ASK1 (pIC50 = 8.3)

Selonsertib (GS-4997) is a clinical-stage ASK1 inhibitor that has been assessed as a potential treatment for kidney fibrosis and diabetic nephropathy[1]. A once-daily oral ASK1 inhibitor with high selectivity and potency, selonsertib (GS-4997), competes with ATP in the ASK1 catalytic kinase domain.

An oral bioavailable inhibitor of apoptosis signal-regulating kinase 1 (ASK1) that may have anti-inflammatory, anti-tumor, and anti-fibrotic properties. The ATP-competitive ASK1 inhibitor GS-4997 targets and binds to the catalytic kinase domain of ASK1 after oral administration, preventing its phosphorylation and activation. This stops downstream kinases, including p38 MAPK and c-Jun N-terminal kinases (JNKs), from becoming phosphorylated. GS-4997 inhibits cellular proliferation, down-regulates the expression of fibrosis-related genes, and suppresses excessive apoptosis by preventing the activation of ASK1-dependent signal transduction pathways[2]. Inflammatory cytokines are also prevented from being produced.

ASK1 Enzyme Inhibition Assay [3]
Material: ASKI recombinant protein and HTRF® KinEASE™ STK substrate 3 kit were used. The kinase assay was conducted in 250mM Hepes buffer containing NaN3 0.1%, 0.05% BSA, 0.5mM Orthovanedate, 5mM MgCl2,1mM DTT, 1% DMSO (after compound addition) at pH 7.0 according to the protocol provided by the vendor. [3]
Methods: IC50 determination – the IC50 value for each compound was determined in the presence of compound (various concentrations, from 0 to 10uM) and a fixed amount of ATP (200uM, final concentration), substrate STK3 (1uM, final concentration). The enzymatic reaction was initiated by adding ASK1 (10nM, final concentration). The assay was conducted at room temperature (~ 22°C). After 60 min, the enzymatic reaction was stopped using the stop reagents provided by the CisBio kit. All the reagents were dispensed using a Multidrop Combi reagent dispenser into white 384 SV Greiner plates. The release of product was detected using a BMG PHERAstar plate reader at 337 nm (excitation wavelength) and measuring the ratio of fluorescence 665/620 nm (emission wavelengths). [3]

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Human and Rat Liver Microsomal stability assay: [3]
Pooled rat or human liver microsomes were quickly thawed before further processing. The reactions were initiated on a Tecan liquid handler by adding room temperature NADPH solution into pre-warmed wells containing test compound or control compound as well as liver microsomes. The final incubation solutions (20 μL) contained 1 μM test compound or control, liver microsomes (0.5 mg/mL), 2 mM NADPH, and 50 mM potassium phosphate buffer (pH 7.4). Plates were incubated at 37°C for 0, 5, 15 and 30 minutes. The reactions were terminated by the addition of 40 μL of 0.1 M trichloroacetic acid (TCA) quenching solution. The plates were centrifuged at 4500g for 20 min. The peak areas of test compound or controls in the supernatants were measured by LC-MS/MS. A Waters Quattro Premier UPLC-MS/MS System consisting of an Acquity Binary Solvent manager; Waters Acquity sample manager and Waters Acquity Sample Organizer was used for liver microsomal sample analysis. Waters MassLynx software was used for instrument control, data acquisition, data processing and chromatogram/spectrum browsing. Liquid chromatography was performed using a Waters BEH C18, 1.7 μm, ID 2.1 x 50 mm at 50°C column eluting with mobile phase A: 0.04% formic acid in water and mobile phase B: 0.04% formic acid in acetonitrile with a flow rate of 0.35 mL min-1 and a gradient of 1% to 99% B over 1.0 min. The metabolic half life (t1/2) values were calculated using Equation 1, where A is the initial peak area expressed as 100%, k is the firstorder rate constant, and t is time in minutes.[3]

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Selonsertib (Also known as GS-4997) is a highly selective and potent once-daily orally available ASK1 (apoptosis signal-regulating kinase 1) inhibitor that is highly potent and selective, with a pIC50 of 8.3–0.07. A once-daily oral ASK1 inhibitor with high selectivity and potency, selonsertib (GS-4997), competes with ATP in the catalytic kinase domain of ASK1.

