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Description: BioE-1115 (BioE 1115) is a novel, potent and highly selective PAS kinase (PASK) inhibitor with an IC50 of ~4 nM, it is also a potent casein kinase 2α inhibitor with an IC50 of ~10 μM. It is a dual inhibitor of PAS kinase (PASK) and casein kinase 2α (CK2α).
| Targets |
PAS kinase (PASK)
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|---|---|
| ln Vitro |
With an IC50 of approximately 1 μM in HEK293 cells, a dose that is thought to cause a loss of PASK phosphorylation was demonstrated in the presence of BioE-1115 [1]. When BioE-1115 was applied at a dose of 10 μM, HepG2 cell growth rate and cell shape did not exhibit any discernible changes, but SREBP activity was significantly reduced [1].
In vitro: BioE-1115 specifically inhibited PASK with an IC50 of ~4 nM in a kinase activity assay against a panel of 50 protein kinases. Among the tested kinases, casein kinase 2a was the next most potently inhibited, with an IC50 of ~10 µM, representing roughly 2500-fold selectivity for PASK [1]. In cell-based assays, BioE-1115 treatment (10-50 µM) dose-dependently reduced PASK autophosphorylation at Thr-307, with an IC50 of ~1 µM for PASK inhibition in HEK293 cells expressing Flag-tagged PASK [1]. In HepG2 cells, BioE-1115 (10-50 µM) suppressed SRE-driven luciferase activity (SREBP activity) in a dose-dependent manner, with significant reduction observed at concentrations above 10 µM [1]. Treatment with BioE-1115 (30-50 µM) suppressed SREBP-1c maturation, as evidenced by a decrease in the mature:precursor SREBP-1c ratio in HepG2 cells [1]. BioE-1115 treatment (30 µM) did not impair insulin-stimulated phosphorylation of Akt or S6K in HepG2 cells, indicating no effect on canonical insulin signaling [1]. |
| ln Vivo |
Doses of Gpat1, Fasn, and all other SREBP-1c target gene expression examined in BioE-1115 (1-100 mg/kg; borderline gavage; daily; for 7 days; cosmetic Sprague-Dawley construct) were found to be dose regulators. SREBP-1 saturation in Manhattan was also inhibited at doses of 10, 30, and 100 mg/kg treated with 1115. BioE-1115 was applied in a dose-dependent manner to decrease HOMA-IR, a computed marker of insulin resistance. By using BioE-1115, liver and serum TAG were decreased in a dose-dependent manner. Serum blood glucose levels significantly dropped after receiving BioE-1115 therapy. At the highest dose, the mRNA for SREBP-1c and SREBP-1a fell consistently.
In vivo: In high-fructose diet (HFrD)-fed Sprague-Dawley rats, oral administration of BioE-1115 (10, 30, 100 mg/kg once daily for 7 days) caused a dose-dependent suppression of hepatic SREBP-1c target gene expression (Gpat1, Fasn, and others) [1]. In the same model, BioE-1115 (10-100 mg/kg) suppressed SREBP-1 maturation in liver, decreased hepatic triglycerides, and dose-dependently lowered serum triglycerides without affecting serum cholesterol [1]. BioE-1115 treatment (10-100 mg/kg) significantly decreased serum glucose and HOMA-IR (a measure of insulin resistance) in HFrD-fed rats, with slightly lower serum insulin levels, suggesting improved insulin sensitivity [1]. In a 90-day study, BioE-1115 (3-100 mg/kg) suppressed Fasn and Acc1 expression, and at 10-100 mg/kg restored expression to normal chow levels; serum triglycerides were dose-responsively decreased at 45 and 90 days; glycated hemoglobin (HbA1c) was also decreased [1]. No significant changes in body weight or liver weight were observed with BioE-1115 treatment at any dose up to 100 mg/kg for 7 or 90 days [1]. No effect on SREBP-1c or its target gene expression was observed in abdominal fat or gastrocnemius muscle after 90 days of BioE-1115 treatment [1]. |
| Enzyme Assay |
Enzyme Assay: The in vitro kinase inhibitory activity of BioE-1115 was assessed by measuring the activity of purified PASK and 50 other protein kinases (selected to represent the breadth of the human kinase family) in the presence of the compound. The IC50 values were determined from dose-response inhibition curves. Specifically, PASK activity was measured in the presence of varying concentrations of BioE-1115, and the percent activity relative to vehicle-treated control was calculated. The IC50 for PASK inhibition was approximately 4 nM. For selectivity profiling, the activity of each of the 50 kinases was measured in the presence of BioE-1115, and IC50 values were calculated. Casein kinase 2a was inhibited with an IC50 of ~10 µM [1].
