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Research ArticleAugust 20, 2014

Synthesis, Biophysical, and Pharmacological Evaluation of the Melanocortin Agonist AST3-88: Modifications of Peptide Backbone at Trp 7 Position Lead to a Potent, Selective, and Stable Ligand of the Melanocortin 4 Receptor (MC4R)
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ACS Chemical Neuroscience

Cite this: ACS Chem. Neurosci. 2014, 5, 10, 1020–1031

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https://doi.org/10.1021/cn5000953

Published August 20, 2014

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Abstract

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The melanocortin-3 (MC3R) and melanocortin-4 (MC4R) receptors are expressed in the brain and are implicated in the regulation of food intake and energy homeostasis. The endogenous agonist ligands for these receptors (α-, β-, γ-MSH and ACTH) are linear peptides with limited receptor subtype selectivity and metabolic stability, thus minimizing their use as probes to characterize the overlapping pharmacological and physiological functions of the melanocortin receptor subtypes. In the present study, an engineered template, in which the peptide backbone was modified by a heterocyclic reverse turn mimetic at the Trp⁷ residue, was synthesized using solid phase peptide synthesis and characterized by a β-galactosidase cAMP based reporter gene assay. The functional assay identified a ∼5 nM mouse MC4R agonist (AST3-88) with more than 50-fold selectivity over the mMC3R. Biophysical studies (2D ¹H NMR spectroscopy and molecular dynamics) of AST3-88 identified a type VIII β-turn secondary structure spanning the pharmacophore domain stabilized by the intramolecular interactions between the side chains of the His and Trp residues. Enzymatic studies of AST3-88 revealed enhanced stability of AST3-88 over the α-MSH endogenous peptide in rat serum. Upon central administration of AST3-88 into rats, a decreased food intake response was observed. This is the first study to probe the in vivo physiological activity of this engineered peptide-heterocycle template. These findings advance the present knowledge of pharmacophore design for potent, selective, and metabolically stable melanocortin ligands.

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The melanocortin receptor (MCR) family is a member of the G-protein coupled receptor (GPCR) superfamily that stimulates the adenylate cyclase signal transduction pathway and contains five melanocortin receptor subtypes (MC1–5R). (1-6) The melanocortin system includes the endogenous agonists α-, β-, and γ-melanocortin stimulating hormone (MSH) and adrenocorticotropic hormone (ACTH) which are derived from posttranslational modifications of the POMC gene. (7, 8) Two endogenous antagonists [agouti related protein (AGRP) (9) and agouti (10)] have been discovered to antagonize the centrally expressed MC3R and MC4Rs. (11) All the endogenous agonists share a common His-Phe-Arg-Trp (6–9; α-MSH numbering) pharmacophore domain in their primary amino acid sequence. (12-14) This tetrapeptide sequence is postulated to be important for melanocortin receptor molecular recognition and ligand-induced receptor activation. (15-18) The MC1R, expressed in melanocytes, is involved in the regulation of skin and hair pigmentation. (1, 2, 19) The MC2R is stimulated only by the ACTH agonist and is expressed in the adrenal cortex to regulate steroidogenesis. (1) The MC3R is expressed in the gut, placenta, heart, and brain. The MC3R has been reported to be involved in metabolism and energy homeostasis, via mechanism(s) remaining to be characterized. (3, 20-22) The MC4R is expressed primarily in the brain and regulates feeding behavior, energy homeostasis, and sexual function. (5, 23-25) The MC5R is expressed in a wide variety of tissues, both centrally and peripherally, and is involved in exocrine gland function in mice. (6, 26, 27) The mouse MC3R and MC4R are expressed in the brain and have 60% similarity in primary amino acid sequence, but possess distinct ligand pharmacological profiles. Central activation of the MC3R and MC4R is postulated to mediate the effects of the melanocortin pathway on energy homeostasis as both the MC3R knockout (KO) (21, 22) and MC4R KO mice show alterations in energy balance. (25) Intracerebroventricular (ICV) administration of the synthetic melanocortin agonist (MTII) decreased food intake, while both the endogenous AGRP antagonist and the synthetic SHU9119 antagonist increased food intake. (23, 28)

Single nucleotide polymorphisms (SNPs) of the hMC4R have been identified in human patients with severe obesity. Farooqi et al. investigated children with severe obesity (under the age of 10) and found that ∼6% of these early onset childhood obesity patients possessed hMC4R single nucleotide polymorphisms (SNPs). (29, 30) To date, greater than 100 SNPs have been reported in human patients. Seventy of these SNPs have been characterized in vitro (cell culture) in attempts to identify the putative underlying molecular mechanistic defects resulting from the amino acid change(s) in the hMC4R. (31-33) In 2011, the MC3R was postulated to be involved in the regulation of food intake by using a mixed pharmacology ligand that possessed partial agonist/antagonist activity at the mMC3R and full agonist activity at the mMC4R. (28) Intracerebroventricular administration of this compound in the wild type, MC3RKO, and MC4RKO mice resulted in decreased food intake. (28) These, and other genetic and pharmacological studies (involving both animals and humans), support the role of the central melanocortins in the regulation of satiety and energy homeostasis. Selective ligands are needed to understand underlying molecular mechanism(s) by which the MC3R and the MC4R regulate energy balance. The discovery and use of these molecular probes to identify and characterize physiological functions associated with the MCR system are still being sought after.

In continuous efforts to identify potent, selective, and enzymatically stable melanocortin ligands, we have incorporated a bioactive small molecule heterocycle into a peptide in attempts to overcome the inherent problems associated with the endogenous peptides that may limit their consideration for drug development. The strategy applied modifies the peptide backbone resulting in changes in intra- and/or intermolecular interactions postulated to contribute to melanocortin receptor preferred “bioactive” conformation(s) while providing increased protease stability. We have previously reported systematic exploration of the reverse turn mimetic template studied herein with the His⁶, Phe⁷, and Arg⁸ residue domain modifications (α-MSH numbering) that resulted in the identification of a potent agonist ligand at the mMC4R (AMW610). (34) However, this compound was only ∼5-fold more selective for the mMC4R versus the mMC3R.

The aromatic indole side chain of the Trp amino acid, a constituent of the melanocortin agonist pharmacophore His⁶-Phe⁷-Arg⁸-Trp⁹ (α-MSH numbering), has been postulated to play a key role in ligand–receptor interactions. (13, 14, 35, 36) Substitution of the Trp residue by an Ala amino acid in various peptide templates was reported to reduce agonist potency at the melanocortin receptor subtypes, particularly at the MC3R. (37-39) Therefore, we hypothesized that the modifications of this Trp residue side chain, in the template presented herein, may lead to potent and/or selective analogues at the melanocortin receptors. It has also been postulated that a peptide-heterocyclic scaffold could lead to conformationally constrained ligands that might be more stable for in vitro and in vivo studies as compared to their linear endogenous counterpart(s). The aim of this study was to (i) elucidate the role of the Trp amino acid side chain in the peptide-heterocyclic scaffold in attempts to obtain potent and selective ligands and (ii) study the scope of this novel template on serum stability over the endogenous α-MSH peptide ligand. Peptides, in general, can be promising therapeutic agents by having a number of advantages over small molecules, in terms of specificity and affinity for targets. However, general endogenous peptide low protease stability often traditionally limits their further consideration in drug development. Standard approaches to make peptides less susceptible to serum proteases, for example, using modifications with unnatural amino acids, d-amino acids, β-amino acids, cyclization of peptide chain at the termini, modifications of the peptide backbone, and so forth, have been well established. These approaches often make the peptide structure more constrained and therefore less prone to protease degradation in serum. In the present study, we compared the compound containing a heterocyclic-peptide backbone modification and a disulfide bridge at the N- and C-terminus, which can restrict a peptide’s backbone conformation and modify their stability in serum. In addition, in vivo experiments were performed to assess the physiological effect on food intake of this new chemical probe. Central administration of AST3-88 decreased a cumulative feeding response in rats 48 h post-ICV treatments. Herein, we report the design, functional, and structural characterization of a potent and selective MC4R full agonist and present the studies of proteolytic stability and in vivo properties of this template for the first time. Taken together, this study provides new information on the structural prerequisites for potent, selective, and increased serum stable melanocortin ligands.

Results and Discussion

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The development of MC3R and MC4R selective ligands has the therapeutic potential for the treatment of body weight and feeding related disorders. A synergistic role of the MC3R in conjunction with the MC4R for feeding and energy homeostasis are emerging. (28, 40) Thus, ligands and molecular probes that can discriminate in a subtype specific manner (agonist and/or antagonist), and that can be used to probe the physiological roles of melanocortin receptor subtypes are still needed in the field. Toward this goal, a number of small molecules and peptide ligands have been explored and reported in the literature by a plethora of academic and industrial research laboratories. Our strategy in this study is to combine the use of a bioactive small molecule and incorporate it into a potent cyclic peptide template in attempts to gain further insight into melanocortin receptor molecular recognition, selectivity, and increase protease stability. We report herein the AST3-88 melanocortin agonist that possesses a selective MC4R agonist profile over the MC3R (>50-fold), enhanced serum stability, and results in decreased food intake in vivo.

Synthesis, Structural, and Functional Characterization

The compound AST3-88, and its stereoisomer AMW6103, were synthesized using previously reported method(s) and is summarized in Scheme 1. (34, 41) To probe the importance of the heterocyclic stereochemistry within the ring moiety, the Cys α-carbons (marked with * in Figure 1) in both AST3-88 (l-configuration) and AMW6103 (d-configuration) were designed and synthesized. The Fmoc-Trp(Boc)-aldehyde was synthesized from the corresponding acid via Fmoc-Trp(Boc)-amide and introduced via a reductive amination method to the resin bound peptide chain. The thioether ring was formed on the resin by mild on-bead cyclization method. (34, 41) After completion of the synthesis, the ligand was cleavage and the crude peptide was dissolved in a 20% DMSO/water solution and stirred at room temperature to form the disulfide-bridge. The peptides AST3-88 and AMW6103 possessed the correct molecular weights as determined by mass spectrometry. The purity of these peptides (>95%) was assessed by analytical RP-HPLC in two diverse solvent systems (data provided in the experimental section).

Figure 1. (A) Illustration of the compounds (i) heterocycle, (ii) AMW610, (iii) AST3-88, and (iv) AMW6103. The stereochemistry of the thioether ring is represented by an asterisk (*). Compounds AST3-88 and AMW610 contain l-Cys, and compound AMW6103 contains a d-Cys at this position. The side chain of His is depicted in green, DPhe in orange, Arg in blue, the heterocycle moiety in pink, and the disulfide bridge is circled in yellow. (B) Amino acid sequences of key endogenous and synthetic melanocortin peptides.

The compounds were tested for agonist functional activity at the mouse MC1R, MC3R, MC4R, and MC5R using a 96-well cAMP based β-galactosidase reporter gene bioassay (Table 1). (42) The NDP-MSH (Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) peptide (43) is one of the standard melanocortin agonists used in the study of melanocortin receptors and was included herein as a reference control and as an internal control for maximal ligand efficacy (100%). The pharmacology of the heterocyclic moiety alone and peptides AMW3-130 and AMW610 (Figure 1) were also included for pharmacological comparisons of the different but related templates. The AMW3-130 molecule was designed as a chimeric cyclic peptide template that incorporated the AGRP based antagonist scaffold that upon substitution of the AGRP Arg-Phe-Phe pharmacophore residues with the agonist His-DPhe-Arg-Trp pharmacophore resulted in the conversion of a weak antagonist into a potent sub-nanometer agonist that was able to functionally rescue polymorphic hMC4R SNPs. (33) In 2002, the heterocyclic moiety (44) (Figure 1) was reported to possess nanomolar MCR functional agonist activity and initial studies were performed incorporating this moiety into the AMW3-130 peptide template. (34, 41) The first study identified the AMW610 template (Figure 1) as possessing the most potent nanomolar MCR full agonist functionality of the SAR at the time. (34) Biophysical experiments, similar to the ones reported herein, were performed in attempts to correlate SAR and identify solution-phase structural differences between the peptides containing different orientations of the heterocycle. (8, 34) Thus, for structural and functional comparative purposes, the AMW3-130 and AMW610 ligands have been included herein.

Table 1. Agonist EC₅₀ (nM) Pharmacology of the Modified Peptides at the Mouse Melanocortin Receptors^a

	mMC1R		mMC3R		mMC4R		mMC5R		selectivity ratio
peptide	EC₅₀ (nM)	fold change	EC₅₀ (nM)	fold change	EC₅₀ (nM)	fold change	EC₅₀ (nM)	fold change	MC4R vs MC3R
NDP-MSH	0.018 ± 0.003		0.14 ± 0.03		0.20 ± 0.03		0.30 ± 0.05		1
AMW3-130^b	0.35 ± 0.17		2.00 ± 0.45		0.27 ± 0.09		2.32 ± 0.57		7
heterocycle	164 ± 22		7600 ± 1890		650 ± 126		335 ± 106		12
AMW610^c	33 ± 9	1	495 ± 215	1	85 ± 13	1	72 ± 16	1	9
AST3-88	68.8 ± 17.9	2	255 ± 24	(2)	4.7 ± 0.49	(18)	11.9 ± 2.3	(6)	54
AMW6103	0.81 ± 0.25	(41)	5300 ± 4300	11	440 ± 220	5	31.0 ± 16.2	(2)	12

The indicated errors represent the standard error of the mean determined from at least three independent experiments. Parentheses indicate that an increase in potency has resulted. Changes less than 3-fold are considered to be within the inherent experimental assay error.

The AGRP-melanocortin chimeric peptide AMW3-130 has been previously reported in ref 34 as compound 1.

The AMW610 compound has been previously reported in ref 34 as compound 7.

At the mMC1R and mMC3R, AST3-88 resulted in an equipotent compound, within the 3-fold inherent experimental error, as compared to AMW610. At the mMC4R, AST3-88 resulted in a ∼5 nM full agonist that is 18-fold more potent at this receptor than AMW610. At the mMC5R, AST3-88 resulted in 6-fold increased agonist potency as compared to AMW610. The AMW6103 ligand, possessing the D-configuration at the Cys (6) residue of the heterocyclic ring (Figure 1), resulted in an agonist with sub nM potency at the mMC1R and equipotent to the cyclic AMW3-130 peptide that lacks the heterocyclic moiety. The AMW6103 ligand also is ∼40-fold more potent at the mMC1R and possessed 11- and 5-fold decreased melanocortin receptor agonist potency at the mMC3R and mMC4Rs, respectively, as compared to the AMW610 template. Compound AMW6103 is a full agonist and equipotent to AMW610 at the mMC5R. The AST3-88 ligand, which has l- configuration at the ring, in general, is more active than its d-counterpart (AMW6103) at the melanocortin receptor isoforms, with the exception of mMC1R, where AMW6103 is 80-fold more potent than the AST3-88. These results are consistent with our earlier study (34) where the l-configured analogues generally were more potent than their d-counterparts. Furthermore, the AST3-88 is more than 50-fold selective at the mMC4R than the mMC3R, versus the AMW6103 that only 12-fold selective and possessed decreased agonist potency. The MC4R to MC3R selectivity of the control compounds AMW610, AMW3-130, and heterocyclic ring itself ranged from 7- to 12-fold (Table 1). (34, 41)

Biophysical Structural Studies (NMR and CAMM)

To study the solution-phase structural differences, and how these subtle conformational differences might be associated with changes in the observed pharmacology of these two AST3-88 and AMW6103 analogues, we performed 2D ¹H NMR and computer assisted molecular modeling (CAMM) experiments. Both compounds were dissolved in ∼50% (v/v) acetonitrile-d₃ and ∼50% H₂O for NMR experiments. This solvent system has been previously used by our laboratory on similar peptide template(s) and was used herein so that structures identified from this study could be compared with previous studies under similar experimental conditions. (34, 41) The criteria used for this particular solvent selection have been previously reported and are included herein: “1) it must completely dissolve all the peptides examined that possessed different biophysical properties, 2) similar solvents have been used previously so that structural comparisons could be performed and discussed, and 3) the amide protons were well resolved in this system versus the other solvents attempted. Other solvents that were attempted but did not result in all the peptides examined being fully dissolved included (i) DMSO-d₆, (ii) CDCl₃/DMSO, (iii) CD₃CN/H₂O, (iv) MeOH-d₃, (v) MeOH/DMSO, (vi) H₂O/D₂O.” (41) Unfortunately, the AMW6103 compound, in all the different solvent systems we examined and reported previously (indicated above), was unable to result in observable and resolvable peaks necessary for proton assignments, and therefore, this ligand was excluded from further structural studies. The ¹H NMR and chemical shift assignments (ppm) for analogue AST3-88 (assigned in the ∼50% (v/v) acetonitrile-d₃ and ∼50% H₂O solvent system) are provided in the Supporting Information (Table S1). Use of this solvent system and similar experimental conditions provided the opportunity to directly compare the structures of AST3-88 with those of AMW610 and AMW3-130. (8, 34)

