Akt1 (Ki = 0.08 nM); Akt2 (Ki = 2 nM); Akt3 (Ki = 2.6 nM); Akt1 E17K mutant (IC50 = 0.2 nM); PKCη (IC50 = 210 nM); PKC-βI (IC50 = 430 nM); ROCK (IC50 = 100 nM); PKCθ (IC50 = 510 nM)

Afuresertib (GSK 2110183) exhibits favorable tumor-suppressive effects on malignant pleural mesothelioma (MPM) cells.Afuresertib significantly raises the activity of caspase-3 and caspase-7 as well as the proportion of apoptotic cells in ACC-MESO-4 and MSTO-211H cells. The cell cycle is strongly stopped by afuresertib in the G1 phase. Afuresertib increases the expression of p21WAF1/CIP1 and decreases the phosphorylation of Akt substrates, such as GSK-3 and FOXO family proteins, according to Western blotting analysis. By stimulating FOXO activity, afuresertib-induced p21 expression encourages G1 phase arrest. Afuresertib significantly increases the cytotoxicity that cisplatin causes. Afuresertib alters the expression of the genes MYC and E2F1, which are related to the fibroblast core serum response[1].

GSK2110183 is administered orally to mice bearing BT474 breast tumor xenografts at doses of 10, 30, or 100 mg/kg every day for 21 days, resulting in 8, 37, or 61% TGI, respectively. Mice tolerated GSK2110183 well; after 5 days of dosing, there was a 1-3% loss of body weight, which recovered throughout the course of the study. To further demonstrate the effectiveness of the compound, other tumor xenograft models with activated Akt pathways are investigated. Mice given GSK2110183 at doses of 10, 30, and 100 mg/kg develop SKOV3 xenografts with TGIs of 23, 37, and 97%, respectively[2].

MPM cells are plated in 96-well plates at a cell density of 2.5 103 per well, and then they are left to grow for 24 hours at 37°C. Following that, the cells are incubated for 72 hours in a medium containing the Akt inhibitors at the indicated concentrations (e.g., Afuresertib; 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1, and 0.01 M). The cells are then incubated for 4 hours with MTT solution added to each well. The cells are then given an overnight incubation in lysis buffer (10% SDS in 0.01 mol/L hydrogen chloride). Absorbance is measured at 550 nm using SpectraMAX M5 spectrophotometer[1].

Apoptosis assay[1]
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[1]
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.[2]

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[2]
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 population/average growth of vehicle-treated control population].[2]

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In vivo dose response pharmacodynamic assay[2]
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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Dissolved in 20% polyethylene glycol (PEG) 400/1% DMSO; 25, 50, 100 mg/kg; p.o.

Female athymic nude and SCID mice bearing SKOV3 or BT474 tumors

[1]. Novel ATP-competitive Akt inhibitor Afuresertib suppresses the proliferation of malignant pleural mesothelioma cells. Cancer Med. 2017 Nov;6(11):2646-2659.

[2]. Discovery of novel AKT inhibitors with enhanced anti-tumor effects in combination with the MEK inhibitor. PLoS One. 2014 Jun 30;9(6):e100880.

N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methyl-3-pyrazolyl)-2-thiophenecarboxamide is a member of amphetamines.

Afuresertib has been used in trials studying the treatment of Cancer and Neoplasms, Haematologic.

Afuresertib is an orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity. Afuresertib binds to and inhibits the activity of Akt, which may result in inhibition of the PI3K/Akt signaling pathway and tumor cell proliferation and the induction of tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is frequently associated with tumorigenesis and dysregulated PI3K/Akt signaling may contribute to tumor resistance to a variety of antineoplastic agents.

