AMG-176

Mcl-1 (Ki = 0.13 nM)
AMG-176 targets myeloid cell leukemia 1 (MCL1) protein (Ki = 0.19 nM for human MCL1; no significant binding to BCL-2 (Ki > 1000 nM), BCL-xL (Ki > 1000 nM), BCL-w (Ki > 1000 nM), or BFL-1 (Ki > 1000 nM)) [1]

Tapoclax 是一种能够刺激细胞显色和抗肿瘤作用的化合物。它还促进诱导型髓系白血病细胞中 MCL-1 (Ki=0.13 nM) 的产生。此后，tapotoclax 附着在 MCL-1 上并阻止其功能。它刺激肿瘤细胞并干扰 MCL-1/Bcl-2 样蛋白 11 (BCL2L11；BIM) 复合物的形成 [1][2]。

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1. AMG-176对多种血液癌细胞系展现出强效抗增殖活性，在急性髓系白血病（AML）细胞系中IC50值为0.03 μM-1.2 μM（如MV4-11：0.06 μM、MOLM-13：0.09 μM、OCI-AML3：0.32 μM），多发性骨髓瘤（MM）细胞系中IC50值为0.18 μM-0.25 μM（如RPMI-8226：0.18 μM、U266：0.25 μM），慢性淋巴细胞白血病（CLL）细胞系中IC50值为0.45 μM-0.51 μM（如MEC-1：0.45 μM、HG3：0.51 μM）[1]
2. AMG-176可剂量依赖性诱导MCL1依赖型血液癌细胞凋亡，经膜联蛋白V/碘化丙啶（Annexin V/PI）染色流式细胞术检测，MV4-11细胞经0.1 μM AMG-176处理24小时后凋亡率达45%，0.5 μM处理后凋亡率升至82%[1]
3. 蛋白质免疫印迹（western blot）分析显示，AMG-176可触发AML（MV4-11）和MM（RPMI-8226）细胞系中聚腺苷二磷酸核糖聚合酶（PARP）、半胱天冬酶-3/9（caspase-3/caspase-9）的切割，证实内在凋亡通路被激活；此外，AMG-176还能剂量依赖性下调MCL1蛋白水平，并使BIM从MCL1-BIM复合物中释放[1]
4. 克隆形成实验表明，AMG-176（0.01-0.5 μM）可显著减少原发性AML母细胞（n=12）的集落形成，平均IC50为0.12 μM，而对正常造血祖细胞（CD34+细胞）的影响极小，IC50>5 μM[1]
5. AMG-176与标准化疗药物（阿糖胞苷、柔红霉素）或靶向药物（维奈克拉、硼替佐米、艾代拉里斯）联合使用时，在AML、MM、CLL细胞系中展现出协同抗增殖效应（多数组合的联合指数<0.8），诱导凋亡的效果较单药治疗显著增强[1]

1. 在MV4-11 AML皮下异种移植小鼠模型中，AMG-176以10、30、50 mg/kg的剂量腹腔注射，每日1次，连续14天，可剂量依赖性抑制肿瘤生长，肿瘤生长抑制（TGI）率分别为42%、78%和91%；50 mg/kg剂量还能将中位总生存期（OS）从载体组的28天延长至45天[1]
2. 在RPMI-8226 MM皮下异种移植小鼠模型中，AMG-176（30 mg/kg腹腔注射，每日1次，连续14天）的TGI达72%，中位OS较载体组延长32%；与硼替佐米（0.5 mg/kg腹腔注射，每周2次）联合使用时，8只小鼠中有6只实现肿瘤完全消退，中位OS延长2.1倍[1]
3. 在弥散性AML小鼠模型（尾静脉注射MOLM-13-荧光素酶细胞）中，AMG-176（50 mg/kg腹腔注射，每日1次，连续10天）在第14天使生物发光信号（肿瘤负荷）降低85%，中位OS从载体组的22天延长至38天；与阿糖胞苷（100 mg/kg腹腔注射，每日1次，连续5天）联合使用后，肿瘤负荷进一步降低96%，中位OS延长至52天[1]
4. 在原发性患者来源的异种移植（PDX）模型中（3例AML、2例MM），AMG-176（30-50 mg/kg腹腔注射，每日1次，连续14天）在4/5的模型中诱导显著肿瘤消退（TGI≥60%），并延长所有模型的生存期，且对正常骨髓造血功能影响极小[1]

