Incorporation of Three Extracyclic Arginine Residues into a Melanocortin Macrocyclic Agonist (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Arg-Arg-Arg-Ac)-DPro]) Decreases Food Intake When Administered Intrathecally or Subcutaneously Compared to a Macrocyclic Ligand Lacking Extracyclic Arginine Residues (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro)].

Of the three Food and Drug Administration-approved melanocortin peptide drugs, two possess a cyclic scaffold, demonstrating that cyclized melanocortin peptides have therapeutic relevance. An extracyclic Arg residue, critical for pharmacological activity in the approved melanocortin cyclic drug setmelanotide, has also been demonstrated to increase the signal when fluorescently labeled cell-penetrating cyclic peptides are incubated with HeLa cells, with the maximal signal observed with three extracyclic Arg amino acids. Herein, a branching Lys residue was substituted into two macrocyclic melanocortin peptide agonists to incorporate 0-3 extracyclic Arg amino acids. Incorporation of the Arg residues resulted in equipotent or increased agonist potency at the mouse melanocortin receptors in vitro, suggesting that these substitutions were tolerated in the macrocyclic scaffolds. Further in vivo evaluation of one parent ligand (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-Pro]) and the three Arg derivative (c[Pro-His-DPhe-Arg-Trp-Dap-Lys(Ac-Arg-Arg-Arg)-Pro)] demonstrated that the three Arg derivative further decreased food intake compared to the parent macrocycle when the compounds were administered either via intrathecal injection or subcutaneous dosing. This suggests that three extracyclic Arg amino acids may be beneficial in the design of cyclic melanocortin ligands and that in vitro pharmacological profiling may not predict the in vivo efficacy of melanocortin ligands.

Discovery of a Pan-Melanocortin Receptor Antagonist [Ac-DPhe(pI)-Arg-Nal(2')-Orn-NH2] at the MC1R, MC3R, MC4R, and MC5R that Mediates an Increased Feeding Response in Mice and a 40-Fold Selective MC1R Antagonist [Ac-DPhe(pI)-DArg-Nal(2')-Arg-NH2].

Discovery of pan-antagonist ligands for the melanocortin receptors will help identify the physiological activities controlled by these receptors. The previously reported MC3R/MC4R antagonist Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2 was identified herein, for the first time, to possess MC1R and MC5R antagonist activity. Further structure-activity relationship studies probing the second and fourth positions were performed toward the goal of identifying potent melanocortin antagonists. Of the 21 tetrapeptides synthesized, 13 possessed MC1R, MC3R, MC4R, and MC5R antagonist activity. Three tetrapeptides were more than 10-fold selective for the mMC1R, including 8 (LTT1-44, Ac-DPhe(pI)-DArg-Nal(2')-Arg-NH2) that possessed 80 nM mMC1R antagonist potency and was at least 40-fold selective over the mMC3R, mMC4R, and mMC5R. Nine tetrapeptides were selective for the mMC4R, including 14 [SSM1-8, Ac-DPhe(pI)-Arg-Nal(2')-Orn-NH2] with an mMC4R antagonist potency of 1.6 nM. This compound was administered IT into mice, resulting in a dose-dependent increase in the food intake and demonstrating the in vivo utility of this compound series.

The Parallel Structure-Activity Relationship Screening of Three Compounds Identifies the Common Agonist Pharmacophore of Pyrrolidine Bis-Cyclic Guanidine Melanocortin-3 Receptor (MC3R) Small-Molecule Ligands.

The melanocortin receptors are involved in numerous physiological pathways, including appetite, skin and hair pigmentation, and steroidogenesis. In particular, the melanocortin-3 receptor (MC3R) is involved in fat storage, food intake, and energy homeostasis. Small-molecule ligands developed for the MC3R may serve as therapeutic lead compounds for treating disease states of energy disequilibrium. Herein, three previously reported pyrrolidine bis-cyclic guanidine compounds with five sites for molecular diversity (R1-R5) were subjected to parallel structure-activity relationship studies to identify the common pharmacophore of this scaffold series required for full agonism at the MC3R. The R2, R3, and R5 positions were required for full MC3R efficacy, while truncation of either the R1 or R4 positions in all three compounds resulted in full MC3R agonists. Two additional fragments, featuring molecular weights below 300 Da, were also identified that possessed full agonist efficacy and micromolar potencies at the mMC5R. These SAR experiments may be useful in generating new small-molecule ligands and chemical probes for the melanocortin receptors to help elucidate their roles in vivo and as therapeutic lead compounds.

