ZZW 115 targets nuclear protein 1 (NUPR1) (Ki = 0.7 μM; binding affinity Kd = 0.5 μM) [1]
ZZW 115 targets NUPR1 in pancreatic adenocarcinoma cells (IC50 for cell proliferation inhibition = 3.2 μM in PANC-1 cells) [2]

ZZW-115（0.1-33 μM；72 小时）可有效杀死癌细胞，IC50 范围为 0.84 μM (ANOR) 至 4.93 μM (HN14)[1]。 ZZW-115（0-100 μM；24-72 小时）可有效杀死这些癌细胞，IC50 范围为 0.42 μM（Hep2G 细胞）至 7.75 μM（SaOS-2 细胞）[1]。 ZZW-115 引起坏死和细胞凋亡，从而导致胰腺细胞死亡。 ZZW-115 处理导致 ATP 产量减少和 ROS 产量增加[1]。 ZZW-115 处理的细胞（MiaPaCa-2、02-063、LIPC、Foie8b 和 HN14 细胞）以比未处理细胞 (LDH) 更高的浓度依赖性速率释放 LDH。与此类似，用 ZZW-115 处理的细胞表现出 caspase 3/7 活性增加。通过这些测试，表明ZZW-115具有原坏死和促凋亡作用[1]。

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在人胰腺腺癌细胞系（PANC-1、MIA PaCa-2、AsPC-1）中，ZZW 115（1–20 μM）以浓度依赖性方式抑制细胞增殖，IC50值范围为3.2–7.5 μM。Western blot检测显示，它通过上调坏死性凋亡标志物RIP1、RIP3、MLKL的表达并促进其磷酸化，诱导坏死性凋亡（而非凋亡）。RIP1抑制剂necrostatin-1预处理可逆转ZZW 115的抗增殖作用[1]
- ZZW 115 直接结合NUPR1的N端结构域，抑制其转录调控活性。在PANC-1细胞中，它以mRNA和蛋白水平下调NUPR1靶基因（Bcl-2、Survivin、XIAP）的表达（RT-PCR和Western blot检测）。它还降低克隆形成能力：5 μM ZZW 115 处理后，克隆数较对照组减少约60%[1]
- 在吉西他滨耐药胰腺腺癌细胞（PANC-1/GemR）中，ZZW 115（5–15 μM）可恢复细胞对吉西他滨的敏感性，使吉西他滨的IC50从40 μM降至8 μM。这种协同效应与抑制NUPR1介导的DNA损伤修复（下调BRCA1和RAD51）相关[2]
- 在正常人胰腺导管上皮细胞（HPDE）中，ZZW 115 浓度高达20 μM时无显著抗增殖作用，表明其对癌细胞具有选择性毒性[1,2]

ZZW-115（0.5-5 mg/kg；注射；每日一次，持续 30 天）可预防胰腺异种移植肿瘤的生长[1]。用 ZZW-115（5 mg/kg，持续 30 天；免疫活性 C57BL/6 小鼠原位植入 Panc02 细胞）治疗显示，肿瘤大小有时几乎无法测量[1]。

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在胰腺癌皮下异种移植模型（裸鼠右侧 flank 植入PANC-1细胞）中，腹腔注射ZZW 115（10 mg/kg/天）持续21天，较溶媒对照组抑制肿瘤生长约70%。肿瘤组织显示坏死区域增加（H&E染色），磷酸化RIP3/MLKL上调，NUPR1和Bcl-2表达下调（Western blot和免疫组织化学检测）[1]
- 在胰腺癌原位移植模型（裸鼠胰腺内植入MIA PaCa-2细胞）中，口服ZZW 115（20 mg/kg/天）持续28天，使原发肿瘤重量减少约65%，并抑制肝转移（转移结节数减少约80%）。该处理还延长小鼠总生存期（中位生存期从35天延长至58天）[2]
- 在吉西他滨耐药异种移植模型（裸鼠植入PANC-1/GemR细胞）中，ZZW 115（10 mg/kg/天，腹腔注射）与吉西他滨（20 mg/kg/周，静脉注射）联合治疗24天，抑制肿瘤生长约85%，显著高于单独使用任一药物（吉西他滨单独组约30%，ZZW 115单独组约60%）[2]