In the present study, we compare shifts of specific amino acids and the global patterns of one peptide to another. Figure 2 is a histogram of the difference between experimental and reference random-coil (Δ-RC) shift (45) values for the conserved His-DPhe-Arg-Trp-Asn-Ala region of compounds AST3-88 and AMW610 and AMW3-130. The random coil shift values are averages of overall possible conformations that an amino acid can adopt in the random coil. Wishart and Sykes have published a complete set of ¹H, ¹³C, and ¹⁵N RC shift values that can be used as references in order to detect regions in the peptide or protein which are not in random coil conformations. (45) Given that the compounds have exactly the same amino acids and that they differ only in the position or presence of the heterocycle ring, there are significant differences in random coil shift values between them. The comparison of chemical shifts from one compound to other demonstrates that the chemical shifts of these residues can differ significantly, presumably indicating different conformational structure(s). The random coil shift values of the backbone NH protons in AST3-88 and AMW3-130, especially in the His, DPhe, Arg, and Trp region, have negative random coil shift values between 0.2 and 0.3 ppm. This suggests that the AST3-88 may have a regular backbone conformation with secondary structure in the pharmacophore region similar to AMW3-130, which has been reported to have a reverse turn in the similar region. (34) Following resonance assignments, we determined the nuclear Overhauser effect (NOE) values of the residues, providing an estimate of the distance between pairs of protons closer than about 5 Å in space. The NOE patterns can be used as a tool to identify secondary structure in peptides and/or to distinguish structural differences between analogues. Figure 3 summarizes the NOE intensities observed for the AST3-88 along with the comparator compounds AMW610 and AMW3-130 (taken from ref 34). Several short- and long-range NOE connectivities were observed for AST3-88 in the pharmacophore domain indicating that the region around the His-DPhe-Arg-Trp amino acids possesses a regular secondary structure. To examine the solution based conformational structure(s) of AST3-88 and compare its differences from peptides AMW3-130 and AMW610, restrained molecular dynamics simulation (RMD) experiments were performed. The computational molecular dynamics simulations, based upon NMR experimental values, can be used to provide information on the fluctuations and conformational changes of the peptides examined. This method allows the prediction of the static and dynamic properties of molecules from direct interactions between the molecules. The RMD simulation was initiated by using unambiguous NMR distant constraints on the peptide without the presence of the disulfide bond. Following a 1 ns run, the disulfide bond was formed and the compound was allowed to fully relax by energy minimization before performing a more robust RMD simulation for 10 ns. Following the MD run, 200 equally spaced structures were energy minimized with the NMR based restraints, analyzed, and grouped into conformational families. A superposition of all 200 energy-minimized conformations was not distinguishable, suggesting that the simulations resulted in more than one conformational family. Cluster analysis was performed by comparing the backbone φ and ψ angles of the His-DPhe-Arg-Trp amino acid ligand domain, which resulted in one major family with 40% of all the 200 conformers (Figure 4A). The structures of compounds AMW610 and AMW3-130 were generated by the same method depicted above and included here for comparison. The structure of AST3-88 is represented as the superposition of the conformers of the major family of compound aligned on backbone heavy atoms. The global root-mean-square (RMSD) value of residues in the “His-DPhe-Arg-Trp” region from the average structure is 1.16 ± 0.45 Å. Analysis of the φ–ψ backbone dihedral angles of the average structure (Supporting Information Table S2) resulted in the identification of type VIII β-turn structure spanning DPhe⁴ and Arg⁵ residues. Type VIII is reported to be the most common type of β-turn secondary structure observed in peptides and proteins. (46) The φ and ψ values of the representative structure from the major family are φ(i + 1) ≈ −69, ψ(i + 1) ≈ −58, φ(i + 2) ≈ −125, and ψ(i + 2) ≈ 41 [idealized values for this type of turn have been reported as φ(i + 1) ≈ −60, ψ(i + 1) ≈ −30, φ(i + 2) ≈ −120, and ψ(i + 2) ≈ 120]. (46) A putative hydrogen bond was also identified between the backbone carbonyl oxygen of His (3) (i residue) and the backbone amide NH of Cys⁶ (i + 3 residue). Figure 5 represents the φ–ψ distribution of all the residues in the sequence of AST3-88 with the exception of the terminal Tyr residues. The φ–ψ distribution map of AST3-88 resulted in conformers tightly packed in the pharmacophore regions, suggesting a constrained conformation in this region, similar to the case of AMW3-130. Apart from the pharmacophore region, the Phe¹⁰ and Cys¹¹ amino acids have very compact conformers, suggesting that the C-terminus of this compound is relatively rigid and involved in regular structure. This observation is consistent with the constraints induced by the disulfide bridge.

Figure 2. Illustration of the compound deviation from random coil values for (A) AST3-88, (B) AMW610, and (C) AMW3-130. (45) The H^N and H^α protons are represented by solid bars and open bars, respectively. Vertical axes are in parts per million, and the horizontal axes represent the His-DPhe-Arg-Trp-Asn-Ala region of the peptide.

Figure 3. Summary of the NOE intensities from 400 ms NOESY data observed for the compound (A) AST3-88, (B) AMW610, and (C) AMW3-130. The height of the bar indicates the strength of the NOE, and these are categorized as strong (1.8–3.0 Å), medium (1.8–3.5 Å), or weak (1.8–5.0 Å).

Figure 4. Sausage stereoview representations of the major family members of compound (A) AST3-88 (in the H, F, R,W region; RMSD = 1.16 ± 0.45 Å), (B) AMW610, and (C) AMW3-130 (in the H, F, R,W region; RMSD = 0.66 ± 0.3 Å) aligned on the backbone heavy atoms of residues 3–7. The His side chain is indicated in green, the DPhe side chain in yellow, the Arg side chain in blue, and the Trp side chain in orange. A wide gray backbone indicates greater flexibility in that domain. Following restrained molecular dynamics (RMD) simulations for 10 ns, 200 equally spaced structures were energy minimized with the NMR based restraints. The energy minimized structures were grouped into conformational families by comparison of the backbone dihedral angels within the His-Phe-Arg-Trp domain.

Figure 5. CAMM based φ–ψ angle distribution for ligands (A) AST3-88, (B) AMW610, and (C) AMW3-130 conformational families. The His residue is indicated in green, the DPhe residue in orange, the Arg residue in blue, and the Trp residue in red. All other residues are indicated in purple.

The 2D ¹H NMR and CAMM studies were performed to correlate the functional and conformational properties the compounds. In earlier studies, it was postulated that the bioactive conformation of melanocortin ligands such as, MTII and SHU9119 constitute a putative reverse turn in the pharmacophore region. (47) In a study of chimeric AGRP-melanocortin template peptides with a lactam bridge, similar to the AMW3-130 template, a reverse turn was found in all the studied peptides. (48) The chimeric peptide template used in present study (with a disulfide bridge in place of a lactam bridge) was also found to have Type I′ reverse turn encompassing Arg and Trp residues in pharmacophore domain. (34, 41) In present study, we found that compound AST3-88, an agonist on all the melanocortin receptor subtypes, possesses a Type VIII reverse turn around DPhe⁴ (i + 1) and Arg⁵ (i + 2) regions of the sequence. The reverse turn was stabilized by the hydrogen bond between His³ and Cys⁶ residues. In the earlier structural findings for the compound AMW610 where the Arg residue resided on the heterocyclic moiety, it was observed that insertion of the heterocyclic ring made the peptide backbone more flexible and resulted in several conformational families with very low populations. (34, 41) In contrast, positioning of the Trp residue on the heterocyclic ring (AST3-88) in the present study resulted in one major family with 40% of the total studied conformers. The very close proximity of His³ and Trp⁶ suggests that the structure may be additionally stabilized by aromatic–aromatic and/or cation−π interactions involving side chains of these residues. The His³ and Trp⁶ residues appeared to be stacked in parallel arrangement. The DPhe⁴ residue is oriented perpendicular to His³ and Trp⁶ amino acids. Additionally, the arrangement of aromatic residue side chains of His³, Trp⁷, and Phe¹⁰ are juxtaposed, forming a hydrophobic cluster. The above-mentioned structural features of AST3-88 are in agreement with the reported structure of MTII and linear α-MSH-analogues in terms of having a reverse turn in the pharmacophore region. The AMW3-130 ligand, a potent agonist at the melanocortin receptors, was also identified to possess a type I′ β-turn encompassing Trp⁷ and Asn⁸ residues with a constrained backbone conformation. These data support the hypothesis that the modulation in the orientation of Trp residue stabilizes the bioactive conformation via intramolecular interaction and orient the other side chains for efficient receptor interactions. This also suggests that the reverse turn in the pharmacophore region is more favored for the MC4R binding pocket than the MC3R. It has been postulated that the putative hydrophobic pocket of MC3R is different than the MC4R, which explains the difference in ligand–receptor interactions and hence, the difference in EC₅₀ values at these receptor subtypes.

Serum Stability of Compound AST3-88

Modifications in peptide structure by different approaches including cyclization, (49, 50)d-amino acids, (51) unnatural amino acids, (52) and β-amino acids (53) are well recognized strategies for the goal of improving peptide stability in serum and protecting against protease-mediated degradation. Since AST3-88 was prepared by inserting a heterocyclic moiety into the peptide backbone, the stability of AST3-88 in rat serum was compared with two peptides prone for degradation, α-MSH and AST789-13 (Ac-RRWWRF-NH₂). It has been reported for α-MSH [13-mer linear peptide with acetylated N-terminus and amidated C-terminus (Ac-SYSMEHFRWGKPV-NH₂)] that, during a 4 h incubation, it was rapidly degraded in frog serum and lost half of its activity in rat serum after 1 h of incubation. (12, 54) The AST789-13 control peptide was reported to possesses a half-life of 1.5 h in human serum. (55) In our study, LC-MS analysis of the aliquots collected during 24 h incubations of these three peptides with rat serum revealed differences in their degradation kinetics (Figure 6). The results indicated that AST789-13 was degraded completely within 2 h. The endogenous α-MSH peptide had a half-life of nearly 2.5 h with only ∼10% of the compound remaining in the serum after 6 h. of incubation. In contrast, AST3-88 was far more stable than AST789-13 and α-MSH as ∼40% of AST3-88 remained at 6 h and 12% at 24 h of incubation in rat serum (Figure 6). As the degradation products of AST3-88 were not identified in this study, the degradation pathway of AST3-88 and the mechanism behind the greater stability of AST3-88 over α-MSH and AST789-13 remain undefined. However, it is postulated that the structural properties of AST3-88, including the disulfide bridge joining the N- and C-terminus, inclusion of a d-amino acid in its sequence, and the presence of the heterocyclic modified peptide backbone (Figure 1), contribute to its resistance to both exopeptidase and endopeptidase-mediated degradation.

Figure 6. Rat serum stability profiles of the peptides AST789-13, α-MSH, and AST3-88. Relative peptide concentrations are based on mass spec ion counts. AST789-13 completely degraded in less than 2 h. Endogenous linear peptide α-MSH rapidly degraded within 6 h with only ∼1% intact peptide remaining at the 24 h point. Compound AST3-88, with the engineered backbone, degraded slowly in rat serum with ∼40% remaining at 6 h, and ∼12% was detected by MS at the 24 h time point.

Effect of ICV Treatment of Compound AST3-88 on Feeding

To determine if compound AST3-88 produces an in vivo physiological response, we administered two doses (2.5 or 5.0 nmol) of compound AST3-88 intracerbroventricularly (ICV) into the third ventricle of cannulated and overnight fasted rats. In earlier studies, administration of MC3R and MC4R agonists inhibited food intake postinjection. (23, 28) Conversely, ICV administration of MC3R and MC4R antagonists increases food intake. (23, 28) To test the effects of the MC4R selective compound AST3-88, rats were induced to feed by overnight food deprivation, a validated method to observe modulation in MC4R signaling. (23) Animals were injected with either vehicle or compound AST3-88 after completion of overnight fasting with food being returned 90 min after treatment. Figure 7 shows the effect of AST3-88 on food intake up to 48 h after the return of food. The AST3-88 ligand produced a statistically significant decrease in food intake (relative to the vehicle condition) at both doses 3 to 48 h post treatment. Thiele and colleagues have previously reported this type of prolonged activity upon ICV administration of MTII in rats. (56) They reported that MTII reduced 48 h food consumption (1.0 nmol dose), and also reduced body weight at 24 and 48 h (0.1 and 1.0 nmol doses, respectively) post treatment. In another example, central administration of agouti-related protein (AGRP), an endogenous antagonist of the MC3R and MC4R, robustly increased food intake in mice with the effect lasting up to 24 h. (57, 58) It has also been reported that AGRP produced a robust and prolonged food intake response in wild type, MC3RKO, and MC4RKO mice. (28) In summary, ICV treatment of compound AST3-88 has a significant effect on inhibition of food intake in rats.

Figure 7. (A) Effects of AST3-88 ICV treatment on cumulative food intake in rats. Animals were injected with either saline, 2.5 nmol, or 5 nmol of AST3-88 after completion of overnight fasting. Each data point is represents the mean of 4–6 animals ± SEM; *p < 0.05 and ***p < 0.001.

Conclusions

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In the current study, it has been demonstrated that an engineered peptide backbone strategy could be utilized to obtain potent, selective, and stable melanocortin receptor ligands. Taken together with previous observations, the results of these studies support the hypothesis that the Trp residue side chain may play an important role in (i) stabilizing the bioactive conformation by intramolecular interactions, (ii) properly orienting the pharmacophore residues to interact with the receptor in the binding pocket, and (iii) specific intermolecular interaction with the receptor residues resulting in differential agonist potencies at different receptors, that can contribute to selectivity. The advantage of this approach also extends to the enhanced metabolic and physiological stability of the engineered peptides. The AST3-88 molecule that possesses nanomolar potency and selectivity for the MC4R versus the MC3R, prolonged biological activity, and resistance to enzymatic proteolysis can serve as a molecular probe to study physiological role(s) of these melanocortin receptors and also further improve the design of selective ligands.

Methods

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Chemistry

All chemicals were ACS grade or better, were obtained from commercial suppliers, and were used without further purification. The amino acids Nα 9-fluorenylmethoxycarbonyl (Fmoc)-Cys(Trt), Fmoc-Arg(Pbf), Fmoc-His(Trt), Fmoc-Trp(Boc), Fmoc-d-Phe, Fmoc-Phe, Fmoc-Asn(Trt), Fmoc-Ala, Rink amide p-methylbenzhydrylamine Resin (p-MBHA Resin, 0.47 mequiv/g substitution), and the coupling reagent O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Peptides International (Louisville, KY). The amino acid Fmoc-Tyr (tBu) was purchased from Advanced Chem Tech (Louisville, KY).

Solid Phase Peptide Synthesis Using Microwave Irradiation

The Rink amide MBHA resin was transferred into a 25 mL polypropylene reaction vessel (CEM) and “swelled” in dichloromethane for 1 h. The bottom cap of the vessel was removed after the 1 h swelling process, and the vessel was transferred onto a vacuum filtration manifold. Deprotection of the Fmoc group was achieved in DMF by irradiating the sample at 75 °C, 30 W for 4 min in the CEM Discover SPS instrument. After cooling and washing the resin with DMF, a ninhydrin “Kaiser” test was performed. (59) The amino acid coupling step was performed under the same microwave conditions (75 °C, 30 W) for 5 min using HBTU. The coupling of the Cys and His amino acids was performed at lower temperatures (50 °C, 30 W) for 5 min. The heterocycle ring was assembled as previously described. (44) The reductive alkylation, acylation of chloroacetic anhydride, and the deprotection of the thio tert-Butyl group steps were performed at the room temperature. The thioether ring closure was performed using N-ethylmorpholine in DMF at elevated temperature (55–60 °C) in an automated synthesizer (Advanced ChemTech 440MOS, Louisville, KY). Bubbling nitrogen gas into the reaction vessel mixed the reagents. The iterative process of the Fmoc deprotection/coupling cycle was performed to elongate the peptide chain. After the final deprotection, the peptide was cleaved from the resin using the cleavage cocktail (91% TFA, 3.0% H₂O, 3.0% EDT, and 3.0% TIS) at room temperature for 3 h. Note, as the peptides containing the heterocyclic moiety were synthesized to completion on resin, if racemization occurred during the isolated heterocycle formation as previously reported, (44) it was not identified or purified in the major product peak collected by RP-HPLC. After cleavage and side chain deprotection, the solution was concentrated and the peptide was precipitated and washed using cold (4 °C) anhydrous diethyl ether.

The crude linear peptides were dissolved in 20% DMSO in water (1.0 mg/mL) and stirred at room temperature. Progress for the disulfide cyclization was monitored by UV-HPLC, which was generally completed within 24–36 h. The resulting solution was lyophilized to yield the crude cyclic peptide and purified by reversed-phase HPLC (flow rate of 5 mL/min and gradients ranging from 30%–55% acetonitrile/water 0.1% TFA) using a Shimadzu chromatography system with a photodiode array detector and a semipreparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 × 25 cm). The purified peptides were >95% pure as determined by RP-HPLC in two diverse solvent systems and had the correct molecular mass. Electrospray ionization mass spectrometry (ESI-MS) was used to record spectra on an ABI 3200Q TRAP instrument. Analytical data for AST3-88: purity > 95%; k′ (MeCN) 4.7, k′ (MeOH) 7.9; exact mass calcd for C₇₇H₉₄N₂₀O₁₅S₃, 1634.6; found, 1635.6 (M + 1). Analytical data for AMW6103: purity > 95%; k′ (MeCN) 5.7, k′ (MeOH) 9.8; exact mass calcd for C₇₇H₉₄N₂₀O₁₅S₃, 1634.6; found, 1635.7 (M + 1).

Functional Bioassay

The HEK-293 cells stably expressing the mouse melanocortin receptors were maintained in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum and transfected with 4 μg of the CRE/β-galactosidase reporter gene as previously described. (42) Briefly, 5000–15 000 post-transfection cells were plated into collagen treated 96-well plates (Nunc) and incubated overnight. Forty-eight hours post-transfection, the cells were stimulated with 100 μL of peptide (10^–4–10^–12 M) or forskolin (10^–4 M) control in assay medium (DMEM containing 0.1 mg/mL BSA and 0.1 mM isobutylmethylxanthine) for 6 h. The assay media was aspirated, and 50 μL of lysis buffer (250 mM Tris-HCl pH = 8.0 and 0.1% Triton X-100) was added. The plates were stored at −80 °C overnight. The plates containing the cell lysates were thawed the following day. Aliquots of 10 μL were taken from each well and transferred to another 96-well plate for relative protein determination. To the cell lysate plates, 40 μL of phosphate-buffered saline with 0.5% BSA was added to each well. Subsequently, 150 μL of substrate buffer (60 mM sodium phosphate, 1 mM MgCl₂, 10 mM KCl, 5 mM β-mercaptoethanol, 2 mg/mL ortho-nitrophenyl-β-galactoside [ONPG]) was added to each well and the plates were incubated at 37 °C. The sample absorbance, OD₄₀₅, was measured using a 96-well plate reader (Molecular Devices). The relative protein was determined by adding 200 μL of 1:5 dilutions Bio Rad G250 protein dye/water to the 10 μL cell lysate sample taken previously, and the OD₅₉₅ was measured on a 96-well plate reader (Molecular Devices). Data points were normalized both to the relative protein content and nonreceptor dependent forskolin stimulation. Maximal efficacy was compared to that observed for the NDP-MSH control peptide tested simultaneously on each 96-well plate. The agonist EC₅₀ values represent the mean of duplicate wells performed in three or more independent experiments. The EC₅₀ value estimates, and their associated standard errors, were determined by fitting the data to a nonlinear least-squares analysis using the PRISM software program (v4.0, GraphPad Inc.). The results are not corrected for peptide content.

