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1.  α-Melanocyte-stimulating hormone regulates vascular NO availability and protects against endothelial dysfunction 
Cardiovascular Research  2012;97(2):360-368.
α-Melanocyte-stimulating hormone (α-MSH), derived from the precursor molecule pro-opiomelanocortin, exerts potent anti-inflammatory actions in the vasculature, but its role in circulatory regulation remains unclear. Therefore, we sought to investigate whether α-MSH could regulate the local control of blood vessel tone.
Methods and results
Using in vivo and ex vivo methods to assess vascular reactivity, we found that α-MSH improved endothelium-dependent vasodilatation in the mouse aorta and coronary circulation without directly contracting or relaxing blood vessels. α-MSH promoted vasodilatation by enhancing endothelial nitric oxide (NO) formation and by improving sensitivity to endothelium-independent blood vessel relaxation. Using cultured human endothelial cells to elucidate the involved molecular mechanisms, we show that α-MSH increased the expression and phosphorylation of endothelial NO synthase in these cells. The observed effects were regulated by melanocortin 1 (MC1) receptors expressed in the endothelium. In keeping with the vascular protective role of α-MSH, in vivo treatment with stable analogues of α-MSH ameliorated endothelial dysfunction associated with aging and diet-induced obesity in mice.
The present study identifies α-MSH and endothelial MC1 receptors as a new signalling pathway contributing to the regulation of NO availability and vascular function. These findings suggest applicability of α-MSH analogues for therapeutic use in pathological conditions that are characterized by vascular dysfunction.
PMCID: PMC3543993  PMID: 23131503
Vasodilation; Nitric oxide; Endothelial function; Endothelial nitric oxide synthase; Melanocortin
2.  An Unusual Conformation of MSH Analogues Leads to a Selective Human Melanocortin 1 Receptor Antagonist for Targeting Melanoma Cells 
Biochemistry  2013;52(4):752-764.
γ-MSH (γ-melanocyte-stimulating hormone: H-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-OH), with its exquisite specificity and potency, has recently created much excitement as a drug lead. However, this peptide like most peptides susceptible to proteolysis in vivo which potentially decreases its beneficial activities. In our continued effort to design a proteolytically stable with specific receptor binding ligand, we have engineered peptides by cyclizing γ-MSH using a thioether bridge. A number of novel cyclic truncated γ-MSH analogues were designed and synthesized, in which a thioether bridge was incorporated between a cysteine side chain and an N-terminal bromoacyl group. One of these peptides, cyclo-[(CH2)3CO-Gly1-His2-D-Phe3-Arg4-D-Trp5-Cys(S-)6]-Asp7-Arg8-Phe9-Gly10-NH2, demonstrated potent antagonist activity and receptor selectivity for the human melanocortin 1 receptor (hMC1R) (IC50 = 17 nM). This novel peptide is the most selective antagonist for the human hMC1R to date. Further pharmacological studies have shown that this peptide can specifically target melanoma cells. The NMR analysis of this peptide in a membrane–like environment revealed a new turn structure, specific to the hMC1R antagonist, at the C terminal, wherein the side chain and backbone conformation of D-Trp5 and Phe9 of the peptide are contributors to the hMC1R selectivity. Cyclization strategies represent an approach for stabilizing bioactive peptides while keeping their full potencies and should boost applications of peptide-based drugs in human medicine.
PMCID: PMC3641192  PMID: 23276279
α-MSH; γ-MSH; melanocortin receptors; melanotropin antagonist; hMC1R; steric constraints; selective ligands; melanoma
3.  Utilize conjugated melanotropins for the earlier diagnosis and treatment of Melanoma 
European journal of pharmacology  2011;660(1):188-193.
Peptides serve as effective drugs and the contrast agents in the clinic today. However the inherent drawbacks of peptide structures can limit their efficacy as drugs. To overcome this we have been developing new methods to create ‘tailor-made’ peptides and peptide mimetics with improved pharmacological and physical properties. In this work we introduce novel peptide and small molecule conjugated molecules for the earlier diagnosis and treatment of melanoma.
PMCID: PMC3095766  PMID: 21315067
Melanotropin; Human Melanocortin Receptors; Melanoma; Selective Ligands for MC receptors; MTII; THIQ
4.  Design of novel melanocortin receptor ligands: multiple receptors, complex pharmacology, the challenge 
European journal of pharmacology  2011;660(1):88-93.
