 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C110H170N34O24 |
| Molecular Weight | 2352.7402 |
| Exact Mass | 2351.31 |
| CAS # | 158859-98-4 |
| Related CAS # | GR231118 TFA |
| PubChem CID | 24868178 |
| Appearance | Typically exists as solid at room temperature |
| LogP | 6.664 |
| Hydrogen Bond Donor Count | 36 |
| Hydrogen Bond Acceptor Count | 30 |
| Rotatable Bond Count | 62 |
| Heavy Atom Count | 168 |
| Complexity | 4870 |
| Defined Atom Stereocenter Count | 18 |
| SMILES | NC(NCCC[C@@H](C(N[C@@H](CC1C=CC(O)=CC=1)C(=O)N)=O)NC([C@@H](NC([C@@H](NC([C@@H](NC(C1CNC(=O)CC[C@H](NC([C@H]([C@H](CC)C)N)=O)C(=O)N2CCC[C@H]2C(=O)NC(C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@H](C(N[C@@H](CC2C=CC(O)=CC=2)C(=O)N)=O)CCCNC(=N)N)=O)CC(C)C)=O)CCCNC(=N)N)=O)CC2C=CC(O)=CC=2)=O)CNC(=O)CC[C@H](NC([C@H]([C@H](CC)C)N)=O)C(=O)N2CCC[C@H]2C(=O)N1)=O)CC1C=CC(O)=CC=1)=O)CCCNC(=N)N)=O)CC(C)C)=O)=N |
| InChi Key | RJRBRCCJETZJLT-GSICZYLSSA-N |
| InChi Code | InChI=1S/C110H170N34O24/c1-9-59(7)87(111)103(165)133-73-39-41-85(149)127-55-81(99(161)139-79(53-63-27-35-67(147)36-28-63)97(159)131-71(19-13-45-125-109(119)120)93(155)137-77(49-57(3)4)95(157)129-69(17-11-43-123-107(115)116)91(153)135-75(89(113)151)51-61-23-31-65(145)32-24-61)142-102(164)84-22-16-48-144(84)106(168)74(134-104(166)88(112)60(8)10-2)40-42-86(150)128-56-82(141-101(163)83-21-15-47-143(83)105(73)167)100(162)140-80(54-64-29-37-68(148)38-30-64)98(160)132-72(20-14-46-126-110(121)122)94(156)138-78(50-58(5)6)96(158)130-70(18-12-44-124-108(117)118)92(154)136-76(90(114)152)52-62-25-33-66(146)34-26-62/h23-38,57-60,69-84,87-88,145-148H,9-22,39-56,111-112H2,1-8H3,(H2,113,151)(H2,114,152)(H,127,149)(H,128,150)(H,129,157)(H,130,158)(H,131,159)(H,132,160)(H,133,165)(H,134,166)(H,135,153)(H,136,154)(H,137,155)(H,138,156)(H,139,161)(H,140,162)(H,141,163)(H,142,164)(H4,115,116,123)(H4,117,118,124)(H4,119,120,125)(H4,121,122,126)/t59-,60-,69-,70-,71-,72-,73-,74-,75-,76-,77-,78-,79-,80-,81?,82?,83-,84-,87-,88-/m0/s1 |
| Chemical Name | (3S,12S,18S,27S)-9-N,24-N-bis[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-amino-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]-3,18-bis[[(2S,3S)-2-amino-3-methylpentanoyl]amino]-2,6,11,17,21,26-hexaoxo-1,7,10,16,22,25-hexazatricyclo[25.3.0.012,16]triacontane-9,24-dicarboxamide |
| Synonyms | [125I]GR231118; [125I]GW1229; [125I]1229U91; GTPL1531 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | NPY Y4 receptor |
| ln Vitro | GR231118, BW1911U90, Bis(31/31')[[Cys31, Trp32, Nva34] neuropeptide Y(31-36)] (T-190) and [Trp-Arg-Nva-Arg-Tyr]2-NH2 (T-241) are peptide analogs of the C-terminus of neuropeptide Y that have recently been shown to be antagonists of the neuropeptide Y Y1 receptor. In this study, the activity of these peptides at each of the cloned neuropeptide Y receptor subtypes is determined in radioligand binding assays and in functional assays (inhibition of forskolin-stimulated cAMP formation). GR231118 is a potent antagonist at the human and rat neuropeptide Y Y1 receptors (pA2 = 10.5 and 10.0, respectively; pKi = 10.2 and 10.4, respectively), a potent agonist at the human neuropeptide Y Y4 receptor (pEC50 = 8.6; pKi = 9.6) and a weak agonist at the human and rat neuropeptide Y Y2 and Y5 receptors. GR231118 also has high affinity for the mouse neuropeptide Y Y6 receptor (pKi = 8.8). Therefore, GR231118 is a relatively selective neuropeptide Y Y1 receptor antagonist, but has appreciable activity at the neuropeptide Y Y4 and Y6 receptors as well. BW1911U90, T-190 and T-241 are moderately potent neuropeptide Y Y1 receptor antagonists (pA2 = 7.1, 5.8 and 6.5, respectively; pKi = 8.3, 6.5 and 6.8, respectively) and neuropeptide Y Y4 receptor agonists (pEC50 = 6.8, 6.3 and 6.6, respectively; pKi; 8.3, 7.7 and 8.3, respectively). These data suggest that the C-terminus of neuropeptide Y and related peptides is sufficient for activation of the neuropeptide Y Y4 receptor, but is not sufficient for activation of the neuropeptide Y Y1 receptor. Because BW1911U90, T-190 and T-241 are significantly less potent at the cloned human neuropeptide Y Y1 receptor than at the neuropeptide Y receptor in human erythroleukemia cells, these cells may express a novel neuropeptide Y receptor with high affinity for these peptides. [1] |
