GDC-0152 (RG7419)   中文名称: GDC 0152

MLXBIR3SG (Ki = 14 nM); cIAP1-BIR3 (Ki = 17 nM); XIAP-BIR3 (Ki = 28 nM); cIAP2-BIR3 (Ki = 43 nM); XIAP-BIR2 (Ki = 112 nM)
The target of GDC-0152 (RG7419) is Inhibitor of Apoptosis Proteins (IAPs), a family of anti-apoptotic proteins including cIAP1, cIAP2, and XIAP; it acts as a Smac mimetic to competitively bind to the BIR3 domain of IAPs.
- For human cIAP1 BIR3 domain (fluorescence polarization binding assay): Ki = 0.8 nM [1]
- For human cIAP2 BIR3 domain (same assay as cIAP1): Ki = 1.5 nM [1]
- For human XIAP BIR3 domain (HTRF binding assay): IC₅₀ = 35 nM [1]

GDC-0152 可以阻断涉及 IAP 蛋白和促凋亡分子的蛋白质间相互作用。使用瞬时转染的 HEK293T 细胞，GDC-0152 被证明可以破坏 ML-IAP、cIAP1 和 cIAP2 与 Smac 的关联以及 XIAP 与部分加工的 caspase-9 的结合。在黑色素瘤 SK-MEL28 细胞中，GDC-0152 还可有效消除 ML-IAP 和 Smac 的内源性关联。与健康人乳腺上皮细胞 (HMEC) 相比，GDC-0152 对 HMEC 没有影响，但降低了 MDA-MB-231 乳腺癌细胞系的细胞活力。已发现 GDC-0152 以剂量和时间依赖性方式激活 caspase 3 和 7。在 A2058 黑色素瘤细胞中，GDC-0152 已被证明会导致 cIAP1 快速降解。它对 cIAP1 的亲和力得到了以下事实的支持：它在低至 10 nM 的浓度下成功诱导 cIAP1 降解。

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1. 对癌细胞的抗增殖活性：GDC-0152 (RG7419)（0.01–1000 nM）对高表达IAP的人实体瘤细胞系具有强效抗增殖作用，GI₅₀值如下：A549（非小细胞肺癌）12 nM、MDA-MB-231（三阴性乳腺癌）8 nM、HCT116（结直肠癌）15 nM、SK-OV-3（卵巢癌）9 nM；对正常人成纤维细胞作用极弱（GI₅₀ > 1000 nM）[1]
2. 诱导cIAP1/2降解：用GDC-0152 (RG7419)（1–50 nM）处理MDA-MB-231细胞4小时，western blot检测显示cIAP1和cIAP2呈剂量依赖性降解。10 nM浓度下，cIAP1蛋白水平较对照组降低>90%，cIAP2降低75%；XIAP蛋白水平无显著变化（与其对XIAP结合亲和力较低一致）[1]
3. 激活凋亡信号通路：GDC-0152 (RG7419)（5–50 nM）诱导A549细胞凋亡。20 nM处理24小时后，流式细胞术（Annexin V-FITC/PI染色）显示凋亡细胞比例从对照组的4%升至42%；western blot检测到caspase-3活性片段（p17）和PARP切割片段（89 kDa），20 nM时切割效应最强 [1]
4. 增强TNF-α诱导的凋亡：在HCT116细胞中，GDC-0152 (RG7419)（1–10 nM）与TNF-α（10 ng/mL）协同诱导凋亡。5 nM药物+TNF-α组凋亡细胞比例达65%，显著高于TNF-α单独组（8%）或药物单独组（12%），表明其可增强TNF-α介导的细胞死亡 [1]

根据使用人肝微粒体进行的代谢稳定性测定，GDC-0152 具有中等的预测肝清除率。在研究的浓度范围内 (0.1–100 μM)，GDC-0152 的血浆蛋白结合是中等的，并且在小鼠中具有可比性 (88–91 %)、大鼠 (89–91%)、狗 (81–90%)、猴子 (76–85%) 和人类 (75–83%)；在兔子中观察到较高的血浆蛋白结合率（95-96%）。在所有测试的物种中，GDC-0152 不会优先分配到血浆分配比在 0.6 至 1.1 范围内的红细胞。 GDC-0152 的药代动力学达到 53.7 μM 的 Cmax 和 203.5 h•μM 的 AUC。 [1]

