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| Targets |
Serum- and glucocorticoid-regulated kinase 1 (SGK1).
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| ln Vitro |
SGK1-IN-2 (14h) is a selective inhibitor of serum- and glucocorticoid-regulated kinase 1 (SGK1), with an IC50 of 5 nM at an ATP concentration of 10 uM. The compound is designed to be selective for SGK1 over other closely related kinases, such as AKT, PKC, and p70S6K, although the full selectivity profile is not explicitly detailed. By binding to the ATP-binding pocket of SGK1, it prevents the phosphorylation and activation of downstream substrates. These substrates include N-myc downstream-regulated gene 1 (NDRG1) and the ubiquitin ligase NEDD4-2, which regulate ion channels and transporters. Inhibition of SGK1 by SGK1-IN-2 has been shown to reduce the viability and proliferation of several cancer cell lines in vitro, including those derived from breast, prostate, and colon cancers. It also promotes apoptosis in certain contexts. The compound is a valuable tool for dissecting the specific contributions of SGK1 signaling to cellular physiology.
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| ln Vivo |
The in vivo efficacy of SGK1-IN-2 has been evaluated in preclinical models of cancer and metabolic disease. In mouse xenograft models of human tumors that express high levels of SGK1, such as triple-negative breast cancer (TNBC), oral administration of SGK1-IN-2 resulted in significant inhibition of tumor growth. This effect was associated with reduced phosphorylation of NDRG1 (a biomarker of SGK1 activity) in the tumor tissue, confirming on-target engagement. In models of metabolic syndrome, SGK1-IN-2 treatment has been shown to improve insulin sensitivity and reduce blood pressure. These studies support the concept that SGK1 is a valid target for cancer and metabolic diseases. However, the compound is still in the preclinical research phase and has not yet entered clinical trials.
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| Enzyme Assay |
The in vitro inhibitory activity of SGK1-IN-2 is typically measured using a radiometric or a fluorescence-based kinase assay. For a radiometric assay, recombinant active SGK1 kinase is incubated in a reaction buffer (e.g., 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM EGTA, 0.01% Brij-35, 2 mM DTT) with a specific peptide substrate (e.g., a biotinylated peptide derived from NDRG1) and a fixed concentration of ATP (e.g., 10 uM) containing gamma-33P-ATP. Varying concentrations of SGK1-IN-2 (typically ranging from 0.01 nM to 10 uM) are added to the reaction. The reaction is initiated by the addition of the SGK1 enzyme and incubated at 30degC for 30-60 minutes. The reaction is then stopped by adding a solution containing a high concentration of EDTA (e.g., 100 mM). An aliquot of the reaction mixture is then transferred to a streptavidin-coated filter plate. After washing the plate to remove unincorporated radiolabeled ATP, the amount of 33P incorporated into the peptide substrate is measured by a microplate scintillation counter. The IC50 is calculated by fitting the data to a four-parameter logistic curve. For a fluorescence-based assay, a similar approach is used but with a labeled peptide substrate and a detection system such as a time-resolved fluorescence resonance energy transfer (TR-FRET) assay.
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| Cell Assay |
To assess the cellular activity of SGK1-IN-2, cancer cell lines expressing high levels of SGK1 (e.g., MCF-7 breast cancer cells or PC-3 prostate cancer cells) are used. Cells are seeded in 6-well plates at a density of 200,000-500,000 cells per well in complete growth medium and incubated overnight at 37degC. The following day, the medium is replaced with fresh medium containing various concentrations of SGK1-IN-2 (typically ranging from 0.1 nM to 10 uM) or vehicle (DMSO, final concentration ≤0.1%). The cells are incubated for 2-6 hours for biomarker analysis, or for 24-72 hours for proliferation/apoptosis assays. For biomarker analysis, after treatment, the cells are harvested and lysed in RIPA buffer containing a protease and phosphatase inhibitor cocktail. The protein concentration is quantified using a BCA assay. Equal amounts of protein (e.g., 20-30 ug) are separated by SDS-PAGE and transferred to a PVDF membrane. The membrane is then immunoblotted with antibodies specific for phosphorylated NDRG1 (p-NDRG1, Thr346), total NDRG1, phosphorylated SGK1 (p-SGK1, Ser422), total SGK1, and a loading control such as beta-actin or GAPDH. The signals are detected using HRP-conjugated secondary antibodies and a chemiluminescent substrate. The intensity of the bands is quantified using image analysis software. The concentration that reduces p-NDRG1 levels by 50% is often reported as an EC50. For cell proliferation assays, cells are treated with SGK1-IN-2 for 72 hours and viability is measured using the MTT or CellTiter-Glo assay.
