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Purity: ≥98%
GSK2110183 analog, an analog of Afuresertib, is a potent, orally bioavailable and ATP-competitive Akt inhibitor with Ki of 0.08 nM, 2 nM, and 2.6 nM for Akt1, Akt2, and Akt3, respectively. GSK2110183 is a protein kinase B (Akt) inhibitor with potential anticancer properties. 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 |
Akt1 (Ki = 0.08 nM); Akt2 (Ki = 2 nM); Akt3 (Ki = 2.6 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 M). In response to afuresertib, 21% of tested solid tumor cell lines have an EC50 1 M. [1]
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| ln Vivo |
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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| Enzyme Assay |
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. |
| Cell Assay |
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.
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
100 mg/kg p.o. 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 |
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.[1]
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| Molecular Formula |
C₁₈H₁₆CL₂F₂N₄OS
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|---|---|
| Molecular Weight |
445.31
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| Exact Mass |
444.039
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| Elemental Analysis |
C, 48.55; H, 3.62; Cl, 15.92; F, 8.53; N, 12.58; O, 3.59; S, 7.20
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| CAS # |
1047634-63-8
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| Related CAS # |
GSK2110183 analog 1 hydrochloride;2070009-64-0; 1047644-62-1 ; 1047645-82-8 (HCl); 1047634-63-8 (Afuresertib-F free base)
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| PubChem CID |
57826797
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| Appearance |
Solid powder
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| Density |
1.5±0.1 g/cm3
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| Boiling Point |
535.1±50.0 °C at 760 mmHg
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| Flash Point |
277.4±30.1 °C
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| Vapour Pressure |
0.0±1.4 mmHg at 25°C
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| Index of Refraction |
1.674
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| LogP |
3.29
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
28
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| Complexity |
552
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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(=C(C=2[H])F)F)=O)S1
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| InChi Key |
AHDFWNJLFALBJP-JTQLQIEISA-N
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| InChi Code |
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
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| Chemical Name |
N-[(2S)-1-amino-3-(3,4-difluorophenyl)propan-2-yl]-5-chloro-4-(4-chloro-2-methylpyrazol-3-yl)thiophene-2-carboxamide
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| Synonyms |
Afuresertib-F; Afuresertib-F free base; GSK-2110183-analog; GSK 2110183-analog; GSK2110183-analog
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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 |
| 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) |
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.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] Oral Formulations
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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2456 mL | 11.2281 mL | 22.4563 mL | |
| 5 mM | 0.4491 mL | 2.2456 mL | 4.4913 mL | |
| 10 mM | 0.2246 mL | 1.1228 mL | 2.2456 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 |
| NCT01531894 | Completed | Drug: GSK2110183 (afuresertib) |
Cancer | Novartis Pharmaceuticals | February 8, 2012 | Phase 2 |
| NCT01428492 | Completed | Drug: GSK2110183 Drug: Bortezomib |
Multiple Myeloma | Novartis | December 2011 | Phase 1 |
| NCT01476137 | Completed | Drug: GSK1120212 Drug: GSK2110183 |
Cancer | GlaxoSmithKline | October 26, 2011 | Phase 1 |
| NCT00881946 | Completed | Drug: GSK21110183 | Hematologic Malignancies | Accenture | July 2009 | Phase 1 Phase 2 |
| NCT01395004 | Completed | Drug: GSK2110183 | Langerhans Cell Histiocytosis | GlaxoSmithKline | November 2011 | Phase 2 |
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