ASK1-HEK293 P-JNK Cellular Assay. [3]
Human ASK1/HEK-293 cells were engineered to express human ASK1 utilizing a tetracycline-inducible gene expression (Tet-on) system, in which the gene of interest is activated by adding tetracycline (Tet) to the culture medium. Cells were maintained in DMEM high glucose medium containing 10% Fetal Bovine Serum, 100units/ml Penicillin G/100ug/mL Streptomycin Sulfate, 500ug/ml Geneticin and 5ug/ml Blasticidin. Cells were seeded at 300,000 cell/ml, 145ul/well, in 96 well black clear plates coated with PolyD Lysine and incubated at 37°C under 5% CO2 for 5 hours before ASK1 expression was induced by adding 15ul of tetracycline (11ug/ml) to all wells (except for no Tet/No H2O2 negative controls). Cells were incubated overnight at 37°C under 5% CO2. Five-fold concentrated serial diluted compound plates were prepared the next morning and 0.040 mL from each dilution was transferred to the cell plates. Plates were then incubated for 30 minutes at 37°C under 5% CO2 before activating ASK-1 by adding 20ul of 10mM H2O2 for 30 more minutes to all wells (except for negative controls). At the end of incubation with H2O2, cell supernatants were gently aspirated, and cells were lysed by adding 70ul/well of complete MSD lysis buffer. Phospho- and total JNK levels were determined by MESO Scale ELISA. [3]

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The mouse macrophage cell line RAW264.7 was cultured in 1640 medium containing 10% foetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin and maintained at 37 °C in a humidified atmosphere containing 5% CO2. The cells were preincubated with Selonsertib (GS-4997) (5 µM) or mdivi (10 µM) for 6 h and then incubated with LPS (500 ng/ml) for 4 h. The supernatant and cell samples were then collected for further analysis[4].

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An oral bioavailable inhibitor of apoptosis signal-regulating kinase 1 (ASK1) that may have anti-inflammatory, anti-tumor, and anti-fibrotic properties. The ATP-competitive ASK1 inhibitor GS-4997 targets and binds to the catalytic kinase domain of ASK1 after oral administration, preventing its phosphorylation and activation. This stops downstream kinases, including p38 MAPK and c-Jun N-terminal kinases (JNKs), from becoming phosphorylated. GS-4997 suppresses excessive apoptosis, inhibits cellular proliferation, down-regulates the expression of fibrosis-related genes, and prevents the production of inflammatory cytokines by preventing the activation of ASK1-dependent signal transduction pathways.

