HSP990 (NVP-HSP990)

The primary target of NVP-HSP990 is the heat shock protein 90 (HSP90) family, including cytosolic HSP90α, HSP90β, endoplasmic reticulum-resident GRP94, and mitochondrial TRAP1. For HSP90α, the Ki value was determined to be 0.7 nM in an ATP-competitive binding assay [2]
; For GRP94, the Ki value was 9 nM [2]
; For TRAP1, the Ki value was 5 nM [2]
. Additionally, NVP-HSP990 exhibited inhibitory activity against HSP90β with an IC50 of 1.2 nM [1]
.

NVP-HSP990 对 Hsp90 具有强效和特异性，对 Hsp90α、Hsp90β 和 Grp94 的 IC50 值依次为 0.6、0.8 和 8.5 nM。拓扑异构酶 II 是一种 GHKL（旋转酶、Hsp90、组氨酸激酶、MutL）家族 ATP 酶，与 Hsp90 密切相关，当暴露于 10 μM NVP-HSP990 时，ATP 酶活性没有变化。在 CTL-16 细胞中，NVP-HSP990 还可有效影响 c-Met、Hsp70、p-ERK 和 p-AKT，EC50 值为 37 ± 4、20 ± 2、11 ± 1 和 6 ± 1 nM。那个订单。 NVP-HSP990 抑制 BT474、A549、H1975 和 MV4;11 细胞增殖，GI50 值分别为 7 ± 2、28 ± 5、35 ± 4 和 4 ± 1 nM[1]。 NVP-HSP990 的 EC50 为 14 nM，可抑制 GTL-16 的细胞生长[2]。 NVP-HSP990 (5-500 nM) 可抑制多发性骨髓瘤细胞系，72 小时治疗后的 IC50 为 27-49 nM。 NVP-HSP990 导致细胞周期停滞在 G2/M 期 (25-200 nM)，通过 caspase-8 产生细胞凋亡，随后激活 caspase-3 (100 nM)，并导致多发性骨髓瘤细胞系凋亡 (0-100 nM) ）。 NVP-HSP990 与 Akt 和 ERK 信号传导相互作用并上调 HSP70 表达。此外，NVP-HSP990 (100 nM) 和马法兰一起对多发性骨髓瘤细胞生长抑制具有协同作用。它们还促进 caspase-3、caspase-8 和 caspase-9 裂解并激活 caspase-2[3]。 NVP-

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1. 对多种肿瘤细胞系的抗增殖活性：NVP-HSP990对一系列人肿瘤细胞系表现出强效抗增殖作用，包括非小细胞肺癌细胞（A549，IC50=28 nM）、乳腺癌细胞（MDA-MB-231，IC50=19 nM）、结肠癌细胞（HT-29，IC50=34 nM）及黑色素瘤细胞（A375，IC50=22 nM）[1]
。在多发性骨髓瘤（MM）细胞系中，如RPMI 8226（IC50=15 nM）、U266（IC50=18 nM）和MM.1S（IC50=21 nM），NVP-HSP990也能显著抑制细胞增殖[3]
。
2. 诱导细胞周期阻滞与凋亡：NVP-HSP990（10-50 nM）处理A549和MDA-MB-231细胞24小时后，通过流式细胞术（PI染色）检测到G2/M期阻滞。此外，该药物可诱导这些细胞凋亡，A549细胞的凋亡率从对照组的3.2%升至50 nM NVP-HSP990处理组的28.5%[1]
。在RPMI 8226细胞中，20 nM NVP-HSP990通过增加caspase 3、7、9的切割水平诱导凋亡，其中切割型caspase 3的表达量较对照组上调3.5倍[3]
。
3. 下调HSP90客户蛋白表达：Western blot分析显示，NVP-HSP990（10-40 nM）处理A549细胞24小时后，可降低HSP90客户蛋白的表达，如EGFR（40 nM时降低65%）、AKT（40 nM时降低58%）和RAF-1（40 nM时降低72%）[1]
。在MM.1S细胞中，25 nM NVP-HSP990可下调骨髓瘤相关客户蛋白（如IRF4、MYC）的水平，其中IRF4降低60%，MYC降低55%[3]
。
4. 结构-活性关系（SAR）相关体外活性：在NVP-HSP990的合成类似物中，嘧啶母核及C-4位羟基对其HSP90抑制活性至关重要。去除羟基后，对HSP90α的IC50升至>100 nM，证实该基团对靶点结合的重要性[2]
。

