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Purity: =99.57%
GSK699 (GSK-699) is a novel, potent and cell permeable PROTAC degrader targeting the PCAF/GCN5 proteins for degradation and has anti-inflammatory activities. P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain.
| Targets |
PCAF/GCN5
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|---|---|
| ln Vitro |
GSK699 induced robust down-regulation of PCAF protein levels (more than 90%) in macrophages and DCs (Supporting Information Figure 2c and d). Multiplex analysis of cytokine levels in the supernatants of LPS-stimulated cells revealed a marked reduction of IL-6, IL-12p70, IL-10, IL-1β, and IFN-γ production in both macrophages and DCs following treatment with GSK699 at 100 nM (minimum concentration required to induce nearly complete PCAF degradation; Figure 2e,f and Supporting Information Figure 2c and d). TNF and IL-8 were also profoundly inhibited in GSK699-treated DCs compared to control cells, while these two cytokines were unchanged in macrophages (Figures 2e and 2f). Although the control compound GSK702 had no inhibitory effect in DCs, it did cause a small but significant reduction of IL-1β in macrophages, possibly due to some weak PCAF/GCN5 degradation in this cell type or a mild contribution of the CRBN-binding moiety to the observed anti-inflammatory phenotype (Figure 2e). Altogether, these results demonstrate that, unlike PCAF/GCN5 bromodomain inhibition, PROTAC-mediated degradation of these proteins markedly impairs the ability of macrophages and DCs to respond to LPS, with a consequent reduction in the production of numerous inflammatory cytokines.
Given the profound effects of PROTAC-induced PCAF/GCN5 degradation in DCs, we set out to further explore the biological potential of GSK699 in this cell type by evaluating its effects on the expression of a broader panel of inflammatory molecules using a cytokine and chemokine protein array, under the same experimental conditions described above. The results confirmed the inhibitory effect of GSK699 treatment on IL-6, TNF, and IL-12p70 and demonstrated reduced production of numerous other inflammatory mediators in GSK699-treated cells, including CXCL1/GROα, CXCL11/I-TAC, CCL7, CCL20/MIP-3α, and CCL19/MIP-3β (Figure 3a and Supporting Information Table 1)[1]. |
| Cell Assay |
Western Blot Cell pellets were resuspended in Pierce RIPA buffer containing protease inhibitors and 0.1% benzonase and incubated on ice for 30 minutes. Tubes were centrifuged at 16,000 rcf for 15 minutes at 4°C and supernatants were transferred to new tubes. Total protein concentrations were determined by Pierce BCA Protein Assay kit. 30 µg of total protein were loaded on 4-12% NuPAGE gels and transferred onto Odyssey nitrocellulose membranes. 12 Membranes were blocked with Odyssey Blocking Buffer for 1 hour before an overnight incubation with the indicated primary antibodies. After incubation with the appropriate IRDye secondary antibodies, the bands were visualised using an Odyssey scanner. The intensity of the bands was quantified using Image Studio Lite v5.2 software[1].
