GS-444217 is a selective ATP-competitive inhibitor of apoptosis signal-regulating kinase 1 (ASK1, IC50: 2.87 nM).

ASK1:2.87 nM (cell free)

GS-444217 对 ASK1 的结合具有高选择性，与面板中的其他激酶相比。GS-444217 对 ASK1 的亲和力（KD = 4.1 nM）是对 DYRK1A（KD = 220 nM）的 53 倍，是对 RSK4（KD = 430 nM）的 104 倍。GS-444217 的处理降低了 ASK1 的磷酸化，并防止了在 0.3 μM 及以上浓度下 MKK3/6、MKK4、p38 和 JNK 的磷酸化，1 μM 时完全抑制了 ASK1 活性。GS-444217 降低 ASK1 活性，在加入培养物后 5 分钟内达到，并在 30 分钟内达到最大抑制水平。从培养物中移除 GS-444217 后，ASK1 自磷酸化在 10 分钟内重新激活，并在化合物清洗后 2 小时内近乎完全恢复[1]。

使用GS-444217处理能够抑制糖尿病肾脏中p38 MAPK的激活，但对Nos3(-/-)小鼠的高血压无影响。GS-444217的早期干预显著抑制了糖尿病性肾小球硬化，减少了肾功能障碍，但对白蛋白尿的发展无效。GS-444217的晚期干预改善了肾功能，阻止了肾小球硬化、肾脏炎症和肾小管损伤的进展，尽管对已确立的白蛋白尿无影响[2]。GS-444217（30 mg/kg）单剂量给药，在给予金刚烷胺（30 mg/kg）前30分钟，能够抑制肾皮质中ASK1、p38和JNK的激活。金刚烷胺的给药引起了炎症细胞因子（Il1b、Ccl2和Cxcl2）mRNA表达的增加，并在肾脏中增加了caspase活性。这些ASK1激活的下游效应被GS-444217所抑制。通过比较GS-444217的血浆浓度与肾脏中相应的磷酸化p38（p-p38）信号，GS-444217对抑制啮齿动物肾脏中的ASK1通路具有大约1.6 μM的体内EC50[1]。

Human embryonic kidney cells (HEK293T) were infected with full-length human ASK1 adenovirus or with an inactive truncated ASK1 adenovirus (K709R mutant with N-terminally truncated protein) as a negative control using the following conditions: (a) dose response: cells were infected for 24 hours followed by incubation for 2 hours with 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μM GS-444217; (b) kinetics: after cells were infected for 24 hours, 1 μM GS-444217 was added to cells for 1, 5, 10, or 30 minutes or 1, 2, or 4 hours; (c) off-rate kinetics: cells were infected for 24 hours followed by incubation with GS-444217 for 30 minutes. After 30 minutes, medium was replaced with serum-free medium without compound and incubated for 0, 10, or 30 minutes or 1, 2, or 4 hours [1].

Male Sprague-Dawley rats (176–200 g, 7–8 weeks old) were randomly assigned to weight-matched treatment groups: (a) sham procedure, n = 8; (b) ischemia 30 minutes, n = 8; (c) ischemia 30 minutes plus GS-444217 (30 mg/kg, p.o.), n = 8. Bilateral renal occlusion was performed on anesthetized rats held at 37°C for 30 minutes. After recovering from anesthesia, rats were placed in metabolic cages for a 24-hour collection of urine. Sham rats underwent midline incision with surgery duration of 30 minutes but were not subjected to occlusion. Necropsy was performed on all rats 24 hours after surgery to collect kidneys and blood. Renal I/R studies were performed at Physiogenix Inc. Serum was analyzed for creatinine and blood urea nitrogen concentrations on a clinical chemistry analyzer. The right kidney was fixed in formalin and stained with H&E to assess tubular necrosis by pathology and with TUNEL to detect apoptotic cells [1].