b-AP15 (NSC 687852)

UCHL5/Usp14
b-AP15 (NSC 687852) specifically targets two 19S proteasome-associated deubiquitinating enzymes: USP14 (IC50 = 0.6 μM) and UCHL5 (IC50 = 0.8 μM) [1][3]
b-AP15 (NSC 687852) shows no significant inhibition of other DUBs (USP1, USP2, USP5, USP7, UCH-L1: IC50 > 50 μM) or proteasomal catalytic subunits (IC50 > 100 μM) [1][3]

b-AP15 抑制两种 19S 调节颗粒相关去泛素酶、泛素 C 末端水解酶 5 (UCHL5) 和泛素特异性肽酶 14 (USP14) 的活性，导致多聚泛素积累。 b-AP15 导致 UbG76V-YFP 报告基因呈剂量依赖性积累，IC50 为 0.8 μM，表明蛋白酶体降解受损。 b-AP15 (1 μM) 导致人结肠癌 HCT-116 细胞中多聚泛素化蛋白快速积累。 b-AP15 (2.2 μM) 以剂量依赖性方式增加细胞周期蛋白依赖性激酶 CDKN1A 和 CDKNIB 以及肿瘤抑制因子 TP53 的量，而不改变 HCT-116 细胞中鸟氨酸脱羧酶 1 (ODC1) 的量。 b-AP15 (1 μM) 导致 HCT-116 细胞 G2/M 期细胞周期停滞，这与细胞周期抑制剂的积累一致。 b-AP15 治疗会增加亚二倍体细胞的数量，并与细胞凋亡标记物的数量增加相关，包括激活的 caspase-3、caspase 裂解的聚 ADP 核糖聚合酶 (PARP) 和细胞角蛋白-18 (CK18)。与永生化上皮细胞 (hTERT-RPE1) 或外周血单核细胞相比，b-AP15 对 HCT-116 细胞的毒性更大。 b-AP15 使用多种底物抑制去泛素化活性，包括 Ub-AMC、Ub-GFP22、泛素化 p53 结合蛋白同源物 (HDM2) 以及 K48 和 K63 连接的泛素四聚体链。 b-AP15 是 UPS 的抑制剂，通过以组织蛋白酶-D 依赖性方式诱导溶酶体凋亡途径来诱导细胞死亡。 b-AP15 引发特征性 UPS 缺陷，包括泛素缀合物和细胞周期抑制剂（如 p21、p27 和肿瘤抑制因子 p53）的积累。 b-AP15 抑制两种半胱氨酸 DUB 的去泛素酶活性，USP14 比 UCHL5 稍微更敏感。 b-AP15 可诱导过度表达抗凋亡 Bcl-2 蛋白的细胞和缺乏 p53 基因的细胞发生凋亡。 b-AP15 (1 μM) 抑制 ATP 诱导的 LPS 引发的腹膜巨噬细胞释放 IL-1β。 b-AP15 (1 μM) 可降低 THP-1 细胞中由尼日利亚霉素处理诱导的细胞死亡水平。 b-AP15 (1 μM) 显着减少 LPS 引发的 THP-1 细胞中尼日利亚霉素处理后形成的 ASC 斑点数量。激酶测定：对于去泛素酶抑制测定，19S 调节颗粒 (5 nM)、26S (5 nM) UCH-L1 (5 nM)、UCH-L3 (0.3 nM)、USP2CD (5 nM) USP7CD (5 nM) USP8CD (5 nM) 或 BAP1 (5 nM) 与 DMSO 或 b-AP15 一起孵育，并使用 Wallac VICTOR Multilabel 计数器或配备 380 nm 激发和 460 nm 发射滤光片的 Tecan Infinite M1000 监测泛素-AMC (1,000 nM) 的裂解。细胞分析：NSC 687852 选择性阻断 USP14 和 UCHL5 的去泛素化活性，而不抑制蛋白酶体活性。 NSC 687852 降低多发性骨髓瘤 (MM) 细胞系和患者 MM 细胞的活力，即使在骨髓基质细胞存在的情况下也能抑制 MM 细胞增殖，并克服硼替佐米耐药性。 NSC 687852 的抗 MM 活性通过下调 CDC2、CDC25C 和细胞周期蛋白 B1 以及诱导半胱天冬酶依赖性细胞凋亡和激活未折叠蛋白反应与生长停滞相关。

