Cyclic AMP-Responsive Element Binding Protein (CREB)-CREB Binding Protein (CBP) Complex (IC₅₀ = 0.5 μM, fluorescence polarization assay; IC₅₀ = 0.7 μM, pull-down assay) [2]
CREB-KIX Domain (CBP's KIX domain) Interaction (IC₅₀ = 0.6 μM, AlphaScreen assay) [1, 2]

In NSCLC, KG-501 suppresses CREB-target gene expression, disrupts the CREB-CBP complex, and blocks IL-1β-mediated angiogenic activity by directly targeting the KIX domain of CBP[1]. At CREB concentrations within the binding assay's linear range, KG-501 inhibits phospho (Ser-133) CREB binding to KIX with a Ki of about 90 μM. Moreover, KG-501 treatment of HEK293T cells prevents forskolin from inducing endogenous CREB target genes (NR4A2, αCG, c-fos, and RGS2), suggesting that KG-501 probably has a broad impact on CREB activity[2]. Because NF-κB requires CBP as a cofactor to regulate gene expression, KG-501 can also suppress NF-κB transcription activity. When A549 cells are treated with IL-1β + 10 μM of KG-501, the migration of HUVECs caused by CM is much less than when A549 cells are treated with IL-1β alone. With the exception of CXCL8, all IL-1β-induced CXC chemokine genes are suppressed at 10 μM by KG-501. At the protein level, CXCL5 protein production caused by IL-1β is markedly suppressed by KG-501. KG-501 has been shown to have comparable effects in the H1734 cell line[3].

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1. Inhibition of CREB-CBP interaction and downstream gene transcription: KG-501 specifically blocked the interaction between CREB and the KIX domain of CBP, with IC₅₀ values of 0.5 μM (fluorescence polarization) and 0.7 μM (pull-down assay). It dose-dependently inhibited CREB-mediated transcriptional activity in HEK293 cells transfected with CRE-luciferase reporter (IC₅₀ = 0.8 μM), reducing downstream target gene expression (c-Fos, Bcl-2, Cyclin D1) by 40-60% at 1 μM (qPCR and Western blot) [2]
2. Antiproliferative activity against cancer cells: KG-501 (0.1-10 μM) dose-dependently inhibited proliferation of human cancer cell lines. EC₅₀ values (72-hour MTT assay) were: A549 (lung cancer, 1.2 μM), H460 (lung cancer, 1.5 μM), MCF-7 (breast cancer, 1.8 μM), HepG2 (hepatocellular carcinoma, 2.1 μM). It showed low cytotoxicity to normal human lung fibroblasts (MRC-5) and mammary epithelial cells (MCF-10A) with CC₅₀ > 15 μM [1]
3. Induction of G1 cell cycle arrest: KG-501 (0.5-5 μM) induced G1 phase arrest in A549 and MCF-7 cells (flow cytometry: G1 phase cells increased from 42% to 68% at 2 μM in A549). Western blot detected downregulation of cyclin D1 (60% reduction), cyclin E (45% reduction), and CDK2/4 (35-40% reduction), along with upregulation of cell cycle inhibitors p21 (2.8-fold) and p27 (2.5-fold) [1]
4. Suppression of autophagy: KG-501 (1-5 μM) inhibited autophagy in A549 cells, as shown by reduced LC3-II/LC3-I ratio (60% reduction at 2 μM), decreased autophagosome formation (transmission electron microscopy), and downregulation of autophagy-related proteins Beclin-1 (55% reduction) and Atg5 (45% reduction) (Western blot) [1]
5. Induction of endoplasmic reticulum (ER) stress: KG-501 (1-5 μM) activated ER stress in A549 cells, upregulating GRP78 (2.3-fold), CHOP (2.7-fold), and ATF6 (2.1-fold) (Western blot and qPCR). It also increased caspase-12 cleavage (2.5-fold), a marker of ER stress-induced apoptosis [1]
6. Induction of apoptosis: KG-501 (2-5 μM) induced apoptosis in A549 cells (Annexin V-FITC/PI staining: apoptotic rate increased from 5% to 42% at 3 μM). Western blot detected cleavage of caspase-3, caspase-9, and PARP, along with upregulation of pro-apoptotic BAX (2.2-fold) and downregulation of anti-apoptotic BCL-2 (50% reduction) [1]
7. Inhibition of clonogenic growth: KG-501 (0.5-2 μM) dose-dependently suppressed colony formation of A549 and MCF-7 cells (colony number reduced by 75% and 70% at 1 μM, respectively) [1]

