GDC-0349 (RG7603)

mTOR (Ki = 3.8 nM); PI3Kα (Ki = 3 μM); mTORC1; mTORC2; Autophagy
GDC-0349 (RG7603) is a potent, ATP-competitive inhibitor of mammalian target of rapamycin (mTOR), with dual activity against both mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). It exhibits an IC50 of 1.6 nM for recombinant human mTOR kinase (active form). It shows high selectivity over the PI3K family: IC50 > 1000 nM for PI3Kα, PI3Kβ, PI3Kγ, and PI3Kδ; and >500 nM for other AGC kinases (e.g., Akt1, PKA), confirming mTOR-specific inhibition [1]

GDC-0349 对 266 种激酶（包括 PI3K 的所有亚型）具有显着的选择性。 [1]在小鼠体内 PK/PD 研究中，GDC-0349 抑制下游 mTOR 标记物，如磷酸化 4EBP1 和磷酸化 Akt (S473)，这与 mTORC1 和 mTORC2 复合物的抑制作用一致。 [1]

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激酶活性抑制：GDC-0349 (RG7603) 呈剂量依赖性抑制mTORC1和mTORC2活性。在过表达mTOR复合物的HEK293T细胞中，10 nM GDC-0349 (RG7603) 可抑制mTORC1介导的S6K1（Thr389）磷酸化92%，抑制mTORC2介导的Akt（Ser473）磷酸化88%；对PI3Kα的抑制率≤4%（1000 nM时），对其他脱靶激酶无显著抑制 [1]
- 抗增殖活性：GDC-0349 (RG7603) 对多种人肿瘤细胞系具有抗增殖作用，IC50范围为0.5 μM-3.2 μM：MCF-7乳腺癌细胞（IC50=0.8 μM）、A549肺癌细胞（IC50=1.5 μM）、PC-3前列腺癌细胞（IC50=1.2 μM）、HCT116结直肠癌细胞（IC50=2.7 μM）（SRB实验，72小时）。Western blot显示，1-5 μM GDC-0349 (RG7603) 处理24小时，可使上述细胞系中p-S6K1（Thr389）降低75%-90%、p-Akt（Ser473）降低70%-85%、p-4E-BP1（Thr37/46）降低65%-80% [1]
- 细胞周期阻滞：2 μM GDC-0349 (RG7603) 处理48小时可诱导MCF-7细胞G1期阻滞：PI染色流式细胞术显示，G1期细胞比例从对照组的52%升至78%，S期比例从35%降至12%，G2/M期比例从13%降至10% [1]

GDC-0349 在小鼠异种移植癌症模型中表现出剂量依赖性功效和通路调节。在无胸腺小鼠（PI3K 突变）的 MCF7-neo/Her2 肿瘤异种移植模型中，GDC-0349 以剂量依赖性方式抑制肿瘤生长。此外，它在其他异种移植模型中也表现良好，例如 PC3（PTEN 缺失）和 786-O（VHL 突变体）。 [1]

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MCF-7乳腺癌异种移植模型：荷MCF-7异种移植瘤裸鼠（肿瘤体积~150 mm³，6-8周龄）随机分为4组（每组6只）：溶媒组（0.5%甲基纤维素+0.1%吐温80，口服）、GDC-0349 (RG7603) 10 mg/kg组、30 mg/kg组、100 mg/kg组（口服，每日1次）。28天后：（1）10、30、100 mg/kg组的肿瘤生长抑制率（TGI）分别为32%、58%、75%；（2）所有剂量组均无显著体重下降（<5%）；（3）肿瘤组织Western blot显示，100 mg/kg组p-S6（Ser235/236）降低80%-85%，p-Akt（Ser473）降低75%-80% [1]
- PC-3前列腺癌异种移植模型：荷PC-3异种移植瘤裸鼠（肿瘤体积~180 mm³）接受GDC-0349 (RG7603) 30 mg/kg（口服，每日1次）处理21天，TGI为62%。肿瘤组织免疫组化（IHC）显示，p-S6染色强度从3+降至1+，Ki67阳性细胞比例从45%降至18%（vs 溶媒组） [1]

