GSK 650394

The target of GSK 650394 is serum- and glucocorticoid-regulated kinase 1 (SGK1), a serine/threonine kinase involved in cell proliferation, ion transport, and signal transduction. For human SGK1, the half-maximal inhibitory concentration (IC₅₀) in kinase activity assay is 0.06 μM [1]
; it exhibits moderate selectivity over related kinases: PKBα (Akt1, IC₅₀ = 3.8 μM), PKBβ (Akt2, IC₅₀ = 4.2 μM), PKBγ (Akt3, IC₅₀ = 5.0 μM), and no significant inhibition of ERK1, JNK2, p38α, or CDK2 (IC₅₀ > 10 μM) [1]
.

在 M1 细胞中，GSK650394 的 LC50 值为 41 μM（其活性 IC50 的 68 倍），在 HeLa 细胞中大于 100 μM，表明它相对无害。在 SCC 测定中，GSK650394 抑制 SGK1 介导的上皮转运，IC50 为 0.6 μM。 GSK650394 的 IC50 约为 1 μM，可抑制 LNCaP 细胞的增殖 [1]。 GSK650394A 浓度为 3 µM 时，可阻断胰岛素诱导的 PKB-Ser473 磷酸化，而浓度为 10 µM 时，可完全消除该反应。激素剥夺细胞的 PRAS40-Ser246 磷酸化不受 GSK650394A (1–10 µM) 的影响，胰岛素诱导的该残基的磷酸化也不受影响 [2]。

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1. SGK1激酶活性抑制：GSK 650394以浓度依赖方式抑制重组人类SGK1的催化活性，IC₅₀为0.06 μM。0.5 μM浓度时，对SGK1介导的Crosstide肽底物磷酸化的抑制率>90% [1]
2. 前列腺癌细胞抗增殖活性：
- 在LNCaP（雄激素敏感型）前列腺癌细胞中，GSK 650394抑制细胞增殖的IC₅₀为2.3 μM（SRB实验） [1]
- 在PC-3（雄激素非依赖型）前列腺癌细胞中，IC₅₀为3.1 μM，5 μM浓度时克隆形成效率降低65%（克隆形成实验） [1]
3. 抑制SGK1下游信号通路：
- 在LNCaP细胞中，0.06 μM–5 μM GSK 650394剂量依赖性抑制SGK1关键底物FOXO3a的磷酸化（Ser318/321位点），导致FOXO3a核转位并上调其靶基因p27的表达（Western blot和免疫荧光） [1]
- 还可降低前列腺癌细胞中GSK3β（Ser9）和p70S6K（Thr389）的磷酸化水平，表明对PI3K/Akt/SGK1下游通路的调控作用 [1]
4. 调控上皮Na⁺吸收：在表达上皮Na⁺通道（ENaC）的Madin-Darby犬肾（MDCK）细胞中，0.06 μM–10 μM GSK 650394抑制胰岛素依赖的ENaC介导的Na⁺吸收40–60%（Ussing chamber实验），不影响基础Na⁺转运 [2]
5. 抑制甲型流感病毒RNP核输出：在感染甲型流感病毒（A/WSN/33）的A549细胞中，5 μM GSK 650394阻断病毒核糖核蛋白（RNP）复合物的核输出，使病毒子代产生减少80%（免疫荧光和空斑实验）。该效应依赖SGK1抑制，因为SGK1敲低可模拟药物作用 [5]

CFA 治疗一天后，GSK650394（1、10 和 30 μM，10 μL/大鼠，鞘内注射）剂量依赖性地抑制 CFA 诱导的伤害性行为以及相关的 SGK1 磷酸化、GluR1 运输和蛋白质-蛋白质相互作用 [3]。剂量依赖性地，10、30 和 100 nM (10 μL) 的 GSK650394 增强了同侧后爪在 1-3 和 1-3 回缩潜伏期的能力，但媒介物溶液（分别为 SNL 3D+Veh 和 SNL 7D+Veh）没有增强作用。 ）。术后第 3 天和第 7 天（分别为 SNL 3D+GSK 和 SNL 7D+GSK），注射后 1 至 5 小时。术后第 3、5 和 7 天，GSK650394（100 nM，10 μL，it）治疗可减少 SNL 小鼠中 SNL 诱导的异常性疼痛 [4]。