The major pharmacophore for the melanocortin 1,3,4 and 5 receptors is the sequence -His-Phe-Arg-Trp-. There is a need for potent, biologically stable, receptor selective ligands, both agonists and antagonists, for these receptors. In this report we briefly examine the structural and biophysical approaches we have taken to develop selective agonist and antagonist ligands that can cross (or not) the blood brain barrier. Remaining questions and unmet needs are also discussed.
PMCID: PMC3138524  PMID: 21208601
Melanocortin Receptor Ligands SAR; Selective Ligands for MCRs; Melanocortin Receptor Selective Ligands; Orthosteric and Allosteric Ligands; Agonists and Antagonists for MCRs
5.  Cyclic Lactam Hybrid α-MSH/Agouti-Related Protein (AGRP) Analogues with Nanomolar Range Binding Affinities at the Human Melanocortin Receptors 
A novel hybrid melanocortin pharmacophore was designed based on the topographical similarities between the pharmacophores of Agouti related protein (AGRP) an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. When employed in two different 23-membered macrocyclic lactam peptide templates, the designed hybrid AGRP/MSH pharmacophore yielded non-competitive ligands with nanomolar range binding affinities. The topography-based pharmacophore hybridization strategy will prove useful in development of unique non-competitive melanocortin receptor modulators.
PMCID: PMC3216836  PMID: 21486697
α-MSH; Agouti-related protein; human melanocortin receptors; hybrid pharmacophore; macrocyclic peptide
6.  Key amino acid residues in the Melanocortin-4 Receptor for nonpeptide THIQ Specific Binding and Signaling 
Regulatory peptides  2009;155(1-3):46-54.
Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and glucose homeostasis. Synthetic nonpeptide compound N- (3R)-1 4-tetrahydroisoquinolinium-3-ylcarbonyl -(1R)-1-(4-chlorobenzyl)-2- 4-cyclohexyl-4-(1H-1,2,4-triazol-1-ylmethyl)piperidin-1-yl -2-oxoethylamine (THIQ) is a potent agonist at MC4R but not at hMC2R. In this study, we utilized two approaches (chimeric receptor and site-directed mutagenesis) to narrow down the key amino acid residues of MC4R responsible for THIQ binding and signaling. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TMs) of the human MC4R (hMC4R) with the homologous regions of hMC2R were constructed. Our results indicate that the cassette substitutions of these TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter THIQ binding affinity and potency except the substitution of the hMC4R TM3, suggesting that the conserved amino acid residues in these TMs of the hMC4R are main potential candidates for THIQ binding and signaling while non conserved residues in TM3 of MC4R may also be involved. Nineteen MC4R mutants were then created, including 13 conserved amino acid residues and 6 non-conserved amino acid residues. Our results indicate that seven conserved residue [E100 (TM2), D122 (TM3), D126 (TM3), F254 (TM6), W258 (TM6), F261 (TM6), H264 (TM6)] are important for THIQ binding and three non-conserved residues [N123 (TM3), I129 (TM3) and S131 (TM3)] are involved in THIQ selectivity. In conclusion, our results suggest that THIQ utilize both conserved and non-conserved amino acid residues for binding and signaling at hMC4R and non conserved residues may be responsible for MC4R selectivity.
PMCID: PMC3216638  PMID: 19303903
7.  Solid-Phase Peptide Head-to-Side Chain Cyclodimerization: Discovery of C2-Symmetric Cyclic Lactam Hybrid α-Melanocyte-Stimulating Hormone (MSH)/Agouti-Signaling Protein (ASIP) Analogues with Potent Activities at the Human Melanocortin Receptors 
Peptides  2010;31(10):1894-1905.
A novel hybrid melanocortin pharmacophore was designed based on the pharmacophores of the Agouti signaling protein (ASIP), an endogenous melanocortin antagonist, and α-melanocyte-stimulating hormone (α-MSH), an endogenous melanocortin agonist. The designed hybrid ASIP/MSH pharmacophore was explored in monomeric cyclic, and cyclodimeric templates. The monomeric cyclic disulfide series yielded peptides with hMC3R-selective non-competitive binding affinities. The direct on-resin peptide lactam cyclodimerization yielded nanomolar range (25-120 nM) hMC1R-selective full and partial agonists in the cyclodimeric lactam series which demonstrates an improvement over the previous attempts at hybridization of MSH and agouti protein sequences. The secondary structure-oriented pharmacophore hybridization strategy will prove useful in development of unique allosteric and orthosteric melanocortin receptor modulators. This report also illustrates the utility of peptide cyclodimerization for the development of novel GPCR peptide ligands.
