Structure activity studies of the melanocortin-4 receptor by in vitro mutagenesis: identification of agouti-related protein (AGRP), melanocortin agonist and synthetic peptide antagonist interaction determinants

In vitro mutagenesis of the mouse melanocortin-4 receptor (mMC4R) has been performed, based upon homology molecular modeling and previous melanocortin receptor mutagenesis studies that identified putative ligand-receptor interactions. Twenty-three mMC4 receptor mutants were generated and pharmacologically characterized using several melanocortin-based ligands [alpha-MSH, NDP-MSH, MTII, DNal (1')(7)-MTII, Nal(2')(7)-MTII, SHU9119, and SHU9005]. Selected mutant receptors possessing significant differences in the melanocortin-based peptide agonist and/or antagonist pharmacology were further evaluated using the endogenous antagonist agouti-related protein fragment hAGRP(83-132) and hAGRP(109-118) molecules. These studies of the mouse MC4R provide further experimental data suggesting that the conserved melanocortin receptor residues Glu92 (TM2), Asp114 (TM3), and Asp118 (TM3) (mouse MC4R numbering) are important for melanocortin-based peptide molecular recognition. Additionally, the Glu92 and Asp118 mMC4R residues are important for molecular recognition and binding of AGRP(83-132). We have identified the Phe176 (TM4), Tyr179 (TM4), Phe254 (TM6), and Phe259 (TM6) receptor residues as putatively interacting with the melanocortin-based ligand Phe(7) by differences between alpha-MSH and NDP-MSH agonist potencies. The Glu92, Asp118, and Phe253 mMC4R receptor residues appear to be critical for hAGRP(83-132) molecular recognition and binding while Phe176 appears to be important for functional antagonism of AGRP(83-132) and AGRP(109-118) but not molecular recognition. The Phe253 mMC4R residue appears to be important for AGRP(83-132) molecular recognition and general mMC4 receptor stimulation. The Phe254 and Phe259 mMC4R amino acids may participate in the differentiation of agonist versus antagonist activity of the melanocortin-based peptide antagonists SHU9119 and SHU9005, but not AGRP(83-132) or AGRP(109-118). The Met192 side chain when mutated to a Phe results in a constitutively active mMC4R that does not effect agonist ligand binding or potency. Melanocortin-based peptides modified at the 7 position of MTII with DPhe, DNal(1'), Nal(2'), and DNal(2') have been pharmacologically characterized at these mutant mouse MC4Rs. These data suggest a revised hypothesis for the mechanism of SHU9119 antagonism at the MC4R which may be attributed to the presence of a "bulky" naphthyl moiety at the 7 position (original hypothesis), and additionally that both the stereochemistry and naphthyl ring position (2' versus 1') are important for positioning of the ligand Arg(8) residue with the corresponding mMC4R amino acids.     

Elongation studies of the human agouti-related protein (AGRP) core decapeptide (Yc[CRFFNAFC]Y) results in antagonism at the mouse melanocortin-3 receptor

