Novel Cocrystal Structures of Peptide Antagonists Bound to the Human Melanocortin Receptor 4 Unveil Unexplored Grounds for Structure-Based Drug Design.

Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders.

Targeting the central melanocortin system for the treatment of metabolic disorders.

A large body of preclinical and clinical data shows that the central melanocortin system is a promising therapeutic target for treating various metabolic disorders such as obesity and cachexia, as well as anorexia nervosa. Setmelanotide, which functions by engaging the central melanocortin circuitry, was approved by the FDA in 2020 for use in certain forms of syndromic obesity. Furthermore, the FDA approvals in 2019 of two peptide drugs targeting melanocortin receptors for the treatment of generalized hypoactive sexual desire disorder (bremelanotide) and erythropoietic protoporphyria-associated phototoxicity (afamelanotide) demonstrate the safety of this class of peptides. These approvals have also renewed excitement in the development of therapeutics targeting the melanocortin system. Here, we review the anatomy and function of the melanocortin system, discuss progress and challenges in developing melanocortin receptor-based therapeutics, and outline potential metabolic and behavioural disorders that could be addressed using pharmacological agents targeting these receptors.

Demonstration of a Common DPhe7 to DNal(2')7 Peptide Ligand Antagonist Switch for Melanocortin-3 and Melanocortin-4 Receptors Identifies the Systematic Mischaracterization of the Pharmacological Properties of Melanocortin Peptides.

Melanocortin peptides containing a 3-(2-naphthyl)-d-alanine residue in position 7 (DNal(2')7), reported as melanocortin-3 receptor (MC3R) subtype-specific agonists in two separate publications, were found to lack significant MC3R agonist activity. The cell lines used at the University of Arizona for pharmacological characterization of these peptides, consisting of HEK293 cells stably transfected with human melanocortin receptor subtypes MC1R, MC3R, MC4R, or MC5R, were then obtained and characterized by quantitative polymerase chain reaction (PCR). While the MC1R cell line correctly expressed only hMCR1, the three other cell lines were mischaracterized with regard to receptor subtype expression. The demonstration that a 3-(2-naphthyl)-d-alanine residue in position 7, irrespective of the melanocortin peptide template, results primarily in the antagonism of MC3R and MC4R then allowed us to search the published literature for additional errors. The erroneously characterized DNal(2')7-containing peptides date back to 2003; thus, our analysis suggests that systematic mischaracterization of the pharmacological properties of melanocortin peptides occurred.

Shared genetic risk between eating disorder- and substance-use-related phenotypes: Evidence from genome-wide association studies.

Eating disorders and substance use disorders frequently co-occur. Twin studies reveal shared genetic variance between liabilities to eating disorders and substance use, with the strongest associations between symptoms of bulimia nervosa and problem alcohol use (genetic correlation [rg ], twin-based = 0.23-0.53). We estimated the genetic correlation between eating disorder and substance use and disorder phenotypes using data from genome-wide association studies (GWAS). Four eating disorder phenotypes (anorexia nervosa [AN], AN with binge eating, AN without binge eating, and a bulimia nervosa factor score), and eight substance-use-related phenotypes (drinks per week, alcohol use disorder [AUD], smoking initiation, current smoking, cigarettes per day, nicotine dependence, cannabis initiation, and cannabis use disorder) from eight studies were included. Significant genetic correlations were adjusted for variants associated with major depressive disorder and schizophrenia. Total study sample sizes per phenotype ranged from ~2400 to ~537 000 individuals. We used linkage disequilibrium score regression to calculate single nucleotide polymorphism-based genetic correlations between eating disorder- and substance-use-related phenotypes. Significant positive genetic associations emerged between AUD and AN (rg = 0.18; false discovery rate q = 0.0006), cannabis initiation and AN (rg = 0.23; q < 0.0001), and cannabis initiation and AN with binge eating (rg = 0.27; q = 0.0016). Conversely, significant negative genetic correlations were observed between three nondiagnostic smoking phenotypes (smoking initiation, current smoking, and cigarettes per day) and AN without binge eating (rgs = -0.19 to -0.23; qs < 0.04). The genetic correlation between AUD and AN was no longer significant after co-varying for major depressive disorder loci. The patterns of association between eating disorder- and substance-use-related phenotypes highlights the potentially complex and substance-specific relationships among these behaviors.