PMCID: PMC3041174  PMID: 20688117
Agouti-signalling protein; melanocortin receptors; cyclodimerization; C2-symmetry; macrocyclic peptide
8.  Multiple N-methylation of MT-II backbone amide bonds leads to melanocortin receptor subtype hMC1R selectivity; pharmacological and conformational studies 
Multiple N-methylation is a novel technology to improve bioavailability of peptides and increase receptor subtype selectivity. This technique has been applied here to the superpotent but non-selective cyclic peptide MT-II. A library of all possible 31 backbone N-methylated derivatives has been synthesized and tested for binding and activation at melanocortin receptor subtypes 1, 3, 4 and 5. It turned out that selectivity is improved with every introduced N-methyl group, resulting in several N-methylated selective and potent agonists for the hMC1R. The most potent of these derivatives is N-methylated on four out of five amide bonds in the cyclic structure. Its solution structure indicates a strongly preferred backbone conformation which resembles other a-MSH analogs but possesses much less flexibility and in addition distinct differences in the spatial arrangement of individual amino acid side chains.
PMCID: PMC2895553  PMID: 20496895
9.  Novel Binding Motif of ACTH Analogues at the Melanocortin Receptors 
Biochemistry  2009;48(41):9775-9784.
The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily and each of them has a different pharmacological profile regarding the relative potency of the endogenous and synthetic melanocortin peptides. α-MSH and ACTH are endogenous nonselective agonists for MC1R, MC3R, MC4R and MC5R. In this study, we examined the role of Phe7 in ACTH on Human (h)MC1R, MC3R and MC4R binding and signaling. Our results indicate that substitution of the Phe7 with DNal (2’)7 in ACTH1-24 has different pharmacological profile from that of substitution of the Phe7 with DNal (2’)7 in MSH at hMC1R, hMC3R and hMC4R. NDNal (2’)7-ACTH1-24 is an agonist at hMC3R and hMC4R which did not switch peptide from agonist to antagonist at hMC3R and hMC4R. Further experiments indicate that NDNal (2’)7-ACTH1-17 is the minimal peptide required for hMC3R and hMC4R activation. Single amino acid substitution studies of DNal (2’)7 ACTH1-17 indicate that the amino acid residues 15, 16 and 17 in NDNal (2’)7-ACTH1-17 are crucial for hMC3R and hMC4R activation. Substitutions of these amino acid residues reduced or abolished agonist activity at hMC3R and hMC4R. Conformational studies revealed a new β-turn (-Arg8-Trp9-Gly10-Lys11-) in NDNal (2’)7-ACTH1-17, compared to the β-turn like structure at NDP-α-MSH (–His6-DPhe7-Arg8-Trp9-). Our results suggest that NDP-α-MSH and NDNal (2’)7-ACTH1-17 does not share the same binding site; highly basic C terminal fragment Lys15-Lys16-Arg17 of NDNal (2’)7-ACTH1-17 induced a new β-turn and this shift contributed the selective agonist activity at hMC3R and hMC4R.
PMCID: PMC2775456  PMID: 19743876
POMC; MCR; agonist; GPCR; receptor activation
10.  Substitution of Arginine with Proline and Proline Derivatives in Melanocyte-Stimulating Hormones Leads to Selectivity for Human Melanocortin 4 Receptor 
Journal of medicinal chemistry  2009;52(12):3627-3635.
A new series of melanotropin analogues with His or Arg residues in the core pharmacophores of MTII, SHU9119 and Ac-NDP-γ-MSH-NH2 replaced by Pro or trans-/cis- 4-guanidinyl-Pro derivatives were designed and synthesized to introduce selectivity toward the human melanocortin 4 receptor (hMC4R). Analogues 1, 2, 3, 6, 7, 8 were found to be hMC4R selective. Second messenger studies have demonstrated that analogues 1 and 2 are insurmountable inhibitors of MTII agonist activity at the hMC4R. Molecular modeling studies suggest that the hMC4R selectivity is due to a β-turn shift induced by the Pro ring that makes the global minimum structures of these analogues resemble the NMR solution structure of the hASIP melanocortin receptor binding motif. Substitution of His in MTII also provided functional selectivity for the hMC3R or the hMC4R. These findings are important for a better understanding of the selectivity mechanism at the hMC3R/hMC4R, and the development of therapeutic ligands selectively targeting the hMC4R.