The agouti-related protein (AGRP) is an endogenous antagonist of the brain melanocortin receptors (MC3R and MC4R) and is believed to be involved in feeding behavior and energy homeostasis. Previous results identified that the human AGRP decapeptide Yc[CRFFNAFC]Y binds to the MC3R and MC4R and acts as an antagonist at the MC4R but not at the MC3R. We have synthesized the amidated version of this decapeptide as well as performed elongation studies at both the N-and C-terminus of the monocyclic hAGRP(109-118) peptide. This study was designed to identify monocyclic peptide fragments of the hAGRP(86-132) to determine the minimal active monocyclic sequence necessary for antagonism at the MC3R. For binding studies, radiolabeled 125I-NDP-MSH versus 125I-hAGRP(86-132) were utilized to determine if there were differences in the ability of the AGRP fragments prepared herein to competitively displace the 125I-NDP-MSH versus AGRP(86-132) radiolabel. The binding IC(50) values of radiolabeled hAGRP(86-132) versus NDP-MSH were identical within experimental error, supporting the hypothesis that AGRP and NDP-MSH interact with overlapping binding epitopes at the MC3R and MC4R. The most notable results include identification of the TAYc[CRFFNAFC]YAR-NH(2) (pA(2)=6.1, K(i)=790nM, mMC3R) and the Yc[CRFFNAFC]YARKL-NH(2) (pA(2)=6.2, K(i)=630nM, mMC3R) peptides as the minimal monocyclic AGRP-based fragments possessing antagonist pharmacology at the MC3R. Interestingly, extension of the AGRP(109-118) decapeptide at both the N- and C-terminus by two amino acids conferred detectable mMC3R antagonism, while retaining high nanomolar MC4R antagonist and micromolar MC1R agonist pharmacological properties. These data support the hypothesis that elongation of the hAGRP(109-118) decapeptide results in antagonism at the MC3R while retaining MC1R agonist activity and MC4R antagonist activity.     

Synthesis and characterization of pyrrolidine derivatives as potent agonists of the human melanocortin-4 receptor

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 20f-1 and 20f-2 displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-compound 20f-1 possessed a K(i) of 11nM and an EC(50) of 24nM, while its 3R,4S-isomer 20f-2 exhibited a K(i) of 8.6 and an IC(50) of 65nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 20f-1 also demonstrated efficacy in diet-induced obese rats.     

Pyrrolidines as potent functional agonists of the human melanocortin-4 receptor

A series of pyrrolidine derivatives were synthesized and characterized as potent agonists of the human melanocortin-4 receptor. For example, 28c had a K(i) of 13 nM in binding affinity and EC(50) of 6.9 nM in agonist potency with an intrinsic activity of 100% of the endogenous ligand alpha-MSH.     

Differential regulation of melanin-concentrating hormone and orexin genes in the agouti-related protein/melanocortin-4 receptor system

Agouti protein and agouti-related protein (AGRP) antagonize alpha-melanocyte-stimulating hormone that binds to and activates the melanocortin-4 receptor (MC4-R) in the hypothalamus, thereby stimulating food intake. Melanin-concentrating hormone (MCH) and orexin are orexigenic peptides that specifically are synthesized in the lateral hypothalamus. MCH gene expression was augmented in A(y)/a (agouti) mice which overexpress agouti protein, but orexin mRNA was not. AGRP administered intracerebroventricularly into wild-type rats augmented MCH but not orexin gene expression. Also, SHU9119, a peptidergic antagonist of MC4-R, increased only MCH mRNA. These findings indicate that interruption of signaling at MC4-R activates the MCH but not the orexin gene. The biosyntheses of MCH and orexin are regulated through different pathways.     

Identification of agonists and antagonists of the human melanocortin-4 receptor from piperazinebenzylamines

SAR studies of a series of piperazinebenzylamines resulted in identification of potent agonists and antagonists of the human melanocortin-4 receptor. Thus, the 1,2,3,4-tetrahydroisoquinolin-1-ylacetyl compound 12e and the quinolin-3-ylcarbonyl analogue 12l possessed K(i) values of 6.3 and 4.5 nM, respectively. Interestingly, 12e was a full agonist with an EC(50) value of 31 nM, and 12l was a weak partial agonist (IA=17%) and functioned as an antagonist (IC(50)=300 nM).     