Determination of the melanocortin-4 receptor structure identifies Ca2+ as a cofactor for ligand binding.

The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte-stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)-independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein-coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

G protein-coupled receptors differentially regulate glycosylation and activity of the inwardly rectifying potassium channel Kir7.1.

Kir7.1 is an inwardly rectifying potassium channel with important roles in the regulation of the membrane potential in retinal pigment epithelium, uterine smooth muscle, and hypothalamic neurons. Regulation of G protein-coupled inwardly rectifying potassium (GIRK) channels by G protein-coupled receptors (GPCRs) via the G protein ßγ subunits has been well characterized. However, how Kir channels are regulated is incompletely understood. We report here that Kir7.1 is also regulated by GPCRs, but through a different mechanism. Using Western blotting analysis, we observed that multiple GPCRs tested caused a striking reduction in the complex glycosylation of Kir7.1. Further, GPCR-mediated reduction of Kir7.1 glycosylation in HEK293T cells did not alter its expression at the cell surface but decreased channel activity. Of note, mutagenesis of the sole Kir7.1 glycosylation site reduced conductance and open probability, as indicated by single-channel recording. Additionally, we report that the L241P mutation of Kir7.1 associated with Lebers congenital amaurosis (LCA), an inherited retinal degenerative disease, has significantly reduced complex glycosylation. Collectively, these results suggest that Kir7.1 channel glycosylation is essential for function, and this activity within cells is suppressed by most GPCRs. The melanocortin-4 receptor (MC4R), a GPCR previously reported to induce ligand-regulated activity of this channel, is the only GPCR tested that does not have this effect on Kir7.1.

Characterization of MC4R Regulation of the Kir7.1 Channel Using the Tl+ Flux Assay.

The family of inward rectifying potassium channels (Kir channels) plays crucial roles in the regulation of heart rhythms, renal excretion, insulin release, and neuronal activity. Their dysfunction has been attributed to numerous diseases such as cardiac arrhythmia, kidney failure and electrolyte imbalance, diabetes mellitus, epilepsy, retinal degeneration, and other neuronal disorders. We have recently demonstrated that the melanocortin-4 receptor (MC4R), a Gαs-coupled GPCR, regulates Kir7.1 activity through a mechanism independent of Gαs and cAMP. In contrast to the many other members of the Kir channel family, less is known about the biophysical properties, regulation, and physiological functions of Kir7.1. In addition to using conventional patch clamp techniques, we have employed a high-throughput Tl+ flux assay to further investigate the kinetics of MC4R-Kir7.1 signaling in vitro. Here, we discuss the employment of the Tl+ flux assay to study MC4R -mediated regulation of Kir7.1 activity and to screen compounds for drug discovery.

Loss of the melanocortin-4 receptor in mice causes dilated cardiomyopathy.

Haploinsufficiency of the melanocortin-4 receptor, the most common monogenetic obesity syndrome in humans, is associated with a reduction in autonomic tone, bradycardia, and incidence of obesity-associated hypertension. Thus, it has been assumed that melanocortin obesity syndrome may be protective with respect to obesity-associated cardiovascular disease. We show here that absence of the melanocortin-4 receptor (MC4R) in mice causes dilated cardiomyopathy, characterized by reduced contractility and increased left ventricular diameter. This cardiomyopathy is independent of obesity as weight matched diet induced obese mice do not display systolic dysfunction. Mc4r cardiomyopathy is characterized by ultrastructural changes in mitochondrial morphology and cardiomyocyte disorganization. Remarkably, testing of myocardial tissue from Mc4r-/- mice exhibited increased ADP stimulated respiratory capacity. However, this increase in respiration correlates with increased reactive oxygen species production - a canonical mediator of tissue damage. Together this study identifies MC4R deletion as a novel and potentially clinically important cause of heart failure.

Leptin signaling regulates glucose homeostasis, but not adipostasis, in the zebrafish.

