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Purity: ≥98%
GW 6471 (GW6471; GW-6471) is a novel and potent PPARα antagonist with an IC50 of 0.24 μ M. GW6471 can enhance the binding affinity of the PPAR α ligand-binding domain to the co-repressor proteins SMRT and NCoR.
| Targets |
PPARα
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|---|---|
| ln Vitro |
GW6471 significantly reduced GW409544-induced PPARα activation in a cell-based gene reporter test, with an IC50 of 0.24 μM [1]. Using the MTT assay, the functional impact of PPARα on the viability of renal cell carcinoma (RCC) cells was evaluated. For a duration of 72 hours, Caki-1 (VHL wild-type) and 786-O (VHL mutant) cells were sterilely treated with either the specific PPARα-selective antagonist GW6471 or the specific PPARα agonist WY14,643 at doses ranging from 12.5 to 100 μM. The vitality of the cells was then evaluated. WY14,643 did not affect cell viability or cause a gradual increase in it, but GW6471 dramatically reduced cell viability in both cell lines in a foot-dependent manner, up to almost 80% [2].
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| ln Vivo |
In vivo anti-activity of PPARα antagonist was evaluated using a tumor subcutaneous xenograft model. Nude mice (Nu/Nu) were given subcutaneous injections of Caki-1 cells. The medication GW6471 was given intraperitoneally every day for four weeks at a dose of 20 mg/kg mouse body weight, which was found to be effective in an in vivo dose-response study and validated here, once the tumor mass had grown to a diameter of approximately 5 mm. The tumor growth of the animals treated with GW6471 and those treated with vehicle differed significantly. The animals' body weight indicated that there was no toxicity at the GW6471 dosage, and laboratory results, such as liver and renal function tests, showed no negative effects. c-Myc levels in tumors were assessed to illustrate GW6471's inhibitory effect, and results indicated a significant decrease in tumors in animals treated with GW6471 [3].
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| Enzyme Assay |
Binding assays[1]
The effects of GW6471 on the interaction of coactivator and co-repressor peptides with PPARα were determined by chemical-mediated fluorescence energy transfer assays using the AlphaScreen Technology from Packard BioScience30. The experiments were conducted with 5 nM PPARα LBD of biotinylated peptide containing individual motifs (Fig. 3a), following the manufacturer's instructions for the hexahistidine detection kit in a buffer containing 50 mM MOPS, pH 7.4, 50 mM NaF, 0.05 mM CHAPS, 0.1 mg ml-1 bovine serum albumin, and 10 mM dithiothreitol (DTT). The binding signals were detected with the increasing concentrations of GW6471, and the results from four repeated experiments were normalized as a percentage of the binding in the absence of GW6471. The effects of GW6471 on the affinity of the SMRT or N-CoR peptides with purified PPARα LBD were determined by fluorescence polarization in a buffer containing 10 mM HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate-20, 5 mM DTT and 2.5% DMSO. Varied concentration of PPARα LBD in the presence or absence of 40 µM GW6471 were incubated at room temperature with 10 nM of a fluorescein-labelled peptide of N-CoR2 or SMRT2 (Fig. 3a). The fluorescence polarization values for each concentration of receptor were determined using a BMG PolarStar Galaxy fluorescence reader with 485 nm excitation and 520 nm emission filters. The apparent dissociation constant (Kd) values were determined by the binding curves derived from a nonlinear least-squares-fit of the data for a simple 1:1 interaction. Mutational analysis of the SMRT co-repressor motif interaction with the PPARα and TRβ LBDs was also performed by fluorescence polarization. To determine the importance of each amino acid in the SMRT motif for binding to nuclear receptors, SMRT peptides with alanine substitution at each position were added to inhibit the binding of 1 µM TRβ LBD or 2 µM PPARα to the fluorescent N-CoR2 peptide. For the PPARα experiments we added 10 µM GW6471. The inhibition curves were constructed and IC50 values were determined by nonlinear least-squares-fit of the data to a simple 1:1 interaction. |
| Cell Assay |
Cell-based assays for GW6471 as an antagonist were performed in CV-1 cells using the (UAS)5-tk-SPAP reporter and the human PPARα–GAL4 chimaeras as described previously11. We carried out transfections using lipofectamine with a β-galactosidase vector as a normalization control. The mammalian two-hybrid assays were performed according the published procedures29. Transfection mixes included 8 ng (UAS)5-tk-CAT reporter plasmid, 8 ng VP16-human PPARα expression plasmid, 2 ng of either GAL4–N-CoR or GAL4–SMRT expression plasmid, 25 ng β-galactosidase expression plasmid as internal control, and 35 ng carrier plasmid. The synthesis of GW6471 and GW409544 will be described elsewhere.[1]
