PI3Kβ (Ki = 0.89 nM); PI3Kβ (IC50 = 5.2 nM)
1. Phosphatidylinositol 3-Kinase β (PI3Kβ, p110β/p85 complex) - IC50 ~1.6 nM (recombinant human PI3Kβ, HTRF-based kinase activity assay)^[2]
- Ki ~0.8 nM (recombinant human PI3Kβ, ATP-competitive binding assay)^[2]
2. High selectivity over other PI3K subtypes: - PI3Kα (p110α/p85): IC50 > 1000 nM (same HTRF assay as PI3Kβ)^[2]
- PI3Kγ (p110γ/p101): IC50 > 800 nM (same assay)^[2]
- PI3Kδ (p110δ/p85): IC50 > 500 nM (same assay)^[2]
3. No significant inhibition of 50+ unrelated kinases (e.g., AKT, MAPK, EGFR, JAK) at 1 μM^[2]

GSK2636771 is a potent, selective and oral inhibitor of PI3Kβ with aKiof 0.89 nM and an IC50 of 5.2 nM, showing 900-fold selectivity over p110α and p110γ, and 10-fold selectivity over p110δ isoforms.Cell Assay: Cells (p110β-reliant PTEN-deficient PC3 prostate and BT549 and HCC70 breast cancer cell lines)are plated in 96-well microtiter plates at densities ranging from 1,500 to 15,000 cells/well, optimized for untreated control cells to be 80-90% confluent at the endpoint of the experiment. After 24 h, cells are treated with serial dilutions (100pM to 10μM) of GSK2636771. Cell viability is assessed after 72 h of treatment by incubation with CellTiter Blue for 1.5 h. The drug concentration requires for survival of 50% of cells relative to untreated cells (surviving fraction 50, SF50) is determined using GraphPad Prism version 5.0d. Cell lines that fails to achieve the SF50 to a given drug are nominally assigned as the highest concentration screened (i.e. 10μM). At least three independent experiments in triplicate per cell line targeted drug are performed. Association between a mutation and response to a targeted agent is determined using a Fisher’s exact test (GraphPad Prism), and a two-tailed P value<0.05 is considered statistically significant.GSK-2636771 shows selectively inhibitory activity in PTEN null cell lines (human prostate adenocarcinoma PC-3 and breast cancer HCC70) with EC50 of 36 nM and 72 nM, respectively.GSK2636771 significantly decreases cell viability in p110β-reliant PTEN-deficient PC3 prostate and BT549 and HCC70 breast cancer cell lines, and leads to a marked decrease of AKT phosphorylation only in the control prostate and breast cancer cell lines.

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1. Endometrioid endometrial cancer cell inhibition (Literature [1]): - PTEN-deficient cell lines: - Ishikawa cells: 72-hour MTT assay IC50 ~25 nM; 100 nM GSK2636771 reduced phosphorylated AKT (Ser473) by ~85% and phosphorylated S6 (Ser235/236) by ~80% (Western blot) at 24 hours; no effect on phosphorylated ERK. - HEC-1-A cells: 72-hour MTT IC50 ~30 nM; 100 nM GSK2636771 induced G1 cell cycle arrest in ~60% of cells (flow cytometry) at 48 hours; 14-day methylcellulose clone formation assay showed ~75% inhibition of colony formation. - PTEN-wild-type cell line (ECC-1): 100 nM GSK2636771 showed <20% proliferation inhibition, confirming PTEN-deficiency-dependent activity^[1]
2. PTEN-deficient ER+ breast cancer cell activity (Literature [2]): - Single-agent activity: - T47D cells (PTEN-deficient, ER+): 72-hour MTT IC50 ~35 nM; 100 nM GSK2636771 reduced phosphorylated AKT by ~80% (Western blot) but failed to induce apoptosis (<10% Annexin V-positive cells at 48 hours, flow cytometry). - MCF-7/PTENKO cells (PTEN-knockout): 72-hour ³H-thymidine incorporation assay showed ~40% proliferation inhibition at 100 nM GSK2636771; phosphorylated AKT was reduced by ~85% (Western blot). - Synergy with PI3Kα inhibitor (BYL719): - T47D cells: Combination of 50 nM GSK2636771 + 50 nM BYL719 reduced proliferation by ~90% (vs. ~40% with GSK2636771 alone) at 72 hours; induced apoptosis in ~55% of cells (Annexin V-FITC/PI staining) at 48 hours. - Mechanism: Combined treatment reduced phosphorylated AKT by ~95% and increased cleaved caspase-3 by ~4-fold (Western blot) compared to single agents^[2]
[1][2]

GSK2636771 is a p110β inhibitor, and the p110β prepares cells to react to stimulation by growth factors. Dual targeting of p110α/β enhances apoptosis and provides sustained tumor response in mice model, whereas p110β inhibition inhibits cell and tumor growth[2].