PMCID: PMC2775485  PMID: 19473029
11.  Plasmon-waveguide resonance studies of ligand binding to integral proteins in membrane fragments derived from bacterial and mammalian cells 
Analytical biochemistry  2009;387(1):95-101.
A procedure has been developed for directly depositing membrane fragments derived from bacterial (chromatophores from Rhodopseudomonas sphaeroides) and mammalian cells (μ-opioid receptor- and MC4 receptor-transfected HEK cells, and rat trigeminal ganglion cells) onto the silica surface of a plasmon-waveguide resonance (PWR) spectrometer. Binding of ligands (cytochrome c2 for the chromatophores, the peptide agonists DAMGO and Melanotan-II that are specific for the μ-opioid and MC4 receptors, and two non-peptide agonists that are specific for the CB1 receptor) to these membrane fragments has been observed and characterized with high sensitivity using PWR spectral shifts. The KD values obtained are in excellent agreement with conventional pharmacological assays and with prior PWR studies using purified receptors inserted into deposited lipid bilayer membranes. These studies provide a new tool for obtaining useful biological information about receptor-mediated processes in real biological membranes.
PMCID: PMC2783692  PMID: 19454250
G-protein coupled receptors; bacterial chromatophores; transfected HEK cells; rat trigeminal ganglion; μ-opioid receptor; cannabinoid CB1 receptor; melanocortin-4 receptor
12.  Design and Microwave-Assisted Synthesis of Novel Macrocyclic Peptides Active at Melanocortin Receptors: Discovery of Potent and Selective hMC5R Receptor Antagonists 
Journal of medicinal chemistry  2008;51(9):2701-2707.
Differentiation of the physiological role of the melanocortin receptor 5 MC5R from that of other melanocortin receptors will require development of high affinity and selective antagonists. To date, a few synthetic antagonist ligands active at hMC5 receptor are available, but most do not have appreciable selectivity. With the aim to gain more potent and selective antagonists for the MC5R ligands, we have designed, synthesized, and pharmacologically characterized a series of alkylthioaryl-bridged macrocyclic peptide analogues derived from MT-II and SHU9119. These 20-membered macrocycles were synthesized by a tandem combination using solid phase peptide synthesis and microwave-assisted reactions. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues, compounds, 9, 4, and 7, are selective antagonists at the hMC5 receptor. In particular, compound 9 (PG-20N) is a selective and competitive hMC5R antagonist, with IC50 of 130 ± 11 nM, and a pA2 value of 8.3, and represents an important tool for further biological investigations of the hMC5R. Compounds 4 and 7 (PG14N, PG17N) show potent and selective allosteric inhibition at hMC5R with IC50 values of 38 ± 3 nM and 58 ± 6 nM, respectively. Compound 9 will be used to further investigate and more clearly understand the physiological roles played by the MC5 receptor in humans and other animals.
PMCID: PMC2756086  PMID: 18412316
13.  Contribution of the Conserved Amino Acids of the Melanocortin-4 Receptor in D-[Nle4,Phe7]-α-Melanocyte-stimulating Hormone Binding and Signaling□S 
The Journal of biological chemistry  2007;282(30):21712-21719.
Melanocortin 4 receptor (MC4R) plays an important role in the regulation of food intake and body weight. To determine the molecular basis of human MC4R (hMC4R) responsible for α-melanocortin-stimulating hormone (α-MSH) binding, in this study, we utilized both receptor domain exchange and site-directed mutagenesis studies to investigate the molecular determinants of hMC4R responsible for α-MSH binding and signaling. α-MSH is a potent agonist at hMC4R but not at hMC2R. Cassette substitutions of the second, third, fourth, fifth, and sixth transmembrane regions (TM) of the hMC4R with the homologous regions of hMC2R were performed and α-MSH binding and signaling were examined. Our results indicate that each chimeric receptor was expressed at the cell surface and the expression levels remain similar to that of the wild-type receptor. The cassette substitutions of the second, fourth, fifth, and sixth TMs of the hMC4R with homologous regions of the hMC2R did not significantly alter α-MSH binding affinity and potency except substitution of the TM3 of the hMC4R, suggesting that the conserved residues in TMs of the hMC4R are crucial for α-MSH binding and signaling. Further mutagenesis studies indicate that conserved residues Glu100 in TM2, Asp122, Asp126 in TM3 and Trp258, Phe261, His264 in TM6 are involved in α-MSH binding and signaling. In conclusion, our results suggest that the conserved residues in the TM2, TM3, and TM6 of the hMC4R are responsible for α-MSH binding and signaling.