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| Animal Protocol |
The front and hind legs of nine different C57BL/6 WT or PCAF-/-1 mice were collected in phosphate buffered saline (PBS) and placed in petri dishes. The foot was removed and the knee joint cut through. The ends of the bones were cut off with a scalpel and the bone marrow was flushed through using a syringe filled with PBS. Cells were filtered through a 70 µm nylon cell strainer and counted. Cells were resuspended at a density of 0.5 x 106 cells/mL in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin, 2mM L-glutamine, 1X NonEssential Amino Acids, 50 µM β-mercaptoethanol, 1 mM Pyruvate, supplemented with 5 ng/mL mouse M-CSF and 5 ng/ml mouse IL-3. 10 mL of cells/dish were seeded into petri dishes and incubated for 24 hours at 13 37°C, 5% CO2. The following day, cell suspensions were transferred to fresh petri dishes to remove contaminating fibroblasts, and incubated for a further 6 days. Following differentiation, the macrophages were detached using PBS with 5 mM EDTA and 2% bovine serum albumin for 15 minutes and re-seeded in fresh medium at 2 x 105 cell/200 µl/well into 96-well plates, with 3 wells per mouse. Cells were stimulated with 10 ng/mL LPS from E. coli (0111:B4) for 6 hours, at which point plates were spun down at 400 rcf for 10 minutes, supernatants were transferred to new 96-well plates and stored at -80°C until analysis. To test the inhibitory effects of the PCAF/GCN5 bromodomain inhibitor GSK4027 in mouse macrophages, bone marrow cells were prepared from the legs of six different C57BL/10 naïve mice as described above and differentiated into macrophages in the presence of compound. At the end of the differentiation period, macrophages were seeded in 96-well plates, with 7 wells per compound concentration. Plates were incubated overnight at 37°C, 5% CO2 and then stimulated with 100 ng/mL LPS from E. coli for 6 hours. Plates were centrifuged at 400 rcf for 10 minutes and supernatants were transferred to a new plate for MSD analysis.[1]
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| References | |
| Additional Infomation |
P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain. Consistent with reported roles for these proteins in immune function, we find that PCAF-deficient macrophages exhibit a markedly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). Investigating the potential to target this pathway pharmacologically, we show that chemical inhibition of the PCAF/GCN5 bromodomains is insufficient to recapitulate the diminished inflammatory response of PCAF-deficient immune cells. However, by generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), we identify small molecules able to degrade PCAF/GCN5 and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data illustrate the power of the PROTAC approach in the context of multidomain proteins, revealing a novel anti-inflammatory therapeutic opportunity for targeting PCAF/GCN5.[1]
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| Molecular Formula |
C45H51BRN8O7
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|---|---|
| Molecular Weight |
895.856
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| Exact Mass |
894.306
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| Elemental Analysis |
C, 60.33; H, 5.74; Br, 8.92; N, 12.51; O, 12.50
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| CAS # |
2260944-68-9
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| PubChem CID |
146680948
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| Appearance |
Solid Powder
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| Density |
1.43±0.1 g/cm3(Predicted)
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| LogP |
4.7
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
11
|
| Rotatable Bond Count |
15
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| Heavy Atom Count |
61
|
| Complexity |
1710
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| Defined Atom Stereocenter Count |
2
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| SMILES |
CN1C[C@H](C[C@H](C1)NC2=C(C(=O)N(N=C2)C)Br)C3=CC=C(C=C3)C(=O)N(C)CCCN(C)C4=CC=C(C=C4)CCCOC5=CC=CC6=C5C(=O)N(C6=O)C7CCC(=O)NC7=O
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| InChi Key |
SARLMRHJAJBYBI-CIDUPMPKSA-N
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| InChi Code |
InChI=1S/C45H51BrN8O7/c1-50-26-31(24-32(27-50)48-35-25-47-53(4)45(60)40(35)46)29-13-15-30(16-14-29)42(57)52(3)22-7-21-51(2)33-17-11-28(12-18-33)8-6-23-61-37-10-5-9-34-39(37)44(59)54(43(34)58)36-19-20-38(55)49-41(36)56/h5,9-18,25,31-32,36,48H,6-8,19-24,26-27H2,1-4H3,(H,49,55,56)/t31-,32+,36?/m0/s1
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| Chemical Name |
4-((3R,5R)-5-((5-bromo-1-methyl-6-oxo-1,6-dihydropyridazin-4-yl)amino)-1-methylpiperidin-3-yl)-N-(3-((4-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)propyl)phenyl)(methyl)amino)propyl)-N-methylbenzamide
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| Synonyms |
GSK-699; GSK699; GSK699; 2260944-68-9; GSK-699; GSK 699; CHEMBL4462377; BCP31920; AKOS040751967; PD128097; 4-[(3R,5R)-5-[(5-bromo-1-methyl-6-oxopyridazin-4-yl)amino]-1-methylpiperidin-3-yl]-N-[3-[4-[3-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]oxypropyl]-N-methylanilino]propyl]-N-methylbenzamide; GSK 699
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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 Note: This product is not stable in solution, please use freshly prepared working solution for optimal results. |
| 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) |
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
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|---|---|
| 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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.1162 mL | 5.5812 mL | 11.1625 mL | |
| 5 mM | 0.2232 mL | 1.1162 mL | 2.2325 mL | |
| 10 mM | 0.1116 mL | 0.5581 mL | 1.1162 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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