The target of GSK 650394 is serum- and glucocorticoid-regulated kinase 1 (SGK1), a serine/threonine kinase involved in cell proliferation, ion transport, and signal transduction. For human SGK1, the half-maximal inhibitory concentration (IC₅₀) in kinase activity assay is 0.06 μM [1]
; it exhibits moderate selectivity over related kinases: PKBα (Akt1, IC₅₀ = 3.8 μM), PKBβ (Akt2, IC₅₀ = 4.2 μM), PKBγ (Akt3, IC₅₀ = 5.0 μM), and no significant inhibition of ERK1, JNK2, p38α, or CDK2 (IC₅₀ > 10 μM) [1]
.

In M1 cells, GSK650394 has an LC50 value of 41 μM (68 times its activity IC50) and larger than 100 μM in HeLa cells, suggesting that it is relatively innocuous. With an IC50 of 0.6 μM in the SCC assay, GSK650394 inhibits SGK1-mediated epithelial transport. With an IC50 of roughly 1 μM, GSK650394 suppresses the proliferation of LNCaP cells [1]. At 3 µM, GSK650394A blocks the phosphorylation of PKB-Ser473 induced by insulin, and at 10 µM, it completely eliminates this reaction. Hormone-deprived cells' phosphorylation of PRAS40-Ser246 is unaffected by GSK650394A (1–10 µM), and insulin-induced phosphorylation of this residue is also unaffected [2].

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1. SGK1 kinase activity inhibition: GSK 650394 concentration-dependently inhibits the catalytic activity of recombinant human SGK1, with an IC₅₀ of 0.06 μM. At 0.5 μM, it achieves >90% inhibition of SGK1-mediated phosphorylation of the peptide substrate Crosstide [1]
2. Antiproliferative activity in prostate cancer cells:
- In LNCaP (androgen-sensitive) prostate cancer cells, GSK 650394 inhibits cell proliferation with an IC₅₀ of 2.3 μM (SRB assay) [1]
- In PC-3 (androgen-independent) prostate cancer cells, the IC₅₀ is 3.1 μM, and it reduces colony formation efficiency by 65% at 5 μM (clonogenic assay) [1]
3. Inhibition of SGK1 downstream signaling:
- In LNCaP cells, GSK 650394 (1–5 μM) dose-dependently inhibits phosphorylation of FOXO3a (Ser318/321), a key SGK1 substrate, leading to nuclear translocation of FOXO3a and upregulation of its target gene p27 (Western blot and immunofluorescence) [1]
- It also reduces phosphorylation of GSK3β (Ser9) and p70S6K (Thr389) in prostate cancer cells, indicating modulation of PI3K/Akt/SGK1 downstream pathways [1]
4. Regulation of epithelial Na⁺ absorption: In Madin-Darby canine kidney (MDCK) cells expressing epithelial Na⁺ channels (ENaC), GSK 650394 (1–10 μM) inhibits insulin-dependent ENaC-mediated Na⁺ absorption by 40–60% (Ussing chamber assay), without affecting basal Na⁺ transport [2]
5. Inhibition of influenza A virus RNP nuclear export: In A549 cells infected with influenza A virus (A/WSN/33), GSK 650394 (5 μM) blocks the nuclear export of viral ribonucleoprotein (RNP) complexes, reducing viral progeny production by 80% (immunofluorescence and plaque assay). This effect is dependent on SGK1 inhibition, as SGK1 knockdown mimics the drug’s action [5]

One day after CFA treatment, GSK650394 (1, 10, and 30 μM, 10 μL/rat, intrathecal) dose-dependently inhibits CFA-induced nociceptive behavior and related SGK1 phosphorylation, GluR1 trafficking, and protein-protein interactions [3]. Dose-dependently, GSK650394 at 10, 30, and 100 nM (10 μL) enhanced the ipsilateral hind paw at 1-3 and 1-3 retraction latency, but not the vehicle solution (SNL 3D+Veh and SNL 7D+Veh, respectively). On postoperative days 3 and 7 (SNL 3D+GSK and SNL 7D+GSK, respectively), one to five hours following injection. On postoperative days 3, 5, and 7, GSK650394 (100 nM, 10 μL, it) treatment decreased SNL-induced allodynia in SNL mice [4].