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Malignant pleural mesothelioma (MPM), an asbestos‐related occupational disease, is an aggressive and incurable tumor of the thoracic cavity. Despite recent advances in MPM treatment, overall survival of patients with MPM is very low. Recent studies have implicated that PI3K/Akt signaling is involved in MPM cell survival and development. To investigate the effects of Akt inhibitors on MPM cell survival, we examined the effects of nine selective Akt inhibitors, namely, afuresertib, Akti‐1/2, AZD5363, GSK690693, ipatasertib, MK‐2206, perifosine, PHT‐427, and TIC10, on six MPM cell lines, namely, ACC‐MESO‐4, Y‐MESO‐8A, MSTO‐211H, NCI‐H28, NCI‐H290, and NCI‐H2052, and a normal mesothelial cell line MeT‐5A. Comparison of IC 50 values of the Akt inhibitors showed that afuresertib, an ATP‐competitive specific Akt inhibitor, exerted tumor‐specific effects on MPM cells. Afuresertib significantly increased caspase‐3 and caspase‐7 activities and apoptotic cell number among ACC‐MESO‐4 and MSTO‐211H cells. Moreover, afuresertib strongly arrested the cell cycle in the G1 phase. Western blotting analysis showed that afuresertib increased the expression of p21WAF 1/ CIP 1 and decreased the phosphorylation of Akt substrates, including GSK‐3β and FOXO family proteins. These results suggest that afuresertib‐induced p21 expression promotes G1 phase arrest by inducing FOXO activity. Furthermore, afuresertib significantly enhanced cisplatin‐induced cytotoxicity. Interestingly, results of gene set enrichment analysis showed that afuresertib modulated the expression E2F1 and MYC, which are associated with fibroblast core serum response. Together, these results suggest that afuresertib is a useful anticancer drug for treating patients with MPM.[1]

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Tumor cells upregulate many cell signaling pathways, with AKT being one of the key kinases to be activated in a variety of malignancies. GSK2110183 and GSK2141795 are orally bioavailable, potent inhibitors of the AKT kinases that have progressed to human clinical studies. Both compounds are selective, ATP-competitive inhibitors of AKT 1, 2 and 3. Cells treated with either compound show decreased phosphorylation of several substrates downstream of AKT. Both compounds have desirable pharmaceutical properties and daily oral dosing results in a sustained inhibition of AKT activity as well as inhibition of tumor growth in several mouse tumor models of various histologic origins. Improved kinase selectivity was associated with reduced effects on glucose homeostasis as compared to previously reported ATP-competitive AKT kinase inhibitors. In a diverse cell line proliferation screen, AKT inhibitors showed increased potency in cell lines with an activated AKT pathway (via PI3K/PTEN mutation or loss) while cell lines with activating mutations in the MAPK pathway (KRAS/BRAF) were less sensitive to AKT inhibition. Further investigation in mouse models of KRAS driven pancreatic cancer confirmed that combining the AKT inhibitor, GSK2141795 with a MEK inhibitor (GSK2110212; trametinib) resulted in an enhanced anti-tumor effect accompanied with greater reduction in phospho-S6 levels. Taken together these results support clinical evaluation of the AKT inhibitors in cancer, especially in combination with MEK inhibitor.[2]

C₁₈H₁₈CL₃FN₄OS

463.78

462.025

C, 46.62; H, 3.91; Cl, 22.93; F, 4.10; N, 12.08; O, 3.45; S, 6.91

1047645-82-8

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

46843056

White to off-white solid powder

5.787

3

5

6

28

520

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([H])=C(C=2[H])F)=O)S1.Cl[H]

YFQJOPFTGMHYNV-YDALLXLXSA-N

InChI=1S/C18H17Cl2FN4OS.ClH/c1-25-16(14(19)9-23-25)13-7-15(27-17(13)20)18(26)24-12(8-22)6-10-3-2-4-11(21)5-10;/h2-5,7,9,12H,6,8,22H2,1H3,(H,24,26);1H/t12-;/m0./s

N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methylpyrazol-3-yl)thiophene-2-carboxamide;hydrochloride

GSK2110183; GSK 2110183; ASB183; ASB-183; GSK 2110183C; Afuresertib (GSK2110183); GSK-2110183; GSK2110183B; GSK2110183B; GSK 2110183B; 1047645-82-8; GSK2110183B; Afuresertib hydrochloride [USAN]; UNII-0FC27E442O; 0FC27E442O; Afuresertib HCl; Afuresertib hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage.  (2). Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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: ≥ 2.5 mg/mL (5.39 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 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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Solubility in Formulation 2: ≥ 2.5 mg/mL (5.39 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), clear solution.
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 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: ≥ 2.5 mg/mL (5.39 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 25.0 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.)