for quantification of A beta(40) and A beta(42) accumulation in brains of APP695SWE transgenic mice. These over-express human beta APP(swe), beta-amyloid precursor protein (beta-APP) containing the K670N/M671L 'Swedish' familial Alzheimer's disease (FAD) mutation. Both peptides start to accumulate in this line from about 260 to 280 days of age. Co-expression of a human presenilin-1 (PS1) transgene containing the A246E FAD mutation accelerates deposition and also favors-at least initially-accumulation of A beta(42) so that the A beta(2):A beta(40) ratio of peptides from 7- to 12-month-old APP695SWE x PS1A246E animals is significantly elevated above that observed throughout the lifetime of APP695SWE mice. These findings, supported by parallel immunohistochemical staining and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry data, offer important longitudinal characterization of two mouse models of cerebral amyloidosis. Application of the same extraction and quantitation procedures to samples of temporal cortex from AD sufferers indicates however that A beta(40) is only a minor component of beta-amyloid in humans.

We have developed an expression, refolding, and purification protocol for the catalytic domain of human Phosphodiesterase 3B (PDE3B). High level expression in Escherichia coli has been achieved with yields of up to 20mg/L. The catalytic domain of the enzyme was purified by affinity chromatography utilizing a novel affinity ligand. PDE3B, purified by affinity chromatography, with no single impurity #10878;1% as determined by SDS-PAGE, has a specific activity of 2210+/-442nmol/min/mg and a KM for cAMP of 44+/-4.5nM. Reducing the size of the expressed catalytic domain from residues 387-1112 to residues 654-1086 greatly reduced the aggregation phenomena observed with the affinity purified PDE3B. The definition of the N-terminus of the catalytic core was examined through the generation of several truncation mutants spanning amino acid residues 636-674. Constructs starting at E665 and M674 were fully active and devoid of activity, respectively. A construct starting at D668 had a Vmax reduced by approximately 10-fold relative to the longer constructs, yet the KM was not affected. This indicates the minimal N-terminus of the catalytic core lies between E665 and Y667. Refolding and affinity purification of the 654-1073 catalytic core of PDE3B has been employed to produce large quantities of highly pure enzyme for structural studies.

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.

Phosphodiesterases (PDEs) are enzymes that modulate cyclic nucleotide signaling and as such are clinical targets for a range of disorders including congestive heart failure, erectile dysfunction, and inflammation. The PDE3 family comprises two highly homologous subtypes expressed in different tissues, and inhibitors of this family have been shown to increase lipolysis in adipocytes. A specific PDE3B (the lipocyte-localized subtype) inhibitor would be a very useful tool to evaluate the effects of PDE3 inhibition on lipolysis and metabolic rate and might become a novel tool for treatment of obesity. We report here the three-dimensional structures of the catalytic domain of human PDE3B in complex with a generic PDE inhibitor and a novel PDE3 selective inhibitor. These structures explain the dual cAMP/cGMP binding capabilities of PDE3, provide the molecular basis for inhibitor specificity, and can supply a valid platform for the design of improved compounds.

The discovery and optimization of a new class of non-peptidyl, pyridazinone derived melanocortin subtype-4 receptor agonists is disclosed.

Aryldihydropyridazinones and aryldimethylpyrazolones with 2-benzyl vinylogous amide substituents have been identified as potent PDE3B subtype selective inhibitors. Dihydropyridazinone 8a (PDE3B IC(50)=0.19 nM, 3A IC(50)=1.3 nM) was selected for in vivo evaluation of lipolysis induction, metabolic rate increase, and cardiovascular effects.

The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His6 by Pro6 in the well-known antagonist SHU-9119 resulted in a potent agonist at the hMC5R (EC50 = 0.072 nm) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT-II and SHU-9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac-Nle-c[Asp-Pro-D-Phe-Arg-Trp-Lys]-Pro-Val-NH2 (6, PG-931) which will be useful in further biologic investigations of the hMC4R. PG-931 was about 100-fold more selective for the hMC4R vs. the hMC3R (IC50 = 0.58 and 55 nm, respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.