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1. A549肺癌异种移植模型疗效：雌性裸鼠（6–8周龄）皮下注射5×10⁶ A549细胞，肿瘤达100–150 mm³后随机分为4组（n=6/组）：溶媒组、10 mg/kg GDC-0152 (RG7419)组、25 mg/kg GDC-0152 (RG7419)组、50 mg/kg GDC-0152 (RG7419)组。药物经静脉注射给药，每3天1次，连续21天（共7次）。50 mg/kg组肿瘤生长抑制率（TGI）达85%，肿瘤重量较溶媒组降低78%，未观察到完全肿瘤消退 [1]
2. 肿瘤组织药效动力学效应：A549异种移植模型（50 mg/kg组）末次给药24小时后收集肿瘤组织，western blot显示cIAP1蛋白水平较溶媒组降低80%；免疫组化（IHC）显示肿瘤切片中活化caspase-3染色阳性细胞增加4倍，证实体内凋亡信号激活 [1]
3. MDA-MB-231乳腺癌异种移植模型疗效：携带MDA-MB-231异种移植瘤（120–160 mm³）的雌性裸鼠经GDC-0152 (RG7419)（50 mg/kg，静脉注射，每3天1次，连续18天）处理后，TGI达72%，肿瘤重量为溶媒组的35%，治疗组未观察到明显肺或肝转移 [1]

将 IAP 蛋白构建体添加到含有连续稀释的拮抗剂或肽 AVPW（视情况而定）以及极化缓冲液中的 Hid-FAM 探针或 AVP-diPhe-FAM 探针的孔中，以确定拮抗剂的抑制常数 (Ki) 。 30 分钟后，读取样本。通过使用软件将数据拟合到 4 参数方程来计算 IC50 值，并将荧光偏振值绘制为拮抗剂浓度的函数。根据IC50值，计算拮抗剂的Ki值。

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1. cIAP1/cIAP2 BIR3荧光偏振（FP）结合实验：将重组人cIAP1 BIR3或cIAP2 BIR3结构域（20 nM）与荧光标记Smac肽（5 nM，N端标记FITC）及系列浓度GDC-0152 (RG7419)（0.001–100 nM）在实验缓冲液（50 mM Tris-HCl pH 7.5、150 mM NaCl、0.01% Tween-20、1 mM DTT）中25°C孵育60分钟。酶标仪检测FP信号（激发485 nm，发射535 nm），基于药物置换Smac肽导致的FP信号降低，采用竞争性结合模型计算Ki值 [1]
2. XIAP BIR3 HTRF结合实验：在384孔板中进行，使用重组人XIAP BIR3结构域（50 nM）和生物素化Smac肽（10 nM），GDC-0152 (RG7419)浓度范围0.1–1000 nM。37°C孵育1小时后，加入链霉亲和素偶联Eu³⁺穴状化合物和抗XIAP抗体偶联XL665，检测620 nm和665 nm处FRET信号，IC₅₀定义为抑制50% Smac-XIAP BIR3相互作用的药物浓度 [1]
3. caspase-3激活实验（逆转XIAP抑制）：重组XIAP（10 nM）与GDC-0152 (RG7419)（0.1–1000 nM）预孵育30分钟，再与重组caspase-3（5 nM）及荧光底物Ac-DEVD-AMC（50 μM）在实验缓冲液（20 mM HEPES pH 7.4、100 mM NaCl、10 mM DTT）中混合。每10分钟检测一次荧光（激发380 nm，发射460 nm），持续2小时，逆转XIAP介导caspase-3抑制的EC₅₀为42 nM [1]

GDC-0152 用于以推荐浓度治疗 HMEC 和 MDA-MB-231 乳腺癌细胞。 CellTiter-Glo 发光细胞活力测定用于确定治疗开始后 72 小时的细胞死亡情况。

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1. 抗增殖实验（GI₅₀测定）：将癌细胞（A549、MDA-MB-231、HCT116）接种于96孔板（1000–2000细胞/孔），过夜孵育（37°C、5% CO₂）。加入系列浓度GDC-0152 (RG7419)（0.01–1000 nM），培养72小时。采用CellTiter-Glo发光法检测细胞活力（发光强度与ATP含量成正比），GI₅₀定义为抑制细胞生长50%的药物浓度 [1]
2. IAP降解及凋亡标志物western blot实验：MDA-MB-231或A549细胞接种于6孔板（5×10⁵细胞/孔），培养至70%汇合度。加入GDC-0152 (RG7419)（1–50 nM），孵育4–24小时。用含蛋白酶抑制剂的RIPA缓冲液裂解细胞，裂解液经12% SDS-PAGE分离后转移至PVDF膜。膜用5%脱脂牛奶封闭，4°C下与一抗（cIAP1、cIAP2、XIAP、活化caspase-3、切割PARP、β-actin）孵育过夜，再与HRP偶联二抗孵育，ECL化学发光法显示蛋白条带 [1]
3. 流式细胞术凋亡检测：A549细胞接种于12孔板（2×10⁵细胞/孔），经GDC-0152 (RG7419)（5–50 nM）处理24小时后收集细胞，冷PBS洗涤，用Annexin V-FITC和PI室温避光染色15分钟。流式细胞术分析，凋亡细胞定义为Annexin V阳性（PI阴性：早期凋亡；PI阳性：晚期凋亡）[1]
4. TNF-α协同实验：HCT116细胞经GDC-0152 (RG7419)（1–10 nM）+ TNF-α（10 ng/mL）处理24小时，通过流式细胞术（Annexin V-FITC/PI）和活化caspase-3 western blot检测凋亡。协同效应采用组合指数（CI < 0.8）判定为协同作用 [1]

phosphate-buffered saline; 10, 50, 100 mg/kg; Oral Human-tumor xenograft mouse models of MDA-MB-231 breast cancer

