ASK1-IN-1

ASK1 (IC50 = 2.87 nM)
ASK1-IN-1 (GS-444217) targets MAP Kinase Kinase Kinase 5 (ASK1, MAP3k5), a redox-sensitive serine threonine kinase; the in vivo EC50 for inhibition of the renal ASK1 pathway in rats is 1.6 μM [1]
ASK1-IN-1 (GS-444217) targets ASK1 (MAP3k5), which activates p38 and JNK MAPKs[2]

ASK1-IN-1 是凋亡信号调节激酶 1 (ASK1) 抑制剂（专利 WO2016025474A1 中的化合物 4）的有效抑制剂。在体内肺动脉高压（PH）模型中，它以剂量依赖性方式降低右心室（RV）肥大和肺动脉压。肺血管疾病的特征，例如降低的肺血管阻力(PVR)、改善的肺压、减少的肺血管重塑、改善的血管功能、减少的适应不良的RV肥大和改善的RV功能，都可以通过抑制ASK1来减轻。与PH相关的病理变化也可以通过抑制ASK1信号传导来减缓、避免和/或逆转。

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1. 在腺病毒过表达人源ASK1（AdASK1）的HEK293T细胞中，给予浓度范围为0.001~10 μM的ASK1-IN-1 (GS-444217) 处理2小时，可剂量依赖性抑制ASK1及其下游底物（MKK3/6、MKK4、p38、JNK）的磷酸化水平（Western blot检测）；给予1 μM ASK1-IN-1 (GS-444217) 处理1分钟至4小时，可时间依赖性抑制ASK1活性；洗脱药物并更换为无血清培养基后（0~240分钟），ASK1抑制作用具有可逆性 [1]
2. 采用KINOME scan结合实验对442种激酶进行检测，1 μM ASK1-IN-1 (GS-444217) 对ASK1具有高度选择性，仅对核糖体s6激酶4（RSK4）和双特异性酪氨酸磷酸化调节激酶1A（DYRK1A）有轻微亲和力（实验中球体半径代表抑制剂亲和力，ASK1对应的点尺寸最大） [1]
3. 在腺病毒过表达ASK1的大鼠心肌细胞中，ASK1-IN-1 (GS-444217) 可降低由ASK1激活诱导的p38和JNK磷酸化水平 [2]
4. 在原代小鼠心脏成纤维细胞和肺动脉高压（PAH）患者来源的人肺外膜成纤维细胞中，ASK1-IN-1 (GS-444217) 可降低这些成纤维细胞的活化和迁移能力 [2]

在肺动脉高压 (PH) 体内模型中，ASK1-IN-1 剂量依赖性地降低肺动脉压和右心室 (RV) 肥厚。

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1. 在金诺芬（30 mg/kg，腹腔注射）诱导氧化应激（OS）激活ASK1通路的SD大鼠模型中，单次口服30 mg/kg ASK1-IN-1 (GS-444217) 可降低肾脏p-ASK1、p-p38、p-JNK水平（Western blot检测），并下调肾脏炎症细胞因子（Il1b、Ccl2、Cxcl2）的mRNA表达及半胱天冬酶活性 [1]
2. 在大鼠肾脏缺血再灌注（I/R）损伤模型中，肾脏双侧缺血30分钟前口服给予30 mg/kg ASK1-IN-1 (GS-444217)，可降低再灌注24小时后血清肌酐和血尿素氮浓度，并减少肾小管坏死（HE染色）和凋亡/坏死（TUNEL染色）评分 [1]
3. 在大鼠单侧输尿管梗阻（UUO）模型中，术前1小时口服给予30 mg/kg ASK1-IN-1 (GS-444217)，并持续每日两次给药7天，可降低肾脏p-p38和p-JNK水平（Western blot检测），减少胶原IV沉积、皮质间质α-平滑肌肌动蛋白（α-SMA）阳性肌成纤维细胞数量、肾脏上皮细胞凋亡/坏死（TUNEL）及Col1a1 mRNA表达 [1]
4. 在10周龄db/db eNOS⁻/⁻ 糖尿病肾病（DKD）小鼠模型中，给予含0.3% ASK1-IN-1 (GS-444217) 的饲料喂养8周，可阻止肾小球滤过率（GFR）下降，降低蛋白尿（尿白蛋白/肌酐比值）、肾小球硬化评分、胶原IV沉积及足细胞丢失（WT-1染色） [1]
5. 在SD大鼠5/6肾切除慢性肾小球损伤模型中，ASK1-IN-1 (GS-444217)（饲料中0.3%添加+每日一次口服30 mg/kg）与依那普利（饮用水中50 mg/L）联合给药4周，相比单药治疗可更显著降低蛋白尿并逆转肾小球硬化；ASK1-IN-1 (GS-444217) 单药也可降低收缩压并延缓肾小球硬化进展 [1]
6. 在两种大鼠肺动脉高压（PAH）模型（野百合碱模型、Sugen/低氧模型）中，口服给予ASK1-IN-1 (GS-444217) 可剂量依赖性降低肺动脉压和右心室（RV）肥厚；即使在疾病已建立的动物中给药仍有疗效，且与ASK1磷酸化水平降低、肺动脉肌化程度减轻及RV纤维化基因表达下调相关 [2]
7. 在肺动脉结扎诱导的小鼠RV压力超负荷模型中，ASK1-IN-1 (GS-444217) 可直接减少心脏纤维化并改善心脏功能 [2]