| ln Vivo | GR231118 (also known as 1229U91 and GW1229), a purported Y(1) antagonist and Y(4) agonist was radiolabelled using the chloramine T method. [(125)I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [(125)I]-GR231118 binds with very high affinity (K(d) of 0.09 - 0.24 nM) in transfected HEK293 cells with the rat Y(1) and Y(4) receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y(2) or Y(5) receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [(125)I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y(1), but not rat Y(4), receptor cDNA. Autoradiographic studies demonstrated that [(125)I]-GR231118 binding sites were fully inhibited by the Y(1) antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [(125)I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [(125)I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y(4) receptor subtype. In summary, [(125)I]-GR231118 is a new radiolabelled probe to investigate the Y(1) and Y(4) receptors; its major advantage being its high affinity. Using highly selective Y(1) antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [(125)I]-GR231118 to the Y(1) receptor allowing for the characterization and visualization of the purported Y(4) subtype. [2] |
| Enzyme Assay |
Binding assays [2] All binding assays were initiated by adding 100 μl of membrane preparations in a final volume of 500 μl of KRP containing 0.1% (w v−1) BSA, 0.05% (w v−1) bacitracin, [125I]-GR231118 and unlabelled peptide or competitor as needed. Time dependency was established using 25 pM [125I]-GR231118 at both 4°C and room temperature. Isotherm saturations and competition binding assays were performed at room temperature. Saturation experiments were performed in the presence of increasing concentrations of [125I]-GR231118 whereas competition binding experiments were performed in the presence of 25–30 pM [125I]-GR231118 and various competitors (pNPY, hPYY, [Leu31,Pro34]-pNPY, [Leu31,Pro34]-pPYY, pNPY2–36, pNPY13–36, hPYY3–36, hPYY13–36, rPP, hPP, GR231118, BIBO3304 and BIBP3226) at concentrations ranging from 10−13 M to 10−6 M. Non-specific binding was determined in the presence of 1 μM GR231118. After a 1 h incubation, the binding reaction was terminated by rapid filtration through Schleicher and Schuell #32 glass filters (previously soaked in 1.0% polyethyleneimine) using a cell harvester filtering apparatus. Filters were rinsed three times with 3 ml cold KRP and the radioactivity remaining on filters was quantified using a gamma counter with 85% efficiency. All binding experiments were repeated three to six times (each in triplicate), and results (mean±s.e.mean) expressed as percentage of specific binding or fmol mg−1 protein. All data obtained from the saturation isotherm experiments were subtracted for [125I]-GR231118 values found on filters in absence of membrane homogenates. Kd, Bmax and half time association values were calculated from data using the GraphPad Prism. IC50 values (i.e. concentration of unlabelled competitor required to compete for 50% of specific binding of the radioligand) for the various competitors were calculated using the GraphPad Prism. Radioligand binding assays [1] Binding of porcine peptide YY (2200 Ci/mmol) to the neuropeptide Y receptors expressed in CHO, COS1 or 293 cell membranes was performed in binding buffer (50 mM HEPES [pH 7.3], 0.1% bovine serum albumin, 2.5 mM CaCl2, 1 mM MgCl2). Saturation binding assays were performed in binding buffer containing 0.01–2 nM peptide YY and 2.5–5 μg of membrane protein (50 μl final volume). Competition binding assays were carried out in binding buffer containing 5–10 μg membrane protein, 0.1 nM peptide YY and various concentrations of competing peptides (200 μl final volume). In all cases, nonspecific binding was defined as binding in the presence of 1 μM unlabelled human neuropeptide Y. Binding assays were incubated at room temperature for 90 min and were terminated by rapid vacuum filtration through 0.3% polyethyleneimine pretreated glass fiber filters in a 96-well format). Each filter was then washed 3 times with 100 μl of phosphate buffered saline and subsequently counted in a gamma counter. All data were analyzed by nonlinear regression analysis. |
| Cell Assay |
