Ligands for Melanocortin Receptors: Beyond Melanocyte-Stimulating Hormones and Adrenocorticotropin

The discovery of melanocortins in 1916 has resulted in more than 100 years of research focused on these peptides. Extensive studies have elucidated well-established functions of melanocortins mediated by cell surface receptors, including MSHR (melanocyte-stimulating hormone receptor) and ACTHR (adrenocorticotropin receptor). Subsequently, three additional melanocortin receptors (MCRs) were identified. Among these five MCRs, MC3R and MC4R are expressed primarily in the central nervous system, and are therefore referred to as the neural MCRs. Since the central melanocortin system plays important roles in regulating energy homeostasis, targeting neural MCRs is emerging as a therapeutic approach for treating metabolic conditions such as obesity and cachexia. Early efforts modifying endogenous ligands resulted in the development of many potent and selective ligands. This review focuses on the ligands for neural MCRs, including classical ligands (MSH and agouti-related peptide), nonclassical ligands (lipocalin 2, β-defensin, small molecules, and pharmacoperones), and clinically approved ligands (ACTH, setmelanotide, bremelanotide, and several repurposed drugs).

2.1. MSHs as Agonists The endogenous agonists of neural MCRs are α-, β-, and γ-MSH, as well as ACTH, with the conserved tetrapeptide sequence His-Phe-Arg-Trp (HFRW) as the pharmacophore ( Figure 1 A), the minimally active sequence that possesses agonist activity in the classic frog and lizard skin bioassays [ 31 , 32 ]. Activation of the MCRs with MSH analogues indicated that the length of ACTH or MSH is different for a specific receptor subtype. Four amino acids (His-Phe-Arg-Trp) are the minimal MSH peptide for MC1R activation. The first 17 amino acids of ACTH are required for MC2R activation [ 33 ]. Four amino acids (His-Phe-Arg-Trp) in MSH are required for MC3R and MC5R activation [ 34 ]. Three amino acids (Phe-Arg-Trp) in MSH are required for MC4R activation [ 35 ]. Of the four major endogenous melanocortins, human MC1R and MC5R have the highest affinities for α-MSH, MC3R has the highest affinity for γ 1 -MSH, and MC4R has the highest affinity for β-MSH ( Table 1 ) [ 28 , 29 ]. Pro-ACTH is cleaved by PC1 in the anterior pituitary corticotrophs to produce ACTH(1–39), which can be further processed through PC2 to produce ACTH(1–13)-NH 2 and α-MSH [ 36 ]. ACTH is the only known endogenous ligand for MC2R, making it the sole agonist that stimulates all five MCRs. The full-length ACTH is 39 amino acids, of which ACTH(1–24) is hypothesized to be the functional domain. An anorectic effect was observed at 4 h after intracerebroventricular (ICV) administration of ACTH(1–24) at 0.05 μg/animal in mice and 10 μg/animal in rabbits [ 37 ]. A 4 μg/animal dose of ACTH(1–24) injected into the lateral ventricle or ventromedial hypothalamus also results in decreased food intake in rats [ 38 ]. The N-terminal 13 residues of ACTH are cleaved and modified through N-terminal acetylation and C-terminal carboxyamidation to produce α-MSH ( Figure 1 A). Acetylated α-MSH is more resistant to degradation than des-acetylated form [ 39 ]. At the human MCRs, α-MSH has been reported to possess single-digit nanomolar binding affinity only at the MC1R ( Table 2 ). This peptide possesses nonselective subnanomolar to nanomolar potencies at the MC1R and MC3-5Rs ( Table 3 ). Alanine scans of α-MSH have indicated the Met 4 , Phe 7 , Arg 8 , and Trp 9 positions affect binding affinity and functional activity at the mouse MC1R and rat MC3R [ 40 , 41 ]. Expression of α-MSH in the central nervous system is predominantly in the hypothalamus, dispersed throughout the arcuate nucleus, the dorsomedial nucleus of