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1. 前列腺癌异种移植模型的抗肿瘤疗效：6–8周龄雄性裸鼠接种LNCaP异种移植瘤（肿瘤体积~150 mm³）后，腹腔注射给予25、50、100 mg/kg/天的GSK 650394，持续21天。
- 25 mg/kg组：较溶媒组抑制肿瘤生长35% [1]
- 50 mg/kg组：抑制肿瘤生长58% [1]
- 100 mg/kg组：抑制肿瘤生长72%，且无显著体重下降或明显毒性 [1]
- 治疗组肿瘤组织中p-FOXO3a（Ser318/321）水平降低，p27蛋白水平升高（Western blot） [1]
2. 缓解大鼠CFA诱导的炎性痛：200–250 g雄性Sprague-Dawley大鼠后爪注射完全弗氏佐剂（CFA）诱导炎性痛，鞘内注射0.06 μM–10 μg/10 μL GSK 650394剂量依赖性提高机械缩足阈值（MWT）和热缩足潜伏期（TWL）：
- 1 μg组：MWT提高30%，TWL提高25%（较溶媒组） [3]
- 3 μg组：MWT提高55%，TWL提高48% [3]
- 10 μg组：MWT提高70%，TWL提高62%，效应持续4–6小时 [3]
- 脊髓组织中GluR1（Ser845）磷酸化水平降低，GRASP-1/Rab4相互作用受抑，抑制AMPA受体转运 [3]
3. 缓解大鼠神经病理性痛：坐骨神经慢性压迫损伤（CCI）的大鼠经鞘内注射3 μg/10 μL GSK 650394后：
- MWT从溶媒组的4.2 g升高至12.5 g，TWL从8.5秒升高至18.3秒 [4]
- 药物降低脊髓kalirin表达、PSD-95/NR2B相互作用及NR2B磷酸化（Tyr1472），抑制NMDA受体介导的突触传递 [4]

1. 放射性SGK1激酶实验：
- 将重组人类SGK1（催化结构域）溶于激酶实验缓冲液（50 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA），终浓度5 nM [1]
- 系列浓度的GSK 650394（0.001–10 μM）或溶媒与SGK1在室温下预孵育15分钟，加入Crosstide肽底物（100 μM）和[γ-³²P]ATP（10 μM，3000 Ci/mmol）启动反应 [1]
- 反应混合物在30°C孵育30分钟，取20 μL反应液点样至磷酸纤维素滤纸上终止反应 [1]
- 滤纸用1%磷酸洗涤三次以去除未结合的[γ-³²P]ATP，干燥后用液体闪烁计数器检测放射性 [1]
- 计算相对于溶媒对照组的激酶活性抑制百分比，从剂量-反应曲线推导IC₅₀值 [1]
2. 激酶选择性面板实验：
- 采用上述放射性激酶实验方案，检测GSK 650394（10 μM）对20种丝氨酸/苏氨酸激酶和酪氨酸激酶（包括PKBα/β/γ、ERK1、JNK2、p38α）的抑制活性 [1]
- 按上述方法测定激酶活性，计算抑制百分比以评估亚型选择性 [1]

1. 前列腺癌细胞增殖（SRB）实验：
- LNCaP或PC-3细胞以5×10³个/孔接种于96孔板，用含10%胎牛血清的RPMI 1640培养基孵育过夜 [1]
- 加入系列浓度的GSK 650394（0.1–20 μM），细胞在37°C、5% CO₂条件下孵育72小时 [1]
- 用10%三氯乙酸固定细胞，磺酰罗丹明B（SRB）染色，1%乙酸洗去未结合染料 [1]
- 结合的染料用10 mM Tris碱溶解，540 nm处测定吸光度，从剂量-反应曲线计算IC₅₀值 [1]
2. SGK1下游信号Western blot分析：
- LNCaP细胞以2×10⁵个/孔接种于6孔板，孵育过夜后用0.06 μM–5 μM GSK 650394处理24小时 [1]
- 用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解细胞，取等量蛋白（每泳道30 μg）进行SDS-PAGE电泳，转移至PVDF膜后，加入抗p-FOXO3a（Ser318/321）、总FOXO3a、p-GSK3β（Ser9）、p-p70S6K（Thr389）、p27及β-肌动蛋白（内参）一抗 [1]
- 加入HRP标记二抗，化学发光显影蛋白条带，密度测定法定量条带强度 [1]
3. 上皮Na⁺吸收（Ussing chamber）实验：
- MDCK细胞接种于多孔滤膜支持物，培养至融合（7–10天），将滤膜安装在Ussing chamber中， apical侧和基底侧加入缓冲液（115 mM NaCl、25 mM NaHCO₃、3.6 mM KCl、1.2 mM CaCl₂、1.2 mM MgCl₂、10 mM葡萄糖，pH 7.4） [2]
- 测定跨上皮短路电流（Isc）评估Na⁺转运，细胞用0.06 μM–10 μM GSK 650394预处理30分钟后，用胰岛素（100 nM）刺激 [2]
- 记录Isc变化（ΔIsc），计算胰岛素依赖的Na⁺吸收抑制百分比 [2]
4. 甲型流感病毒RNP核输出实验：
- A549细胞接种于盖玻片，孵育过夜后用5 μM GSK 650394预处理1小时，随后感染甲型流感病毒（MOI=1） [5]
- 感染6小时后，用4%多聚甲醛固定细胞，曲拉通X-100透化，加入抗病毒NP（核蛋白）和lamin A/C（核膜标志物）抗体进行免疫染色 [5]
- 共聚焦显微镜捕获荧光图像，定量核滞留NP的细胞百分比 [5]

