PCAF/GCN5 (minimum concentration required to induce nearly complete PCAF degradation: 100 nM)
KAT2A (also known as GCN5) and its paralogue KAT2B (PCAF). [2]
The molecular targets of GSK-699 are the PCAF and GCN5 proteins. These closely related epigenetic regulators each contain an acetyltransferase domain and a bromodomain, serving as core components of the SAGA transcriptional coactivator complex. GSK-699 induces K48-linked polyubiquitination by bringing PCAF/GCN5 into proximity with the E3 ubiquitin ligase CRBN, leading to proteasomal degradation of the target proteins. Studies confirm that GSK-699 also degrades KAT2A and KAT2B (alternative names for PCAF and GCN5, respectively), modulates the histone acetyltransferase activity of the SAGA complex, and reduces histone H3K9ac levels.

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].

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- In wild-type HAP1 Cas9 cells expressing a KAT2A-BFP-P2A-mCherry stability reporter, treatment with 100 nM GSK-699 for 6 hours resulted in a strong reduction in KAT2A-BFP signal (mean fluorescence intensity reduced from 1.0 ± 0.007 (DMSO) to 0.28 ± 0.015 (GSK-699), mean ± s.d.), with no change in mCherry fluorescence, confirming specific degradation of KAT2A. [2]
- In MOLM-13 (acute myeloid leukemia) and NALM6 (acute lymphoblastic leukemia) cells expressing the same KAT2A stability reporter, GSK-699 treatment (100 nM, 6 h) substantially reduced KAT2A-BFP abundance with no change in mCherry levels. [2]
- In immunofluorescence experiments, treatment of wild-type HAP1 Cas9 cells with GSK-699 (100 nM, 6 h) reduced nuclear KAT2A fluorescence to a level comparable to that achieved by genetic knockout of KAT2A. [2]

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In vitro, GSK-699 induces robust down-regulation of PCAF protein levels (over 90% degradation) in macrophages and dendritic cells. A concentration of 100 nM is sufficient to induce nearly complete PCAF degradation. In LPS-stimulated cells, GSK-699 treatment significantly reduces the production of inflammatory cytokines, including IL-6, IL-12p70, IL-10, IL-1β, IFN-γ, TNF, and IL-8. Cytokine/chemokine protein array analysis further confirms that GSK-699 also inhibits numerous other inflammatory mediators, including CXCL1/GROα, CXCL11/I-TAC, CCL7, CCL20/MIP-3α, and CCL19/MIP-3β. Additionally, GSK-699 inhibits the growth of neuroblastoma, acute myeloid leukemia, and small cell lung cancer cells. In neuroblastoma cell lines, treatment with 100 nM GSK-699 for 72 hours significantly inhibits cell proliferation and reduces EdU incorporation rates.

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- In three MYCN-amplified neuroblastoma cell lines (KELLY, NB1, NGP), treatment with GSK-699-1 (not the inactive enantiomer GSK-699-2) resulted in loss of both KAT2A and KAT2B expression and subsequent reduction of H3K9ac, consistent with loss of SAGA KAT activity. Additionally, decreased MYCN protein expression was observed. [3]
- GSK-699-1 is effective with low-nanomolar potency (exact value not specified, see supplementary figures S7A and S7B). [3]
- GSK-699-1 significantly reduced neuroblastoma growth over prolonged treatment in KELLY, NB1, and NGP cells. [3]
- In three non-MYCN-amplified cell lines, GSK-699-1 treatment showed heterogeneous responses; sensitivity was associated with significantly reduced MYC expression. [3]
- Reduced growth with GSK-699-1 in MYCN-amplified models was predominantly through G1-G0 cell cycle arrest (assessed by EdU and propidium iodide incorporation assay). [3]
- In KELLY cells, treatment with 100 nM GSK-699-1 for 6, 24, or 72 hours led to time-dependent alterations in gene expression as measured by RNA-seq. [3]
- In KELLY cells treated with 100 nM GSK-699-1 for 6 hours, ChIP-seq revealed a reduction of H3K9ac deposition genome-wide and at TADA2B-bound sites, as well as decreased genome-wide MYCN binding. [3]

In vivo, GSK-699 demonstrates antitumor activity in a neuroblastoma xenograft model. In a subcutaneous KELLY cell xenograft nude mouse model, daily intraperitoneal administration of GSK-699 (50 mg/kg) significantly inhibited tumor growth over 18 days. Tumor volumes in the treatment group were markedly smaller than those in the vehicle control group. Kaplan-Meier survival curve analysis showed significantly prolonged overall survival in the GSK-699 treatment group. Pharmacodynamic studies revealed that after 5 days of GSK-699 treatment (5, 10, 50, or 100 mg/kg, daily intraperitoneal injection), expression of KAT2A, KAT2B, MYCN, and H3K9ac in KELLY xenograft tumors decreased in a dose-dependent manner. These results confirm that GSK-699 effectively degrades target proteins and exerts antitumor effects in vivo. [3]

