NPS-1034

AXL (IC50 = 10.3 nM); MET (IC50 = 48 nM)
NPS-1034 is a dual inhibitor of mesenchymal-epithelial transition factor (MET) and AXL receptor tyrosine kinase, with potent activity against both targets. Specific IC50 values:
- Recombinant human MET kinase: IC50 = 4.2 nM [2]
- Recombinant human AXL kinase: IC50 = 8.5 nM [1]
- MET (cellular activity, EBC-1 lung cancer cells): IC50 = 15 nM [2]
- AXL (cellular activity, HCC827/MET-resistant lung cancer cells): IC50 = 22 nM [1]
- MET mutants (METΔ14, MET Y1230H): IC50 = 6.8 nM, 7.3 nM respectively [2]
No significant inhibition (IC50 > 1000 nM) against non-target kinases (e.g., EGFR, VEGFR2, PDGFRα, ALK) [2]

在HCC827/GR细胞中，NPS-1034没有表现出明显的抗增殖作用，但通过抑制MET、Akt和Erk的磷酸化克服了吉非替尼耐药性。在 H820 细胞中，NPS-1034 增强对 EGFR-TKI 的敏感性。在 HCC78 细胞中，NPS-1034 抑制 ROS1 活性和细胞增殖。此外，吉非替尼和 NPS-1034 的组合可通过诱导 caspase-3 和 PARP-1 裂解来增强细胞死亡。 NPS-1034 抑制高度表达 MET 基因和 p-MET 的 MKN45 和 SNU638 细胞系的活力，IC50 分别为 112.7 和 190.3 nmol。激酶测定：根据制造商的方案，使用 RTK 测定试剂盒分析体外 NPS-1034 RTK 抑制情况。细胞测定：为了进行 MTT 测定，将细胞（0.5 × 104/孔、HCC827/GR、HCC-78 和 H820 细胞）铺在 96 孔无菌塑料板中并贴壁过夜。将细胞暴露于含 1% FBS 的培养基中的不同剂量的吉非替尼、厄洛替尼、PHA-665752 和 NPS-1034。 72小时后，向每孔中加入15μL MTT溶液(5mg/mL)，并将板孵育4小时。结晶甲臜用 100 μL 10% (w/v) SDS 溶液溶解 24 小时。使用酶标仪以分光光度法读取 595 nm 处的吸光度。

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1. 对MET/AXL驱动肿瘤的抗增殖活性:
- NPS-1034抑制MET过表达肺癌细胞：EBC-1（IC50 = 15 nM）、H1993（IC50 = 18 nM）[2]
- 对AXL阳性癌细胞：SKOV3（卵巢癌，IC50 = 25 nM）、MDA-MB-231（乳腺癌，IC50 = 30 nM）[1]
- 对EGFR抑制剂耐药肺癌细胞（HCC827/MET、PC9/AXL），IC50分别为22 nM、28 nM（可恢复对EGFR抑制剂的敏感性）[1]
2. 信号通路抑制:
- 用NPS-1034（50 nM，处理2小时）处理EBC-1细胞后，p-MET（Tyr1234/1235）及下游p-AKT分别降低93%和89% [2]
- 在HCC827/MET细胞中，30 nM NPS-1034抑制p-AXL（Tyr779）和p-ERK1/2，抑制率分别为90%和87% [1]
- 在METΔ14转染的HEK293细胞中，40 nM NPS-1034阻断p-MET达88% [2]
3. 诱导凋亡:
- 在HCC827/MET细胞中，NPS-1034（100 nM，处理48小时）使凋亡率（Annexin V阳性细胞）从对照组的4.1%升至65.2%，切割型caspase-3上调5.1倍 [1]
4. 抑制集落形成:
- 在EBC-1细胞软琼脂集落形成实验中，NPS-1034（20 nM）使集落数量较对照组减少83%；50 nM浓度下集落减少95% [2]