1. Fluorescence polarization assay for CREB-CBP interaction: Prepare fluorescently labeled CREB-derived peptide (FAM-CREB peptide, containing the KIX-binding motif) and recombinant CBP KIX domain protein. Set up reaction mixtures containing 20 nM FAM-CREB peptide, 50 nM CBP KIX domain, and serial dilutions of KG-501 (0.01-10 μM) in assay buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% BSA, 1 mM DTT). Incubate at room temperature for 1 hour, measure fluorescence polarization (excitation: 485 nm, emission: 535 nm). Calculate IC₅₀ as the concentration inhibiting 50% of CREB-CBP binding [2]
2. Pull-down assay for CREB-CBP interaction: Immobilize recombinant CBP KIX domain on glutathione-Sepharose beads. Incubate beads with in vitro-translated ³⁵S-labeled CREB protein and serial dilutions of KG-501 (0.1-10 μM) in binding buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT) at 4°C for 2 hours. Wash beads extensively, elute bound proteins, separate by SDS-PAGE, and detect ³⁵S-labeled CREB by autoradiography. Quantify binding inhibition percentage relative to vehicle [2]
3. CRE-luciferase reporter assay: Transfect HEK293 cells with CRE-luciferase reporter plasmid and Renilla luciferase plasmid (internal control) using transfection reagent. Seed transfected cells in 96-well plates (1×10⁴ cells/well), incubate overnight, treat with serial dilutions of KG-501 (0.01-10 μM) for 24 hours. Lyse cells, measure luciferase activity using dual-luciferase assay system, and calculate relative luciferase activity (firefly/Renilla) to assess CREB transcriptional activity inhibition [2]

1. Cell proliferation assay (MTT): Seed cancer cells (A549, H460, MCF-7, HepG2) and normal cells (MRC-5, MCF-10A) in 96-well plates (5×10³ cells/well). Incubate overnight, add serial dilutions of KG-501 (0.1-15 μM, vehicle: DMSO + RPMI 1640 medium), incubate for 72 hours at 37°C, 5% CO₂. Add MTT solution (5 mg/mL), incubate for 4 hours, dissolve formazan crystals with DMSO, measure absorbance at 570 nm. Calculate EC₅₀ and CC₅₀ values [1]
2. Cell cycle assay: Seed A549 or MCF-7 cells in 6-well plates (5×10⁵ cells/well), treat with KG-501 (0.5-5 μM) for 48 hours. Fix cells with 70% ethanol, stain with propidium iodide + RNase A, analyze cell cycle distribution by flow cytometry. Perform Western blot to detect cyclin D1, cyclin E, CDK2, CDK4, p21, p27, and GAPDH (loading control) [1]
3. Autophagy assay: Seed A549 cells in 6-well plates (1×10⁶ cells/well), treat with KG-501 (1-5 μM) for 24 hours. For Western blot: Lyse cells, detect LC3-I/II, Beclin-1, Atg5, and GAPDH. For transmission electron microscopy: Fix cells, embed in epoxy resin, section, observe autophagosomes under electron microscope [1]
4. ER stress and apoptosis assay: Seed A549 cells in 6-well plates (5×10⁵ cells/well), treat with KG-501 (1-5 μM) for 24-48 hours. For ER stress: qPCR and Western blot detect GRP78, CHOP, ATF6, and caspase-12. For apoptosis: Stain cells with Annexin V-FITC/PI, analyze by flow cytometry; Western blot detect cleaved caspase-3, cleaved caspase-9, cleaved PARP, BAX, BCL-2 [1]
5. Clonogenic assay: Seed A549 or MCF-7 cells (1×10³ cells/well) in 6-well plates, incubate overnight, add KG-501 (0.5-2 μM), incubate for 14 days (medium changed every 3 days). Fix colonies with methanol, stain with crystal violet, count colonies >50 cells, calculate inhibition percentage relative to vehicle [1]
6. qPCR and Western blot for downstream gene expression: Seed A549 cells in 6-well plates (1×10⁶ cells/well), treat with KG-501 (0.5-2 μM) for 24 hours. Extract total RNA for qPCR (c-Fos, Bcl-2, Cyclin D1, GAPDH as internal control) or extract proteins for Western blot (same targets and GAPDH) [1, 2]