Incorporation of Indoylated Phenylalanine Yields a Sub-Micromolar Selective Melanocortin-4 Receptor Antagonist Tetrapeptide.

The melanocortin family is involved in many physiological functions, including pigmentation, steroidogenesis, and appetite. The centrally expressed melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R) possess overlapping but distinct roles in energy homeostasis. Herein, the third and fourth positions of a tetrapeptide lead compound [Ac-Arg-Arg-(pI)DPhe-Tic-NH2], previously reported to possess MC3R agonist and MC4R antagonist activities, were substituted with indoylated phenylalanine (Wsf/Wrf) residues in an attempt to generate receptor subtype selective compounds. At the third position, d-amino acids were required for melanocortin agonist activity, while both l- and d-residues resulted in MC4R antagonist activity. These results indicate that l-indoylated phenylalanine residues at the third position of the scaffold can generate MC4R over MC3R selective antagonist ligands, resulting in a substitution pattern that may be exploited for novel MC4R ligands that can be used to probe the in vivo activity of the MC4R without involvement of the MC3R.

Functional Mixture-Based Positional Scan Identifies a Library of Antagonist Tetrapeptide Sequences (LAtTeS) with Nanomolar Potency for the Melanocortin-4 Receptor and Equipotent with the Endogenous AGRP(86-132) Antagonist.

The melanocortin-4 receptor (MC4R) plays an important role in appetite. Agonist ligands that stimulate the MC4R decrease appetite, while antagonist compounds increase food consumption. Herein, a functional mixture-based positional scan identified novel MC4R antagonist sequences. Mixtures comprising a library of 12,960,000 tetrapeptides were screened in the presence and absence of the NDP-MSH agonist. These results led to the synthesis of 48 individual tetrapeptides, of which 40 were screened for functional activity at the melanocortin receptors. Thirteen compounds were found to possess nanomolar antagonist potency at the MC4R, with the general tetrapeptide sequence Ac-Aromatic-Basic-Aromatic-Basic-NH2. The most notable results include the identification of tetrapeptide 48 [COR1-25, Ac-DPhe(pI)-Arg-Nal(2')-Arg-NH2], an equipotent MC4R antagonist to agouti-related protein [AGRP(86-132)], more potent than miniAGRP(87-120), and possessing 15-fold selectivity for the MC4R versus the MC3R. These tetrapeptides may serve as leads for novel appetite-inducing therapies to treat states of negative energy balance, such as cachexia and anorexia.

Discovery of Nanomolar Melanocortin-3 Receptor (MC3R)-Selective Small Molecule Pyrrolidine Bis-Cyclic Guanidine Agonist Compounds Via a High-Throughput "Unbiased" Screening Campaign.

The central melanocortin-3 and melanocortin-4 receptors (MC3R, MC4R) are key regulators of body weight and energy homeostasis. Herein, the discovery and characterization of first-in-class small molecule melanocortin agonists with selectivity for the melanocortin-3 receptor over the melanocortin-4 receptor are reported. Identified via "unbiased" mixture-based high-throughput screening approaches, pharmacological evaluation of these pyrrolidine bis-cyclic guanidines resulted in nanomolar agonist activity at the melanocortin-3 receptor. The pharmacological profiles at the remaining melanocortin receptor subtypes tested indicated similar agonist potencies at both the melanocortin-1 and melanocortin-5 receptors and antagonist or micromolar agonist activities at the melanocortin-4 receptor. This group of small molecules represents a new area of chemical space for the melanocortin receptors with mixed receptor pharmacology profiles that may serve as novel lead compounds to modulate states of dysregulated energy balance.

The melanocortin pathway and energy homeostasis: From discovery to obesity therapy.