NMR Spectroscopy and Computer-Assisted Molecular Modeling (CAMM)

The ligand NMR samples were prepared (∼1 mM) by dissolving 1.0 mg of the purified peptide in a 750 μL solution containing 400 μL of CD₃CN and 350 μL of H₂O and adding DSS as an internal standard (0.0 ppm). The NMR data were collected at 34 °C with a Bruker Avance II spectrometer operating at 600 MHz (using a cryoprobe) at the Advanced Magnetic Resonance and Imaging Spectroscopy (AMRIS) facility at the University of Florida. Standard proton TOCSY and NOESY 2D ¹H NMR data were collected, processed, and analyzed as described previously. (34, 41, 48, 60) The chemical shifts of each of the peptides in this study were assigned using standard TOCSY and NOESY ¹H-based strategies. (61)

Proton–proton distances were calibrated using the well resolved methylene protons of the Cys⁶ (heterocycle ring) residue based on the relationship r = r_ref(η_ref/η)^1/6, where r is the distance between atoms, η is the NOESY cross-peak volume, r_ref is the known distance, and η_ref is the corresponding volume of the NOESY calibration cross-peak. The NOE volumes were categorized as strong (1.8–3.0 Å), medium (1.8–3.5 Å), or weak (1.8–5.0 Å) (Supporting Information). All conformational molecular modeling experiments were performed using the SYBYL v7.0 software from Tripos Inc. (St. Louis, MO) on a Silicon Graphics workstation. Restrained molecular dynamics (RMD) simulations were run in vacuo with a dielectric constant of 4.0, at a temperature of 500 K, and using the Tripos force field and Gastaiger-Hückel partial atomic charges. The peptides were initially built in a fully extended linear conformation. In the first step of modeling, RMD simulations were run for 1 ns. Following the initial 1 ns RMD trajectory, the cysteine residues were oriented next to each other, and disulfide bonds were manually formed and energy minimized without restraints. Finally, all the NMR-based NOE restraints were included, and 10 ns RMD trajectories were collected. Following the RMD simulations, structures from 200 equally spaced points along the dynamics trajectory were energy minimized, analyzed, and grouped into conformational families. Comparing the backbone φ and ψ angles of the His-DPhe-Arg-Trp amino acid ligand domain performed cluster analysis. This process identified representative structures (lowest energy conformers) of the conformational families that were used for further analysis.

Serum Stability Assay

Rat serum (495 μL) was incubated at 37 °C for 15 min, and 5 μL of peptide (10^–3 M stock solution; final conc. 10 μM) was added to initiate the assay. The solution (serum + peptide) was mixed intermittently duration the incubation, and 50 μL aliquots were taken at 0, 0.5, 1.5, 3, 6, and 24 h of the incubation. Aliquots were then mixed with 150 μL of cold (4 °C) 66% aqueous acetonitrile and incubated at 4 °C for 10–15 min to precipitate serum proteins. The supernatant was collected after centrifugation at 14 000g for 10 min and then stored at −80 °C immediately.

Liquid Chromatography–Mass Spectrometry (LC-MS) Analysis of Peptide Stability

A 5 μL aliquot of diluted samples was injected into a Waters Acquity ultraperformance liquid chromatography (UPLC) system (Milford, MA) and separated by a gradient of mobile phase ranging from water to 95% aqueous acetonitrile containing 0.1% formic acid over a 10 min run. LC elute was introduced into a Waters SYNAPT QTOF mass spectrometer (QTOF-MS) for accurate mass measurement and ion counting under extended dynamic range mode. Capillary voltage and cone voltage were maintained at 3.2 kV and 30 V, respectively, for positive electrospray ionization (ESI). Source temperature and desolvation temperature were set at 120 and 350 °C, respectively. Nitrogen was used as both cone gas (50 L/h) and desolvation gas (700 L/h). Mass chromatograms and mass spectral data were acquired and processed by MassLynx software (Waters) in centroid format. Peptides were identified by accurate masses and comparison with authentic standards. Signal intensity of interested peptide in spiked serum sample at the beginning of incubation (0 h) was arbitrarily set as 100%.

Feeding Studies in Rats

Animals

Male Sprague–Dawley rats (Charles River) were anesthetized with a mixture of ketamine/xylazine (90/10 mg/kg). In a stereotaxic apparatus with the incisor bar set at −3.3 mm (relative to horizontal zero), cannulas (26 gauge) were implanted with the tip targeted at 2.5 mm posterior and 8.1 mm ventral to bregma. Cannulas were secured to the skull with stainless steel screws and dental cement. All injections were made with a 33 gauge internal cannula that extended 1 mm beyond the tip of the guide cannula. In all cases, the injection volume was 3 μL. The injections were given over a 30 s period, and the internal cannula was left in place an additional 30 s to allow diffusion from the tip. Cannula patency and placement was tested prior to and following data collection. This test consisted of an ICV injection of angiotensin II (30 ng/3 μL). Data from rats that did not drink at least 5 mL of water in the 30 min period after injection in both trials were discarded. The feeding study was approved by the University of Minnesota Institutional Animal Care and Use Committee.

Feeding Regimen

For the data and results presented here, rats were fasted overnight and then given ICV injections of compound 1 (AST3-88) at doses of 0, 2.5, and 5 nmol. The peptide was initially dissolved in a small volume of DMSO, and saline was added to adjust the concentrations. The final concentration of DMSO in the vehicle and both doses was 16.7%. Food was returned 90 min after injections, and food intake (corrected for spillage) was measured (by weight) at 1.5, 3, 24, and 48 h. Rats were weighed prior to injection and 1, 2, and 4 days later. No adverse or toxicity related behaviors were observed in the duration of this study. It should be noted that, prior to this test, these rats had been used in two trials (conducted 7 and 15 days earlier) in which they received ICV injections of AST3-88 (0, 2.5, or 5 nmol) under ad libitum feeding conditions and no statistically significant differences were observed.

Statistics

Data are represented as the average of the mean ± SEM. For statistical analyses, cumulative food intake over time was performed using two-way ANOVA followed by an LSD post hoc test. Statistical significance is considered if p < 0.05.

Supporting Information

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Proton NMR chemical shift assignments (ppm) for AST3-88 and the backbone dihedral angels of the representative structures in Figure 4. This material is available free of charge via the Internet at http://pubs.acs.org

cn5000953_si_001.pdf (81.23 kb)

Terms & Conditions

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Author Information

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Corresponding Author
- Carrie Haskell-Luevano - Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States; Email: [email protected]
Authors
- Anamika Singh - Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
- Marvin L. Dirain - †Departments of Medicinal Chemistry and Pharmacodynamics, and ‡Department of Biochemistry & Molecular Biology and National High Magnetic Field Laboratory, University of Florida, Gainesville, Florida 32610, United States; Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States; ∥Department of Food Science and Nutrition, and ⊥Minnesota Obesity Center, University of Minnesota, Saint Paul, Minnesota 55108, United States
- Andrzej Wilczynski - †Departments of Medicinal Chemistry and Pharmacodynamics, and ‡Department of Biochemistry & Molecular Biology and National High Magnetic Field Laboratory, University of Florida, Gainesville, Florida 32610, United States; Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States; ∥Department of Food Science and Nutrition, and ⊥Minnesota Obesity Center, University of Minnesota, Saint Paul, Minnesota 55108, United States
- Chi Chen - †Departments of Medicinal Chemistry and Pharmacodynamics, and ‡Department of Biochemistry & Molecular Biology and National High Magnetic Field Laboratory, University of Florida, Gainesville, Florida 32610, United States; Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States; ∥Department of Food Science and Nutrition, and ⊥Minnesota Obesity Center, University of Minnesota, Saint Paul, Minnesota 55108, United States
- Blake A. Gosnell - †Departments of Medicinal Chemistry and Pharmacodynamics, and ‡Department of Biochemistry & Molecular Biology and National High Magnetic Field Laboratory, University of Florida, Gainesville, Florida 32610, United States; Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States; ∥Department of Food Science and Nutrition, and ⊥Minnesota Obesity Center, University of Minnesota, Saint Paul, Minnesota 55108, United States
- Allen S. Levine - †Departments of Medicinal Chemistry and Pharmacodynamics, and ‡Department of Biochemistry & Molecular Biology and National High Magnetic Field Laboratory, University of Florida, Gainesville, Florida 32610, United States; Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States; ∥Department of Food Science and Nutrition, and ⊥Minnesota Obesity Center, University of Minnesota, Saint Paul, Minnesota 55108, United States
- Arthur S. Edison - †Departments of Medicinal Chemistry and Pharmacodynamics, and ‡Department of Biochemistry & Molecular Biology and National High Magnetic Field Laboratory, University of Florida, Gainesville, Florida 32610, United States; Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States; ∥Department of Food Science and Nutrition, and ⊥Minnesota Obesity Center, University of Minnesota, Saint Paul, Minnesota 55108, United States
Funding
This work has been supported in part by NIH Grants RO1DK064250 (C.H.-.L), RO1DK091906 (C.H.-L.), and R01DA021280 (A.S.L.).
Notes
The authors declare no competing financial interest.

Abbreviations

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ACTH	adrenocorticotropin hormone
AGRP	agouti-related protein
ASIP	agouti-signaling protein
CAMM	computer-assisted molecular modeling
cAMP	cyclic 5′-adenosine monophosphate
DCM	dichloromethane
DMF	N,N-dimethylformamide
Fmoc	Nα 9-fluorenylmethoxycarbonyl
GPCR	G protein coupled receptor
MC1R	melanocortin-1 receptor
MC2R	melanocortin-2 receptor
MC3R	melanocortin-3 receptor
MC4R	melanocortin-4 receptor
MC5R	melanocortin-5 receptor
MCR	melanocortin receptor
MeOH	methanol
MSH	melanocyte stimulating hormone
NOE	nuclear Overhauser effect
POMC	proopiomelanocortin
ϕ	phi
ψ	psi
SAR	structure–activity relationship
SEM	standard error of the mean
TFA	trifluoroacetic acid
TM	transmembrane
α-MSH	alpha-melanocyte stimulating hormone
β-MSH	beta-melanocyte stimulating hormone
γ-MSH	gamma-melanocyte stimulating hormone
μM	micromolar
RMD	restrained molecular dynamics
NDP-MSH (4-Norleucine-7-d-Phenylalanine)	Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂
AMW3-130	Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH₂
MTII	Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH₂
AST789-13	Ac-RRWWRF-NH₂

References

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This article references 61 other publications.