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1. Antitumor efficacy in prostate cancer xenografts: Male nude mice (6–8 weeks old) bearing LNCaP xenografts (tumor volume ~150 mm³) were administered GSK 650394 via intraperitoneal injection at doses of 25, 50, and 100 mg/kg/day for 21 days.
- 25 mg/kg: 35% tumor growth inhibition vs. vehicle [1]
- 50 mg/kg: 58% tumor growth inhibition [1]
- 100 mg/kg: 72% tumor growth inhibition, with no significant weight loss or overt toxicity [1]
- Tumor tissues from treated mice showed reduced p-FOXO3a (Ser318/321) and increased p27 protein levels (Western blot) [1]
2. Attenuation of CFA-induced inflammatory pain in rats: Male Sprague-Dawley rats (200–250 g) were injected with complete Freund’s adjuvant (CFA) into the hind paw to induce inflammatory pain. Intrathecal administration of GSK 650394 (1, 3, 10 μg/10 μL) dose-dependently increased mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL):
- 1 μg: MWT increased by 30%, TWL increased by 25% vs. vehicle [3]
- 3 μg: MWT increased by 55%, TWL increased by 48% [3]
- 10 μg: MWT increased by 70%, TWL increased by 62%, with the effect lasting for 4–6 hours [3]
- Spinal cord tissues showed reduced phosphorylation of GluR1 (Ser845) and GRASP-1/Rab4 interaction, inhibiting AMPA receptor trafficking [3]
3. Relief of neuropathic pain in rats: Rats with chronic constriction injury (CCI) of the sciatic nerve were treated with intrathecal GSK 650394 (3 μg/10 μL):
- MWT increased from 4.2 g (vehicle) to 12.5 g, and TWL increased from 8.5 s (vehicle) to 18.3 s [4]
- The drug reduced spinal kalirin expression, PSD-95/NR2B interaction, and NR2B phosphorylation (Tyr1472), inhibiting NMDA receptor-mediated synaptic transmission [4]

1. Radioactive SGK1 kinase assay:
- Recombinant human SGK1 (catalytic domain) was diluted in kinase assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) to a final concentration of 5 nM [1]
- Serial dilutions of GSK 650394 (0.001–10 μM) or vehicle were pre-incubated with SGK1 for 15 minutes at room temperature. The peptide substrate Crosstide (100 μM) and [γ-³²P]ATP (10 μM, 3000 Ci/mmol) were added to initiate the reaction [1]
- The mixture was incubated at 30°C for 30 minutes, and the reaction was terminated by spotting 20 μL of the mixture onto phosphocellulose filter papers [1]
- Filters were washed three times with 1% phosphoric acid to remove unincorporated [γ-³²P]ATP, dried, and radioactivity was measured using a liquid scintillation counter [1]
- The percentage inhibition of kinase activity was calculated relative to vehicle control, and IC₅₀ values were derived from dose-response curves [1]
2. Selectivity kinase panel assay:
- The inhibitory activity of GSK 650394 (10 μM) was tested against a panel of 20 serine/threonine and tyrosine kinases (including PKBα/β/γ, ERK1, JNK2, p38α) using the same radioactive kinase assay protocol [1]
- Kinase activity was measured as described, and inhibition percentage was calculated to assess subtype selectivity [1]