PMCID: PMC2704061  PMID: 17545153
14.  Contribution of the transmembrane domain 6 of melanocortin-4 receptor to peptide [Pro5, DNaI (2′)8]-γ-MSH selectivity 
Biochemical pharmacology  2008;77(1):114-124.
The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily and each of them has a different pharmacological profile regarding the relative potency of the endogenous and synthetic melanocortin peptides. Substitution of Trp with DNaI (2′) in -γ-MSH resulted in the loss of binding affinity and potency at hMC4R. However, the molecular mechanism of this ligand selectivity is unclear. In this study, we utilized chimeric receptors and site-directed mutagenesis approaches to investigate the molecular basis of MC4R responsible for peptide [Pro5, DNaI (2′)8]-γ-MSH selectivity. Cassette substitutions of the second, third, fourth, fifth, and sixth TM of the human MC4R (hMC4R) with the homologous regions of hMC1R were constructed and the binding affinity of peptide [Pro5, DNaI (2′)8]-γ-MSH at these chimeric receptors was evaluated. Our results indicate that the cassette substitutions of TM2, TM3, TM4 and TM5 of hMC4R with homologous regions of the hMC1R did not significantly increase peptide [Pro5, DNaI (2′)8]-γ-MSH binding affinity and potency but substitution of the TM6 of the hMC4R with the same region of the hMC1R significantly enhances [Pro5, DNaI (2′)8]-γ-MSH binding affinity and potency. Further site-directed mutagenesis study indicates that four amino acid residues, Phe267, Tyr268, Ile269 and Ser270, in TM6 of the hMC4R may play an important role in [Pro5, DNaI (2′)-γ-MSH selective activity at MC4R.
PMCID: PMC2701352  PMID: 18930713
MC4R; γ-MSH; MC1R; Obesity; GPCR; Agonist
15.  Opioid and Melanocortin Receptors: Do They Have Opioid Overlapping Pharmacophores? 
Biopolymers  2008;90(3):433-438.
We have identified compound 1 as a novel ligand for opioid and melanocortin (MC) receptors, which is derived from the overlapping of a well known structure for the δ opioid receptor, 2,6-dimethyltyrosine (Dmt)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic), and a small molecule for the MC receptor, Tic-DPhe(p-Cl)-piperidin-4-yl-N-phenyl-propionamide. Ligand 1 showed that there is an overlapping pharmacophore between opioid and MC receptors through the Tic residue. The ligand displayed high biological activities at the δ opioid receptor (Ki = 0.38 nM in binding assay, EC50 = 0.48 nM in GTP-γ-S binding assay, IC50 = 74 nM in MVD) as an agonist instead of an antagonist and showed selective binding affinity (IC50 = 2.3 μM) at the MC-3 receptor rather than at the MC-5 receptor. A study of the structure-activity relationships demonstrated that the residues in positions 2, 3, and the C-terminus act as a pharmacophore for the MC receptors, and the residues in positions 1 and 2 act as a pharmacophore for the opioid receptors. Thus, this structural construct can be used to prepare chimeric structures with adjacent or overlapping pharmacophores for opioid and MC receptors.
PMCID: PMC2693099  PMID: 17657709
opioid receptor; melanocortin receptor; anti-opioid effect; multi-target drug; overlapping pharmacophores; antinociception; side effect; Dmt-Tic; fentanyl
16.  Effects of Selective Modulation of the Central Melanocortin-3-Receptor on Food Intake and Hypothalamic POMC Expression 
Peptides  2007;29(3):440-447.
Hypothalamic POMC neurons regulate energy balance via interactions with brain melanocortin receptors (MC-Rs). POMC neurons express the MC3-R which can function as an inhibitory autoreceptor in vitro. We now demonstrate that central activation of MC3-R with icv infusion of the specific MC3-R agonist, [D-Trp8]-γ-MSH, transiently suppresses hypothalamic Pomc expression and stimulates food intake in rats. Conversely, we also show that icv infusion of a low dose of a selective MC3-R antagonist causes a transient decrease in feeding and weight gain. These data support a functional inhibitory role for the MC3-R on POMC neurons that leads to changes in food intake.
