Akt1 (Ki = 0.08 nM); Akt2 (Ki = 2 nM); Akt3 (Ki = 2.6 nM)
Afuresertib inhibits the kinase activity of the E17K AKT1 mutant protein with EC50 of 0.2 nM. Afuresertib has a concentration-dependent impact on the phosphorylation levels of several AKT substrates, including GSK3b, PRAS40, FOXO, and Caspase 9. Afuresertib has an overall sensitivity of 65% for hematological cell lines (EC50 1 M). In response to afuresertib, 21% of tested solid tumor cell lines have an EC50 1 M. [1]

Afuresertib inhibits the kinase activity of the E17K AKT1 mutant protein with EC50 of 0.2 nM. Afuresertib has a concentration-dependent impact on the phosphorylation levels of several AKT substrates, including GSK3b, PRAS40, FOXO, and Caspase 9. Afuresertib has an overall sensitivity of 65% for hematological cell lines (EC50 1 M). In response to afuresertib, 21% of tested solid tumor cell lines have an EC50 1 M. [1]

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GSK2110183 exhibits potent anti-proliferative activity against a panel of 290 human cancer cell lines, with greater sensitivity observed in cell lines harboring PIK3CA mutations or PTEN loss.
In BT474 breast cancer cells (PIK3CA K111N, ERBB2+), GSK2110183 dose-dependently decreases phosphorylation of AKT substrates GSK3β (Ser9/21), PRAS40 (Thr246), FOXO (Thr24/32), and Caspase 9 (Ser196) after 1-hour treatment, while inducing feedback hyperphosphorylation of AKT at Ser473 and Thr308.
In LNCaP prostate cancer cells (PTEN null), GSK2110183 similarly inhibits phosphorylation of the same AKT substrates.
GSK2110183 does not affect phosphorylation of MEK1/2 (Ser217/221) or ERK1/2 (Tyr204) in BT474 or LNCaP cells at 1 hour, indicating selective AKT pathway inhibition.
In a FOXO3A-GFP nuclear translocation assay using MDA-MB-468 cells, GSK2110183 induces FOXO3A nuclear translocation with an EC₅₀ of 53 nM.
Cell cycle analysis in BT474 cells treated with GSK2110183 for 24 hours shows G1 arrest at 1 µM and an increase in sub-G1 population (indicative of cell death) at 10 µM.
Cell cycle analysis in LNCaP cells shows a dose-dependent increase in the sub-G1 population.
GSK2110183 shows increased anti-proliferative potency in hematological malignancy cell lines (particularly B- and T-cell origin ALL, NHL, and CLL) and in solid tumor cell lines from breast and lung cancers.
Cell lines with activating mutations in KRAS or BRAF are generally less sensitive to GSK2110183. [1]

Afuresertib (p. o.) doses of 10, 30, or 100 mg/kg per day cause 8, 37, or 61% TGI in mice with BT474 breast tumor xenografts. Treatments with 10, 30, and 100 mg/kg of afuresertib result in 23, 37, and 97% TGI, respectively, in mice with SKOV3 ovarian tumor xenografts. [1]

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In female SCID mice bearing established BT474 breast tumor xenografts, daily oral administration of GSK2110183 at 10, 30, and 100 mg/kg for 21 days resulted in tumor growth inhibition (TGI) of 8%, 37%, and 61%, respectively, compared to vehicle control.
In female athymic nude mice bearing SKOV3 ovarian tumor xenografts, daily oral administration of GSK2110183 at 10, 30, and 100 mg/kg for 21 days resulted in TGI of 23%, 37%, and 97%, respectively. [1]