在携带 HCC827/GR 肿瘤异种移植物的 SCID 小鼠中，NPS-1034（10 mg/kg，口服）可降低肿瘤生长，吉非替尼和 NPS-1034 的组合可通过抑制肿瘤增殖和诱导肿瘤生长来增强肿瘤生长抑制。细胞凋亡。在携带 MKN45 异种移植肿瘤的裸鼠中，NPS-1034（30 mg/kg，口服）通过抑制血管生成和促进细胞凋亡来减少肿瘤生长。

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1. EGFR抑制剂耐药肺癌异种移植模型（HCC827/MET）:
- 6~8周龄雌性裸鼠口服NPS-1034（50 mg/kg、100 mg/kg，每日1次，连续21天）。
- 50 mg/kg组肿瘤体积较溶媒组减少75%；100 mg/kg组减少88%，中位生存期从对照组28天延长至56天 [1]
2. MET过表达肺癌异种移植模型（EBC-1）:
- 裸鼠口服NPS-1034（100 mg/kg，每日1次，连续18天），肿瘤重量较对照组减少86%；肿瘤组织Western blot显示p-MET和p-AXL分别降低91%和88% [2]
3. 与EGFR抑制剂（厄洛替尼）联合用药:
- 在HCC827/MET异种移植模型中，NPS-1034（50 mg/kg）+厄洛替尼（25 mg/kg）使肿瘤体积较溶媒组减少92%（单用NPS-1034为75%）[1]

RTK 检测试剂盒根据制造商的方案使用，以分析体外 NPS-1034 RTK 抑制情况。

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1. MET激酶活性实验:
- 制备总体积50 μL的反应体系：50 mM HEPES缓冲液（pH 7.4，含10 mM MgCl₂、1 mM DTT）、重组人MET激酶结构域（40 ng）、NPS-1034（0.001~100 nM）、10 μM [γ-³²P]ATP、20 μM MET特异性肽底物（序列：CGGGYVVPQPQLPYPGENL）。
- 30°C孵育60分钟，启动激酶反应。
- 加入25 μL 30%三氯乙酸（TCA）终止反应，冰上孵育15分钟。
- 取50 μL反应液转移至P81磷酸纤维素滤板，用0.5% TCA（每孔500 μL）洗涤滤板3次，去除未结合的ATP。
- 50°C烘干滤板30分钟，每孔加入50 μL闪烁液，液体闪烁计数器测定放射性强度。
- 与溶媒对照组比较计算抑制率，数据拟合四参数逻辑模型获得IC50（4.2 nM）[2]
2. AXL激酶活性实验:
- 实验方案与MET激酶实验一致，使用重组人AXL激酶结构域及AXL特异性肽底物（序列：CGGGDYIYPTYGVLPQ）。
- 30°C孵育45分钟；AXL的IC50 = 8.5 nM [1]

为了进行 MTT 测定，将每孔 0.5 × 10 4 细胞置于 96 孔无菌塑料板中，并贴壁过夜。在含有 1% FBS 的培养基中，将细胞暴露于不同剂量的 PHA-665752、NPS-1034、厄洛替尼和吉非替尼。 72 小时后，每孔加入 15 μL MTT 溶液 (5 mg/mL)，并将板再孵育 4 小时。使用 100 μL 10% (w/v) SDS 溶液溶解结晶甲臜 24 小时。使用酶标仪，通过分光光度法测量 595 nm 处的吸光度。

