PNU-74654

Wnt/β-catenin (IC50 = 129.8 μM, NCI-H295 cell)

PNU-74654 结合 β-连环蛋白的 KD 为 450 nM。 β-连环蛋白的 Tcf3/Tcf4 结合表面上存在一个明显的热点，特别是在残基 K435 和 R469 处。该热点两侧的两个小口袋是 PNU-74654 [2] 装订设计的一部分。在 NCI 中，PNU-74654 显着抑制细胞增殖，增强早期和晚期细胞计数，减少核 β-catenin 积累，抑制治疗后 96 小时的 CTNNB1/β-catenin 表达，并增强治疗后 48 小时的早期和晚期细胞计数。 H295细胞。 β-连环蛋白靶基因的小时数增加。对 HeLa 细胞没有观察到影响。 PNU-74654 治疗后 24 小时和 48 小时可降低 NCI-H295 细胞中的苦杏仁酮和大豆醇。 48 小时治疗后 NCI-H295 细胞中 STAR、醛固酮合酶蛋白以及 SF1 和 CYP21A2 mRNA 表达。水平下降。 PNU-74654 治疗后 24 小时可降低 Y1 细胞中的皮质酮，但不会降低细胞存活率 [1]。

通过将CT-26细胞注射到小鼠侧腹建立CRC体内模型，然后用PNU-74654 /5-FU/ PNU-74654−5-FU的混合物处理这些模型（图5a）。为了进一步研究，肿瘤样本的组织学染色显示除了炎症区域（图5b， d）外，血管紊乱以及大量红细胞外渗（图5b， a），细胞坏死（图5b，b）和肿瘤间质（图5b， c）。我们观察到，与对照组以及PNU-74654 + 5-FU组相比，接受PNU-74654或5-FU组相比，肿瘤生长明显受到抑制（图5C）。同时，肿瘤组织的组织学染色表明，联合用药可显著提高组织坏死。[3]

核磁共振检查[2]
WaterLOGSY是一种鉴定与大分子相互作用的化合物的方法。28,29在本实验中，大量的水磁化部分通过蛋白质-配体复合物以选择性的方式转移到自由配体上。结合化合物（例如PNU-74654）很容易在光谱中通过观察它们的共振符号来识别。不结合的化合物显示负峰或根本没有峰，而结合物显示正峰。这些化合物是在由四到五个分子组成的混合物中筛选出来的。给出阳性信号的混合物被反卷积以识别单个粘合剂。核磁共振实验中使用的β-catenin犰狳重复浓度为2 μM， 5 mM Tris, 10 mM NaCl pH 7.3。Tcf4浓度为25 μM。将化合物溶解在浓度为40 mM的氘化二甲基亚砜（DMSO）中。首先在浓度为50 μM的混合物中在20°C下筛选。竞争实验的样品在PBS中缓冲。核磁共振实验采用瓦里安Inova 600 MHz光谱仪，配备5毫米三共振探头和样品管理系统（SMS）自动进样器。记录每个样品的参考光谱和WaterLOGSY光谱。

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ITC测量[2]
ITC测量在20°C下使用VP-ITC滴定量热计（MicroCal）进行。样品（如PNU-74654）广泛透析PBS， 1mm DTT。每次滴定实验初始注射2 μL，随后20次注射5 μL。通过将Tcf4 （1-53 W-His6）注射到用于每个特定实验的缓冲液中，在空白滴定中测量稀释热。这些是从结合热中减去的。在3 μM的β-catenin和60 μM的β-catenin下进行直接结合实验。竞争分析的浓度为100 μM的Tcf4 （1-53 W-His6）和5 μM的β-catenin。每次滴定实验均在四种化合物的混合物中进行，每种化合物的浓度为100 μM。结合热采用Origin软件包 对实验测量值进行非线性最小二乘拟合，并假设单一结合位点模型。选择至少三倍降低Tcf4结合亲和力的化合物混合物进行进一步表征。

PNU-74654对细胞系的作用[1]
将NCI-H295细胞以每孔200,000个细胞的速度在24孔板中进行基因表达、蛋白分析和肾上腺激素测量。48 h后，细胞分别用载药（0.1%-0.4% DMSO）或10、50、100和200 μM PNU-74654处理。24和48 h后，收集培养基上清液用于测量肾上腺激素。将贴壁细胞固定用于免疫荧光或收获用于RNA和蛋白质分离。至少进行了三个独立的实验。
将Y1细胞以每孔200,000个细胞的速度在24孔板中进行基因表达和皮质酮测定。24 h后，分别用10、50、100、200 μM PNU-74654处理细胞。24和48 h后，收集培养基上清液用于皮质酮测量，收集贴壁细胞用于RNA分离。至少进行了两个独立的实验。
将HeLa细胞以每孔50000个细胞的速度在24孔板中进行基因表达和蛋白分析。24 h后，用50、100和200个PNU-74654培养液处理细胞。24和48 h后，收获贴壁细胞，分离RNA和蛋白。至少进行了两个独立的实验。

Female inbred BALB/c mice were purchased from Pasteur Institute, and divided into 4 groups of 3 mice as follows: administrated with PNU-74654, 5-FU, and 5-FU/ PNU-74654 admixture, and untreated (i.e, control group). The mice were housed under standard laboratory conditions. CT-26 cells were injected subcutaneously in the flanks of the mice. The treatments were administrated when the tumor grew up to a volume of 100mm3. Every 2 days, the size of the tumors was measured. [3]

[1]. Inhibition of the Tcf/beta-catenin complex increases apoptosis and impairs adrenocortical tumor cell proliferation and adrenal steroidogenesis. Oncotarget. 2015 Dec 15;6(40):43016-32.