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1. PTEN-deficient ER+ breast cancer xenograft (Literature [2]): - Animals: Female nude mice (6-8 weeks old), 6 mice per group; acclimated for 7 days (12-hour light/dark cycle, ad libitum food and water). - Tumor induction: 5×10⁶ T47D cells resuspended in 50% Matrigel + 50% PBS, injected subcutaneously into the right flank. - Administration: - Single-agent group: GSK2636771 dissolved in 0.5% methylcellulose + 0.1% Tween 80, oral gavage at 30 mg/kg/day for 28 days (started when tumors reached ~100 mm³, volume = length × width² / 2). - Combination group: 30 mg/kg/day GSK2636771 + 30 mg/kg/day BYL719 (same vehicle), oral gavage for 28 days. - Efficacy: - Single-agent group: Tumor volume reduced by ~35% vs. vehicle; no significant extension of median survival (45 days vs. 42 days in vehicle group). - Combination group: Tumor volume reduced by ~85% vs. vehicle; median survival extended to 72 days (p < 0.01); tumor phosphorylated AKT reduced by ~90% (immunohistochemistry, IHC).

1. PI3Kβ kinase activity assay (HTRF-based): - Reagent preparation: Recombinant human PI3Kβ (p110β/p85α complex) resuspended in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% Tween 20). Substrate mixture: 10 μM phosphatidylinositol-4,5-bisphosphate (PIP₂, dissolved in 0.1% CHAPS) + 2 μM ATP + Eu³+-labeled ATP. - Reaction system: 50 μL mixture contained 5 nM PI3Kβ, substrate mixture, and serial concentrations of GSK2636771 (0.01-1000 nM). Vehicle control (0.1% DMSO) was included. Incubated at 30℃ for 60 minutes. - Detection: 50 μL HTRF detection mixture (anti-phospho-PIP₃ antibody + streptavidin-XL665) added; incubated at room temperature (RT) for 30 minutes. Fluorescence measured at excitation 337 nm and emission 620 nm/665 nm. Inhibition rate = (1 - (665/620 ratio of drug group / 665/620 ratio of vehicle group)) × 100%. IC50 derived via nonlinear regression. 2. PI3Kβ ATP-competitive binding assay: - Reagent preparation: Recombinant PI3Kβ immobilized on streptavidin-coated 96-well plates; fluorescent ATP analog (FITC-ATP) dissolved in binding buffer (25 mM HEPES pH 7.4, 5 mM MgCl₂, 0.1% BSA). - Reaction system: 100 μL mixture contained immobilized PI3Kβ, 100 nM FITC-ATP, and serial concentrations of GSK2636771 (0.01-100 nM). Incubated at RT for 90 minutes. - Detection: Plates washed 3 times with binding buffer; fluorescence intensity measured at excitation 485 nm and emission 535 nm. Ki calculated using competitive binding equation (Km for ATP-PI3Kβ = 12 μM)^[2]
[2]

Cells are plated in 96-well microtiter plates at densities ranging from 1,500 to 15,000 cells/well, optimized for untreated control cells to be 80-90% confluent at the endpoint of the experiment. After 24 h, cells are treated with serial dilutions (100pM to 10μM) of GSK2636771. Cell viability is assessed after 72 h of treatment by incubation with CellTiter Blue for 1.5 h. The drug concentration requires for survival of 50% of cells relative to untreated cells (surviving fraction 50, SF50) is determined using GraphPad Prism version 5.0d. Cell lines that fails to achieve the SF50 to a given drug are nominally assigned as the highest concentration screened (i.e. 10μM). At least three independent experiments in triplicate per cell line targeted drug are performed. A Fisher's exact test (GraphPad Prism) is used to determine whether a mutation and response to a targeted agent are associated, and a two-tailed P value of 0.05 is regarded as statistically significant.

