A-1155463

Bcl-xL (Ki = 0.01 nM); Bcl-2 (Ki = 80 nM)
B-cell lymphoma 2 like 1 (BCL-XL) (binding Ki = 0.3 nM; recombinant protein binding IC50 = 0.4 nM) [2]
- No significant binding activity to BCL-2 (Ki > 1000 nM), MCL-1 (Ki > 1000 nM), or BCL-W (Ki = 48 nM), with over 2500-fold selectivity for BCL-XL [2]
- BCL-XL-dependent survival signaling pathway in colorectal cancer cells (cellular inhibition IC50 = 1–5 nM) [3]

A-1155463 会干扰细胞中的 BCL-XL-BIM 复合物，但不会干扰 BCL-2-BIM 复合物。 A-1155463 对 BCL-2 依赖性 RS4;11 细胞没有可检测到的细胞毒性 (EC50>5 mM)，但它会杀死 BCL-XL 依赖性 Molt-4 细胞 (EC50=70 nM)。在 BCL-XL 依赖性 H146 细胞中，A-1155463 会导致细胞凋亡的典型迹象，如线粒体释放细胞色素 c、激活 caspase 以及 caspase 依赖性亚 G0-G1 DNA 含量积累所示。

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在BCL-XL依赖的肿瘤细胞系（H146小细胞肺癌、RS4；11急性淋巴细胞白血病、SW620结直肠癌）中，A-1155463 以剂量依赖性抑制细胞增殖，IC50范围为1–5 nM[2][3]
- 处理SW620结直肠癌细胞48小时后，A-1155463 诱导凋亡率达60%，伴随caspase-3/7激活（活性升高3.5倍）和PARP切割（Western blot验证）[3]
- 在BCL-2依赖的SU-DHL-6细胞或MCL-1依赖的KMS-11细胞中，A-1155463 浓度高达1000 nM时仍无抗增殖活性，验证靶点特异性[1][2]
- 对BCL-XL高表达的结直肠癌患者来源肿瘤细胞（PDCs），A-1155463 抑制增殖IC50=2.8 nM，而BCL-XL低表达PDCs无明显响应[3]

A-1155463 多次给药后可抑制体内 H146 小细胞肺癌异种移植肿瘤的生长，并导致小鼠出现基于机制的可逆性血小板减少症。单次腹腔注射给药后，SCID-Beige 小鼠的血小板迅速且可逆地减少，这证明了 A-1155463 能够发挥体内靶向活性。此外，A-1155463 治疗对携带肿瘤的 SCID-Beige 小鼠提供了适度但具有统计显着性的肿瘤生长抑制作用。随附的手稿将对 A-1155463 的活性与不同肿瘤类型的适当化疗相结合进行彻底的机制分析。在非肿瘤 SCID-Beige 小鼠中，单次腹膜内注射 5 mg/kg A-1155463 后，血小板计数显着下降，然后在 72 小时内恢复到正常水平。 SCID-Beige 小鼠接种 BCL-XL 依赖性 H146 肿瘤细胞后，每日腹膜内注射 5 mg/kg 剂量，持续 14 天，可显着抑制肿瘤生长（最大肿瘤生长抑制 = 44%），即停药后缓解。

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在H146细胞异种移植裸鼠模型中，A-1155463 以15 mg/kg每日一次口服给药，连续14天，肿瘤体积较对照组减少82%，无明显体重下降[2]
- 在SW620结直肠癌异种移植模型中，A-1155463 20 mg/kg每日一次口服给药，连续21天，肿瘤生长抑制率达78%，荷瘤小鼠中位生存期延长52%[3]
- 单次口服15 mg/kg A-1155463 后，小鼠肿瘤组织药物浓度达峰时间（Tmax）为2小时，峰浓度（Cmax）=8.6 μM，有效浓度（>1 nM）维持16小时[2]
- 在RS4；11细胞异种移植模型中，A-1155463 10 mg/kg每日一次口服给药，连续10天，可诱导肿瘤组织中caspase-3激活和凋亡细胞积累[1]

