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)
Afuresertib (GSK-2110183) is a highly selective ATP-competitive inhibitor of the Akt kinase family. In recombinant enzyme assays, it exhibits IC50 values of 1.2 nM for Akt1, 3.8 nM for Akt2, and 2.5 nM for Akt3. It shows minimal cross-reactivity with other kinases (e.g., PKA, PKCα, EGFR) with IC50 values > 1000 nM [1]
- Afuresertib retains inhibitory activity against Akt mutants (e.g., Akt1 E17K, a common activating mutation in solid tumors) with an IC50 of 4.1 nM for Akt1 E17K, compared to 1.2 nM for wild-type Akt1 [2]

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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In human ovarian cancer cell lines (SKOV3, OVCAR-3) with constitutively activated Akt, Afuresertib (0.01-10 μM) inhibited cell proliferation in a dose-dependent manner. The IC50 values were 0.8 μM for SKOV3 cells and 1.3 μM for OVCAR-3 cells after 72 hours of treatment (MTT assay). Western blot analysis showed that 1 μM Afuresertib reduced phosphorylation of Akt (Ser473 and Thr308) by > 90% within 24 hours, and downregulated downstream targets including phospho-GSK-3β (Ser9, -75%) and phospho-mTOR (Ser2448, -80%) [1]
- In human breast cancer cell lines (MDA-MB-468, BT-474) harboring PI3K/Akt pathway alterations, Afuresertib (0.1-5 μM) induced apoptosis after 48 hours. Flow cytometry with Annexin V-FITC/PI staining revealed that 2 μM Afuresertib increased the apoptotic rate from 3% (control) to 40% in MDA-MB-468 cells. Additionally, 1 μM Afuresertib inhibited colony formation of BT-474 cells by 70% compared to vehicle control (crystal violet staining, 14-day culture) [2]
- In ovarian cancer SKOV3 cells co-treated with Afuresertib (0.5 μM) and paclitaxel (10 nM), the combination showed synergistic antiproliferative activity with a combination index (CI) of 0.58 (CI < 1 indicates synergy). The apoptotic rate in the combination group was 55%, significantly higher than 22% (afuresertib alone) and 18% (paclitaxel alone) [1]

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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In a nude mouse xenograft model of human ovarian cancer (SKOV3), Afuresertib was administered orally at doses of 25 mg/kg and 50 mg/kg once daily for 21 days. Compared to the vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80), the 25 mg/kg group showed a 55% reduction in tumor volume, and the 50 mg/kg group showed a 72% reduction. Immunohistochemical staining of tumor tissues demonstrated decreased phospho-Akt (Ser473) expression (-85%) and Ki-67 (proliferation marker) positive cells (-60%) in the 50 mg/kg group [1]
- In a nude mouse xenograft model of human breast cancer (MDA-MB-468), Afuresertib was administered intraperitoneally (i.p.) at 10 mg/kg and 20 mg/kg once daily for 14 days. The 10 mg/kg group had a 40% reduction in tumor weight, and the 20 mg/kg group had a 60% reduction. Western blot analysis of tumor lysates confirmed reduced phospho-mTOR (Ser2448) and increased cleaved caspase-3 (apoptosis marker) in treated groups [2]
- In the SKOV3 xenograft model, combination treatment with Afuresertib (25 mg/kg oral, once daily) and paclitaxel (10 mg/kg i.p., once weekly) for 21 days resulted in an 85% reduction in tumor volume, which was significantly greater than monotherapy effects (55% for afuresertib alone, 45% for paclitaxel alone) [1]

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.

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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.

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Akt1 Kinase Inhibition Assay: Recombinant human Akt1 (0.1 μg per reaction) was mixed with 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 10 μM ATP (including [γ-32P]ATP), 20 μM Crosstide (Akt-specific substrate peptide), and serial dilutions of Afuresertib (0.1 nM-100 nM) in a total volume of 50 μL. The reaction mixture was incubated at 30°C for 30 minutes, then terminated by adding 25 μL of 30% trichloroacetic acid. The precipitated phosphorylated peptide was transferred to P81 phosphocellulose filters, washed three times with 1% phosphoric acid, and dried. Radioactivity was measured using a liquid scintillation counter, and IC50 was calculated via four-parameter logistic regression [1]
- Akt1 E17K Mutant Kinase Assay: Recombinant human Akt1 E17K mutant (0.1 μg per reaction) was incubated with the same buffer system, ATP, and substrate as the wild-type Akt1 assay, along with Afuresertib (0.5 nM-50 nM). The reaction was conducted at 30°C for 45 minutes, terminated with trichloroacetic acid, and processed identically to the wild-type assay. IC50 was determined by plotting the percentage of remaining kinase activity against drug concentration [2]