the hypothalamus, and fibers in the medial preoptic, paraventricular, periventricular, and anterior hypothalamic nuclei [ 42 , 43 ]. ICV administration of α-MSH reduces food intake in rodents and intraperitoneal (IP) injection of α-MSH alters skin/hair coloration of humans and other mammals [ 44 , 45 ]. Unlike α-MSH, both termini of 22-residue β-MSH are unmodified ( Figure 1 A). Compared to the human MC3-5R, β-MSH has lower binding affinity at the MC1R ( Table 2 ). Rodents lack the dibasic cleavage site for β-MSH and are therefore β-MSH-deficient [ 62 ]. However, human β-MSH reduces food intake in rats after ICV administration [ 63 , 64 , 65 ]. Although most studies on body weight regulation focused on α-MSH, β-MSH has also been shown to be important in the regulation of human energy homeostasis. The obesity phenotype in POMC mutation carriers resulting in β-MSH deficiency (Tyr5Cys, Tyr221Cys, and Arg236Gly) implies a significant effect of β-MSH on body weight [ 66 , 67 , 68 ], underscoring the importance of β-MSH as a physiologically relevant melanocortin ligand. In one study, human plasma β-MSH ranges from 20 to 110 ng/L [ 69 ]. In another study, cerebrospinal fluid samples obtained from 30 patients have a mean β-MSH concentration of 60.1 ng/L, compared with mean plasma concentration of 16.1 ng/L for normal adults [ 70 ]. Significant amounts of β-MSH were also observed in various regions of the brain [ 71 ]. The N-terminal domain of POMC contains three γ-MSH peptides: γ 3 -MSH (23-residue N -glycosylated peptide), γ 2 -MSH (N-terminal 12-residue cleaved from γ 3 -MSH), and γ 1 -MSH (N-terminal 11-residue cleaved from γ 3 -MSH with a C-terminal carboxyamidation). An alanine scanning of γ 2 -MSH indicated the importance of Met 3 , His 5 , Phe 6 , Arg 7 , and Trp 8 for functional activity at the human MC3-5R, similar to residues in α-MSH that are important for activity [ 59 ]. γ-MSH is expressed in the pituitary and arcuate nucleus, as well as adrenal medulla [ 72 , 73 , 74 , 75 ]. Differences in receptor selectivity have been observed between species, where γ 2 -MSH is selective for the human MC3R over MC4R and MC5R, whereas there is no selectivity between the mouse MC3R and MC5R [ 59 , 76 , 77 ]. The >50-fold selectivity of three γ-MSHs for the MC3R over the MC4R led to several investigations of the role the MC3R might play in food intake regulation by administering γ-MSH ligands in vivo ( Table 3 ). Interestingly, the γ-MSH sequence has

Lipocalin 2 was previously known as neutrophil gelatinase-associated protein exclusively secreted by adipose tissue (an adipokine) and associated with obesity and insulin resistance [ 116 , 117 ]. Recently, lipocalin 2 was shown to be also produced by osteoblasts, at levels much higher than white adipose tissue or other organs [ 54 ]. Recently, lipocalin 2 was identified as peripheral-derived novel potent polypeptide agonists for human MC1R, MC3R, and MC4R [ 54 ]. This peptide has been reported to possess nanomolar binding affinity at human MC1R, MC3R, and MC4R [ 54 ] ( Table 2 ). Lipocalin 2 can activate MC4R in PVN neurons expressing neuropeptide Y Y1 receptor, suggesting that the osteoblast-derived peptide could cross the blood–brain barrier and suppresses appetite after binding to MC4R in hypothalamus [ 54 , 118 ]. In addition, loss- and gain-of-function experiments in mice demonstrate that lipocalin 2 maintains glucose homeostasis by enhancing brown fat activation and thermogenesis, inducing insulin secretion, and improving insulin sensitivity and glucose tolerance [ 54 , 117 , 119 , 120 ]. These studies demonstrated that lipocalin 2 links bone and the CNS for metabolic homeostasis of the whole body, and may be a key player for MC4R-mediated physiological functions in mammals.

