Melanocortin receptor activation alleviates amyloid pathology and glial reactivity in an Alzheimer's disease transgenic mouse model.

Alzheimer's disease (AD) is a devastating neurodegenerative disorder with no disease-modifying treatment. AD progression is characterized by cognitive decline, neuroinflammation, and accumulation of amyloid-beta (Aß) and neurofibrillary tangles in the brain, leading to neuronal and glial dysfunctions. Neuropeptides govern diverse pathophysiological processes and represent key players in AD pathogenesis, regulating synaptic plasticity, glial cell functions and amyloid pathology. Activation of the pro-opiomelanocortin (POMC)-derived neuropeptide and its receptor from the melanocortin receptor (MCR) family have previously been shown to rescue the impairment in hippocampus-dependent synaptic plasticity in the APP/PS1 mouse model of AD. However, the functional roles of MCR signaling in AD conditions, particularly in glial functions, are largely unknown. In this study, we investigated the potential benefits of MCR activation in AD. In APP/PS1 transgenic mice, we demonstrate that MCR activation mediated by the central administration of its agonist D-Tyr MTII substantially reduces Aß accumulation, while alleviating global inflammation and astrocytic activation, particularly in the hippocampus. MCR activation prominently reduces the A1 subtype of reactive astrocytes, which is considered a key source of astrocytic neurotoxicity in AD. Concordantly, MCR activation suppresses microglial activation, while enhancing their association with amyloid plaques. The blunted activation of microglia may contribute to the reduction in the neurotoxic phenotypes of astrocytes. Importantly, transcriptome analysis reveals that MCR activation restores the impaired homeostatic processes and microglial reactivity in the hippocampus in APP/PS1 mice. Collectively, our findings demonstrate the potential of MCR signaling as therapeutic target for AD.

Distinct binding and signaling activity of Acthar Gel compared to other melanocortin receptor agonists.

PURPOSE: To compare the binding and agonistic activity of Acthar® Gel and synthetic melanocortin receptor (MCR) agonists and examine how the activity of select agonists affects the in vivo production of corticosterone. MATERIALS AND METHODS: In vitro binding was determined using concentration-dependent displacement of the ligand [125I]Nle4, D-Phe7-α-melanocyte-stimulating hormone (α-MSH) on cells expressing MC1R, MC3R, MC4R, or MC5R. Functional activity was determined using a time-resolved fluorescence cyclic adenosine monophosphate (cAMP) assay in cells expressing MC1R, MC2R, MC3R, MC4R, or MC5R. In vivo corticosterone analyses were performed by measuring plasma corticosterone levels in Sprague Dawley rats. RESULTS: Acthar Gel and synthetic MCR agonists exhibited the highest binding at MC1R, lowest binding at MC5R, and moderate binding at MC3R and MC4R. Acthar Gel stimulated the production of cAMP in all 5 MCR-expressing cell lines, with MC2R displaying the lowest level of full agonist activity, 3-, 6.6-, and 10-fold lower than MC1R, MC3R, and MC4R, respectively. Acthar Gel was a partial agonist at MC5R. The synthetic MCR agonists induced full activity at all 5 MCRs, with the exception of α-MSH having no activity at MC2R. Acthar Gel treatment had less of an impact on in vivo production of corticosterone compared with synthetic ACTH1-24 depot. CONCLUSIONS: Acthar Gel bound to and activated each MCR tested in this study, with partial agonist activity at MC5R and the lowest level of full agonist activity at MC2R, which distinguished it from synthetic MCR agonists. The minimal activity of Acthar Gel at MC2R corresponded to lower endogenous corticosteroid production.

Activation of Melanocortin Receptors as a Potential Strategy to Reduce Local and Systemic Reactions Induced by Respiratory Viruses.

The clinical hallmarks of infections caused by critical respiratory viruses consist of pneumonia, which can progress to acute lung injury (ALI), and systemic manifestations including hypercoagulopathy, vascular dysfunction, and endotheliitis. The disease outcome largely depends on the immune response produced by the host. The bio-molecular mechanisms underlying certain dire consequences of the infection partly arise from an aberrant production of inflammatory molecules, an event denoted as "cytokine storm". Therefore, in addition to antiviral therapies, molecules able to prevent the injury caused by cytokine excess are under investigation. In this perspective, taking advantage of melanocortin peptides and their receptors, components of an endogenous modulatory system that exerts marked anti-inflammatory and immunomodulatory influences, could be an effective therapeutic strategy to control disease evolution. Exploiting the melanocortin system using natural or synthetic ligands can form a realistic basis to counteract certain deleterious effects of respiratory virus infections. The central and peripheral protective actions exerted following melanocortin receptor activation could allow dampening the harmful events that trigger the cytokine storm and endothelial dysfunction while sustaining the beneficial signals required to elicit repair mechanisms. The long standing evidence for melanocortin safety encourages this approach.