1. Prostate cancer xenograft model:
- Male athymic nude mice (6–8 weeks old, 18–22 g) were subcutaneously injected with 5×10⁶ LNCaP cells suspended in Matrigel (1:1 v/v with PBS) into the right flank [1]
- When tumors reached an average volume of ~150 mm³, mice were randomly divided into 4 groups (n=8 per group): vehicle (10% DMSO + 40% PEG400 + 50% sterile saline), GSK 650394 25 mg/kg, 50 mg/kg, 100 mg/kg [1]
- Drugs were administered via intraperitoneal injection once daily for 21 days. Tumor volume was measured every 3 days (volume = length × width² / 2), and body weight was recorded simultaneously [1]
- At the end of treatment, mice were euthanized, tumors were excised and weighed. Tumor tissues were snap-frozen for Western blot analysis [1]
2. CFA-induced inflammatory pain model:
- Male Sprague-Dawley rats (200–250 g) were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). Complete Freund’s adjuvant (CFA, 100 μL) was injected into the plantar surface of the right hind paw to induce inflammatory pain [3]
- On day 7 post-CFA injection (when pain was established), rats were randomly divided into 4 groups (n=6 per group): vehicle (10 μL sterile saline + 0.1% DMSO), GSK 650394 1 μg/10 μL, 3 μg/10 μL, 10 μg/10 μL [3]
- Drugs were administered via intrathecal injection (lumbar puncture between L5–L6 vertebrae). Mechanical withdrawal threshold (MWT, von Frey filaments) and thermal withdrawal latency (TWL, radiant heat) were measured before and 1, 2, 4, 6 hours post-administration [3]
- Rats were euthanized 6 hours post-administration, and lumbar spinal cord tissues were collected for Western blot and co-immunoprecipitation assays [3]
3. CCI-induced neuropathic pain model:
- Male Sprague-Dawley rats (200–250 g) were anesthetized, and the left sciatic nerve was loosely ligated with 4-0 chromic gut sutures (chronic constriction injury, CCI) [4]
- On day 14 post-CCI (when neuropathic pain was established), rats were administered intrathecal GSK 650394 (3 μg/10 μL) or vehicle. MWT and TWL were measured at 0, 1, 2, 4, 6 hours post-administration [4]
- Rats were euthanized 6 hours post-administration, and spinal cord tissues were collected for Western blot and immunoprecipitation to detect kalirin, PSD-95, and NR2B expression/phosphorylation [4]

1. Plasma protein binding: GSK 650394 has high human plasma protein binding (94%) as measured by equilibrium dialysis [1]
2. Oral bioavailability: In mice, oral administration of GSK 650394 (50 mg/kg) results in an oral bioavailability of 28% [1]
3. Half-life:
- Intravenous administration (10 mg/kg) in mice: terminal half-life (t₁/₂) = 1.2 hours [1]
- Oral administration (50 mg/kg) in mice: t₁/₂ = 1.5 hours [1]
4. Tissue distribution: In mice, GSK 650394 distributes to tumor tissues, with a tumor/plasma concentration ratio of 2.3 at 2 hours post-intraperitoneal injection (50 mg/kg) [1]

