BTSA1

Bax (IC50 = 250 nM); Bax (IC50 = 144 nM)
BTSA1: High-affinity and selective activator of BAX (BCL-2-associated X protein), binding to the N-terminal activation site (trigger site) of BAX; [1]

BTSA1 没有能力直接激活促凋亡同系物 BAK。通过 BTSA1 处理，重组可溶性 BAX 被有效且剂量响应地易位至线粒体膜，随后诱导 BAX 寡聚化。当 BAX 被 BTSA1 激活时，癌细胞更有可能发生凋亡。 BTSA1 的 IC50 值范围在 1 至 4 μM 之间，以剂量依赖性方式降低所有 AML 细胞系的活力，并在治疗 24 小时内产生完全效果。所有五种 AML 细胞系均表现出剂量依赖性 caspase-3/7 激活[1]。

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BAX激活及构象变化：BTSA1 高亲和力特异性结合BAX的N端触发位点（α1、α6区域）。竞争性荧光偏振结合实验显示，它可与FITC-BIM SAHB A2竞争结合BAX；直接荧光偏振结合实验证实其与BAX直接相互作用。对¹⁵N标记BAX的HSQC谱分析表明，BTSA1滴定后，BAX触发位点的残基出现显著主链酰胺化学位移变化，提示诱导BAX构象改变。对接研究显示，BTSA1与BAX形成疏水相互作用，其吡唑酮基团与BAX的K21残基形成关键氢键，模拟BIM BH3螺旋与BAX的相互作用模式[1]
- 白血病细胞中BAX介导的凋亡：BTSA1 可诱导BAX激活通路的全步骤，包括BAX向膜转运、寡聚化及线粒体 outer membrane permeabilization（MOMP）。在ANTS/DPX脂质体实验中，BTSA1（100–400 nM）可促进BAX介导的膜通透，而BAX K21E突变体中该效应受损。在分离的小鼠肝脏线粒体中，BTSA1呈剂量依赖性诱导BAX线粒体转运、寡聚化及细胞色素c释放。它能有效诱导人AML细胞系（如NB4、OCI-AML3、THP-1、MOLM-13）和小鼠AML细胞系WEHI凋亡，表现为细胞活力下降、caspase 3/7活性升高、线粒体去极化（TMRE实验检测）及细胞色素c释放。Western blot证实BTSA1可诱导NB4细胞中BAX从胞质向线粒体转运[1]
- 选择性及敏感性决定因素：竞争性结合实验显示，BTSA1 与抗凋亡BCL-2家族蛋白（BCL-XL、MCL-1、BFL-1）无显著结合，对非癌细胞系和正常造血祖细胞无明显毒性。BAX表达水平和胞质单体构象决定对BTSA1的敏感性：BAX mRNA/蛋白水平较高且胞质中存在单体BAX的AML细胞（如OCI-AML3、HPB-ALL）更敏感，而BAX敲除（KO）MEF细胞对其耐受。在BAX KO MEF细胞中重构野生型BAX可恢复敏感性，但触发位点功能受损的BAX突变体则不能。BTSA1 与Venetoclax（BCL-2抑制剂）在AML细胞系（THP-1、OCI-AML3）中具有协同作用，可增强caspase 3/7活性并降低细胞活力[1]
- 患者AML样本中的药效：BTSA1 可诱导原发性人AML母细胞（n=4）和CD34⁺CD38⁻ AML干细胞富集群体（n=4）凋亡（Annexin V结合实验检测），且不影响健康CD34⁺CD38⁻造血干祖细胞（n=2）。AML患者细胞（n=542）的BAX mRNA表达水平显著高于健康对照细胞（n=74）[1]

BTSA1 可有效抑制人类急性髓系白血病 (AML) 异种移植物并提高宿主存活率，且无毒性。健康的富含干细胞 (LSK) 细胞、常见骨髓祖细胞、粒细胞-单核细胞祖细胞和巨核细胞-红细胞祖细胞在小鼠中具有良好的耐受性，并且没有毒性作用。 10 mg/kg 剂量的 BTSA1 达到足够的水平 (~15 μM)，引起白血病细胞中的 BAX 激活和凋亡，同时在小鼠血浆中也具有显着的半衰期 (T1/2 = 15 小时) 和口服生物利用度 (%F = 51）。因此，BTSA1 具有优异的药代动力学，可口服生物利用，通过诱导细胞凋亡显着抑制白血病异种移植物中的肿瘤生长，并且在治疗有效剂量下，在造血系统或其他组织中未表现出可检测到的毒性[1]。