PMCID: PMC2278043  PMID: 18155809
POMC; MSH; melanocortin-3-receptor; feeding
17.  Structure–Activity Relationships of Cyclic Lactam Analogues of α-Melanocyte-Stimulating Hormone (α-MSH) Targeting the Human Melanocortin-3 Receptor 
Journal of medicinal chemistry  2007;51(2):187-195.
A variety of dicarboxylic acid linkers introduced between the α-amino group of Pro6 and the ε-amino group of Lys10 of the cyclic lactam α-melanocyte-stimulating hormone (α-MSH)-derived Pro6-D-Phe7/D-Nal(2′)7-Arg8-Trp9-Lys10-NH2 pentapeptide template lead to nanomolar range and selective hMC3R agonists and antagonists. Replacement of the Pro6 residue and the dicarboxylic acid linker with 2,3-pyrazine-dicarboxylic acid furnished a highly selective nanomolar range hMC3R partial agonist (analogue 12, c[CO-2,3-pyrazine-CO-D-Phe-Arg-Trp-Lys]-NH2, EC50 = 27 nM, 70% max cAMP) and an hMC3R antagonist (analogue 13, c[CO-2,3-pyrazine-CO-D-Nal(2′)-Arg-Trp-Lys]-NH2, IC50 = 23 nM). Modeling experiments suggest that 2,3-pyrazinedicarboxylic acid stabilizes a β-turn-like structure with the D-Phe/D-Nal(2′) residues, which explains the high potency of the corresponding peptides. Placement of a Nle residue in position 6 produced a hMC3R/hMC5R antagonist (analogue 15, c[CO-(CH2)2-CO-Nle-D-Nal(2′)-Arg-Trp-Lys]-NH2, IC50 = 12 and 17 nM, respectively), similarly to the previously described cyclic γ-melanocyte-stimulating hormone (γ-MSH)-derived hMC3R/hMC5R antagonists. These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin-3 receptor.
PMCID: PMC2587288  PMID: 18088090
18.  Cell Signaling and Trafficking of Human Melanocortin Receptors in Real Time Using Two-photon Fluorescence and Confocal Laser Microscopy: Differentiation of Agonists and Antagonists 
Chemical biology & drug design  2006;68(4):183-193.
Melanocortin hormones and neurotransmitters regulate a vast array of physiologic processes by interacting with five G-protein-coupled melanocortin receptor types. In the present study, we have systematically studied the regulation of individual human melanocortin receptor wild subtypes using a synthetic rhodamine-labeled human melanotropin agonist and antagonist, arrestins fused to green fluorescent protein in conjunction with two-photon fluorescence laser scanning microscopy and confocal microscopy. Stimulation of the melanocortin receptors by its cognate agonist triggered rapid arrestin recruitment and receptor internalization for all four human melanocortin receptors examined. Antagonists-bound melanocortin receptors, on the other hand, did not recruit β-arrestins, and remained in the cell membrane even after long-term (30 min) treatment. Agonist-mediated internalization of all melanocortin receptor subtypes was sensitive to inhibitors of clathrin-dependent endocytosis, but not to caveolae inhibitors. In summary, agonist-mediated internalization of all subtypes of melanocortin receptors are dependent upon β-arrestin-mediated clathrin-coated pits, whereas, β-arrestin-2 conjugated green fluorescence protein (β-arrestin-2-GFP) recruitment is not dependent on protein kinase A activation. Real time two-photon fluorescence laser scanning microscopy is a most powerful tool to study the dynamic processes in living cells and tissues, without inflicting significant and often lethal damage to the specimen.
PMCID: PMC2547351  PMID: 17105482
β-arrestin; green fluorescence protein; melanocortin receptors; MTII; rhodamine; SHU-9119; two-photon fluorescence laser scanning microscopy
19.  Novel selective human melanocortin-3 receptor ligands: Use of the 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one (Aba) scaffold 
In search of new selective antagonists and/or agonists for the human melanocortin receptor subtypes hMC1R to hMC5R to elucidate the specific biological roles of each GPCR, we modified the structures of the superagonist MT-II (Ac-Nle-c[Asp-His-D-Phe-Arg-Trp-Lys]-NH2) and the hMC3R/hMC4R antagonist SHU9119 (Ac-Nle-c[Asp-His-D-Nal(2′)-Arg-Trp-Lys]-NH2) by replacing the His-D-Phe and His-D-Nal(2′) fragments in MT-II and SHU9119, respectively, with Aba-Xxx (4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one-Xxx) dipeptidomimetics (Xxx = D-Phe/pCl-D-Phe/D-Nal(2′)). Employment of the Aba mimetic yielded novel selective high affinity hMC3R and hMC3R/hMC5R antagonists.