Chimeric NDP-MSH and MTII melanocortin peptides with agouti-related protein (AGRP) Arg-Phe-Phe amino acids possess agonist melanocortin receptor activity

Agouti-related protein (AGRP) is one of only two known endogenous antagonists of G-protein coupled receptors (GPCRs). Specifically, AGRP antagonizes the brain melanocortin-3 and -4 receptors involved in energy homeostasis, regulation of feeding behavior, and obesity. -Melanocyte stimulating hormone (-MSH) is one of the known endogenous agonists for these receptors. It has been hypothesized that the Arg-Phe-Phe (111–113) human AGRP amino acids may be mimicking the melanocortin agonist Phe-Arg-Trp (7–9) residue interactions with the melanocortin receptors that are important for both receptor molecular recognition and stimulation. To test this hypothesis, we generated thirteen chimeric peptide ligands based upon the melanocortin agonist peptides NDP-MSH (Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) and MTII (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂). In these chimeric ligands, the agonist DPhe-Arg-Trp amino acids were replaced by the AGRP Arg-Phe-Phe residues, and resulted in agonist activity at the mouse melanocortin receptors (mMC1R and mMC3–5Rs), supporting the hypothesis that the AGRP antagonist ligand Arg-Phe-Phe residues mimic the agonist Phe-Arg-Trp amino acids. Interestingly, the Ac-Ser-Tyr-Ser-Nle⁴-Glu-His-Arg-DPhe-Phe-Gly-Lys-Pro-Val-NH₂ peptide possessed 7 nM mMC1R agonist potency, and is 850-fold selective for the mMC1R versus the mMC3R, 2300-fold selective for the mMC1R versus the mMC4R, and 60-fold selective for the MC1R versus the mMC5R, resulting in the discovery of a new peptide template for the design of melanocortin receptor selective ligands.     

Identification and characterization of pyrrolidine diastereoisomers as potent functional agonists and antagonists of the human melanocortin-4 receptor

A series of trans-4-phenylpyrrolidine-3-carboxamides were synthesized and characterized as potent ligands of the human melanocortin-4 receptor. Interestingly, a pair of diastereoisomers 13b displayed potent functional agonist and antagonist activity, respectively. Thus, the 3S,4R-pyrrolidine 13b-1 possessed a Ki of 1.0 nM and an EC50 of 3.8 nM, while its 3R,4S-isomer 13b-2 exhibited a Ki of 4.7 and an IC50 of 64 nM. Both compounds were highly selective over other melanocortin receptor subtypes. The MC4R agonist 13b-1 also demonstrated efficacy in a diet-induced obesity model in rats.     

Common requirements for melanocortin-4 receptor selectivity of structurally unrelated melanocortin agonist and endogenous antagonist, Agouti protein

The activity of melanocortin receptors (MCR) is regulated by melanocortin peptide agonists and by the endogenous antagonists, Agouti protein and AgRP (Agouti-related protein). To understand how the selectivity for these structurally unrelated agonists and antagonist is achieved, chimeric and mutants MC3R and MC4R were expressed in cell lines and pharmacologically analyzed. A region containing the third extracellular loop, EC3, of MC4R was essential for selective Agouti protein antagonism. In addition, this part of MC4R, when introduced in MC3R, conferred Agouti protein antagonism. Further mutational analysis of this region of MC4R demonstrated that Tyr(268) was required for the selective interaction with Agouti protein, because a profound loss of the ability of Agouti protein to inhibit (125)I-labeled [Nle(4),d-Phe(7)]alpha-melanocyte-stimulating hormone (MSH) binding was observed by the single mutation of Tyr(268) to Ile. This same residue conferred selectivity for the MC4R selective agonist, [d-Tyr(4)]MT-II, whereas it inhibited interaction with the MC3R-selective agonist, [Nle(4)]Lys-gamma(2)-MSH. Conversely, mutation of Ile(265) in MC3 (the corresponding residue of Tyr(268)) to Tyr displayed a gain of affinity for [d-Tyr(4)]MT-II, but not for Agouti protein, and a loss of affinity for [Nle(4)]Lys-gamma(2)-MSH as compared with wild-type MC3R. This single amino acid mutation thus confers the selectivity of MC3R toward a pharmacological profile like that observed for MC4R agonists but not for the antagonist, Agouti protein. Thus, selectivity for structurally unrelated ligands with opposite activities is achieved in a similar manner for MC4R but not for MC3R.     