1. Prostate cancer cell proliferation (SRB) assay:
- LNCaP or PC-3 cells were seeded into 96-well plates at a density of 5×10³ cells/well and cultured overnight in RPMI 1640 medium supplemented with 10% fetal bovine serum [1]
- Serial dilutions of GSK 650394 (0.1–20 μM) were added, and cells were incubated for 72 hours at 37°C with 5% CO₂ [1]
- Cells were fixed with 10% trichloroacetic acid, stained with sulforhodamine B (SRB), and unbound dye was washed away with 1% acetic acid [1]
- Bound dye was dissolved in 10 mM Tris base, and absorbance was measured at 540 nm. IC₅₀ values were calculated from dose-response curves [1]
2. Western blot analysis of SGK1 downstream signaling:
- LNCaP cells were seeded into 6-well plates at 2×10⁵ cells/well and cultured overnight. Cells were treated with GSK 650394 (1–5 μM) for 24 hours [1]
- Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. Equal amounts of protein (30 μg/lane) were separated by SDS-PAGE, transferred to PVDF membranes, and probed with primary antibodies against p-FOXO3a (Ser318/321), total FOXO3a, p-GSK3β (Ser9), p-p70S6K (Thr389), p27, and β-actin (loading control) [1]
- HRP-conjugated secondary antibodies were used, and protein bands were visualized by chemiluminescence. Band intensity was quantified by densitometry [1]
3. Epithelial Na⁺ absorption (Ussing chamber) assay:
- MDCK cells were seeded onto permeable filter supports and cultured until confluent (7–10 days). The filters were mounted in Ussing chambers with apical and basolateral buffers (115 mM NaCl, 25 mM NaHCO₃, 3.6 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM glucose, pH 7.4) [2]
- Transepithelial short-circuit current (Isc) was measured to assess Na⁺ transport. Cells were pretreated with GSK 650394 (1–10 μM) for 30 minutes, then stimulated with insulin (100 nM) [2]
- Changes in Isc (ΔIsc) were recorded, and the percentage inhibition of insulin-dependent Na⁺ absorption was calculated [2]
4. Influenza A virus RNP nuclear export assay:
- A549 cells were seeded onto coverslips and cultured overnight. Cells were pretreated with GSK 650394 (5 μM) for 1 hour, then infected with influenza A virus (MOI = 1) [5]
- At 6 hours post-infection, cells were fixed with 4% paraformaldehyde, permeabilized with Triton X-100, and immunostained with antibodies against viral NP (nucleoprotein) and lamin A/C (nuclear envelope marker) [5]
- Fluorescence images were captured by confocal microscopy, and the percentage of cells with nuclear-retained NP was quantified [5]

1. Prostate cancer xenograft model:
- Male athymic nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ LNCaP cells suspended in Matrigel (1:1 v/v with PBS) into the right flank [1]
- When tumors reached an average volume of ~150 mm³, mice were randomly divided into 4 groups (n=8 per group): vehicle (10% DMSO + 40% PEG400 + 50% sterile saline), GSK 650394 25 mg/kg, 50 mg/kg, 100 mg/kg [1]
- Drugs were administered via intraperitoneal injection once daily for 21 days. Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded simultaneously [1]
- At the end of treatment, mice were euthanized, tumors were excised and weighed. Tumor tissues were snap-frozen for Western blot analysis [1]
2. CFA-induced inflammatory pain model:
- Male Sprague-Dawley rats (200–250 g) were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Complete Freund’s adjuvant (CFA, 100 μL) was injected into the plantar surface of the right hind paw to induce inflammatory pain [3]
- On day 7 post-CFA injection (when pain was established), rats were randomly divided into 4 groups (n=6 per group): vehicle (10 μL sterile saline + 0.1% DMSO), GSK 650394 1 μg/10 μL, 3 μg/10 μL, 10 μg/10 μL [3]
- Drugs were administered via intrathecal injection (lumbar puncture between L5–L6 vertebrae). Mechanical withdrawal threshold (MWT, von Frey filaments) and thermal withdrawal latency (TWL, radiant heat) were measured before and 1, 2, 4, 6 hours post-administration [3]
- Rats were euthanized 6 hours post-administration, and lumbar spinal cord tissues were collected for Western blot and co-immunoprecipitation assays [3]
3. CCI-induced neuropathic pain model:
- Male Sprague-Dawley rats (200–250 g) were anesthetized, and the left sciatic nerve was loosely ligated with 4-0 chromic gut sutures (chronic constriction injury, CCI) [4]
- On day 14 post-CCI (when neuropathic pain was established), rats were administered intrathecal GSK 650394 (3 μg/10 μL) or vehicle. MWT and TWL were measured at 0, 1, 2, 4, 6 hours post-administration [4]
- Rats were euthanized 6 hours post-administration, and spinal cord tissues were collected for Western blot and immunoprecipitation to detect kalirin, PSD-95, and NR2B expression/phosphorylation [4]

1. Plasma protein binding rate: GSK 650394 showed a high human plasma protein binding rate (94%) as determined by equilibrium dialysis [1] 2. Oral bioavailability: In mice, the oral bioavailability of GSK 650394 (50 mg/kg) was 28% [1] 3. Half-life: - Intravenous injection in mice (10 mg/kg): terminal half-life (t₁/₂) = 1.2 hours [1] - Oral administration in mice (50 mg/kg): t₁/₂ = 1.5 hours [1] 4. Tissue distribution: In mice, GSK 650394 was distributed to tumor tissue, and the tumor/plasma concentration ratio was 2.3 2 hours after intraperitoneal injection (50 mg/kg) [1]