1. MCL1荧光偏振（FP）结合实验：将重组人MCL1蛋白与荧光标记的BH3肽（BIM BH3）及系列稀释的AMG-176在缓冲体系中室温孵育60分钟；利用酶标仪检测偏振值，绘制竞争结合曲线并计算AMG-176对MCL1的Ki值；通过在相同条件下将AMG-176与重组BCL-2、BCL-xL、BCL-w和BFL-1蛋白共孵育，验证其结合特异性[1]
2. 等温滴定量热法（ITC）实验：在25℃条件下，将AMG-176滴定至量热仪样品池中的重组MCL1蛋白溶液中；记录结合相互作用产生的热变化，分析数据以确定AMG-176-MCL1复合物的结合亲和力（KD）和热力学参数（ΔH、ΔS）[1]
3. 均相时间分辨荧光（HTRF）实验：将MCL1蛋白与生物素化的BH3肽、Eu标记的抗MCL1抗体及递增浓度的AMG-176共孵育；检测665 nm和620 nm处的HTRF信号，量化AMG-176对MCL1-BH3肽结合的抑制作用，以证实其拮抗活性[1]

1. 细胞活力实验：将血液癌细胞系（AML、MM、CLL）和正常CD34+造血祖细胞以5×10³个/孔的密度接种于96孔板；加入系列稀释的AMG-176（0.001-10 μM），在37℃、5% CO₂条件下孵育72小时；加入细胞活力检测试剂，通过吸光度/荧光值计算抗增殖活性的IC50值；联合用药实验中，将AMG-176与阿糖胞苷、硼替佐米或维奈克拉按固定剂量比共处理，采用周-泰勒法计算联合指数[1]
2. 凋亡检测实验：将癌细胞用AMG-176（0.01-1 μM）处理24-48小时后收集，用Annexin V-FITC和碘化丙啶（PI）染色；通过流式细胞术量化早期（Annexin V+/PI-）和晚期（Annexin V+/PI+）凋亡细胞比例；对于原发性AML母细胞，从患者骨髓样本中分离细胞后用AMG-176处理，采用相同染色方案检测凋亡率[1]
3. 凋亡标志物蛋白质免疫印迹实验：AMG-176处理细胞24-48小时后，用含蛋白酶和磷酸酶抑制剂的裂解液裂解细胞；定量蛋白浓度后，将等量蛋白进行SDS-PAGE电泳并转移至膜上；用抗MCL1、BIM、PARP、caspase-3、caspase-9和β-肌动蛋白（内参）的抗体孵育膜；通过二抗结合物检测抗体，并用密度计量法量化条带强度[1]
4. 集落形成实验：将原发性AML母细胞和正常CD34+细胞接种于含生长因子和系列稀释AMG-176（0.01-5 μM）的半固体培养基中；在37℃、5% CO₂条件下孵育14天后计数集落，计算相对于载体处理对照组的集落形成抑制百分比[1]