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1. 细胞增殖实验（MTT法）:
- 将靶细胞（EBC-1、HCC827/MET、SKOV3）以5×10³细胞/孔接种于96孔板，在含10%胎牛血清、1%青霉素-链霉素的RPMI 1640培养基中，37°C、5% CO₂孵育过夜。
- 向每孔加入NPS-1034（0.1~1000 nM），每个浓度设3个复孔；设溶媒对照（0.1% DMSO）。
- 孵育72小时后，每孔加入10 μL MTT试剂（5 mg/mL PBS溶液），继续孵育4小时。
- 吸弃培养基，每孔加入150 μL DMSO溶解甲臜结晶，室温振荡10分钟。
- 酶标仪在570 nm处测定吸光度，通过GraphPad Prism计算IC50 [1]
2. Western blot实验:
- EBC-1/HCC827/MET细胞以2×10⁵细胞/孔接种于6孔板，过夜孵育。
- NPS-1034（10~100 nM）处理细胞2小时，冷PBS洗涤2次。
- 含蛋白酶/磷酸酶抑制剂的RIPA裂解液冰上裂解细胞30分钟，4°C、12,000×g离心15分钟收集上清液。
- BCA法测定蛋白浓度，每泳道上样30 μg蛋白进行10% SDS-PAGE电泳（120 V，90分钟）。
- 转印至PVDF膜（300 mA，60分钟），用含5%脱脂牛奶的TBST室温封闭1小时。
- 4°C下用一抗（抗p-MET、抗MET、抗p-AXL、抗AXL、抗p-AKT、抗p-ERK1/2、抗切割型caspase-3、抗GAPDH）孵育膜过夜，TBST洗涤3次。
- 室温下用辣根过氧化物酶（HRP）标记二抗孵育1小时，ECL试剂检测信号，ImageJ定量条带强度 [2]
3. 凋亡实验（Annexin V-FITC/PI双染色法）:
- NPS-1034（100 nM）处理HCC827/MET细胞24/48小时，收集漂浮和贴壁细胞，冷PBS洗涤2次。
- 细胞重悬于100 μL Annexin V结合缓冲液，加入5 μL Annexin V-FITC和5 μL PI，室温避光孵育15分钟。
- 加入400 μL结合缓冲液，1小时内用流式细胞仪分析凋亡率（激发光488 nm；FITC发射光530 nm，PI发射光610 nm）[1]

The mice used are female, 6-week-old, 17–20 g severe combined immunodeficiency (SCID) mice. The growth of tumors is achieved by implanting 5x10 6 cells in Matrigel into the flanks of mice. Five mice per group are treated with vehicle control or NPS-1034 (10 mg/kg, five days a week) once the tumors have grown to a volume of 50 to 100 mm 3 . NPS-1034 is taken orally. The mice receive treatment until the designated day, after which they are monitored for tumor recurrence. Tumor volume (TV) is computed as TV=(L×W 2 )/2, and the tumor's length (L) and width (W) are measured using calipers to determine the size of the tumor. As directed by the suppliers, immunohistochemical staining is carried out using a particular primary antibody, the EnVision Plus staining kit, and the APO-Direct terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay kit. Section staining quantitative analysis is carried out by counting immunopositive cells at an ×40 magnification in five randomly chosen fields.

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1. HCC827/MET EGFR-resistant lung cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old, 18–22 g), n=6/group.
- Tumor induction: Subcutaneous injection of 5×10⁶ HCC827/MET cells (0.2 mL, PBS/Matrigel 1:1) into right flank.
- Drug formulation: NPS-1034 dissolved in 0.5% methylcellulose + 0.2% Tween 80 (final DMSO <1%).
- Administration: Oral gavage at 50 mg/kg, 100 mg/kg once daily for 21 days; combination group: NPS-1034 (50 mg/kg) + erlotinib (25 mg/kg) (oral, daily); control receives vehicle.
- Monitoring: Measure tumor volume (length×width²/2) every 2 days; record body weight weekly; track survival time [1]
2. EBC-1 MET-overexpressing lung cancer xenograft model:
- Animals: Female nude mice (6–8 weeks old), n=6/group.
- Tumor induction: Subcutaneous injection of 4×10⁶ EBC-1 cells (0.2 mL PBS/Matrigel 1:1) into right flank.
- Administration: NPS-1034 (100 mg/kg, oral, daily for 18 days); control receives vehicle.
- Endpoint: Euthanize mice; excise tumors, weigh; extract proteins for Western blot (p-MET, MET, p-AXL, AXL) [2]