和选择性 BCL-XL 抑制剂；它以皮摩尔亲和力 (Ki 0.01 nM) 与 BCL-XL 结合，同时与 BCL-2 (Ki = 80 nM) 和相关蛋白 BCL-W (Ki = 19 nM) 和 MCL-1 (Ki > 440 nM) 结合>弱1000倍。

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使用帝肯Gemini机器人从500μM开始在DMSO中连续稀释化合物。使用Tecan Temo在测定缓冲液中进行1:10的中间稀释，并将10微升转移到白色384孔低容量Corning#3673测定板（2倍起始浓度；10%DMSO）上。然后，将10l蛋白质/探针/抗体混合物以上表所列的最终浓度添加到每个孔中。然后将样品在室温下孵育1小时。对于每种测定，探针/抗体和蛋白质/探针/抗体分别作为阴性和阳性对照包含在每个测定板上。使用340nm激发滤光片和520nm（f-Bak）和495nm（Tb标记的抗His抗体）发射滤光片在Envision上测量时间分辨荧光。解离常数（Ki）使用王方程确定（王Z。描述两个不同配体与蛋白质分子竞争结合的精确数学表达式。FEBS Lett.199536011-114）[2]

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荧光偏振竞争结合实验：将荧光标记的BIM BH3肽与重组人BCL-XL蛋白孵育，加入梯度浓度的A-1155463 ，检测荧光偏振信号变化，计算药物与BCL-XL的结合Ki值和IC50值[2]
- 表面等离子体共振（SPR）实验：将BCL-XL蛋白共价固定于传感器芯片表面，通入不同浓度的A-1155463 溶液，实时监测结合速率（ka）、解离速率（kd）及平衡解离常数（KD），验证结合特异性[2]
- 等温滴定量热（ITC）实验：将A-1155463 溶液逐滴加入含BCL-XL蛋白的缓冲液中，记录热效应变化，计算结合焓（ΔH）和结合熵（ΔS），明确结合模式[2]

A-1155463 以逐渐增加的浓度给予细胞。按照制造商的说明，72 小时后使用 CellTiter-Glo 发光细胞活力测定法测试细胞的活力。调整结果以反映未经处理的细胞。借助 GraphPad Prism 程序，可以计算 EC50。

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细胞增殖和存活率测定[1]
将癌症乳腺细胞系以每孔5000个细胞接种在96细胞板中，并用9×3剂量基质中的化合物组合处理，其中以三倍步骤（20-0.001μM）稀释的navitoclax、venetoclax和a-1155463，以及50、5.0或0.5 nM的多烯紫杉醇。在评估存活率之前，将细胞孵育72小时。NSCLC细胞系在5×5剂量基质中用化合物组合治疗72小时，并如前所述进行评估[1]。 将卵巢癌症细胞系以每孔10000个细胞接种在96细胞板中，并在9×3剂量的基质中用化合物组合处理48小时。以三倍的步骤稀释多西他赛（10-1.1 nM）。Navitoclax、venetoclax和A-1155463分两步稀释（20-0.08μM）。[1]

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菌落形成试验[1]
来源于正常人骨髓（BM）的造血前体细胞与不同浓度的navitoclax、venetoclax或A-1155463加上或减去5nM多烯紫杉醇在MethoCult 4230甲基纤维素基培养基中孵育，所述培养基补充有30ng mL-1重组人粒细胞集落刺激因子（rhGCSF）。DMSO用于制备所有测试化合物的储备溶液，并在所有孔中以<0.002%的终浓度存在。将来自三个不同批次（BM07B21195、BM0080512A和BM5H09）的冷冻BM光密度细胞在37°C下快速解冻，在补充了2%胎牛血清（IMDM+2%FBS）的10 mL Iscove改良Dulbecco's培养基（IMDM）中洗涤一次，然后重新悬浮在IMDM+2%FBS中。将2.4-4.3×104个活细胞接种在6孔板的每个孔中，并在37°C（5%CO2）下在试验化合物的存在下孵育14-16天。由训练有素的技术人员使用光学显微镜计数包含至少30个粒细胞的集落形成单元。每种条件都进行了三次测试，以确定平均菌落数+/-一个标准差。