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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Ovarian Cancer Cell Proliferation Assay (MTT Method): SKOV3 or OVCAR-3 cells were seeded in 96-well plates at a density of 5×10³ cells/well and cultured overnight at 37°C with 5% CO2. Afuresertib was added at concentrations ranging from 0.01 nM to 10 μM (10-point serial dilution), and cells were incubated for 72 hours. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added, followed by 4 hours of incubation. The medium was aspirated, 150 μL of DMSO was added to dissolve formazan crystals, and absorbance was measured at 570 nm. IC50 was defined as the concentration of Afuresertib that inhibited proliferation by 50% relative to vehicle control [1]
- Breast Cancer Apoptosis Assay (Annexin V-FITC/PI Staining): MDA-MB-468 cells were seeded in 6-well plates at 2×10⁵ cells/well and treated with Afuresertib (0.1-5 μM) for 48 hours. Cells were harvested by trypsinization, washed twice with cold PBS, and resuspended in 100 μL of Annexin V binding buffer. Five microliters of Annexin V-FITC and 5 μL of propidium iodide (PI) were added, and the mixture was incubated in the dark at room temperature for 15 minutes. Apoptotic cells were analyzed via flow cytometry within 1 hour, with early apoptosis defined as Annexin V-positive/PI-negative and late apoptosis as Annexin V-positive/PI-positive [2]
- Western Blot Analysis (Cellular): Cells were treated with Afuresertib (0.1-5 μM) for 24 hours, then lysed in RIPA buffer containing protease and phosphatase inhibitors. Protein concentration was determined using a BCA assay kit. Equal amounts of protein (30 μg per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes. Membranes were blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated overnight at 4°C with primary antibodies against phospho-Akt (Ser473, Thr308), total Akt, phospho-GSK-3β (Ser9), phospho-mTOR (Ser2448), cleaved caspase-3, or β-actin. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 1 hour, and protein bands were visualized using an ECL detection system. Band intensity was quantified via ImageJ software [1]

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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Ovarian Cancer Xenograft Model (SKOV3): Female nude mice (6-8 weeks old, n=6 per group) were subcutaneously injected with 2×10⁶ SKOV3 cells (suspended in 100 μL of PBS + 50% Matrigel) into the right hind flank. When tumors reached an average volume of 100 mm³, mice were randomly divided into four groups: vehicle control (0.5% carboxymethyl cellulose sodium + 0.1% Tween 80), Afuresertib 25 mg/kg, Afuresertib 50 mg/kg, and Afuresertib 25 mg/kg + paclitaxel 10 mg/kg. Afuresertib was suspended in the vehicle and administered orally once daily for 21 days; paclitaxel was dissolved in normal saline and administered intraperitoneally once weekly for 3 weeks. Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded weekly [1]
- Breast Cancer Xenograft Model (MDA-MB-468): Female nude mice (6-8 weeks old, n=5 per group) were subcutaneously injected with 3×10⁶ MDA-MB-468 cells (in 100 μL of PBS + 50% Matrigel) into the left flank. When tumors reached ~120 mm³, mice were assigned to three groups: vehicle control (5% DMSO + 95% normal saline), Afuresertib 10 mg/kg, and Afuresertib 20 mg/kg. Afuresertib was dissolved in the vehicle and administered intraperitoneally once daily for 14 days. At the end of the experiment, mice were euthanized, tumors were excised and weighed, and tumor lysates were prepared for Western blot analysis [2]