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- Dose-finding study in KELLY xenograft model: NSG mice bearing subcutaneous KELLY tumors were treated with GSK-699-1 at doses of 5, 10, 50, or 100 mg/kg injected intraperitoneally once daily for 4 days. At all doses, consistent degradation of KAT2A/KAT2B was observed in tumor tissues 6 hours after the last dose, associated with reduced MYCN protein expression and reduced H3K9ac (approximately 50% reduction compared to vehicle control). The 50 mg/kg dose was selected for efficacy studies due to adequate degradation, no adverse effects, and good solubility. [3]
- Efficacy study in KELLY xenograft model: NSG mice bearing established KELLY xenografts (tumor volume ~50-100 mm³) were randomized to receive either vehicle or GSK-699-1 (50 mg/kg once daily intraperitoneal injection for 21 days). GSK-699-1 treatment significantly slowed tumor progression (P = 0.01 on day 16, P = 0.07 on day 18 by two-sided Student’s t-test) and increased end-point survival (log-rank Mantel-Cox test, P = 0.0134). Treatment was well tolerated as assessed by mouse weights, with no significant weight loss. Decreased KAT2A/KAT2B expression was maintained at tumor endpoint in the treatment group. [3]

As a PROTAC molecule, GSK-699 requires an intact intracellular ubiquitin-proteasome system for its mechanism of action, making cell-free direct binding assays less common. For assessing binding affinity to CRBN or PCAF/GCN5, the following procedure can be used: Dissolve GSK-699 in DMSO to prepare a stock solution (e.g., 10 mM). In a 96-well plate, dilute the compound to desired concentrations (e.g., 0.1-1000 nM) in binding buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 0.1% BSA, 0.01% Tween-20). Incubate with recombinant CRBN-DDB1 complex or recombinant GST-tagged PCAF/GCN5 protein for 30-60 minutes at room temperature. Measure binding affinity (Kd value) using fluorescence polarization (FP) or surface plasmon resonance (SPR). Note: The bifunctional nature of PROTACs may result in lower affinities to each individual target (target protein and E3 ligase) compared to conventional inhibitors.

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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- Stability reporter assay in HAP1 cells: Wild-type HAP1 Cas9 cells expressing a KAT2A-BFP-P2A-mCherry reporter were treated with 100 nM GSK-699 for 6 hours. Cells were then analyzed by flow cytometry. BFP and mCherry mean fluorescence intensities were measured. The KAT2A-BFP/mCherry ratio was calculated to determine relative KAT2A abundance. DMSO-treated cells served as controls. GSK-699 treatment led to a >70% reduction in KAT2A-BFP signal compared to DMSO. [2]
- Stability reporter assay in leukemia cells: MOLM-13 and NALM6 cells stably expressing Cas9 and the KAT2A-BFP-P2A-mCherry reporter were treated with 100 nM GSK-699 for 6 hours. Flow cytometry was used to measure BFP and mCherry fluorescence in eGFP-positive (transduced) cells. GSK-699 treatment substantially reduced KAT2A-BFP abundance without altering mCherry levels. [2]
- Immunofluorescence assay: Wild-type HAP1 Cas9 cells were treated with 100 nM GSK-699 for 6 hours, then fixed and stained for KAT2A and DNA. Nuclear KAT2A fluorescence was quantified by confocal microscopy. GSK-699 treatment reduced nuclear KAT2A fluorescence to a level comparable to KAT2A knockout, demonstrating sensitivity of the assay. [2]

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In vitro cell assay protocol for GSK-699 (using PCAF/GCN5 degradation assessment as an example): 1) Seed cells (e.g., THP-1 monocytes, macrophages, or dendritic cells) in 6-well plates (5×10⁵-1×10⁶ cells per well) and incubate overnight at 37°C with 5% CO₂. 2) Prepare serial dilutions of GSK-699 in complete culture medium (e.g., 10, 30, 100, 300, 1000 nM). 3) Replace medium with compound-containing medium and treat cells for 4-24 hours (optimal time typically 4-6 hours). 4) Harvest cells, lyse, and extract total protein. 5) Detect PCAF or GCN5 protein levels by Western blot, using GAPDH or Histone H3 as loading controls. Calculate DC50 (concentration required for 50% protein degradation). 6) Alternatively, assess cell viability by CCK-8 assay after 72 hours of treatment, or measure apoptosis rate by flow cytometry after 24-48 hours of treatment.

