Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01189 (beta-endorphin)
 21,003 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A novel approach to specifically target tumor cells for detection and treatment is the proposed use of heteromultivalent ligands, which are designed to interact with, and noncovalently crosslink, multiple different cell surface receptors. Although enhanced binding has been shown for synthetic homomultivalent ligands, proof of cross-linking requires the use of ligands with two or more different binding moieties. As proof-of-concept, we have examined the binding of synthetic heterobivalent ligands to cell lines that were engineered to coexpress two different G-protein-coupled human receptors, i.e., the human melanocortin 4 receptor (MC4R) expressed in combination with either the human delta-opioid receptor (deltaOR) or the human cholecystokinin-2 receptor (CCK2R). Expression levels of these receptors were characterized by time-resolved fluorescence saturation binding assays using Europium-labeled ligands; Eu-DPLCE, Eu-NDP-alpha-MSH, and Eu-CCK8 for the deltaOR, MC4R, and CCK2R, respectively. Heterobivalent ligands were synthesized to contain a MC4R agonist connected via chemical linkers to either a deltaOR or a CCK2R agonist. In both cell systems, the heterobivalent constructs bound with much higher affinity to cells expressing both receptors, compared with cells with single receptors or to cells where one of the receptors was competitively blocked. These results indicate that synthetic heterobivalent ligands can noncovalently crosslink two unrelated cell surface receptors, making feasible the targeting of receptor combinations. The in vitro cell models described herein will lead to the development of multivalent ligands for target combinations identified in human cancers.
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PMID:Enhanced targeting with heterobivalent ligands. 1967 49

The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily, and each of them has a different pharmacological profile with regard to the relative potency of the endogenous and synthetic melanocortin peptides. Alpha-MSH and ACTH are endogenous nonselective agonists for MC1R, MC3R, MC4R, and MC5R. In this study, we examined the role of Phe(7) in ACTH on human (h) MC1R, MC3R, and MC4R binding and signaling. Our results indicate that substitution of Phe(7) with d-Nal(2')(7) in ACTH1-24 yields a pharmacological profile different from that for substitution of Phe(7) with d-Nal(2')(7) in MSH in hMC1R, hMC3R, and hMC4R. N-d-Nal(2')(7)-ACTH1-24 is an agonist at hMC3R and hMC4R which did not change the peptide from an agonist to an antagonist at hMC3R and hMC4R. Further experiments indicate that N-d-Nal(2')(7)-ACTH1-17 is the minimal peptide required for hMC3R and hMC4R activation. Single-amino acid substitution studies of d-Nal(2')(7)-ACTH1-17 indicate that amino acid residues 15-17 in N-d-Nal(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 beta-turn (Arg(8)-Trp(9)-Gly(10)-Lys(11)) in N-d-Nal(2')(7)-ACTH1-17, compared to the beta-turn-like structure at NDP-alpha-MSH (His(6)-d-Phe(7)-Arg(8)-Trp(9)). Our results suggest that NDP-alpha-MSH and N-d-Nal(2')(7)-ACTH1-17 do not share the same binding site; the highly basic C-terminal fragment (Lys(15)-Lys(16)-Arg(17)) of N-d-Nal(2')(7)-ACTH1-17 induced a new beta-turn, and this shift contributed the selective agonist activity at hMC3R and hMC4R.
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PMID:Novel binding motif of ACTH analogues at the melanocortin receptors. 1974 76

alpha-Melanocyte-stimulating hormone (alpha-MSH) is a pro-opiomelanocortin (POMC)-derived peptide that exerts multiple protective effects on host cells. Previous investigations showed that treatment with alpha-MSH or synthetic melanocortin agonists reduces heart damage in reperfusion injury and transplantation. The aim of this preclinical research was to determine whether melanocortin treatment induces preconditioning-like cardioprotection. In particular, the plan was to assess whether melanocortin administration causes phenotype changes similar to those induced by repetitive ischemic events. The idea was conceived because both ischemic preconditioning and melanocortin signaling largely depend on cAMP response element binding protein (CREB) phosphorylation. Rats received single i.v. injections of 750microg/kg of the alpha-MSH analogue Nle(4),DPhe(7)-alpha-MSH (NDP-MSH) or saline and were sacrificed at 0.5, 1, 3, or 5h. Western blot analysis showed that rat hearts expressed melanocortin 1 receptor (MC1R) protein. Treatment with NDP-MSH was associated with early and marked increase in interleukin 6 (IL-6) mRNA. This was followed by signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling 3 (SOCS3). There were no changes in expression of other cytokines of the IL-6 family. Expression of IL-10, IL-1beta, and TNF-alpha was likewise unaltered. In hearts of rats treated with NDP-MSH there was increased expression of the orphan nuclear receptor Nur77. The data indicate that NDP-MSH induces phenotype changes that closely resemble ischemic preconditioning and likely contribute to its established protection against reperfusion injury. In addition, the increased expression of Nur77 and SOCS3 could be part of a broader anti-inflammatory effect.
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PMID:The peptide NDP-MSH induces phenotype changes in the heart that resemble ischemic preconditioning. 1979 52