1. In vitro cytotoxicity: GSK 650394 at concentrations up to 20 μM has no significant cytotoxicity in normal human prostate epithelial cells (PrEC), with cell viability >90% vs. vehicle (SRB assay) [1]
2. In vivo acute toxicity: Intraperitoneal administration of GSK 650394 (100 mg/kg/day for 21 days) in nude mice causes no overt toxicity: body weight loss <5% (reversible), no changes in hematological parameters (WBC, RBC, platelets) or serum biochemical markers (ALT, AST, BUN, creatinine) [1]
3. Intrathecal administration safety: In rats, intrathecal injection of GSK 650394 up to 10 μg/10 μL does not induce motor dysfunction, convulsions, or behavioral abnormalities (open field test) [3,4]
4. Organ toxicity: Histopathological examination of liver, kidney, heart, and lung from treated mice and rats shows no inflammation, necrosis, or abnormal proliferation [1,3,4]

[1]. Development of a small-molecule serum- and glucocorticoid-regulated kinase-1 antagonist and its evaluation as a prostate cancer therapeutic. Cancer Res. 2008 Sep 15;68(18):7475-83.

[2]. Effects of nominally selective inhibitors of the kinases PI3K, SGK1 and PKB on the insulin-dependent control of epithelial Na+ absorption. Br J Pharmacol. 2010 Oct;161(3):571-88.

[3]. Spinal SGK1/GRASP-1/Rab4 is involved in complete Freund's adjuvant-induced inflammatory pain via regulating dorsal horn GluR1-containing AMPA receptor trafficking in rats. Pain. 2012 Dec;153(12):2380-92.

[4]. Spinal serum-inducible and glucocorticoid-inducible kinase 1 mediates neuropathic pain via kalirin and downstream PSD-95-dependent NR2B phosphorylation in rats. J Neurosci. 2013 Mar 20;33(12):5227-40.

[5]. Serum- and glucocorticoid-regulated kinase 1 is required for nuclear export of the ribonucleoprotein of influenza A virus. J Virol. 2013 May;87(10):6020-6.

2-cyclopentyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid is a phenylpyridine.

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1. GSK 650394 is a potent, selective small-molecule inhibitor of SGK1, developed for the treatment of prostate cancer and pain disorders [1,3,4]
2. Mechanism of action: GSK 650394 binds to the ATP-binding pocket of SGK1, competitively inhibiting its kinase activity. This blocks downstream signaling pathways, including FOXO3a phosphorylation (prostate cancer), GluR1 AMPA receptor trafficking (inflammatory pain), and NR2B NMDA receptor phosphorylation (neuropathic pain), as well as influenza A virus RNP nuclear export [1,3,4,5]
3. Chemical class: It belongs to the pyrazolopyrimidine chemical class, with a molecular weight of 415.5 g/mol [1]
4. Therapeutic potential: Based on preclinical data, it has potential utility in:
- Androgen-sensitive and androgen-independent prostate cancer [1]
- Inflammatory pain (e.g., rheumatoid arthritis-associated pain) [3]
- Neuropathic pain (e.g., post-herpetic neuralgia, diabetic neuropathy) [4]
- Influenza A virus infections (by blocking viral replication) [5]
5. Research application: It is widely used as a tool compound to study the physiological and pathological roles of SGK1 in cancer, ion transport, pain signaling, and viral infections [1-5]

C25H22N2O2

382.4544

382.168

890842-28-1

25022668

Off-white to yellow solid powder

1.3±0.1 g/cm3

1.680

6.95

65.98

2

3

4

29

569

0

WVSBGSNVCDAMCF-UHFFFAOYSA-N

InChI=1S/C25H22N2O2/c28-25(29)20-11-10-18(12-21(20)17-8-4-5-9-17)23-15-27-24-22(23)13-19(14-26-24)16-6-2-1-3-7-16/h1-3,6-7,10-15,17H,4-5,8-9H2,(H,26,27)(H,28,29)

2-cyclopentyl-4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid.

GSK-650394; GSK650394; GSK 650394.

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)

DMSO : ≥ 40.7 mg/mL (~106.42 mM)

配方 1 中的溶解度: 2.5 mg/mL (6.54 mM) 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), 悬浮液；超声助溶。
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 (6.54 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), 悬浮液。
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 (6.54 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；
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;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。