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Compound 7
An highly selective antagonist for MC3R

more information for Alpha MSH, Gramma-MSH, Beta-MSH, AGRP, VA-beta-MSH, MT II, SHU9119
Structure-activity relationships of novel cyclic alpha-MSH/beta-MSH hybrid analogues that lead to potent and selective ligands for the human MC3R and human MC5R.
It has been shown by extensive studies that alpha-MSH bioactivity is critically dependent on the core or central tetrapeptide sequence, His-Phe-Arg-Trp, however with poor selectivity for the human MC3R-MC5R. The structure-activity relationships study here is aimed at identifying lead structures or templates of this core sequence by the use of different conformational constraints that might impart changes in its topography and thus promote differences in potency and selectivity at these receptors. Our peptide library consists of a novel series of cyclic alpha-MSH analogues that have disulfide bridges between Cys or Cys-like residues at positions 4 and 10, giving rise to 23-membered rings fused at the C-terminal end with the C-terminal fragment of beta-MSH (Pro-Pro-Lys-Asp). While such constraints of the peptide backbone with disulfide bridges of different chirality affect potency and selectivity at these receptors, further changes in the hydrophobicity at position 7 with either a D-Phe or D-Nal(2') and replacement of a His with a Pro in position 6 cause additional effects. Thus, the most interesting lead compounds that emerged from this study are (1) compound 5, Ac-c[Cys-Glu-His-D-Phe-Arg-Trp-D-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 10 nM), which is the first potent and highly selective antagonist ligand for the hMC5R (560-fold vs the MC3R and 1000-fold vs the MC4R); (2) compound 7, Ac-c[Cys-Glu-Pro-D-Nal(2')-Arg-Trp-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 31 nM), which is a highly selective antagonist analogue for the MC3R (560-fold vs the hMC4R and about 3000-fold vs the hMC5R; and (3) compound 9, Ac-c[Pen-Glu-His-D-Nal(2')-Arg-Trp-Cys]-Pro-Pro-Lys-Asp-NH(2) (IC(50) = 3 nM), which is more potent than 7 at the MC3R but not as selective.
Balse-Srinivasan P, Grieco P, Cai M, Trivedi D, Hruby VJ. J Med Chem. 2003 Aug 14;46(17):3728-33

 
Ligand
MC3R(IC50) MC3R/MC4R MC3R/MC5R
Compound 7
31 nM 560 fold 3000 fold
 

We report the isolation of a novel human circulating proopiomelanocortin-derived peptide named VA-▀-MSH from hemofiltrate and its pharmacological characterization. Screening for lipolytic activity in differentiated 3T3-L1 adipocytes led to the isolation from a hemofiltrate peptide library by alternating reversed-phase and cation-exchange chromatography. In the course of this isolation, we also identified human ▀-MSH (1-22). We synthesized VA-▀-MSH by the Fmoc (N-(9-fluorenyl)-methoxycarbonyl) solid phase method and used synthetic▀-MSH (1-22) to confirm that both isolated peptides are lipolytically active in a dosedependent manner in differentiated 3T3-L1 adipocytes in the nanomolar range. Using cAMPELISA, we demonstrate that stimulation with both peptides caused a strong cAMP elevation in this cell system. Furthermore, we show that the selective inhibitors of cAMP-dependent protein kinase, Rp-8-CPT-cAMPS and H89, significantly reduce VA-▀-MSH- and▀-MSH (1-22)-mediated lipolysis. Although isolated here following its lipolytic activity on 3T3-L1 cells, this newly identified circulating human melanocortin may also serve other functions in human physiology. Moreover, the fact that these peptides have been identified following a functional assay but have been overseen in large proteomic approaches underscore the importance for such approaches in order to identify previously undescribed circulating bioactive molecules.
Isolation and characterisation of a novel POMC-derived peptide from hemofiltrate of chronic renal failure patients.
Fricke K., et al. Endocrinology. First published January 13, 2005 as doi:10.1210/en.2004-1097