Lanthanide-based luminescent ligand binding assays are superior to traditional radiolabel assays due to improving sensitivity and affordability in high-throughput screening while eliminating the use of radioactivity. Despite significant progress using lanthanide(III)-coordinated chelators such as diethylenetriaminepentaacetic acid (DTPA) derivatives, dissociation-enhanced lanthanide fluoroimmunoassays (DELFIAs) have not yet been successfully used with more stable chelators (e.g., tetraazacyclododecyltetraacetic acid [DOTA] derivatives) due to the incomplete release of lanthanide(III) ions from the complex. Here a modified and optimized DELFIA procedure incorporating an acid treatment protocol is introduced for use with Eu(III)-DOTA-labeled peptides. Complete release of Eu(III) ions from DOTA-labeled ligands was observed using hydrochloric acid (2.0M) prior to the luminescent enhancement step. [Nle(4),d-Phe(7)]-alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) labeled with Eu(III)-DOTA was synthesized, and the binding affinity to cells overexpressing the human melanocortin-4 (hMC4) receptor was evaluated using the modified protocol. Binding data indicate that the Eu(III)-DOTA-linked peptide bound to these cells with an affinity similar to its DTPA analogue. The modified DELFIA procedure was further used to monitor the binding of an Eu(III)-DOTA-labeled heterobivalent peptide to the cells expressing both hMC4 and cholecystokinin-2 (CCK-2) receptors. The modified assay provides superior results and is appropriate for high-throughput screening of ligand libraries.
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PMID:Optimization of time-resolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acid-labeled ligand-receptor interactions. 1985 24

As alpha-melanocyte-stimulating hormone (alpha-MSH) is released by immunocompetent cells and has potent immunosuppressive properties, it was determined whether human dendritic cells (DCs) express the receptor for this hormone. Reverse transcription-polymerase chain reaction detected messenger RNA specific for all of the known melanocortin receptors in DCs. Mixed lymphocyte reactions also revealed that treatment with [Nle(4), DPhe(7)]-alpha-MSH (NDP-MSH), a potent alpha-MSH analogue, significantly reduced the ability of DCs to stimulate allogeneic T cells. The expression of various cell surface adhesion, maturation and costimulatory molecules on DCs was also investigated. Although treatment with NDP-MSH did not alter the expression of CD83 and major histocompatibility complex class I and II, the surface expression of CD86 (B7.2), intercellular adhesion molecule (ICAM-1/CD54) and CD1a was reduced. In summary, our data indicate that NDP-MSH inhibits the functional activity of DCs, possibly by down-regulating antigen-presenting and adhesion molecules and that these events may be mediated via the extracellular signal-regulated kinase 1 and 2 pathway.
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PMID:The melanocortin receptor agonist NDP-MSH impairs the allostimulatory function of dendritic cells. 2007 7

Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC(4)) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligands, Cy3B-NDP-alpha-MSH and TAMRA-NDP-alpha-MSH, were used and compared in assays that monitored their binding to MC(4) receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC(4) receptors was saturable and with high affinity. All studied MC(4) receptor-specific nonlabeled ligands displaced fluoroligands' binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters.
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PMID:Fluorescence anisotropy assay for pharmacological characterization of ligand binding dynamics to melanocortin 4 receptors. 2030 39

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low microM affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-dPhe-Arg-Trp-NH(2), exhibited a K(d) for hMC4R of 9.1+/-1.4 microM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-alpha-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects.
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PMID:Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors. 2030 40

Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs), which constitute the largest family of membrane proteins, mediate responses to diverse physiological stimuli. The presence of melanocortin 2 receptors (MC2Rs) on the plasma membrane requires the presence of either MC2R accessory protein (MRAP) or MRAP2, which are homologous accessory proteins. Here, we show that, whereas MRAP was essential for activation of MC2R signaling, MRAP2 was an endogenous inhibitor that competed with MRAP for binding to MC2R and decreased the potency of adrenocorticotropic hormone (ACTH), the endogenous agonist for MC2Rs, in stimulating the production of adenosine 3',5'-monophosphate (cAMP). ACTH bound with high affinity to MC2Rs in the presence of MRAP, but not MRAP2. The ability of MRAP and MRAP2 to influence ligand-binding affinity was specific to MC2R, because these proteins had little effect on the binding of NDP-alpha-melanocyte-stimulating hormone to MC4R or on its stimulation of cAMP responses. These results demonstrate that the balance of stimulatory and inhibitory accessory proteins can control the sensitivity of a GPCR to its natural agonist.
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PMID:Regulation of G protein-coupled receptor signaling: specific dominant-negative effects of melanocortin 2 receptor accessory protein 2. 2037 71