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in Escherichia coli generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαß structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.

Melanocortin 5 receptor signaling pathway in health and disease.

Melanocortin hormone system plays a key role in maintaining the homeostasis of our body via their neuro-immune-endocrine activities and regulates a diverse array of physiological functions, including melanogenesis, inflammation, immunomodulation, adrenocortical steroidogenesis, hemodynamics, natriuresis, energy homeostasis, sexual function, and exocrine secretion. The pathobiologic actions of all melanocortins are conveyed by melanocortin receptors. As the last melanocortin receptor to be cloned and characterized, melanocortin receptor 5 (MC5R) is widely expressed in both central nervous system and a number of peripheral organ systems in man. However, the exact effect of the MC5R mediated melanocortinergic signaling remains largely uncertain. Owing to the recent advances in developing highly selective peptidomimetic agonists and antagonists of MC5R and also to studies in MC5R knockout animals, our understanding of MC5R pathobiology has been greatly expanded and strengthened. Evidence suggests that MC5R plays a key role in governing immune reaction and inflammatory response, and is pivotal for the regulation of sexual behavior, thermoregulation, and exocrine secretion, like sebogenesis, lacrimal secretion and release of sex pheromones. As such, recent translational efforts have focused on developing novel sebum-suppressive therapies for seborrhoea and acne vulgaris based on antagonizing MC5R. Conversely, selective MC5R agonists have demonstrated promising beneficial effects in immune-mediated diseases, metabolic endocrinopathies and other disease conditions, such as glomerular diseases and dry eyes, skin and mouth. Thus, MC5R-mediated signaling is essential for health. Therapeutic targeting of MC5R represents a promising and pragmatic therapeutic strategy for diverse diseases. This review article delineates the biophysiology of MC5R-mediated biophysiology of the melanocortin hormone system, discusses the existing data on MC5R-targeted therapy in experimental disease models, and envisages the translational potential for treating human diseases.

Melanotan II: a possible cause of renal infarction: review of the literature and case report.

Renal infarction is an uncommon condition resulting from an acute disruption of renal blood flow and it is potentially life-threatening disease. The cause and outcome of renal infarction is not well established and is frequently misdiagnosed or diagnosed late. Melanotan II is a non-selective melanocortin-receptor agonist and its effect on humans is an increasing of skin pigmentation, producing of spontaneous penile erection and sexual stimulation. Melanotan II inducing rhabdomyolysis and renal failure have been described previously. We present a review of Melanotan II and the possible effects of this drug on the kidneys by including a case of a renal infarction most likely attributed to Melanotan II. In the mechanism of renal injury with Melanotan II, thrombotic pharmacological influence and possible direct toxic effect on renal parenchyma must be considered.

Therapeutic Strategies for Erectile Dysfunction With Emphasis on Recent Approaches in Nanomedicine.

This review addressed erectile dysfunction, regarding pathophysiology and therapeutic strategies. The line of treatment includes phosphodiesterase type-5 inhibitors and other types of therapy like topical and stem-cell transplant. Scientific literature was assessed to investigate the impact of nanotechnology on erectile dysfunction therapy. Various nanotechnology approaches were applied, like vesicular systems, lipid-based carriers, nanocrystals, dendrimers, liquid crystalline systems and nanoemulsions. Smart nano-systems can alter the landscape of the modern pharmaceutical industry by re- investigation of pharmaceutically suboptimal but biologically active entities for treatment of erectile dysfunction which were previously considered undeveloped.

Structure⁻Activity Relationships of the Tetrapeptide Ac-His-Arg-(pI)DPhe-Tic-NH2 at the Mouse Melanocortin Receptors: Modification at the (pI)DPhe Position Leads to mMC3R Versus mMC4R Selective Ligands.