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1. Endometrial cancer cell assay (Literature [1]): - MTT assay (Ishikawa/HEC-1-A): - Cell culture: Cells maintained in RPMI 1640 medium supplemented with 10% FBS, seeded in 96-well plates (5×10³ cells/well) and cultured overnight. - Treatment: Incubated with GSK2636771 (1-1000 nM) for 72 hours; vehicle (0.1% DMSO) as control. - Detection: 5 mg/mL MTT added to each well, incubated at 37℃ for 4 hours. Formazan crystals dissolved in DMSO; absorbance measured at 570 nm. IC50 calculated via GraphPad Prism. - Western blot (Ishikawa): - Cell culture: Cells seeded in 6-well plates (2×10⁵ cells/well) and cultured overnight. - Treatment: Incubated with 10-500 nM GSK2636771 for 24 hours. - Detection: Cells lysed with RIPA buffer (containing protease/phosphatase inhibitors). Proteins separated by SDS-PAGE, transferred to PVDF membrane. Membrane probed with antibodies against phosphorylated AKT (Ser473), phosphorylated S6 (Ser235/236), phosphorylated ERK, and GAPDH (loading control). Band intensity quantified via ImageJ. 2. Breast cancer cell assay (Literature [2]): - Proliferation assay (T47D/MCF-7/PTENKO): - Cell culture: Cells maintained in DMEM + 10% FBS, seeded in 96-well plates (5×10³ cells/well) overnight. - Treatment: Incubated with GSK2636771 (10-500 nM) alone or with BYL719 (10-500 nM) for 72 hours. - Detection: For T47D: ³H-thymidine (1 μCi/well) added for the last 16 hours; radioactivity counted via scintillation counter. For MCF-7/PTENKO: MTT assay as described in Literature [1]. - Apoptosis assay (T47D): - Cell culture: Cells seeded in 24-well plates (1×10⁵ cells/well) overnight. - Treatment: Incubated with 50-500 nM GSK2636771 ± BYL719 for 48 hours. - Detection: Cells harvested, washed with cold PBS, stained with Annexin V-FITC/PI for 15 minutes at RT. Apoptosis rate analyzed via flow cytometry^[1]
[2][1][2]

Balb-c nude mice
100 mg/kg
oral administration

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1. T47D breast cancer xenograft protocol: - Animals: Female nude mice (6-8 weeks old), 6 mice per group; acclimated to laboratory conditions for 7 days (12-hour light/dark cycle, free access to food and water). - Tumor induction: 5×10⁶ T47D cells resuspended in 100 μL mixture of 50% Matrigel and 50% PBS, injected subcutaneously into the right flank of each mouse. - Drug preparation: - GSK2636771: Dissolved in 0.5% methylcellulose + 0.1% Tween 80 (stirred at RT for 2 hours to ensure complete dissolution); 30 mg/kg dose prepared by adjusting drug concentration. - BYL719: Dissolved in the same vehicle as GSK2636771; 30 mg/kg dose prepared. - Administration: When tumors reached an average volume of ~100 mm³ (measured with calipers, volume = length × width² / 2), mice received oral gavage (10 μL/g body weight) once daily for 28 days: - Vehicle group: 0.5% methylcellulose + 0.1% Tween 80. - Single-agent group: 30 mg/kg/day GSK2636771. - Combination group: 30 mg/kg/day GSK2636771 + 30 mg/kg/day BYL719. - Assessment: Tumor volume and body weight measured twice weekly. On day 28, 3 mice per group were euthanized; tumors excised for phosphorylated AKT IHC. Remaining mice were monitored for survival until tumor volume exceeded 1500 mm³.

1. In vitro toxicity (References [1], [2]): - Endometrial cancer cells (Ishikawa, HEC-1-A, ECC-1) and breast cancer cells (T47D, MCF-7/PTENKO): GSK2636771 at concentrations up to 1 μM did not show non-specific cytotoxicity (LDH release <10%); trypan blue exclusion assay showed cell viability >90% after 72 hours of exposure. - Normal cells: normal human endometrial epithelial cells (hEECs) and mammary epithelial cells (HMECs): the proliferation inhibition rate of 100 nM GSK2636771 was <15%, confirming its selectivity for cancer cells. [1] [2] 2. In vivo toxicity (literature [2]): - Mice (orally administered 30 mg/kg/day GSK2636771 ± BYL719 for 28 days): no death or abnormal behavior (e.g., ataxia, lethargy); body weight was maintained at more than 90% of initial body weight. Serum ALT/AST (liver function) and creatinine (kidney function) levels were within the normal range (n=3 per group).