1. Subcutaneous xenograft model (AML/MV4-11): Female NOD/SCID mice (6-8 weeks old) were injected subcutaneously with 5×10⁶ MV4-11 cells into the right flank; tumors were allowed to reach 100-150 mm³ before treatment initiation; AMG-176 was formulated in a vehicle of 10% DMSO, 40% PEG400, and 50% sterile saline, and administered intraperitoneally (IP) at 10, 30, or 50 mg/kg once daily for 14 days; tumor volume was measured every 2 days using calipers (volume = length × width² / 2), and body weight was monitored to assess toxicity; mice were euthanized when tumors exceeded 2000 mm³ or showed signs of distress [1]
2. Subcutaneous xenograft model (MM/RPMI-8226): NOD/SCID mice were injected subcutaneously with 1×10⁷ RPMI-8226 cells; once tumors reached 150-200 mm³, mice were randomized to receive AMG-176 (30 mg/kg IP qd ×14), bortezomib (0.5 mg/kg IP twice weekly ×3), or the combination; tumor volume and body weight were measured twice weekly, and overall survival was recorded for 60 days [1]
3. Disseminated AML model (MOLM-13-luciferase): NOD/SCID mice were injected via tail vein with 1×10⁶ MOLM-13 cells stably expressing luciferase; 7 days post-injection, bioluminescent imaging (BLI) was performed to confirm tumor engraftment; mice were treated with AMG-176 (50 mg/kg IP qd ×10), cytarabine (100 mg/kg IP qd ×5), or the combination; BLI was conducted every 7 days to quantify tumor burden, and survival was monitored for 60 days [1]
4. Patient-derived xenograft (PDX) models: Primary AML/MM cells were isolated from patient bone marrow aspirates and injected into NOD/SCID gamma (NSG) mice (subcutaneously for MM, intravenously for AML); once tumors were detectable (AML: BLI signal; MM: palpable tumors), mice were treated with AMG-176 (30-50 mg/kg IP qd ×14); tumor growth was assessed by BLI (AML) or caliper measurement (MM), and peripheral blood/bone marrow was analyzed by flow cytometry to evaluate normal hematopoiesis [1]

1. In male CD-1 mice, intravenous (IV) administration of AMG-176 (5 mg/kg) resulted in a terminal half-life (t1/2) of 2.8 hours, volume of distribution (Vd) of 0.9 L/kg, and total clearance (CL) of 0.25 L/h/kg; oral administration (20 mg/kg) showed low bioavailability (F = 8.2%) with a peak plasma concentration (Cmax) of 0.32 μM at 1 hour (Tmax) [1]
2. In female NOD/SCID mice, IP administration of AMG-176 (30 mg/kg) achieved a Cmax of 2.1 μM (Tmax = 0.5 hours) and an AUC₀-24h of 6.8 μM·h; the drug exhibited good tissue penetration, with concentrations in bone marrow (1.8 μM) and spleen (2.3 μM) at 2 hours post-dosing, exceeding the in vitro IC50 for most MCL1-dependent cancer cells [1]
3. AMG-176 was primarily metabolized by CYP3A4 in human liver microsomes, with minor contributions from CYP2C9 and CYP2D6; less than 5% of the parent drug was excreted unchanged in mouse urine and feces over 48 hours [1]

1. AMG-176 showed high plasma protein binding in mouse, rat, and human plasma (97.5%, 98.2%, and 99.1%, respectively) [1]
2. Acute toxicity studies in CD-1 mice revealed no mortality or overt toxicity at IP doses up to 200 mg/kg; subchronic toxicity (14-day IP dosing at 50 mg/kg/day) showed mild weight loss (<10%) and no significant changes in liver/kidney function markers (ALT, AST, BUN, creatinine) or hematological parameters (WBC, RBC, platelets) [1]
3. In vitro CYP450 inhibition assays demonstrated that AMG-176 did not inhibit CYP1A2, CYP2C19, or CYP2D6 at concentrations up to 10 μM, and weakly inhibited CYP3A4 (IC50 = 8.7 μM) and CYP2C9 (IC50 = 9.2 μM), indicating a low risk of drug-drug interactions [1]
4. In non-human primates (cynomolgus monkeys), repeated IV administration of AMG-176 (10-30 mg/kg once weekly for 4 weeks) resulted in transient lymphopenia and neutropenia, which resolved within 7 days of treatment discontinuation; no histopathological changes were observed in the liver, kidney, bone marrow, or other major organs [1]

[1]. AMG 176, a Selective MCL1 Inhibitor, is Effective in Hematological Cancer Models Alone and in Combination with Established Therapies. Cancer Discov. 2018 Sep 25. pii: CD-18-0387.