Kinase Assays[1]
The potency of compounds against AKT enzymes was measured as described before. Since GSK2110183 and GSK2141795 are highly potent inhibitors of the 3 isoforms of AKT, the true potency (Ki *) of the inhibitors was initially determined at low enzyme concentrations (0.1 nM AKT1, 0.7 nM AKT2, and 0.2 nM AKT3) using a filter binding assay and then confirmed with progress curve analysis. In the filter binding assay, a pre-mix of enzyme plus inhibitor was incubated for 1 h and then added to a GSKα peptide (Ac-KKGGRARTSSFAEPG-amide) and [γ33P] ATP. Reactions were terminated after 2 h and the radio labeled AKT peptide product was captured in a phospho-cellulose filter plate. Progress curve analysis utilized continuous real-time fluorescence detection of product formation using the Sox-AKT-tide substrate (Ac-ARKRERAYSF-d-Pro-Sox-Gly-NH2).[1]
 GSK2110183 and GSK2141795 were tested against a diverse panel of kinase assays. Initially, the compounds were tested at 0.5 and 10 µM in all available kinase assays and were followed up with full IC50 curves against a subset of enzymes that showed strong inhibition against 0.5 µM, for which in-house assay were not available.

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The potency of GSK2110183 against AKT enzymes was measured using a filter binding assay. A pre-mix of enzyme and inhibitor was incubated for 1 hour, then added to a GSK3α peptide substrate and [γ³³P]ATP. Reactions were terminated after 2 hours, and radiolabeled phosphorylated peptide product was captured on a phospho-cellulose filter plate for quantification.
Progress curve analysis was also performed using continuous real-time fluorescence detection of product formation with a Sox-AKT-tide substrate.
GSK2110183 was tested against a diverse panel of kinase assays (261 kinases) at concentrations of 0.5 µM and 10 µM. Full IC₅₀ curves were generated for kinases showing >50% inhibition at 0.5 µM. [1]

To measure the growth inhibition caused by the compounds at 0–30 M, a 3-day proliferation assay using CellTiter-Glo is carried out. The rate of cell growth is measured in comparison to untreated (DMSO) controls. In the Assay Client application, EC50 values are calculated from inhibition curves using a 4- or 6-parameter fitting algorithm.

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Apoptosis assay[2]
Apoptosis was evaluated by performing AxV–FITC/PI double staining‐based FACS analysis, as described previously 25. Briefly, ACC‐MESO‐4 and MSTO‐211H cells were seeded in six‐well plates (cell density, 1 × 105 cells/well) and were incubated for 24 h at 37°C. Next, the cells were incubated with indicated concentrations of afuresertib, followed by incubation with AxV–FITC and PI (10 μg/mL) for 15 min at room temperature. Fluorescence intensities were determined by performing FACS with FACSCantoII.

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Cell cycle analysis[2]
Cell cycle was evaluated by performing PI‐staining‐based FACS analysis, as described previously 26. ACC‐MESO‐4 and MSTO‐211H cells were seeded in a six‐well culture plate (cell density, 1 × 105 cells/well) and were incubated for 24 h. Next, the cells were incubated with the indicated concentrations of afuresertib for 24 h. For FACS analysis, the cells were detached using trypsin after 24 h of serum treatment and were fixed overnight in ice‐cold 70% ethanol. After fixation, the cells were treated with RNase A (100 μg/mL) and stained with PI (10 μg/mL). The percentages of cells in the sub‐G1, G1, S, and G2‐M phases of the cell cycle were measured using FlowJo software.

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A 3-day proliferation assay using CellTiter-Glo is performed to measure the growth inhibition by the compounds at 0-30 μM. The rate of cell growth is measured in comparison to untreated (DMSO) controls. In the Assay Client application, EC50 values are calculated from inhibition curves using a 4- or 6-parameter fitting algorithm.[1]