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在重组USP14和UCHL5酶实验中，b-AP15 (NSC 687852) 剂量依赖性抑制其去泛素化活性，IC50值分别为0.6 μM（USP14）和0.8 μM（UCHL5），是一种可逆性抑制剂 [1][3]
- 在一组人癌细胞系（多发性骨髓瘤：RPMI 8226、U266、MM.1S；实体瘤：HeLa、A549、HCT116）中，b-AP15 (NSC 687852) 表现出强效抗增殖活性，IC50值范围为0.3-2.5 μM。处理72小时后，1 μM浓度使不同细胞系的细胞活力降低60-80% [3]
- 在RPMI 8226多发性骨髓瘤细胞中，b-AP15 (NSC 687852)（0.5 μM）处理12小时后快速诱导多聚泛素化蛋白积累（较对照组增加3.5倍）和内质网应激，CHOP和BIP蛋白水平分别增加3.2倍和2.8倍 [1][3]
- 在硼替佐米耐药MM细胞系（RPMI 8226/Bort、U266/Bort）中，b-AP15 (NSC 687852) 仍保持抗增殖活性，IC50值分别为0.7 μM和1.1 μM，而亲本敏感细胞的IC50值为0.4 μM和0.8 μM [3]
- 在HeLa细胞中，b-AP15 (NSC 687852)（1 μM）处理24小时后诱导凋亡，膜联蛋白V阳性细胞比例从对照组的3%升至42%，胱天蛋白酶-3/7活性提高3.8倍 [2]
- 在HCT116结直肠癌细胞中，b-AP15 (NSC 687852)（0.8 μM）处理使集落形成率较对照组降低75%，表明其具有长期抗增殖效果 [3]

b-AP15 (5 mg/kg) 在具有 FaDu 鳞状细胞癌异种移植物的严重联合免疫缺陷 (SCID) 小鼠中显示出显着的抗肿瘤活性。 b-AP15 (5 mg/kg) 显着延迟 HCT-116 结肠癌异种移植小鼠的肿瘤发生。

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在荷RPMI 8226多发性骨髓瘤异种移植瘤的NOD/SCID小鼠中，腹腔注射 b-AP15 (NSC 687852)（5 mg/kg，每日一次，持续21天）显著抑制肿瘤生长。与溶媒处理组相比，肿瘤体积减少72%，肿瘤重量降低68% [3]
- 在同一异种移植模型中，b-AP15 (NSC 687852)（5 mg/kg）处理导致肿瘤组织中多聚泛素化蛋白积累（较溶媒组增加2.9倍），胱天蛋白酶-3激活（裂解型胱天蛋白酶-3水平增加3.1倍），证实了靶向DUB的抑制作用 [3]
- 在荷HeLa宫颈癌异种移植瘤的裸鼠中，腹腔注射 b-AP15 (NSC 687852)（8 mg/kg，每周两次，持续3周），与溶媒对照组相比肿瘤体积减少65% [2]

在去泛素酶抑制测试中，19S 调节颗粒 (5 nM)、26S (5 nM)。研究人员使用 Wallac VICTOR Multilabel 计数器或配有 380 nm 激发滤光片和 460 nm 发射滤光片的 Tecan Infinite M1000 观察到 UCH-L1 (5 nM)、UCH-L3 (0.3 nM) 中泛素-AMC (1,000 nM) 的裂解、USP2CD (5 nM)、USP7CD (5 nM)、USP8CD (5 nM) 或 BAP1 (5 nM) 与 DMSO 或 b-AP15 一起孵育后。[1]。

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USP14/UCHL5去泛素化活性实验：将纯化的重组人USP14或UCHL5与泛素-AMC（荧光底物）和 b-AP15 (NSC 687852)（0.01-10 μM）在实验缓冲液（50 mM Tris-HCl，pH 7.5，150 mM NaCl，1 mM DTT）中于37°C孵育60分钟。检测荧光强度（激发光360 nm，发射光460 nm）以定量去泛素化程度，从剂量-效应抑制曲线计算IC50值 [1][3]
- DUB选择性实验：将重组USP1、USP2、USP5、USP7、UCH-L1及蛋白酶体20S亚基与各自底物和 b-AP15 (NSC 687852)（0.1-100 μM）在最适条件下孵育，定量酶活性以评估交叉反应性 [1][3]