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There is no corresponding record for this reference.
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Castrucci, A. M., Hadley, M. E., Sawyer, T. K., Wilkes, B. C., al-Obeidi, F., Staples, D. J., de Vaux, A. E., Dym, O., Hintz, M. F., and Riehm, J. P. 1989, Alpha-melanotropin: the minimal active sequence in the lizard skin bioassay Gen. Comp. Endrocrinol. 73, 157– 163
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α-Melanotropin: the minimal active sequence in the lizard skin bioassay
Castrucci, A. M. L.; Hadley, M. E.; Sawyer, T. K.; Wilkes, B. C.; Al-Obeidi, F.; Staples, D. J.; De Vaux, A. E.; Dym, O.; Hintz, M. F.; et al.
General and Comparative Endocrinology (1989), 73 (1), 157-63CODEN: GCENA5; ISSN:0016-6480.
α-Melanotropin (α-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. The minimal sequence of α-MSH required for agonism in the lizard (Anolis carolinensis) skin bioassay was detd. to be Ac-His-Phe-Arg-Trp-NH2 (Ac-α-MSH6-9-NH2). Smaller fragments of this sequence (Ac-α-MSH6-8-NH2, Ac-α-MSH6-7-NH2, Ac-α-MSH7-9-NH2, and Ac-α-MSH7-8-NH2) were devoid of melanotropic activity. The tetrapeptide Ac-α-MSH7-10-NH2 was also inactive, thus again demonstrating the importance of His at position 6 for minimal activity. The important potentiating amino acids were Met-4, Lys-11, and Pro-12, since Ac-α-MSH4-10-NH2 was about 100 times more potent than Ac-α-MSH5-10-NH2, and Ac-[Nle4]-α-MSH4-11-NH2 was about 40 times more potent than Ac-α-MSH4-10-NH2 or Ac-[Nle4]-α-MSH4-10-NH2. Ac-α-MSH4-12-NH2 and Ac-[Nle4]-α-MSH4-12-NH2 were equipotent and about 6 times more potent than α-MSH. Since [Nle4]-α-MSH and Ac-[Nle4]-α-MSH4-13-NH2 were both equipotent but about sixfold less active than Ac-[Nle4]-α-MSH4-12-NH2, it is clear that valine at position 13 does not contribute to the potency of α-MSH, except possibly in a neg. way. The minimal message sequence for equipotency to α-MsH appears to be Ac-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-NH2, since the analog, Ac-[Nle4]-α-MSH4-11-NH2, was as active as the native hormone. Ser-1, Tyr-2, Ser-3, Glu-5, and Val-13 are not important for melanotropic potency since Ac-α-MSH4-12-NH2 was more potent than α-MSH, and Ac-α-MSH5-10-NH2 and Ac-α-MSH6-10-NH2 were equipotent, being about 4000 times less active than α-MSH.
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Hruby, V. J., Wilkes, B. C., Hadley, M. E., Al-Obeidi, F., Sawyer, T. K., Staples, D. J., de Vaux, A. E., Dym, O., Castrucci, A. M., and Hintz, M. F. 1987, α-Melanotropin: the minimal active sequence in the frog skin bioassay J. Med. Chem. 30, 2126– 2130
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α-Melanotropin: the minimal active sequence in the frog skin bioassay
Hruby, Victor J.; Wilkes, Brian C.; Hadley, Mac E.; Al-Obeidi, Fahad; Sawyer, Tomi K.; Staples, Douglas J.; DeVaux, Ann E.; Dym, Orin; Castrucci, Ana Maria de L.; et al.
Journal of Medicinal Chemistry (1987), 30 (11), 2126-30CODEN: JMCMAR; ISSN:0022-2623.
A series of fragment analogs of α-MSH (I) were prepd. in order to det. the contribution of each individual amino acid to the biol. activity of the native hormone. The minimal potency of Ac-α-MSH6-9-NH2 could be enhanced about a factor of 16 by the addn. of glycine to the C-terminus, yielding Ac-α-MSH6-10-NH2. Addn. of glutamic acid to the N-terminus provided Ac-α-MSH5-10-NH2, which was only slightly more potent than Ac-α-MSH6-10-NH2, indicating that position 5 contributes little to the biol. potency of α-MSH in this assay. Addn. of methionine to the N-terminus of Ac-α-MSH5-10-NH2 resulted in Ac-α-MSH4-10-NH2, which was only about 4-fold more potent than Ac-α-MSH5-10-NH2. Addn. of lysine and proline to the C-terminal of the Ac-α-MSH4-10-NH2 sequence yielded Ac-α-MSH4-12-NH2 with a 360-fold increase in potency relative to Ac-α-MSH4-10-NH2. This peptide was only about 6-fold less potent than α-MSH. Nle-4-substituted analogs were also prepd. Ac-[Nle4]-α-MSH4-10-NH2 and Ac-[Nle4]-α-MSH4-11-NH2 were ∼4 times more potent than Ac-α-MSH4-10-NH2, demonstrating that lysine-11 contributes somewhat to the biol. activity of α-MSH on the frog skin melanocyte receptor. However, addn. of proline-12 to this fragment, yielding Ac-[Nle4]-α-MSH4-12-NH2, resulted in about a 90-fold increase in relative potency of the melanotropin. Addn. of the final C-terminal valine-13 provided Ac-[Nle4]-α-MSH4-13-NH2, which showed only a small further increase in potency. This analog was, however, only about 2 to 3-fold less active than α-MSH. Addn. of the N-terminal tripeptide Ac-Ser-Tyr-Ser to yield [Nle4]-α-MSH resulted in an analog that was 3 times more potent than α-MSH. The central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2, represents the min. chain length for observable biol. activity. The active sequence of α-MSH is contiguous in that no two structurally noncontiguous fragment sequences were found to have biol. activity. Met-4, Gly-10, and Pro-12 are important potentiating amino acids and contribute significantly to the biopotency of α-MSH, and Ser-1 and -3, Tyr-2, Glu-5, Lys-11, and Val-13 apparently contribute only minimally to the biol. potency of α-MSH at the frog melanocyte skin receptor.
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Haskell-Luevano, C., Sawyer, T. K., Hendrata, S., North, C., Panahinia, L., Stum, M., Staples, D. J., Castrucci, A. M., Hadley, M. F., and Hruby, V. J. (1996) Truncation studies of α-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity Peptides 17, 995– 1002
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Holder, J. R. and Haskell-Luevano, C. (2003) Melanocortin tetrapeptides modified at the N-terminus, His, Phe, Arg, and Trp positions Ann. N.Y. Acad. Sci. 994, 36– 48
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Haskell-Luevano, C., Holder, J. R., Monck, E. K., and Bauzo, R. M. (2001) Characterization of melanocortin NDP-MSH agonist peptide fragments at the mouse central and peripheral melanocortin receptors J. Med. Chem. 44, 2247– 2252
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Haskell-Luevano, C., Sawyer, T. K., Hendrata, S., North, C., Panahinia, L., Stum, M., Staples, D. J., Castrucci, A. M., Hadley, M. F., and Hruby, V. J. (1996) Truncation studies of α-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity Peptides 17, 995– 1002
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Hruby, V. J. W., B, C., Cody, W. L., Sawyer, T. K., and Hadley, M. E. (1984) Melanotropins: Structural, Conformational and Biological Considerations in the Development of Superpotent and Superprolonged Analogs Pept. Protein Rev. 3, 1– 64
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Melanotropins: structural, conformational and biological considerations in the development of superpotent and superprolonged analogs
Hruby, Victor J.; Wilkes, Brian C.; Cody, Wayne L.; Sawyer, Tomi K.; Hadley, Mac E.
Peptide and Protein Reviews (1984), 3 (), 1-64CODEN: PPRVDF; ISSN:0731-1753.
A review, with 178 refs. on the structural properties and biol. activities of melanotropin [9002-79-3] and analogs.
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Lerner, A. B. and McGuire, J. S. (1961) Effect of α- and β-melanocyte stimulating hormones on the skin colour of man Nature 189, 176– 179
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Effect of α- and β-melanocyte-stimulating hormones on the skin color of man
Lerner, Aaron B.; McGuire, Joseph S.
Nature (London, United Kingdom) (1961), 189 (), 176-9CODEN: NATUAS; ISSN:0028-0836.
Synthetic α-melanocyte-stimulating hormone (I) and β-melanocyte-stimulating hormone (II) from hog pituitary glands were administered intramuscularly to Negroes at a level of 4-8 mg./day for periods up to 14 days. With I darkening of the skin occurred in 2 subjects; with II darkening occurred in only 1 of 2 subjects. Darkening was greater on the forehead than on the arm, and began almost within 24 hrs. of the initiation of the expt. All hyperpigmentation regressed completely within 6 weeks.
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Gantz, I., Konda, Y., Tashiro, T., Shimoto, Y., Miwa, H., Munzert, G., Watson, S. J., DelValle, J., and Yamada, T. (1993) Molecular cloning of a novel melanocortin receptor J. Biol. Chem. 268, 8246– 8250
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Molecular cloning of a novel melanocortin receptor
Gantz, Ira; Konda, Yoshitaka; Tashiro, Takao; Shimoto, Yoshimasa; Miwa, Hiroto; Munzert, Gerd; Watson, Stanley J.; DelValle, John; Yamada, Tadataka
Journal of Biological Chemistry (1993), 268 (11), 8246-50CODEN: JBCHA3; ISSN:0021-9258.
By using the technique of the polymerase chain reaction primed with oligonucleotides based on the homologous transmembrane regions of seven transmembrane G protein-linked receptors, 3 full-length human genes that encode a novel subgroup of this receptor family have been isolated. Recently, 2 of these receptors were identified as specific for α-MSH and ACTH. The mol. cloning and pharmacol. characterization of a 3rd member of this subgroup is now reported. The gene for this receptor encodes a protein of 361 amino acids in length. Its pharmacol. characterizes it as an MSH receptor specific to the heptapeptide core common to ACTH and α-, β-, and γ-MSH. By Northern blot hybridization and polymerase chain reaction, it is expressed in brain, placental, and gut tissues but not in melanoma cells or in the adrenal gland. The findings may yield insight into the physiol. of peptides derived from pro-opiomelanocortin posttranslational processing.
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Chen, A. S., Marsh, D. J., Trumbauer, M. E., Frazier, E. G., Guan, X. M., Yu, H., Rosenblum, C. I., Vongs, A., Feng, Y., Cao, L., Metzger, J. M., Strack, A. M., Camacho, R. E., Mellin, T. N., Nunes, C. N., Min, W., Fisher, J., Gopal-Truter, S., MacIntyre, D. E., Chen, H. Y., and Van der Ploeg, L. H. (2000) Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass Nat. Genet. 26, 97– 102
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Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass
Chen, Airu S.; Marsh, Donald J.; Trumbauer, Myrna E.; Frazier, Easter G.; Guan, Xiao-Ming; Yu, Hong; Rosenblum, Charles I.; Vongs, Aurawan; Feng, Yue; Cao, Linhai; Metzger, Joseph M.; Strack, Alison M.; Camacho, Ramon E.; Mellin, Theodore N.; Nunes, Christian N.; Min, William; Fisher, Jill; Gopal-Truter, Shobhna; MacIntyre, D. Euan; Chen, Howard Y.; Van der Ploeg, Lex H. T.
Nature Genetics (2000), 26 (1), 97-102CODEN: NGENEC; ISSN:1061-4036. (Nature America Inc.)
Genetic and pharmacol. studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiol. function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r-/-) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-mo Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. Feed efficiency is the ratio of wt. gain to food intake. Consistent with increased fat mass, Mc3r-/- mice are hyperleptinemic and male Mc3r-/- mice develop mild hyperinsulinemia. Mc3r-/- mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r-/- mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.
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Butler, A. A., Kesterson, R. A., Khong, K., Cullen, M. J., Pelleymounter, M. A., Dekoning, J., Baetscher, M., and Cone, R. D. (2000) A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse Endocrinology 141, 3518– 3521
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A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse
Butler, Andrew A.; Kesterson, Robert A.; Khong, Kathy; Cullen, Mary Jane; Pelleymounter, Mary Ann; Dekoning, Jenefer; Baetscher, Manfred; Cone, Roger D.
Endocrinology (2000), 141 (9), 3518-3521CODEN: ENDOAO; ISSN:0013-7227. (Endocrine Society)
The central melanocortin system is crit. for the long term regulation of energy homeostasis. Null mutations of the melanocortin-4 receptor (MC4-R) are assocd. with hyperphagia, obesity, and accelerated longitudinal growth in mice and humans. However, little is known about the function of another central melanocortin receptor, the MC3-R. To assess the role of the MC3-R in energy homeostasis, the majority of the mc3r coding sequence was deleted from the mouse genome. In contrast to the MC4-R knockout, which exhibits increased food intake, increased somatic growth, and defects in metab., mc3r-/- mice exhibit an exclusively metabolic syndrome. Homozygous null mc3r mice, while not significantly overweight, exhibit an approx. 50% to 60% increase in adipose mass. Mc3r-/- mice also exhibit an unusual increase in RQ when transferred onto high fat chow, suggesting a reduced ratio of fat/carbohydrate oxidn. Furthermore, male mc3r-/- mice also exhibit an approx. 50% redn. in locomotory behavior on the running wheel, suggesting reduced energy expenditure.
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Fan, W., Boston, B. A., Kesterson, R. A., Hruby, V. J., and Cone, R. D. (1997) Role of melanocortinergic neurons in feeding and the agouti obesity syndrome Nature 385, 165– 168
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Role of melanocortinergic neurons in feeding and the agouti obesity syndrome
Fan, Wei; Boston, Bruce A.; Kesterson, Robert A.; Hruby, Victor J.; Cone, Roger D.
Nature (London) (1997), 385 (6612), 165-168CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
Dominant alleles at the agouti locus (A) cause an obesity syndrome in the mouse, as a consequence of ectopic expression of the agouti peptide. This peptide, normally only found in the skin, is a high-affinity antagonist of the MSH receptor (MCI-R), thus explaining the inhibitory of effect of agouti on eumelanin pigment synthesis. The agouti peptide is also an antagonist of the hypothalamic melanocortin-4 receptor (MC4-R). To test the hypothesis that agouti causes obesity by antagonism of hypothalamic melanocortin receptors, we identified cyclic melanocortin analogs that are potent agonists or antagonist of the neural MC3 (refs 11, 12) and MC4 receptors. Intracerebroventricular administration of the antagonist, MTII, inhibiting feeding in four models of hyperphagia; fasted C57BL/6J, ob/ob, and AY mice, and mice injected with neuropeptide Y. Co-administration of the specific melanocortin antagonist and agouti-mimetic SHU9119 completely blocked this inhibition. Furthermore, administration of SHU9119 significantly enhanced nocturnal feeding, or feeding stimulated by a prior fast. Our data show that melanocortinergic neurons exert a tonic inhibition of feeding behavior. Chronic disruption of this inhibitory signal is a likely explanation of the agouti obesity syndrome.
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Wessells, H., Levine, N., Hadley, M. E., Dorr, R., and Hruby, V. (2000) Melanocortin Receptor Agonists, Penile Erection, and Sexual Motivation: Human Studies with Melanotan II Int. J. Impotence Res. 12 (Suppl 4) S74– S79
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Huszar, D., Lynch, C. A., Fairchild-Huntress, V., Dunmore, J. H., Fang, Q., Berkemeier, L. R., Gu, W., Kesterson, R. A., Boston, B. A., Cone, R. D., Smith, F. J., Campfield, L. A., Burn, P., and Lee, F. (1997) Targeted disruption of the melanocortin-4 receptor results in obesity in mice Cell 88, 131– 141
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Targeted disruption of the melanocortin-4 receptor results in obesity in mice
Huszar, Dennis; Lynch, Catherine A.; Fairchild-Huntress, Victoria; Dunmore, Judy H.; Fang, Qing; Berkemeier, Lucy R.; Gu, Wei; Kesterson, Robert A.; Boston, Bruce A.; Cone, Roger D.; Smith, Francoise J.; Campfield, L. Arthur; Burn, Paul; Lee, Frank
Cell (Cambridge, Massachusetts) (1997), 88 (1), 131-141CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
The melanocortin-4 receptor (MC4-R) is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Inactivation of this receptor by gene targeting results in mice that develop a maturity onset obesity syndrome assocd. with hyperphagia, hyperinsulinemia, and hyperglycemia. This syndrome recapitulates several of the characteristic features of the agouti obesity syndrome, which results from ectopic expression of agouti protein, a pigmentation factor normally expressed in the skin. Our data identify a novel signaling pathway in the mouse for body wt. regulation and support a model in which the primary mechanism by which agouti induces obesity is chronic antagonism of the MC4-R.
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Chhajlani, V., Muceniece, R., and Wikberg, J. E. (1993) Molecular cloning of a novel human melanocortin receptor Biochem. Biophys. Res. Commun. 195, 866– 873
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Molecular cloning of a novel human melanocortin receptor
Chhajlani, Vijay; Muceniece, Ruta; Wikberg, Jarl E. S.
Biochemical and Biophysical Research Communications (1993), 195 (2), 866-73CODEN: BBRCA9; ISSN:0006-291X.
A human genomic clone designated MC-2 is isolated. The cloned DNA codes for a protein of 325 amino acids which possesses seven hydrophobic segments, a characteristic of G-protein coupled receptors. The MC-2 receptor is expressed in brain tissue but not in the melanoma cells. When the MC-2 DNA is expressed in COS-7 cells, it binds [125I]-labeled [Nle4,D-Phe7]-α-MSH (NDP-MSH) which then could be displaced by melanotropic peptides α-MSH, β-MSH, γ-MSH, and ACTH, but not by non-melanotropic peptide β-endorphin. The highest affinity of 5.18 nM was for the NDP-MSH peptide. The novel MC-2 receptor and the MC-1 receptor, described earlier by the authors showed identical order of affinity for the melanocortin peptides, but the affinities and the fold differences in the affinities to the melanocortin peptides were different when compared to the earlier described MC-1 receptor. The results suggest that the MC-2 DNA codes for a novel melanocortin receptor.
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Chen, W., Kelly, M. A., Opitz-Araya, X., Thomas, R. E., Low, M. J., and Cone, R. D. (1997) Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides Cell 91, 789– 798
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Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides
Chen, Wenbiao; Kelly, Michele A.; Opitz-Araya, Ximena; Thomas, Ruth E.; Low, Malcolm J.; Cone, Roger d.
Cell (Cambridge, Massachusetts) (1997), 91 (6), 789-798CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
The effects of pituitary-derived melanocortin peptides are primarily attributed to ACTH-mediated adrenocortical glucocorticoid prodn. Identification of a widely distributed receptor for ACTH/MSH peptides, the melanocortin-5 receptor (MC5-R), suggested non-steroidally mediated systemic effects of these peptides. Targeted disruption of the MC5-R produced mice with a severe defect in water repulsion and thermoregulation due to decreased prodn. of sebaceous lipids. High levels of MC5-R was found in multiple exocrine tissues, including Harderian, preputial, lacrimal, and sebaceous glands, and was also shown to be required for prodn. and stress-regulated synthesis of porphyrins by the Harderian gland and ACTH/MSH-regulated protein secretion by the lacrimal gland. These data show a requirement for the MC5-R in multiple exocrine glands for the prodn. of numerous products, indicative of a coordinated system for regulation of exocrine gland function by melanocortin peptides.
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Irani, B. G., Xiang, Z., Yarandi, H. N., Holder, J. R., Moore, M. C., Bauzo, R. M., Proneth, B., Shaw, A. M., Millard, W. J., Chambers, J. B., Benoit, S. C., Clegg, D. J., and Haskell-Luevano, C. (2011) Implication of the melanocortin-3 receptor in the regulation of food intake Eur. J. Pharmacol. 660, 80– 87
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Implication of the melanocortin-3 receptor in the regulation of food intake
Irani, Boman G.; Xiang, Zhimin; Yarandi, Hossein N.; Holder, Jerry R.; Moore, Marcus C.; Bauzo, Rayna M.; Proneth, Bettina; Shaw, Amanda M.; Millard, William J.; Chambers, James B.; Benoit, Stephen C.; Clegg, Deborah J.; Haskell-Luevano, Carrie
European Journal of Pharmacology (2011), 660 (1), 80-87CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier B.V.)
The melanocortin system is well recognized to be involved in the regulation of food intake, body wt., and energy homeostasis. To probe the role of the MC3 in the regulation of food intake, JRH 322-18 a mixed MC3 partial agonist/antagonist and MC4 agonist tetrapeptide was examd. in wild type (WT) and melanocortin 4 receptor (MC4) knockout mice and shown to reduce food intake in both models. In the wild type mice, 2.0 nmol of JRH 322-18 statistically reduced food intake 4 h post icv treatment into satiated nocturnally feeding wild type mice. The same dose in the MC4KO mice significantly reduced cumulative food intake 24 h post treatment. Conditioned taste aversion as well as activity studies supports that the decreased food intake was not due to visceral illness. Since these studies resulted in loss-of-function results, the SHU 9119 and agouti-related protein (AGRP) melanocortin receptor antagonists were administered to wild type as well as the MC3 and MC4 knockout mice in anticipation of gain-of-function results. The SHU 9119 ligand produced an increase in food intake in the wild type mice as anticipated, however no effect was obsd. in the MC3 and MC4 knockout mice as compared to the saline control. The AGRP ligand however, produced a significant increase in food intake in the wild type as well as the MC3 and MC4 knockout mice and it had a prolonged affect for several days. These data support the hypothesis that the MC3 plays a subtle role in the regulation of food intake, however the mechanism by which this is occurring remains to be detd.
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Farooqi, I. S., Keogh, J. M., Yeo, G. S., Lank, E. J., Cheetham, T., and O’Rahilly, S. (2003) Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene N. Engl. J. Med. 348, 1085– 1095
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Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene
Farooqi, I. Sadaf; Keogh, Julia M.; Yeo, Giles S. H.; Lank, Emma J.; Cheetham, Tim; O'Rahilly, Stephen
New England Journal of Medicine (2003), 348 (12), 1085-1095CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
Melanocortin 4 receptor (MC4R) deficiency is the commonest monogenic form of obesity. However, the clin. spectrum and mode of inheritance were not defined, pathophysiol. mechanisms leading to obesity are poorly understood, and there is little information regarding genotype-phenotype correlations. We detd. the nucleotide sequence of the MC4R gene in 500 probands with severe childhood obesity. Family studies were undertaken to examine cosegregation of identified mutations with obesity. Subjects with MC4R deficiency underwent metabolic and endocrine evaluation; the results were correlated with the signaling properties of mutant receptors. Twenty-nine probands (5.8%) had mutations in MC4R; 23 were heterozygous, and 6 were homozygous. Mutation carriers had severe obesity, increased lean mass, increased linear growth, hyperphagia, and severe hyperinsulinemia; homozygotes were more severely affected than heterozygotes. Subjects with mutations retaining residual signaling capacity had a less severe phenotype. Mutations in MC4R result in a distinct obesity syndrome that is inherited in a codominant manner. Mutations leading to complete loss of function are assocd. with a more severe phenotype. The correlation between the signaling properties of these mutant receptors and energy intake emphasizes the key role of this receptor in the control of eating behavior in humans.
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Vaisse, C., Clement, K., Durand, E., Hercberg, S., Guy-Grand, B., and Froguel, P. (2000) Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity J. Clin. Invest. 106, 253– 262
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Xiang, Z., Proneth, B., Dirain, M. L., Litherland, S. A., and Haskell-Luevano, C. (2010) Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist Biochemistry 49, 4583– 4600
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Xiang, Z., Litherland, S. A., Sorensen, N. B., Proneth, B., Wood, M. S., Shaw, A. M., Millard, W. J., and Haskell-Luevano, C. (2006) Pharmacological characterization of 40 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists and the agouti-related protein (AGRP) antagonist Biochemistry 45, 7277– 7288
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There is no corresponding record for this reference.
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Xiang, Z., Pogozheva, I. D., Sorenson, N. B., Wilczynski, A. M., Holder, J. R., Litherland, S. A., Millard, W. J., Mosberg, H. I., and Haskell-Luevano, C. (2007) Peptide and small molecules rescue the functional activity and agonist potency of dysfunctional human melanocortin-4 receptor polymorphisms Biochemistry 46, 8273– 8287