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For phospho-GSK3β ELISA, cells were seeded at 25,000 cells/well in a 96-well plate overnight, then treated with GSK2110183 or DMSO for 1 hour. Cells were lysed, and lysates were transferred to ELISA plates coated with anti-GSK3β antibody. After overnight incubation, plates were washed, incubated with rabbit anti-phospho-GSK3β (Ser9) antibody, developed using HRP-linked secondary antibody and TMB substrate, and absorbance was measured at 450 nm.
A similar ELISA methodology was used for phospho-PRAS40 (Thr246) detection using a commercial kit.
For cell proliferation assays, various tumor cell lines were grown and treated with a titration of GSK2110183 for 3 days. Cell viability was measured using the CellTiter-Glo luminescent assay, and EC₅₀ values were calculated.
For Western blot analysis, cells treated with GSK2110183 were lysed in RIPA buffer with protease and phosphatase inhibitors. Equal protein amounts were resolved by SDS-PAGE, transferred to PVDF membranes, and probed with specific primary antibodies overnight. Blots were incubated with IRDye-labeled secondary antibodies and imaged using an infrared imaging system.
For cell cycle analysis, cells treated with GSK2110183 for 24 hours were analyzed by flow cytometry following propidium iodide staining.
For the FOXO3A-GFP nuclear translocation assay, MDA-MB-468 cells stably expressing FOXO3A-GFP were treated with GSK2110183 for 1 hour and analyzed using a high-content imager. [1]

Female athymic nude and SCID mice bearing SKOV3 or BT474 tumors
100 mg/kg
p.o.

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Female athymic nude and SCID mice bearing SKOV3 or BT474 tumors[1]
100 mg/kg
p.o.

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In vivo Xenograft experiments[1]
Tumors were initiated by injecting either cells (SKOV3, CAPAN-2 and HPAC) or a tumor fragments (BT474) subcutaneously into 6–8 week female athymic nude (SKOV3) and SCID (all others) mice. Once tumors reached between 120 and 300 mm3, mice were randomized according to tumor volume into groups of n = 7–10 mice per treatment. GSK2110183 and GSK2141795 were administered daily at various doses by oral gavage. In combination experiments, GSK1120212 was also administered daily by oral gavage. Tumor volumes and body weight were measured twice weekly, tumor volume was measured with calipers and calculated using equation: Tumor volume (mm3)  =  (length x width)2/2. Results are represented as percent inhibition on completion of dosing  = 100 x [1- average growth of drug-treated populatioverage growth of vehicle-treated control population].[1]

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In vivo dose response pharmacodynamic assay[1]
SCID mice bearing BT474 tumor xenografts were treated with either vehicle, GSK2110183 or GSK2141795 daily for 7 days prior to harvesting tissue 2 h post the last dose. Protein lysates were analyzed by phospho-PRAS40 ELISA according to the methods described above. Concentration of the test compounds in the tissue and blood was analyzed using protein precipitation with acetonitrile, followed by HPLC/MS/MS analysis using positive ion atmospheric pressure chemical ionization or Turbo ionspray ionization. The lower level of detection of compound was 10 ng/mL and the assays were linear over a 100- to a 1000-fold drug concentration range.

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For in vivo xenograft efficacy studies, tumors were established in female nu/nu CD-1 or SCID mice by subcutaneous injection of cancer cells or tumor fragments. When tumors reached 120–300 mm³, mice were randomized into groups (n=7-10).
GSK2110183 was formulated in 20% polyethylene glycol (PEG) 400 / 1% DMSO and administered daily by oral gavage at specified doses (e.g., 10, 30, 100 mg/kg) for 21 days.
Tumor volumes were measured twice weekly using calipers, and body weight was monitored. [1]

GSK2110183 has a high plasma protein binding rate (>95% in both human and rodent plasma). In BT474 tumor-bearing mice, a single oral dose of 100 mg/kg resulted in a plasma concentration of approximately 3–4 µM (Cmax), which inhibited approximately 60% of phosphorylated PRAS40 in the tumor, and the inhibitory effect lasted for 24 hours. The exposure of GSK2110183 in the tumor was consistently higher than the plasma concentration (≥3 times). Time-course experiments showed that the inhibition of phosphorylated PRAS40 returned to baseline levels 48 hours after administration. [1]

Following a single oral administration of 100 mg/kg of GSK2110183 to mice, both blood glucose (peak at approximately 211 mg/dL 2 hours after administration) and plasma insulin (peak at approximately 105.6 ng/mL 4 hours after administration) showed transient increases. All parameters returned to normal 8 hours after administration. In a 21-day efficacy study, mouse body weight showed only a slight decrease (1-3%), which gradually recovered during the study. [1]

[1]. PLoS One . 2014 Jun 30;9(6):e100880.