Leptin is the primary adipostatic factor in mammals. Produced largely by adipocytes in proportion to total adipose mass, the hormone informs the brain regarding total energy stored as triglycerides in fat cells. The hormone acts on multiple circuits in the brain to regulate food intake, autonomic outflow, and endocrine function to maintain energy balance. In addition to regulating adipose mass, mammalian leptin also plays a role in the regulation of glucose homeostasis and as a gating factor in reproductive competence. Leptin-deficient mice and people exhibit early onset profound hyperphagia and obesity, diabetes, and infertility. Although leptin and the leptin receptor are found in fish, the hormone is not expressed in adipose tissue, but is found in liver and other tissues. Here, we show that adult zebrafish lacking a functional leptin receptor do not exhibit hyperphagia or increased adiposity, and exhibit normal fertility. However, leptin receptor-deficient larvae have increased numbers of ß-cells and increased levels of insulin mRNA. Furthermore, larval zebrafish have been shown to exhibit ß-cell hyperplasia in response to high fat feeding or peripheral insulin resistance, and we show here that leptin receptor is required for this response. Adult zebrafish also have increased levels of insulin mRNA and other alterations in glucose homeostasis. Thus, a role for leptin in the regulation of ß-cell mass and glucose homeostasis appears to be conserved across vertebrates, whereas its role as an adipostatic factor is likely to be a secondary role acquired during the evolution of mammals.

Regulation of orexigenic AgRP neurons: A third way?

Arcuate AgRP neurons are critical for food intake. Two pathways leading to AgRP neuron activation and food intake include regulation by peripheral hormones leptin and ghrelin, and neuronal regulation via glutamatergic inputs. In a recent article in Cell Reports, Yang et al. demonstrate 'a third way,' regulation by resident astrocytes.

Gender-specific roles for the melanocortin-3 receptor in the regulation of the mesolimbic dopamine system in mice.

The melanocortin-3 receptor (MC3R) and MC4R are known to play critical roles in energy homeostasis. However, the physiological functions of the MC3R remain poorly understood. Earlier reports indicated that the ventral tegmental area (VTA) is one of the highest sites of MC3R expression, and we sought to determine the function of the receptor in this brain region. A MC3R-green-fluorescent protein transgenic mouse and a MC3R knockout mouse strain were used to characterize the neurochemical identity of the MC3R neurons in the VTA and to determine the effects of global MC3R deletion on VTA dopamine (DA) homeostasis. We demonstrate that the MC3R, but not MC4R, is expressed in up to a third of dopaminergic neurons of the VTA. Global deletion of the MC3R increases total dopamine by 42% in the VTA and decreases sucrose intake and preference in female but not male mice. Ovariectomy restores dopamine levels to normal, but aberrant decreased VTA dopamine levels are also observed in prepubertal female mice. Because arcuate Agouti-related peptide/neuropeptide Y neurons are known to innervate and regulate VTA signaling, the MC3R in dopaminergic neurons provides a specific input for communication of nutritional state within the mesolimbic dopamine system. Data provided here suggest that this input may be highly sexually dimorphic, functioning as a specific circuit regulating effects of estrogen on VTA dopamine levels and on sucrose preference. Overall, this data support a sexually dimorphic function of MC3R in regulation of the mesolimbic dopaminergic system and reward.

Melanocortin-4 receptor mutations paradoxically reduce preference for palatable foods.

Haploinsufficiency of the melanocortin-4 receptor (MC4R) results in melanocortin obesity syndrome, the most common monogenic cause of severe early onset obesity in humans. The syndrome, which produces measurable hyperphagia, has focused attention on the role of MC4R in feeding behavior and macronutrient intake. Studies show that inhibition of MC4R signaling can acutely increase the consumption of high-fat foods. The current study examines the chronic feeding preferences of mice with deletion of one or both alleles of the MC4R to model the human syndrome. Using two-choice diet paradigms with high-fat or high-carbohydrate foods alongside normal chow, we show, paradoxically, that deletion of one allele has no effect, whereas deletion of both alleles of the MC4R actually decreases preference for palatable high-fat and high-sucrose foods, compared with wild-type mice. Nonetheless, we observed hyperphagic behavior from increased consumption of the low-fat standard chow when either heterozygous or homozygous mutant animals were presented with dietary variety. Thus, decreased MC4R signaling in melanocortin obesity syndrome consistently yields hyperphagia irrespective of the foods provided, but the hyperphagia appears driven by variety and/or novelty, rather than by a preference for high-fat or high-carbohydrate foodstuffs.

Melanocortin-4 receptor signaling is required for weight loss after gastric bypass surgery.