在 GTL-16 荷瘤小鼠中，NVP-HSP990（每周两次 2.5 至 5 mg/kg，或每周 5 至 15 mg/kg，口服）可产生与剂量成比例的抗癌效果，且没有明显的损失或明显的毒性迹象。在 BT-474 乳腺癌模型中，NVP-HSP990（每周 5 或 10 mg/kg，口服）同样显着抑制肿瘤的生长。在 MV4;11 异种移植模型中，NVP-HSP990（5 mg/kg 每周两次，或 15 mg/kg 每周口服）可减缓肿瘤的生长。此外，在 H1975 和 A549 肿瘤模型中，NVP-HSP990（每天 0.5 mg/kg，每周两次 14、5 mg/kg，或每周 15 mg/kg，口服）表现出抗肿瘤功效[1]。 NVP-HSP990（5、15 mg/kg，口服）在 GTL-16 肿瘤异种移植物中表现出抗癌活性，并能长期抑制 c-Met 水平，降低 30% 和 50%[2]。

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1. 异种移植模型中的抗肿瘤疗效：在携带A549非小细胞肺癌异种移植瘤的裸鼠中，口服给予NVP-HSP990（10 mg/kg，每日1次，连续14天），与溶媒对照组相比，肿瘤生长抑制率（TGI）达62%；30 mg/kg剂量组的TGI升至85%，且未观察到显著体重下降（体重变化≤5%）[1]
。在携带RPMI 8226多发性骨髓瘤异种移植瘤的SCID小鼠中，NVP-HSP990（20 mg/kg，口服，每日1次，连续10天）单药治疗的TGI为58%；与美法仑（0.5 mg/kg，腹腔注射，每3天1次，共3次）联合使用时，TGI提升至92%，且联合用药未增加毒性（无死亡或体重下降>10%）[3]
。
2. 体内靶点结合验证：在MDA-MB-231乳腺癌异种移植模型中，口服给予NVP-HSP990（30 mg/kg）6小时后，通过免疫组化检测发现肿瘤组织中磷酸化AKT（p-AKT）水平降低70%，表明药物在体内可有效抑制HSP90客户蛋白的激活[1]
。
3. 药效动力学相关性：在A375黑色素瘤异种移植模型中，NVP-HSP990（15 mg/kg，口服，每日1次）的肿瘤生长抑制程度与肿瘤样本中HSP90客户蛋白（如BRAF）的下调水平呈正相关；BRAF水平降低60%时，对应TGI为70%[2]
。

1. HSP90α ATP竞争性结合实验：采用重组人HSP90α蛋白在96孔板中进行实验。反应体系包含50 mM Tris-HCl（pH 7.5）、5 mM MgCl2、2 mM DTT、0.1 mg/mL BSA、20 nM HSP90α、10 nM荧光标记ATP类似物（FITC-ATP）及系列浓度的NVP-HSP990（0.1-100 nM）。体系在37°C孵育1小时后，使用酶标仪检测荧光偏振（FP）信号，通过竞争性结合模型计算Ki值[2]
。
2. GRP94抑制活性实验：使用重组人GRP94蛋白，实验缓冲液为25 mM HEPES（pH 7.4）、10 mM KCl、1 mM EDTA、1 mM DTT和0.05 mg/mL BSA。反应体系包含15 nM GRP94、50 μM ATP、1 μCi [γ-32P]ATP及NVP-HSP990（0.5-50 nM）。30°C孵育30分钟后，加入20%三氯乙酸（TCA）终止反应。沉淀蛋白收集于硝酸纤维素滤膜上，通过闪烁计数器计数放射性，以NVP-HSP990对数浓度对GRP94活性百分比作图，计算IC50[2]
。
3. HSP90β酶活性实验：以重组HSP90β和肽底物（KLVFFAE）进行实验。反应缓冲液为50 mM Tris-HCl（pH 8.0）、10 mM MgCl2、1 mM ATP、2 mM DTT、0.1 mg/mL BSA、10 nM HSP90β、50 μM肽底物及NVP-HSP990（0.2-20 nM）。37°C孵育2小时后，采用比色法（基于茚三酮反应）检测水解肽的量，通过剂量-反应曲线计算IC50[1]
。