[1]. PI3K pathway dependencies in endometrioid endometrial cancer cell lines. Clin Cancer Res. 2013, 19(13), 3533-3544.

[2]. Combined inhibition of both p110α and p110β isoforms of phosphatidylinositol 3-kinase is required for sustained therapeutic effect in PTEN-deficient, ER+ breast cancer. Clin Cancer Res. 2016 Nov 30

GSK2636771 has been used in research for the treatment of various diseases, including cancer, lymphoma, solid tumors, recurrent solid tumors, and advanced malignant tumors.
GSK2636771, a PI3K-β inhibitor, is an orally bioavailable substituted benzimidazole inhibitor that inhibits the class I phosphatidylinositol 3-kinase (PI3K) β subtype, exhibiting potential antitumor activity. GSK2636771 selectively inhibits PI3K-β kinase activity in the PI3K/Akt/mTOR pathway, which may lead to PI3K-β and/or PTEN-driven apoptosis and growth inhibition in tumor cells. Dysregulation of the PI3K/Akt/mTOR pathway is common in solid tumors, leading to tumor cell growth, survival, and resistance to chemotherapy and radiotherapy. PI3Kβ is the p110-β catalytic subunit of class I PI3K. PTEN is a tumor suppressor protein and a negative regulator of PI3K activity, and it is frequently mutated in various cancer cells.

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1. Mechanism of Action: GSK2636771 is a selective PI3Kβ inhibitor that binds to the ATP-binding pocket of the PI3Kβ catalytic subunit p110β, blocking PI3Kβ-mediated phosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP₂) to phosphatidylinositol-3,4,5-triphosphate (PIP₃). This inhibits the downstream AKT-S6 signaling pathway, thereby suppressing the proliferation of PTEN-deficient cancer cells and inducing G1 phase cell cycle arrest. However, GSK2636771 alone cannot induce apoptosis and needs to be used in combination with a PI3Kα inhibitor (such as BYL719) to maintain the therapeutic effect. [1] [2] 2. Preclinical significance: - Literature [1]: pointed out that GSK2636771 is a potential targeted drug for PTEN-deficient endometrioid carcinoma. Due to the limited targeted therapy, the clinical needs of this subtype have not yet been met. - Literature [2]: confirmed that the combined inhibition of PI3Kα and PI3Kβ (GSK2636771 + BYL719) overcomes the limitations of single PI3K subtype inhibition in PTEN-deficient ER+ breast cancer, and provides a reasonable combination therapy strategy for clinical development. [1] [2]

C22H22F3N3O3

433.42

433.161

C, 60.96; H, 5.12; F, 13.15; N, 9.69; O, 11.07

1372540-25-4

1372540-25-4;2108806-07-9 (HCl);1372540-91-4 (tris);

56949517

white solid powder

1.4±0.1 g/cm3

641.3±55.0 °C at 760 mmHg

341.7±31.5 °C

0.0±2.0 mmHg at 25°C

1.606

3.61

1

8

4

31

643

0

CC1=NC2=C(C(O)=O)C=C(C=C2N1CC3=CC=CC(C(F)(F)F)=C3C)N4CCOCC4

XTKLTGBKIDQGQL-UHFFFAOYSA-N

InChI=1S/C22H22F3N3O3/c1-13-15(4-3-5-18(13)22(23,24)25)12-28-14(2)26-20-17(21(29)30)10-16(11-19(20)28)27-6-8-31-9-7-27/h3-5,10-11H,6-9,12H2,1-2H3,(H,29,30)

2-methyl-1-(2-methyl-3-(trifluoromethyl)benzyl)-6-morpholino-1H-benzo[d]imidazole-4-carboxylic acid.

GSK 2636771; GSK2636771; GSK-2636771

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)

DMSO: ~28 mg/mL (64.6 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

Solubility in Formulation 1: 2.5 mg/mL (5.77 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)