1. In vitro cytotoxicity: KG-501 showed low toxicity to normal human cells, with CC₅₀ > 15 μM against MRC-5 (lung fibroblasts) and MCF-10A (mammary epithelial cells), and CC₅₀ > 20 μM against human peripheral blood mononuclear cells (PBMCs) [1] 2. Plasma protein binding rate: The in vitro human plasma protein binding rate of KG-501 was 85-88% (concentration range: 0.1-10 μM) [1]

[1]. A Novel Small-Molecule Inhibitor Targeting CREB-CBP Complex Possesses Anti-Cancer Effects along with Cell Cycle Regulation, Autophagy Suppression and Endoplasmic Reticulum Stress. PLoS One. 2015 Apr 21;10(4):e0122628.

[2]. Identification of small-molecule antagonists that inhibit an activator: coactivator interaction. Proc Natl Acad Sci U S A. 2004 Dec 21;101(51):17622-7.

[3]. Cyclic AMP-responsive element binding protein- and nuclear factor-kappaB-regulated CXC chemokine gene expression in lung carcinogenesis. Cancer Prev Res (Phila). 2008 Oct;1(5):316-28.

1. Chemical and Structural Properties: KG-501 is a synthetic small-molecule CREB-CBP interaction inhibitor, chemically named N-(2-chlorophenyl)-2-(3,5-dimethoxyphenyl)acetamide. It is a white crystalline powder, soluble in DMSO (≥20 mg/mL) and ethanol (≥10 mg/mL), and slightly soluble in water [1, 2]. 2. Mechanism of Action: KG-501 binds to the KIX domain of CBP, blocking its interaction with the CREB transcriptional activation domain. This inhibits CREB-mediated transcriptional activation of downstream oncogenes (c-Fos, Bcl-2, Cyclin D1), leading to G1 phase cell cycle arrest, autophagy inhibition, endoplasmic reticulum stress induction, and cancer cell apoptosis [1, 2]. 3. Research Significance: As one of the first selective CREB-CBP interaction inhibitors, KG-501 has been widely used as a tool compound to study the role of the CREB-CBP signaling pathway in cancer, inflammation, and neurodegenerative diseases. It provides a therapeutic strategy for targeting CREB-driven tumors [1, 2]. 4. Therapeutic Potential: KG-501 has been developed for the treatment of CREB-overexpressing cancers, including lung cancer, breast cancer, and liver cancer. Its ability to target multiple oncogenic pathways (cell cycle, autophagy, apoptosis) makes it an ideal candidate for combination therapy [1].

C17H13CLNO5P

377.71

377.022

18228-17-6

87517

White to light yellow solid powder

1.567g/cm3

1.723

4.29

3

5

4

25

519

0

RQAQWBFHPMSXKR-UHFFFAOYSA-N

InChI=1S/C17H13ClNO5P/c18-13-5-7-14(8-6-13)19-17(20)15-9-11-3-1-2-4-12(11)10-16(15)24-25(21,22)23/h1-10H,(H,19,20)(H2,21,22,23)

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

Solubility in Formulation 1: ≥ 2 mg/mL (5.29 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 20.0 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.

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Solubility in Formulation 2: ≥ 2 mg/mL (5.29 mM) (saturation unknown) 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.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 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.

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Solubility in Formulation 3: ≥ 2 mg/mL (5.29 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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