PMCID: PMC2442462  PMID: 17314042
Human melanocortin receptors; 4-Amino-1,2,4,5-tetrahydro-2-benzazepin-3-ones; Cyclic lactam analogues; Conformational restrictions; hMC3R/hMC5R antagonists
20.  Further structure-activity studies of lactam derivatives of MT-II and SHU-9119: Their activity and selectivity at human melanocortin receptors 3, 4 and 5 
Peptides  2007;28(6):1191-1196.
Recently we have demonstrated that replacing His6 by constrained amino acids in the well known antagonist SHU9119 resulted in potent and selective antagonist ligands especially at the hMC3R and hMC4 receptors. With the aim to further explore position 6 in the sequence of SHU9119 and MT-II, we have designed, synthesized, and pharmacologically characterized a series of peptide analogues of MT-II and SHU9119 at the human melanocortin receptors subtypes MC3R, MC4R and MC5R. All these peptides were modified at position 6 with constrained amino acids which are commercially available. In this study we have identified new selective ligands for the hMC4R, and an antagonist for the hMC3/hMC4 receptors. Additionally, we have discovered an interesting new selective antagonist at the hMC3R, Ac-Nle-c[Asp-βAla-DNal(2′)-Arg-Trp-Lys]-NH2 (2, PG-106) which represents an important tool in further biological investigations of the hMC3R. PG-106 will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.
PMCID: PMC1955225  PMID: 17482720
Melanocortin receptors; Melanotropins; Structure-activity relationships; Cyclic melanotropins; Receptor selective melanotropin antagonists
21.  Design, Synthesis and Biological Evaluation of Ligands Selective for the Melanocortin-3 Receptor 
The processed products of the proopiomelanocortin gene (ACTH, α-MSH, β-MSH, γ-MSH, etc.) interact with five melanocortin receptors, the MC1R, MC2R, MC3R, MC4R, and MC5R to modulate and control many important biological functions crucial for good health both peripherally (as hormones) and centrally (as neurotransmitters). Pivotal biological functions include pigmentation, adrenal function, response to stress, fear/flight, energy homeostasis, feeding behavior, sexual function and motivation, pain, immune response, and many others, and are believed to be involved in many disease states including pigmentary disorders, adrenal disorders, obesity, anorexia, prolonged and neuropathic pain, inflammatory response, etc. The roeianocortin-3 receptor (MC3R) is found primarily in the brain and spinal cord and also in the periphery, and its biological functions are still not well understood. Here we review some of the biological functions attributed to the MC3R, and then examine in more detail efforts to design and synthesize ligands that are potent and selective for the MC3R, which might help resolve the many questions still remaining about its function. Though some progress has been made, there is still much to be done in this critical area.
PMCID: PMC2274922  PMID: 17584128
22.  Effects of Macrocycle Size and Rigidity on Melanocortin Receptor-1 and -5 Selectivity in Cyclic Lactam α-Melanocyte-Stimulating Hormone Analogs 
Chemical biology & drug design  2006;67(5):329-335.
The effects of the linker arm rigidity and size on melanocortin receptor selectivity were explored in a series of compounds using cyclic lactam α-melanocyte-stimulating hormone template. A variety of dicarboxylic acid linkers introduced between the α-amino group of His6 and the ɛ-amino group of Lys10 lead to high-affinity, selective human melanocortin receptor-1 and -5 (hMC1R and hMC5R) antagonists. The incorporation of hydrophilic functions into the linker arm was found to be unfavorable for both binding potency and receptor selectivity. Analogs 8 and 9 containing highly conformationally constrained hydrophobic linkers (m- and p-phthalic acids) were found to be selective nanomolar range hMC1R antagonists (IC50 = 7 and 4 nM, respectively), whereas the employment of a small conformationally constrained linker (maleic acid) resulted in a high-affinity (IC50 = 19 nM) and selective hMC5R antagonist (analog 12). These newly developed melanotropins will serve as critical biochemical tools for elucidating the full spectrum of functions performed by the physiologically important melanocortin-1 and -5 receptors.