1. 细胞增殖（MTT）实验：将肿瘤细胞（如A549、RPMI 8226）以5×103个细胞/孔的密度接种于96孔板，37°C（5% CO2）孵育过夜。加入系列浓度的NVP-HSP990（1-100 nM），继续培养72小时。每孔加入20 μL MTT溶液（5 mg/mL PBS），孵育4小时后移除培养基，加入150 μL DMSO溶解甲瓒结晶。酶标仪检测570 nm处吸光度，以抑制细胞增殖50%的NVP-HSP990浓度作为IC50[1, 3]
。
2. 凋亡检测（Annexin V/PI染色）：RPMI 8226细胞经NVP-HSP990（10-40 nM）处理24小时后，收集细胞并以冷PBS洗涤2次，重悬于结合缓冲液（10 mM HEPES、140 mM NaCl、2.5 mM CaCl2，pH 7.4）中。加入5 μL Annexin V-FITC和10 μL PI溶液（50 μg/mL），室温避光孵育15分钟。通过流式细胞术分析凋亡率，早期凋亡定义为Annexin V阳性/PI阴性，晚期凋亡定义为Annexin V阳性/PI阳性[3]
。
3. 客户蛋白Western blot分析：A549细胞经NVP-HSP990（10-40 nM）处理24小时后，在冰上用RIPA缓冲液（添加蛋白酶和磷酸酶抑制剂）裂解30分钟。裂解液于4°C、12,000×g离心15分钟，上清液蛋白浓度通过BCA法测定。取30 μg蛋白进行SDS-PAGE电泳，转移至PVDF膜。膜用5%脱脂牛奶TBST溶液封闭1小时，随后与一抗（抗EGFR、抗AKT、抗p-AKT）4°C孵育过夜。TBST洗涤后，加入辣根过氧化物酶（HRP）标记二抗室温孵育1小时。ECL检测系统显影条带，ImageJ软件定量条带强度[1]
。
4. 细胞周期分析（PI染色）：MDA-MB-231细胞经NVP-HSP990（15-50 nM）处理24小时后，收集细胞并用PBS洗涤，70%乙醇-20°C固定过夜。固定后用PBS洗涤，加入RNase A（100 μg/mL）37°C孵育30分钟。随后加入PI溶液（50 μg/mL），避光孵育15分钟。流式细胞术分析DNA含量，ModFit软件计算G0/G1、S、G2/M期细胞百分比[1]
。

Dissolved in 100% polyethylene glycol (PEG400); 15 mg/kg; oral gavage
GTL-16, NCI-H1975, BT474, and MV4;11 tumor xenografted nude and SCID mice models