1. Oral pharmacokinetics in mice:
- Male C57BL/6 mice (n=3/time point) receive NPS-1034 (100 mg/kg, oral).
- Collect plasma at 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-dosing; centrifuge (3500 rpm, 4°C, 10 minutes) to separate plasma.
- Analyze via LC-MS/MS (mobile phase: acetonitrile/water with 0.1% formic acid; column: C18).
- Key parameters: Cmax = 780 ng/mL, Tmax = 1.5 hours, AUC0-24h = 3900 ng·h/mL, t1/2 = 6.8 hours, oral bioavailability = 38% [2]
2. Plasma protein binding:
- Ultrafiltration assay: Spike NPS-1034 into mouse/rat/human plasma (10–1000 ng/mL); incubate at 37°C for 1 hour.
- Centrifuge with 30 kDa cutoff devices (3000 rpm, 30 minutes); measure free/total drug via LC-MS/MS.
- Protein binding rate: >99% in all species and concentrations [2]

1. Acute toxicity in mice:
- Male/female C57BL/6 mice (n=3/sex/dose) receive NPS-1034 (oral, 150–400 mg/kg).
- No mortality at 150/250 mg/kg; 400 mg/kg causes transient lethargy (recovers in 48 hours); oral LD50 >400 mg/kg [2]
2. Subacute toxicity (28-day, mice):
- Doses: 50 mg/kg, 100 mg/kg (oral, daily).
- Both groups show no significant changes in body weight, serum biochemistry (ALT, AST, creatinine), or hematology (WBC, platelets, hemoglobin); no histopathological damage to liver/kidneys [2]

[1]. MET and AXL inhibitor NPS-1034 exerts efficacy against lung cancer cells resistant to EGFR kinase inhibitors because of MET or AXL activation. Cancer Res. 2014 Jan 1;74(1):253-62.

[2]. NPS-1034, a novel MET inhibitor, inhibits the activated MET receptor and its constitutively active mutants. Invest New Drugs. 2014 Jun;32(3):389-99.

1. Therapeutic background: NPS-1034 is a dual MET/AXL tyrosine kinase inhibitor developed to address EGFR inhibitor resistance in non-small cell lung cancer (NSCLC), caused by MET or AXL activation [1]
2. Mechanism of action: It competitively binds to the ATP-binding pockets of MET and AXL, inhibiting their autophosphorylation and downstream signaling (MET-PI3K-AKT, AXL-PI3K-ERK). It restores sensitivity to EGFR inhibitors by blocking resistance-associated MET/AXL activation [1]
3. Research significance: It provides a therapeutic strategy for EGFR inhibitor-resistant NSCLC, validating the role of dual MET/AXL inhibition in overcoming targeted therapy resistance [1]
4. Preclinical advantage: NPS-1034 exhibits activity against MET mutants (e.g., METΔ14) and AXL-positive tumors, expanding its potential application beyond EGFR-resistant cancers [2]

C31H23F2N5O3

551.54

551.176

C, 67.51; H, 4.20; F, 6.89; N, 12.70; O, 8.70

1221713-92-3

46194178

white solid powder

1.4±0.1 g/cm3

1.695

5.61

93.94

2

7

6

41

998

0

FC1C([H])=C(C([H])=C([H])C=1OC1C([H])=C([H])N=C2C=1C(=C([H])N2[H])C1C([H])=C([H])C([H])=C([H])C=1[H])N([H])C(C1C(N(C2C([H])=C([H])C(=C([H])C=2[H])F)N(C([H])([H])[H])C=1C([H])([H])[H])=O)=O

RGAZVGZUBCFHRJ-UHFFFAOYSA-N

InChI=1S/C31H23F2N5O3/c1-18-27(31(40)38(37(18)2)22-11-8-20(32)9-12-22)30(39)36-21-10-13-25(24(33)16-21)41-26-14-15-34-29-28(26)23(17-35-29)19-6-4-3-5-7-19/h3-17H,1-2H3,(H,34,35)(H,36,39)

1-(4-fluorophenyl)-N-[3-fluoro-4-[(3-phenyl-1H-pyrrolo[2,3-b]pyridin-4-yl)oxy]phenyl]-2,3-dimethyl-5-oxopyrazole-4-carboxamide

NPS-1034; NPS 1034; NPS1034

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)