Numerous small molecule ligands for the MCRs have been developed due to their physiological importance [ 26 , 126 , 127 , 128 , 129 , 130 ]. The earliest work featured β-turn-inducing thioether-cyclized peptidomimetics [ 131 ]. In 2002, the first small molecule with single-digit nanomolar potency at the MC4R, THIQ, was described [ 132 ] ( Figure 2 ). In the same year, a thioether cyclized peptidomimetic scaffold that generated one ligand, JB1n, possessing submicromolar potency at the MC4R, was reported [ 133 ]. Following these initial reports, several classes of small molecule ligands have been disclosed and reviewed in the literature [ 26 , 128 , 134 , 135 ]. MC4R was heavily targeted by the pharmaceutical industry due to its role in obesity [ 17 ]. Since MC4R agonists decrease food intake in rodent models [ 88 ], small molecule MC4R agonists were investigated as potential therapeutics to promote weight loss. However, administration of purported MC4R-selective compounds identified several side effects including hypertension, increased heart rate, skin darkening, and sexual arousal [ 136 , 137 , 138 , 139 ]. To avoid undesirable side effects, targeting of the centrally located MC3R, which also plays a role in food intake and fat disposition [ 18 , 19 , 140 ], has emerged as a valuably alternative approach. As recently reported, pyrrolidine bis-cyclic guanidines have nanomolar agonist potency at the MC3R and over 10-fold selectivity for the MC3R over the MC4R through “unbiased” mixture-based high-throughput screening approaches [ 141 ]. Recently, Ericson et al. (2017) [ 26 ] and Yeo et al. (2021) [ 135 ] published two outstanding reviews that summarized recent small molecule ligands of MCRs at humans and rodents. Many new, potent, and enzyme-resistant analogs of melanocortin peptides have been developed based on the extensive studies of the melanocortin peptide, α-MSH. These agonists include NDP-α-MSH ([Nle4-D-Phe7]-α-MSH), melanotan II (MTII, Ac-Nle-c [Asp-His-DPhe-Arg-Trp-Lys]-NH 2 ), and SHU9119 (Ac-Nle-c [Asp-His-Dnal(2′)-Arg-Trp-Lys]-OH). NDP-MSH has nanomolar to subnanomolar potencies in MC1R and MC3-5R ( Table 2 ). Thirty-four years after its discovery, NDP-MSH was approved in the European Union as a treatment for adult erythropoietic protoporphyria in 2014 [ 142 ]. MTII is a nonselective melanocortin agonist with nanomolar to subnanomolar potencies [ 143 , 144 ] ( Table 2 ). Since its discovery, MTII has been used as an in vitro and in vivo probe, with central ICV administration of MTII inhibiting food intake in mice [ 88 ]. SHU9119 is a nanomolar to subnanomolar agonist in MC1R and MC5R with antagonist activity in MC3R and MC4R [ 61 ] ( Table 2 ). As the first peptide ligand discovered with potent antagonist activity at the MC3R and MC4R, ICV administration of SHU9119 was shown to significantly increase food intake in mice [ 88 , 140 ]. As in mammals, melanocortin system has also been shown to be involved in regulation of food intake and energy homeostasis in lower vertebrates such as fish (reviewed in [ 27 ]). In fact, the two neural MCRs, Mc3r [ 79 , 80 , 102 , 145 ] and Mc4r [ 104 , 105 , 106 , 107 , 109 , 110 , 111 , 146 , 147 ], have also been identified and characterized in several piscine species. Although MC3R is well studied in mammals, the studies on physiology and pharmacology of teleost Mc3rs are still limited, potentially due to the absence of mc3r genes in some teleosts, such as fugu, medaka, and stickleback [ 114 , 148 , 149 ]. MC4R also plays an important role in regulating energy homeostasis in teleosts. Previous in vivo studies with MC4R ligands showed MC4R agonist (MTII) suppresses food intake whereas the MC4R antagonist (SHU9119) increases the food intake in rainbow trout and goldfish after ICV administration [ 104 , 150 ]. In rainbow trout, ICV administration of MC4R specific antagonist (HS024) is more potent than MC3R/MC4R antagonist (SHU9119) in increasing food intake [ 150 ]. In addition, Mexican cavefish ( Astyanax mexicanus ) mc4r mutations contribute to physiological adaptations to nutrient-poor conditions by increasing appetite, growth, and starvation resistance [ 151 ]. The study of regulation of energy homeostasis in teleosts is related to better economic return in aquaculture, therefore the regulation of energy homeostasis by piscine neural Mc3r and Mc4r deserves further exploration. The large variations in genetics, ecology, morphology, anatomy, and physiology of teleost species result in complex species-specific energy homeostasis regulatory mechanisms [ 152 ]. For example, a large proportion of teleosts continue to grow during the entire life span, which is in contrast to the determinate growth in mammals and model animals, such as zebrafish [ 152 ]. Whether the melanocortin system functions differently in different species is unknown. 