The five melanocortin receptors (MC1R-MC5R) are involved in numerous biological pathways, including steroidogenesis, pigmentation, and food intake. In particular, MC3R and MC4R knockout mice suggest that the MC3R and MC4R regulate energy homeostasis in a non-redundant manner. While MC4R-selective agonists have been utilized as appetite modulating agents, the lack of MC3R-selective agonists has impeded progress in modulating this receptor in vivo. In this study, the (pI)DPhe position of the tetrapeptide Ac-His-Arg-(pI)DPhe-Tic-NH2 (an MC3R agonist/MC4R antagonist ligand) was investigated with a library of 12 compounds. The compounds in this library were found to have higher agonist efficacy and potency at the mouse (m) MC3R compared to the MC4R, indicating that the Arg-DPhe motif preferentially activates the mMC3R over the mMC4R. This observation may be used in the design of new MC3R-selective ligands, leading to novel probe and therapeutic lead compounds that will be useful for treating metabolic disorders.

ARPE-19-derived VEGF-containing exosomes promote neovascularization in HUVEC: the role of the melanocortin receptor 5.

ARPE-19 retinal pigment epithelial cells cultured in a medium containing 35 mM D-glucose led to an augmented ROS formation and release of vascular endothelial factor (VEGF)-containing exosomes compared to ARPE-19 cells cultured in a medium containing 5 mM D-glucose (standard medium). Exposing these cells to the melanocortin 5 receptor agonist (MCR5) PG-901 (10-10M), for 9 d reduced ROS generation, the number of exosomes released and their VEGF content. In contrast, incubating the cells with the melanocortin receptor MCR1 agonist BMS-470539 (10-5 M) or with the mixed MCR3/4 agonist MTII (0.30 nmol) did not produce any significant decrease in ROS levels. ARPE-19-derived VEGF-containing exosomes promoted neovascularization in human umbilical vein endothelial cells (HUVEC), an effect that was markedly reduced by PG-901 (10-10M) but not by the MCR3/4 agonist MTII (0.30 nmol) or the MCR1 agonist BMS-470539 (10-5 M). The MCR5-related action in the ARPE-19 cells was accompanied by the increased expression of two coupled factors, cytochrome p4502E1 (CYP2E1) and nuclear factor kappa b (Nf-κB). These are both involved in high glucose signalling, in ROS generation and, interestingly, were reduced by the MCR5 agonist in the ARPE-19 cells. Altogether, these data suggest that MCR5 is a modulator of the responses stimulated by glucose in ARPE-19 cells, which might possibly be translated into a modulation of the retinal pigment epithelium response to diabetes in vivo.

Developing a Biased Unmatched Bivalent Ligand (BUmBL) Design Strategy to Target the GPCR Homodimer Allosteric Signaling (cAMP over ß-Arrestin 2 Recruitment) Within the Melanocortin Receptors.

Understanding the functional relevance of G protein-coupled receptor (GPCR) homodimerization has been limited by the insufficient tools to assess asymmetric signaling occurring within dimers comprised of the same receptor type. We present unmatched bivalent ligands (UmBLs) to study the asymmetric function of melanocortin homodimers. UmBLs contain one agonist and one antagonist pharmacophore designed to target a melanocortin homodimer such that one receptor is occupied by an agonist and the other receptor by an antagonist pharmacophore. First-in-class biased UmBLs (BUmBLs) targeting the human melanocortin-4 receptor (hMC4R) were discovered. The BUmBLs displayed biased agonism by potently stimulating cAMP signaling (EC50 ∼ 2-6 nM) but minimally activating the ß-arrestin recruitment pathway (≤55% maximum signal at 10 µM). To our knowledge, we report the first single-compound strategy to pharmacologically target melanocortin receptor allosteric signaling that occurs between homodimers that can be applied straightforwardly in vitro and in vivo to other GPCR systems.

Fenoprofen-An Old Drug Rediscovered as a Biased Allosteric Enhancer for Melanocortin Receptors.

It is time-consuming and costly to bring new drugs to market, making it necessary and urgent to exploit existing drugs for new uses. Recently, fenoprofen was demonstrated as an allosteric modulator at melanocortin receptors (MCRs), although the exact mode of action has not been clarified. MCRs regulate multiple functions, including pigmentation, adrenal steroidogenesis, inflammation, energy homeostasis, and exocrine gland secretion. In this study, we showed that fenoprofen failed to displace the orthosteric agonist Nle4-d-Phe7-α-melanocyte stimulating hormone from binding to MC3-5R while possessing positive allosteric modulator activities at these receptors. In addition, fenoprofen induced biased signaling at MC3-5R, as it selectively activated ERK1/2 cascade but not the canonical cAMP signaling. Notably, fenoprofen stimulated biased signaling at MC3-5R, but not at MC1R, hence acting selectively among this highly conserved family of receptors. Moreover, PAM activity and biased signaling induced by fenoprofen were observed not only at wild-type but also at naturally occurring mutant MC3Rs, suggesting that this biased allosteric enhancer action might constitute as novel therapeutic opportunity for obese patients harboring these mutations. Our study might guide novel therapeutic applications for repurposing current drugs or designing new drugs combining allosteric and biased properties.