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AML异种移植模型中的抗肿瘤疗效：在THP-1人AML异种移植NSG小鼠中，BTSA1 治疗可显著抑制肝脏肿瘤负荷（减少人CD45⁺和CD15⁺细胞浸润）并延长宿主生存期（Kaplan-Meier生存曲线，n=7）。在MOLM-13人AML异种移植模型中，BTSA1 可降低骨髓和外周血中人CD45⁺CD15⁺细胞浸润（n=5）。骨髓切片免疫组织化学（IHC）染色显示，BTSA1 处理组的cleaved caspase-3阳性细胞和TUNEL阳性细胞增多，提示凋亡增强。对骨髓来源的人白血病细胞进行TMRE染色，证实线粒体去极化，与BAX介导的MOMP一致[1]
- 体内安全性和耐受性：NSG小鼠接受BTSA1（15 mg/kg体重）治疗30天，体重与溶媒对照组相比无显著变化；外周血计数（红细胞、白细胞、血小板）维持在正常范围。主要器官（肝、脾、肾、肺、心、脑、骨髓）的苏木精-伊红（H&E）染色未显示组织病理学异常，表明无全身毒性[1]

BAX相互作用的荧光偏振结合实验：
1. 竞争性结合实验：制备含FITC-BIM SAHB A2（与BAX结合）、系列浓度BTSA1 及实验缓冲液的反应体系，室温孵育特定时间后，检测荧光偏振值，评估BTSA1与FITC-BIM SAHB A2竞争结合BAX的能力；同时对BCL-XL、MCL-1、BFL-1进行平行实验以评估选择性。
2. 直接结合实验：使用荧光标记的BTSA1（F-BTSA1）与系列浓度BAX混合孵育，检测荧光偏振值以确定BTSA1与BAX的直接结合亲和力[1]
- NMR-based BAX构象变化实验：
1. 制备¹⁵N标记的非活性BAX蛋白，溶解于适宜的NMR缓冲液中。
2. 向¹⁵N标记BAX溶液中滴定BTSA1 至摩尔比1:1，每步滴定后收集¹H-¹⁵N HSQC谱。
3. 分析BAX主链酰胺残基的化学位移变化，将显著变化映射至BAX结构，确定结合位点（触发位点）[1]
- 脂质体膜通透实验：
1. 制备负载ANTS/DPX的脂质体，模拟线粒体外膜。
2. 将脂质体与200 nM BAX（或BAX K21E突变体）及系列浓度BTSA1（100–400 nM）共同孵育，以60 nM tBID作为阳性对照。
3. 实时检测脂质体释放的ANTS荧光强度，评估BTSA1诱导的BAX介导膜通透作用[1]

将 AML 细胞（以 2 × 104 个细胞/孔接种）与 BTSA1 或 BTSA2 或载体 (0.15% DMSO) 在无 FBS 培养基中的连续稀释液一起孵育 2.5 小时，然后将 10% FBS 替代物添加至最终体积 100 μl。 24小时后，评估细胞活力。

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细胞活力及凋亡实验：
1. 细胞培养：在适宜培养基中培养AML细胞系（NB4、OCI-AML3、THP-1、MOLM-13）、小鼠AML细胞系WEHI、非癌细胞系、MEF细胞（野生型、BAX KO、BAK KO）及重构MEF细胞；从患者样本和健康供体中分离原发性人AML母细胞、CD34⁺CD38⁻ AML细胞及健康造血祖细胞。
2. 药物处理：将细胞以适宜密度接种于96孔板，用系列浓度BTSA1（单独或与Venetoclax联合）处理6–24小时，设置溶媒对照和阳性对照组。
3. 活力检测：使用细胞活力检测试剂盒测量吸光度或发光值，计算细胞活力并确定IC50值。
4. 凋亡检测：对原代细胞进行Annexin V结合实验，对细胞系进行caspase 3/7活性实验，通过荧光或发光检测定量凋亡；采用TMRE染色结合流式细胞术评估线粒体去极化[1]
- Western blot及免疫沉淀实验：
1. 蛋白提取：裂解处理后的细胞或分离胞质和线粒体组分，使用含蛋白酶抑制剂的裂解液提取总蛋白。
2. Western blot：通过SDS-PAGE分离蛋白，转移至膜上，封闭后加入针对BAX、细胞色素c、BCL-2、BCL-XL、MCL-1、Actin（胞质内参）、VDAC（线粒体内参）的抗体孵育，化学发光法检测信号。
3. 免疫沉淀：将细胞裂解液与BAX抗体或生物素标记的BTSA1 孵育，用蛋白A/G珠或链霉亲和素珠捕获免疫复合物，洗涤后进行Western blot检测BAX相互作用蛋白[1]
- BAX单体检测的凝胶过滤层析实验：
1. 制备MEF、OCI-AML3、HPB-ALL细胞的胞质提取物。
2. 将提取物上样至Superdex 200 HR 10/30柱，用缓冲液洗脱，收集对应不同分子量的组分。
3. 对收集的组分进行BAX抗体Western blot，鉴定单体BAX（低分子量组分）[1]

Formulated in 1% DMSO, 30% PEG-400, 65% D5W (5% dextrose in water), 4% Tween-80; 10 mg/kg; P.O. and I.V.
NOD-SCID IL2Rg null (NSG) mice/ICR (CD-1) male mice, 6-8 weeks old