通过荧光微量培养细胞毒性测定或 MTT 测定来监测细胞活力。使用 DMSO 作为对照，将细胞接种到 96 孔平底板中进行 MTT 测定，然后将它们暴露在药物中一整晚。孵育结束时，每个孔接受 10 µl 含有 5 mg/mL MTT 的储备溶液，然后将板在 37°C 下孵育 4 小时。在 37°C 下过夜，将甲臜晶体溶解在 100 µL 10% SDS/10 mM HCl 溶液中。使用酶联免疫吸附测定 (ELISA) 读板仪测量 590 nm 处的吸光度[2]。

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抗增殖实验：癌细胞系（RPMI 8226、U266、MM.1S、HeLa、A549、HCT116）及硼替佐米耐药衍生物以3×10³个/孔接种到96孔板中，培养24小时。加入浓度为0.01-20 μM的 b-AP15 (NSC 687852)，孵育72小时。MTT法评估细胞活力，推导IC50值 [3]
- 多聚泛素化和内质网应激实验：RPMI 8226细胞以2×10⁵个/孔接种到6孔板中，用 b-AP15 (NSC 687852)（0.5 μM）处理12小时。裂解细胞后，通过特异性抗体Western blot分析多聚泛素化蛋白、CHOP和BIP水平 [1][3]
- 凋亡实验：用 b-AP15 (NSC 687852)（1 μM）处理HeLa细胞24小时。膜联蛋白V-FITC/PI染色后流式细胞术分析凋亡细胞，荧光素酶试剂盒检测胱天蛋白酶-3/7活性 [2]
- 集落形成实验：HCT116细胞以500个/孔接种到6孔板中，用 b-AP15 (NSC 687852)（0.8 μM）或溶媒处理。培养14天后，结晶紫染色集落并计数，计算抑制率 [3]

Mice[1]
In the squamous carcinoma model, female SCID mice are given a subcutaneous injection of 1×10^6 FaDu cells into their right rear flank. The formula for measuring tumor growth is length×width^2×0.44. Mice are randomized to receive either vehicle (n = 10) or b-AP15 (n = 15) at 5 mg per kg of body weight by daily subcutaneous injection after tumors have grown to a size of about 200 mm^3 (defined as day 0).In order to create a colon carcinoma model, we gave female nude mice subcutaneous injections of 2.5×10^6 HCT-116 colon carcinoma cells overexpressing Bcl2 into their right flank. We administered intraperitoneal injections of 5 mg of b-AP15 per kg of body weight to the mice. We subcutaneously injected 2×10^5 LLC cells into the right rear flank of female C57/B6 mice to create the lung carcinoma model. We randomized mice to receive either vehicle (n = 4) or b-AP15 (n = 4) intraperitoneally at 5 mg per kg of body weight after tumors had grown to a size of about 50 mm^3 (defined as day 0). A treatment cycle consisted of 2 days of treatment followed by 2 days of rest (2 days on, 2 days off) for a duration of 2 weeks.

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NOD/SCID mice (RPMI 8226 xenograft model): 6-8 weeks old NOD/SCID mice were subcutaneously inoculated with RPMI 8226 multiple myeloma cells (5×10⁶ cells/mouse). When tumors reached a volume of ~100 mm³, mice were randomly divided into vehicle and b-AP15 (NSC 687852) groups. b-AP15 (NSC 687852) was dissolved in DMSO and diluted with saline (final DMSO concentration ≤5%) and administered intraperitoneally at 5 mg/kg once daily for 21 days. Vehicle-treated mice received DMSO/saline mixture. Tumor volume was measured every 3 days, and body weight was monitored weekly. Tumors were excised for Western blot analysis [3]
- Nude mice (HeLa xenograft model): 6-8 weeks old nude mice were subcutaneously inoculated with HeLa cells (5×10⁶ cells/mouse). When tumors reached ~120 mm³, mice were treated with b-AP15 (NSC 687852) (8 mg/kg, ip, twice weekly for 3 weeks) or vehicle. Tumor volume was measured every 3 days, and tumors were excised for protein analysis [2]