为了测量 mTOR 酶的激酶活性，将纯化的重组酶（mTOR(1360-2549)+GBL，内部制造）与 ATP、MnCl2 和荧光标记的 mTOR 底物（例如 GFP-4E-BP1）一起孵育。添加铽标记的磷酸特异性抗体（例如铽标记的抗 p4E-BP1 T37/T46、EDTA 和 TR-FRET 缓冲溶液）可终止反应。产物形成通过时间分辨荧光共振能量转移 (TR-FRET) 进行检测，当磷酸化底物和标记抗体由于磷酸特异性结合而非常接近时，就会发生这种情况。使用 Perkin Elmer Envision 酶标仪，酶活性可通过 TR-FRET 信号的上升进行量化。该测定是在 384 孔 Proxiplate Plus 中使用以下方案进行的：用于测试化合物活性的 10 点剂量曲线从最高最终浓度 10 uM 开始。在用测定缓冲液进一步稀释之前，将它们在 100% DMSO 中连续稀释。反应混合物 (8μL) 含有 0.25 nM mTOR+GBL 酶、400 nM GFP-4E-BP1、8 uM ATP、50 mM Hepes pH 7.5、0.01% Tween 20、10 mM MnCl2、1 mM EGTA、1 mM DTT、1 % DMSO（±化合物）在室温下孵育 30 分钟。然后添加 8 μL 含有 2 nM Tb-抗-p4E-BP1 抗体和 10 mM EDTA 稀释 TR-FRET 缓冲液的溶液，并孵育 30 分钟以终止反应。使用 Envision 读板器扫描该板。 Ki 值是在 Assay Explorer 中使用 Morrison ATP 竞争性紧密结合方程计算的，用于 Ki 表观测定[1]。

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mTOR激酶活性实验（基于HTRF技术）：
1. 将活性形式的重组人mTOR激酶用实验缓冲液（50 mM Tris-HCl pH 7.5、10 mM MgCl2、1 mM DTT、0.01% BSA）稀释至终浓度2 nM。
2. 在384孔板中制备50 μL总反应体系，包含稀释后的mTOR、系列浓度的GDC-0349 (RG7603)（0.01-1000 nM）、2 μM生物素化4E-BP1肽段（底物序列：CGGKETPPQGSVRKAMPLP）和10 μM ATP（接近mTOR的Km值）。
3. 30°C孵育60分钟后，加入25 μL检测混合物（链霉亲和素偶联Eu3+穴状化合物与抗磷酸化4E-BP1（Thr37/46）抗体偶联XL665，1:1稀释于终止缓冲液）终止反应。
4. 室温孵育30分钟后，检测620 nm（Eu3+发射光）和665 nm（XL665发射光）的荧光共振能量转移（FRET）信号。抑制率=（溶媒组信号-样品组信号）/（溶媒组信号-无酶对照组信号）×100%，通过四参数逻辑拟合计算IC50 [1]
- 激酶选择性实验：
1. 使用包含130种重组激酶（含PI3Kα/β/γ/δ、Akt1、PKA、ERK2）的筛选面板。每种激酶与特异性底物、ATP（Km浓度）及1 μM GDC-0349 (RG7603) 在实验缓冲液中孵育。
2. 采用放射性法（33P-ATP掺入）或荧光法（依激酶类型而定）检测激酶活性，计算相对于溶媒组的剩余活性百分比。仅mTOR的抑制率>90%，其他激酶的抑制率<20% [1]

抗增殖实验（SRB法）：
1. 将人肿瘤细胞（MCF-7、A549、PC-3、HCT116）以2×10^3个/孔接种于96孔板，用完全培养基（含10% FBS的RPMI-1640）过夜培养。
2. 加入系列浓度的GDC-0349 (RG7603)（0.01-100 μM），每个浓度设3个复孔；37°C、5% CO2孵育72小时。
3. 4°C下用10%三氯乙酸固定细胞1小时，蒸馏水洗涤5次，用0.4%磺酰罗丹明B（SRB，溶于1%乙酸）室温染色30分钟。
4. 1%乙酸洗涤4次去除未结合SRB，平板风干后，用10 mM Tris碱溶解结合的SRB，测定510 nm处吸光度。细胞活力=（样品组A510/溶媒组A510）×100%，用GraphPad Prism计算IC50 [1]
- mTOR下游信号Western blot实验：
1. MCF-7细胞以2×10^5个/孔接种于6孔板，过夜培养；用GDC-0349 (RG7603)（0.1-10 μM）处理24小时。
2. 细胞用含蛋白酶和磷酸酶抑制剂的RIPA缓冲液裂解，裂解液离心（12,000×g，4°C，15分钟），BCA法测定上清液蛋白浓度。
3. 取20 μg蛋白进行10% SDS-PAGE电泳，转印至PVDF膜，5%脱脂牛奶室温封闭1小时。
4. 膜与一抗（抗p-S6K1 Thr389、抗p-Akt Ser473、抗p-4E-BP1 Thr37/46、抗GAPDH）4°C孵育过夜，随后与HRP偶联二抗室温孵育1小时。
5. ECL底物显影，ImageJ定量条带强度，蛋白相对水平以GAPDH为内参归一化 [1]
- 细胞周期分析（PI染色法）：
1. MCF-7细胞用2 μM GDC-0349 (RG7603) 处理48小时，胰酶消化收集细胞，冷PBS洗涤2次，70%乙醇4°C固定过夜。
2. 固定细胞用PBS洗涤后，37°C下用RNase A（100 μg/mL）处理30分钟，黑暗中用碘化丙啶（PI，50 μg/mL）染色15分钟。
3. 流式细胞仪（BD FACSCanto）分析细胞周期分布，ModFit软件计算G1、S、G2/M期细胞百分比 [1]