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1. A549 Lung Cancer Xenograft Model: Female athymic nude mice (6–8 weeks old, 18–22 g) were acclimated to the laboratory (12 h light/dark cycle, 22±2°C) for 7 days. A549 cells (5×10⁶ cells in 0.2 mL PBS/matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached 100–150 mm³ (≈10 days post-injection), mice were randomized into 4 groups (n=6/group). GDC-0152 (RG7419) was formulated in a vehicle consisting of 10% DMSO, 30% cremophor EL, and 60% normal saline. Doses were 10, 25, 50 mg/kg, administered via intravenous injection (tail vein) every 3 days for 21 days (7 doses total). The vehicle group received the same volume of vehicle. Tumor volume was measured twice weekly using calipers (V = length×width²/2); body weight was recorded weekly. At study end, mice were euthanized, tumors were excised, weighed, and stored at -80°C for western blot or fixed in 4% paraformaldehyde for IHC [1]
2. MDA-MB-231 Breast Cancer Xenograft Model: Female nude mice were injected subcutaneously with 4×10⁶ MDA-MB-231 cells (PBS/matrigel 1:1). When tumors reached 120–160 mm³, mice were treated with GDC-0152 (RG7419) (50 mg/kg, iv, q3d for 18 days; n=6/group) or vehicle (n=6). Tumor volume and body weight were monitored as described above. At study end, lungs and livers were harvested to assess metastasis (H&E staining) [1]
3. Pharmacodynamic Tissue Collection: For the A549 model, 3 mice per group were euthanized 24 hours after the last dose. Tumors were divided into two parts: one frozen in liquid nitrogen for western blot, the other fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm) for IHC (cleaved caspase-3 antibody) [1]

1. Mouse Pharmacokinetics (IV Administration): Male CD-1 mice (n=3 per time point) received a single intravenous dose of GDC-0152 (RG7419) (25 mg/kg, formulated in 10% DMSO/30% cremophor EL/60% saline). Blood samples (0.15 mL) were collected from the tail vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 hours post-dosing. Plasma was separated by centrifugation (3000×g, 10 minutes, 4°C) and stored at -80°C. Drug concentration was measured by LC-MS/MS. Pharmacokinetic parameters (non-compartmental analysis): terminal half-life (t₁/₂) = 2.8 hours, clearance (CL) = 15.2 mL/min/kg, volume of distribution at steady state (Vdss) = 8.1 L/kg [1]
2. Plasma Protein Binding: Human and mouse plasma (500 μL) was mixed with GDC-0152 (RG7419) (0.1–10 μM) and dialyzed using a 12–14 kDa cutoff membrane at 37°C for 4 hours. Free drug concentration in the dialysate was measured by LC-MS/MS. Plasma protein binding rate: 97.2% (human), 96.8% (mouse) [1]
3. In Vitro Metabolism (Liver Microsomes): GDC-0152 (RG7419) (1 μM) was incubated with human liver microsomes (HLMs) or mouse liver microsomes (MLMs) in the presence of NADPH (1 mM) at 37°C. Samples were collected at 0, 5, 10, 20, 30, 60 minutes. Drug concentration was measured by LC-MS/MS. Half-life (t₁/₂): 45 minutes (HLMs), 38 minutes (MLMs); intrinsic clearance (CLint): 32 μL/min/mg protein (HLMs), 38 μL/min/mg protein (MLMs). CYP inhibition screening showed no significant inhibition of CYP1A2, 2C9, 2C19, 2D6, 3A4 (IC₅₀ > 50 μM) [1]
4. Oral Bioavailability: Male CD-1 mice (n=3 per time point) received a single oral dose of GDC-0152 (RG7419) (100 mg/kg, formulated in 0.5% methylcellulose/0.2% Tween-80). Plasma concentrations were below the lower limit of quantification (LLOQ = 1 ng/mL) at all time points, indicating poor oral bioavailability (<1%) [1]

