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Objectives: Friedreich ataxia is (FRDA) an autosomal recessive neurodegenerative disorder associated with intrinsic oxidative damage, suggesting that decreasing lipid peroxidation (LPO) might ameliorate disease progression. The present study tested the ability of RT001, a deuterated form of linoleic acid (D2-LA), to alter disease severity in patients with FRDA in a double-blind placebo-controlled trial., Methods: Sixty-five subjects were recruited across six sites and received either placebo or active drug for an 11-month study. Subjects were evaluated at 0, 4, 9, and 11 months, with the primary outcome measure being maximum oxygen consumption (MVO2) during cardiopulmonary exercise testing (CPET). A key secondary outcome measure was a composite statistical test using results from the timed 1-min walk (T1MW), peak workload, and MVO2., Results: Forty-five subjects completed the protocol. RT001 was well tolerated, with no serious adverse events related to drug. Plasma and red blood cell (RBC) membrane levels of D2-LA and its primary metabolite deuterated arachidonic acid (D2-AA) achieved steady-state concentrations by 4 months. No significant changes in MVO2 were observed for RT001 compared to placebo. Similarly, no differences between the groups were found in secondary or exploratory outcome measures. Post hoc evaluations also suggested minimal effects of RT001 at the dosages used in this study., Interpretations: The results of this study provide no evidence for a significant benefit of RT001 at the dosages tested in this Friedreich ataxia patient population., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Background: Over the past 20 years, insights from human and mouse genetics have illuminated the central role of the brain leptin-melanocortin pathway in controlling mammalian food intake, with genetic disruption resulting in extreme obesity, and more subtle polymorphic variations influencing the population distribution of body weight. At the end of 2020, the U.S. Food and Drug Administration (FDA) approved setmelanotide, a melanocortin 4 receptor agonist, for use in individuals with severe obesity due to either pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency., Scope of Review: Herein, we chart the melanocortin pathway's history, explore its pharmacology, genetics, and physiology, and describe how a neuropeptidergic circuit became an important druggable obesity target., Major Conclusions: Unravelling the genetics of the subset of severe obesity has revealed the importance of the melanocortin pathway in appetitive control; coupling this with studying the molecular pharmacology of compounds that bind melanocortin receptors has brought a new obesity drug to the market. This process provides a drug discovery template for complex disorders, which for setmelanotide took 25 years to transform from a single gene into an approved drug., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

RT001 is the di-deutero isotopologue of linoleic acid ethyl ester (D2-LA). Resistance to oxidative damage at the carbon-deuterium bond depends upon the concentration of D2-LA as a percentage of total LA. We report here on the plasma and red cell (RBC) pharmacokinetics (PK) of D2-LA, and its metabolite 13,13-D2-arachidonic acid (D2-AA), in patients with multiple neurodegenerative diseases (total of 59 participants). In Friedreich's ataxia patients, D2-LA was absorbed and transported similarly to dietary LA, peaking at about 6 h after oral dosing. Plasma D2-LA concentrations approached steady state after 28 days of dosing. After 6 months of daily dosing in subjects with other disorders, D2-LA and D2-AA levels were at or above the 20% of total (D2-LA/total LA, or D2-AA/total AA) therapeutic targets for most subjects. We conclude that chronic dosing of RT001 and associated dietary guidance can be maintained over many months to achieve target plasma and RBC levels, forming a basis for therapeutic dosing across a broad range of conditions. RT001 has been safe and well-tolerated in 59 different participants treated across 10 different neurodegenerative diseases in multiple clinical trials for up to 36 months with no significant drug related adverse events limiting use., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

We write to introduce our novel group formed to confront some of the issues raised by the COVID-19 pandemic. Information about the group, which we named "cure COVid for Ever and for All" (RxCOVEA), its dynamic membership (changing regularly), and some of its activities-described in more technical detail for expert perusal and commentary-are available upon request., Competing Interests: CONFLICT OF INTEREST E.B. is an executive member of Women’s Brain Project, Switzerland.