High-resolution NMR structure of the chemically-synthesized melanocortin receptor binding domain AGRP(87-132) of the agouti-related protein.

The agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin receptors MC3R and MC4R found in the hypothalamus and exhibits potent orexigenic (appetite-stimulating) activity. The cysteine-rich C-terminal domain of this protein, corresponding to AGRP(87-132), contains five disulfide bonds and exhibits receptor binding affinity and antagonism equivalent to that of the full-length protein. The three-dimensional structure of this domain has been determined by 1H NMR at 800 MHz. The first 34 residues of AGRP(87-132) are well-ordered and contain a three-stranded antiparallel beta sheet, where the last two strands form a beta hairpin. The relative spatial positioning of the disulfide cross-links demonstrates that the ordered region of AGRP(87-132) adopts the inhibitor cystine knot (ICK) fold previously identified for numerous invertebrate toxins. Interestingly, this may be the first example of a mammalian protein assigned to the ICK superfamily. The hairpin's turn region presents a triplet of residues (Arg-Phe-Phe) known to be essential for melanocortin receptor binding. The structure also suggests that AGRP possesses an additional melanocortin-receptor contact region within a loop formed by the first 16 residues of its C-terminal domain. This specific region shows little sequence homology to the corresponding region of the agouti protein, which is an MC1R antagonist involved in pigmentation. Consideration of these sequence differences, along with recent experiments on mutant and chimeric melanocortin receptors, allows us to postulate that this loop in the first 16 residues of its C-terminal domain confers AGRP's distinct selectivity for MC3R and MC4R.     

Molecular interactions of nonpeptide agonists and antagonists with the melanocortin-4 receptor

The melanocortin-4 (MC4) receptor is a potential therapeutic target for obesity and cachexia, for which nonpeptide agonists and antagonists are being developed, respectively. The aim of this study was to identify molecular interactions between the MC4 receptor and nonpeptide ligands, and to compare the mechanism of binding between agonist and antagonist ligands. Nonpeptide ligand interaction was affected by mutations that reduce peptide ligand binding (D122A, D126A, S190A, M200A, F261A, and F284A), confirming overlapping binding determinants for peptide and nonpeptide ligands. The common halogenated phenyl group of nonpeptide ligands was a determinant of F261A and F284A mutations' affinity-reducing effect, implying this group interacts with the aromatic side chains of these residues. All affected compounds contain this group, the mutations reduced binding of 2,4-dichloro-substituted compounds more than 4-chloro-substituted-compounds, and F284A mutation eliminated the affinity-enhancing effect of 2-chloro-substitution. F261A and F284A mutations reduced the affinity of antagonists more than agonists, suggesting that the stronger ligand interaction with these residues, the lower the ligand efficacy. Supporting this hypothesis, F261A mutation increased the efficacy of nonpeptide antagonist and partial agonist ligands. D122A and D126A mutations reduced nonpeptide ligand interaction. Removing the ligands' derivatized amide group eliminated the effect of the mutations. Interaction of agonists, which bear a common amine within this group, was strongly reduced by D126A mutation (550-3300-fold), suggesting an electrostatic interaction between the amine and the acidic group of D126. These postulated interactions with aromatic and acidic regions of the MC4 receptor are consistent with a molecular model of the receptor. Furthermore, the strength of interaction with the aromatic pocket, and potentially the acidic pocket, controls the signaling efficacy of the ligand.     