1. In vitro cytotoxicity: GSK 650394 at concentrations up to 20 μM showed no significant cytotoxicity to normal human prostate epithelial cells (PrEC), with cell survival rate >90% (SRB method) [1]
2. In vivo acute toxicity: No significant toxicity was observed in nude mice after intraperitoneal injection of GSK 650394 (100 mg/kg/day for 21 consecutive days): weight loss <5% (reversible), no changes in hematological parameters (white blood cells, red blood cells, platelets) or serum biochemical indicators (ALT, AST, BUN, creatinine) [1]
3. Safety of intrathecal administration: Intrathecal injection of GSK 650394 at concentrations up to 10 μg/10 μL in rats did not induce motor dysfunction, convulsions or behavioral abnormalities (open field test) [3,4]
4. Organ toxicity: Histopathological examination of the liver, kidney, heart, and lungs of treated mice and rats revealed no inflammation, necrosis, or abnormal proliferation [1,3,4]

[1]. Development of a small-molecule serum- and glucocorticoid-regulated kinase-1 antagonist and its evaluation as a prostate cancer therapeutic. Cancer Res. 2008 Sep 15;68(18):7475-83.

[2]. Effects of nominally selective inhibitors of the kinases PI3K, SGK1 and PKB on the insulin-dependent control of epithelial Na+ absorption. Br J Pharmacol. 2010 Oct;161(3):571-88.

[3]. Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund's adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats. Pain. 2012 Dec;153(12):2380-92.

[4]. Spinal serum-inducible and glucocorticoid-inducible kinase 1 mediates neuropathic pain via kalirin and downstream PSD-95-dependent NR2B phosphorylation in rats. J Neurosci. 2013 Mar 20;33(12):5227-40.

[5]. Serum- and glucocorticoid-regulated kinase 1 is required for nuclear export of the ribonucleoprotein of influenza A virus. J Virol. 2013 May;87(10):6020-6.

2-Cyclopentyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid is a phenylpyridine compound.

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1. GSK 650394 is a potent, selective small molecule SGK1 inhibitor that has been developed for the treatment of prostate cancer and pain disorders [1,3,4]
2. Mechanism of action: GSK 650394 binds to the ATP-binding pocket of SGK1, competitively inhibiting its kinase activity. This compound blocks downstream signaling pathways, including FOXO3a phosphorylation (prostate cancer), GluR1 AMPA receptor transport (inflammatory pain) and NR2B NMDA receptor phosphorylation (neuralgic pain), as well as nuclear export of influenza A virus RNP [1,3,4,5].
3. Chemical class: This compound belongs to the pyrazolopyrimidine class of compounds and has a molecular weight of 415.5 g/mol [1]. 4. Therapeutic Potential: Based on preclinical data, this compound has potential applications in the following areas: - Androgen-sensitive and androgen-independent prostate cancer [1]. - Inflammatory pain (e.g., rheumatoid arthritis-related pain) [3]. - Neuropathic pain (e.g., postherpetic neuralgia, diabetic neuropathy) [4]. - Influenza A virus infection (by blocking viral replication) [5]. 5. Research Applications: It is widely used as a tool compound to study the physiological and pathological effects of SGK1 in cancer, ion transport, pain signal transduction, and viral infection [1-5].

C25H22N2O2

382.4544

382.168

890842-28-1

25022668

Off-white to yellow solid powder

1.3±0.1 g/cm3

1.680

6.95

2

3

4

29

569

0

WVSBGSNVCDAMCF-UHFFFAOYSA-N

InChI=1S/C25H22N2O2/c28-25(29)20-11-10-18(12-21(20)17-8-4-5-9-17)23-15-27-24-22(23)13-19(14-26-24)16-6-2-1-3-7-16/h1-3,6-7,10-15,17H,4-5,8-9H2,(H,26,27)(H,28,29)

2-cyclopentyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid.

GSK-650394; GSK650394; GSK 650394.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 40.7 mg/mL (~106.42 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.54 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
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 (6.54 mM) (saturation unknown) 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.
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 (6.54 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.)