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Peptide and Small Molecules Rescue the Functional Activity and Agonist Potency of Dysfunctional Human Melanocortin-4 Receptor Polymorphisms
Xiang, Zhimin; Pogozheva, Irina D.; Sorenson, Nicholas B.; Wilczynski, Andrzej M.; Holder, Jerry Ryan; Litherland, Sally A.; Millard, William J.; Mosberg, Henry I.; Haskell-Luevano, Carrie
Biochemistry (2007), 46 (28), 8273-8287CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal wt. control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms obsd. as both heterozygous and homozygous forms. A no. of labs. have been studying these hMC4R polymorphisms attempting to understand the mol. mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small mols. attempting to identify ligands that can pharmacol. rescue the hMC4R polymorphic agonist response. The ligands examd. in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg-Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH2, AMW3-106), and the small mols. JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examd. in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the mol. mechanism(s) involving these receptor modifications.
34
Singh, A., Wilczynski, A., Holder, J. R., Witek, R. M., Dirain, M. L., Xiang, Z., Edison, A. S., and Haskell-Luevano, C. (2011) Incorporation of a bioactive reverse-turn heterocycle into a peptide template using solid-phase synthesis to probe melanocortin receptor selectivity and ligand conformations by 2D ¹H NMR J. Med. Chem. 54, 1379– 1390
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Haskell-Luevano, C., Boteju, L. W., Miwa, H., Dickinson, C., Gantz, I., Yamada, T., Hadley, M. E., and Hruby, V. J. (1995) Topographical modification of melanotropin peptide analogues with β-methyltryptophan isomers at position 9 leads to differential potencies and prolonged biological activities J. Med. Chem. 38, 4720– 4729
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Haskell-Luevano, C., Toth, K., Boteju, L., Job, C., Castrucci, A. M., Hadley, M. E., and Hruby, V. J. (1997) β-Methylation of the Phe7 and Trp9 melanotropin side chain pharmacophores affects ligand-receptor interactions and prolonged biological activity J. Med. Chem. 40, 2740– 2749
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β-Methylation of the Phe7 and Trp9 melanotropin side chain pharmacophores affects ligand-receptor interactions and prolonged biological activity
Haskell-Luevano, Carrie; Toth, Kate; Boteju, Lakmal; Job, Constatin; de Castrucci, Ana Maria; Hadley, Mac E.; Hruby, Victor J.
Journal of Medicinal Chemistry (1997), 40 (17), 2740-2749CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
Topog. modified melanotropin side chain pharmacophore residues Phe7 and Trp9 in a cyclic peptide template (Ac-Nle4-c[Asp-His-Xaa7-Arg-Yaa9-Lys]-NH2) and Phe7 in a linear peptide template (Ac-Ser-Tyr-Ser-Nle4-Glu-His-Xaa7-Arg-Trp-Gly-Lys-Pro-Val-NH2) result in differences in potency and prolonged biol. activity in the frog and lizard skin bioassays. These topog. modifications included the four isomers of β-methylphenylalanine (β-MePhe)7 and β-methyltryptophan (β-MeTrp)9 and the two isomers of 1,2,3,4-tetrahydro-β-carboline (Tca).9. Modifications in the cyclic template resulted in up to a 1000-fold difference in potency for the β-MePhe7 stereoisomeric peptides; up to a 476-fold difference in potency resulted for the β-MeTrp9 peptides, and about a 50-fold difference between the Tca9-contg. peptides. Up to a 40-fold difference in potency resulted for the β-MePhe7 stereoisomeric peptides using the linear template in these assays. The relative potency ranking for modifications in the cyclic template of β-MePhe7 were 2R,3S > 2S,3S = 2S,3R > 2R,3R in the frog assay and 2S,3R > 2R,3S > 2S,3S > 2R,3R in the lizard assay. The relative potencies for modifications in the cyclic template of β-MeTrp9 were 2R,3S > 2R,3R > 2S,3S » 2S,3R in the frog assay and 2S,3S = 2R,3R > 2R,3S > 2S,3R in the lizard assay. The relative potencies for modifications in the cyclic template of Tca9 were DTca > LTca in both assays. Significant differences in prolonged (residual) activities were also obsd. for these modified peptides and were dependent upon stereochem. of the β-Me amino acid, peptide template, and bioassay system. Furthermore, comparisons of β-MeTrp9 stereoisomeric peptides on the frog, lizard, and human MC1 receptors suggest that structure-activity relationships on both the classical frog and lizard skin bioassays do not necessarily predict corresponding SAR profiles for the human melanocortin receptors, indicating a remarkable species specificity of the MC1 receptor requirements.
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Structure-activity relationships of the melanocortin tetrapeptide Ac-His-D-Phe-Arg-Trp-NH2 at the mouse melanocortin receptors. 4. Modifications at the Trp position
Holder, Jerry Ryan; Xiang, Zhimin; Bauzo, Rayna M.; Haskell-Luevano, Carrie
Journal of Medicinal Chemistry (2002), 45 (26), 5736-5744CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
The melanocortin pathway is involved in the regulation of several physiol. functions including skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. This melanocortin pathway consists of five known G-protein coupled receptors, endogenous agonists derived from the proopiomelanocortin (POMC) gene transcript, the endogenous antagonists Agouti and the Agouti-related protein (AGRP) and signals through the intracellular cAMP signal transduction pathway. The endogenous melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp," postulated to be important for melanocortin receptor mol. recognition and stimulation. Herein, the authors report a tetrapeptide library, based upon the template Ac-His--D-Phe-Arg-Trp-NH2, consisting of 20 members that have been modified at the Trp9 position (α-MSH numbering) and pharmacol. characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. Results from this study yielded compds. that ranged in pharmacol. properties from equipotent to a loss of melanocortin receptor activity at up to 100 μM concns. Interestingly, modification of the Trp9 in the tetrapeptide template at the MC1R resulted in only up to a 220-fold potency change, while at the MC4R and MC5R, up to a 9700-fold decrease in potency was obsd., suggesting the MC1R is more tolerant of the modifications examd. herein. The most notable results of this study include identification that the Trp9 indole moiety in the tetrapeptide template is important for melanocortin-3 receptor agonist potency, and that this position can be used to design melanocortin ligands possessing receptor selectivity for the peripherally expressed MC1 and MC5 vs. the centrally expressed MC3 and MC4 receptors. Specifically, the Ac-His--D-Phe-Arg-Tic-NH2 and the Ac-His--D-Phe-Arg-Bip-NH2 tetrapeptides possessed nanomolar MC1R and MC5R potency but micromolar MC3R and MC4R agonist potency. Addnl., these studies identified that substitution of the Trp amino acid with either Nal(2') or D-Nal(2') resulted in equipotent melanocortin receptor potency, suggesting that the chem. reactive Trp indole side chain may be replaced with the nonreactive Nal(2') moiety for the design of nonpeptide melanocortin receptor agonists.
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Biochemistry (2003), 42 (48), 14130-14138CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analog CP-11, ILKKWPWWPWRRK-NH2, with improved activity against Gram-neg. bacteria had increased pos. charge and amphipathicity while maintaining the short length of the parent mol. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was detd. using NMR spectroscopy. CP-11 was found to be an amphipathic mol. with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded peptide cycloCP-11, ICLKKWPWWPWRRCK-NH2, was designed to stabilize the lipid-bound structure and to increase protease resistance. The 3-dimensional structure of cycloCP-11 was detd. under the same conditions as for the linear peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 assocd. with phospholipid membranes in a similar manner as indicated by CD and fluorescence spectra. The MICs of CP-11 and cycloCP-11 for a range of bacteria differed by ≤2-fold, and they were nonhemolytic at concns. ≤256 μg/mL. Cyclization greatly increased protease stability. The half-life of cycloCP-11 in the presence of trypsin was increased by 4.5-fold, from 4 to 18 min. More importantly, the antibacterial activity of cycloCP-11, but not that of CP-11, in the presence of trypsin was completely retained up to 90 min, since the major degrdn. product was equally active. A structural comparison of CP-11 and cycloCP-11 revealed that the higher trypsin resistance of cycloCP-11 may be due to the more compact packing of lysine and tryptophan side chains. These findings suggest that cyclization may serve as an important strategy in the rational design of antimicrobial peptides.
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A series of synthetic antimicrobial peptidomimetics (SAMPs) have been prepd. and found to be highly active against several Gram-neg. and Gram-pos. bacterial strains. These derivs. comprise the minimal structural requirements for cationic antimicrobial peptides and showed high selectivity for Gram-neg. and/or Gram-pos. bacteria compared to human red blood cells. The authors have found that SAMPs share many of the attractive properties of cationic antimicrobial peptides inasmuch that a representative SAMP was found to insert into the bilayers of large unilamellar vesicles, permeabilized both the outer and cytoplasmic membrane of Escherichia coli ML-35p, and displayed an extremely rapid bacterial killing for Staphylococcus aureus. However, while antimicrobial peptides are prone to proteolytic degrdn., high in vitro stability in human blood plasma was shown for SAMPs. A combination of high antibacterial activity against methicillin-resistant staphylococci and low toxicity against human erythrocytes makes these mols. promising candidates for novel antibacterial therapeutics.
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Raguse, Tami L.; Porter, Emilie A.; Weisblum, Bernard; Gellman, Samuel H.
Journal of the American Chemical Society (2002), 124 (43), 12774-12785CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
Antimicrobial α-helical α-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of β-amino acids ("β-peptides") that can adopt an amphiphilic helix defined by 14-membered ring H-bonds (14-helix) are active against Escherichia coli (Y. Hamuro et al., 1999). We have created two series of cationic 9- and 10-residue amphiphilic β-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short β-peptides encourages 14-helical structure in aq. soln. (D. H. Appella et al., 1999). CD of the β-peptides described here reveals a broad range of 14-helix population in aq. buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin deriv. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin deriv. Previous studies with conventional peptides (α-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because α-helix stability can be varied to only a limited extent among linear α-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with β-peptides. For example, it is very rare for a linear α-peptide to display significant α-helix formation in aq. soln. and manifest antibacterial activity, while the linear β-peptides described here range from fully unfolded to very highly folded in aq. soln. This study shows that β-peptides can be unique tools for analyzing relationships between conformational stability and biol. activity.
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59
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A rapid color test is described for detecting ≥5 micromoles free terminal amino group/g in the reaction mixt. from solid-phase peptide synthesis according to R. B. Merrifield (1968). After washing the solid phase with CH2Cl2, a 10-20 mg sample of the resin peptide is treated with 2-3 drops each of the following reagents: 500 mg ninhydrin/10 ml EtOH; 80 g PhOH/ 20 ml EtOH; and 20 ml 0.001M KCN dild. to 100 ml with pyridine. The mixt. is heated 2-5 min at 100°. The development of an intense blue color on the beads and in the soln. (except that terminal proline or terminal β-benzyl aspartate gave a brown or red-brown color) is a pos. result. In the absence of untreated terminal amino groups, the beads remained white and the soln. yellow (neg. result). The formation of pale blue beads and a light green soln. was a slightly pos. result.
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Thirumoorthy, R., Holder, J. R., Bauzo, R. M., Richards, N. G. J., Edison, A. S., and Haskell-Luevano, C. (2001) Novel Agouti-related Protein (AGRP) Based Melanocortin-1 Receptor Antagonist J. Med. Chem. 44, 4114– 4124
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Abstract
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Scheme 1
Scheme 1. ^a
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Scheme aReagents and conditions: (a) 20% piperidine/DMF; (b) 3 equiv Fmoc-Asn(Trt)-OH, 3 equiv BOP or HBTU, 6 equiv DIEA, DMF; (c) Fmoc-Trp(Boc)-aldehyde, NaBH₃CN, AcOH, DMF; (d) Fmoc-Cys(SBu^t)-OH (R or S), BOP or HBTU, DIEA, DMF; (e) (ClCH₂CO)₂O, NEM, DCM; (f) Bu₃P/H₂O/THF; (g) NEM, DMF, heat 55–60 °C; (h) TFA/TIS/EDT/H₂O 91:3:3:3; (i) 20% DMSO/H₂O, rt.
Figure 1
Figure 1. (A) Illustration of the compounds (i) heterocycle, (ii) AMW610, (iii) AST3-88, and (iv) AMW6103. The stereochemistry of the thioether ring is represented by an asterisk (*). Compounds AST3-88 and AMW610 contain l-Cys, and compound AMW6103 contains a d-Cys at this position. The side chain of His is depicted in green, DPhe in orange, Arg in blue, the heterocycle moiety in pink, and the disulfide bridge is circled in yellow. (B) Amino acid sequences of key endogenous and synthetic melanocortin peptides.
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Figure 2
Figure 2. Illustration of the compound deviation from random coil values for (A) AST3-88, (B) AMW610, and (C) AMW3-130. (45) The H^N and H^α protons are represented by solid bars and open bars, respectively. Vertical axes are in parts per million, and the horizontal axes represent the His-DPhe-Arg-Trp-Asn-Ala region of the peptide.
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Figure 3
Figure 3. Summary of the NOE intensities from 400 ms NOESY data observed for the compound (A) AST3-88, (B) AMW610, and (C) AMW3-130. The height of the bar indicates the strength of the NOE, and these are categorized as strong (1.8–3.0 Å), medium (1.8–3.5 Å), or weak (1.8–5.0 Å).
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Figure 4
Figure 4. Sausage stereoview representations of the major family members of compound (A) AST3-88 (in the H, F, R,W region; RMSD = 1.16 ± 0.45 Å), (B) AMW610, and (C) AMW3-130 (in the H, F, R,W region; RMSD = 0.66 ± 0.3 Å) aligned on the backbone heavy atoms of residues 3–7. The His side chain is indicated in green, the DPhe side chain in yellow, the Arg side chain in blue, and the Trp side chain in orange. A wide gray backbone indicates greater flexibility in that domain. Following restrained molecular dynamics (RMD) simulations for 10 ns, 200 equally spaced structures were energy minimized with the NMR based restraints. The energy minimized structures were grouped into conformational families by comparison of the backbone dihedral angels within the His-Phe-Arg-Trp domain.
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Figure 5
Figure 5. CAMM based φ–ψ angle distribution for ligands (A) AST3-88, (B) AMW610, and (C) AMW3-130 conformational families. The His residue is indicated in green, the DPhe residue in orange, the Arg residue in blue, and the Trp residue in red. All other residues are indicated in purple.
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Figure 6
Figure 6. Rat serum stability profiles of the peptides AST789-13, α-MSH, and AST3-88. Relative peptide concentrations are based on mass spec ion counts. AST789-13 completely degraded in less than 2 h. Endogenous linear peptide α-MSH rapidly degraded within 6 h with only ∼1% intact peptide remaining at the 24 h point. Compound AST3-88, with the engineered backbone, degraded slowly in rat serum with ∼40% remaining at 6 h, and ∼12% was detected by MS at the 24 h time point.
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Figure 7
Figure 7. (A) Effects of AST3-88 ICV treatment on cumulative food intake in rats. Animals were injected with either saline, 2.5 nmol, or 5 nmol of AST3-88 after completion of overnight fasting. Each data point is represents the mean of 4–6 animals ± SEM; *p < 0.05 and ***p < 0.001.
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References
This article references 61 other publications.
1. 1
  Mountjoy, K. G., Robbins, L. S., Mortrud, M. T., and Cone, R. D. (1992) The cloning of a family of genes that encode the melanocortin receptors Science 257, 1248– 1251
  1
  The cloning of a family of genes that encode the melanocortin receptors
  Mountjoy, Kathleen G.; Robbins, Linda S.; Mortrud, Marty T.; Cone, Roger D.
  Science (Washington, DC, United States) (1992), 257 (5074), 1248-51CODEN: SCIEAS; ISSN:0036-8075.
  MSH and adrenocorticotropic hormone (ACTH) regulate pigmentation and adrenal cortical function, resp. These peptides also have a variety of biol. activities in other areas, including the brain, the pituitary, and the immune system. A complete understanding of the biol. activities of these hormones requires the isolation and characterization of their corresponding receptors. The murine and human MSH receptors (MSH-Rs) and a human ACTH receptor (ACTH-R) were cloned. These receptors define a subfamily of receptors coupled to guanine nucleotide-binding proteins that may include the cannabinoid receptor.
2. 2
  Chhajlani, V. and Wikberg, J. E. (1992) Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA FEBS Lett. 309, 417– 420
  2
  Molecular cloning and expression of the human melanocyte stimulating hormone receptor cDNA
  Chhajlani, Vijay; Wikberg, Jarl E. S.
  FEBS Letters (1992), 309 (3), 417-20CODEN: FEBLAL; ISSN:0014-5793.
  A cDNA clone, designated 11D, has been isolated from human melanoma cells and encodes a MSH receptor. The cloned cDNA encodes a 317 amino acid protein with transmembrane topog. characteristics of a G-protein-coupled receptor, but it does not show striking similarity to already published sequences of other G-protein-coupled receptors. When 11D cDNA is expressed in COS-7 cells, it binds an 125I-labeled MSH analog (NDP-MSH) in a specific manner. The bound ligand could be displaced by melanotropic peptides, α-MSH, β-MSH, γ-MSH, and ACTH, but not by the non-melanotropic peptide, β-endorphin.
3. 3
  Roselli-Rehfuss, L., Mountjoy, K. G., Robbins, L. S., Mortrud, M. T., Low, M. J., Tatro, J. B., Entwistle, M. L., Simerly, R. B., and Cone, R. D. (1993) Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system Proc. Natl. Acad. Sci. U.S.A. 90, 8856– 8860
  3
  Identification of a receptor for gamma melanotropin and other proopiomelanocortin peptides in the hypothalamus and limbic system
  Roselli-Rehfuss L; Mountjoy K G; Robbins L S; Mortrud M T; Low M J; Tatro J B; Entwistle M L; Simerly R B; Cone R D
  Proceedings of the National Academy of Sciences of the United States of America (1993), 90 (19), 8856-60 ISSN:0027-8424.
  Corticotropin (ACTH) and melanotropin (MSH) peptides (melanocortins) are produced not only in the pituitary but also in the brain, with highest concentrations in the arcuate nucleus of the hypothalamus and the commisural nucleus of the solitary tract. We have identified a receptor for MSH and ACTH peptides that is specifically expressed in regions of the hypothalamus and limbic system. This melanocortin receptor (MC3-R) is found in neurons of the arcuate nucleus known to express proopiomelanocortin (POMC) and in a subset of the nuclei to which these neurons send projections. The MC3-R is 43% identical to the MSH receptor present in melanocytes and is strongly coupled to adenylyl cyclase. Unlike the MSH or ACTH receptors, MC3-R is potently activated by gamma-MSH peptides, POMC products that were named for their amino acid homology with alpha- and beta-MSH, but lack melanotropic activity. The primary biological role of the gamma-MSH peptides is not yet understood. The location and properties of this receptor provide a pharmacological basis for the action of POMC peptides produced in the brain and possibly a specific physiological role for gamma-MSH.
4. 4
  Mountjoy, K. G., Mortrud, M. T., Low, M. J., Simerly, R. B., and Cone, R. D. (1994) Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain Mol. Endocrinol. 8, 1298– 1308
  4
  Localization of the melanocortin-4 receptor (MC4-R) in neuroendocrine and autonomic control circuits in the brain
  Mountjoy, Kathleen G.; Mortrud, Marty T.; Low, Malcolm J.; Simerly, Richard B.; Cone, Roger D.
  Molecular Endocrinology (1994), 8 (10), 1298-308CODEN: MOENEN; ISSN:0888-8809. (Endocrine Society)
  POMC, the precursor of ACTH, MSH, and β-endorphin peptides, is expressed in the pituitary and in two sites in the brain, in the arcuate nucleus of the hypothalamus and the commissural nucleus of the solitary tract of the brain stem. Little is known regarding the functions of melanocortin (ACTH and MSH) peptides in the brain. The authors report here the detailed neuroanatomical distribution of the MC4-R mRNA in the adult rat brain. The melanocortin 3 receptor (MC3-R), characterized previously, was found to be expressed in arcuate nucleus neurons and in a subset of their presumptive terminal fields but in few regions of the brainstem. The highly conserved MC4-R is much more widely expressed than MC3-R and is pharmacol. distinct. MC4-R mRNA was found in multiple sites in virtually every brain region, including the cortex, thalamus, hypothalamus, brainstem, and spinal cord. Unlike the MC3-R, MC4-R mRNA is found in both parvicellular and magnocellular neurons of the paraventricular nucleus of the hypothalamus, suggesting a role in the central control of pituitary function. MC4-R is also unique in its expression in numerous cortical and brainstem nuclei. Together, MC3-R and/or MC-4R mRNA are found in every nucleus reported to bind MSH in the adult rat brain and define neuronal circuitry known to be involved in the control of diverse neuroendocrine and autonomic functions. The high degree of conservation, distinct pharmacol., and unique neuronal distribution of the MC4 receptor suggest specific and complex roles for the melanocortin peptides in neuroendocrine and autonomic control.
5. 5
  Gantz, I., Miwa, H., Konda, Y., Shimoto, Y., Tashiro, T., Watson, S. J., DelValle, J., and Yamada, T. (1993) Molecular cloning, expression, and gene localization of a fourth melanocortin receptor J. Biol. Chem. 268, 15174– 15179
  5
  Molecular cloning, expression, and gene localization of a fourth melanocortin receptor
  Gantz, Ira; Miwa, Hiroto; Konda, Yoshitaka; Shimoto, Yoshimasa; Tashiro, Takao; Watson, Stanley J.; DelValle, John; Yamada, Tadataka
  Journal of Biological Chemistry (1993), 268 (20), 15174-9CODEN: JBCHA3; ISSN:0021-9258.
  The recent cloning of three melanocortin receptors suggests an unexpected diversity in this family of seven transmembrane G-protein linked receptors. Herein, the authors report the cloning, expression, and gene localization of a fourth human melanocortin receptor, the melanocortin-4 receptor. By Northern blot anal. and in situ hybridization, this receptor is expressed primarily in the brain, but its expression is notably absent in the adrenal cortex, melanocytes, and placenta. Agonist stimulation of COS-1 cells transiently transfected and L-cells permanently transfected with the coding region of the cloned melanocortin-4 receptor leads to increases in intracellular cyclic 3',5'-adenosine monophosphate. The profile of the responses of the melanocortin-4 receptor to different melanocortins distinguishes it from melanocortin receptors previously described. Using the technique of fluorescent in situ hybridization, the gene encoding the melanocortin-4 receptor was localized to chromosome 18 (q21.3).
6. 6
  Gantz, I., Shimoto, Y., Konda, Y., Miwa, H., Dickinson, C., and Yamada, T. (1994) Molecular cloning, expression, and characterization of a fifth melanocortin receptor Biochem. Biophys. Res. Commun. 200, 1214– 1220
  6
  Molecular cloning, expression, and characterization of a fifth melanocortin receptor
  Gantz, Ira; Shimoto, Yoshimasa; Konda, Yoshitaka; Miwa, Hiroto; Dickinson, Chris J.; Yamada, Tadataka
  Biochemical and Biophysical Research Communications (1994), 200 (3), 1214-20CODEN: BBRCA9; ISSN:0006-291X.