Transfected cells [2] HEK 293 cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% foetal calf serum and antibiotics (penicillin G sodium, streptomycin sulphate and amphotericin B). Cultured cells were transfected with either of the rat Y1, Y2, Y4 or Y5 receptor cDNA using a calcium phosphate method (Tong et al., 1995). Briefly, 125 μl of 2.5 M calcium phosphate was added to 1.125 ml water containing 50 μg of either rat Y1, Y2, Y4 or Y5 receptor cDNA which was previously inserted in expressing pcDNA3 vectors and was slowly mixed with 1.25 ml 2× HEPES buffer at pH 7.05 and left at room temperature for 20 min. The mixture was added to a 150 mm dish containing HEK293 cells at 30% confluent and returned to the incubator. The medium was changed the next morning. Forty-eight h later, cells were washed with KRP buffer pH 7.4 and scratched. Detached cells were then centrifuged at 400×g for 10 min and the pellet washed with KRP buffer (pH 7.4), recentrifuged twice, and resuspended in 8 ml of KRP buffer pH 7.4 and used for receptor binding assay. cAMP assays [1] CHO or 293 cells expressing neuropeptide Y receptors were seeded into 96-well, flat-bottom tissue culture plates at a density of 20 000 cells per well. After approximately 48 h, the cell monolayers were rinsed twice with Hank's balanced salt solution, then preincubated for 10 min at 37°C with assay buffer (Hank's balanced salt solution supplemented with 4 mM MgCl2, 10 mM HEPES [pH 7.4], 0.2% bovine serum albumin, 1 mM 3-isobutyl-1-methylxanthine). When the antagonist effects of peptides were being tested, the peptide antagonists were included in this 10 min preincubation. Subsequently, the assay buffer was removed and replaced with assay buffer containing 1 μM forskolin and various concentrations of peptides. After 10 min at 37°C, the medium was removed and ethanol was added to the cell monolayers. The tissue culture plates were agitated on a platform shaker for 15 min and the plates were then transferred to a warm water bath to evaporate the ethanol. The cell residues were dissolved in FlashPlate assay buffer and the amount of cAMP in each well was quantified using the cAMP FlashPlate kit according to the manufacturer's protocol. To calculate EC50 values for agonists, all data were analyzed by nonlinear regression analysis |
| Animal Protocol |
Quantitative receptor autoradiography [2] Receptor autoradiography was performed as described in details elsewhere (Dumont et al., 1996a; 1998a). Briefly, rats were sacrificed by decapitation, and their brains rapidly removed from the skull, frozen in 2-methylbutane at −40°C for 15 s, and then kept at −80°C until needed. Sections (20 μM) were obtained using a cryomicrotome at −17°C, mounted on gelatin-chrome-alum-coated slides, dried overnight in a desiccator at 4°C, and then kept at −80°C until use. On the days of the experiments, adjacent coronal sections were preincubated for 60 min at room temperature in KRP buffer at pH 7.4 and then incubated for 60 min in a fresh preparation of KRP buffer containing 0.1% BSA, 0.05% bacitracin, 25 pM [125I]-GR231118 in the presence and absence of 100 nM BIBO3304 (Y1 antagonist), hPP(Y4/Y5) or GR231118. Following a 1 h incubation, sections were washed four times, 2 min each in ice-cold KRP buffer then dipped in deionized water to remove salts and rapidly dried. Non-specific binding was determined using 100 nM GR231118. Incubated sections were apposed against 3H-Hyperfilms for 4 days alongside radioactive standards. Films were developed and quantified as described in details elsewhere (Dumont et al., 1996a; 1998a). |
| References |
[1]. GR231118 (1229U91) and other analogues of the C-terminus of neuropeptide Y are potent neuropeptide Y Y1 receptor antagonists and neuropeptide Y Y4 receptor agonists. Eur J Pharmacol. [2]. [(125)I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y(1) and Y(4) receptors. Br J Pharmacol. 2000 Jan;129(1):37-46. |
| Additional Infomation |