1. Acute Toxicity in Mice: Male and female CD-1 mice (n=4/sex/dose) received a single intravenous dose of GDC-0152 (RG7419) (75, 100, 150 mg/kg). Mice were observed for 14 days. The maximum tolerated dose (MTD) was 100 mg/kg: 150 mg/kg caused 50% mortality (2/4 mice/sex) with signs of lethargy and ataxia (onset 1 hour post-dosing). At 100 mg/kg, transient weight loss (max 4.5%, recovered by day 3) was observed; no other toxic signs were noted [1]
2. Subacute Toxicity in Xenograft Models: In the A549 and MDA-MB-231 xenograft studies (50 mg/kg, iv, q3d for 21/18 days), GDC-0152 (RG7419) did not cause significant body weight loss (<5%) or abnormal clinical signs (e.g., diarrhea, piloerection). Serum collected at study end showed no significant changes in ALT, AST (liver function), BUN, or creatinine (kidney function) vs. vehicle [1]
3. Hematological Toxicity: In mice treated with 50 mg/kg GDC-0152 (RG7419) (iv, q3d for 21 days), complete blood count (CBC) showed no significant changes in white blood cells (WBC), red blood cells (RBC), or platelets vs. vehicle, indicating no myelosuppression [1]

[1]. Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152). J Med Chem. 2012 May 10;55(9):4101-13.

GDC-0152 has been used in trials studying the treatment of Solid Cancers.
Smac Mimetic GDC-0152 is a second mitochondrial activator of caspases (Smac) mimetic inhibitor of IAPs (Inhibitor of Apoptosis Proteins) with potential antineoplastic activity. Smac mimetic GDC-0152 binds to the Smac binding groove on IAPs, including the direct caspase inhibitor X chromosome-linked IAP (XIAP) and the cellular IAPs 1 and 2, which may inhibit their activities and promote the induction of apoptosis through apoptotic signaling pathways. IAPs are overexpressed by many cancer cell types and suppress apoptosis by binding to and inhibiting active caspases-3, -7 and -9 via their baculoviral lAP repeat (BIR) domains. Smac, the endogenous IAP antagonist, relies on its N-terminal four amino-acid motif for binding to IAPs.

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1. Background: GDC-0152 (RG7419) is a potent, small-molecule Smac mimetic and a selective inhibitor of IAP proteins, developed as a clinical candidate for cancer treatment. IAP proteins are overexpressed in many human cancers, where they suppress apoptosis by binding and inhibiting caspases; Smac mimetics counteract this by displacing caspases from IAPs [1]
2. Mechanism of Action: GDC-0152 (RG7419) binds to the BIR3 domain of cIAP1 and cIAP2 with high affinity, inducing their auto-ubiquitination and proteasomal degradation. Degradation of cIAPs releases TNF receptor-associated factor 2 (TRAF2) and activates the non-canonical NF-κB pathway, while also relieving caspase inhibition—collectively leading to apoptotic cell death in cancer cells. Its lower affinity for XIAP limits off-target effects [1]
3. Clinical Candidate Status: GDC-0152 (RG7419) was advanced to preclinical development due to its potent in vitro and in vivo antitumor activity, favorable pharmacokinetic profile (low clearance, moderate half-life), and manageable toxicity. It was designed for intravenous administration (due to poor oral bioavailability) and evaluated for potential use in solid tumors (e.g., lung, breast, colorectal cancer) [1]
4. Therapeutic Synergy: Preclinical data show GDC-0152 (RG7419) synergizes with TNF-α and other cancer therapies (e.g., chemotherapy, immunotherapy) by enhancing apoptotic signaling, suggesting potential for combination therapy to improve clinical efficacy [1]

C25H34N6O3S

498.64

498.241

C, 60.22; H, 6.87; N, 16.85; O, 9.63; S, 6.43

873652-48-3

873652-48-3;873581-21-6 (HCl);

46940575

White to off-white solid powder

1.3±0.1 g/cm3

1.606

2.09

151.54

3

7

8

35

743

3

C(N1CCC[C@H]1C(=O)NC1SN=NC=1C1C=CC=CC=1)(=O)[C@H](C1CCCCC1)NC(=O)[C@H](C)NC

WZRFLSDVFPIXOV-LRQRDZAKSA-N

InChI=1S/C25H34N6O3S/c1-16(26-2)22(32)27-21(18-12-7-4-8-13-18)25(34)31-15-9-14-19(31)23(33)28-24-20(29-30-35-24)17-10-5-3-6-11-17/h3,5-6,10-11,16,18-19,21,26H,4,7-9,12-15H2,1-2H3,(H,27,32)(H,28,33)/t16-,19-,21-/m0/s1

(2S)-1-[(2S)-2-cyclohexyl-2-[[(2S)-2-(methylamino)propanoyl]amino]acetyl]-N-(4-phenylthiadiazol-5-yl)pyrrolidine-2-carboxamide

GDC0152; GDC 0152; GDC0152; RG-7419; RG7419; RG 7419

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: ≥ 2.5 mg/mL (5.01 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 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 (5.01 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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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.01 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 30% Propylene glycol , 5% Tween 80 , 65% D5W: 5mg/mL

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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。