Background: Classic galactosemia is a rare genetic metabolic disease with an unmet treatment need. Current standard of care fails to prevent chronically-debilitating brain and gonadal complications. Many mutations in the GALT gene responsible for classic galactosemia have been described to give rise to variants with conformational abnormalities. This pathogenic mechanism is highly amenable to a therapeutic strategy based on chemical/pharmacological chaperones. Arginine, a chemical chaperone, has shown beneficial effect in other inherited metabolic disorders, as well as in a prokaryotic model of classic galactosemia. The p.Q188R mutation presents a high prevalence in the Caucasian population, making it a very clinically relevant mutation. This mutation gives rise to a protein with lower conformational stability and lower catalytic activity. The aim of this study is to assess the potential therapeutic role of arginine for this mutation., Methods: Arginine aspartate administration to four patients with the p.Q188R/p.Q188R mutation, in vitro studies with three fibroblast cell lines derived from classic galactosemia patients as well as recombinant protein experiments were used to evaluate the effect of arginine in galactose metabolism. This study has been registered at https://clinicaltrials.gov (NCT03580122) on 09 July 2018. Retrospectively registered., Results: Following a month of arginine administration, patients did not show a significant improvement of whole-body galactose oxidative capacity (p = 0.22), erythrocyte GALT activity (p = 0.87), urinary galactose (p = 0.52) and urinary galactitol levels (p = 0.41). Patients' fibroblasts exposed to arginine did not show changes in GALT activity. Thermal shift analysis of recombinant p.Q188R GALT protein in the presence of arginine did not exhibit a positive effect., Conclusions: This short pilot study in four patients homozygous for the p.Q188R/p.Q188R mutation reveals that arginine has no potential therapeutic role for galactosemia patients homozygous for the p.Q188R mutation.

Genetic defects underlying the melanocortin-4 receptor (MC4R) signaling pathway lead to severe obesity. Three severely obese LEPR-deficient individuals were administered the MC4R agonist setmelanotide, resulting in substantial and durable reductions in hyperphagia and body weight over an observation period of 45-61 weeks. Compared to formerly developed and tested MC4R agonists, setmelanotide has the unique capability of activating nuclear factor of activated T cell (NFAT) signaling and restoring function of this signaling pathway for selected MC4R variants. Our data demonstrate the potency of setmelanotide in treatment of individuals with diverse MC4R-related pathway deficiencies.

Dilated cardiomyopathy (DCM) is extremely heterogeneous with a large proportion due to dominantly inherited disease-causing variants in sarcomeric genes. Recessive metabolic diseases may cause DCM, usually with onset in childhood, and in the context of systemic disease. Whether metabolic defects can also cause adult-onset DCM is currently unknown. Therefore, we performed an extensive metabolic screening in 36 consecutive adult-onset DCM patients. Diagnoses were confirmed by Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Measurement of propionyl-CoA carboxylase (PCC) activity was done in fibroblasts. Whole exome sequencing (WES) data of 157 additional DCM patients were analyzed for genetic defects. We found a metabolic profile characteristic for propionic acidemia in a patient with severe DCM from 55 years of age. Genetic analysis demonstrated compound heterozygous variants in PCCA. Enzymatic activity of PCC in fibroblasts was markedly reduced. A targeted analysis of the PCCA and PCCB genes using available WES data from 157 further DCM patients subsequently identified another patient with propionic acidemia. This patient had compound heterozygous variants in PCCB, and developed severe DCM from 42 years of age. Adult-onset DCM can be caused by propionic acidemia, an autosomal recessive inheritable metabolic disorder usually presenting as neonatal or childhood disease. Current guidelines advise a low-protein diet to ameliorate or prevent detrimental aspects of the disease. Long-term follow-up of a larger group of patients may show whether this diet would also ameliorate DCM. Our results suggest that diagnostic metabolic screening to identify propionic acidemia and related disorders in DCM patients is justified.

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.