CONTEXT: Roux-en-Y gastric bypass (RYGB) is one of the most effective long-term therapies for the treatment of severe obesity. Recent evidence indicates that RYGB effects weight loss through multiple physiological mechanisms, including changes in energy expenditure, food intake, food preference, and reward pathways. OBJECTIVE: Because central melanocortin signaling plays an important role in the regulation of energy homeostasis, we investigated whether genetic disruption of the melanocortin-4 receptor (MC4R) in rodents and humans affects weight loss after RYGB. METHODS AND RESULTS: Here we report that MC4R(-/-) mice lost substantially less weight after surgery than wild-type animals, indicating that MC4R signaling is necessary for the weight loss effects of RYGB in this model. Mice heterozygous for MC4R remain fully responsive to gastric bypass. To determine whether mutations affect surgically induced weight loss in humans, we sequenced the MC4R gene in 972 patients undergoing RYGB. Patients heterozygous for MC4R mutations exhibited the same magnitude and distribution of postoperative weight loss as patients without such mutations, suggesting that although two normal copies of the MC4R gene are necessary for normal weight regulation, a single normal copy of the MC4R gene is sufficient to mediate the weight loss effects of RYGB. CONCLUSIONS: MC4R is the first gene identified that is required for the sustained effects of bariatric surgery. The need for MC4R signaling for the weight loss effects of RYGB in mice underscores the physiological mechanisms of action of this procedure and demonstrates that RYGB both influences and is dependent on the normal pathways that regulate energy balance.

Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.

The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z'=0.50). A pilot screen run on the Microsource Spectrum compound library (n=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: ß(2)AR agonists - the ß(2)AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor.

Characterization of the hyperphagic response to dietary fat in the MC4R knockout mouse.

Defective melanocortin signaling causes hyperphagic obesity in humans and the melanocortin-4 receptor knockout mouse (MC4R(-/-)). The human disease most commonly presents, however, as haploinsufficiency of the MC4R. This study validates the MC4R(+/-) mouse as a model of the human disease in that, like the MC4R(-/-), the MC4R(+/-) mouse also exhibits a sustained hyperphagic response to dietary fat. Furthermore, both saturated and monounsaturated fats elicit this response. N-acylphosphatidylethanolamine (NAPE) is a signaling lipid induced after several hours of high-fat feeding, that, if dysregulated, might explain the feeding behavior in melanocortin obesity syndrome. Remarkably, however, MC4R(-/-) mice produce elevated levels of NAPE and are fully responsive to the anorexigenic activity of NAPE and oleoylethanolamide. Interestingly, additional differences in N-acylethanolamine (NAE) biochemistry were seen in MC4R(-/-) animals, including reduced plasma NAE levels and elevated hypothalamic levels of fatty acid amide hydrolase expression. Thus, while reduced expression of NAPE or NAE does not explain the high-fat hyperphagia in the melanocortin obesity syndrome, alterations in this family of signaling lipids are evident. Analysis of the microstructure of feeding behavior in response to dietary fat in the MC4R(-/-) and MC4R(+/-) mice indicates that the high-fat hyperphagia involves defective satiation and an increased rate of food intake, suggesting defective satiety signaling and enhanced reward value of dietary fat.

Obesity-induced inflammation in white adipose tissue is attenuated by loss of melanocortin-3 receptor signaling.

Metabolic syndrome, a complex of highly debilitating disorders that includes insulin resistance, hypertension, and dyslipidemia, is associated with the development of obesity in humans as well as rodent models. White adipose tissue (WAT) inflammation, caused in part by macrophage infiltration, and fat accumulation in the liver are both linked to development of the metabolic syndrome. Despite large increases in body fat, melanocortin 3-receptor (MC3-R)-deficient mice do not get fatty liver disease or severe insulin resistance. This is in contrast to obese melanocortin 4-receptor (MC4-R)-deficient mice and diet-induced obese (DIO) mice, which show increased adiposity, fatty liver disease, and insulin resistance. We hypothesized that defects in the inflammatory response to obesity may underlie the protection from metabolic syndrome seen in MC3-R null mice. MC4-R mice fed a chow diet show increased proinflammatory gene expression and macrophage infiltration in WAT, as do wild-type (WT) DIO mice. In contrast, MC3-R-deficient mice fed a normal chow diet show neither of these inflammatory changes, despite their elevated adiposity and a comparable degree of adipocyte hypertrophy to the MC4-R null and DIO mice. Furthermore, even when challenged with high-fat chow for 4 wk, a period of time shown to induce an inflammatory response in WAT of WT animals, MC3-R nulls showed an attenuated up-regulation in both monocyte chemoattractant protein-1 (MCP-1) and TNFalpha mRNA in WAT compared with WT high-fat-fed animals.