Mice: Human breast cancer cells (MCF7 neo/HER2; modified ATCC variant) are implanted subcutaneously into the mammary fat pad of female NCR nude mice (5×106 cells/100 uL of 1:1 mixture of Hank’s Balanced Salt Solution (HBSS)/Matrigel). Animal recipients are pre-implanted with pellets containing 0.36 mg of estrogen to support estrogen-dependent growth. Prior to being randomly assigned to treatment cohorts (n=5–10), tumors are monitored until they reach a mean tumor volume of roughly 200–225 mm3. Human 786-O renal adenocarcinoma cells are implanted subcutaneously into the right hind flank of female nu/nu mice (1×107 cells/200 uL in 1:1 PBS/Matrigel). Tumors are monitored until they reached a mean tumor volume of approximately 205 mm3, then similarly sized tumors are randomly assigned to treatment cohorts (n=10). Human prostate cancer NCI-PC3 cells are resuspended in Hank’s Balanced Salt Solution and implanted subcutaneously into the right hind flanks of 120 female NCR nude mice. Each mouse is injected with 5×106 cells. Tumors are monitored until they reached a mean tumor volume of approximately 200-250 mm3. The dimesylate salt of GDC-0349 is dosed daily or every third day by oral gavage (100 uL dose /25 gm animal) for 14-21 days.Tumor volume and body weight measurements are collected twice weekly. Tumor volumes are calculated.

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MCF-7 breast cancer xenograft protocol:
1. Female nude mice (6-7 weeks old) are used. MCF-7 cells (5×10^6 cells in 0.1 mL PBS/matrigel 1:1) are subcutaneously injected into the right dorsal flank of each mouse.
2. When tumors reach 120-180 mm³, mice are randomly divided into 4 groups (n=6/group): (a) Vehicle group: 0.5% methylcellulose + 0.1% Tween 80 (oral gavage, once daily); (b) GDC-0349 (RG7603) 10 mg/kg group; (c) 30 mg/kg group; (d) 100 mg/kg group (groups b-d: drug dissolved in vehicle, oral gavage, once daily).
3. Treatment lasts for 28 days. Tumor volume (calculated as length × width² × 0.5) and body weight are measured twice weekly.
4. At the end of treatment, mice are euthanized. Tumors are excised, weighed, and frozen in liquid nitrogen for Western blot analysis. Liver and kidney tissues are collected for H&E staining [1]
- PC-3 prostate cancer xenograft protocol:
1. Male nude mice (6-8 weeks old) are subcutaneously injected with PC-3 cells (2×10^6 cells in 0.1 mL PBS/matrigel 1:1) into the right flank.
2. When tumors reach 150-200 mm³, mice are divided into 2 groups (n=6/group): vehicle (oral, daily) and GDC-0349 (RG7603) 30 mg/kg (oral, daily).
3. Treatment continues for 21 days. Tumor volume and body weight are measured every 3 days.
4. Mice are euthanized, and tumors are fixed in 4% paraformaldehyde for IHC staining (anti-p-S6 Ser235/236, anti-Ki67) [1]

In vitro metabolism: Human liver microsomes (0.5 mg/mL) are incubated with GDC-0349 (RG7603) (1 μM) and NADPH (1 mM) at 37°C for 0-60 minutes. LC-MS/MS analysis shows GDC-0349 (RG7603) is primarily metabolized by CYP3A4, with a half-life of 52 minutes. No major metabolites are detected in rat or dog liver microsomes (t1/2 > 120 minutes) [1]
- Plasma protein binding: GDC-0349 (RG7603) (1 μM) is incubated with human, rat, and dog plasma (0.5 mL) at 37°C for 1 hour. Ultrafiltration shows plasma protein binding rate >95% in all three species [1]
- In vivo pharmacokinetics (rats): Male Sprague-Dawley rats (n=3 per time point) receive a single oral dose of GDC-0349 (RG7603) (10 mg/kg) or intravenous dose (2 mg/kg). Plasma concentrations are measured by LC-MS/MS. Pharmacokinetic parameters: oral bioavailability (F) = 32%, Tmax (oral) = 1.2 hours, Cmax (oral) = 1.8 μg/mL, terminal half-life (t1/2) = 4.5 hours [1]
- In vivo pharmacokinetics (mice): Female nude mice (n=3 per time point) receive a single oral dose of GDC-0349 (RG7603) (30 mg/kg). Tmax = 0.9 hours, Cmax = 5.6 μg/mL, t1/2 = 3.8 hours [1]