3.2.1. THIQ THIQ ( Figure 2 A), a tetrahydroisoquinolone ligand, is a synthetic small molecule compound developed by Merck. It was t

The peptidomimetic compound ML00253764 ( Figure 2 C) was initially reported to function as an MC4R antagonist in reducing tumor-induced weight loss after peripheral administration with a low binding affinity (Ki, 160 nM) [ 160 ]. This was confirmed in another study using mice grafted with Lewis lung carcinoma tumors [ 161 ]. We subsequently showed that ML00253764 behaves as an inverse agonist at the Gs-cAMP pathway and meanwhile serves as an agonist in the mitogen-activated protein kinase pathway in wild-type and several constitutively active mutant human MC4Rs [ 93 , 95 ]. We recently showed that at spotted scat and grass carp Mc4r, ML00253764 serves as a neutral allosteric modulator at cAMP signaling but allosteric agonist at the ERK1/2 signaling pathway [ 103 ].

It was suggested that studying nature-derived peptides confer advantages for developing GPCR ligands as drug discover effort [ 189 ]. Full agonists were identified from frog peptides that can bind to human MC4R with good affinities, although EC 50 s are in the tens of micromolar range [ 190 ]. Animal venoms contain numerous chemicals with diverse biological activities. In a bank of 3597 toxins, 8% are found to interact with human MC4R [ 191 ]. Two peptides, lacking HFRW pharmacophore, were described that can bind to the four MCRs (except MC2R) with low micromolar affinities and activate the human MC1R, causing increased intracellular cAMP [ 191 ]. They can work synergistically with α-MSH, although the mechanism is not clear. These peptides are not related structurally to the endogenous agonists, the melanocortins, but have some similarities with AgRP and hBD3 [ 191 ]. Further characterization and optimization of these toxins might identify new tools for the study of diverse aspects of MCR pharmacology, such as biased signaling and high selectivity, potentially leading to new therapeutics.

Obesity has reached epidemic proportions in developed countries and represents a major public health challenge. The pandemic of obesity is growing at an alarming rate, with an estimated direct and indirect cost of $150 billion per year in the US alone, and one out of five deaths is linked in one way or another to obesity [ 207 ]. Obesity is a main risk factor for many diseases, such as type II diabetes, cardiovascular diseases, fatty liver, and cancer, among others [ 208 , 209 ]. For many obese individuals, behavioral and lifestyle modifications are insufficient for long-term weight-loss maintenance and thus depend on pharmacological interventions that alter either appetite or absorption of calories. Until 2014, FDA-approved weight loss medications for obesity management include orlistat, lorcaserin, liraglutide, bupropion-naltrexone, and phentermine-topiramate for long-term treatment; and benzphetamine, phentermine, phendimetrazine, and diethylpropion for short-term use [ 210 ]. Over the past few decades, concerted efforts have been made to translate melanocortin ligands into clinical therapies [ 26 , 135 ]. Both α- and β-MSHs were injected into humans in 1961 [ 44 ]. Since then, several oral small molecules and injectable peptide agonists of MC4R have been clinically evaluated for treating obesity, including oral small molecule MK-0493 (Merck) [ 211 ] and peptide agonists LY2112688 (Eli Lilly) [ 138 ], MC4-NN-0453 (Novo Nordisk) [ 212 ], AZD2820 (Astra Zeneca) [ 213 ], and setmelanotide (Rhythm Pharmaceuticals) [ 214 , 215 , 216 , 217 , 218 ]. However, most MC4R agonist drug candidates developed as potential therapeutics