Akt3 (IC50 = 38 nM); Akt1 (IC50 = 180 nM); Akt2 (IC50 = 328 nM); ROCK1 (IC50 = 1570 nM); ROCK2 (IC50 = 1850 nM); CDK7 (IC50 = 2100 nM)
GSK2141795 targets Akt1, Akt2, and Akt3 (pan-Akt inhibitor; IC50 for growth inhibition: 5–15 μM in EGFR-TKI-resistant NSCLC cell lines); downstream inhibition includes phospho-PRAS40 and phospho-FOXO1/3a.

In both BT474 and LNCaP cells, uprosertib inhibits multiple AKT substrate phosphorylation levels, including GSK3β, PRAS40, FOXO, and Caspase 9. When the AKT pathway is activated in human cancer cell lines, uprosertib preferentially prevents cell proliferation. Uprosertib also results in cell cycle arrest in the cell lines LNCaP, BT474, A3, and I9.2. [2] Uprosertib inhibits growth as a single agent in SKOV3 and PEO4 cells and increases cisplatin-induced apoptosis.

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Kinase Inhibitor Selectivity Profile [1]
To demonstrate the utility of the new kinobeads KBβ, we subjected the Akt inhibitors GSK690693 (phase I, terminated) and GSK2141795 (active phase I trials in patients with solid tumors or lymphomas) to kinase selectivity profiling in mixed lysates of four cancer cells (see Materials and Methods section). Briefly, KBβ pulldowns were performed after preincubation of separate lysates with increasing concentrations of the respective drug. Protein targets that bind the drug in the lysate show a dose-dependent reduction in binding to the kinobeads, while proteins unaffected by the drug show no reduction in binding. Proteins were eluted from kinobeads, digested with trypsin into peptides, and analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Following protein identification by database searching, proteins were quantified by use of their LC-MS/MS intensities, and dose–response curves were generated from the resulting data (Figure S5, Supporting Information). The results of these experiments show clear similarities as well as differences in the selectivity profiles of the two compounds. Both expectedly show inhibition of Akt1 and 2 (Figure 3A,B and Table 2; Akt3 was detected only in assays with GSK2141795), and IC50 values of 138 nM (Akt1) and 128 nM (Akt2) were determined for GSK690693. GSK2141795 inhibited kinobead binding with IC50 values of 180 nM for Akt1, 328 nM for Akt2, and 38 nM for Akt3 (Table 2). We note here that IC50 values for target affinities determined in such competition binding assays are often affected by the partial depletion of individual target proteins from the lysate. This can be compensated for by calculating a target-specific KD value using a correction factor derived from the Cheng–Prusoff equation. This results in KD values of 16 nM for Akt1, 49 nM for Akt2, and 5 nM for Akt3 (for GSK2141795), which are in line with literature data from biochemical assays that report potencies of 2 nM for Akt1, 2–13 nM for Akt2, and 3–9 nM Akt3 for GSK690693.

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In Vitro [2]
GSK2141795 inhibited cell growth as a single agent in EGFR-TKI-resistant NSCLC cell lines (e.g., PC9-derived GR1-5 and ER lines, 11–18-derived GR1-GR6 lines) with IC50 values ranging from 5 to 15 μM, as measured by MTT, crystal violet, and CellTiterBlue assays. Growth inhibition was assessed over 72 hours to 8 days.

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Combined with EGFR TKIs (erlotinib or gefitinib), GSK2141795 induced synergistic growth inhibition (CI < 1) in resistant cell lines. Synergy was quantified via crystal violet viability assays (8-day incubation) and BrdU incorporation assays (48- and 96-hour incubation), showing significant reduction in cell viability and proliferation compared to monotherapy (p < 0.05, ANOVA).

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GSK2141795 enhanced apoptosis in resistant cell lines when combined with EGFR TKIs, as demonstrated by DNA fragmentation ELISA assays after 96-hour treatment. Apoptosis levels increased significantly versus single-agent treatments (p < 0.05).

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Western blot analysis confirmed that GSK2141795 alone suppressed phosphorylation of Akt downstream targets (pPRAS40 and pFOXO1/3a) in PC9 and 11–18 cell lines within 4 hours. Combined with EGFR TKIs, it achieved simultaneous inhibition of EGFR, pPRAS40, and pFOXO1/3a, while increasing pAkt S473 due to feedback hyperphosphorylation.

Uprosertib (100 mg/kg, p.o.) results in a 61% inhibition of tumor growth in mice with BT474 breast tumor xenografts. Uprosertib (30 mg/kg, p.o.) results in a 61% inhibition of tumor growth in mice with SKOV3 ovarian tumor xenografts.

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In Vivo [2]
In CB17 SCID mouse xenograft models (subcutaneously inoculated with EGFR-TKI-resistant PC9-ER cells), GSK2141795 monotherapy (10 µg/g bodyweight, oral gavage, 5 days/week) showed modest tumor growth inhibition versus vehicle.