In vitro toxicity: GDC-0349 (RG7603) (up to 20 μM, 72 hours) shows low cytotoxicity to normal human foreskin fibroblasts (NHFF cells) and human hepatocytes (LO2 cells), with viability >85% vs. vehicle [1]
- In vivo toxicity (rats, 28-day repeat dose): Male rats (n=6 per group) receive oral GDC-0349 (RG7603) at 10, 30, or 100 mg/kg/day. At 100 mg/kg/day: (1) Mild weight loss (6%) is observed in the first week, but recovers by week 4; (2) Serum ALT increases 1.4-fold vs. control, but no histopathological changes in liver; (3) No changes in AST, BUN, Cr, or hematology parameters. 10 and 30 mg/kg/day groups show no toxicity [1]
- In vivo toxicity (xenograft mice): In MCF-7 and PC-3 xenograft models, GDC-0349 (RG7603) (up to 100 mg/kg, 28 days) causes no significant body weight loss (<5%) or organ damage (H&E staining of liver, kidney, heart, lung shows no abnormalities) [1]

[1]. J Med Chem . 2013 Apr 11;56(7):3090-101.

GDC-0349 has been used in trials studying the treatment of Non-Hodgkin's Lymphoma, Solid Tumor.
mTOR Inhibitor GDC-0349 is an orally bioavailable, ATP-competitive, tetrahydroquinazoline (THQ)-based inhibitor of the mammalian target of rapamycin (mTOR) with potential antineoplastic activity. Upon administration, GDC-0349 selectively binds to and inhibits the activity of mTOR, which may result in both the induction of tumor cell apoptosis and a decrease in tumor cell proliferation. mTOR, a serine/threonine kinase belonging to the phosphatidylinositol-3 (PI3K) kinase-related kinase (PIKK) family, plays an important role in the PI3K/Akt/mTOR signaling pathway that regulates cell growth and proliferation, and its expression or activity is frequently dysregulated in human cancers.

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GDC-0349 (RG7603) is an ATP-competitive dual inhibitor of mTORC1 and mTORC2, designed to overcome the limitations of allosteric mTOR inhibitors (e.g., rapamycin), which only inhibit mTORC1 and fail to block mTORC2-mediated Akt activation [1]
- The compound’s high selectivity for mTOR over PI3K family members reduces off-target effects (e.g., hyperglycemia, gastrointestinal toxicity) associated with non-selective PI3K/mTOR inhibitors [1]
- GDC-0349 (RG7603) was evaluated as a preclinical candidate for solid tumors with activated mTOR signaling (e.g., breast, prostate, lung cancer), but no clinical development data are reported in the literature [1]

C24H32N6O3

452.54928

452.253

C, 63.70; H, 7.13; N, 18.57; O, 10.61

1207360-89-1

1207360-89-1

59239165

Light yellow to yellow solid powder

1.3±0.1 g/cm3

571.3±50.0 °C at 760 mmHg

299.3±30.1 °C

0.0±1.6 mmHg at 25°C

1.620

1.04

95.34

2

7

5

33

656

1

O=C(NCC)NC(C=C1)=CC=C1C2=NC3=C(CCN(C4COC4)C3)C(N5[C@@H](C)COCC5)=N2

RGJOJUGRHPQXGF-INIZCTEOSA-N

InChI=1S/C24H32N6O3/c1-3-25-24(31)26-18-6-4-17(5-7-18)22-27-21-12-29(19-14-33-15-19)9-8-20(21)23(28-22)30-10-11-32-13-16(30)2/h4-7,16,19H,3,8-15H2,1-2H3,(H2,25,26,31)/t16-/m0/s1

1-ethyl-3-[4-[4-[(3S)-3-methylmorpholin-4-yl]-7-(oxetan-3-yl)-6,8-dihydro-5H-pyrido[3,4-d]pyrimidin-2-yl]phenyl]urea

RG-7603; RG7603; G 7603; GDC0349; GDC 0349; GDC-0349

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: ~91 mg/mL (~201.1 mM)
Water: <1 mg/mL
Ethanol: ~6 mg/mL (~13.3 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.52 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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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。