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Purity: ≥98%
Afuresertib (also named as GSK2110183C) is a potent, orally bioavailable and ATP-competitive Akt inhibitor with potential anticancer activity. With Kis of 0.08 nM, 2 nM, and 2.6 nM, respectively, it inhibits Akt1, Akt2, and Akt3. Afuresertib may have anti-cancer effects because it inhibits the serine/threonine protein kinase Akt (protein kinase B). The PI3K/Akt signaling pathway, tumor cell proliferation, and tumor cell apoptosis may all be inhibited as a result of the Akt inhibitor GSK2110183's binding to and inhibition of Akt activity. The PI3K/Akt signaling pathway is frequently involved in the development of tumors, and aberrant PI3K/Akt signaling may play a role in the development of tumor resistance to various antineoplastic agents.
| Targets |
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); PKCθ (IC50 = 510 nM); ROCK (IC50 = 100 nM)
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|---|---|
| ln Vitro |
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 μM). In response to afuresertib, 21% of tested solid tumor cell lines have an EC50 < 1 μM. [1]
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| ln Vivo |
Afuresertib (p.o.) is dosed to mice with BT474 breast tumor xenografts at 10, 30, or 100 mg/kg per day, resulting in 8, 37, or 61% TGI, respectively. Treatment with 10, 30, and 100 mg/kg afuresertib causes 23, 37, and 97% TGI in mice with SKOV3 ovarian tumor xenografts, respectively. [1]
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| Enzyme Assay |
The true potency (Ki*) of the inhibitor is 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, an enzyme and an inhibitor pre-mix are incubated for 1 hour before being added to a GSK peptide (Ac-KKGGRARTSS-FAEPG-amide) and [33P] ATP. In a phospho-cellulose filter plate, the radio-labeled AKT peptide product is collected after the reaction has been shut down for two hours. By using the Sox-AKT-tide substrate (Ac-ARKRERAYSF-d-Pro-Sox-Gly-NH2), progress curve analysis continuously monitors the fluorescence of the product as it is formed.
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). 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. |
| Cell Assay |
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. 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. 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] |
| Animal Protocol |
Female athymic nude and SCID mice bearing SKOV3 or BT474 tumors[1]
100 mg/kg p.o. 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 population/average growth of vehicle-treated control population].[1] 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. |
| References | |
| Additional Infomation |
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. |
| Molecular Formula |
C18H17CL2FN4OS
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|---|---|
| Molecular Weight |
427.32
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| Exact Mass |
426.048
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| Elemental Analysis |
C, 50.59; H, 4.01; Cl, 16.59; F, 4.45; N, 13.11; O, 3.74; S, 7.50
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| CAS # |
1047644-62-1
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| Related CAS # |
Afuresertib hydrochloride;1047645-82-8; 1047644-62-1; 1047634-63-8 (Afuresertib-F free base); 2070009-64-0 (Afuresertib-F HCl)
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| PubChem CID |
46843057
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| Appearance |
White to off-white solid powder
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| LogP |
4.985
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
5
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
27
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| Complexity |
520
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| Defined Atom Stereocenter Count |
1
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| SMILES |
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
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| InChi Key |
AFJRDFWMXUECEW-LBPRGKRZSA-N
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| InChi Code |
InChI=1S/C18H17Cl2FN4OS/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)/t12-/m0/s1
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| Chemical Name |
N-[(2S)-1-amino-3-(3-fluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methylpyrazol-3-yl)thiophene-2-carboxamide
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| Synonyms |
Afuresertib free base; GSK 2110183; ASB183; ASB-183; GSK 2110183C; Afuresertib (GSK2110183); GSK2110183; GSK-2110183; GSK 2110183C; GSK2110183C; GSK2110183C
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| HS Tariff Code |
2934.99.9001
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| Storage |
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. |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.85 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. Solubility in Formulation 2: 2.5 mg/mL (5.85 mM) 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; with ultrasonication. 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (5.85 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.3402 mL | 11.7008 mL | 23.4017 mL | |
| 5 mM | 0.4680 mL | 2.3402 mL | 4.6803 mL | |
| 10 mM | 0.2340 mL | 1.1701 mL | 2.3402 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
| NCT Number | Status | Interventions | Conditions | Sponsor/Collaborators | Start Date | Phases |
| NCT05383482 | Recruiting | Drug: Afuresertib Drug: Docetaxel |
Solid Tumor NSCLC |
Laekna Limited | June 30, 2022 | Phase 1 Phase 2 |
| NCT05390710 | Recruiting | Drug: Nab-paclitaxel | Solid Tumor | Laekna Limited | June 12, 2021 | Phase 1 Phase 2 |
| NCT04851613 | Active Recruiting |
Drug: Afuresertib | Breast Cancer | Laekna LLC | February 18, 2022 | Phase 1 |
| NCT04060394 | Active Recruiting |
Drug: Phase I and Phase II:LAE001/prednisone + afuresertib |
Metastatic Castration- resistant Prostate Cancer |
Laekna Limited | September 13, 2019 | Phase 1 Phase 2 |
| NCT04374630 | Active Recruiting |
Drug: Paclitaxel Drug: Afuresertib |
Platinum-resistant Ovarian Cancer |
Laekna Limited | June 9, 2020 | Phase 2 |
Effect of GSK2110183 on AKT signaling and growth inhibition in human cancer cell lines. PLoS One, 2014, 9(6):e100880. |
The impact of GSK2110183 and GSK2141795 on glucose homeostasis in vivo. |
Combination anti-tumor effect of AKT and MEK inhibitors in mouse models of pancreatic cancer. |
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