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Combined with erlotinib (25 µg/g bodyweight), GSK2141795 significantly suppressed tumor growth (p = 0.0017 vs. vehicle; p = 0.03 vs. erlotinib alone; p = 0.0295 vs. GSK2141795 alone). Tumor volumes were measured weekly with calipers and calculated as (length × width²)/2, with excision at day 36 confirming reduced volumes.

The lysates (5 mg of total protein each) are preincubated for 45 minutes at 4 °C with the following free compounds: DMSO control, 2.5 nM, 25 nM, 250 nM, 2.5 M, or 25 M (GSK690693 or GSK2141795). For both qualitative and quantitative experiments, lysates are then incubated with beads (coupled Akt probe or kinobeads) for 1 h at 4 °C. The beads are centrifuged to separate them after being cleaned with a 1 CP buffer. Using 2 NuPAGE LDS sample buffer, bound proteins are eluted, and the eluates are then reduced and alkylated with 50 mM dithiothreitol and 55 mM iodoacetamide.

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Affinity Purification [1]
Akt probe and kinobead pulldowns were performed as described previously. Briefly, lysates of a cell mix were diluted with equal volumes of 1× compound pulldown (CP) buffer [50 mM Tris-HCl, pH 7.5, 5% glycerol, 1.5 mM MgCl2, 150 mM NaCl, 25 mM NaF, 1 mM dithiothreitol, and freshly added protease inhibitors and phosphatase inhibitors (5× phosphatase inhibitor cocktail 1, Germany; 5× phosphatase inhibitor cocktail 2, 1 mM sodium orthovanadate; and 20 nM calyculin A)]. If necessary, lysates were further diluted to a final protein concentration of 5 mg/mL in 1× CP buffer supplemented with 0.4% NP-40. For selectivity profiling experiments, the lysates (5 mg of total protein each) were preincubated with 0 (DMSO control), 2.5 nM, 25 nM, 250 nM, 2.5 μM or 25 μM free compound (GSK690693 or GSK2141795) on an end-over-end shaker for 45 min at 4 °C. Subsequently, lysates were incubated with beads (coupled Akt probe or kinobeads) for 1 h at 4 °C, for both qualitative and quantitative experiments. The beads were washed with 1× CP buffer and collected by centrifugation. Bound proteins were eluted with 2× NuPAGE LDS sample buffer, and eluates were reduced and alkylated by 50 mM dithiothreitol and 55 mM iodoacetamide. Samples were then run into a 4–12% NuPAGE gel for about 0.5 cm to concentrate the sample prior to in-gel tryptic digestion, which was performed according to standard procedures.

Cell lines are typically grown in 10% FBS-RPMI 160 medium. Some cell lines are grown in media that the vendor specifies. 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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For viability assays, cells (e.g., PC9, PC9-ER, 11–18, and resistant derivatives) were seeded at 4,000–10,000 cells/well in 96- or 24-well plates, allowed to attach for 6–24 hours, and treated with sub-inhibitory concentrations of GSK2141795 (1.25–3 μM), EGFR TKIs (e.g., gefitinib 5–40 nM for sensitive lines, 5–10 μM for resistant lines), or combinations. Viability was assessed after 48–96 hours using MTT (absorbance at 495 nm), crystal violet (absorbance at 570 nm after dissolution in Na-citrate buffer), or CellTiterBlue (metabolic activity).

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Proliferation was measured via BrdU incorporation assays: Cells were incubated with BrdU for 48–96 hours, fixed, and detected using anti-BrdU antibodies, with absorbance quantified.

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Apoptosis was evaluated using the Cell Death Detection ELISA^Plus kit (Roche): Cells were lysed after 96-hour treatment, and cytoplasmic histone-associated DNA fragments were quantified via spectrophotometry.

Mice bearing either BT474 or SKOV3 tumors
100 mg/kg
p.o.

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CB17 SCID mice (female, 16 weeks old) were subcutaneously inoculated with 1.5 × 10⁶ PC9-ER cells (resuspended in 1:1 Matrigel/RPMI medium) bilaterally. When tumors reached 2–3 mm diameter, mice were randomized into treatment groups (n = 9).

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GSK2141795 was formulated at 10 µg/g bodyweight in DMSO, diluted in 15% Capitsol (final DMSO ≤ 10%), and administered via oral gavage 5 days/week for 3 weeks. Erlotinib (25 µg/g bodyweight in 15% Capitsol) was co-administered for combination groups. Tumor volumes and body weights were monitored weekly; mice were euthanized for adverse events (e.g., weight loss, discomfort).[2]
Dosing: GSK2141795 (10 µg/g bodyweight) formulated in DMSO + 15% Captisol (final DMSO ≤ 10%); erlotinib (25 µg/g bodyweight) in 15% Captisol. [2]

[1]. Characterization of a chemical affinity probe targeting Akt kinases. J Proteome Res. 2013 Aug 2;12(8):3792-800.