Discovery of Melanocortin Ligands via a Double Simultaneous Substitution Strategy Based on the Ac-His-dPhe-Arg-Trp-NH2 Template.

The melanocortin system regulates an array of diverse physiological functions including pigmentation, feeding behavior, energy homeostasis, cardiovascular regulation, sexual function, and steroidogenesis. Endogenous melanocortin agonist ligands all possess the minimal messaging tetrapeptide sequence His-Phe-Arg-Trp. Based on this endogenous sequence, the Ac-His1-dPhe2-Arg3-Trp4-NH2 tetrapeptide has previously been shown to be a useful scaffold when utilizing traditional positional scanning approaches to modify activity at the various melanocortin receptors (MC1-5R). The study reported herein was undertaken to evaluate a double simultaneous substitution strategy as an approach to further diversify the Ac-His1-dPhe2-Arg3-Trp4-NH2 tetrapeptide with concurrent introduction of natural and unnatural amino acids at positions 1, 2, or 4, as well as an octanoyl residue at the N-terminus. The designed library includes the following combinations: (A) double simultaneous substitution at capping group position (Ac) together with position 1, 2, or 4, (B) double simultaneous substitution at positions 1 and 2, (C) double simultaneous substitution at positions 1 and 4, and (D) double simultaneous substitution at positions 2 and 4. Several lead ligands with unique pharmacologies were discovered in the current study including antagonists targeting the neuronal mMC3R with minimal agonist activity and ligands with selective profiles for the various melanocortin subtypes. The results suggest that the double simultaneous substitution strategy is a suitable approach in altering melanocortin receptor potency or selectivity or converting agonists into antagonists and vice versa.

Human ß-Defensin 1 and ß-Defensin 3 (Mouse Ortholog mBD14) Function as Full Endogenous Agonists at Select Melanocortin Receptors.

ß-Defensin 3 (BD3) was identified as a ligand for the melanocortin receptors (MCRs) in 2007, although the pharmacology activity of BD3 has not been clearly elucidated. Herein, it is demonstrated that human BD3 and mouse BD3 are full micromolar agonists at the MCRs. Furthermore, mouse ß-defensin 1 (BD1) and human BD1 are also MCR micromolar agonists. This work identifies BD1 as an endogenous MCR ligand and clarifies the controversial role of BD3 as a micromolar agonist.

Melanocortin receptor subtypes are expressed on cells in the oligodendroglial lineage and signal ACTH protection.

ACTH, a melanocortin peptide used to treat multiple sclerosis (MS) relapses, acts by stimulating adrenal corticosteroid (CS) production via melanocortin receptor 2 (MC2R), but it may also exert a therapeutic effect independent of CS by stimulating other melanocortin receptors (MCR) distributed in many tissues, including the brain. We reported that oligodendroglia (OL) and oligodendroglial precursor cells (OPC) express MC4R, and that ACTH 1-39 protects OL and OPC in vitro from cell death induced by mechanisms likely involved in white matter damage in MS. This study investigates expression of MC1R, MC2R, MC3R and MC5R in OL and MC4R in OPC using immunocytochemistry with MCR subtype specific antibodies. OL express surface MC1R, MC3R and MC5R, in addition to MC4R. To investigate whether these receptors are functional, we asked if signaling through MCR is involved in ACTH protection of cultured rat OL from apoptosis (staurosporine), or cell death induced by excitotoxicity (glutamate), reactive oxygen species (ROS), or an inflammatory mediator (quinolinic acid). Like ACTH 1-39, MCR subtype specific agonists for MC1R, MC3R, MC4R and MC5R all protected OL from these insults. Conversely, antagonists for MC3R and MC4R blocked ACTH protection of OL. We then investigated the role of MC4R, as a prototype MCR, in protection and proliferation of OPC; MC4R agonists protected OPC and increased their proliferation, while antagonists blocked these effects. Our results demonstrate that MCR on OL and OPC are functional and activate signaling pathways that protect against mechanisms involved in OL damage in MS, suggesting potential beneficial effects in neurologic diseases.