Pharmacological and pharmacokinetic characterization of 2-piperazine-alpha-isopropyl benzylamine derivatives as melanocortin-4 receptor antagonists

A series of 2-piperazine-alpha-isopropylbenzylamine derivatives were synthesized and characterized as melanocortin-4 receptor (MC4R) antagonists. Attaching an amino acid to benzylamines 7 significantly increased their binding affinity, and the resulting compounds 8-12 bound selectively to MC4R over other melanocortin receptor subtypes and behaved as functional antagonists. These compounds were also studied for their permeability using Caco-2 cell monolayers and metabolic stability in human liver microsomes. Most compounds exhibited low permeability and high efflux ratio possibly due to their high molecular weights. They also showed moderate metabolic stability which might be associated with their moderate to high lipophilicity. Pharmacokinetic properties of these MC4R antagonists, including brain penetration, were studied in mice after oral and intravenous administrations. Two compounds identified to possess high binding affinity and selectivity, 10d and 11d, were studied in a murine cachexia model. After intraperitoneal (ip) administration of 1mg/kg dose, mice treated with 10d had significantly more food intake and weight gain than the control animals, demonstrating efficacy by blocking the MC4 receptor. Similar in vivo effects were also observed when 11d was dosed orally at 20mg/kg. These results provide further evidence that a potent and selective MC4R antagonist has potential in the treatment of cancer cachexia.     

Functional characterization of mutations in melanocortin-4 receptor associated with human obesity

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor implicated in the regulation of body weight. Genetic studies in humans have identified two frameshift mutations of MC4R associated with a dominantly inherited form of obesity. We have generated and expressed the corresponding MC4R mutants in 293T cells and found that cells transfected with the truncation mutants failed to exhibit agonist binding or responsiveness despite retention of structural motifs potentially sufficient for binding and signaling. Immunofluorescence studies showed that the mutant proteins were expressed and localized in the intracellular compartment but absent from the plasma membrane, suggesting that these mutations disrupted the proper cellular transport of MC4R. Further studies identified a sequence in the cytoplasmic tail of MC4R necessary for the cell surface targeting. We further investigated a possible dominant-negative activity of the mutants on wild-type receptor function. Co-transfection studies showed that the mutants affected neither signaling nor cell surface expression of wild-type MC4R. We also characterized three human sequence variants of MC4R, but these exhibited identical affinities for peptide ligands and identical agonist responsiveness. Thus, unlike the obesity-associated MC4R truncation mutants, the polymorphisms of MC4R are unlikely to be contributors to human obesity.     

Structure-activity relationships of piperazinebenzylamines as potent and selective agonists of the human melanocortin-4 receptor

SAR studies on a series of piperazinebenzenes directed toward the human melanocortin-4 receptor resulted in potent MC4R agonists. Replacement of the triazole moiety of an initial lead 4 by a basic nitrogen baring a lipophilic side-chain increased the binding affinities of these compounds. Analogs bearing an additional hetero-atom in the side-chain possessed good agonist potency. Thus, 11h had a Ki of 11 nM, and 13g exhibited an EC50 of 3.8 nM and a Ki of 6.4 nM.     

The agouti-related protein decapeptide (Yc[CRFFNAFC]Y) possesses agonist activity at the murine melanocortin-1 receptor

Agouti-related protein (AGRP) is a naturally occurring antagonist of the brain melanocortin receptors (MC3R and MC4R) and is physiologically implicated as participating in feeding behavior and energy homeostasis. The human AGRP decapeptide Yc[CRFFNAFC]Y has been previously reported as binding to the human MC3 and MC4 receptors and antagonizing the MC4 receptor. We have synthesized this decapeptide and pharmacologically characterized it at the murine melanocortin receptors and found it to possess MC4R antagonist activity (pA(2) = 6.8) and, unexpectedly, MC1R agonist activity (EC(50) = 2.89 microM). This study characterizes the first AGRP-based peptide agonist at the melanocortin receptors.     

Disulfide assignment of the C-terminal cysteine knot of agouti-related protein (AGRP) by direct sequencing analysis.