Recently we reported that an efferent vagal fibre-mediated cholinergic protective pathway, activated by melanocortins acting at brain melanocortin MC(3) receptors, is operative in a condition of short-term myocardial ischemia/reperfusion associated with a high incidence of severe arrhythmias and death. Here we investigated melanocortin effects, and the role of the vagus nerve-mediated cholinergic protective pathway, in a rat model of prolonged myocardial ischemia/reperfusion associated with marked inflammatory and apoptotic reactions, and a large infarct size. Ischemia was produced in rats by ligature of the left anterior descending coronary artery for 30 min. At the end of the 2-h reperfusion, western blot analysis of the inflammatory and apoptotic markers tumor necrosis factor-alpha (TNF-alpha), c-jun N-terminal kinases (JNK) and caspase-3, as well as of the anti-apoptotic extracellular signal-regulated kinases (ERK 1/2), was performed in the left ventricle. In saline-treated ischemic rats there was an increase in TNF-alpha levels and in the activity of JNK and caspase-3 accompanied, despite an appreciable ERK 1/2 activation, by a large infarct size. Intravenous treatment, during coronary artery occlusion, with the melanocortin analog [Nle(4), D-Phe(7)]alpha-melanocyte-stimulating hormone (NDP-alpha-MSH) produced a reduction in TNF-alpha levels and in the activity of JNK and caspase-3, associated with marked activation of the pro-survival kinases ERK 1/2, and consequent attenuation of infarct size. Bilateral cervical vagotomy blunted the protective effects of NDP-alpha-MSH. These results indicate that melanocortins modulate the inflammatory and apoptotic cascades triggered by prolonged myocardial ischemia/reperfusion, and reduce infarct size, seemingly by activation of the vagus nerve-mediated cholinergic protective pathway.
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PMID:Melanocortins counteract inflammatory and apoptotic responses to prolonged myocardial ischemia/reperfusion through a vagus nerve-mediated mechanism. 2038 18

The melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor (GPCR) that is expressed in the central nervous system and has a role in regulating feeding behavior, obesity, energy homeostasis, male erectile response, and blood pressure. Since the report of the MC4R knockout mouse in 1997, the field has been searching for links between this genetic biomarker and human obesity and type 2 diabetes. More then 80 single nucleotide polymorphisms (SNPs) have been identified from human patients, both obese and nonobese controls. Many significant studies have been performed examining the pharmacological characteristics of these hMC4R SNPs in attempts to identify a molecular defects/insights that might link a genetic factor to the obese phenotype observed in patients possessing these mutations. Our laboratory has previously reported the pharmacological characterization of 40 of these polymorphic hMC4 receptors with multiple endogenous and synthetic ligands. The goal of the current study is to perform a similar comprehensive side-by-side characterization of 30 additional human hMC4R with single nucleotide polymorphisms using multiple endogenous agonists [alpha-, beta-, and gamma(2)-melanocyte stimulating hormones (MSH) and adrenocorticotropin (ACTH)], the antagonist agouti-related protein hAGRP(87-132), and synthetic agonists [NDP-MSH, MTII, and the tetrapeptide Ac-His-dPhe-Arg-Trp-NH(2) (JRH887-9)]. These in vitro data, in some cases, provide a putative molecular link between dysfunctional hMC4R's and human obesity. These 30 hMC4R SNPs include R7H, R18H, R18L, S36Y, P48S, V50M, F51L, E61K, I69T, D90N, S94R, G98R, I121T, A154D, Y157S, W174C, G181D, F202L, A219 V, I226T, G231S, G238D, N240S, C271R, S295P, P299L, E308K, I317V, L325F, and 750DelGA. All but the N240S hMC4R were identified in obese patients. Additionally, we have characterized a double I102T/V103I hMC4R. In addition to the pharmacological characterization, the hMC4R variants were evaluated for cell surface expression by flow cytometry. The F51L, I69T, and A219V hMC4Rs possessed full agonist activity and significantly decreased endogenous agonist ligand potency. At the E61K, D90N, Y157S, and C271R hMC4Rs, all agonist ligands examined were only partially efficacious in generating a maximal signaling response (partial agonists) and possessed significantly decreased endogenous agonist ligand potency. Only the A219V, G238D, and S295P hMC4Rs possessed significantly decreased AGRP(87-132) antagonist potency. These data provide new information for use in GPCR computational development as well as insights into MC4R structure ad function.
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PMID:Pharmacological characterization of 30 human melanocortin-4 receptor polymorphisms with the endogenous proopiomelanocortin-derived agonists, synthetic agonists, and the endogenous agouti-related protein antagonist. 2046 74


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