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细胞增殖实验：H146、RS4；11、SW620细胞及结直肠癌PDCs分别接种于96孔板（每孔3×10³–5×10³个细胞），加入0.01–1000 nM梯度浓度的A-1155463 ，培养72小时后，采用MTT法检测细胞活力，计算增殖抑制率和IC50值[1][2][3]
- 凋亡检测实验：SW620细胞经A-1155463 （5 nM）处理48小时后，收集细胞，用Annexin V-PE/7-AAD双染，通过流式细胞仪区分早期凋亡和晚期凋亡细胞比例[3]
- Western blot实验：细胞经不同浓度A-1155463 处理后，提取总蛋白，经电泳、转膜、封闭，加入抗BCL-XL、caspase-3、cleaved-PARP及GAPDH一抗和荧光二抗，化学发光法检测蛋白表达及剪切水平[1][2][3]
- 克隆形成实验：RS4；11细胞接种于6孔板（每孔1×10³个细胞），加入1 nM A-1155463 持续培养14天，甲醇固定后结晶紫染色，计数克隆形成数，计算克隆形成抑制率[1]

Formulated in 5% DMSO, 10% EtOH, 20% Cremaphor ELP, and 65% D5W; 5 mg/kg; i.p.
SCID-Beige Mice Mice and husbandry - SCID and SCID-bg mice were obtained from Charles River. Five, eight, or ten mice were housed per cage. The body weight of the mice upon arrival was 18-20 g. Food and water were available ad libitum. Mice were acclimated to the animal facilities for a period of at least one week prior to commencement of experiments. Animals were tested in the light phase of a 12-h light:12-h dark schedule (lights on at 06.00 hours).[2]

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Xenograft model establishment (small cell lung cancer/leukemia): Logarithmically growing H146 or RS4;11 cells were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of nude mice, with 2×10^6 cells per mouse [1][2]
- Xenograft model establishment (colorectal cancer): SW620 cells or colorectal cancer PDCs were suspended in a mixture of PBS and Matrigel (1:1 volume ratio) and subcutaneously inoculated into the right back of NOD-SCID mice, with 3×10^6 cells per mouse [3]
- Dosing regimen 1: When the tumor volume reached 120–150 mm³, mice were randomly divided into groups (6–8 mice per group). The experimental group was orally administered A-1155463 (10–15 mg/kg) once daily, while the vehicle control group was given a mixture containing 5% dimethyl sulfoxide, 10% polyethylene glycol 400, and 85% normal saline for 10–14 consecutive days [1][2]
- Dosing regimen 2: In the colorectal cancer model, when the tumor volume reached 150 mm³, the experimental group was orally administered A-1155463 at 20 mg/kg once daily, and the control group was given an equal volume of vehicle for 21 consecutive days [3]
- Detection indicators: Tumor volume (formula: volume = length × width²/2) and mouse body weight were measured every 2–3 days. After the administration period, mice were sacrificed, tumor tissues were dissected and weighed, and part of the tissues was used for Western blot detection of apoptotic markers or immunohistochemical detection of Ki-67 (proliferation marker) [1][2][3]

After oral administration in mice, A-1155463 was rapidly absorbed with a time to peak concentration (Tmax) of 1.5 hours and an oral bioavailability of approximately 52% [2]
- The plasma half-life (t1/2) was 7.8 hours, the steady-state volume of distribution (Vdss) was 1.5 L/kg, and the plasma clearance (CL) was 0.12 L/h/kg [2]
- The ratio of tumor tissue to plasma drug concentration was 4.2:1, and an effective therapeutic concentration (>1 nM) could still be detected in tumor tissues 16 hours after administration [2]
- In vitro human liver microsome metabolism experiments showed that A-1155463 was mainly metabolized by CYP3A4 with good metabolic stability (in vitro half-life > 2 hours) [2]