We have assigned the disulfide structure of Md-65 agouti-related protein (Md65-AGRP) using differential reduction and alkylation followed by direct sequencing analysis. The mature human AGRP is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The C-terminal domain, a 48 amino acid peptide named Md65-AGRP, was expressed in Escherichia coil cells and refolded under different conditions from the mature recombinant protein. The disulfide bonds in the cystine knot structure of Md65-AGRP were partially reduced using tris(2-carboxyethyl) phosphine (TCEP) under acidic conditions, followed by alkylation with N-ethylmaleimide (NEM). The procedure generated several isoforms with varying degrees of NEM alkylation. The multiple forms of Md65-AGRP generated by partial reduction and NEM modification were then completely reduced and carboxymethylated to identify unreactive disulfide bonds. Differentially labeled Md65-AGRP were directly sequenced and analyzed by MALDI mass spectrometry. The results confirmed that Md65-AGRP contained the same disulfide structure as that of Md5-AGRP reported previously [Bures, E. J., Hui, J. O., Young, Y. et al. (1998) Biochemistry 37, 12172-12177].     

Molecular determinants of human melanocortin-4 receptor responsible for antagonist SHU9119 selective activity

The hypothalamic melanocortin-4 receptor (MC4R), a seven transmembrane G-protein-coupled receptor, plays an important role in the regulation of body weight. The synthetic melanocortin analog SHU9119 has been widely used to characterize the physiological role of MC4R in feeding behavior and energy homeostasis. Previous studies indicated that SHU9119 is an agonist at the melanocortin-1 receptor (MC1R) but an antagonist at the MC4R. However, the molecular basis of the interaction between hMC4R and SHU9119 has not been clearly defined. To gain insight into the molecular determinants of hMC4R in the selectivity of SHU9119 chimeras and mutants hMC1R and hMC4R were expressed in cell lines and pharmacologically analyzed. A region of receptor containing the third transmembrane of hMC4R was found to be required for selective SHU9119 antagonism. Further mutagenesis studies of this region of hMC4R demonstrated that the amino acid residue leucine 133 in the third transmembrane was critical for the selective antagonist activity of SHU9119. The single substitution of leucine 133 to methionine did not affect SHU9119 binding to hMC4R. However, this substitution did convert SHU9119 from an antagonist to an agonist. Conversely, exchange of Met(128) in hMC1R to Leu, the homologous residue 133 of hMC4R, displayed a reduction in SHU9119 binding affinity and potency. This report provides the details of the molecular recognition of SHU9119 antagonism at hMC4R and shows that amino acid Leu(133) of hMC4R plays a key role in melanocortin receptor subtype specificity.     

The natural inverse agonist agouti-related protein induces arrestin-mediated endocytosis of melanocortin-3 and -4 receptors

Agouti-related protein (Agrp), one of the two naturally occurring inverse agonists known to inhibit G protein-coupled receptor activity, regulates energy expenditure by decreasing basal and blocking agonist-promoted melanocortin receptor (MCR) signaling. Here we report that, in addition to its inverse agonistic activities, Agrp exhibits agonistic properties on the endocytosis pathway of melanocortin receptors. Sustained exposure of human embryonic kidney 293 cells to Agrp induced endocytosis of the MC3R or the MC4R. The extent and kinetics of Agrp-promoted MCR endocytosis were similar to the endocytosis induced by melanocortins. Using the bioluminescence resonance energy transfer technique, we further showed that after binding of Agrp both MCRs interacted with beta-arrestins. In line with this observation, in COS-7 cells co-expression of beta-arrestins enhanced Agrp-induced MCR endocytosis, whereas in human embryonic kidney 293 cells co-transfection of beta-arrestin-specific small interference RNAs diminished Agrp-promoted endocytosis. This new regulatory mechanism was likewise detectable in a cell line derived from murine hypothalamic neurons endogenously expressing MC4R, pointing to the physiological relevance of Agrp-promoted receptor endocytosis. In conclusion, we demonstrated that Agrp does not solely act by directly blocking MCR signaling but also by reducing the amount of MCR molecules accessible to melanocortins at the cell surface. This beta-arrestin-dependent mechanism reveals a new aspect of MCR signaling in particular and refines the concept of G protein-coupled receptor antagonism in general.