ASK1抑制改善血管功能并减少肺血管疾病的体征和症状，例如肺血管阻力(PVR)、肺压力、肺动脉重塑、适应不良的RV肥大和RV功能。通过抑制ASK1信号传导可以减缓、避免或什至逆转与PH相关的病理变化。两种 ASK1-IN-1 剂量（0.1% 为 0.1+0.05 和 0.2% 为 0.1±0.1 ng/mL）均可降低 B 型钠尿肽 (BNP) 的血浆水平。 ASK1-IN-1 治疗使 Su/Hx 大鼠完全肌化的小动脉数量分别减少 35.6% 或 32.6%。

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1. 酶促竞争实验：通过在递增浓度的ASK1-IN-1 (GS-444217)（0~24 nM）和ATP（0~2400 μM）存在下检测ASK1酶活性，分析药物与ATP的相互作用，证实ASK1-IN-1 (GS-444217) 是ASK1的ATP竞争性抑制剂 [1]
2. 表面等离子体共振（SPR）实验：采用SPR技术测定ASK1-IN-1 (GS-444217) 与ASK1的结合动力学和亲和力；将实验数据（黑色轨迹）拟合至简单的1:1相互作用模型（红色轨迹），以分析结合特征 [1]
3. KINOME scan结合实验：检测1 μM ASK1-IN-1 (GS-444217) 对442种激酶（涵盖主要激酶家族：TK、TKL、STE、CK1、AGC、CAMK、CMGC及其他家族）的选择性；该实验定量绘制相互作用模式图，球体半径代表抑制剂亲和力，点的尺寸反映药物对ASK1、RSK4和DYRK1A的相对亲和力 [1]

1. HEK293T细胞ASK1抑制实验：将腺病毒载体转染HEK293T细胞以过表达人源ASK1（AdASK1）；给予细胞1:3系列稀释的ASK1-IN-1 (GS-444217)（0.001~10 μM）处理2小时，制备细胞裂解液后通过Western blot检测ASK1、MKK3/6、MKK4、p38、JNK的磷酸化水平，以GAPDH作为内参 [1]
2. HEK293T细胞时间进程实验：将过表达AdASK1的HEK293T细胞用1 μM ASK1-IN-1 (GS-444217) 处理1分钟至4小时；通过Western blot检测ASK1通路激活标志物，以明确药物的时间依赖性作用 [1]
3. HEK293T细胞可逆性实验：过表达AdASK1的HEK293T细胞经ASK1-IN-1 (GS-444217) 处理后，洗脱药物并更换为无血清培养基；在洗脱后0~240分钟的时间点收集细胞裂解液，通过Western blot评估ASK1抑制作用的可逆性 [1]
4. 心肌细胞实验：将腺病毒载体转染大鼠心肌细胞以过表达ASK1，随后给予ASK1-IN-1 (GS-444217) 处理；检测p38和JNK的磷酸化水平，以评估药物对ASK1下游信号通路的作用 [2]
5. 成纤维细胞活化/迁移实验：培养原代小鼠心脏成纤维细胞和PAH患者来源的人肺外膜成纤维细胞，给予ASK1-IN-1 (GS-444217) 处理；采用相关细胞功能实验评估这些成纤维细胞的活化状态和迁移能力 [2]