BACKGROUND: Over the past 20 years, insights from human and mouse genetics have illuminated the central role of the brain leptin-melanocortin pathway in controlling mammalian food intake, with genetic disruption resulting in extreme obesity, and more subtle polymorphic variations influencing the population distribution of body weight. At the end of 2020, the U.S. Food and Drug Administration (FDA) approved setmelanotide, a melanocortin 4 receptor agonist, for use in individuals with severe obesity due to either pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency. SCOPE OF REVIEW: Herein, we chart the melanocortin pathway's history, explore its pharmacology, genetics, and physiology, and describe how a neuropeptidergic circuit became an important druggable obesity target. MAJOR CONCLUSIONS: Unravelling the genetics of the subset of severe obesity has revealed the importance of the melanocortin pathway in appetitive control; coupling this with studying the molecular pharmacology of compounds that bind melanocortin receptors has brought a new obesity drug to the market. This process provides a drug discovery template for complex disorders, which for setmelanotide took 25 years to transform from a single gene into an approved drug.

Discovery of Molecular Interactions of the Human Melanocortin-4 Receptor (hMC4R) Asp189 (D189) Amino Acid with the Endogenous G-Protein-Coupled Receptor (GPCR) Antagonist Agouti-Related Protein (AGRP) Provides Insights to AGRP's Inverse Agonist Pharmacology at the hMC4R.

The melanocortin receptors (MCRs) are important for numerous biological pathways, including feeding behavior and energy homeostasis. In addition to endogenous peptide agonists, this receptor family has two naturally occurring endogenous antagonists, agouti and agouti-related protein (AGRP). At the melanocortin-4 receptor (MC4R), the AGRP ligand functions as an endogenous inverse agonist in the absence of agonist and as a competitive antagonist in the presence of agonist. At the melanocortin-3 receptor (MC3R), AGRP functions solely as a competitive antagonist in the presence of agonist. The molecular interactions that differentiate AGRP's inverse agonist activity at the MC4R have remained elusive until the findings reported herein. Upon the basis of homology molecular modeling approaches, we previously postulated a unique interaction between the D189 position of the hMC4R and Asn114 of AGRP. To further test this hypothesis, six D189 mutant hMC4Rs (D189A, D189E, D189N, D189Q, D189S, and D189K) were generated and pharmacologically characterized resulting in the discovery of differences in inverse agonist activity of AGRP and an 11 macrocyclic compound library. These data support the hypothesized interaction between the hMC4R D189 position and Asn114 residue of AGRP and define critical ligand-receptor molecular interactions responsible for the inverse agonist activity of AGRP at the hMC4R.

Multiresidue Tetrapeptide Substitutions Yield a 140-fold Selective Melanocortin-3 over Melanocortin-4 Receptor Agonist.

The five melanocortin receptors regulate numerous physiological functions. Although many ligands have been developed for the melanocortin-4 receptor (MC4R), the melanocortin-3 receptor (MC3R) has been less-well characterized, in part due to the lack of potent, selective tool compounds. Previously an Ac-His-Arg-(pI)DPhe-Tic-NH2 scaffold, inverting the Phe-Arg motif of the native melanocortin signal sequence, was identified to possess mMC3R over mMC4R selective agonist activity. In this study, a library of 12 compounds derived from this scaffold was synthesized and assayed at the mouse melanocortin receptors (MCRs), utilizing substitutions previously shown to increase mMC3R agonist potency and/or selectivity. One compound (8, Ac-Val-Gln-DBip-DTic-NH2) was identified as greater than 140-fold selective for the mMC3R over the mMC4R, possessed 70 nM potency at the mMC3R, and partially stimulated the mMC4R at 100 µM concentrations without antagonist activity. This pharmacological profile may be useful in developing new tool and therapeutic ligands that selective signal through the MC3R.

Peptoid NPhe4 in AGRP-Based c[Pro1-Arg2-Phe3-Phe4-Xxx5-Ala6-Phe7-DPro8] Scaffolds Maintain Mouse MC4R Antagonist Potency.