Central administration of the neuropeptide melanin-concentrating hormone (MCH) stimulates feeding in rodents. We studied the effects of intracerebroventricular (i.c.v.) administration of an MCH-1 receptor agonist (Compound A) and an MCH-1 receptor antagonist (Compound B) on feeding in satiated rats. Compound B (10 microg, i.c.v.) blocked the acute orexigenic effect of Compound A (5 microg, i.c.v.). In an experiment designed to either stimulate or inhibit MCH-1 receptor signaling over an extended period, rats received continuous i.c.v. infusions of vehicle (saline), Compound A (30 microg/day), Compound B (30 or 48 microg/day) or neuropeptide Y (24 microg/day, as positive control) via implantable infusion pumps. Continuous MCH-1 receptor activation recapitulated the obese phenotype of MCH-over-expressor mice, manifest as enhanced feeding (+23%, P<0.001), caloric efficiency and body weight gain (+38%, P<0.005) over the 14-day period relative to controls. Chronic MCH-1 receptor activation also elevated plasma insulin and leptin levels significantly. Conversely, continuous MCH-1 receptor antagonism led to sustained reductions in food intake (-16%, P<0.001), body weight gain (-35%, P<0.01), and body fat gain relative to controls, without an effect on lean mass. Antagonism of the MCH-1 receptor may be an effective approach for the treatment of obesity.

The catalytic domain of recombinant human PDE3B was expressed in Escherichia coli as inclusion bodies and refolded to form active enzyme. A mutation at tryptophan 1072 in PDE3B disrupts inhibitor binding, but has minimal effect on cAMP hydrolysis. The W1072A mutation caused a 158-fold decrease in affinity for cilostamide, a 740-fold decrease for cGMP, and a 15-fold decrease in affinity for IBMX. The corresponding tyrosine mutation had a smaller effect. However, the K(m) of cAMP for the W1072A mutation was only increased by about 7-fold. The data indicate that the inhibitor binding region is not completely coincident with the substrate binding region. The homologous residue in PDE4B is located on helix 16 within 7A of the predicted bound substrate. A model of PDE3B was constructed based on the X-ray crystal structure of PDE4B.

Melanotan-II (MT-II), a cyclic heptapeptide, is a potent, non-selective melanocortinergic agonist. When administered centrally or systemically, MT-II elicited a profound inhibitory effect on food intake in rodents, presumably via activation of melanocortin-4-receptor (MC4R). In this study, we sought to investigate whether penetration of MT-II and iodo-MT-II into brain parenchyma is required for the anorectic effect following intravenous (IV) administration. Firstly, both MT-II and iodo-MT-II were effective at suppressing appetite in rats following their IV administration. We next surveyed by in vitro autoradiographic studies the distribution of selective (125)I-MT-II binding sites in multiple brain regions including areas important for feeding regulation such as the hypothalamus and caudal brainstem. Upon IV administration of (125)I-MT-II, significant radioactivity could not be detected in various brain regions by autoradiography except for a group of circumventricular organs (CVOs), which are anatomically situated outside the blood-brain barrier (BBB). The most intensely labeled CVOs include the subfornical organ, median eminence, area postrema and choroid plexus, and accumulation of radioactivity at these sites can be blocked by co-injection of excess unlabeled MT-II. Direct measurement of MT-II in the brain and plasma by LC-MS-MS following IV injection confirmed that the degree of MT-II penetration into the brain is negligible. Furthermore, when given peripherally under conditions that suppressed food intake, MT-II did not result in a detectable induction of c-Fos-like immunoreactivity in brain regions where a significantly elevated c-Fos expression was observed following intracerebroventricular injection of this peptide. Our results indicate that MT-II has a very limited brain penetration capability, and its effect on feeding behavior following systemic administration may be mediated by either the brain regions in close proximity to the CVOs or sites outside of the BBB, including CVOs or other peripheral systems.