Structure-Activity Relationship Studies on a Macrocyclic Agouti-Related Protein (AGRP) Scaffold Reveal Agouti Signaling Protein (ASP) Residue Substitutions Maintain Melanocortin-4 Receptor Antagonist Potency and Result in Inverse Agonist Pharmacology at the Melanocortin-5 Receptor.

The melanocortin system consists of five reported receptors, agonists from the proopiomelanocortin gene transcript, and two antagonists, agouti-signaling protein (ASP) and agouti-related protein (AGRP). For both ASP and AGRP, the hypothesized Arg-Phe-Phe pharmacophores are on exposed ß-hairpin loops. In this study, the Asn and Ala positions of a reported AGRP macrocyclic scaffold (c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro]) were explored with 14-compound and 8-compound libraries, respectively, to generate more potent, selective melanocortin receptor antagonists. Substituting diaminopropionic acid (Dap), DDap, and His at the Asn position yielded potent MC4R ligands, while replacing Ala with Ser maintained MC4R potency. Since these substitutions correlate to ASP loop residues, an additional Phe to Ala substitution was synthesized and observed to maintain MC4R potency. Seventeen compounds also possessed inverse agonist activity at the MC5R, the first report of this pharmacology. These findings are useful in developing molecular probes to study negative energy balance conditions and unidentified functions of the MC5R.

Chronic intrahypothalamic rather than subcutaneous liraglutide treatment reduces body weight gain and stimulates the melanocortin receptor system.

BACKGROUND: The GLP-1 receptor agonist liraglutide is marketed for obesity treatment where it induces body weight reduction possibly via the hypothalamus, which regulates energy homeostasis. In animal studies, acute liraglutide treatment triggers satiety, weight loss and activates thermogenesis in adipose tissue. However, the precise mechanisms how liraglutide affects in particular chronic weight loss are still under investigation. OBJECTIVES: We aimed to evaluate whether chronic hypothalamic or chronic subcutaneous administration of liraglutide induces sustained weight loss through altered adipose tissue function and to what extent hypothalamic neuronal appetite regulators are involved in the liraglutide-induced weight loss in healthy lean rats on a normal diet. MATERIALS/METHODS: We continuously administered liraglutide either intrahypothalamically (10 µg per day) or subcutaneously (200 µg kg-1 per day) for 28 days to lean Sprague Dawley rats (n=8 each). We assessed changes in body weight, adipose tissue mass, adipocyte size and adipose tissue volume in the abdominal region by using micro-CT. We analyzed genetic expression patterns of browning, thermogenic and adipocyte differentiation regulators in adipose tissues as well as particular neuronal appetite regulators in the hypothalamus. RESULTS: Intrahypothalamic liraglutide administration induced an 8% body weight reduction at day 9 compared with the control group (P<0.01) and a 7% body weight loss at day 9 compared with subcutaneous liraglutide treatment (P<0.01), supported by a significant reduction in adipose tissue mass and volume with intrahypothalamic liraglutide administration (P<0.05). Our data show that chronic intrahypothalamic liraglutide treatment triggered an 18-fold induction of the hypothalamic mc4r gene (P<0.01) accompanied by a significant increase in circulating thyroxine (T4) levels (P<0.05). CONCLUSIONS: Chronic intrahypothalamic liraglutide administration resulted in a profound reduction in body weight and fat mass loss most likely mediated by the hypothalamic melanocortin system rather than by adipose tissue browning or improved thermogenesis.

Molecular signatures of human melanocortin receptors for ligand binding and signaling.

Human melanocortin receptors (hMCRs) belong to the seven-transmembrane (TM) domain proteins. There are five hMCR subtypes and each of these receptor subtypes has different patterns of tissue expression and physiological function. The endogenous agonists for hMCRs are α-, ß-, and γ-MSH and ACTH and endogenous antagonists are Agouti and AGRP which are the only known naturally occurring antagonists for the receptors. These peptides have their own profiles regarding the relative potency for specific hMCR subtype. Extensive studies have been performed to examine the molecular basis of the hMCRs for different ligand binding affinity and potency. Studies indicate that natural ligand α-MSH utilizes conserved amino acid residues for MCR specific binding (orthosteric binding) while synthetic ligands utilize non-conserved amino acid residues for receptor subtype specific binding (allosteric binding). ACTH is the only endogenous agonist for hMC2R and more amino acid residues at hMC2R are required for ACTH binding and signaling. HMCR computer modeling provides the detailed information of ligand and MCR interaction. This review provides the latest understanding of the molecular basis of the hMCRs for ligand binding and signaling. This article is part of a Special Issue entitled: Melanocortin Receptors - edited by Ya-Xiong Tao.

