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 trials studying the treatment of Cancer, Melanoma, Solid Tumours, Cervical Cancer, and HER2/Neu Negative, among others.
Uprosertib is an orally bioavailable inhibitor of the serine/threonine protein kinase Akt (protein kinase B) with potential antineoplastic activity. Uprosertib 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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Protein kinases are key regulators of cellular processes, and aberrant function is often associated with human disease. Consequently, kinases represent an important class of therapeutic targets and about 20 kinase inhibitors (KIs) are in clinical use today. Detailed knowledge about the selectivity of KIs is important for the correct interpretation of their pharmacological and systems biological effects. Chemical proteomic approaches for systematic kinase inhibitor selectivity profiling have emerged as important molecular tools in this regard, but the coverage of the human kinome is still incomplete. Here, we describe a new affinity probe targeting Akt and many other members of the AGC kinase family that considerably extends the scope of KI profiling by chemical proteomics. In combination with the previously published kinobeads, the synthesized probe was applied to selectivity profiling of the Akt inhibitors GSK690693 and GSK2141795 in human cancer cells. The results confirmed the inhibition of all Akt isoforms and of a number of known as well as CDC42BPB as a novel putative target for GSK690693. This work also established, for the first time, the kinase selectivity profile of the clinical phase I drug GSK2141795 and identified PRKG1 as a low nanomolar kinase target as well as the ATP-dependent 5'-3' DNA helicase ERCC2 as a potential new non-kinase off-target. [1]

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Non-small-cell lung cancer patients with activating epidermal growth factor receptor (EGFR) mutations typically benefit from EGFR tyrosine kinase inhibitor treatment. However, virtually all patients succumb to acquired EGFR tyrosine kinase inhibitor resistance that occurs via diverse mechanisms. The diversity and unpredictability of EGFR tyrosine kinase inhibitor resistance mechanisms presents a challenge for developing new treatments to overcome EGFR tyrosine kinase inhibitor resistance. Here, we show that Akt activation is a convergent feature of acquired EGFR tyrosine kinase inhibitor resistance, across a spectrum of diverse, established upstream resistance mechanisms. Combined treatment with an EGFR tyrosine kinase inhibitor and Akt inhibitor causes apoptosis and synergistic growth inhibition in multiple EGFR tyrosine kinase inhibitor-resistant non-small-cell lung cancer models. Moreover, phospho-Akt levels are increased in most clinical specimens obtained from EGFR-mutant non-small-cell lung cancer patients with acquired EGFR tyrosine kinase inhibitor resistance. Our findings provide a rationale for clinical trials testing Akt and EGFR inhibitor co-treatment in patients with elevated phospho-Akt levels to therapeutically combat the heterogeneity of EGFR tyrosine kinase inhibitor resistance mechanisms.EGFR-mutant non-small cell lung cancer are often resistant to EGFR tyrosine kinase inhibitor treatment. In this study, the authors show that resistant tumors display high Akt activation and that a combined treatment with AKT inhibitors causes synergistic tumour growth inhibition in vitro and in vivo. [2]

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Mechanistically, GSK2141795 blocks Akt-dependent survival pathways (e.g., PRAS40/mTORC1 and FOXO1/3a-mediated apoptosis), converging diverse resistance mechanisms (e.g., T790M, MET/AXL upregulation, EMT).

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Efficacy was demonstrated preclinically: Combined with EGFR TKIs, it synergistically inhibited growth and induced apoptosis in resistant models, with phospho-Akt as a predictive biomarker. Clinical relevance was supported by elevated pAkt in 60% of post-progression patient samples.

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Potential indications include EGFR-mutant NSCLC with high pAkt levels. No FDA approvals or warnings were noted.

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.)