in vivo model of pulmonary hypertension (PH

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1. Rat auranofin-induced ASK1 activation model: Sprague-Dawley rats were administered a single oral dose of 30 mg/kg ASK1-IN-1 (GS-444217) or vehicle (equal volume); 30 minutes later, rats were challenged with auranofin (30 mg/kg, i.p.) to induce OS-mediated ASK1 activation. Kidney cortex samples were collected 30 minutes after auranofin administration for Western blot, mRNA expression, and caspase activity analysis (n=8 for drug group, n=5 for vehicle group) [1]
2. Rat renal I/R injury model: Sprague-Dawley rats received oral administration of 30 mg/kg ASK1-IN-1 (GS-444217) or vehicle just before 30 minutes of bilateral renal ischemia. After a 24-hour reperfusion period, serum samples were collected to measure creatinine and blood urea nitrogen, and kidney tissues were harvested for pathological scoring of tubular necrosis (H&E) and apoptosis/necrosis (TUNEL) (n=5–8 per group) [1]
3. Rat UUO model: Sprague-Dawley rats underwent sham or UUO surgery; ASK1-IN-1 (GS-444217) (30 mg/kg) or vehicle was orally administered 1 hour before surgery and continued twice daily for 7 days. Kidney lysates were analyzed by Western blot for p-p38 and p-JNK, and renal sections were stained for collagen IV, α-SMA, and TUNEL to assess fibrosis and cell death; Col1a1 mRNA was measured by RT-PCR (n=4 for sham, n=8 for UUO group) [1]
4. Mouse DKD model (db/db eNOS⁻/⁻): 10-week-old db/db eNOS⁻/⁻ mice were fed standard chow (vehicle) or chow containing 0.3% ASK1-IN-1 (GS-444217) for 8 weeks; a baseline group was euthanized at 10 weeks. GFR was measured by inulin-FITC clearance, proteinuria by urinary albumin to creatinine ratio (UACR) at weeks 10, 14, 18, and kidney tissues were analyzed for glomerulosclerosis (PAS staining), collagen IV deposition, and podocyte loss (WT-1 staining) (n=9–12 per group) [1]
5. Rat 5/6 nephrectomy model: 8 weeks after 5/6 nephrectomy, Sprague-Dawley rats were randomized by sclerosis index (SI) scores and assigned to control (standard chow), ASK1-IN-1 (GS-444217) (0.3% in chow plus 30 mg/kg once daily p.o.), enalapril (50 mg/l in drinking water), or combination groups for 4 weeks. Systolic blood pressure, UACR, and glomerulosclerosis severity (PAS staining) were assessed (n=12–14 per group) [1]
6. Rat PAH models (monocrotaline and Sugen/hypoxia): ASK1-IN-1 (GS-444217) was administered orally to rats with established PAH ; pulmonary arterial pressure, RV hypertrophy, ASK1 phosphorylation, pulmonary artery muscularization, and RV fibrotic gene expression were measured [2]
7. Murine RV pressure overload model (pulmonary artery banding): ASK1-IN-1 (GS-444217) was administered orally to mice with RV pressure overload; cardiac fibrosis and cardiac function were evaluated [2]

[1]. ASK1 contributes to fibrosis and dysfunction in models of kidney disease. J Clin Invest. 2018 Oct 1;128(10):4485-4500.

[2]. ASK1 Inhibition Halts Disease Progression in Preclinical Models of Pulmonary Arterial Hypertension. Am J Respir Crit Care Med. 2018 Feb 1;197(3):373-385.

1. ASK1-IN-1 (GS-444217) is a potent and selective small-molecule inhibitor of ASK1, designed via structure-based drug design to optimize biochemical potency and lipophilic ligand efficiency (LLE) while maintaining low lipophilicity [1]
2. ASK1 activation is observed in glomerular and tubular compartments of renal biopsies from diabetic kidney disease (DKD) patients; ASK1-IN-1 (GS-444217) reduces ASK1 pathway activation in rodent models of kidney injury/fibrosis mimicking DKD pathology, and combination with enalapril enhances proteinuria reduction and glomerulosclerosis regression [1]
3. Oxidative stress drives pathological remodeling in PAH via MAPK activation; ASK1-IN-1 (GS-444217) inhibits ASK1 (a redox-sensitive apical MAPK) to halt PAH progression by reducing pulmonary vascular and RV remodeling, representing the first evidence of a causal role of ASK1 in PAH pathogenesis [2]

C₂₃H₂₁N₇O

411.46

411.18

C, 67.14; H, 5.14; N, 23.83; O, 3.89

1262041-49-5

57504987

White to off-white solid powder

2

90.5

1

5

6

31

654

0

ZGCMQKWOUIMBEP-UHFFFAOYSA-N

InChI=1S/C23H21N7O/c31-23(20-11-19(8-9-24-20)29-12-21(25-13-29)15-4-5-15)27-17-3-1-2-16(10-17)22-28-26-14-30(22)18-6-7-18/h1-3,8-15,18H,4-7H2,(H,27,31)

4-(4-cyclopropylimidazol-1-yl)-N-[3-(4-cyclopropyl-1,2,4-triazol-3-yl)phenyl]pyridine-2-carboxamide

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: ≥ 2.5 mg/mL (6.08 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 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；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 2 中的溶解度: ≥ 2.5 mg/mL (6.08 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 25.0 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 3 中的溶解度: ≥ 2.5 mg/mL (6.08 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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；
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。