Biased signaling at neural melanocortin receptors in regulation of energy homeostasis.

The global prevalence of obesity highlights the importance of understanding on regulation of energy homeostasis. The central melanocortin system is an important intersection connecting the neural pathways controlling satiety and energy expenditure to regulate energy homeostasis by sensing and integrating the signals of external stimuli. In this system, neural melanocortin receptors (MCRs), melanocortin-3 and -4 receptors (MC3R and MC4R), play crucial roles in the regulation of energy homeostasis. Recently, multiple intracellular signaling pathways and biased signaling at neural MCRs have been discovered, providing new insights into neural MCR signaling. This review attempts to summarize biased signaling including biased receptor mutants (both naturally occurring and lab-generated) and biased ligands at neural MCRs, and to provide a better understanding of obesity pathogenesis and new therapeutic implications for obesity treatment.

Bench-top to clinical therapies: A review of melanocortin ligands from 1954 to 2016.

The discovery of the endogenous melanocortin agonists in the 1950s have resulted in sixty years of melanocortin ligand research. Early efforts involved truncations or select modifications of the naturally occurring agonists leading to the development of many potent and selective ligands. With the identification and cloning of the five known melanocortin receptors, many ligands were improved upon through bench-top in vitro assays. Optimization of select properties resulted in ligands adopted as clinical candidates. A summary of every melanocortin ligand is outside the scope of this review. Instead, this review will focus on the following topics: classic melanocortin ligands, selective ligands, small molecule (non-peptide) ligands, ligands with sex-specific effects, bivalent and multivalent ligands, and ligands advanced to clinical trials. Each topic area will be summarized with current references to update the melanocortin field on recent progress. This article is part of a Special Issue entitled: Melanocortin Receptors - edited by Ya-Xiong Tao.

A Direct in Vivo Comparison of the Melanocortin Monovalent Agonist Ac-His-DPhe-Arg-Trp-NH2 versus the Bivalent Agonist Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH2: A Bivalent Advantage.

Bivalent ligands targeting putative melanocortin receptor dimers have been developed and characterized in vitro; however, studies of their functional in vivo effects have been limited. The current report compares the effects of homobivalent ligand CJL-1-87, Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH2, to monovalent ligand CJL-1-14, Ac-His-DPhe-Arg-Trp-NH2, on energy homeostasis in mice after central intracerebroventricular (ICV) administration into the lateral ventricle of the brain. Bivalent ligand CJL-1-87 had noteworthy advantages as an antiobesity probe over CJL-1-14 in a fasting-refeeding in vivo paradigm. Treatment with CJL-1-87 significantly decreased food intake compared to CJL-1-14 or saline (50% less intake 2-8 h after treatment). Furthermore, CJL-1-87 treatment decreased the respiratory exchange ratio (RER) without changing the energy expenditure indicating that fats were being burned as the primary fuel source. Additionally, CJL-1-87 treatment significantly lowered body fat mass percentage 6 h after administration (p < 0.05) without changing the lean mass percentage. The bivalent ligand significantly decreased insulin, C-peptide, leptin, GIP, and resistin plasma levels compared to levels after CJL-1-14 or saline treatments. Alternatively, ghrelin plasma levels were significantly increased. Serum stability of CJL-1-87 and CJL-1-14 (T1/2 = 6.0 and 16.8 h, respectively) was sufficient to permit physiological effects. The differences in binding affinity of CJL-1-14 compared to CJL-1-87 are speculated as a possible mechanism for the bivalent ligand's unique effects. We also provide in vitro evidence for the formation of a MC3R-MC4R heterodimer complex, for the first time to our knowledge, that may be an unexploited neuronal molecular target. Regardless of the exact mechanism, the advantageous ability of CJL-1-87 compared to CJL-1-14 to increase in vitro binding affinity, increase the duration of action in spite of decreased serum stability, decrease in vivo food intake, decrease mice's body fat percent, and differentially affect mouse hormone levels demonstrates the distinct characteristics achieved from the current melanocortin agonist bivalent design strategy.