To clarify the role of the neuropeptide Y (NPY) Y5 receptor subtype in energy homeostasis, the effect of the intracerebroventricular infusion of a selective Y5 agonist, D-Trp(34)NPY, was investigated in C57BL/6J mice. Intracerebroventricular infusion of D-Trp(34)NPY (5 and 10 microg/d) produced hyperphagia and body weight gain, accompanied by increased adipose tissue weight, hypercholesterolemia, hyperinsulinemia, and hyperleptinemia. Oral administration of a selective Y5 antagonist at a dose of 100 mg/kg twice a day completely suppressed all of these D-Trp(34)NPY-induced changes, indicating that chronic activation of the Y5 receptor produces hyperphagia and obesity. In addition, D-Trp(34)NPY still resulted in an increase in adipose tissue weight accompanied by hyperleptinemia and hypercholesterolemia, although D-Trp(34)NPY-induced food intake was restricted by pair-feeding. Under the pair-fed condition, D-Trp(34)NPY decreased hormone-sensitive lipase activity in white adipose tissue and uncoupling protein-1 mRNA expression in brown adipose tissue. These findings indicate that Y5-mediated obesity may involve metabolic changes, such as decreased lipolysis and thermogenesis, as well as hyperphagia. Therefore, the Y5 receptor can play a key role in regulating energy homeostasis.

We have designed and synthesized several novel cyclic SHU9119 analogues (Ac-Nle4-[Asp5-His6-DNal(2')7-Arg8-Trp9-Lys10]-NH2) modified in position 6 with nonconventional amino acids. SHU9119 is a high affinity nonselective antagonist at hMC3R and hMC4R with potent agonist activity at hMC1R and hMC5R. We measured the binding affinity and agonist potency of the novel analogues at cloned hMC3R, hMC4R, and hMC5R receptors and identified several selective, high affinity hMC3R and hMC4R antagonists. Compound 4 containing Che substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.48 nM) with 100-fold selectivity over hMC3R antagonist. Analogue 7 with a Cpe substitution in position 6 is a high affinity hMC4R antagonist (IC50 = 0.51 nM) with a 200-fold selectivity vs the hMC3R. Interestingly, analogue 9 with an Acpc residue in position 6 is a high affinity hMC3R antagonist (IC50 = 2.5 nM) with 100-fold selectivity vs the hMC4R antagonist based on its binding affinities. This compound represents the first cyclic lactam antagonist with high selectivity for the hMC3R vs hMC4R. To understand the possible structural basis responsible for selectivity of these peptides at hMCR3 and hMCR4, we have carried out a molecular modeling study in order to examine the conformational properties of the cyclic peptides modified in position 6 with conformationally restricted amino acids.

Melanocortin peptide agonists, alpha-melanocyte stimulating hormone (alpha-MSH) and melanotan-II, stimulate erectile activity in a variety of species, including man. Since neither peptide discriminates amongst melanocortin receptors, it is not clear which subtype mediates these pro-erectile effects. Here, we present data that melanocortin-induced erectogenesis is mediated by melanocortin MC(4) receptors. Systemic administration of a melanocortin MC(4) receptor agonist (N-[(3R)-1,2,3,4-tetrahydroisoquinolinium-3-ylcarbonyl]-(1R)-1-(4-chlorobenzyl)-2-[4-cyclohexyl-4-(1H-1,2,4-triazol-1ylmethyl)piperidin-1-yl]-2-oxoethylamine; THIQ) with high selectivity over other melanocortin receptors enhanced intracavernosal pressure and stimulated erectile activity in rats ex copula. THIQ dose-dependently (1-5 mg/kg, i.v.) increased the total number of erections, to an extent comparable or greater than that produced by apomorphine (0.025 mg/kg, s.c.). Central administration of THIQ (20 microg, intracerebroventricular (i.c.v.)) increased the number of reflexive penile erections; whereas administration of both a nonselective endogenous melanocortin MC(4) receptor antagonist (agouti-related protein (AgRP), 5.5. microg, i.c.v.) and a melanocortin MC(4) receptor preferring antagonist (MPB10, 1 mg/kg, i.v.) blocked THIQ-induced erectogenesis. These pro-erectile effects were also attenuated by systemic or central administration of an oxytocin antagonist (L-368899, 1 mg/kg, i.v.). Thus, melanocortin MC(4) receptor activation is sufficient for erectogenesis and these effects may involve oxytocinergic pathways.

Synthetic and natural peptides that act as nonselective melanocortin receptor agonists have been found to be anorexigenic and to stimulate erectile activity. We report the design and development of 1, a potent, selective (1184-fold vs MC3R, 350-fold vs MC5R), small-molecule agonist of the MC4 receptor. Pharmacological testing confirms the food intake lowering effects of MC4R agonism and suggests another role for the receptor in the stimulation of erectile activity.