ALF mouse model and treatment[4]
C57BL/6J mice were intraperitoneally injected with LPS (10 μg/kg, Sigma-Aldrich, St Louis, MO, USA) and D-GalN (400 mg/kg, Sigma-Aldrich) to establish a mouse model of LPS/GalN-induced ALF. Selonsertib (GS-4997) (15, 30, and 60 mg/kg, MCE) was administered via i.p. injection 30 min prior to LPS/GalN injection or at 0.5, 1, 2, and 4 h after LPS/GalN injection. A JNK activator, anisomycin (20 mg/kg, MCE), was administered via i.p. injection combined with Selonsertib (GS-4997) to further investigate the role of the JNK pathway in mediating the protective effect of Selonsertib (GS-4997). For another therapy, mdivi (30 mg/kg, MCE) was administered via i.p. injection 30 min prior to LPS/GalN injection. The control group was administered vehicle (n = 6). At 0.5, 1, 2, 4, and 6 h after LPS/GalN injection, the mice were sacrificed, and serum and liver samples were collected to assess the extent of liver injury. Serum was evaluated for biochemical parameters. The liver samples were evaluated for histochemistry and Western blot analysis. Rat Pharmacokinetics: [3]
Cannulated male Sprague-Dawley (SD) rats were fasted overnight then treated with 19 (analog of Selonsertib (GS-4997)) formulated in 20% (2-hydroxypropyl)-β-cyclodextrin in 0.05M methanesulfonic acid at 5 mg/kg by oral gavage. Dose volume was 5 ml/kg and 19 was dosed as a solution at a concentration of 1 mg/ml. Residual dose form was saved and analyzed for exact dose concentration. Following administration of test article, 200 ul of blood was collected through the jugular vein catheter from conscious animals at 0.25, 0.5, 1, 2, 4, 7 and 24 hours. Blood samples were kept on ice until processed for plasma. Plasma was prepared by centrifugation at 5C, frozen and stored at -70C until analyzed. Analysis of 19 plasma concentrations was performed by LC-MS/MS analysis. Briefly, 25 ul of plasma was mixed with 100 ul acetonitrile containing an appropriate internal standard. Samples were vortexed for 1 minute then centrifuged at 300 rpm for 5 min at 2-8C. Forty ul of supernatant was then diluted 1:2 with water and vortexed for 5 min. Samples were analyzed with a AB Sciex API-4000 triple quadropole mass spectrometer equipped with a Shimadzu LC System and a LEAP autosampler. A reverse-phase gradient method running at a flow rate of 0.500 mL/min on an Phenomenex, Kinetic C18 column (2.1 mm ID  50 mm; particle size 5.0 m) was used for the test article separation. The mobile phase used was water (A) and acetonitrile (B), and both were supplemented with formic acid (0.04%, volume-to-volume ratio [v:v]). Samples were ionized and detected in multiple reactions monitoring (MRM) mode by monitoring the transition m/z 394.051→ 336.100. Samples were quantitated by use of analyte standards prepared in pooled rat plasma with internal standard. The lower limit of quantitation in this assay was 1.00 ng/mL and linearity was achieved in the concentration range of 1.00 ng/mL to 2500 ng/mL. Langendorff Perfused Heart Model: [3]
Male Sprague Dawley rats were obtained from Harlan laboratories and were allowed to acclimate at least 48 hours before being used in the study. Rats were housed 2-3 animals/cage and had free access to food and water throughout the study. Rats were dosed with vehicle or compound 1, 4, 6, or 8 hours prior to ex vivo ischemia/reperfusion (I/R). Global no-flow ex vivo I/R was performed on a constant pressure recirculating Langendorff apparatus. Rats were treated with 500 U/kg heparin 10 min prior to administration of ketamine (60 mg/kg), xylazine (7.5 mg/kg) via intraperitoneal injection. Analgesics (buprenorphine hydrochloride, 0.05 mg/kg) were administered prior to surgery via subcutaneous injection. Once appropriate depth of anesthesia and analgesia were confirmed, animals were sacrificed and the hearts were quickly removed and placed in ice-cold modified Krebs-Henseleit buffer. The aorta was then cannulated and the heart was mounted on a Langendorff apparatus and perfused with oxygenated Krebs-Henseleit buffer at a constant pressure of 80 mmHg. Hearts were submerged in buffer warmed to 37°C at all times. Following a 30 minutes equilibration period, hearts were subjected to 30 minutes of no flow ischemia followed by 90 minutes of reperfusion. At the completion of I/R, hearts were flash frozen on dry ice and sectioned coronally into 3mm pieces (6 in total) using a rat heart matrix and razor blades. Hearts were then stained with 1% 2,3,5-triphenyltetrazolium chloride (TTC) for 10 minutes to visualize viable tissue and then fixed overnight in 10% formalin.

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[1]. Nephron . 2015;129(1):29-33.

[2]. NCI Drug Dictionary. ASK1 inhibitor GS-4997

[3]. ACS Med Chem Lett . 2017 Feb 8;8(3):316-320.

[4]. Cell Biosci . 2021 Jan 7;11(1):9.

Selonsertib is under investigation in clinical trial NCT03053050 (Safety and Efficacy of Selonsertib in Adults With Nonalcoholic Steatohepatitis (NASH) and Bridging (F3) Fibrosis).
Selonsertib is an orally bioavailable inhibitor of apoptosis signal-regulating kinase 1 (ASK1), with potential anti-inflammatory, antineoplastic and anti-fibrotic activities. Upon oral administration, selonsertib targets and binds to the catalytic kinase domain of ASK1 in an ATP-competitive manner, thereby preventing its phosphorylation and activation. This prevents the phosphorylation of downstream kinases, such as c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinase (p38 MAPK). By preventing the activation of ASK1-dependent signal transduction pathways, GS-4997 prevents the production of inflammatory cytokines, down-regulates the expression of genes involved in fibrosis, suppresses excessive apoptosis and inhibits cellular proliferation. ASK1, also called mitogen-activated protein kinase kinase kinase 5 (MAP3K5), is activated in response to oxidative and endoplasmic reticulum (ER) stress, calcium influx and infection. It plays a key role in the development of certain cardiovascular and neurodegenerative diseases, diabetes, as well as certain types of cancer.