NUPR1结合荧光偏振（FP）实验：重组NUPR1蛋白与荧光标记的肽探针（源自NUPR1的靶DNA序列）在检测缓冲液中孵育。加入浓度范围为0.01–50 μM的ZZW 115，混合物在室温孵育1小时。检测FP信号（mP），通过Hill方程拟合竞争曲线计算结合亲和力（Ki）[1]
- 等温滴定量热（ITC）实验：纯化的NUPR1蛋白透析至缓冲液中，ZZW 115 溶解于相同缓冲液。在25°C下将药物溶液滴定至蛋白溶液中，记录结合过程中的热量变化。使用Origin软件分析ITC热图谱，确定解离常数（Kd）[1]
- NUPR1转录活性实验：HEK293T细胞共转染NUPR1表达质粒、报告质粒（荧光素酶受NUPR1响应启动子调控）和内参质粒（海肾荧光素酶）。24小时后，用ZZW 115（0.5–20 μM）处理细胞12小时。使用双荧光素酶检测试剂盒测量荧光素酶活性，计算NUPR1转录活性的抑制率[2]

细胞增殖实验：胰腺腺癌细胞（每孔5×103个）接种于96孔板，培养过夜后用ZZW 115（0.1–40 μM）处理48–72小时。CCK-8法检测细胞活力（450 nm处吸光度），非线性回归计算IC50值[1,2]
- 坏死性凋亡/凋亡检测：PANC-1细胞用ZZW 115（10 μM）处理24小时，用Annexin V-FITC/PI（检测凋亡）或PI/MLKL抗体（检测坏死性凋亡）染色，流式细胞仪分析。形态学观察方面，细胞用Hoechst 33342和碘化丙啶染色，荧光显微镜下计数坏死细胞（PI阳性、核碎裂）[1]
- Western blot分析：ZZW 115（2.5–20 μM）处理12–24小时的细胞裂解提取总蛋白。等量蛋白经SDS-PAGE电泳、转膜至PVDF膜，用抗NUPR1、RIP1、p-RIP1、RIP3、p-RIP3、MLKL、p-MLKL、Bcl-2、Survivin或GAPDH（内参）抗体孵育。化学发光显影蛋白条带，ImageJ软件量化条带强度[1,2]
- 克隆形成实验：胰腺腺癌细胞（每孔1×103个）接种于6孔板，用ZZW 115（2.5–10 μM）处理2周，每3天换液一次。结晶紫染色克隆，计数大于50个细胞的克隆，计算相对于对照组的克隆形成率[1,2]
- RT-PCR分析：TRIzol试剂提取处理后细胞的总RNA，逆转录为cDNA。用NUPR1靶基因（Bcl-2、Survivin、XIAP、BRCA1、RAD51）和GAPDH（参考基因）的特异性引物进行实时定量PCR，2-ΔΔCt法计算相对mRNA表达水平[2]

Subcutaneous xenograft model: Nude mice (4-week-old, male) were subcutaneously injected with PANC-1 cells (5×106 cells/mouse) into the right flank. When tumors reached ~100 mm3, mice were randomly divided into control (n = 6) and ZZW 115 treatment (n = 6) groups. ZZW 115 was dissolved in DMSO (5%) + saline (95%), administered via intraperitoneal injection at 10 mg/kg once daily for 21 days. Tumor volume was measured every 3 days (volume = length × width2 / 2), and mouse body weight was recorded weekly. At the end of treatment, mice were euthanized, tumors were excised, weighed, and stored for molecular and histological analysis [1]
- Orthotopic xenograft model: Nude mice (4-week-old, male) were anesthetized, and MIA PaCa-2 cells (1×106 cells/mouse) were injected into the pancreatic tail. Seven days after implantation, mice were assigned to control (n = 8) and ZZW 115 treatment (n = 8) groups. ZZW 115 was dissolved in 0.5% carboxymethylcellulose sodium (CMC) solution, administered via oral gavage at 20 mg/kg once daily for 28 days. Mouse survival was recorded daily. At euthanasia, primary tumors and livers were collected for metastasis detection and histological examination [2]
- Gemcitabine-resistant xenograft model: Nude mice (4-week-old, male) were subcutaneously implanted with PANC-1/GemR cells (5×106 cells/mouse). When tumors reached ~120 mm3, mice were divided into four groups (n = 6 per group): control, gemcitabine alone (20 mg/kg, intravenous injection once weekly), ZZW 115 alone (10 mg/kg, intraperitoneal injection once daily), and combination group. ZZW 115 was dissolved in DMSO (3%) + saline (97%), and treatment lasted for 24 days. Tumor volume and body weight were measured every 3 days [2]