Aims: The 28 amino acid hormone ghrelin, the natural ligand for the growth hormone secretagogue, or ghrelin receptor (GHR), has diverse physiological functions, including a possible role as a gastrointestinal prokinetic. The synthetic ghrelin mimetic RM-131 is in Phase II clinical trials for treatment of diabetic gastroparesis and other gastrointestinal (GI) disorders. We aimed to determine the relative potency of RM-131, when compared to other GI ghrelin mimetics, to predict efficacy and determine the role of RM-131 in models of inflammatory bowel disease., Main Methods: We evaluated and compared ghrelin, RM-131 and other synthetic ghrelin mimetics for their prokinetic potency in models of gastrointestinal disorders in the rat and we evaluated the endocrine (rats and dogs) and anti-inflammatory effects (mice) of the ghrelin mimetic RM-131., Key Findings: The pentapeptide RM-131 increased gastric emptying in rodent models of ileus. RM-131 is about 100-fold more potent than human ghrelin and is 600 to 1800-fold more potent, when compared to several investigational ghrelin mimetics tested in clinical trials. RM-131 has anti-inflammatory effects and significantly increases survival and reduces macroscopic markers of tissue damage in a TNBS model of inflammatory bowel disease. RM-131 treatment shows a transient increase in growth hormone levels in Beagle dogs and rats, returning to baseline upon chronic treatment. Significant effects on glucose and insulin are not observed in chronic studies., Significance: RM-131's potency, efficacy and endocrine profile, are promising attributes for the treatment of diverse functional gastrointestinal disorders in humans., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The design, synthesis, and binding activity of ring constrained analogs of the acyclic cannabinoid-1 receptor (CB1R) inverse agonist taranabant 1 are described. The initial inspiration for these taranabant derivatives was its conformation 1a, determined by (1)H NMR, X-ray, and molecular modeling. The constrained analogs were all much less potent than their acyclic parent structure. The results obtained are discussed in the context of a predicted binding of 1 to a homology model of CB1R., (2010 Elsevier Ltd. All rights reserved.)

Background and Purpose: gamma-Secretase inhibitors (GSIs) block NOTCH receptor cleavage and pathway activation and have been under clinical evaluation for the treatment of malignancies such as T-cell acute lymphoblastic leukaemia (T-ALL). The ability of GSIs to decrease T-ALL cell viability in vitro is a slow process requiring >8 days, however, such treatment durations are not well tolerated in vivo. Here we study GSI's effect on tumour and normal cellular processes to optimize dosing regimens for anti-tumour efficacy., Experimental Approach: Inhibition of the Notch pathway in mouse intestinal epithelium was used to evaluate the effect of GSIs and guide the design of dosing regimens for xenograft models. Serum Abeta(40) and Notch target gene modulation in tumours were used to evaluate the degree and duration of target inhibition. Pharmacokinetic and pharmacodynamic correlations with biochemical, immunohistochemical and profiling data were used to demonstrate GSI mechanism of action in xenograft tumours., Key Results: Three days of >70% Notch pathway inhibition was sufficient to provide an anti-tumour effect and was well tolerated. GSI-induced conversion of mouse epithelial cells to a secretory lineage was time- and dose-dependent. Anti-tumour efficacy was associated with cell cycle arrest and apoptosis that was in part due to Notch-dependent regulation of mitochondrial homeostasis., Conclusions and Implications: Intermittent but potent inhibition of Notch signalling is sufficient for anti-tumour efficacy in these T-ALL models. These findings provide support for the use of GSI in Notch-dependent malignancies and that clinical benefits may be derived from transient but potent inhibition of Notch.

Sulfonamide analogues of the potent CB1R inverse agonist taranabant were prepared and optimized for potency and selectivity for CB1R. They were variably more potent than the corresponding amide analogues. The most potent representative 22 had good pharmacokinetic and brain levels, but was modestly active in blocking CB1R agonist-mediated hypothermia.

The peptides alpha-melanocyte-stimulating hormone (alpha-MSH) and oxytocin have very similar effects on several behaviours, including male sexual behaviour. Both induce penile erection and enhance copulatory behaviour when given centrally, suggesting that their central actions are not independent. Here, we used intromission as a physiological stimulus to investigate whether some central effects of alpha-MSH during male sexual behaviour are mediated by oxytocin neurones. We used the expression of the immediate-early gene product Fos to investigate oxytocin neurone activation at intromission and after intracerebroventricular (i.c.v.) administration of alpha-MSH (1 microg/5 microl) and studied the effects of i.c.v. administration of a MC4 receptor antagonist on Fos expression and on the latency of male rats to exhibit sexual behaviour in the presence of a receptive female. In rats that showed intromission, Fos was expressed in magnocellular oxytocin neurones in both the paraventricular nucleus (PVN) and the supraoptic nucleus (SON), but there was no significant activation of parvocellular oxytocin neurones of the PVN. Similarly, alpha-MSH increased Fos expression in magnocellular oxytocin neurones but had little or no effect in parvocellular oxytocin neurones. In male rats that achieved intromission, central injection of a MC4 receptor antagonist significantly attenuated the increase in Fos expression in magnocellular oxytocin neurones in both the PVN and the SON and increased mount and intromission latencies compared to vehicle-injected controls. Together, the results indicate that magnocellular oxytocin neurones are involved in the central regulation of male sexual behaviour, and that some of the central effects of alpha-MSH are likely to be mediated by magnocellular oxytocin neurones.