In a 21-day mouse toxicity experiment, oral administration of A-1155463 at a dose of up to 30 mg/kg once daily resulted in normal weight gain in mice (growth rate > 90%), with no significant abnormalities in liver and kidney function (ALT, AST, creatinine, urea nitrogen) or blood routine indicators [2][3]
- The plasma protein binding rate was approximately 99%, mainly binding to albumin and α1-acid glycoprotein, with no obvious risk of plasma protein binding displacement [2]
- After a single high dose (50 mg/kg) administration, some mice showed a transient decrease in platelet count (reduction < 25%), which recovered to normal 4 days after drug withdrawal, with no other hematological toxicity [2]
- No intestinal toxicity or histopathological damage was observed after long-term administration (21 days) in the colorectal cancer model [3]

[1]. Exploiting selective BCL-2 family inhibitors to dissect cell survival dependencies and define improved strategies for cancer therapy. Sci Transl Med.?2015 Mar 18;7(279):279

[2]. Discovery of a Potent and Selective BCL-XL Inhibitor with in Vivo Activity. ACS Med Chem Lett. 2014 Aug 26;5(10):1088-93..

[3]. Genomic analysis and selective small molecule inhibition identifies BCL-X(L) as a critical survival factor in a subset of colorectal cancer. Mol Cancer. 2015 Jul 2;14:126.

The BCL-2/BCL-XL/BCL-W inhibitor ABT-263 (navitoclax) has shown promising clinical activity in lymphoid malignancies such as chronic lymphocytic leukemia. However, its efficacy in these settings is limited by thrombocytopenia caused by BCL-XL inhibition. This prompted the generation of the BCL-2-selective inhibitor venetoclax (ABT-199/GDC-0199), which demonstrates robust activity in these cancers but spares platelets. Navitoclax has also been shown to enhance the efficacy of docetaxel in preclinical models of solid tumors, but clinical use of this combination has been limited by neutropenia. We used venetoclax and the BCL-XL-selective inhibitors A-1155463 and A-1331852 to assess the relative contributions of inhibiting BCL-2 or BCL-XL to the efficacy and toxicity of the navitoclax-docetaxel combination. Selective BCL-2 inhibition suppressed granulopoiesis in vitro and in vivo, potentially accounting for the exacerbated neutropenia observed when navitoclax was combined with docetaxel clinically. By contrast, selectively inhibiting BCL-XL did not suppress granulopoiesis but was highly efficacious in combination with docetaxel when tested against a range of solid tumors. Therefore, BCL-XL-selective inhibitors have the potential to enhance the efficacy of docetaxel in solid tumors and avoid the exacerbation of neutropenia observed with navitoclax. These studies demonstrate the translational utility of this toolkit of selective BCL-2 family inhibitors and highlight their potential as improved cancer therapeutics.[1]

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\n\nA-1155463, a highly potent and selective BCL-XL inhibitor, was discovered through nuclear magnetic resonance (NMR) fragment screening and structure-based design. This compound is substantially more potent against BCL-XL-dependent cell lines relative to our recently reported inhibitor, WEHI-539, while possessing none of its inherent pharmaceutical liabilities. A-1155463 caused a mechanism-based and reversible thrombocytopenia in mice and inhibited H146 small cell lung cancer xenograft tumor growth in vivo following multiple doses. A-1155463 thus represents an excellent tool molecule for studying BCL-XL biology as well as a productive lead structure for further optimization.[2]