[2]. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer. Nat Commun. 2017 Sep 4;8(1):410.

Uprosertib has been used in research for the treatment of various cancers, including melanoma, solid tumors, cervical cancer, and HER2/Neu-negative tumors. Uprosertib is an orally bioavailable serine/threonine protein kinase Akt (protein kinase B) inhibitor with potential antitumor activity. Uprosertib binds to Akt and inhibits its activity, thereby suppressing the PI3K/Akt signaling pathway and tumor cell proliferation, and inducing tumor cell apoptosis. Activation of the PI3K/Akt signaling pathway is commonly associated with tumorigenesis, while dysregulation of the PI3K/Akt signaling pathway may lead to resistance to multiple antitumor drugs in tumors. Protein kinases are key regulators of cellular processes, and their dysfunction is often associated with human diseases. Therefore, kinases are an important class of therapeutic targets, and approximately 20 kinase inhibitors (KIs) are currently used clinically. A thorough understanding of the selectivity of KIs is crucial for correctly interpreting their pharmacological and systems biology effects. Chemical proteomics has become an important molecular tool for the selective analysis of systemic kinase inhibitors, but its coverage of the human kinaseome remains incomplete. This article introduces a novel affinity probe targeting Akt and other members of the AGC kinase family, which significantly expands the application scope of chemical proteomics in kinase analysis. Combined with previously reported kinase magnetic beads, we applied the synthesized probe to the selective analysis of the Akt inhibitors GSK690693 and GSK2141795 in human cancer cells. The results confirmed the inhibitory effect of GSK690693 on all Akt subtypes and multiple known targets, and identified CDC42BPB as a novel potential target for GSK690693. This work also identifies for the first time the kinase selectivity profile of the phase I clinical drug GSK2141795, and identifies PRKG1 as a low nanomolar kinase target, and the ATP-dependent 5'-3' DNA helicase ERCC2 as a potential novel non-kinase off-target target. [1]

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Patients with epidermal growth factor receptor (EGFR) activating mutations typically benefit from EGFR tyrosine kinase inhibitor (EGFR) therapy. However, almost all patients eventually die from acquired EGFR tyrosine kinase inhibitor (EGFR) resistance developed through multiple mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor (EGFR) resistance mechanisms present challenges in developing new therapies to overcome EGFR tyrosine kinase inhibitor (EGFR) resistance. This study demonstrates that Akt activation is a common feature of acquired EGFR tyrosine kinase inhibitor (EGFR) resistance, spanning multiple known upstream resistance mechanisms. In various EGFR tyrosine kinase inhibitor (EGFR) resistant NSCLC models, combination therapy with EGFR tyrosine kinase inhibitors and Akt inhibitors induces apoptosis and produces synergistic growth inhibition. Furthermore, phosphorylated Akt levels are elevated in clinical specimens from most patients with EGFR-mutant NSCLC who are resistant to acquired EGFR tyrosine kinase inhibitors. Our findings provide a theoretical basis for conducting clinical trials of combination therapy with Akt and EGFR inhibitors in patients with elevated phosphorylated Akt levels, aiming to address the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms. EGFR-mutant non-small cell lung cancer is usually resistant to EGFR tyrosine kinase inhibitor therapy. This study shows that resistant tumors exhibit high levels of Akt activation, and that combination therapy with AKT inhibitors can produce synergistic tumor growth inhibition in vitro and in vivo. [2] Mechanistically, GSK2141795 blocks Akt-dependent survival pathways (e.g., PRAS40/mTORC1 and FOXO1/3a-mediated apoptosis), thereby inhibiting multiple resistance mechanisms (e.g., T790M mutation, MET/AXL upregulation, EMT). Preclinical studies have confirmed its efficacy: when used in combination with EGFR-TKIs, it can synergistically inhibit cell growth and induce apoptosis in resistant models, and phosphorylated Akt can serve as a predictive biomarker. Elevated pAkt levels were observed in 60% of patient samples after disease progression, supporting its clinical relevance. Potential indications include EGFR-mutant non-small cell lung cancer with high pAkt levels. No FDA approvals or warnings have been identified to date.

C18H16CL2F2N4O2

429.25

428.062

C, 50.37; H, 3.76; Cl, 16.52; F, 8.85; N, 13.05; O, 7.45

1047634-65-0

Uprosertib hydrochloride;1047635-80-2

51042438

White to off-white solid powder

4.656

2

6

6

28

550

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)O1

AXTAPYRUEKNRBA-JTQLQIEISA-N

InChI=1S/C18H16Cl2F2N4O2/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)furan-2-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: 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 (5.82 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 (5.82 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 (5.82 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.)