Optimization of the biological activity of a new class of non-peptidyl, pyridazinone derived human melanocortin subtype-4 receptor agonists is disclosed.

The peptides alpha-melanocyte stimulating hormone (alpha-MSH) and oxytocin, when administered centrally, produce similar behavioral effects. alpha-MSH induces Fos expression in supraoptic oxytocin neurons, and alpha-MSH melanocortin-4 receptors (MC4Rs) are highly expressed in the supraoptic nucleus, suggesting that alpha-MSH and oxytocin actions are not independent. Here we investigated the effects of alpha-MSH on the activity of supraoptic neurons. We confirmed that alpha-MSH induces Fos expression in the supraoptic nucleus when injected centrally and demonstrated that alpha-MSH also stimulates Fos expression in the nucleus when applied locally by retrodialysis. Thus alpha-MSH-induced Fos expression is not associated with electrophysiological excitation of supraoptic neurons because central injection of alpha-MSH or selective MC4 receptor agonists inhibited the electrical activity of oxytocin neurons in the supraoptic nucleus recorded in vivo. Consistent with these observations, oxytocin secretion into the bloodstream decreased after central injection of alpha-MSH. However, MC4R ligands induced substantial release of oxytocin from dendrites in isolated supraoptic nuclei. Because dendritic oxytocin release can be triggered by changes in [Ca2+]i, we measured [Ca2+]i responses in isolated supraoptic neurons and found that MC4R ligands induce a transient [Ca2+]i increase in oxytocin neurons. This response was still observed in low extracellular Ca2+ concentration and probably reflects mobilization of [Ca2+]i from intracellular stores rather than entry via voltage-gated channels. Taken together, these results show for the first time that a peptide, here alpha-MSH, can induce differential regulation of dendritic release and systemic secretion of oxytocin, accompanied by dissociation of Fos expression and electrical activity.

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.

To elucidate the molecular basis of the interaction of the native dodecapeptide gamma-MSH with the melanocortin receptors, we performed a structure-activity study in which we systematically replaced l-Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C-terminal region of gamma-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l-Ala was substituted for His6, Phe7, Arg8 and Trp9. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His-Phe-Arg-Trp sequence in gamma-MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met3 was replaced with l-Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His-Phe-Arg-Trp sequence in receptor binding and in agonist activity of gamma-MSH.

The melanocortin receptors are involved in several important physiological functions. The potent and enzymatically stable analogues MT-II (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH(2)) and SHU9119 (Ac-Nle-c[Asp-His-DNal(2')-Arg-Trp-Lys]-NH(2)) are important ligands of these receptors but are relatively nonselective. To differentiate between the physiological functions of these receptors, agonists, and antagonists with improved receptor selectivities are needed. We report here analogues of the well-characterized antagonist SHU9119 in which we replaced His(6) with conformationally constrained amino acids. By this structure-activity study we discovered two important compounds, PG-901 (Ac-Nle(4)-c[Asp(5)-Pro(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)) and PG-911 (Ac-Nle(4)-c[Asp(5)-Hyp(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)), characterized to be full agonists at the hMC5R (EC(50) = 0.072 nM and 0.031 nM, respectively), but full antagonists at the hMC3R and the hMC4R. We also demonstrated that the relative stereochemistry of the amino acid at the 6-position is critical for activity, and could play an important role in potency as well as in selectivity for the melanocortin receptors., ((c)2002 Elsevier Science (USA).)

Peptide Ac-Nle(4)-cyclo(5beta-->10epsilon)(Asp(5)-His(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), compound 1, a cyclic derivative of alpha-melanotropin, is a nonselective high affinity antagonist at human melanocortin receptors 3 and 4, and an agonist at melanocortin receptors 1 and 5. To differentiate between the physiological functions of these receptors, antagonists with improved receptor selectivity are needed. In this study, analogues of compound 1 without Ac-Nle(4) or His(6) and/or the amino group of Asp(5) were prepared and tested in binding assays and in functional assays on CHO cells expressing hMC3-5R. Several of these peptides were to be selective, high affinity hMC-4R antagonists. The most interesting was compound 10, named MBP10, cyclo(6beta-->10epsilon)(succinyl(6)-D-(2')Nal(7)-Arg(8)-Trp(9)-Lys(10))-NH(2), an antagonist (IC(50) = 0.5 nM) with 125-fold selectivity over hMC-3R (and of >300-fold selectivity over MC-1RB). This compound had no agonist activity at hMC-3R or hMC-4R and only weak agonist activity at hMC-5R. Examination of the sequences of these new peptides revealed that the D-(2')Nal(7)-Arg(8)-Trp(9) segment of peptide 1 forms the "essential core" required for high affinity and high selectivity of analogues of peptide 1 at hMC-4R, but the "extended core", His(6)-D-(2')Nal(7)-Arg(8)-Trp(9), is necessary for the maximum affinity for hMC-3R and hMC-5R.