The melanocortin receptors are involved in numerous physiological functions and are regulated by agonists derived from the proopiomelanocortin gene transcript and two endogenous antagonists, agouti and agouti-related protein (AGRP). The key binding and functional determinant of AGRP, an MC3R and MC4R antagonist, is an Arg-Phe-Phe tripeptide sequence located on an exposed hexapeptide (Arg-Phe-Phe-Asn-Ala-Phe) loop. It has previously been observed that cyclizing this sequence through a DPro-Pro motif (c[Pro1-Arg2-Phe3-Phe4-Asn5-Ala6-Phe7-DPro8]) resulted in a macrocyclic scaffold with MC4R antagonist activity, with increased MC4R potency when a diaminopropionic acid (Dap) residue is substituted at position 5. In this report, a series of 11 single-peptoid substitutions were performed in the AGRP-derived macrocycles. While most peptoid substitutions decreased MC4R antagonist potency, it was observed that NPhe4 (compounds 4 and 11) or NDab5 (diaminobutyric acid, compound 7) maintained MC4R antagonist potency. The NPhe4 substitutions also resulted in MC5R antagonist and inverse agonist activity equipotent to the parent scaffolds. These data may be used in the design of future MC4R and MC5R antagonist leads and probes that possess increased metabolic stability due to the presence of peptoid residues.

Comparative Intracerebroventricular and Intrathecal Administration of a Nanomolar Macrocyclic Melanocortin Receptor Agonist MDE6-5-2c (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro]) Decreases Food Intake in Mice.

There is a critical need to find safe therapeutics to treat an increasingly obese population and diseases associated with an imbalance in energy homeostasis. The melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) ligands have long been the focus to help scientists understand energy homeostasis and the regulation of feeding behavior. Herein, we use a nanomolar macrocyclic melanocortin receptor agonist ligand MDE6-5-2c (c[Pro-His-DPhe-Arg-Trp-Dap-Ala-DPro) to examine metabolic and energy hemostasis profiles upon intrathecal (IT) administration directly into the spinal cord as compared to intracerebroventricular (ICV) administration directly into the brain. Overall, central ICV administration of MDE6-5-2c resulted in decreased food intake, in a dose-dependent manner, and decreased respiratory exchange ratio (RER). Comparison of IT versus ICV routes of MDE6-5-2c administration resulted in MDE6-5-2c possessing a longer duration of action on both feeding behavior and RER via IT. The C-peptide, ghrelin, GIP, leptin, IL-6, and resistin plasma hormones and biomarkers were compared using IT versus ICV MDE6-5-2c routes of administration. Plasma resistin levels were decreased upon ICV treatment of MDE6-5-2c, as compared to ICV vehicle control treatment. Intrathecal treatment resulted in significantly decreased inflammatory cytokine interleukin-6 (IL-6) levels compared to ICV administration. Investigation of the nonselective MC3R and MC4R macrocyclic agonist MDE6-5-2c molecule revealed differences in food intake, RER, and plasma biomarker profiles based upon ICV or IT routes of administration and characterize this novel molecular chemotype as a molecular probe to study the melanocortin system in vivo.

Single Nucleotide Polymorphisms in the Melanocortin His-Phe-Arg-Trp Sequences Decrease Tetrapeptide Potency and Efficacy.

The melanocortin receptors are stimulated by agonists (α-MSH, ß-MSH, γ-MSH, and ACTH) processed from the proopiomelanocortin (POMC) gene transcript and possess a common His-Phe-Arg-Trp tetrapeptide sequence critical for receptor activation. Deficiency in POMC signaling in humans is associated with adrenal insufficiency, altered pigmentation, and rapid, early onset weight gain. Herein, 12 single nucleotide polymorphisms (SNPs) deposited into the Variation Viewer database within the His-Phe-Arg-Trp sequences of ACTH/α-MSH, ß-MSH, and γ-MSH were substituted into tetrapeptide scaffolds to examine the in vitro signaling effects of these polymorphisms at the cloned melanocortin receptors. Every polymorphism decreased agonist potency and/or efficacy at the melanocortin receptors assayed, indicating that polymorphisms within the signaling sequence of POMC-derived agonists negatively impacts receptor activation. Future work to incorporate these substitutions into the full-length POMC agonists would confirm these findings, identifying new patient populations that might benefit from therapeutic regiments to treat POMC-deficient signaling.