In male Sprague-Dawley (SD) rats, Afuzeilibu was administered via two routes: intravenous (iv) at a dose of 5 mg/kg and oral (po) at a dose of 20 mg/kg. Following intravenous administration, the plasma concentration-time curve conformed to a two-compartment model, with a terminal half-life (t1/2β) of 3.5 h, a steady-state volume of distribution (Vdss) of 2.8 L/kg, and a total clearance (CL) of 0.7 L/h/kg. After oral administration, the peak plasma concentration (Cmax) was 1.9 μg/mL, the time to peak concentration (Tmax) was 1.2 hours, and the oral bioavailability (F) was 22% [2]. In vitro plasma protein binding experiments using the equilibrium dialysis method showed that avzelib had a high plasma protein binding rate: 95% in human plasma, 93% in rat plasma, and 91% in canine plasma; the free fraction was <5% in all tested species [2]. In vitro metabolic studies using human liver microsomes showed that avzelib was metabolized into three minor metabolites (M1-M3), of which about 60% of the total metabolism was mediated by CYP3A4 (confirmed by specific CYP3A4 inhibitors) [2].

In a 28-day repeated-dose toxicity study, male and female Sprague-Dawley rats were orally administered Afuresertib once daily at doses of 10 mg/kg, 20 mg/kg, and 40 mg/kg, respectively. In the 40 mg/kg dose group, both male and female rats experienced a 10% decrease in body weight and a 1.5-fold increase in serum ALT (alanine aminotransferase) levels compared to the control group, but no histopathological changes were observed in the liver. No significant toxicity was observed at doses of 10 mg/kg or 20 mg/kg (no weight loss, no abnormal liver enzymes) [2]
- In the SKOV3 ovarian cancer xenograft model, doses of Afuresertib up to 50 mg/kg (oral, 21 days) did not cause significant changes in weight or significant pathological abnormalities in major organs (liver, kidney, heart, lungs) [1]
- In vitro cytotoxicity assays in normal human peripheral blood mononuclear cells (PBMCs) showed that Afuresertib had a CC50 of 15 μM and a therapeutic index (TI = CC50/IC50) of 18.8 (compared to IC50 = 0.8 μM in SKOV3 cells) [1]

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

[2]. Cancer Med. 2017 Nov;6(11):2646-2659.

N-[(2S)-1-amino-3-(3-fluorophenyl)propyl-2-yl]-5-chloro-4-(4-chloro-2-methyl-3-pyrazolyl)-2-thiophene carboxamide belongs to the amphetamine class of drugs. Afuritinib has been used in clinical trials to investigate the treatment of cancer and tumors, including hematologic malignancies. Afuritinib is an orally bioavailable serine/threonine protein kinase Akt (protein kinase B) inhibitor with potential antitumor activity. Afuritinib binds to Akt and inhibits its activity, thereby inhibiting the PI3K/Akt signaling pathway and tumor cell proliferation, and inducing tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is generally associated with tumorigenesis, and abnormal activation of the PI3K/Akt signaling pathway may lead to tumor resistance to multiple antitumor drugs.

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Afuresertib (GSK-2110183) is a potent, selective ATP-competitive Akt kinase family inhibitor for the treatment of solid tumors (e.g., ovarian, breast, and prostate cancer) with aberrant activation of the PI3K/Akt/mTOR signaling pathway [1][2].
- Preclinical studies have shown that Afuresertib can overcome chemotherapy resistance (e.g., paclitaxel resistance in ovarian cancer) by blocking Akt-mediated survival pathways, which are typically upregulated in chemotherapy-resistant tumors [1].
- Afuresertib is active against Akt activating mutations (e.g., Akt1). E17K is associated with poor prognosis in breast and ovarian cancers and is therefore a candidate target for targeted therapy in patients carrying these mutations [2].

C18H17CL2FN4OS

427.32

426.048

C, 50.59; H, 4.01; Cl, 16.59; F, 4.45; N, 13.11; O, 3.74; S, 7.50

1047644-62-1

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

46843057

White to off-white solid powder

4.985

2

5

6

27

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

AFJRDFWMXUECEW-LBPRGKRZSA-N

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

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

Afuresertib free base; GSK 2110183; ASB183; ASB-183; GSK 2110183C; Afuresertib (GSK2110183); GSK2110183; GSK-2110183; GSK 2110183C; GSK2110183C; GSK2110183C

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 (5.85 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.

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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.

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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.
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.)