In summary, our results demonstrated that [125I]-GR231118 binds with very high affinity to rat brain homogenates, rapidly reaching equilibrium at room temperature. Isotherm saturation experiments revealed that [125I]-GR231118 binds with very high affinity to the Y1 and Y4 receptors while it is basically inactive at the Y2 and Y5 subtypes transfected and expressed in HEK293 cells. Additionally, in the presence of a selective Y1 antagonist, it is possible to discriminate between the Y1 and Y4 subtypes in tissues expressing both receptors such as the rat brain. Hence, [125I]-GR231118 should prove most useful to investigate in detail the respective characteristics of these two NPY receptor subtypes in a variety of tissues.[2] The results demonstrated that [125I]-GR231118 has very high affinity (Kd in the sub-nM range) for the Y1 and Y4 receptor subtypes while being mostly inactive on the Y2 and Y5 receptors. In fact, [125I]-GR231118 is one of the highest affinity radioligand developed thus far to target the Y1 receptor subtype including [125I]-[Leu31,Pro34]-PYY (Dumont et al., 1995) and [3H]-BIBP3226 (Entzeroth et al., 1995). These two radioligands have at least a 10 fold lower affinity for the Y1 receptor compared to [125I]-GR231118. This new radioligand also has very low non-specific binding, this being a major advantage for both homogenate binding assays and receptor autoradiography. Moreover, despite its relatively poor selectivity for the Y1 vs Y4 receptor subtypes, [125I]-GR231118 in the presence of a saturating concentration of a highly selective Y1 antagonist such as BIBO3304, allows for the investigation of the Y4 receptor without interference from the Y5 subtype, in contrast to PP-related radioligands (Gerald et al., 1996; Hu et al., 1996). Hence, [125I]-GR231118 should become a most useful probe to investigate both the Y1 (as a peptide antagonist ligand) and Y4 (as a peptide agonist ligand) receptors in mammalian tissues.[2] GR231118 has been reported to be a potent neuropeptide Y Y1 receptor antagonist with little activity at the neuropeptide Y Y2 receptor (Hegde et al., 1995; Daniels et al., 1995). Because of its very high potency and selectivity, GR231118 has been used to determine the role of the neuropeptide Y Y1 receptor in mediating various physiological effects of neuropeptide Y (Kanatani et al., 1996; Hegde et al., 1995; Lew et al., 1996). The present study demonstrates that GR231118 is not only a potent neuropeptide Y Y1 receptor antagonist, but is also a potent neuropeptide Y Y4 receptor agonist. GR231118 also has high affinity for the mouse neuropeptide Y Y6 receptor, although its functional activity at this receptor has not been determined. GR231118 has weak agonist activity at the neuropeptide Y Y2 and Y5 receptors. GR231118 is 20- to 60-fold more potent in blocking the neuropeptide Y Y1 receptor than in activating the neuropeptide Y Y4 receptor or in binding to the neuropeptide Y Y6 receptor. Thus, GR231118 can be considered to be a relatively selective neuropeptide Y Y1 receptor antagonist in experiments where its concentration can be precisely defined (e.g., isolated tissue experiments). However, the concentration of GR231118 cannot always be precisely defined, particularly in studies in which GR231118 is administered directly into the central nervous system (e.g., Kanatani et al., 1996). Interpretation of data obtained in such studies requires that the activity of GR231118 at the neuropeptide Y Y1, Y4, and Y6 receptors be taken into consideration. Because the physiological roles of the neuropeptide Y Y4 and Y6 receptors have not yet been determined, it is not clear how much the activity of GR231118 at these receptors detracts from its utility as a neuropeptide Y Y1 receptor antagonist. In practice, neuropeptide Y Y1 receptor antagonism may be distinguished from neuropeptide Y Y4 receptor agonism by determining if GR231118 mimics or antagonizes the effects of neuropeptide Y. [1] |
Solubility Data
| Solubility (In Vitro) | May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples |
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples. Injection Formulations (e.g. IP/IV/IM/SC) Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] *Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline) Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline) Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline) Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil) Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Oral Formulations Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). Oral Formulation 3: Dissolved in PEG400 Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose Oral Formulation 6: Mixing with food powders Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.4250 mL | 2.1252 mL | 4.2504 mL | |
| 5 mM | 0.0850 mL | 0.4250 mL | 0.8501 mL | |
| 10 mM | 0.0425 mL | 0.2125 mL | 0.4250 mL |