[2]. Inhibition of protein-protein interactions: the discovery of druglike beta-catenin inhibitors by combining virtual and biophysical screening. Proteins. 2006 Jul 1;64(1):60-7.

[3]. Inhibition of the Wnt/b-catenin pathway using PNU-74654 reduces tumor growth in in vitro and in vivo models of colorectal cancer. Tissue Cell . 2022 Aug:77:101853.

PNU-74654 is beta-catenin - TCF interaction inhibitor
Background: To date, there is no effective therapy for patients with advanced/metastatic adrenocortical cancer (ACC). The activation of the Wnt/beta-catenin signaling is frequent in ACC and this pathway is a promising therapeutic target. Aim: To investigate the effects of the inhibition of the Wnt/beta-catenin in ACC cells. Methods: Adrenal (NCI-H295 and Y1) and non-adrenal (HeLa) cell lines were treated with PNU-74654 (5-200 μM) for 24-96 h to assess cell viability (MTS-based assay), apoptosis (Annexin V), expression/localization of beta-catenin (qPCR, immunofluorescence, immunocytochemistry and western blot), expression of beta-catenin target genes (qPCR and western blot), and adrenal steroidogenesis (radioimmunoassay, qPCR and western blot). Results: In NCI-H295 cells, PNU-74654 significantly decreased cell proliferation 96 h after treatment, increased early and late apoptosis, decreased nuclear beta-catenin accumulation, impaired CTNNB1/beta-catenin expression and increased beta-catenin target genes 48 h after treatment. No effects were observed on HeLa cells. In NCI-H295 cells, PNU-74654 decreased cortisol, testosterone and androstenedione secretion 24 and 48 h after treatment. Additionally, in NCI-H295 cells, PNU-74654 decreased SF1 and CYP21A2 mRNA expression as well as the protein levels of STAR and aldosterone synthase 48 h after treatment. In Y1 cells, PNU-74654 impaired corticosterone secretion 24 h after treatment but did not decrease cell viability. Conclusions: Blocking the Tcf/beta-catenin complex inhibits the Wnt/beta-catenin signaling in adrenocortical tumor cells triggering increased apoptosis, decreased cell viability and impairment of adrenal steroidogenesis. These promising findings pave the way for further experiments inhibiting the Wnt/beta-catenin pathway in pre-clinical models of ACC. The inhibition of this pathway may become a promising adjuvant therapy for patients with ACC.[1]

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The interaction between beta-catenin and Tcf family members is crucial for the Wnt signal transduction pathway, which is commonly mutated in cancer. This interaction extends over a very large surface area (4800 A(2)), and inhibiting such interactions using low molecular weight inhibitors is a challenge. However, protein surfaces frequently contain "hot spots," small patches that are the main mediators of binding affinity. By making tight interactions with a hot spot, a small molecule can compete with a protein. The Tcf3/Tcf4-binding surface on beta-catenin contains a well-defined hot spot around residues K435 and R469. A 17,700 compounds subset of the Pharmacia corporate collection was docked to this hot spot with the QXP program; 22 of the best scoring compounds were put into a biophysical (NMR and ITC) screening funnel, where specific binding to beta-catenin, competition with Tcf4 and finally binding constants were determined. This process led to the discovery of three druglike, low molecular weight Tcf4-competitive compounds with the tightest binder having a K(D) of 450 nM. Our approach can be used in several situations (e.g., when selecting compounds from external collections, when no biochemical functional assay is available, or when no HTS is envisioned), and it may be generally applicable to the identification of inhibitors of protein-protein interactions.[2]

C19H16N2O3

320.348

320.116

C, 71.24; H, 5.03; N, 8.74; O, 14.98

113906-27-7

113906-27-7;

9836739

White to off-white solid powder

1.2±0.1 g/cm3

1.593

3.9

63.83

1

4

5

24

434

0

CC1=CC=C(O1)/C=N/NC(=O)C2=CC=CC=C2OC3=CC=CC=C3

JJEDWBQZCRESJL-DEDYPNTBSA-N

InChI=1S/C19H16N2O3/c1-14-11-12-16(23-14)13-20-21-19(22)17-9-5-6-10-18(17)24-15-7-3-2-4-8-15/h2-13H,1H3,(H,21,22)/b20-13+

N-[(E)-(5-methylfuran-2-yl)methylideneamino]-2-phenoxybenzamide

PNU-74654; 113906-27-7; Benzoic acid, 2-phenoxy-, 2-[(5-methyl-2-furanyl)methylene]hydrazide; PNU 74654; CHEMBL254381; N-[(E)-(5-Methylfuran-2-yl)methylideneamino]-2-phenoxybenzamide; PNU74654; (E)-N'-((5-methylfuran-2-yl)methylene)-2-phenoxybenzohydrazide;

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 : ≥ 30 mg/mL (~93.65 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.80 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 (7.80 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！
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。