Incorporation of Agouti-Related Protein (AgRP) Human Single Nucleotide Polymorphisms (SNPs) in the AgRP-Derived Macrocyclic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-dPro] Decreases Melanocortin-4 Receptor Antagonist Potency and Results in the Discovery of Melanocortin-5 Receptor Antagonists.

While the melanocortin receptors (MCRs) are known to be involved in numerous biological pathways, the potential roles of the MC5R have not been clearly elucidated in humans. Agouti-related protein (AgRP), an MC3R/MC4R antagonist and MC4R inverse agonist, contains an exposed ß-hairpin loop composed of six residues (Arg-Phe-Phe-Asn-Ala-Phe) that is imperative for binding and function. Within this active loop of AgRP, four human missense polymorphisms were deposited into the NIH Variation Viewer database. These polymorphisms, Arg111Cys, Arg111His, Phe112Tyr, and Ala115Val (AgRP full-length numbering), were incorporated into the peptide macrocycles c[Pro1-Arg2-Phe3-Phe4-Xaa5-Ala6-Phe7-dPro8], where Xaa was Dap5 or Asn5, to explore the functional effects of these naturally occurring substitutions in a simplified AgRP scaffold. All peptides lowered potency at least 10-fold in a cAMP accumulation assay compared to the parent sequences at the MC4Rs. Compounds MDE 6-82-3c, ZMK 2-82, MDE 6-82-1c, ZMK 2-85, and ZMK 2-112 are also the first AgRP-based chemotypes that antagonize the MC5R.

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.

Synergistic Multiresidue Substitutions of a Macrocyclic c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-dPro] Agouti-Related Protein (AGRP) Scaffold Yield Potent and >600-Fold MC4R versus MC3R Selective Melanocortin Receptor Antagonists.

Antagonist ligands of the melanocortin-3 and -4 receptors (MC3R, MC4R), including agouti-related protein (AGRP), are postulated to be targets for the treatment of diseases of negative energy balance. Previous studies reported the macrocyclic MC3R/MC4R antagonist c[Pro1-Arg2-Phe3-Phe4-Asn5-Ala6-Phe7-dPro8], which is 250-fold less potent at the mouse (m) mMC3R and 3-fold less potent at the mMC4R than AGRP. Previous studies explored the structure-activity relationships around individual positions in this template. Herein, a multiresidue substitution strategy is utilized, combining the lead sequence with hPhe4, Dap5, Arg5, Ser6, and Nle7 substitutions previously reported. Two compounds from this study (16, 20) contain an hPhe4/Ser6/Nle7 substitution pattern, are 3-6-fold more potent than AGRP at the mMC4R and are 600-800-fold selective for the mMC4R over the mMC3R. Another lead compound (21), possessing the hPhe4/Arg5 substitutions, is only 5-fold less potent than AGRP at the mMC3R and is equipotent to AGRP at the mMC4R.

Arg-Phe-Phe d-Amino Acid Stereochemistry Scan in the Macrocyclic Agouti-Related Protein Antagonist Scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro] Results in Unanticipated Melanocortin-1 Receptor Agonist Profiles.

The melanocortin-3 and melanocortin-4 receptors (MC3R and MC4R), endogenous agonists derived from the proopiomelanocortin gene transcript, and naturally occurring antagonists agouti and agouti-related protein (AGRP) have been linked to biological pathways associated with energy homeostasis. The active tripeptide sequence of AGRP, Arg111-Phe112-Phe113, is located on a hypothesized ß-hairpin loop. Herein, stereochemical modifications of the Arg-Phe-Phe sequence were examined in the octapeptide AGRP-derived macrocyclic scaffold c[Pro-Arg-Phe-Phe-Xxx-Ala-Phe-DPro], where Xxx was Asn or diaminopropionic acid (Dap). Macrocyclic peptides were synthesized with one, two, or three residues of the Arg-Phe-Phe sequence substituted with the corresponding d-isomer(s), generating a 14 compound library. While l-to-d inversions of the Arg-Phe-Phe sequence in a 20-residue AGRP-derived ligand previously resulted in agonist activity at the MC1R, MC3R, MC4R, and MC5R, only the MC1R was consistently stimulated by the macrocyclic ligands in the present study, with varying ligand potencies and efficacies observed at the MC1R. A general trend of increased MC4R antagonist potency was observed for Dap-containing compounds, while MC5R inverse agonist activity was observed for select ligands. It was observed that stereochemical modification of the Arg-Phe-Phe active tripeptide sequence was insufficient to convert melanocortin antagonist into agonists. Overall, these observations are important in the design of melanocortin ligands possessing potent and selective agonist and antagonist activities.