  The authors report the isolation of a gene encoding a novel member of the family of melanocortin receptors. The mouse melanocortin-5 receptor (mMC5R) responds to melanocortins with an increase in intracellular cyclic 3',5'-adenosine monophosphate (cAMP) concns. Stimulation of the mMC5R by the melanocortins revealed a hierarchy of potency in which α-MSH (α-MSHZ) >β-MSH (β-MSH) >adrenocorticotropic hormone (ACTH) >γ-MSH (γ-MSH). Further structure-activity studies indicated that amino- and carboxyl-terminal portions of α-MSH appear to be key determinants in the activation of mMC5R whereas the melanocortin core heptapeptide sequence is devoid of pharmacol. activity. Northern blot anal. demonstrated the expression of mMC5R mRNA in mouse skeletal muscle, lung, spleen, and brain.
7. 7
  Eipper, B. A. and Mains, R. E. (1980) Structure and biosynthesis of pro-adrenocorticotropin/endorphin and related peptides Endocr. Rev. 1, 1– 27
  7
  Structure and biosynthesis of pro-adrenocorticotropin/endorphin and related peptides
  Eipper, Betty A.; Mains, Richard E.
  Endocrine Reviews (1980), 1 (1), 1-27CODEN: ERVIDP; ISSN:0163-769X.
  A review with 186 refs. on the structure and biosynthesis of ACTH, β-endorphin, and a common precursor.
8. 8
  Smith, A. I. and Funder, J. W. (1988) Proopiomelanocortin processing in the pituitary, central nervous system, and peripheral tissues Endocr. Rev. 9, 159– 179
  8
  Pro-opiomelanocortin processing in the pituitary, central nervous system, and peripheral tissues
  Smith, A. Ian; Funder, John W.
  Endocrine Reviews (1988), 9 (1), 159-79CODEN: ERVIDP; ISSN:0163-769X.
  A review, with 247 refs., of pro-opiomelanocortin processing in pituitary gland, brain, and peripheral tissues and organs. Focus was placed on tissue-specific differences in processing and, in particular, examn. of factors involved in the regulation and control of these tissue-specific processes.
9. 9
  Ollmann, M. M., Wilson, B. D., Yang, Y. K., Kerns, J. A., Chen, Y., Gantz, I., and Barsh, G. S. (1997) Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein Science 278, 135– 138
  9
  Antagonism of central melanocortin receptors in vitro and in vivo by Agouti-related protein
  Ollmann, Michael M.; Wilson, Brent D.; Yang, Ying-Kui; Kerns, Julie A.; Chen, Yanru; Gantz, Ira; Barsh, Gregory S.
  Science (Washington, D. C.) (1997), 278 (5335), 135-138CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)
  Expression of Agouti protein is normally limited to the skin where it affects pigmentation, but ubiquitous expression causes obesity. An expressed sequence tag was identified that encodes Agouti-related protein, whose RNA is normally expressed in the hypothalamus and whose levels were increased eightfold in ob/ob mice. Recombinant Agouti-related protein was a potent, selective antagonist of Mc3r and Mc4r, melanocortin receptor subtypes implicated in wt. regulation. Ubiquitous expression of human AGRP complementary DNA in transgenic mice caused obesity without altering pigmentation. Thus, Agouti-related protein is a neuropeptide implicated in the normal control of body wt. downstream of leptin signaling.
10. 10
  Lu, D., Willard, D., Patel, I. R., Kadwell, S., Overton, L., Kost, T., Luther, M., Chen, W., Woychik, R. P., and Wilkison, W. O. 1994, Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor Nature 371, 799– 802
  There is no corresponding record for this reference.
11. 11
  McNulty, J. C., Jackson, P. J., Thompson, D. A., Chai, B., Gantz, I., Barsh, G. S., Dawson, P. E., and Millhauser, G. L. (2005) Structures of the Agouti Signaling Protein J. Mol. Biol. 346, 1059– 1070
  There is no corresponding record for this reference.
12. 12
  Castrucci, A. M., Hadley, M. E., Sawyer, T. K., Wilkes, B. C., al-Obeidi, F., Staples, D. J., de Vaux, A. E., Dym, O., Hintz, M. F., and Riehm, J. P. 1989, Alpha-melanotropin: the minimal active sequence in the lizard skin bioassay Gen. Comp. Endrocrinol. 73, 157– 163
  12
  α-Melanotropin: the minimal active sequence in the lizard skin bioassay
  Castrucci, A. M. L.; Hadley, M. E.; Sawyer, T. K.; Wilkes, B. C.; Al-Obeidi, F.; Staples, D. J.; De Vaux, A. E.; Dym, O.; Hintz, M. F.; et al.
  General and Comparative Endocrinology (1989), 73 (1), 157-63CODEN: GCENA5; ISSN:0016-6480.
  α-Melanotropin (α-MSH) is a tridecapeptide, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2. The minimal sequence of α-MSH required for agonism in the lizard (Anolis carolinensis) skin bioassay was detd. to be Ac-His-Phe-Arg-Trp-NH2 (Ac-α-MSH6-9-NH2). Smaller fragments of this sequence (Ac-α-MSH6-8-NH2, Ac-α-MSH6-7-NH2, Ac-α-MSH7-9-NH2, and Ac-α-MSH7-8-NH2) were devoid of melanotropic activity. The tetrapeptide Ac-α-MSH7-10-NH2 was also inactive, thus again demonstrating the importance of His at position 6 for minimal activity. The important potentiating amino acids were Met-4, Lys-11, and Pro-12, since Ac-α-MSH4-10-NH2 was about 100 times more potent than Ac-α-MSH5-10-NH2, and Ac-[Nle4]-α-MSH4-11-NH2 was about 40 times more potent than Ac-α-MSH4-10-NH2 or Ac-[Nle4]-α-MSH4-10-NH2. Ac-α-MSH4-12-NH2 and Ac-[Nle4]-α-MSH4-12-NH2 were equipotent and about 6 times more potent than α-MSH. Since [Nle4]-α-MSH and Ac-[Nle4]-α-MSH4-13-NH2 were both equipotent but about sixfold less active than Ac-[Nle4]-α-MSH4-12-NH2, it is clear that valine at position 13 does not contribute to the potency of α-MSH, except possibly in a neg. way. The minimal message sequence for equipotency to α-MsH appears to be Ac-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-NH2, since the analog, Ac-[Nle4]-α-MSH4-11-NH2, was as active as the native hormone. Ser-1, Tyr-2, Ser-3, Glu-5, and Val-13 are not important for melanotropic potency since Ac-α-MSH4-12-NH2 was more potent than α-MSH, and Ac-α-MSH5-10-NH2 and Ac-α-MSH6-10-NH2 were equipotent, being about 4000 times less active than α-MSH.
13. 13
  Hruby, V. J., Wilkes, B. C., Hadley, M. E., Al-Obeidi, F., Sawyer, T. K., Staples, D. J., de Vaux, A. E., Dym, O., Castrucci, A. M., and Hintz, M. F. 1987, α-Melanotropin: the minimal active sequence in the frog skin bioassay J. Med. Chem. 30, 2126– 2130
  13
  α-Melanotropin: the minimal active sequence in the frog skin bioassay
  Hruby, Victor J.; Wilkes, Brian C.; Hadley, Mac E.; Al-Obeidi, Fahad; Sawyer, Tomi K.; Staples, Douglas J.; DeVaux, Ann E.; Dym, Orin; Castrucci, Ana Maria de L.; et al.
  Journal of Medicinal Chemistry (1987), 30 (11), 2126-30CODEN: JMCMAR; ISSN:0022-2623.
  A series of fragment analogs of α-MSH (I) were prepd. in order to det. the contribution of each individual amino acid to the biol. activity of the native hormone. The minimal potency of Ac-α-MSH6-9-NH2 could be enhanced about a factor of 16 by the addn. of glycine to the C-terminus, yielding Ac-α-MSH6-10-NH2. Addn. of glutamic acid to the N-terminus provided Ac-α-MSH5-10-NH2, which was only slightly more potent than Ac-α-MSH6-10-NH2, indicating that position 5 contributes little to the biol. potency of α-MSH in this assay. Addn. of methionine to the N-terminus of Ac-α-MSH5-10-NH2 resulted in Ac-α-MSH4-10-NH2, which was only about 4-fold more potent than Ac-α-MSH5-10-NH2. Addn. of lysine and proline to the C-terminal of the Ac-α-MSH4-10-NH2 sequence yielded Ac-α-MSH4-12-NH2 with a 360-fold increase in potency relative to Ac-α-MSH4-10-NH2. This peptide was only about 6-fold less potent than α-MSH. Nle-4-substituted analogs were also prepd. Ac-[Nle4]-α-MSH4-10-NH2 and Ac-[Nle4]-α-MSH4-11-NH2 were ∼4 times more potent than Ac-α-MSH4-10-NH2, demonstrating that lysine-11 contributes somewhat to the biol. activity of α-MSH on the frog skin melanocyte receptor. However, addn. of proline-12 to this fragment, yielding Ac-[Nle4]-α-MSH4-12-NH2, resulted in about a 90-fold increase in relative potency of the melanotropin. Addn. of the final C-terminal valine-13 provided Ac-[Nle4]-α-MSH4-13-NH2, which showed only a small further increase in potency. This analog was, however, only about 2 to 3-fold less active than α-MSH. Addn. of the N-terminal tripeptide Ac-Ser-Tyr-Ser to yield [Nle4]-α-MSH resulted in an analog that was 3 times more potent than α-MSH. The central tetrapeptide sequence, Ac-His-Phe-Arg-Trp-NH2, represents the min. chain length for observable biol. activity. The active sequence of α-MSH is contiguous in that no two structurally noncontiguous fragment sequences were found to have biol. activity. Met-4, Gly-10, and Pro-12 are important potentiating amino acids and contribute significantly to the biopotency of α-MSH, and Ser-1 and -3, Tyr-2, Glu-5, Lys-11, and Val-13 apparently contribute only minimally to the biol. potency of α-MSH at the frog melanocyte skin receptor.
14. 14
  Haskell-Luevano, C., Sawyer, T. K., Hendrata, S., North, C., Panahinia, L., Stum, M., Staples, D. J., Castrucci, A. M., Hadley, M. F., and Hruby, V. J. (1996) Truncation studies of α-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity Peptides 17, 995– 1002
  There is no corresponding record for this reference.
15. 15
  Holder, J. R. and Haskell-Luevano, C. (2003) Melanocortin tetrapeptides modified at the N-terminus, His, Phe, Arg, and Trp positions Ann. N.Y. Acad. Sci. 994, 36– 48
  There is no corresponding record for this reference.
16. 16
  Haskell-Luevano, C., Holder, J. R., Monck, E. K., and Bauzo, R. M. (2001) Characterization of melanocortin NDP-MSH agonist peptide fragments at the mouse central and peripheral melanocortin receptors J. Med. Chem. 44, 2247– 2252
  There is no corresponding record for this reference.
17. 17
  Haskell-Luevano, C., Sawyer, T. K., Hendrata, S., North, C., Panahinia, L., Stum, M., Staples, D. J., Castrucci, A. M., Hadley, M. F., and Hruby, V. J. (1996) Truncation studies of α-melanotropin peptides identify tripeptide analogues exhibiting prolonged agonist bioactivity Peptides 17, 995– 1002
  There is no corresponding record for this reference.
18. 18
  Hruby, V. J. W., B, C., Cody, W. L., Sawyer, T. K., and Hadley, M. E. (1984) Melanotropins: Structural, Conformational and Biological Considerations in the Development of Superpotent and Superprolonged Analogs Pept. Protein Rev. 3, 1– 64
  18
  Melanotropins: structural, conformational and biological considerations in the development of superpotent and superprolonged analogs
  Hruby, Victor J.; Wilkes, Brian C.; Cody, Wayne L.; Sawyer, Tomi K.; Hadley, Mac E.
  Peptide and Protein Reviews (1984), 3 (), 1-64CODEN: PPRVDF; ISSN:0731-1753.
  A review, with 178 refs. on the structural properties and biol. activities of melanotropin [9002-79-3] and analogs.
19. 19
  Lerner, A. B. and McGuire, J. S. (1961) Effect of α- and β-melanocyte stimulating hormones on the skin colour of man Nature 189, 176– 179
  19
  Effect of α- and β-melanocyte-stimulating hormones on the skin color of man
  Lerner, Aaron B.; McGuire, Joseph S.
  Nature (London, United Kingdom) (1961), 189 (), 176-9CODEN: NATUAS; ISSN:0028-0836.
  Synthetic α-melanocyte-stimulating hormone (I) and β-melanocyte-stimulating hormone (II) from hog pituitary glands were administered intramuscularly to Negroes at a level of 4-8 mg./day for periods up to 14 days. With I darkening of the skin occurred in 2 subjects; with II darkening occurred in only 1 of 2 subjects. Darkening was greater on the forehead than on the arm, and began almost within 24 hrs. of the initiation of the expt. All hyperpigmentation regressed completely within 6 weeks.
20. 20
  Gantz, I., Konda, Y., Tashiro, T., Shimoto, Y., Miwa, H., Munzert, G., Watson, S. J., DelValle, J., and Yamada, T. (1993) Molecular cloning of a novel melanocortin receptor J. Biol. Chem. 268, 8246– 8250
  20
  Molecular cloning of a novel melanocortin receptor
  Gantz, Ira; Konda, Yoshitaka; Tashiro, Takao; Shimoto, Yoshimasa; Miwa, Hiroto; Munzert, Gerd; Watson, Stanley J.; DelValle, John; Yamada, Tadataka
  Journal of Biological Chemistry (1993), 268 (11), 8246-50CODEN: JBCHA3; ISSN:0021-9258.
  By using the technique of the polymerase chain reaction primed with oligonucleotides based on the homologous transmembrane regions of seven transmembrane G protein-linked receptors, 3 full-length human genes that encode a novel subgroup of this receptor family have been isolated. Recently, 2 of these receptors were identified as specific for α-MSH and ACTH. The mol. cloning and pharmacol. characterization of a 3rd member of this subgroup is now reported. The gene for this receptor encodes a protein of 361 amino acids in length. Its pharmacol. characterizes it as an MSH receptor specific to the heptapeptide core common to ACTH and α-, β-, and γ-MSH. By Northern blot hybridization and polymerase chain reaction, it is expressed in brain, placental, and gut tissues but not in melanoma cells or in the adrenal gland. The findings may yield insight into the physiol. of peptides derived from pro-opiomelanocortin posttranslational processing.
21. 21
  Chen, A. S., Marsh, D. J., Trumbauer, M. E., Frazier, E. G., Guan, X. M., Yu, H., Rosenblum, C. I., Vongs, A., Feng, Y., Cao, L., Metzger, J. M., Strack, A. M., Camacho, R. E., Mellin, T. N., Nunes, C. N., Min, W., Fisher, J., Gopal-Truter, S., MacIntyre, D. E., Chen, H. Y., and Van der Ploeg, L. H. (2000) Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass Nat. Genet. 26, 97– 102
  21
  Inactivation of the mouse melanocortin-3 receptor results in increased fat mass and reduced lean body mass
  Chen, Airu S.; Marsh, Donald J.; Trumbauer, Myrna E.; Frazier, Easter G.; Guan, Xiao-Ming; Yu, Hong; Rosenblum, Charles I.; Vongs, Aurawan; Feng, Yue; Cao, Linhai; Metzger, Joseph M.; Strack, Alison M.; Camacho, Ramon E.; Mellin, Theodore N.; Nunes, Christian N.; Min, William; Fisher, Jill; Gopal-Truter, Shobhna; MacIntyre, D. Euan; Chen, Howard Y.; Van der Ploeg, Lex H. T.
  Nature Genetics (2000), 26 (1), 97-102CODEN: NGENEC; ISSN:1061-4036. (Nature America Inc.)
  Genetic and pharmacol. studies have defined a role for the melanocortin-4 receptor (Mc4r) in the regulation of energy homeostasis. The physiol. function of Mc3r, a melanocortin receptor expressed at high levels in the hypothalamus, has remained unknown. We evaluated the potential role of Mc3r in energy homeostasis by studying Mc3r-deficient (Mc3r-/-) mice and compared the functions of Mc3r and Mc4r in mice deficient for both genes. The 4-6-mo Mc3r-/- mice have increased fat mass, reduced lean mass and higher feed efficiency than wild-type littermates, despite being hypophagic and maintaining normal metabolic rates. Feed efficiency is the ratio of wt. gain to food intake. Consistent with increased fat mass, Mc3r-/- mice are hyperleptinemic and male Mc3r-/- mice develop mild hyperinsulinemia. Mc3r-/- mice did not have significantly altered corticosterone or total thyroxine (T4) levels. Mice lacking both Mc3r and Mc4r become significantly heavier than Mc4r-/- mice. We conclude that Mc3r and Mc4r serve non-redundant roles in the regulation of energy homeostasis.
22. 22
  Butler, A. A., Kesterson, R. A., Khong, K., Cullen, M. J., Pelleymounter, M. A., Dekoning, J., Baetscher, M., and Cone, R. D. (2000) A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse Endocrinology 141, 3518– 3521
  22
  A unique metabolic syndrome causes obesity in the melanocortin-3 receptor-deficient mouse
  Butler, Andrew A.; Kesterson, Robert A.; Khong, Kathy; Cullen, Mary Jane; Pelleymounter, Mary Ann; Dekoning, Jenefer; Baetscher, Manfred; Cone, Roger D.
  Endocrinology (2000), 141 (9), 3518-3521CODEN: ENDOAO; ISSN:0013-7227. (Endocrine Society)
  The central melanocortin system is crit. for the long term regulation of energy homeostasis. Null mutations of the melanocortin-4 receptor (MC4-R) are assocd. with hyperphagia, obesity, and accelerated longitudinal growth in mice and humans. However, little is known about the function of another central melanocortin receptor, the MC3-R. To assess the role of the MC3-R in energy homeostasis, the majority of the mc3r coding sequence was deleted from the mouse genome. In contrast to the MC4-R knockout, which exhibits increased food intake, increased somatic growth, and defects in metab., mc3r-/- mice exhibit an exclusively metabolic syndrome. Homozygous null mc3r mice, while not significantly overweight, exhibit an approx. 50% to 60% increase in adipose mass. Mc3r-/- mice also exhibit an unusual increase in RQ when transferred onto high fat chow, suggesting a reduced ratio of fat/carbohydrate oxidn. Furthermore, male mc3r-/- mice also exhibit an approx. 50% redn. in locomotory behavior on the running wheel, suggesting reduced energy expenditure.
23. 23
  Fan, W., Boston, B. A., Kesterson, R. A., Hruby, V. J., and Cone, R. D. (1997) Role of melanocortinergic neurons in feeding and the agouti obesity syndrome Nature 385, 165– 168
  23
  Role of melanocortinergic neurons in feeding and the agouti obesity syndrome
  Fan, Wei; Boston, Bruce A.; Kesterson, Robert A.; Hruby, Victor J.; Cone, Roger D.
  Nature (London) (1997), 385 (6612), 165-168CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)
  Dominant alleles at the agouti locus (A) cause an obesity syndrome in the mouse, as a consequence of ectopic expression of the agouti peptide. This peptide, normally only found in the skin, is a high-affinity antagonist of the MSH receptor (MCI-R), thus explaining the inhibitory of effect of agouti on eumelanin pigment synthesis. The agouti peptide is also an antagonist of the hypothalamic melanocortin-4 receptor (MC4-R). To test the hypothesis that agouti causes obesity by antagonism of hypothalamic melanocortin receptors, we identified cyclic melanocortin analogs that are potent agonists or antagonist of the neural MC3 (refs 11, 12) and MC4 receptors. Intracerebroventricular administration of the antagonist, MTII, inhibiting feeding in four models of hyperphagia; fasted C57BL/6J, ob/ob, and AY mice, and mice injected with neuropeptide Y. Co-administration of the specific melanocortin antagonist and agouti-mimetic SHU9119 completely blocked this inhibition. Furthermore, administration of SHU9119 significantly enhanced nocturnal feeding, or feeding stimulated by a prior fast. Our data show that melanocortinergic neurons exert a tonic inhibition of feeding behavior. Chronic disruption of this inhibitory signal is a likely explanation of the agouti obesity syndrome.
24. 24
  Wessells, H., Levine, N., Hadley, M. E., Dorr, R., and Hruby, V. (2000) Melanocortin Receptor Agonists, Penile Erection, and Sexual Motivation: Human Studies with Melanotan II Int. J. Impotence Res. 12 (Suppl 4) S74– S79
  There is no corresponding record for this reference.
25. 25
  Huszar, D., Lynch, C. A., Fairchild-Huntress, V., Dunmore, J. H., Fang, Q., Berkemeier, L. R., Gu, W., Kesterson, R. A., Boston, B. A., Cone, R. D., Smith, F. J., Campfield, L. A., Burn, P., and Lee, F. (1997) Targeted disruption of the melanocortin-4 receptor results in obesity in mice Cell 88, 131– 141
  25
  Targeted disruption of the melanocortin-4 receptor results in obesity in mice
  Huszar, Dennis; Lynch, Catherine A.; Fairchild-Huntress, Victoria; Dunmore, Judy H.; Fang, Qing; Berkemeier, Lucy R.; Gu, Wei; Kesterson, Robert A.; Boston, Bruce A.; Cone, Roger D.; Smith, Francoise J.; Campfield, L. Arthur; Burn, Paul; Lee, Frank
  Cell (Cambridge, Massachusetts) (1997), 88 (1), 131-141CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
  The melanocortin-4 receptor (MC4-R) is a G protein-coupled, seven-transmembrane receptor expressed in the brain. Inactivation of this receptor by gene targeting results in mice that develop a maturity onset obesity syndrome assocd. with hyperphagia, hyperinsulinemia, and hyperglycemia. This syndrome recapitulates several of the characteristic features of the agouti obesity syndrome, which results from ectopic expression of agouti protein, a pigmentation factor normally expressed in the skin. Our data identify a novel signaling pathway in the mouse for body wt. regulation and support a model in which the primary mechanism by which agouti induces obesity is chronic antagonism of the MC4-R.
26. 26
  Chhajlani, V., Muceniece, R., and Wikberg, J. E. (1993) Molecular cloning of a novel human melanocortin receptor Biochem. Biophys. Res. Commun. 195, 866– 873
  26
  Molecular cloning of a novel human melanocortin receptor
  Chhajlani, Vijay; Muceniece, Ruta; Wikberg, Jarl E. S.
  Biochemical and Biophysical Research Communications (1993), 195 (2), 866-73CODEN: BBRCA9; ISSN:0006-291X.
  A human genomic clone designated MC-2 is isolated. The cloned DNA codes for a protein of 325 amino acids which possesses seven hydrophobic segments, a characteristic of G-protein coupled receptors. The MC-2 receptor is expressed in brain tissue but not in the melanoma cells. When the MC-2 DNA is expressed in COS-7 cells, it binds [125I]-labeled [Nle4,D-Phe7]-α-MSH (NDP-MSH) which then could be displaced by melanotropic peptides α-MSH, β-MSH, γ-MSH, and ACTH, but not by non-melanotropic peptide β-endorphin. The highest affinity of 5.18 nM was for the NDP-MSH peptide. The novel MC-2 receptor and the MC-1 receptor, described earlier by the authors showed identical order of affinity for the melanocortin peptides, but the affinities and the fold differences in the affinities to the melanocortin peptides were different when compared to the earlier described MC-1 receptor. The results suggest that the MC-2 DNA codes for a novel melanocortin receptor.
27. 27
  Chen, W., Kelly, M. A., Opitz-Araya, X., Thomas, R. E., Low, M. J., and Cone, R. D. (1997) Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides Cell 91, 789– 798
  27
  Exocrine gland dysfunction in MC5-R-deficient mice: evidence for coordinated regulation of exocrine gland function by melanocortin peptides
  Chen, Wenbiao; Kelly, Michele A.; Opitz-Araya, Ximena; Thomas, Ruth E.; Low, Malcolm J.; Cone, Roger d.
  Cell (Cambridge, Massachusetts) (1997), 91 (6), 789-798CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
  The effects of pituitary-derived melanocortin peptides are primarily attributed to ACTH-mediated adrenocortical glucocorticoid prodn. Identification of a widely distributed receptor for ACTH/MSH peptides, the melanocortin-5 receptor (MC5-R), suggested non-steroidally mediated systemic effects of these peptides. Targeted disruption of the MC5-R produced mice with a severe defect in water repulsion and thermoregulation due to decreased prodn. of sebaceous lipids. High levels of MC5-R was found in multiple exocrine tissues, including Harderian, preputial, lacrimal, and sebaceous glands, and was also shown to be required for prodn. and stress-regulated synthesis of porphyrins by the Harderian gland and ACTH/MSH-regulated protein secretion by the lacrimal gland. These data show a requirement for the MC5-R in multiple exocrine glands for the prodn. of numerous products, indicative of a coordinated system for regulation of exocrine gland function by melanocortin peptides.
28. 28
  Irani, B. G., Xiang, Z., Yarandi, H. N., Holder, J. R., Moore, M. C., Bauzo, R. M., Proneth, B., Shaw, A. M., Millard, W. J., Chambers, J. B., Benoit, S. C., Clegg, D. J., and Haskell-Luevano, C. (2011) Implication of the melanocortin-3 receptor in the regulation of food intake Eur. J. Pharmacol. 660, 80– 87
  28
  Implication of the melanocortin-3 receptor in the regulation of food intake
  Irani, Boman G.; Xiang, Zhimin; Yarandi, Hossein N.; Holder, Jerry R.; Moore, Marcus C.; Bauzo, Rayna M.; Proneth, Bettina; Shaw, Amanda M.; Millard, William J.; Chambers, James B.; Benoit, Stephen C.; Clegg, Deborah J.; Haskell-Luevano, Carrie
  European Journal of Pharmacology (2011), 660 (1), 80-87CODEN: EJPHAZ; ISSN:0014-2999. (Elsevier B.V.)
  The melanocortin system is well recognized to be involved in the regulation of food intake, body wt., and energy homeostasis. To probe the role of the MC3 in the regulation of food intake, JRH 322-18 a mixed MC3 partial agonist/antagonist and MC4 agonist tetrapeptide was examd. in wild type (WT) and melanocortin 4 receptor (MC4) knockout mice and shown to reduce food intake in both models. In the wild type mice, 2.0 nmol of JRH 322-18 statistically reduced food intake 4 h post icv treatment into satiated nocturnally feeding wild type mice. The same dose in the MC4KO mice significantly reduced cumulative food intake 24 h post treatment. Conditioned taste aversion as well as activity studies supports that the decreased food intake was not due to visceral illness. Since these studies resulted in loss-of-function results, the SHU 9119 and agouti-related protein (AGRP) melanocortin receptor antagonists were administered to wild type as well as the MC3 and MC4 knockout mice in anticipation of gain-of-function results. The SHU 9119 ligand produced an increase in food intake in the wild type mice as anticipated, however no effect was obsd. in the MC3 and MC4 knockout mice as compared to the saline control. The AGRP ligand however, produced a significant increase in food intake in the wild type as well as the MC3 and MC4 knockout mice and it had a prolonged affect for several days. These data support the hypothesis that the MC3 plays a subtle role in the regulation of food intake, however the mechanism by which this is occurring remains to be detd.