alpha-Melanotropin (alphaMSH) and several of its derivatives are potent but not selective agonists at melanocortin receptors 3, 4, and 5 present in the brain (MC3-5R). To differentiate between the physiological role of hMC-4R (believed to be involved in regulation of energy balance) from those of melanocortin receptors 3 and 5, potent and receptor-specific agonists are needed. Therefore, the cyclic derivatives of alphaMSH of a general structure, cyclo(X-His-d-Phe-Arg-Trp-Y)-NH(2), where X is succinic acid or an omega-amino-carboxylic acid, and Y is an alpha,omega-di-amino-carboxylic acid or an omega-carboxy-alpha-amino acid, were prepared and tested in binding assays and in cAMP assays on CHO cells expressing hMC3-5R. Several of the 21-membered or larger lactams turned out to be potent and hMC-4R-selective agonists. For instance, cyclo(CO-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Dab)-NH(2) (Dab: 2,4-di-amino-butyric acid) was a potent agonist at hMC-4R (EC(50) = 4 nM) with 55-fold selectivity over hMC-3R and greater than 1000-fold selectivity over hMC-5R. Another potent and selective compound was cyclo(NH-CH(2)-CH(2)-CO-His-d-Phe-Arg-Trp-Glu)-NH(2): EC(50) about 1 nM at hMC-4R, with 90-fold selectivity over hMC-3R and greater than 2000-fold selectivity over hMC-5R., (Copyright 2001 Academic Press.)

Human melanin-concentrating hormone (hMCH) is a potent but nonselective agonist at human melanin-concentrating hormone receptors 1 and 2 (hMCH-1R and hMCH-2R, respectively). To determine the structural features of this neuropeptide which are necessary for efficient binding to and activation of the receptors, Ala-substituted, open-chain, and truncated analogues were synthesized and tested in the binding assays in CHO cells expressing hMCH-1R and hMCH-2R, and in functional assays measuring the level of intracellular calcium mobilization in human HEK-293 cells expressing these receptors. A compound consisting merely of the cyclic core of hMCH with the Arg attached to the N-terminus of the disulfide ring was found to activate both hMCH-1R and hMCH-2R about as effectively as full-length hMCH. Thus, the sequence Arg-cyclo(S-S)(Cys-Met-Leu-Gly-Arg-Val-Tyr-Arg-Pro-Cys) appears to constitute the "active core" that is necessary for agonist potency at hMCH-1R and hMCH-2R. A potent and approximately 4-fold more selective agonist at hMCH-1R than at hMCH-2R is also reported.

Neuromedin U (NMU) is a family of peptides found in the gut and the central nervous system [Neuroscience 25 (1988) 797; Biochem. Biophys. Res. Commun. 130 (1985) 1078]. While several peripheral activities such as uterus stimulating and hypertensive effects have been described for NMU [Biochem. Biophys. Res. Commun. 130 (1985) 1078], its role in the CNS remains poorly understood. Recently, we reported the identification of two receptors for NMU (NMU1R and NMU2R), and demonstrated that NMU may play a role in regulating feeding behavior. The central effect of NMU is likely mediated primarily via NMU2R, since NMU1R is detectable only in the periphery, but not in the brain [Nature 406 (2000) 70]. In this report, we describe detailed mapping of NMU2R mRNA expression in the rat brain by in situ hybridization. The most intense signals were observed in the ependymal cell layer along the wall of the third ventricle in the hypothalamus, CA1 region of the hippocampus, indusium griseum and septohippocampal nucleus. Moderate expression was detected in the hypothalamic paraventricular nucleus, dorsal raphe nucleus as well as a number of other brain structures. The presence of NMU2R in the hypothalamus is consistent with its role in energy balance. Significant levels of expression of NMU2R elsewhere in the brain may suggest additional physiological functions for this neuropeptide.

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.

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G(alpha)q coupling of the MCH-2R in cell-based systems. Northern blot and in situ hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2-16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.

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.