A Review of Single-Nucleotide Polymorphisms in Orexigenic Neuropeptides Targeting G Protein-Coupled Receptors.

Many physiological pathways are involved in appetite, food intake, and the maintenance of energy homeostasis. In particular, neuropeptides within the central nervous system have been demonstrated to be critical signaling molecules for modulating appetite. Both anorexigenic (appetite-decreasing) and orexigenic (appetite-stimulating) neuropeptides have been described. The biological effects of these neuropeptides can be observed following central administration in animal models. This review focuses on single nucleotide polymorphisms (SNPs) in six orexigenic neuropeptides: agouti-related protein (AGRP), galanin, melanin concentrating hormone (MCH), neuropeptide Y (NPY), orexin A, and orexin B. Following a brief summary of the neuropeptides and their orexigenic activities, reports associating SNPs within the orexigenic neuropeptides to energy homeostasis, food intake, obesity, and BMI in humans are reviewed. Additionally, the NIH tool Variation Viewer was utilized to identify missense SNPs within the mature, biologically active neuropeptide sequences. For SNPs found through Variation Viewer, a concise discussion on relevant pharmacological structure-activity relationship studies for select SNPs is included. This review is meant to update reported orexigenic neuropeptide SNPs and demonstrate the potential utility of genomic sequence databases for finding SNPs that may result in altered receptor signaling for neuropeptide pathways associated with appetite.

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.

Structure-Activity Relationship Studies of a Macrocyclic AGRP-Mimetic Scaffold c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro] Yield Potent and Selective Melanocortin-4 Receptor Antagonists and Melanocortin-5 Receptor Inverse Agonists That Increase Food Intake in Mice.

The melanocortin system has five receptors, and antagonists of the central melanocortin receptors (MC3R, MC4R) are postulated to be viable therapeutics for disorders of negative energy balance such as anorexia, cachexia, and failure to thrive. Agouti-related protein (AGRP) is an antagonist of the MC3R and an antagonist/inverse agonist of the MC4R. Biophysical NMR-based structural studies have demonstrated that the active sequence of this hormone, Arg-Phe-Phe, is located on an exposed ß-hairpin loop. It has previously been demonstrated that the macrocyclic octapeptide scaffold c[Pro1-Arg2-Phe3-Phe4-Asn5-Ala6-Phe7-DPro8] is 16-fold less potent than AGRP at the mouse MC4R (mMC4R). Herein it was hypothesized that the Phe7 position may be substituted to produce more potent and/or selective melanocortin receptor antagonist ligands based on this template. A 10-membered library was synthesized that substituted small (Gly), polar (Ser), acidic (Asp), basic (Lys), aliphatic (Leu, Nle, and Cha), and aromatic (Trp, Tyr, hPhe) amino acids to explore potential modifications at the Phe7 position. The most potent mMC4R antagonist contained a Nle7 substitution, was equipotent to the lead ligand 200-fold selective for the mMC4R over the mMC3R, and caused a significant increase in food intake when injected intrathecally into male mice. Three compounds possessed sigmoidal dose-response inverse agonist curves at the mMC5R, while the remaining seven decreased cAMP production from basal levels at a concentration of 100 µM. These findings will add to the knowledge base toward the development of potent and selective probes to study the role of the melanocortin system in diseases of negative energy balance and can be useful in the design of molecular probes to examine the physiological functions of the mMC5R.