Tumor cells activate multiple cellular signaling pathways, among which AKT is one of the key kinases activated in various malignant tumors. GSK2110183 and GSK2141795 are highly bioavailable and potent AKT kinase inhibitors that have entered human clinical trials. Both compounds are selective ATP-competitive AKT1, 2, and 3 inhibitors. Cells treated with these compounds showed reduced phosphorylation levels of multiple downstream substrates of AKT. Both compounds possess desirable pharmacological properties, providing sustained inhibition of AKT activity with daily oral administration and suppressing tumor growth in various mouse tumor models derived from different tissues. Compared to previously reported ATP-competitive AKT kinase inhibitors, GSK2110183 and GSK2141795 exhibit higher kinase selectivity and therefore less impact on glucose homeostasis. In various cell line proliferation screenings, the AKT inhibitors showed higher potency in cell lines with AKT pathway activation (via PI3K/PTEN mutations or deletions), while cell lines with MAPK pathway (KRAS/BRAF) activation mutations were less sensitive to AKT inhibition. Further studies in a KRAS-driven mouse model of pancreatic cancer confirmed that the AKT inhibitor GSK2141795, when used in combination with the MEK inhibitor (GSK2110212; trametinib), enhanced antitumor activity and was accompanied by a significant reduction in phosphorylated S6 levels. In summary, these results support the clinical evaluation of AKT inhibitors in cancer treatment, especially when used in combination with MEK inhibitors. [1]

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GSK2110183 (Afuresertib) is an orally bioavailable ATP-competitive pan-AKT kinase inhibitor that is currently in human clinical trials.
It inhibits the AKT1 E17K activating mutant, which distinguishes it from allosteric AKT inhibitors that bind to the PH domain.
Compared to earlier AKT inhibitors (such as GSK690693), this compound shows higher kinase selectivity, which is associated with a reduced effect on glucose homeostasis.
Preclinical data support the clinical evaluation of GSK2110183. [1]

C₁₈H₁₆CL₂F₂N₄OS

445.31

444.039

C, 48.55; H, 3.62; Cl, 15.92; F, 8.53; N, 12.58; O, 3.59; S, 7.20

1047634-63-8

GSK2110183 analog 1 hydrochloride;2070009-64-0; 1047644-62-1 ; 1047645-82-8 (HCl); 1047634-63-8 (Afuresertib-F free base)

57826797

Solid powder

1.5±0.1 g/cm3

535.1±50.0 °C at 760 mmHg

277.4±30.1 °C

0.0±1.4 mmHg at 25°C

1.674

3.29

2

6

6

28

552

1

ClC1=C(C2=C(C([H])=NN2C([H])([H])[H])Cl)C([H])=C(C(N([H])[C@]([H])(C([H])([H])N([H])[H])C([H])([H])C2C([H])=C([H])C(=C(C=2[H])F)F)=O)S1

AHDFWNJLFALBJP-JTQLQIEISA-N

InChI=1S/C18H16Cl2F2N4OS/c1-26-16(12(19)8-24-26)11-6-15(28-17(11)20)18(27)25-10(7-23)4-9-2-3-13(21)14(22)5-9/h2-3,5-6,8,10H,4,7,23H2,1H3,(H,25,27)/t10-/m0/s1

N-[(2S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methylpyrazol-3-yl)thiophene-2-carboxamide

Afuresertib-F; Afuresertib-F free base; GSK-2110183-analog; GSK 2110183-analog; GSK2110183-analog

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)

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.)