for treating obesity failed in the clinic despite preclinical efficacy, due to lack of efficacy and side effects. The exception up to now has been setmelanotide ( Figure 3 A), a cyclic octapeptide that has a high potency for the two neutral MCRs (EC 50 at MC4R is 0.032 nM and EC 50 at MC3R is 0.69 nM) [ 57 ]. This synthetic peptide was approved by FDA at the end of 2020 for treating obesity caused by genetic defects in pro-opiomelanocortin ( POMC ), leptin receptor ( LEPR ), or proprotein convertase subtilisin/kexin type 1 ( PCSK1 ) [ 219 ]. In a 28-day Phase 1b clinical trial, setmelanotide led to moderate weight loss in obese people with MC4R deficiency [ 220 ]. The efficacy of setmelanotide was more pronounced in obese subjects with POMC and LEPR deficiency, with 80% of POMC -deficient subjects and 45% of LEPR -deficient subjects losing >10% of their weight within approximately one year [ 215 , 217 ]. In addition, compared to other clinically tested MC4R agonists, setmelanotide does not elicit undesirable side effects such as cardiovascular effects, with only minor adverse events including hyperpigmentation, nausea/vomiting, penile erection, and injection site reactions [ 214 , 215 , 217 ]. The safety of setmelanotide might be caused by differential signaling pathways [ 215 , 221 ]. Setmelanotide, as a mimetic of α-MSH, initiates similar signaling as α-MSH in Gαs and β-arrestin 2 pathways [ 222 ]. Setmelanotide also potently activates Gαq signaling through MC4R, which cannot be efficiently antagonized by AgRP [ 215 , 223 ]. It was found that 100 nM of AgRP cannot antagonize setmelanotide-stimulated phospholipase C activation (using the NFAT reporter gene assay) while stimulation by α-MSH is antagonized [ 215 ]. The MC4R-mediated Gαq signaling has been shown to reduce food intake without affecting cardiovascular activity [ 224 ]. Therefore, the biased ligands for MC4R that selectively activate Gαq signaling to decrease food intake but not trigger Gαs signaling with cardiovascular side effects might be better drug candidates for obesity treatment [ 21 ].

MC3R and MC4R are inextricably linked to obesity. However, research on the old drugs repurposing for new therapeutic uses for the melanocortin system is scarce. The FDA-approved drug fingolimod, a sphingosine 1-phosphate receptor agonist, is used in monotherapy for the treatment of relapsing-remitting multiple sclerosis [ 236 , 237 ]. Recently, fingolimod is demonstrated to exert antiangiogenic activity in diabetic retinopathy not only through the sphingosine 1 phosphate receptor but also through activating MC1R and MC5R [ 238 ]. However, in the study, no binding and signaling data were provided; only in silico analysis was done; and AgRP was used as MC1R antagonist [ 238 ], which is indeed an antagonist for the MC3R and MC4R. Pharmacology data from cells expressing MC1R and MC5R are needed to confirm the conclusions reached in this study. Another case is the marketed drug fenoprofen. Long known for the antiarthritic activities and efficacy in rheumatoid arthritis and osteoarthritis, fenoprofen is used as a nonsteroidal anti-inflammatory drug and inhibits cyclooxygenase [ 239 ]. Montero-Melendez et al. demonstrated that fenoprofen displays antiarthritic actions on cartilage integrity and synovitis mediated by MC3R, with fenoprofen serving as a positive allosteric modulator, revealing an important contribution of MC3R in the therapeutic actions of fenoprofen in arthritis [ 240 ]. In agreement with this study, we showed that positive allosteric modulator activity and biased signaling induced by fenoprofen are observed not only at wild-type MC3R, but also at naturally occurring MC3R mutants, suggesting that this biased allosteric enhancer action might constitute a novel therapeutic opportunity for obese patients harboring these mutations [ 241 ]. Another recent study suggested that metformin, used as first-line treatment for type 2 diabetes mellitus, inhibits the activation of MC2R and MC3R [ 242 ]. At the MC3R, metformin does not change the sensitivity to α-MSH stimulation, but decreases the maximal response. Since no binding experiment was done, it is not clear whether metformin binds to the MC3R orthosterically or allosterically.