In this work we summarize our understanding of melanocortin 4 receptor (MC4R) pathway activation, aiming to define a safe and effective therapeutic targeting strategy for the MC4R. Delineation of cellular MC4R pathways has provided evidence for distinct MC4R signaling events characterized by unique receptor activation kinetics. While these studies remain narrow in scope, and have largely been explored with peptidic agonists, the results provide a possible correlation between distinct ligand groups and differential MC4R activation kinetics. In addition, when a set of small-molecule and peptide MC4R agonists are compared, evidence of biased signaling has been reported. The results of such mechanistic studies are discussed.
It is with great pleasure we dedicate this review article to Prof. Victor J. Hruby, in honor of his 80th birthday. Prof. Hruby has been a seminal leader in several areas of peptide research. His research has blazed the trail for melanocortin research efforts, including building understanding of chemistry, biology and pharmacology of melanocortins. Prof. Hruby has made many pivotal contributions to the scientific community, by creating widely used melanocortin receptor agonists, MT-I (NDP-α-MSH), MT-II, and the melanocortin receptor-3 and -4 antagonist SHU9119. These reagents have been instrumental in unraveling the function of melanocortin receptors including their roles in pigmentation, energy homeostasis, body weight regulation and sexual arousal. Prof. Hruby continues to make powerful contributions to the melanocortin research field, through the development of potent and selective agonists and antagonists for various melanocortin receptor subtypes.
The central hypothalamic melanocortin-4 receptor (MC4R) is a uniquely validated therapeutic target for the treatment of obesity based on both pharmacologic and human genetic evidence. Acting in concert with leptin (a satiety hormone), ghrelin (a hunger hormone) and their receptors, the MC4R holds a key position in the regulation of energy homeostasis and body weight. The MC4R and leptin receptor are key components of the MC4R pathway, which, when disrupted by genetic defects in any of these contributing receptor/ligand systems, causes impaired energy balance. A variety of peptide and small molecule MC4R agonists have been developed over the past nearly three decades and have been shown in rodent models to elicit decreases in food intake and body weight. However, the diverse nature of MC4R-driven pharmacological efficacy has posed challenges in developing an MC4R agonist for the treatment of obesity. These hurdles include MC4R-related sympathetic activation leading to elevation of blood pressure (BP) and heart rate (HR), as well as activation of sexual arousal. As a result, the feasibility of targeting the MC4R for treating obesity by peptides and small molecules ligands has been called into question, despite intense drug discovery and development activity which started in the 1990s. There have been some notable successes in creating MC4R agonist compositions, including orally bioavailable leads (for example, Merck compounds MK-0493 and MB243; Pfizer compound-13; and several Neurocrine NBI compounds described in MacNeil et al.; Palucki et al.; Ujjainwalla and Sebhat; Chen et al.; Krishna et al.; He et al.; and Lansdell et al. and reviewed in Todorovic and Haskell-Luevano and Ericson et al.. MK-0493 evaluated in a phase-1 human study was shown to be ineffective in controlling food intake or body weight meaningfully. A few peptide MCR agonist compositions, including LY2112688, MC4R-NN2-0453, and AZD2820, were also explored in early clinical studies for the treatment of obesity (Table 1). However, their development was stopped due to several adverse effects, including increased HR and BP, hyperpigmentation (melanocortin receptor-1 (MC1R)-driven), and sexual arousal, which were seen in early clinical trials. Similarly, development of bremelanotide, an MC4R peptide agonist for the treatment of male erectile dysfunction, was halted following adverse effects, including BP and HR elevation, as well as nausea and vomiting. However, bremelanotide is currently being investigated for the treatment of hypoactive sexual dysfunction in pre-menopausal women.
Table 1. Structures of various melanocortin-4 receptor (MC4R) agonists evaluated in human clinical studies.
Setmelanotide, an eight amino acid cyclic MC4R agonist peptide (Table 1), is being investigated in several clinical studies for the treatment of obesity, including rare genetic disorders of obesity. These genetic deficiencies include subjects with pro-opiomelanocortin (POMC) deficiency, proprotein-convertase (PCSK1) deficiency, leptin receptor (LEPR) deficiency, Prader-Willi syndrome (PWS), Bardet-Biedl syndrome (BBS), Alström syndrome (AS), and selected other genetic forms of early-onset severe obesity arising from defects that impair the MC4R pathway. Earlier results indicate that setmelanotide is generally well tolerated in humans and holds promise for treating obesity and hyperphagia in subjects with these rare genetic disorders of obesity.