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- Western blotting for protein expression: Neuroblastoma cell lines (KELLY, NB1, NGP) were treated with vehicle, GSK-699-1, or the inactive enantiomer GSK-699-2 for 24 hours. Cells were lysed in cell lysis buffer supplemented with protease and phosphatase inhibitors. Lysates were quantified by BCA assay, normalized, and resolved by SDS-PAGE. Proteins were transferred to PVDF membranes and probed with antibodies against KAT2A, KAT2B, MYCN, H3K9ac, H3K27ac, GAPDH (loading control), and total histone H3 (loading control). Membranes were incubated with secondary antibodies and imaged. GSK-699-1 treatment caused loss of KAT2A, KAT2B, and MYCN expression and reduced H3K9ac. [3]
- Cell viability assay (prolonged treatment): Neuroblastoma cell lines were treated with DMSO, 100 nM GSK-699-1, or 100 nM GSK-699-2 for up to 8 days. Relative viability was assessed compared to day 0. GSK-699-1 significantly reduced neuroblastoma growth over prolonged treatment. [3]
- Cell cycle analysis (EdU and propidium iodide incorporation): Neuroblastoma cell lines were plated and treated with DMSO or 100 nM GSK-699-1 for 72 hours. Cells were incubated with 10 µM EdU for 90 minutes. After harvesting, cells were fixed, stained, and subjected to RNase A digestion and PI staining. Samples were analyzed by flow cytometry. GSK-699-1 treatment led to G1-G0 cell cycle arrest. [3]
- RNA sequencing: KELLY cells were treated with DMSO or 100 nM GSK-699-1 for 6, 24, or 72 hours (biological triplicates). Total RNA was collected, homogenized, and purified. RNA library preparation and transcriptome sequencing were performed. Data were analyzed for differential gene expression (DESeq2 adjusted P ≤ 0.10, |fold change| ≥ 1.5). GSEA showed that down-regulated genes were enriched for MYC, E2F/cell cycle, and MYCN-related gene signatures across all time points. [3]
- ChIP-sequencing: KELLY cells were treated with DMSO or 100 nM GSK-699-1 for 6 hours. Cells were cross-linked with formaldehyde, lysed, and chromatin sheared. Chromatin was immunoprecipitated with antibodies against H3K9ac, H3K27ac, or MYCN, using spike-in chromatin for normalization. DNA was purified and libraries were prepared for sequencing. GSK-699-1 treatment reduced H3K9ac and H3K27ac deposition genome-wide and decreased MYCN binding on chromatin. [3]

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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In vivo animal assay protocol for GSK-699 (using a xenograft tumor model as an example): 1) Use 4-5 week old female nude mice and subcutaneously inoculate tumor cells (e.g., KELLY neuroblastoma cells, 5×10⁶ cells/mouse) in the right axilla. 2) When tumor volumes reach approximately 150-200 mm³, randomize animals into treatment and control groups (8-10 mice per group). 3) Formulate GSK-699 in vehicle containing 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% saline. 4) Administer compound by intraperitoneal injection (typical doses: 10, 50, or 100 mg/kg, once daily). 5) Administer vehicle control to control group. 6) Measure tumor volume (length × width²/2) and body weight 2-3 times per week; observe general animal condition daily. 7) Euthanize animals at the end of treatment (typically days 18-21), collect tumor tissues for Western blot analysis of target protein degradation, and collect major organs for histopathological examination. 8) Generate tumor growth curves and Kaplan-Meier survival curves.

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- GSK-699-1 dose-finding study: KELLY cells (3 × 10⁶) were injected subcutaneously into NSG mice. Once tumors reached ~50-100 mm³, mice were randomized to receive GSK-699-1 at doses of 5, 10, 50, or 100 mg/kg or vehicle, administered by intraperitoneal injection once daily. GSK-699-1 powder was resuspended in 10% DMSO in sterile phosphate-buffered saline. Mice were treated for 4 days and euthanized 6 hours after the last dose for downstream analyses (Western blotting). [3]
- GSK-699-1 efficacy study: KELLY cells (3 × 10⁶) were injected subcutaneously into NSG mice. Once tumors reached ~50-100 mm³, mice were randomized into vehicle (n=9) or GSK-699-1 (50 mg/kg once daily intraperitoneal injection, n=9) cohorts. Treatment lasted 21 days. Tumor size was measured three times weekly by digital caliper, and mouse weight was determined three times weekly. Experimental endpoint was defined as tumor diameter ≥20 mm in any direction, maximal tumor burden of ~1800 mm³, or if mice appeared unhealthy. Kaplan-Meier survival curves were generated, and significance was determined by log-rank (Mantel-Cox) test. [3]

As a PROTAC molecule, GSK-699 has a relatively high molecular weight (895.84 Da), which typically limits oral bioavailability and cell membrane permeability. Its predicted logP value is 4.7, indicating moderate lipophilicity. The topological polar surface area (tPSA) is 164 Ų, which is higher than the ideal range for conventional small molecule drugs, potentially affecting cell penetration efficiency via passive diffusion. In vivo studies have employed intraperitoneal administration (50 mg/kg once daily), suggesting that GSK-699 achieves adequate systemic exposure via injection routes. PROTAC molecules generally have prolonged duration of action because they can function catalytically after degrading target proteins; however, specific half-life and clearance pathways require further investigation.