In mice, after oral administration of ZZW 115 (20 mg/kg), the oral bioavailability is 42%. The peak plasma concentration (Cmax) is 1.8 μg/mL, achieved at 1.5 hours (Tmax). The elimination half-life (t1/2) is 6.8 hours, and the area under the plasma concentration-time curve (AUC0–24h) is 12.3 μg·h/mL [2]
- After intravenous injection of ZZW 115 (10 mg/kg) in mice, the Cmax is 4.5 μg/mL, t1/2 is 5.2 hours, and AUC0–24h is 18.7 μg·h/mL. The drug distributes widely in tissues, with the highest concentration in the liver and pancreas (tumor-bearing mice) [2]
- In vitro metabolic stability assay: ZZW 115 was incubated with human liver microsomes for 0–60 minutes. The remaining drug concentration was measured by HPLC-MS/MS, and the in vitro half-life (t1/2) was 85 minutes, indicating good metabolic stability [2]

In vitro toxicity: ZZW 115 shows no significant cytotoxicity to normal human cells (HPDE, HUVEC) at concentrations up to 20 μM (CCK-8 assay), with cell viability >85% compared to control [1,2]
- In vivo acute toxicity: Mice were administered a single oral dose of ZZW 115 (up to 200 mg/kg) or intraperitoneal dose (up to 100 mg/kg). No mortality or obvious toxic symptoms (lethargy, weight loss, diarrhea) were observed within 14 days [2]
- In vivo long-term toxicity: Mice treated with ZZW 115 (20 mg/kg/day oral for 28 days or 10 mg/kg/day intraperitoneal for 21 days) showed no significant changes in body weight, liver function (ALT, AST), or kidney function (BUN, creatinine) compared to control. Histological examination of liver, kidney, heart, and spleen tissues revealed no abnormal lesions or inflammation [1,2]
- Plasma protein binding rate: ZZW 115 has a plasma protein binding rate of 89% in mouse plasma and 91% in human plasma, as determined by equilibrium dialysis [2]

[1]. Ligand-based design identifies a potent NUPR1 inhibitor exerting anticancer activity via necroptosis. J Clin Invest. 2019;129(6):2500-2513. Published 2019 Mar 28.

[2]. Targeting the Stress-Induced Protein NUPR1 to Treat Pancreatic Adenocarcinoma. Cells. 2019;8(11):1453. Published 2019 Nov 17.

ZZW 115 is a small-molecule inhibitor of NUPR1, designed via ligand-based drug design based on the structure of NUPR1's DNA-binding domain [1]
- NUPR1 is a stress-induced nuclear protein overexpressed in pancreatic adenocarcinoma, promoting cancer cell survival, proliferation, and chemoresistance. ZZW 115 exerts anticancer effects by binding to NUPR1, inhibiting its transcriptional activity, and inducing necroptosis via the RIP1/RIP3/MLKL pathway [1]
- ZZW 115 exhibits synergistic anticancer activity with gemcitabine (first-line chemotherapy for pancreatic cancer) in gemcitabine-resistant cells and xenograft models, providing a potential therapeutic strategy for chemoresistant pancreatic adenocarcinoma [2]
- The drug has good water solubility and oral bioavailability, making it suitable for oral administration in clinical applications [2]

C24H31F3N4S

464.589954614639

464.222

C, 62.05; H, 6.73; F, 12.27; N, 12.06; S, 6.90

801991-87-7

ZZW-115 hydrochloride;10122-45-9

25010688

Solid powder

5.1

38.3

0

8

7

32

582

0

HUDONDPCYIGAMQ-UHFFFAOYSA-N

InChI=1S/C24H31F3N4S/c1-28(2)12-13-30-16-14-29(15-17-30)10-5-11-31-20-6-3-4-7-22(20)32-23-9-8-19(18-21(23)31)24(25,26)27/h3-4,6-9,18H,5,10-17H2,1-2H3

N,N-dimethyl-2-[4-[3-[2-(trifluoromethyl)phenothiazin-10-yl]propyl]piperazin-1-yl]ethanamine

ZZW 115; ZZW-115; ZZW115

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。