Lipolytic potency and intracellular cAMP formation of VA-▀-MSH, ▀-MSH (1-22), hACTH (1-39), and forskolin. Results are shown as means (▒ SD) of three independent experiments.
msh
Lipolytic effect of synthetic VA-▀-MSH and ▀-MSH (1-22). Dose-response relationships of increasing concentrations of both peptides. After incubation of 3T3-L1 adipocytes with either peptide for 5 h, glycerol content in the supernatants was measured. Results are expressed as percentages of the lipolytic response of cells to 1 ÁM isoproterenol and are means ▒ SD (n=3). One of three independent experiments is presented.
Effects of specific PKA inhibitors on VA-▀-MSH- and ▀-MSH (1-22)- induced lipolysis. 3T3-L1 adipocytes were preincubated with or without inhibitor for 40 minutes, followed by the incubation with 20 nM VA-▀-MSH or ▀-MSH (1-22) for 5 h. A. Preincubation of 3T3-L1 adipocytes with 0.3 mM and 1 mM Rp-8-CPT-cAMPS significantly decreased VA-▀-MSH-induced lipolysis. B. VA-▀-MSH- and ▀-MSH (1-22)- induced glycerol release were inhibited in the presence of H89. Basal lipolysis was not significantly changed by either inhibitor. Data points are means ▒ SD (n=3). Results shown are representative for three separate experiments. * P < 0.05 versus no inhibitor.
Characterization of the VA-▀-MSH- and ▀-MSH (1-22)- stimulated cAMP production in 3T3-L1 adipocytes. The differentiated adipocytes were transferred into fresh DMEM on the day of the experiment, varying concentrations of ▀-MSH (1-22) and VA-▀- MSH were added and incubated at 37░C for the indicated time points. The supernatants were 27 removed and cells were lysed with seventy percent ethanol. Cell lysates were assayed for cAMP by ELISA. A. Time dependency of VA-▀-MSH- and ▀-MSH (1-22)- stimulated cAMP production in 3T3-L1 cells. Isoproterenol (1 ÁM) was used as a positive control. B Doseresponse relationship of cAMP-production in differentiated 3T3-L1 cells by VA-▀-MSH and▀-MSH (1-22). Each point represents the mean ▒ SD of triplicate values. Results are representative for three (A) and two (B) independent experiments.
Effect of VA-▀-MSH on CREB and perilipin phosphorylation. A 3T3-L1 adipocytes were serum deprived overnight and subsequently stimulated with 300 nM VA-▀- MSH for the times indicated. At each time point, cells were lysed, and equal amounts of lysate protein were subjected to immunoblot analysis. Blots were probed with antibodies against serine 133-phosphorylated CREB (pCREB) and total CREB. One of three independent experiments each with two replicates is shown. The positions of the pCREB and CREB bands are indicated. B After 15 min stimulation with VA-▀-MSH, cell lysates were subjected to immunoblot analysis, and blots were probed with an antibody against perilipin. The characteristic upward shift observed with VA-▀-MSH reveals the phosphorylation of perilipin A in stimulated cells compared to cells treated with control medium (representative blot of three independent experiments).
Protein expression of melanocortin receptor isoforms 2 and 5 in 3T3-L1 adipocytes. Untreated 3T3-L1 adipocytes were lysed and subjected to SDS-PAGE followed by immunoblot analysis. Blots were probed using a specific antiserum directed against MC2R (left side) or an affinity-purified antibody against MC5R (right side). ▀-actin served as a loading control.
Fricke K., et al. Endocrinology. First published January 13, 2005 as doi:10.1210/en.2004-1097


beta-MSH: a functional ligand that regulated energy homeostasis via hypothalamic MC4-R?


alpha-Melanocyte stimulating hormone (MSH) has generally been assumed to be the endogenous ligand acting at the melanocortin-4 receptor (MC4-R), activation of which in the hypothalamus leads to reduced feeding. However, beta-MSH is also capable of activating MC4-R and inhibiting feeding. Here, we investigated the possibility that beta-MSH acts as an endogenous MC4-R agonist and that this melanocortin peptide plays a role in the regulation of feeding and energy balance. We found that beta-MSH had significantly higher affinities than alpha-MSH at both human MC4-R transfected into CHO cells (K(i): beta-MSH, 11.40.4 nmol/l versus alpha-MSH, 32416 nmol/l, P<0.001) and MC4-R in rat hypothalamic homogenates (K(i): beta-MSH, 5.00.4 nmol/l versus alpha-MSH, 22.52.3 nmol/l, P<0.001). Incubation of brain slices with 5 microM beta-MSH significantly increased [35S]GTPgammaS binding by 140-160% (P<0.001), indicating activation of G-protein-coupled receptors (GPCRs), in the hypothalamic ventromedial (VMH), dorsomedial (DMH), arcuate (ARC) and paraventricular (PVN) nuclei. These sites match the distribution of beta-MSH immunoreactive fibres and also the distribution of MC4-R binding sites which we and others previously reported. Food-restriction significantly increased beta-MSH levels in the VMH, DMH and ARC (all P<0.05) above freely-fed controls, whilst alpha-MSH concentrations were unchanged. We propose that increased beta-MSH concentrations reflect blockade of the peptide's release in these sites, consistent with the increased hunger and the known up-regulation of MC4-R in the same nuclei. Thus, we conclude that (1). beta-MSH has higher affinity at MC4-R than alpha-MSH; (2). beta-MSH activates GPCR in these sites, which are rich in MC4-R; and (3). beta-MSH is present in hypothalamic nuclei that regulate feeding and its concentrations alter with nutritional state. We suggest that beta-MSH rather than alpha-MSH is the key ligand at the MC4-R populations that regulate feeding, and that inhibition of tonic release of beta-MSH is one mechanism contributing to hunger in under-feeding.
Harrold JA, et al. Peptides. 2003 Mar;24(3):397-405

 
 
 

Binding Assay (IC50, nM)

Selectivity

  hMC-3R hMC-4R hMC-5R 3/4 5/4
MC-4R Agonist 490+-5 4.3+-.67 4600+-50 114 1070
MT II 1.6+-.09 0.07+-.02 0.89+-.01 23 13
 
 

 

Melanocyte Stimulating Hormones (MSH) Related Products

RAB-043-01;T-043-01;FR-043-01;T-043-12;RAB-043-19;T-043-19;RAB-043-16;T-043-16;WBK-043-17;043-25;043-27;043-32;043-26;043-28;043-31;B-043-02;043-20;043-23;043-29;FR-043-23;043-24

%compound 7%;%043-12%


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