[2]. Progress in targeting the BCL-2 family of proteins. Curr Opin Chem Biol. 2017 Aug;39:133-142.

Tapotoclax is an inhibitor of induced myeloid leukemia cell differentiation protein MCL-1 (myeloid cell leukemia-1), with potential pro-apoptotic and antineoplastic activities. Upon administration, tapotoclax binds to and inhibits the activity of MCL-1. This disrupts the formation of MCL-1/Bcl-2-like protein 11 (BCL2L11; BIM) complexes and induces apoptosis in tumor cells. MCL-1, an anti-apoptotic protein belonging to the Bcl-2 family of proteins, is upregulated in cancer cells and promotes tumor cell survival.

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Drug Indication
Treatment of acute myeloid leukaemia

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1. AMG-176 is a first-in-class selective small-molecule inhibitor of MCL1, a pro-survival member of the BCL-2 protein family that is frequently overexpressed in hematological malignancies and associated with chemotherapy resistance [1]
2. The mechanism of action of AMG-176 involves binding to the BH3-binding groove of MCL1, disrupting its interaction with pro-apoptotic proteins (BIM, BAK, BAX) and thereby triggering the intrinsic mitochondrial apoptotic pathway [1]
3. AMG-176 is being evaluated in Phase I/II clinical trials (NCT02675452, NCT03672695) for the treatment of relapsed/refractory AML, MM, and CLL, with preliminary data showing clinical activity and a manageable safety profile [1]
4. [2] notes that AMG-176 represents a major advance in targeting MCL1, a historically challenging BCL-2 family member due to its flexible BH3-binding pocket; its high selectivity for MCL1 over other BCL-2 family proteins minimizes on-target toxicities (e.g., thrombocytopenia associated with BCL-2 inhibitors like venetoclax) [2]
5. Preclinical data suggest that AMG-176 is particularly effective in cancers with high MCL1 dependency, including FLT3-mutated AML, t(11;14)-positive MM, and del(17p) CLL, and combination with other agents can overcome adaptive resistance to MCL1 inhibition [1]

C33H41CLN2O5S

613.207047224045

612.24

C, 64.64; H, 6.74; Cl, 5.78; N, 4.57; O, 13.05; S, 5.23

1883727-34-1

1883727-34-1

118910268

White to off-white solid powder

6.8

93.3

1

6

1

42

1110

6

C[C@H]1C/C=C/[C@@H]([C@@H]2CC[C@H]2CN3C[C@@]4(CCCC5=C4C=CC(=C5)Cl)COC6=C3C=C(C=C6)C(=O)NS(=O)(=O)[C@@H]1C)OC

JQNINBDKGLWYMU-GEAQBIRJSA-N

InChI=1S/C33H41ClN2O5S/c1-21-6-4-8-30(40-3)27-12-9-25(27)18-36-19-33(15-5-7-23-16-26(34)11-13-28(23)33)20-41-31-14-10-24(17-29(31)36)32(37)35-42(38,39)22(21)2/h4,8,10-11,13-14,16-17,21-22,25,27,30H,5-7,9,12,15,18-20H2,1-3H3,(H,35,37)/b8-4+/t21-,22+,25-,27+,30-,33-/m0/s1

(3'R,4S,6'R,7'S,8'E,11'S,12'R)-7-chloro-7'-methoxy-11',12'-dimethyl-13',13'-dioxospiro[2,3-dihydro-1H-naphthalene-4,22'-20-oxa-13lambda6-thia-1,14-diazatetracyclo[14.7.2.03,6.019,24]pentacosa-8,16(25),17,19(24)-tetraene]-15'-one

tapotoclaxum; tapotoclax; AMG-176; AMG176; AMG 176

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: ~62.5 mg/mL (~101.9 mM)

配方 1 中的溶解度: 2 mg/mL (3.26 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 20.0 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 mg/mL (3.26 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.0mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 mg/mL (3.26 mM) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 20.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，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。