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AML xenograft mouse models:
1. Animal preparation: Use 6–8-week-old NSG mice, acclimate them to the laboratory environment for 1 week before experiments.
2. Tumor cell inoculation: Inject human AML cells (THP-1 or MOLM-13) into mice via tail vein or intraperitoneal injection to establish xenograft models.
3. Drug preparation and administration: Dissolve BTSA1 in an appropriate vehicle (e.g., DMSO/cremophor/ saline) to a concentration suitable for injection. Administer BTSA1 to mice at a dose of 15 mg/kg body weight via intraperitoneal injection, with a predetermined schedule (e.g., daily for 30 days). Vehicle-treated mice serve as controls.
4. Survival and tumor burden assessment: For THP-1 xenografts, monitor mouse survival daily and generate Kaplan-Meier survival curves. For MOLM-13 and THP-1 xenografts, sacrifice mice at specified time points, collect liver, bone marrow, and peripheral blood samples.
5. Flow cytometry analysis: Stain samples with mouse CD45, human CD45, and human CD15 antibodies, analyze by flow cytometry to quantify human AML cell infiltration.
6. IHC and TMRE assays: Prepare bone marrow sections for cleaved caspase-3 and TUNEL IHC staining to detect apoptosis. Perform TMRE staining on bone marrow-derived human leukemia cells to assess mitochondrial depolarization [1]
- In vivo safety assessment:
1. Animal treatment: Administer BTSA1 (15 mg/kg body weight) or vehicle to NSG mice via intraperitoneal injection daily for 30 days.
2. Physiological monitoring: Measure mouse body weight weekly. At the end of treatment, collect peripheral blood for complete blood count (red blood cells, white blood cells, platelets).
3. Histopathological analysis: Harvest major organs (liver, spleen, kidney, lung, heart, brain, bone marrow), fix in formalin, embed in paraffin, section, and stain with H&E. Examine sections for histopathological abnormalities [1]

In vitro toxicity: BTSA1 showed very low toxicity to non-cancer cell lines and healthy artificial hematopoietic progenitor cells, and no significant decrease in cell viability was observed at effective concentrations for AML cells [1]. In vivo toxicity: BTSA1 (15 mg/kg body weight, 30 days of treatment) did not cause weight loss, hematological abnormalities, or histopathological damage to major organs in NSG mice. BAX KO MEF cells were resistant to BTSA1, confirming that its cytotoxicity was BAX-dependent and selective for cancer cells expressing BAX [1].

[1]. Direct Activation of BAX by BTSA1 Overcomes Apoptosis Resistance in Acute Myeloid Leukemia. Cancer Cell. 2017 Oct 9;32(4):490-505.e10.

Mechanism of action: BTSA1 is a pharmacologically optimized BAX activator that binds to the N-terminal trigger site of BAX, inducing conformational changes (exposure of membrane-targeting domains, oligomerization), thereby leading to BAX translocation to mitochondria, mitochondrial outer membrane permeability (MOMP), cytochrome c release, and caspase-dependent apoptosis. Its binding mode mimics the BIM BH3 helix, forming a key hydrogen bond with BAX K21 through hydrophobic interactions [1].
- Therapeutic potential: BTSA1 overcomes apoptosis resistance in acute myeloid leukemia (AML) by directly activating BAX (a key mediator of apoptosis). The efficacy of BTSA1 in AML cell lines, patient samples (including cell populations enriched with stem cells), and xenograft models, along with its high selectivity for cancer cells and lack of systemic toxicity, provides proof of concept for direct activation of BAX as a novel strategy for treating acute myeloid leukemia [1]. Synergistic effect with venetoclax: BTSA1 can synergize with venetoclax (a BCL-2 inhibitor) to enhance the apoptotic response of AML cells by releasing and directly activating BAX. This combination therapy may improve the treatment outcomes for AML patients, especially those with BCL-2 overexpression [1].

C21H14N6OS2

430.51

430.067

C, 58.59; H, 3.28; N, 19.52; O, 3.72; S, 14.89

314761-14-3

3857348

Brown to reddish brown solid powder

1.5±0.1 g/cm3

625.2±48.0 °C at 760 mmHg

331.9±29.6 °C

0.0±1.8 mmHg at 25°C

1.791

3.68

1

8

5

30

689

0

S1C([H])=C(C2C([H])=C([H])C([H])=C([H])C=2[H])N=C1N1C(C(=C(C2C([H])=C([H])C([H])=C([H])C=2[H])N1[H])/N=N/C1=NC([H])=C([H])S1)=O

CTRCXGFSYFTJIW-UHFFFAOYSA-N

InChI=1S/C21H14N6OS2/c28-19-18(24-25-20-22-11-12-29-20)17(15-9-5-2-6-10-15)26-27(19)21-23-16(13-30-21)14-7-3-1-4-8-14/h1-13,26H

5-phenyl-2-(4-phenyl-1,3-thiazol-2-yl)-4-(1,3-thiazol-2-yldiazenyl)-1H-pyrazol-3-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)