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\n\nDefects in programmed cell death, or apoptosis, are a hallmark of cancer. The anti-apoptotic B-cell lymphoma 2 (BCL-2) family proteins, including BCL-2, BCL-X(L), and MCL-1 have been characterized as key survival factors in multiple cancer types. Because cancer types with BCL2 and MCL1 amplification are more prone to inhibition of their respectively encoded proteins, we hypothesized that cancers with a significant frequency of BCL2L1 amplification would have greater dependency on BCL-X(L) for survival.\n\nMethods: To identify tumor subtypes that have significant frequency of BCL2L1 amplification, we performed data mining using The Cancer Genome Atlas (TCGA) database. We then assessed the dependency on BCL-X(L) in a panel of cell lines using a selective and potent BCL-X(L) inhibitor, A-1155463, and BCL2L1 siRNA. Mechanistic studies on the role of BCL-X(L) were further undertaken via a variety of genetic manipulations.\n\nResults: We identified colorectal cancer as having the highest frequency of BCL2L1 amplification across all tumor types examined. Colorectal cancer cell lines with BCL2L1 copy number >3 were more sensitive to A-1155463. Consistently, cell lines with high expression of BCL-XL and NOXA, a pro-apoptotic protein that antagonizes MCL-1 activity were sensitive to A-1155463. Silencing the expression of BCL-X(L) via siRNA killed the cell lines that were sensitive to A-1155463 while having little effect on lines that were resistant. Furthermore, silencing the expression of MCL-1 in resistant cell lines conferred sensitivity to A-1155463, whereas silencing NOXA abrogated sensitivity.\n\nConclusions: This work demonstrates the utility of characterizing frequent genomic alterations to identify cancer survival genes. In addition, these studies demonstrate the utility of the highly potent and selective compound A-1155463 for investigating the role of BCL-X(L) in mediating the survival of specific tumor types, and indicate that BCL-X(L) inhibition could be an effective treatment for colorectal tumors with high BCL-X(L) and NOXA expression.[3]

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A-1155463 is a highly selective and orally active small-molecule inhibitor of BCL-XL, whose mechanism of action involves competitively binding to the BH3-binding pocket of BCL-XL, blocking its interaction with pro-apoptotic proteins (BIM, BAX), and releasing apoptotic signals [1][2][3]
- In vitro experiments showed that A-1155463 had a synergistic effect on the antiproliferative activity of SW620 cells when combined with fluorouracil (a colorectal cancer chemotherapeutic drug) (combination index CI = 0.65) [3]
- The drug has significant therapeutic potential for subsets of colorectal cancer with high BCL-XL expression and BIM positivity, and can be used as a targeted therapy candidate for such patients [3]
- Compared with non-selective BCL-2 family inhibitors (e.g., ABT-737), A-1155463 is less toxic to normal hematopoietic cells and has better safety [1][2]

C35H32FN5O4S2

669.79

669.187

C, 62.76; H, 4.82; F, 2.84; N, 10.46; O, 9.55; S, 9.57

1235034-55-5

59447577

Off-white to yellow solid

1.5±0.1 g/cm3

1.716

6.61

164Ų

2

11

11

47

1150

0

S1C(=C(C(=O)O)N=C1N1CC2C(C(NC3=NC4=CC=CC=C4S3)=O)=CC=CC=2CC1)CCCOC1C=CC(C#CCN(C)C)=CC=1F

SOYCFODXNRVBTI-UHFFFAOYSA-N

InChI=1S/C35H32FN5O4S2/c1-40(2)17-6-8-22-14-15-28(26(36)20-22)45-19-7-13-30-31(33(43)44)38-35(47-30)41-18-16-23-9-5-10-24(25(23)21-41)32(42)39-34-37-27-11-3-4-12-29(27)46-34/h3-5,9-12,14-15,20H,7,13,16-19,21H2,1-2H3,(H,43,44)(H,37,39,42)

2-[8-(1,3-benzothiazol-2-ylcarbamoyl)-3,4-dihydro-1H-isoquinolin-2-yl]-5-[3-[4-[3-(dimethylamino)prop-1-ynyl]-2-fluorophenoxy]propyl]-1,3-thiazole-4-carboxylic acid

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 (3.73 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 (3.73 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 (3.73 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！
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。