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Drug Indication
Treatment of non-alcoholic steatohepatitis (NASH).

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Apoptosis signal-regulating kinase 1 (ASK1/MAP3K) is a mitogen-activated protein kinase family member shown to contribute to acute ischemia/reperfusion injury. Using structure-based drug design, deconstruction, and reoptimization of a known ASK1 inhibitor, a lead compound was identified. This compound displayed robust MAP3K pathway inhibition and reduction of infarct size in an isolated perfused heart model of cardiac injury.[3]

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Background: Acute liver failure (ALF) is associated with a high mortality rate, and there are still no effective treatments except liver transplantation and artificial liver therapies. This study aimed to determine the effects, therapeutic window and mechanisms of selonsertib, a selective inhibitor of ASK1, for ALF therapy. Results: Lipopolysaccharide and D-galactosamine (LPS/GalN) were used to simulate ALF. We found that selonsertib pretreatment significantly ameliorated ALF, as determined by reduced hepatic necrosis and serum alanine aminotransferase, aspartate aminotransferase and inflammatory cytokine levels. However, selonsertib is only effective early after LPS/GalN administration, and the limited therapeutic window is related to the activation and mitochondrial translocation of JNK and DRP1. Further experiments revealed that selonsertib could alleviate LPS-induced mitochondrial damage in macrophages by evaluating the mitochondrial membrane potential and mitochondrial permeability transition pore opening in macrophages. Selonsertib also suppressed the release of inflammatory cytokines from macrophages by reducing DRP1-mediated mitochondrial dysfunction, which was confirmed by using mdivi, a specific DRP1 inhibitor. Conclusions: Selonsertib protected against LPS/GalN-induced ALF by attenuating JNK-mediated DRP1 mitochondrial translocation and then rescuing mitochondrial damage in macrophages and may have therapeutic potential for early ALF patients.[4]

C24H24FN7O

445.49

445.202

C, 64.71; H, 5.43; F, 4.26; N, 22.01; O, 3.59

1448428-04-3

1448428-04-3;1448428-05-4 (HCl);

71245288

White to off-white

1.4±0.1 g/cm3

1.704

3.17

1

6

6

33

692

0

FC1C([H])=C(C([H])([H])[H])C(=C([H])C=1C(N([H])C1=C([H])C([H])=C([H])C(C2=NN=C([H])N2C([H])(C([H])([H])[H])C([H])([H])[H])=N1)=O)N1C([H])=NC(=C1[H])C1([H])C([H])([H])C1([H])[H]

YIDDLAAKOYYGJG-UHFFFAOYSA-N

InChI=1S/C24H24FN7O/c1-14(2)32-13-27-30-23(32)19-5-4-6-22(28-19)29-24(33)17-10-21(15(3)9-18(17)25)31-11-20(26-12-31)16-7-8-16/h4-6,9-14,16H,7-8H2,1-3H3,(H,28,29,33)

5-(4-cyclopropylimidazol-1-yl)-2-fluoro-4-methyl-N-[6-(4-propan-2-yl-1,2,4-triazol-3-yl)pyridin-2-yl]benzamide

Selonsertib free base; GS-4997; GS4997; 5-(4-cyclopropyl-1H-imidazol-1-yl)-2-fluoro-N-(6-(4-isopropyl-4H-1,2,4-triazol-3-yl)pyridin-2-yl)-4-methylbenzamide; Selonsertib [INN]; Selonsertib(GS-4997); 5-(4-cyclopropylimidazol-1-yl)-2-fluoro-4-methyl-N-[6-(4-propan-2-yl-1,2,4-triazol-3-yl)pyridin-2-yl]benzamide; GS 4997; Selonsertib

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)

Solubility in Formulation 1: ≥ 2.62 mg/mL (5.88 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 2: ≥ 2.08 mg/mL (4.67 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 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (4.67 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), 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 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 4: (saturation unknown) in (add these co-solvents sequentially from left to right, and one by one),

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 (Please use freshly prepared in vivo formulations for optimal results.)