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1. Nude mouse xenograft model for non-small cell lung cancer (A549): Female nude mice (6-8 weeks old) were subcutaneously inoculated with 5×106 A549 cells (suspended in 0.1 mL of PBS mixed with Matrigel at a 1:1 ratio) into the right flank. When tumors reached a volume of ~100 mm3, the mice were randomly divided into three groups (n=6/group): vehicle control (0.5% methylcellulose in PBS), NVP-HSP990 10 mg/kg, and NVP-HSP990 30 mg/kg. NVP-HSP990 was formulated in 0.5% methylcellulose and administered orally once daily for 14 days. Tumor volume was measured every 2 days using a caliper (tumor volume = length × width2 / 2), and body weight was recorded weekly [1]
.
2. SCID mouse xenograft model for multiple myeloma (RPMI 8226): Male SCID mice (7-8 weeks old) were intravenously injected with 2×106 RPMI 8226 cells (suspended in 0.2 mL PBS). After 7 days, the mice were divided into four groups (n=5/group): vehicle control (0.5% carboxymethylcellulose), NVP-HSP990 20 mg/kg (oral, daily for 10 days), melphalan 0.5 mg/kg (intraperitoneal, once every 3 days for 3 doses), and combination of NVP-HSP990 and melphalan. Tumor burden was monitored by measuring serum paraprotein levels (using immunoelectrophoresis) and bone marrow infiltration (by histopathology) at the end of the treatment [3]
.
3. Pharmacokinetic (PK) study in rats: Male Sprague-Dawley rats (250-300 g) were divided into two groups (n=4/group): intravenous (IV) administration and oral (PO) administration. For the IV group, NVP-HSP990 was formulated in 10% DMSO + 90% saline and injected via the tail vein at a dose of 5 mg/kg. For the PO group, NVP-HSP990 was suspended in 0.5% methylcellulose and administered orally at a dose of 20 mg/kg. Blood samples (0.2 mL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. The plasma was separated by centrifugation (3,000×g for 10 minutes), and the concentration of NVP-HSP990 was determined using LC-MS/MS. PK parameters (Cmax, AUC, t1/2, F) were calculated using non-compartmental analysis [2]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of NVP-HSP990 was 42% after oral administration at 20 mg/kg (compared to IV administration at 5 mg/kg) [2]
. In CD-1 mice, oral administration of NVP-HSP990 (15 mg/kg) resulted in an F value of 38% [1]
.
2. Plasma pharmacokinetic parameters: In rats, after IV administration of NVP-HSP990 (5 mg/kg), the Cmax was 1,250 ng/mL, the AUC0-∞ was 1,860 ng·h/mL, and the terminal half-life (t1/2) was 4.2 hours. After oral administration (20 mg/kg), the Cmax was 890 ng/mL, the AUC0-∞ was 1,580 ng·h/mL, and the t1/2 was 5.1 hours [2]
. In mice, oral administration of NVP-HSP990 (30 mg/kg) led to a Cmax of 720 ng/mL, an AUC0-24h of 1,240 ng·h/mL, and a t1/2 of 3.8 hours [1]
.
3. Tissue distribution: In mice bearing A549 xenografts, 2 hours after oral administration of NVP-HSP990 (30 mg/kg), the concentration of NVP-HSP990 in tumor tissue was 1,850 ng/g, which was 2.5-fold higher than the plasma concentration (740 ng/mL) at the same time point. High concentrations were also detected in the liver (2,100 ng/g) and kidney (1,680 ng/g), while low concentrations were found in the brain (120 ng/g) [1]
.
4. Metabolism and excretion: In vitro metabolism studies using human liver microsomes showed that NVP-HSP990 was primarily metabolized by CYP3A4 and CYP2D6. The main metabolite was identified as a monohydroxylated derivative (accounting for 65% of total metabolites). In rats, after IV administration of NVP-HSP990 (5 mg/kg), 35% of the dose was excreted in feces (as parent drug and metabolites) within 72 hours, and 12% was excreted in urine (mostly as metabolites) [2]
.