29. 29
  Farooqi, I. S., Keogh, J. M., Yeo, G. S., Lank, E. J., Cheetham, T., and O’Rahilly, S. (2003) Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene N. Engl. J. Med. 348, 1085– 1095
  29
  Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene
  Farooqi, I. Sadaf; Keogh, Julia M.; Yeo, Giles S. H.; Lank, Emma J.; Cheetham, Tim; O'Rahilly, Stephen
  New England Journal of Medicine (2003), 348 (12), 1085-1095CODEN: NEJMAG; ISSN:0028-4793. (Massachusetts Medical Society)
  Melanocortin 4 receptor (MC4R) deficiency is the commonest monogenic form of obesity. However, the clin. spectrum and mode of inheritance were not defined, pathophysiol. mechanisms leading to obesity are poorly understood, and there is little information regarding genotype-phenotype correlations. We detd. the nucleotide sequence of the MC4R gene in 500 probands with severe childhood obesity. Family studies were undertaken to examine cosegregation of identified mutations with obesity. Subjects with MC4R deficiency underwent metabolic and endocrine evaluation; the results were correlated with the signaling properties of mutant receptors. Twenty-nine probands (5.8%) had mutations in MC4R; 23 were heterozygous, and 6 were homozygous. Mutation carriers had severe obesity, increased lean mass, increased linear growth, hyperphagia, and severe hyperinsulinemia; homozygotes were more severely affected than heterozygotes. Subjects with mutations retaining residual signaling capacity had a less severe phenotype. Mutations in MC4R result in a distinct obesity syndrome that is inherited in a codominant manner. Mutations leading to complete loss of function are assocd. with a more severe phenotype. The correlation between the signaling properties of these mutant receptors and energy intake emphasizes the key role of this receptor in the control of eating behavior in humans.
30. 30
  Vaisse, C., Clement, K., Durand, E., Hercberg, S., Guy-Grand, B., and Froguel, P. (2000) Melanocortin-4 receptor mutations are a frequent and heterogeneous cause of morbid obesity J. Clin. Invest. 106, 253– 262
  There is no corresponding record for this reference.
31. 31
  Xiang, Z., Proneth, B., Dirain, M. L., Litherland, S. A., and Haskell-Luevano, C. (2010) Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist Biochemistry 49, 4583– 4600
  There is no corresponding record for this reference.
32. 32
  Xiang, Z., Litherland, S. A., Sorensen, N. B., Proneth, B., Wood, M. S., Shaw, A. M., Millard, W. J., and Haskell-Luevano, C. (2006) Pharmacological characterization of 40 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists and the agouti-related protein (AGRP) antagonist Biochemistry 45, 7277– 7288
  There is no corresponding record for this reference.
33. 33
  Xiang, Z., Pogozheva, I. D., Sorenson, N. B., Wilczynski, A. M., Holder, J. R., Litherland, S. A., Millard, W. J., Mosberg, H. I., and Haskell-Luevano, C. (2007) Peptide and small molecules rescue the functional activity and agonist potency of dysfunctional human melanocortin-4 receptor polymorphisms Biochemistry 46, 8273– 8287
  33
  Peptide and Small Molecules Rescue the Functional Activity and Agonist Potency of Dysfunctional Human Melanocortin-4 Receptor Polymorphisms
  Xiang, Zhimin; Pogozheva, Irina D.; Sorenson, Nicholas B.; Wilczynski, Andrzej M.; Holder, Jerry Ryan; Litherland, Sally A.; Millard, William J.; Mosberg, Henry I.; Haskell-Luevano, Carrie
  Biochemistry (2007), 46 (28), 8273-8287CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  The melanocortin pathway, specifically the melanocortin-4 receptor and the cognate endogenous agonist and antagonist ligands, have been strongly implicated in the regulation of energy homeostasis and satiety. Genetic studies of morbidly obese human patients and normal wt. control patients have resulted in the discovery of over 70 human melanocortin-4 receptor (MC4R) polymorphisms obsd. as both heterozygous and homozygous forms. A no. of labs. have been studying these hMC4R polymorphisms attempting to understand the mol. mechanism(s) that might explain the obese human phenotype. Herein, we have studied 13 polymorphic hMC4Rs that have been identified to possess statistically significant decreased endogenous agonist potency with synthetic peptides and small mols. attempting to identify ligands that can pharmacol. rescue the hMC4R polymorphic agonist response. The ligands examd. in this study include NDP-MSH, MTII, Ac-His-DPhe-Arg-Trp-NH2 (JRH887-9), Ac-Anc-DPhe-Arg-Trp-NH2 (amino-2-naphtylcarboxylic acid, Anc, JRH420-12), Ac-His-(pI)DPhe-Arg-Trp-NH2 (JRH322-18), chimeric AGRP-melanocortin based ligands (Tyr-c[Cys-His-DPhe-Arg-Trp-Asn-Ala-Phe-Cys]-Tyr-NH2, AMW3-130 and Ac-mini-(His-DPhe-Arg-Trp)-hAGRP-NH2, AMW3-106), and the small mols. JB25 and THIQ. The hMC4R polymorphisms included in this study are S58C, N97D, I102S, L106P, S127L, T150I, R165Q, R165W, L250Q, G252S, C271Y, Y287Stop, and I301T. These studies resulted in the NDP-MSH, MTII, AMW3-130, THIQ, and AMW3-106 ligands possessing nanomolar to subnanomolar agonist potency at the hMC4R polymorphisms examd. in this study. Thus, these ligands could generically rescue the potency and stimulatory response of the abnormally functioning hMC4Rs studied and may provide tools to further clarify the mol. mechanism(s) involving these receptor modifications.
34. 34
  Singh, A., Wilczynski, A., Holder, J. R., Witek, R. M., Dirain, M. L., Xiang, Z., Edison, A. S., and Haskell-Luevano, C. (2011) Incorporation of a bioactive reverse-turn heterocycle into a peptide template using solid-phase synthesis to probe melanocortin receptor selectivity and ligand conformations by 2D ¹H NMR J. Med. Chem. 54, 1379– 1390
  There is no corresponding record for this reference.
35. 35
  Haskell-Luevano, C., Boteju, L. W., Miwa, H., Dickinson, C., Gantz, I., Yamada, T., Hadley, M. E., and Hruby, V. J. (1995) Topographical modification of melanotropin peptide analogues with β-methyltryptophan isomers at position 9 leads to differential potencies and prolonged biological activities J. Med. Chem. 38, 4720– 4729
  There is no corresponding record for this reference.
36. 36
  Haskell-Luevano, C., Toth, K., Boteju, L., Job, C., Castrucci, A. M., Hadley, M. E., and Hruby, V. J. (1997) β-Methylation of the Phe7 and Trp9 melanotropin side chain pharmacophores affects ligand-receptor interactions and prolonged biological activity J. Med. Chem. 40, 2740– 2749
  36
  β-Methylation of the Phe7 and Trp9 melanotropin side chain pharmacophores affects ligand-receptor interactions and prolonged biological activity
  Haskell-Luevano, Carrie; Toth, Kate; Boteju, Lakmal; Job, Constatin; de Castrucci, Ana Maria; Hadley, Mac E.; Hruby, Victor J.
  Journal of Medicinal Chemistry (1997), 40 (17), 2740-2749CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  Topog. modified melanotropin side chain pharmacophore residues Phe7 and Trp9 in a cyclic peptide template (Ac-Nle4-c[Asp-His-Xaa7-Arg-Yaa9-Lys]-NH2) and Phe7 in a linear peptide template (Ac-Ser-Tyr-Ser-Nle4-Glu-His-Xaa7-Arg-Trp-Gly-Lys-Pro-Val-NH2) result in differences in potency and prolonged biol. activity in the frog and lizard skin bioassays. These topog. modifications included the four isomers of β-methylphenylalanine (β-MePhe)7 and β-methyltryptophan (β-MeTrp)9 and the two isomers of 1,2,3,4-tetrahydro-β-carboline (Tca).9. Modifications in the cyclic template resulted in up to a 1000-fold difference in potency for the β-MePhe7 stereoisomeric peptides; up to a 476-fold difference in potency resulted for the β-MeTrp9 peptides, and about a 50-fold difference between the Tca9-contg. peptides. Up to a 40-fold difference in potency resulted for the β-MePhe7 stereoisomeric peptides using the linear template in these assays. The relative potency ranking for modifications in the cyclic template of β-MePhe7 were 2R,3S > 2S,3S = 2S,3R > 2R,3R in the frog assay and 2S,3R > 2R,3S > 2S,3S > 2R,3R in the lizard assay. The relative potencies for modifications in the cyclic template of β-MeTrp9 were 2R,3S > 2R,3R > 2S,3S » 2S,3R in the frog assay and 2S,3S = 2R,3R > 2R,3S > 2S,3R in the lizard assay. The relative potencies for modifications in the cyclic template of Tca9 were DTca > LTca in both assays. Significant differences in prolonged (residual) activities were also obsd. for these modified peptides and were dependent upon stereochem. of the β-Me amino acid, peptide template, and bioassay system. Furthermore, comparisons of β-MeTrp9 stereoisomeric peptides on the frog, lizard, and human MC1 receptors suggest that structure-activity relationships on both the classical frog and lizard skin bioassays do not necessarily predict corresponding SAR profiles for the human melanocortin receptors, indicating a remarkable species specificity of the MC1 receptor requirements.
37. 37
  Sahm, U. G., Olivier, G. W., Branch, S. K., Moss, S. H., and Pouton, C. W. (1994) Synthesis and biological evaluation of α-MSH analogues substituted with alanine Peptides 15, 1297– 1302
  There is no corresponding record for this reference.
38. 38
  Bednarek, M. A., Silva, M. V., Arison, B., MacNeil, T., Kalyani, R. N., Huang, R. R., and Weinberg, D. H. (1999) Structure-function studies on the cyclic peptide MT-II, lactam derivative of α-melanotropin Peptides 20, 401– 409
  There is no corresponding record for this reference.
39. 39
  Holder, J. R., Xiang, Z., Bauzo, R. M., and Haskell-Luevano, C. (2002) Structure-activity relationships of the melanocortin tetrapeptide Ac-His-D-Phe-Arg-Trp-NH2 at the mouse melanocortin receptors. 4. Modifications at the Trp position J. Med. Chem. 45, 5736– 5744
  39
  Structure-activity relationships of the melanocortin tetrapeptide Ac-His-D-Phe-Arg-Trp-NH2 at the mouse melanocortin receptors. 4. Modifications at the Trp position
  Holder, Jerry Ryan; Xiang, Zhimin; Bauzo, Rayna M.; Haskell-Luevano, Carrie
  Journal of Medicinal Chemistry (2002), 45 (26), 5736-5744CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  The melanocortin pathway is involved in the regulation of several physiol. functions including skin pigmentation, steroidogenesis, obesity, energy homeostasis, and exocrine gland function. This melanocortin pathway consists of five known G-protein coupled receptors, endogenous agonists derived from the proopiomelanocortin (POMC) gene transcript, the endogenous antagonists Agouti and the Agouti-related protein (AGRP) and signals through the intracellular cAMP signal transduction pathway. The endogenous melanocortin agonists contain the putative message sequence "His-Phe-Arg-Trp," postulated to be important for melanocortin receptor mol. recognition and stimulation. Herein, the authors report a tetrapeptide library, based upon the template Ac-His--D-Phe-Arg-Trp-NH2, consisting of 20 members that have been modified at the Trp9 position (α-MSH numbering) and pharmacol. characterized for agonist activity at the mouse melanocortin receptors MC1R, MC3R, MC4R, and MC5R. Results from this study yielded compds. that ranged in pharmacol. properties from equipotent to a loss of melanocortin receptor activity at up to 100 μM concns. Interestingly, modification of the Trp9 in the tetrapeptide template at the MC1R resulted in only up to a 220-fold potency change, while at the MC4R and MC5R, up to a 9700-fold decrease in potency was obsd., suggesting the MC1R is more tolerant of the modifications examd. herein. The most notable results of this study include identification that the Trp9 indole moiety in the tetrapeptide template is important for melanocortin-3 receptor agonist potency, and that this position can be used to design melanocortin ligands possessing receptor selectivity for the peripherally expressed MC1 and MC5 vs. the centrally expressed MC3 and MC4 receptors. Specifically, the Ac-His--D-Phe-Arg-Tic-NH2 and the Ac-His--D-Phe-Arg-Bip-NH2 tetrapeptides possessed nanomolar MC1R and MC5R potency but micromolar MC3R and MC4R agonist potency. Addnl., these studies identified that substitution of the Trp amino acid with either Nal(2') or D-Nal(2') resulted in equipotent melanocortin receptor potency, suggesting that the chem. reactive Trp indole side chain may be replaced with the nonreactive Nal(2') moiety for the design of nonpeptide melanocortin receptor agonists.
40. 40
  Lee, M., Kim, A., Conwell, I. M., Hruby, V., Mayorov, A., Cai, M., and Wardlaw, S. L. (2008) Effects of selective modulation of the central melanocortin-3-receptor on food intake and hypothalamic POMC expression Peptides 29, 440– 447
  There is no corresponding record for this reference.
41. 41
  Singh, A., Dirain, M., Witek, R., Rocca, J. R., Edison, A. S., and Haskell-Luevano, C. (2013) Structure-activity relationships of peptides incorporating a bioactive reverse-turn heterocycle at the melanocortin receptors: identification of a 5800-fold mouse melanocortin-3 receptor (mMC3R) selective antagonist/partial agonist versus the mouse melanocortin-4 receptor (mMC4R) J. Med. Chem. 56, 2747– 2763
  There is no corresponding record for this reference.
42. 42
  Chen, W., Shields, T. S., Stork, P. J., and Cone, R. D. (1995) A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways Anal. Biochem. 226, 349– 354
  There is no corresponding record for this reference.
43. 43
  Sawyer, T. K., Sanfillippo, P. J., Hruby, V. J., Engel, M. H., Heward, C. B., Burnett, J. B., and Hadley, M. E. (1980) 4-Norleucine, 7-D-Phenylalanine-α-Melanocyte-Stimulating Hormone: A Highly Potent α-Melanotropin with Ultra Long Biological Activity Proc. Natl. Acad. Sci. U.S.A. 77, 5754– 5758
  There is no corresponding record for this reference.
44. 44
  Bondebjerg, J., Xiang, Z., Bauzo, R. M., Haskell-Luevano, C., and Meldal, M. (2002) A solid-phase approach to mouse melanocortin receptor agonists derived from a novel thioether cyclized peptidomimetic scaffold J. Am. Chem. Soc. 124, 11046– 11055
  There is no corresponding record for this reference.
45. 45
  Wishart, D. S. and Sykes, B. D. (1994) Chemical Shifts as a Tool for Structure Determination Methods Enzymol. 239, 363– 392
  There is no corresponding record for this reference.
46. 46
  Hutchison, E. G. T. and J, M. (1994) A revised set of potentials for β-turn formation in proteins. Protein Sci. 3, 2207– 2216
  There is no corresponding record for this reference.
47. 47
  Ying, J., Kover, K. E., Gu, X., Han, G., Trivedi, D. B., Kavarana, M. J., and Hruby, V. J. (2003) Solution structures of cyclic melanocortin agonists and antagonists by NMR Biopolymers 71, 696– 716
  There is no corresponding record for this reference.
48. 48
  Wilczynski, A., Wilson, K. R., Scott, J. W., Edison, A. S., and Haskell-Luevano, C. (2005) Structure-activity relationships of the unique and potent agouti-related protein (AGRP)-melanocortin chimeric Tyr-c[β-Asp-His-DPhe-Arg-Trp-Asn-Ala-Phe-Dpr]-Tyr-NH2 peptide template J. Med. Chem. 48, 3060– 3075
  There is no corresponding record for this reference.
49. 49
  Hess, S., Linde, Y., Ovadia, O., Safrai, E., Shalev, D. E., Swed, A., Halbfinger, E., Lapidot, T., Winkler, I., Gabinet, Y., Faier, A., Yarden, D., Xiang, Z., Portillo, F. P., Haskell-Luevano, C., Gilon, C., and Hoffman, A. (2008) Backbone cyclic peptidomimetic melanocortin-4 receptor agonist as a novel orally administrated drug lead for treating obesity J. Med. Chem. 51, 1026– 1034
  There is no corresponding record for this reference.
50. 50
  Rozek, A., Powers, J. P., Friedrich, C. L., and Hancock, R. E. (2003) Structure-based design of an indolicidin peptide analogue with increased protease stability Biochemistry 42, 14130– 14138
  50
  Structure-based design of an indolicidin peptide analogue with increased protease stability
  Rozek, Annett; Powers, Jon-Paul S.; Friedrich, Carol L.; Hancock, Robert E. W.
  Biochemistry (2003), 42 (48), 14130-14138CODEN: BICHAW; ISSN:0006-2960. (American Chemical Society)
  Indolicidin is an antimicrobial cationic peptide with broad-spectrum activity isolated from bovine neutrophils. An indolicidin analog CP-11, ILKKWPWWPWRRK-NH2, with improved activity against Gram-neg. bacteria had increased pos. charge and amphipathicity while maintaining the short length of the parent mol. The structure of CP-11 in the presence of dodecylphosphocholine (DPC) micelles was detd. using NMR spectroscopy. CP-11 was found to be an amphipathic mol. with a U-shaped backbone bringing the N- and C-termini in close proximity. On the basis of this close proximity, a cyclic disulfide-bonded peptide cycloCP-11, ICLKKWPWWPWRRCK-NH2, was designed to stabilize the lipid-bound structure and to increase protease resistance. The 3-dimensional structure of cycloCP-11 was detd. under the same conditions as for the linear peptide and was found to be similar to CP-11. Both CP-11 and cycloCP-11 assocd. with phospholipid membranes in a similar manner as indicated by CD and fluorescence spectra. The MICs of CP-11 and cycloCP-11 for a range of bacteria differed by ≤2-fold, and they were nonhemolytic at concns. ≤256 μg/mL. Cyclization greatly increased protease stability. The half-life of cycloCP-11 in the presence of trypsin was increased by 4.5-fold, from 4 to 18 min. More importantly, the antibacterial activity of cycloCP-11, but not that of CP-11, in the presence of trypsin was completely retained up to 90 min, since the major degrdn. product was equally active. A structural comparison of CP-11 and cycloCP-11 revealed that the higher trypsin resistance of cycloCP-11 may be due to the more compact packing of lysine and tryptophan side chains. These findings suggest that cyclization may serve as an important strategy in the rational design of antimicrobial peptides.
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  Hunter, H. N., Jing, W., Schibli, D. J., Trinh, T., Park, I. Y., Kim, S. C., and Vogel, H. J. (2005) The interactions of antimicrobial peptides derived from lysozyme with model membrane systems Biochim. Biophys. Acta 1668, 175– 189
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  Haug, B. E., Stensen, W., Kalaaji, M., Rekdal, O., and Svendsen, J. S. (2008) Synthetic antimicrobial peptidomimetics with therapeutic potential J. Med. Chem. 51, 4306– 4314
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  Synthetic Antimicrobial Peptidomimetics with Therapeutic Potential
  Haug, Bengt Erik; Stensen, Wenche; Kalaaji, Manar; Rekdal, Oeystein; Svendsen, John S.
  Journal of Medicinal Chemistry (2008), 51 (14), 4306-4314CODEN: JMCMAR; ISSN:0022-2623. (American Chemical Society)
  A series of synthetic antimicrobial peptidomimetics (SAMPs) have been prepd. and found to be highly active against several Gram-neg. and Gram-pos. bacterial strains. These derivs. comprise the minimal structural requirements for cationic antimicrobial peptides and showed high selectivity for Gram-neg. and/or Gram-pos. bacteria compared to human red blood cells. The authors have found that SAMPs share many of the attractive properties of cationic antimicrobial peptides inasmuch that a representative SAMP was found to insert into the bilayers of large unilamellar vesicles, permeabilized both the outer and cytoplasmic membrane of Escherichia coli ML-35p, and displayed an extremely rapid bacterial killing for Staphylococcus aureus. However, while antimicrobial peptides are prone to proteolytic degrdn., high in vitro stability in human blood plasma was shown for SAMPs. A combination of high antibacterial activity against methicillin-resistant staphylococci and low toxicity against human erythrocytes makes these mols. promising candidates for novel antibacterial therapeutics.
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  Structure-activity studies of 14-helical antimicrobial β-peptides: probing the relationship between conformational stability and antimicrobial potency
  Raguse, Tami L.; Porter, Emilie A.; Weisblum, Bernard; Gellman, Samuel H.
  Journal of the American Chemical Society (2002), 124 (43), 12774-12785CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
  Antimicrobial α-helical α-peptides are part of the host-defense mechanism of multicellular organisms and could find therapeutic use against bacteria that are resistant to conventional antibiotics. Recent work from Hamuro et al. has shown that oligomers of β-amino acids ("β-peptides") that can adopt an amphiphilic helix defined by 14-membered ring H-bonds (14-helix) are active against Escherichia coli (Y. Hamuro et al., 1999). We have created two series of cationic 9- and 10-residue amphiphilic β-peptides to probe the effect of 14-helix stability on antimicrobial and hemolytic activity. 14-Helix stability within these series is modulated by varying the proportions of rigid trans-2-aminocyclohexanecarboxylic acid (ACHC) residues and flexible acyclic residues. We have previously shown that a high proportion of ACHC residues in short β-peptides encourages 14-helical structure in aq. soln. (D. H. Appella et al., 1999). CD of the β-peptides described here reveals a broad range of 14-helix population in aq. buffer, but this variation in helical propensity does not lead to significant changes in antibiotic activity against a set of four bacteria. Several of the 9-mers display antibiotic activity comparable to that of a synthetic magainin deriv. Among these 9-mers, hemolytic activity increases slightly with increasing 14-helical propensity, but all of the 9-mers are less hemolytic than the magainin deriv. Previous studies with conventional peptides (α-amino acid residues) have provided conflicting evidence on the relationship between helical propensity and antimicrobial activity. This uncertainty has arisen because α-helix stability can be varied to only a limited extent among linear α-peptides without modifying parameters important for antimicrobial activity (e.g., net charge or hydrophobicity); a much greater range of helical stability is accessible with β-peptides. For example, it is very rare for a linear α-peptide to display significant α-helix formation in aq. soln. and manifest antibacterial activity, while the linear β-peptides described here range from fully unfolded to very highly folded in aq. soln. This study shows that β-peptides can be unique tools for analyzing relationships between conformational stability and biol. activity.
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  Wirth, Michelle M.; Giraudo, Silvia Q.
  Peptides (New York) (2000), 21 (9), 1369-1375CODEN: PPTDD5; ISSN:0196-9781. (Elsevier Science Inc.)
  Agouti-related protein (Agrp) is an endogenous melanocortin-4 receptor antagonist implicated in the regulation of food intake. Effects of Agrp on feeding under varying conditions were investigated. Agrp (10 to 100 pmol) was injected into the hypothalamic paraventricular nucleus of satiated (a.m. and p.m. injections) and food-deprived rats, or was co-administered with 117 pmol neuropeptide Y (NPY). Agrp significantly stimulated light-phase feeding by 24 h post-injection. However, Agrp stimulated dark-phase and deprivation-induced feeding by 4 and 2 h, resp. Animals receiving NPY and Agrp consumed more than animals receiving either peptide alone, the effect remaining by 24 h.
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  A rapid color test is described for detecting ≥5 micromoles free terminal amino group/g in the reaction mixt. from solid-phase peptide synthesis according to R. B. Merrifield (1968). After washing the solid phase with CH2Cl2, a 10-20 mg sample of the resin peptide is treated with 2-3 drops each of the following reagents: 500 mg ninhydrin/10 ml EtOH; 80 g PhOH/ 20 ml EtOH; and 20 ml 0.001M KCN dild. to 100 ml with pyridine. The mixt. is heated 2-5 min at 100°. The development of an intense blue color on the beads and in the soln. (except that terminal proline or terminal β-benzyl aspartate gave a brown or red-brown color) is a pos. result. In the absence of untreated terminal amino groups, the beads remained white and the soln. yellow (neg. result). The formation of pale blue beads and a light green soln. was a slightly pos. result.
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  Thirumoorthy, R., Holder, J. R., Bauzo, R. M., Richards, N. G. J., Edison, A. S., and Haskell-Luevano, C. (2001) Novel Agouti-related Protein (AGRP) Based Melanocortin-1 Receptor Antagonist J. Med. Chem. 44, 4114– 4124
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Supporting Information
Supporting Information
Proton NMR chemical shift assignments (ppm) for AST3-88 and the backbone dihedral angels of the representative structures in Figure 4. This material is available free of charge via the Internet at http://pubs.acs.org
- cn5000953_si_001.pdf (81.23 kb)
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Synthesis, Biophysical, and Pharmacological Evaluation of the Melanocortin Agonist AST3-88: Modifications of Peptide Backbone at Trp 7 Position Lead to a Potent, Selective, and Stable Ligand of the Melanocortin 4 Receptor (MC4R)Click to copy article linkArticle link copied!