Creation of a genetic model of obesity in a teleost.

The adipostat is the mechanism by which the brain detects and maintains constant levels of energy stored in adipocytes in the form of lipids. Key elements of the adipostat include the adipocyte-derived hormone leptin that is expressed in proportion to energy levels and serves to communicate this information to the central nervous system and the central circuits, which sense and respond to leptin. Blockade of one of these circuits, the central melanocortin system, disrupts leptin action and causes a distinct obesity syndrome in mice and humans, characterized by increased adiposity as well as increased linear growth. We show here that transgenic zebrafish overexpressing the endogenous melanocortin antagonist agouti-related protein (AgRP) also exhibit obesity, increased linear growth, and adipocyte hypertrophy. These findings demonstrate that key elements of the adipostat originated before the evolution of mammals. Furthermore, transgenic overexpression of AgRP in zebrafish yields a new model system for the genetic analysis of energy homeostasis in a simple vertebrate system.

Studies on the physiological functions of the melanocortin system.

The melanocortin system refers to a set of hormonal, neuropeptidergic, and paracrine signaling pathways that are defined by components that include the five G protein-coupled melanocortin receptors; peptide agonists derived from the proopiomelanocortin preprohormone precursor; and the endogenous antagonists, agouti and agouti-related protein. This signaling system regulates a remarkably diverse array of physiological functions including pigmentation, adrenocortical steroidogenesis, energy homeostasis, natriuresis, erectile responses, energy homeostasis, and exocrine gland secretion. There are many complex and unique aspects of melanocortin signaling, such as the existence of endogenous antagonists, the agouti proteins, that act at three of the five melanocortin receptors. However, there is an aspect of melanocortin signaling that has facilitated highly reductionist approaches aimed at understanding the physiological functions of each receptor and peptide: in contrast to many peptides, the melanocortin agonists and antagonists are expressed in a limited number of very discrete locations. Similarly, the melanocortin receptors are also expressed in a limited number of discrete locations where they tend to be involved in rather circumscribed physiological functions. This review examines my laboratory's participation in the cloning of the melanocortin receptors and characterization of their physiological roles.

Mechanisms of disease: Cytokine and adipokine signaling in uremic cachexia.

Clinical wasting is an important risk factor for mortality in uremic patients and is reported to have a prevalence of 30-60%. 'Malnutrition' is often inappropriately used to describe a group of nutritional abnormalities in uremic patients, which are characterized by anorexia, increased basal metabolic rate, loss of lean body mass, and declining levels of serum proteins. This syndrome--more accurately described as 'cachexia'--manifests as growth failure in children with uremia. Acidosis and inflammation are important causes of uremic cachexia but the underlying molecular mechanism is not well understood. Concentrations of circulating cytokines, such as leptin, tumor necrosis factor-alpha, interleukin-1, and interleukin-6, are elevated in patients with end-stage renal disease and correlate with the degree of cachexia in these individuals. Other energy-modulating hormones such as ghrelin, and adipokines such as adiponectin and resistin, are also perturbed in uremia and could contribute to nutritional abnormalities. We recently showed that elevated levels of circulating cytokines might be an important contributor to uremia-associated cachexia via signaling through the central melanocortin system. Small-molecule melanocortin antagonists, which are biologically active when administered orally or intraperitoneally, are now available and have been used successfully to ameliorate experimental cachexia. These findings could form the basis of a novel therapeutic strategy for uremic cachexia.

Interactions between gut peptides and the central melanocortin system in the regulation of energy homeostasis.

Genetic and pharmacological studies have shown that the central melanocortin system plays a critical role in the regulation of energy homeostasis. Animals and humans with defects in the central melanocortin system display a characteristic melanocortin obesity phenotype typified by increased adiposity, hyperphagia, metabolic defects and increased linear growth. In addition to interacting with long-term regulators of energy homeostasis such as leptin, more recent data suggest that the central melanocortin system also responds to gut-released peptides involved in mediating satiety. In this review, we discuss the interactions between these systems, with particular emphasis on cholecystokinin (CCK), ghrelin and PYY(3-36).