This review compares the unique pharmacological and mechanistic profiles of several MC4R agonists based on a series of in vivo and in vitro studies. Special emphasis is placed on comparative pharmacological data obtained for the MC4R agonist peptides setmelanotide and LY2112688. While both setmelanotide and LY2112688 are potent MC4R agonists which can elicit decreases in food intake and body weight in animal models, only setmelanotide lacks adverse increases in cardiovascular activity (HR and BP) in non-human primates and humans. Studies highlighting key differentiating features among these two agonists are discussed.
Recent investigations which may help differentiate various in vivo pharmacological profiles of certain MC4R agonists have revealed an agonist-based bias for the activation of distinct MC4R intracellular signaling pathways. Studies probing differential MC4R signaling have included assays performed under equilibrium ligand binding conditions or temporally dynamic activation of the MC4R followed by the measurement of their impact on internalization and desensitization of the MC4R. Various intracellular pathways that could be invoked in neural MCR signaling, along with biased signaling ligands, have recently been reviewed by Yang and Tao. Various signaling pathways explored with MC4R include the use of different G-proteins, including Gαs, Gαi/Gαo, Gαq, and G-protein dependent mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) activation, as well as G-protein independent effects on the potassium channel Kir7.1 on MC4R neurons. These are discussed in more detail in the following sections.
The most ubiquitous MC4R signaling events described are mediated through Gαs activation, leading to the production of intracellular cyclic adenosine monophosphate (cAMP). However, recent studies using surrogate reporter systems in MC4R-expressing cells have produced indirect evidence for the involvement of a Gαq signaling pathway leading to the activation of phospholipase-C (PLC) and calcium mobilization. Some investigators have explored the involvement of the Gi/o-mediated pathway, without, however, clear evidence of its involvement. This initial evidence of possible divergence in signaling pathways downstream of MC4R is of interest and requires in depth follow-up to understand its impact in physiologically relevant signaling events. The results of these studies are summarized here and include evaluations of setmelanotide, a related compound RM-511, and the LY2112688 MC4R agonist peptide.
It is well established that MC4R signals through Gαs under equilibrium ligand binding conditions using human embryonic kidney (HEK), Chinese hamster ovary (CHO), or monkey kidney tissue derived fibroblast-like cell (COS) cell-based systems stably transfected with MC4R. Under these equilibrium conditions, receptor activation by both peptide and small molecule agonists can be antagonized by MC4R antagonists like SHU9119 or Agouti gene-related peptide (AgRP). While AgRP is an endogenous antagonist of MC4R mediated GαS signaling, it has also been shown in in vitro systems to function as an inverse agonist by decreasing basal levels of cAMP. Table 2 summarizes the effective concentration for 50% stimulation (EC 50) values for cAMP stimulation and Ki (binding constant) values for receptor binding for setmelanotide, LY2112688, Melanotan-II (MT-II) and alpha-melanocyte stimulating hormone (α-MSH, an endogenous agonist) in CHO cells stably transfected with human (h)MC1R, hMC3R, hMC4R, or hMC5R (these agonists do not interact with the MC2R). In these experiments, setmelanotide, MT-II, and LY2112688 displayed approximately similar binding affinities for the MC4R. In cAMP functional assays performed under equilibrium conditions, the potencies for each of these three agonists were also equivalent when compared between setmelanotide and LY2112688 or MT-II, with setmelanotide being about three times less active.
Table 2. Inhibitory constants and 50% effective concentrations of peptide agonists in CHO-K1 cells expressing human melanocortin receptors (data from Kievit et al.).
In prior studies using time-resolved signaling conditions, LY-2112688, MT-II, THIQ, and a setmelanotide analog, RM-511, were shown to differentiate in their dynamic effects during MC4R signaling or antagonism by AgRP.
These studies by Molden et al. employed acute exposure to the MC4R agonist and applied cell-based reporter moiety and receptor tags for tracking receptor activation kinetics. Using a temporally-resolved Förster resonance energy transfer (FRET) based cAMP assay with Neuro2A HA-MC4R-GFP cells (stably expressing epitope-tagged hemagglutinin-MC4R-green fluorescent pro