In an in vivo neuroblastoma model, daily intraperitoneal administration of 50 mg/kg GSK-699 for 18 days of treatment did not report significant treatment-related mortality or severe systemic toxicity. However, because GSK-699 degrades PCAF/GCN5, which are widely expressed epigenetic regulators, long-term use may carry potential toxicity risks. CRBN modulators (such as lenalidomide-like compounds) are known to be associated with thromboembolic events and embryo-fetal toxicity, so GSK-699 may have similar potential risks. This compound is for research use only and is not intended for human or veterinary use. All experiments involving GSK-699 should be conducted in appropriate biosafety cabinets following standard chemical safety practices.

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- In the dose-finding study, GSK-699-1 at doses up to 100 mg/kg (daily intraperitoneal injection for 4 days) showed no adverse effects. The 50 mg/kg dose was described as having no adverse effects and good solubility. [3]
- In the efficacy study (50 mg/kg once daily intraperitoneal injection for 21 days), the treatment was well tolerated as assessed by mouse weights (no significant weight loss observed). [3]

[1]. Modulating PCAF/GCN5 Immune Cell Function through a PROTAC Approach. ACS Chem Biol. 2018 Oct 19;13(10):2862-2867.

[2]. Disruption of the SAGA CORE triggers collateral degradation of KAT2A. Nat Commun. 2026 Apr 20;17(1):3410.

[3]. The KAT module of the SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma. Sci Adv. 2024 May 31;10(22):eadm9449.

P300/CBP-associated factor (PCAF) and general control non-inhibitory 5 (GCN5) are closely related epigenetic proteins, both containing an acetyltransferase domain and a bromine domain. Consistent with the reported roles of these proteins in immune function, we found that PCAF-deficient macrophages had a significantly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). To investigate the possibility of drug therapy targeting this pathway, we found that chemical inhibition of the PCAF/GCN5 bromine domain was insufficient to reproduce the attenuation of the inflammatory response in PCAF-deficient immune cells. However, by constructing the first PCAF/GCN5 proteolytic targeting chimera (PROTAC), we identified a small molecule that could degrade PCAF/GCN5 and effectively modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data demonstrate the powerful role of the PROTAC approach in multi-domain proteins, revealing a novel anti-inflammatory therapeutic opportunity targeting PCAF/GCN5. [1]

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- GSK-699 is described as a “potent, recently developed KAT2A/KAT2B Proteolysis Targeting Chimera (PROTAC)”. It is used as a pharmacological tool to induce acute degradation of KAT2A/B, serving as a positive control for KAT2A protein reduction in various cell lines including HAP1, MOLM-13, and NALM6. [2]
- The compound allows comparison between chemical degradation and genetic knockout of KAT2A, helping to validate the specificity of the KAT2A stability reporter system. [2]

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- GSK-699-1 is a PROTAC that induces proteolysis of both KAT2A and KAT2B. It was described as a "recently developed" compound (reference 39 in the manuscript). [3]
- An inactive enantiomer, GSK-699-2, was used as a negative control in all in vitro experiments to confirm on-target effects. [3]
- GSK-699-1 treatment reduced MYCN protein expression, a key oncogenic driver in MYCN-amplified neuroblastoma. The compound also reduced H3K9ac and H3K27ac levels. [3]
- The study provides evidence that pharmacological targeting of the SAGA complex via degradation of KAT2A and KAT2B may be a therapeutic strategy for MYCN-amplified neuroblastoma. [3]

C45H51BRN8O7

895.856

894.306

C, 60.33; H, 5.74; Br, 8.92; N, 12.51; O, 12.50

2260944-68-9

146680948

Solid Powder

1.43±0.1 g/cm3(Predicted)

4.7

2

11

15

61

1710

2

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

SARLMRHJAJBYBI-CIDUPMPKSA-N

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

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

GSK-699; GSK699; GSK699; GSK-699-1; 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

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Soluble in DMSO (50mg/ml)

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.

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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).

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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)

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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).

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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

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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.

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 (Please use freshly prepared in vivo formulations for optimal results.)