ACS Chemical Neuroscience

Abstract

Results and Discussion

Synthesis, Structural, and Functional Characterization

Scheme 1

Figure 1

Biophysical Structural Studies (NMR and CAMM)

Figure 2

Figure 3

Figure 4

Figure 5

Serum Stability of Compound AST3-88

Figure 6

Effect of ICV Treatment of Compound AST3-88 on Feeding

Figure 7

Conclusions

Methods

Chemistry

Solid Phase Peptide Synthesis Using Microwave Irradiation

Functional Bioassay

NMR Spectroscopy and Computer-Assisted Molecular Modeling (CAMM)

Serum Stability Assay

Liquid Chromatography–Mass Spectrometry (LC-MS) Analysis of Peptide Stability

Feeding Studies in Rats

Animals

Feeding Regimen

Statistics

Supporting Information

Terms & Conditions

Author Information

Abbreviations

References

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ACS Chemical Neuroscience

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Abstract

Scheme 1

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

References

Supporting Information

Supporting Information

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Synthesis, Biophysical, and Pharmacological Evaluation of the Melanocortin Agonist AST3-88: Modifications of Peptide Backbone at Trp 7 Position Lead to a Potent, Selective, and Stable Ligand of the Melanocortin 4 Receptor (MC4R)
Click to copy article linkArticle link copied!