PMCID: PMC1851940  PMID: 16784457
α-melanocyte-stimulating hormone; antagonist; human melanocortin-1 receptor; human melanocortin-5 receptor; macrocyclic; melanocortin; peptide; All, allyl; Alloc, allyloxycarbonyl; Boc, tert-butyloxycarbonyl; Fmoc, fluorenylmethoxycarbonyl; CH3CN, acetonitrile; DCM, dichloromethane; DIPEA, diisopropylethylamine; DMF, N,N-dimethylformamide; DIC, diisopropyl carbodiimide; HBTU, 2-(1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyluronium hexafluorophosphate; HOBt, N-hydroxybenzotriazole; hMCR, human melanocortin receptor; MSH, melanocyte-stimulating hormone; Nal(2′), 2′-naphthylalanine; Pbf, 2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl; PyBOP, benzotriazol-1-yloxy-tris-pyrrolidinophosphonium hexafluorophosphate; TFA, trifluoroacetic acid; Trt, trityl; SPPS, solid-phase peptide synthesis; RP-HPLC, reverse-phase high-performance liquid chromatography; hMC1R, human melanocortin-1 receptor; α-MSH, Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2; NDP-α-MSH, Ac-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2
23.  Design, synthesis, and biological evaluation of a new class of small molecule peptide mimetics targeting the melanocortin receptors 
A new bicyclic template has been developed for the synthesis of peptide mimetics. Straightforward synthetic steps, starting from amino acids, allow the facile construction of a wide range of analogs. This system was designed to target the melanocortin receptors (MCRs), with functional group selection based on a known pharmacophore and guidance from molecular modeling to rationally identify positional and stereochemical isomers likely to be active. The functions of hMCRs are critical to myriad biological activities, including pigmentation, steroidogenesis, energy homeostasis, erectile activity, and inflammation. These G-protein-coupled receptors (GPCRs) are targets for drug discovery in a number of areas, including cancer, pain, and obesity therapeutics. All compounds from this series tested to date are antagonists which bind with high affinity. Importantly, many are highly selective for a particular MCR subtype, including some of the first completely hMC5R-selective antagonists reported.
PMCID: PMC1810397  PMID: 16931008
Melanocortins; Peptide mimetics; GPCRs
24.  Design, Synthesis, and Biological Evaluation of New Cyclic Melanotropin Peptide Analogues Selective for the Human Melanocortin-4 Receptor 
Journal of medicinal chemistry  2006;49(23):6888-6896.
Intensive efforts have been made to develop potent and selective ligands for certain human melanocortin receptors as possible treatments for obesity and sexual dysfunction due to the role of these receptors in feeding behavior, energy homeostasis, sexual function, etc. A number of novel α-MSH analogues were designed and synthesized primarily on the basis of our previous MTII NMR structure. In these peptide analogues, a disulfide or lactam bridge between residues at positions 5 and 8 was used as a conformational constraint to enhance the β-turn spanning His6 and d-Phe7, while the pharmacophore group in Arg8 was mimicked via Nα-alkylation of residues 8 or 9 with the guanidinylbutyl group. Biological assays for binding affinities and adenylate cyclase activities for the hMC1R, hMC3R, hMC4R, and hMC5R showed that three analogues have good binding affinity for the hMC4R (0.7–4.1 nM), but have no binding affinity up to 10 μM at the other three melanocortin receptors. Interestingly, the three hMC4R selective analogues display only 50% binding efficiency, suggesting there is allosteric modulation of the melanocortin-4 receptor. These analogues were found to act as antagonists of the hMC4R. This result represents a discovery of very selective peptide-based antagonists for the hMC4R. The high selectivity may be due to the strong conformational constraint via ring contraction as compared to MTII, and the rigid conformation preferred by these new ligands allows them to recognize only the hMC4R, but not to activate the second messenger. The MTII NMR structure-based design thus not only examined the structural model of melanocortin ligands, but also yielded new biologically unique α-MSH analogues.
PMCID: PMC1764620  PMID: 17154518
25.  New Paradigms and Tools in Drug Design for Pain and Addiction 
The AAPS journal  2006;8(3):E450-E460.
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and δ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
PMCID: PMC1764851  PMID: 17025262
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors

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