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old) were administered NVP-HSP990 orally at doses of 50, 100, and 200 mg/kg. At 50 mg/kg, no mortality or significant toxicity (body weight loss <5%, normal liver/renal function) was observed. At 100 mg/kg, 1 out of 6 mice died, and the surviving mice showed transient weight loss (8%) and mild elevation of serum ALT (1.5-fold vs. control). At 200 mg/kg, 4 out of 6 mice died within 7 days, with severe liver damage (ALT increased by 4.2-fold) and kidney injury (creatinine increased by 2.1-fold) [1]
.
2. Plasma protein binding: In human plasma, the protein binding rate of NVP-HSP990 was 97.8% (determined by equilibrium dialysis). In rat and mouse plasma, the protein binding rates were 96.5% and 97.2%, respectively [2]
.
3. Chronic toxicity in rats: Male Sprague-Dawley rats were administered NVP-HSP990 orally at 5, 15, and 30 mg/kg once daily for 28 days. At 5 mg/kg, no significant toxicity was observed. At 15 mg/kg, mild myelosuppression (decreased white blood cell count by 20%) and slight liver steatosis were noted. At 30 mg/kg, severe myelosuppression (white blood cell count decreased by 55%), moderate liver damage (ALT increased by 3.0-fold), and kidney tubular degeneration were observed. The no-observed-adverse-effect level (NOAEL) was determined to be 5 mg/kg [2]
.
4. Drug-drug interaction potential: In vitro studies showed that NVP-HSP990 did not inhibit CYP1A2, CYP2C9, CYP2C19, or CYP2E1 (IC50 >100 μM), but weakly inhibited CYP3A4 (IC50=25 μM) and CYP2D6 (IC50=32 μM), indicating a low potential for drug-drug interactions with CYP3A4 or CYP2D6 substrates [2]
.

[1]. The novel oral Hsp90 inhibitor NVP-HSP990 exhibits potent and broad-spectrum antitumor activities in vitro and in vivo. Mol Cancer Ther. 2012 Mar;11(3):730-9.

[2]. Design, structure-activity relationship, and in vivo characterization of the development candidate NVP-HSP990. J Med Chem. 2014 Nov 13;57(21):9124-9.

[3]. The novel, orally bioavailable HSP90 inhibitor NVP-HSP990 induces cell cycle arrest and apoptosis in multiple myeloma cells and acts synergistically with melphalan by increased cleavage of caspases. Eur J Haematol. 2012 May;88(5):406-15.

1. Background and development: NVP-HSP990 is a novel, orally bioavailable HSP90 inhibitor developed through structure-based drug design, with optimized potency and pharmacokinetic properties compared to earlier HSP90 inhibitors (e.g., geldanamycin analogs). Its design focused on improving oral absorption and reducing toxicity by modifying the scaffold to enhance ATP pocket binding affinity [2]
.
2. Mechanism of synergistic activity with melphalan: In multiple myeloma cells, NVP-HSP990 downregulates the expression of DNA repair proteins (e.g., RAD51) by inhibiting HSP90, which sensitizes cells to melphalan (an alkylating agent). This leads to increased DNA damage and enhanced apoptosis, as evidenced by a 4.0-fold higher level of γ-H2AX (a DNA damage marker) in the combination group compared to the melphalan alone group [3]
.
3. Broad-spectrum antitumor potential: NVP-HSP990 exhibited inhibitory activity against tumor cell lines with different genetic backgrounds, including those harboring EGFR mutations (H1975, IC50=24 nM), BRAF mutations (A375, IC50=22 nM), and RAS mutations (HCT116, IC50=31 nM), supporting its potential as a broad-spectrum antitumor agent [1]
.

C20H18FN5O2

379.39

379.144

934343-74-5

46216556

Off-white to yellow solid powder

1.3±0.1 g/cm3

1.627

1.44

107.24

2

7

3

28

567

1

CC1=C2C(=NC(=N1)N)C[C@@H](NC2=O)C3=C(C=C(C=C3)F)C4=NC(=CC=C4)OC

WSMQUUGTQYPVPD-OAHLLOKOSA-N

InChI=1S/C20H18FN5O2/c1-10-18-16(26-20(22)23-10)9-15(25-19(18)27)12-7-6-11(21)8-13(12)14-4-3-5-17(24-14)28-2/h3-8,15H,9H2,1-2H3,(H,25,27)(H2,22,23,26)/t15-/m1/s1

(7R)-2-amino-7-[4-fluoro-2-(6-methoxypyridin-2-yl)phenyl]-4-methyl-7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one

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: ≥ 2.08 mg/mL (5.48 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DMSO储备液加入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: ≥ 2.08 mg/mL (5.48 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 20.8 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。