Renal cell carcinoma (RCC) is the sixth most common cancer in the US. While RCC is highly metastatic, there are few therapeutics options available for patients with metastatic RCC, and progression-free survival of patients even with the newest targeted therapeutics is only up to two years. Thus, novel therapeutic targets for this disease are desperately needed. Based on our previous metabolomics studies showing alteration of peroxisome proliferator-activated receptor α (PPARα) related events in both RCC patient and xenograft mice materials, this pathway was further examined in the current study in the setting of RCC. PPARα is a nuclear receptor protein that functions as a transcription factor for genes including those encoding enzymes involved in energy metabolism; while PPARα has been reported to regulate tumor growth in several cancers, it has not been evaluated in RCC. A specific PPARα antagonist, GW6471, induced both apoptosis and cell cycle arrest at G0/G1 in VHL(+) and VHL(-) RCC cell lines (786-O and Caki-1) associated with attenuation of the cell cycle regulatory proteins c-Myc, Cyclin D1, and CDK4; this data was confirmed as specific to PPARα antagonism by siRNA methods. Interestingly, when glycolysis was blocked by several methods, the cytotoxicity of GW6471 was synergistically increased, suggesting a switch to fatty acid oxidation from glycolysis and providing an entirely novel therapeutic approach for RCC.[2] |
| Animal Protocol |
All animal procedures were performed in compliance with the University of California Animal Care and Use Committee. Male athymic Nu/Nu mice (8 wk of age, ∼25 g body wt) were injected with 1 × 105 Caki-1 cells subcutaneously (3:1 DMEM-Matrigel) in the flank region. Tumor progression was monitored weekly by calipers using the following formula: tumor volume (in mm3) = (length × width2)/2. When tumor size reached ∼80–100 mm3, animals were randomly assigned to four groups and treatments were started (day 1). The vehicle group received DMSO (4% in PBS) intraperitoneally and vegetable oil via oral gavage. The PPARα group was injected intraperitoneally with GW6471 in the same vehicle [20 mg/kg body wt; murine dose response is reported elsewhere] every other day. The sunitinib group received sunitinib in vegetable oil via oral gavage (40 mg/kg body wt) 5 days/wk. Another group received GW6471 + sunitinib as described above. To determine any potential toxicity of the treatment(s), body weights of the animals were measured and signs of adverse reactions were monitored. On day 28, the mice were euthanized and the tumor mass was determined. Tumor growth rate was calculated as follows: tumor volume on day x/tumor volume on day 1. Serum samples collected from mice at the end of the experiment were analyzed using the Roche cobas c501 analyzer (6000 series) at Clinical Diagnostic Laboratories in the University of California, Davis, using the Small Animal Chem 2 panel. Frozen tumor tissues were also collected at the end of the experiment and homogenized and extracted in T-PER for c-Myc quantification by Western blotting.[3]
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| References |
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| Molecular Formula |
C35H36F3N3O4
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|---|---|
| Molecular Weight |
619.6733
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| Exact Mass |
619.265
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| Elemental Analysis |
C, 67.84; H, 5.86; F, 9.20; N, 6.78; O, 10.33
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| CAS # |
880635-03-0
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| PubChem CID |
446738
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| Appearance |
Light yellow to yellow solid
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| Density |
1.2±0.1 g/cm3
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| Index of Refraction |
1.556
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| LogP |
7.24
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
14
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| Heavy Atom Count |
45
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| Complexity |
954
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C(C1=C(C)OC(C2C=CC=CC=2)=N1)COC1C=CC(C[C@@H](CNC(=O)CC)N/C(/C)=C\C(C2C=CC(C(F)(F)F)=CC=2)=O)=CC=1
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| InChi Key |
TYEFSRMOUXWTDN-DYQICHDWSA-N
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| InChi Code |
InChI=1S/C35H36F3N3O4/c1-4-33(43)39-22-29(40-23(2)20-32(42)26-12-14-28(15-13-26)35(36,37)38)21-25-10-16-30(17-11-25)44-19-18-31-24(3)45-34(41-31)27-8-6-5-7-9-27/h5-17,20,29,40H,4,18-19,21-22H2,1-3H3,(H,39,43)/b23-20-/t29-/m0/s1
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| Chemical Name |
(S,Z)-N-(3-(4-(2-(5-methyl-2-phenyloxazol-4-yl)ethoxy)phenyl)-2-((4-oxo-4-(4-(trifluoromethyl)phenyl)but-2-en-2-yl)amino)propyl)propionamide
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| Synonyms |
GW6471; GW-6471; GW 6471
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| HS Tariff Code |
2934.99.9001
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| 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)
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| Solubility (In Vitro) |
DMSO : ~83.33 mg/mL (~134.47 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (3.36 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: 2.08 mg/mL (3.36 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. View More
Solubility in Formulation 3: ≥ 2.08 mg/mL (3.36 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6138 mL | 8.0688 mL | 16.1376 mL | |
| 5 mM | 0.3228 mL | 1.6138 mL | 3.2275 mL | |
| 10 mM | 0.1614 mL | 0.8069 mL | 1.6138 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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