In another assay, purified PASK protein kinase activity was assayed in the presence of indicated concentrations of BioE-1115, and the percent of vehicle-treated activity was plotted to determine IC50 [1]. |
| Cell Assay |
Cell Assay: For cell-based efficacy, HEK293 cells expressing Flag-tagged PASK were treated with various concentrations of BioE-1115 (0.1-100 µM) for 16 hours. Cells were then lysed, and PASK activity was analyzed by ELISA using a phospho-Akt substrate antibody (detecting PASK autophosphorylation) and a pan-PASK antibody. The phospho/total PASK signal was quantified, and an IC50 of ~1 µM for BioE-1115 was determined [1].
HepG2 cells were transfected with an SRE-luciferase reporter and Renilla luciferase construct. After transfection, cells were serum-starved overnight and then treated with vehicle or varying concentrations of BioE-1115 (10-50 µM) along with 100 nM insulin for 6 hours. Firefly and Renilla luciferase activities were measured using a dual-reporter assay system. A significant reduction in SREBP activity was observed at all concentrations above 10 µM BioE-1115 [1]. HepG2 cells were treated with BioE-1115 (30-50 µM) along with insulin, and whole cell lysates and nuclear extracts were subjected to immunoblotting to detect precursor and mature forms of SREBP-1c. The ratio of mature to precursor SREBP-1c was decreased, indicating suppression of SREBP-1 maturation [1]. Rat primary hepatocytes were isolated and pre-treated with DMSO or BioE-1115 (actual concentration not specified for this compound in the hepatocyte experiments; the text refers to BioE-1197 for primary hepatocyte studies, and BioE-1115 was used in animal studies. No explicit BioE-1115 cell assay in hepatocytes is described. |
| Animal Protocol |
Animal/Disease Models: Male SD (SD (Sprague-Dawley)) rats (12 weeks old; 129.4 ± 0.63 g) fed a high-fructose diet [1]
Doses: 1 mg of BioE-1115 did not cause significant changes in body weight or weight [1]. /kg, 3 mg/kg, 10 mg/kg, 30 mg/kg and 100 mg/kg Route of Administration: po (oral gavage); daily; 7-day Experimental Results: Treatment with 10, 30 and 100 mg/kg, shown Dose-dependent inhibition of Gpat1, Fasn, and all other SREBP-1c target gene expression analyzed. Hepatic expression of lipogenic SREBP-1c target genes was diminished, serum triglycerides were diminished and insulin resistance was partially reversed. Animal Protocol: Male Sprague-Dawley rats (12 weeks old) were fed a high-fructose diet (60% fructose) for 2 weeks. Then, BioE-1115 was formulated as a fine suspension in a vehicle of 0.5% methylcellulose and 0.025% Tween-80 in distilled water. Animals were orally dosed once daily between 7 am and 9 am for 7 days at doses of 1, 3, 10, 30, and 100 mg/kg body weight. Control animals received vehicle only. On the final day, rats were fasted for 24 hours and then refed for 12 hours prior to termination. All animals received their respective compound dosage three hours prior to termination. Animals were euthanized by CO2 asphyxiation, and cardiac puncture was performed for serum analysis. Liver tissue was harvested, weighed, and snap-frozen in liquid nitrogen [1]. In a separate 90-day study, similarly designed, HFrD-fed rats were treated once daily with vehicle or BioE-1115 at 1, 3, 10, 30, and 100 mg/kg for 90 days. Body weights were taken daily for compound formulation. Animals were fasted for 24 h and refed for 12 h prior to termination. Blood was collected for serum analysis, and liver and other tissues were harvested [1]. In another study, rats were maintained on a high-fat and high-fructose diet for 18 weeks, then treated with BioE-1115 (doses not specified here but presumably as above) for the last 3 weeks. Livers were harvested after fast/refeeding as above [1]. |
| ADME/Pharmacokinetics |
ADME/Pharmacokinetics: The text states that "BioE-1115" was selected for in vivo studies due to its "more favorable in vivo pharmacologic and pharmacokinetic properties" relative to BioE-1197 (see Figure S4A). However, no specific pharmacokinetic parameters (e.g., half-life, bioavailability, clearance, volume of distribution) are provided in the manuscript. Therefore, no quantitative ADME data are available for BioE-1115 in this paper [1].
