ASK1 (pIC50 = 8.3)

Selonsertib (GS-4997) 是一种临床阶段的 ASK1 抑制剂，已被评估为肾纤维化和糖尿病肾病的潜在治疗方法[1]。 Selonsertib (GS-4997) 是一种每日一次的口服 ASK1 抑制剂，具有高选择性和效力，可与 ASK1 催化激酶结构域中的 ATP 竞争。

一种口服生物可利用的凋亡信号调节激酶 1 (ASK1) 抑制剂，可能具有抗炎、抗肿瘤和抗纤维化特性。 ATP 竞争性 ASK1 抑制剂 GS-4997 口服后靶向并结合 ASK1 的催化激酶结构域，防止其磷酸化和激活。这会阻止下游激酶，包括 p38 MAPK 和 c-Jun N 末端激酶 (JNK) 磷酸化。 GS-4997 可抑制细胞增殖，下调纤维化相关基因的表达，并通过阻止 ASK1 依赖性信号转导途径的激活来抑制过度细胞凋亡 [2]。炎症细胞因子的产生也被阻止。

ASK1酶抑制试验[3]
材料：使用ASKI重组蛋白和HTRF®KinEASE™STK底物3试剂盒。根据供应商提供的方案，在pH 7.0的250mM Hepes缓冲液中进行激酶测定，缓冲液中含有NaN3 0.1%, 0.05% BSA， 0.5mM正钒酸盐，5mM MgCl2,1mM DTT, 1% DMSO（复合添加后）。[3]

方法：IC50测定——在化合物（不同浓度，0 ~ 10uM）、固定剂量的ATP （200uM，终浓度）、底物STK3 （1uM，终浓度）存在下测定每种化合物的IC50值。加入ASK1 （10nM，终浓度）启动酶促反应。实验在室温（~ 22℃）下进行。60 min后，使用CisBio试剂盒提供的停止试剂停止酶促反应。使用Multidrop Combi试剂分配器将所有试剂分配到白色384 SV格莱纳板中。利用BMG PHERAstar平板阅读器在337 nm（激发波长）处检测产物的释放度，并测量665/620 nm（发射波长）的荧光比。[3]

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人和大鼠肝微粒体稳定性测定：[3]
混合的大鼠或人肝微粒体在进一步处理前迅速解冻。在Tecan液体处理机上，将室温NADPH溶液加入含有测试化合物或对照化合物以及肝微粒体的预热孔中，开始反应。最终培养液（20 μL）含有1 μM试验化合物或对照物、肝微粒体（0.5 mg/mL）、2 mM NADPH和50 mM磷酸钾缓冲液（pH 7.4）。37℃孵育0、5、15、30分钟。加入40 μL 0.1 M的三氯乙酸（TCA）猝灭液终止反应。在4500g下离心20 min，用LC-MS/MS测定上清液中被试化合物或对照物的峰面积。Waters Quattro Premier UPLC-MS/MS系统包括Acquity二元溶剂管理器使用Waters Acquity样本管理器和Waters Acquity样本管理器进行肝微粒体样本分析。使用Waters MassLynx软件进行仪器控制、数据采集、数据处理和色谱/光谱浏览。液相色谱使用本·C18海域,执行1.7μm, ID 2.1 x 50毫米50°C列与流动相洗脱:0.04%的甲酸水和流动相乙:0.04%的甲酸乙腈流量为0.35毫升最低为1和一个梯度1%到99%的B / 1.0分钟。代谢半衰期(t1/2)值计算使用公式1,一个是初始峰面积表示为100%,一阶速率常数k, t是时间分钟。[3]

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Selonsertib（也称为 GS-4997）是一种高选择性和强效的每日一次口服 ASK1（凋亡信号调节激酶 1）抑制剂，具有高强效和选择性，pIC50 为 8.3-0.07。 Selonsertib (GS-4997) 是一种每日一次的口服 ASK1 抑制剂，具有高选择性和效力，可与 ASK1 催化激酶结构域中的 ATP 竞争。

ASK1-HEK293 P-JNK细胞测定。[3]
利用四环素诱导基因表达（Tet-on）系统对人ASK1/HEK-293细胞进行工程化，使其表达人ASK1，该系统通过在培养基中添加四环素（Tet）来激活感兴趣的基因。细胞保存在含有10%胎牛血清、100units/ml青霉素G/100ug/ ml硫酸链霉素、500ug/ml遗传素和5ug/ml囊胚素的DMEM高糖培养基中。细胞以300,000个细胞/ml， 145ul/孔的剂量接种于96孔黑色透明板上，涂有PolyD Lysine，在37°C下5% CO2下孵育5小时，然后在所有孔中加入15ul四环素（11ug/ml）诱导ASK1表达（没有Tet/ no H2O2阴性对照除外）。细胞在37°C、5% CO2下孵育过夜。第二天早上制备5倍浓度连续稀释的复方板，每次稀释取0.040 mL转移到细胞板上。然后在37°C下5% CO2下孵育30分钟，然后通过在所有孔中添加20ul 10mM H2O2激活ASK-1 30分钟（阴性对照除外）。在H2O2孵育结束时，轻轻抽吸细胞上清，加入70ul/well的全MSD裂解缓冲液裂解细胞。采用MESO Scale ELISA法检测磷酸化和总JNK水平。[3]

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小鼠巨噬细胞RAW264.7在含有10%胎牛血清、100 U/ml青霉素和100 μg/ml链霉素的1640培养基中培养，并在含5% CO2的湿化气氛中37℃保存。将细胞用Selonsertib (GS-4997)（5µM）或mdivi（10µM）预孵育6 h，然后用LPS （500 ng/ml）孵育4 h，收集上清和细胞样本进行进一步分析[4]。

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一种口服生物可利用的凋亡信号调节激酶 1 (ASK1) 抑制剂，可能具有抗炎、抗肿瘤和抗纤维化特性。 ATP 竞争性 ASK1 抑制剂 GS-4997 口服后靶向并结合 ASK1 的催化激酶结构域，防止其磷酸化和激活。这会阻止下游激酶，包括 p38 MAPK 和 c-Jun N 末端激酶 (JNK) 磷酸化。 GS-4997 可抑制过度细胞凋亡、抑制细胞增殖、下调纤维化相关基因的表达，并通过阻止 ASK1 依赖性信号转导途径的激活来阻止炎症细胞因子的产生。

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)

配方 1 中的溶解度: ≥ 2.62 mg/mL (5.88 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.67 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (4.67 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: (饱和度未知) in (这些助溶剂从左到右依次添加，逐一添加),

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。