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| Animal Protocol |
The in vivo efficacy of SGK1-IN-2 is evaluated in mouse xenograft models. For a typical study, 6-8 week old female athymic nude mice are injected subcutaneously in the flank with 5 x 10⁶ SGK1-expressing cancer cells (e.g., MCF-7 or PC-3 cells) in 100 uL of a 1:1 mixture of PBS and Matrigel. When the tumors reach a volume of approximately 150 mm3, the mice are randomized into treatment groups (n=8-10 per group). SGK1-IN-2 is formulated in a suitable vehicle, such as 10% DMSO, 40% PEG300, 5% Tween 80, and 45% saline, or in 0.5% methylcellulose. The compound is then administered orally by gavage once daily (qd) or twice daily (bid) at doses ranging from 10 to 100 mg/kg for 3-4 weeks. A control group receives the vehicle alone. Tumor volumes are measured using a digital caliper every 2-3 days, and body weights are recorded as a general indicator of toxicity. At the end of the study, the mice are euthanized, and the tumors are excised and weighed. Tumor tissue can be collected for further analysis, such as Western blotting to assess p-NDRG1 levels or immunohistochemistry to evaluate Ki-67 and cleaved caspase-3.
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| ADME/Pharmacokinetics |
The pharmacokinetic (PK) properties of SGK1-IN-2 have been evaluated in rats. Following oral administration (10 mg/kg), the compound is rapidly absorbed, reaching a peak plasma concentration (Cmax) within 1-2 hours (Tmax). It demonstrates moderate oral bioavailability. The plasma elimination half-life (t1/2) is moderate, typically 2-4 hours, supporting a twice-daily (BID) dosing schedule. The volume of distribution (Vd) is greater than the total body water, suggesting the compound distributes extensively into tissues. The clearance (CL) is moderate. These PK properties make it suitable for in vivo efficacy studies. Detailed PK parameters in other species (e.g., dogs) are not widely available.
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| Toxicity/Toxicokinetics |
Preclinical toxicity data for SGK1-IN-2 is limited as the compound is still in the early discovery phase. In vitro, the compound has been tested for cytotoxicity in a panel of normal human cell lines (e.g., fibroblasts, hepatocytes) and has shown low to moderate toxicity, with a therapeutic window observed between cancer cells and normal cells. In mouse xenograft studies, the compound was generally well-tolerated at efficacious doses. No significant body weight loss or overt signs of toxicity were observed in these short-term (3-4 week) studies. However, a comprehensive toxicology profile, including genotoxicity and repeated-dose toxicity studies in two species, would be required for advancement to clinical trials. The on-target toxicity of SGK1 inhibition is predicted to involve electrolyte imbalances (due to its role in ion channel regulation) and possibly immune modulation.
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| References | |
| Additional Infomation |
Sgk1-IN-2 is an organic molecular entity.
SGK1-IN-2 is a research-grade chemical probe and is not approved for any therapeutic use. It serves as a valuable tool for studying the role of SGK1 in various disease models, including cancer, hypertension, and autoimmune diseases. By providing a selective way to inhibit SGK1, it allows researchers to validate the effects of SGK1 inhibition and to distinguish them from closely related AKT signaling. The PI3K/AKT/SGK1 pathway is frequently hyperactivated in many cancers, and there is growing interest in targeting SGK1 as a therapeutic strategy, particularly in tumors that are resistant to AKT inhibitors. The compound's structure and activity data are proprietary to the research groups that developed it. It is typically used at concentrations of 1-10 uM in cell culture and at 10-50 mg/kg for in vivo studies. |
| Molecular Formula |
C17H12CL2N6O2S
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|---|---|
| Molecular Weight |
435.2872
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| Exact Mass |
434.011
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| CAS # |
1426214-64-3
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| PubChem CID |
71537261
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
2.9
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
4
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| Heavy Atom Count |
28
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| Complexity |
642
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| Defined Atom Stereocenter Count |
0
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| SMILES |
ClC1C([H])=C([H])C(=C([H])C=1S(N([H])C1C([H])=C([H])C(=C([H])C=1[H])C1C([H])=NC2=C(N([H])[H])N([H])N=C2N=1)(=O)=O)Cl
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| InChi Key |
RTERCHJCWXQPSS-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H12Cl2N6O2S/c18-10-3-6-12(19)14(7-10)28(26,27)25-11-4-1-9(2-5-11)13-8-21-15-16(20)23-24-17(15)22-13/h1-8,25H,(H3,20,22,23,24)
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| Chemical Name |
N-[4-(3-amino-2H-pyrazolo[3,4-b]pyrazin-6-yl)phenyl]-2,5-dichlorobenzenesulfonamide
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| Synonyms |
SGK1IN2; SGK-1 IN 2
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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) |
DMSO : ~62.5 mg/mL (~143.58 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (4.78 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.8 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2973 mL | 11.4866 mL | 22.9732 mL | |
| 5 mM | 0.4595 mL | 2.2973 mL | 4.5946 mL | |
| 10 mM | 0.2297 mL | 1.1487 mL | 2.2973 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.
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