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| Toxicity/Toxicokinetics |
Toxicity/Toxicokinetics: No overt toxicity was observed upon extended treatment with doses of the PASK inhibitor (including BioE-1115) that are 10-30 fold higher than those required to see significant effects on lipogenic gene expression and serum triglycerides. Such a treatment regimen (up to 100 mg/kg for 90 days) had no effect on body weight or liver weight. No other specific toxicity parameters (e.g., histopathology, serum biomarkers of liver/kidney injury) are reported. The text also notes that PASK knockout mice are viable and show no overt toxicity, and that BioE-1115 treatment had no effect on SREBP-1c or its target genes in skeletal muscle or adipose tissue, suggesting tissue-selective action without general toxicity [1].
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| References | |
| Additional Infomation |
Additional Info: BioE-1115 is a selective and potent inhibitor of PAS kinase (PASK). It was developed as a chemical probe to study the role of PASK in SREBP-1c maturation and lipogenesis. The compound was synthesized by Pharmaron. In the study, BioE-1115 was used to demonstrate that pharmacological inhibition of PASK improves lipid and glucose metabolism in dietary animal models of obesity and dyslipidemia, including reversal of hypertriglyceridemia and insulin resistance. The mechanism involves blockade of SREBP-1 proteolytic maturation, leading to decreased expression of lipogenic genes. The authors propose that PASK inhibition, including by BioE-1115, may have therapeutic potential for metabolic syndrome [1].
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| Molecular Formula |
C19H18FN3O2
|
|---|---|
| Molecular Weight |
339.363527774811
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| Exact Mass |
339.138
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| CAS # |
1268863-35-9
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| Related CAS # |
1268863-35-9
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| PubChem CID |
50922674
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.8
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| Hydrogen Bond Donor Count |
1
|
| Hydrogen Bond Acceptor Count |
6
|
| Rotatable Bond Count |
4
|
| Heavy Atom Count |
25
|
| Complexity |
468
|
| Defined Atom Stereocenter Count |
0
|
| SMILES |
FC1C=CC(=CC=1)C1C(=NC2C=C(C(=O)O)C=CC=2N=1)N(C)C(C)C
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| InChi Key |
JKKRYYTVQXUOKL-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C19H18FN3O2/c1-11(2)23(3)18-17(12-4-7-14(20)8-5-12)21-15-9-6-13(19(24)25)10-16(15)22-18/h4-11H,1-3H3,(H,24,25)
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| Chemical Name |
2-(4-fluorophenyl)-3-[methyl(propan-2-yl)amino]quinoxaline-6-carboxylic acid
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| Synonyms |
BioE-1115BioE 1115BioE1115
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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 (~184.17 mM)
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|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.08 mg/mL (6.13 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 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. Solubility in Formulation 2: ≥ 2.08 mg/mL (6.13 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.9467 mL | 14.7336 mL | 29.4672 mL | |
| 5 mM | 0.5893 mL | 2.9467 mL | 5.8934 mL | |
| 10 mM | 0.2947 mL | 1.4734 mL | 2.9467 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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