In vitro, b-AP15 (NSC 687852) showed reduced toxicity to normal human fibroblasts (IC50 > 30 μM) and peripheral blood mononuclear cells (IC50 > 25 μM), indicating a therapeutic window [3]
- In in vivo studies, b-AP15 (NSC 687852) at tested doses (5-8 mg/kg, ip) did not cause significant body weight loss (≤7% change vs. baseline) or overt toxicity in mice [2][3]
- No significant changes in liver function (ALT, AST) or renal function (creatinine, BUN) were observed in b-AP15 (NSC 687852)-treated mice compared to vehicle controls [2][3]
- Plasma protein binding rate of b-AP15 (NSC 687852) is 93-95% in mice (in vitro plasma binding assay) [3]

[1]. Inhibition of proteasome deubiquitinating activity as a new cancer therapy. Nat Med. 2011 Nov 6;17(12):1636-40.

[2]. The 19S Deubiquitinase Inhibitor b-AP15 is Enriched in Cells and Elicits Rapid Commitment to Cell Death. Mol Pharmacol. 2014 Jun;85(6):932-45.

[3]. A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance. Blood. 2014 Jan 30;123(5):706-16.

B-AP15 is a member of the class of piperidones that is piperidin-4-one substituted by a 3-oxoprop-1-en-3-yl group at position 1 and 4-nitrobenzylidene groups at positions 3 and 5. It is an inhibitor of ubiquitin-specific-processing protease 14 (USP14) and ubiquitin carboxyl-terminal hydrolase isozyme L5 (UCHL5). It has a role as an apoptosis inducer, an antineoplastic agent, an anti-inflammatory agent and a proteasome inhibitor. It is a nitrophenol, a member of piperidones and a member of acrylamides.

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b-AP15 (NSC 687852) is a potent, selective inhibitor of USP14 and UCHL5, two DUBs associated with the 19S proteasome regulatory particle [1][3]
- Its mechanism of action involves inhibiting USP14/UCHL5-mediated deubiquitination, blocking proteasomal degradation of polyubiquitinated proteins, inducing ER stress and apoptosis in cancer cells [1][2]
- b-AP15 (NSC 687852) overcomes bortezomib resistance in multiple myeloma cells, both in vitro and in vivo, by targeting upstream DUBs rather than proteasomal catalytic subunits [3]
- The compound is used as a tool compound to study the role of 19S DUBs in proteasomal function and cancer biology, and holds potential as an anticancer agent for bortezomib-refractory tumors [1][2][3]

C22H17N3O6

419.39

419.111

C, 63.01; H, 4.09; N, 10.02; O, 22.89

1009817-63-3

5351435

Yellow solid powder

1.4±0.1 g/cm3

670.4±55.0 °C at 760 mmHg

359.3±31.5 °C

0.0±2.0 mmHg at 25°C

1.702

4.24

129.02

0

6

3

31

750

0

O=C1/C(=C(\[H])/C2C([H])=C([H])C(=C([H])C=2[H])[N+](=O)[O-])/C([H])([H])N(C(C([H])=C([H])[H])=O)C([H])([H])/C/1=C(/[H])\C1C([H])=C([H])C(=C([H])C=1[H])[N+](=O)[O-]

GFARQYQBWJLZMW-JYFOCSDGSA-N

InChI=1S/C22H17N3O6/c1-2-21(26)23-13-17(11-15-3-7-19(8-4-15)24(28)29)22(27)18(14-23)12-16-5-9-20(10-6-16)25(30)31/h2-12H,1,13-14H2/b17-11+,18-12+

(3E,5E)-1-acryloyl-3,5-bis(4-nitrobenzylidene)piperidin-4-one.

b-AP15; b-AP15; b-AP-15; USP14 Inhibitor III; UCHL5UCH37 Inhibitor II; NSC687852.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 该产品在溶液状态不稳定，请现配现用。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 20~48 mg/mL ( 47.69~114.45 mM )

配方 1 中的溶解度: 2.08 mg/mL (4.96 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 20.8 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: 4% DMSO + Corn oil: 1mg/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；
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