3PO   中文名称: 3PO

PFKFB3 (IC50 = 155 nM)

3PO不抑制纯化的PFK-1的活性；相反，它主要通过与Fru-6-P竞争来抑制PFKFB3同工酶。与正常人支气管上皮细胞相比，3PO选择性地抑制ras转化的人支气管上皮细胞，从而显着减弱几种人恶性造血细胞和腺癌细胞系的增殖（IC50，1.4-24μmol/L）。 3PO 可导致 G2-M 的相停滞[1]。

当荷瘤小鼠腹腔注射3PO（0.07 mg/g）时，体内Fru-2,6-BP的细胞内浓度、葡萄糖的摄取和已形成肿瘤的生长均显着降低。它抑制白血病、肺腺癌和乳腺癌细胞在体内生长致瘤的能力[1]。对 C57Bl/6 小鼠静脉注射 3PO 后，研究了以下 PK 特性：清除率 CL=2312 mL/min/kg，T_1/2=0.3 hr，C_max=113 ng/ml，AUC_0-inf=36 ng/hr/ml。据报道，3PO 对白血病小鼠模型表现出很强的活性，与高度相关[2]。

PFKFB3酶法测定[1]
PFKFB3蛋白活性通过结合丙酮酸激酶和乳酸脱氢酶的酶偶联动力学测定来测量，如前所述。用于3PO抑制的对照反应包含在不添加PFKFB3的情况下增加量的3PO。SigmaPlot 9.0的酶动力学模块用于计算PFKFB3和3PO抑制的动力学变量（Vmax、Km和Ki）。所代表的数据是两个独立实验的三次测量的平均值±SD<小时> 用于哺乳动物表达的FLAG-PFKFB3构建体的产生[1]
将含有完整PFKFB3编码序列和其NH2末端的FLAG表位的FLAG-PFKFB3亚克隆到逆转录病毒Tet应答载体pRevTRE内的BamHI/HindIII限制性位点中。通过脂质体介导将pRevTRE-FLAG-PFKFB3构建体转染到PT67包装细胞系中来产生重组逆转录病毒。为了创建稳定整合并表达诱导型FLAG-PFKFB3的Jurkat细胞系，用含有FLAG-PF肯德基B3的重组逆转录病毒感染细胞，并在400μg/mL潮霉素存在下选择稳定的克隆。

在补充有 10% 胎牛血清和 50 μg/mL 硫酸庆大霉素的 RPMI 1640 中，Jurkat 细胞以 1 × 10⁵/mL 的密度铺板。用载体或 10 μmol/L 3PO 处理细胞 0、4、8、16、24 或 36 小时。分析细胞周期。

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体外3PO生长抑制[1]
所有细胞系均以1×105/mL的浓度接种在适当的培养基中。对于悬浮细胞，立即向培养基中加入3PO，而对于贴壁细胞系，第二天开始3PO处理。对于剂量依赖性实验，以递增的浓度添加3PO 36小时。对于时间依赖性试验，在时间0、4、8、16、24或36小时添加10μmol/L 3PO。对于PFKFB3过表达研究，在3PO孵育前24小时通过添加强力霉素（1μg/mL）诱导含有FLAG-PFKFK3表达载体或对照质粒的Jurkat细胞。然后在处理48小时后收集细胞，通过台盼蓝排斥法测定细胞数量和存活率。IC50被计算为50%载体处理细胞生长所需的3PO浓度。所表示的数据是三个独立实验的三次测量的平均值±标准差。

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细胞周期分析和流式细胞术[1]
Jurkat细胞以1×105/mL的浓度接种在添加了10%胎牛血清和50μg/mL硫酸庆大霉素的RPMI 1640中。细胞立即用载体或10μmol/L3PO处理0、4、8、16、24或36小时。根据制造商的方案，使用Vybrant DyeCycle Orange染色进行细胞周期分析。

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果糖-2,6-BP和乳酸的测量[1]
Jurkat细胞以1×105/mL的浓度铺板，并立即与10μmol/L3PO一起孵育0、4、8、16、24或36小时。收集培养基样本，根据制造商的说明，使用基于乳酸氧化酶的比色测定法在540 nm处读取乳酸水平，并将其归一化为蛋白质浓度。Fru-2,6-BP测定如前所述进行。

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2-脱氧葡萄糖摄取量[1]
Jurkat细胞以1×105/mL的浓度接种在添加了10%胎牛血清和50μg/mL硫酸庆大霉素的RPMI 1640中。细胞立即用载体或10μmol/L3PO处理指定时间，随后置于无葡萄糖RPMI 1640中30分钟。再加入14C-2-脱氧葡萄糖（0.25μCi/mL）60分钟，然后用不含葡萄糖的冰冷RPMI 1640洗涤三个细胞。在500μL 0.1%SDS中收集细胞裂解物，并在400μL裂解物上测量闪烁计数（计数/分钟）。计数标准化为蛋白质浓度，数据表示为两个独立实验的重复测量的平均值±标准差。

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全细胞ATP、NAD+和NADH的测定[1]
Jurkat细胞以1×105/mL的浓度铺板，并立即与10μmol/L的3PO一起孵育指定的时间。根据制造商的方案，使用ATP测定试剂盒测定ATP水平，并在所有时间点使用EnzyChrom NAD+/NDH测定试剂盒在1×106个细胞上测量载体和3PO处理样品的NAD+和NADH水平。

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核磁共振代谢物提取[1]
在13C-葡萄糖存在下，用载体或10μmol/L3PO处理Jurkat细胞36小时。对细胞进行计数，将等量的细胞制成丸粒，用冷PBS洗涤两次以去除粘附的培养基，并在液氮中快速冷冻。用10%冰冷的三氯乙酸提取冷颗粒（两次），然后冻干。将干提取物重新溶解在0.35 mL D2O中，并装入5 mm Shigemi管中。

tumor bearing mice (BALB/c nude mice or C57Bl/6 female mice background)
0.07 mg/g
i.p.

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In vivo Studies [1]
Exponentially growing MDA-MB231 and HL-60 cells were collected, washed, and resuspended in PBS at 20 × 107/mL. Cells were then mixed 1:1 with Matrigel, and 0.1 mL of the cell suspension was injected s.c. (1 × 107 cells) into female BALB/c nude mice (20 g). Exponentially growing Lewis lung carcinoma cells were collected, washed twice, and resuspended in PBS (1 × 107/mL). C57Bl/6 female mice (20 g) were injected s.c. with 0.1 mL of the suspension. Body weight and tumor growth were monitored daily throughout the study. Tumor masses were determined by measurement with Vernier calipers using the formula: mass (mg) = [width2 (mm) × length (mm)] / 2. Mice with established tumors (between 130 and 190 mg) were randomized into vehicle control or 3PO-treated groups. Vehicle control groups received i.p. injections of 50 μL DMSO, whereas treated groups received 0.07 mg/g 3PO in 50 μL DMSO at the indicated time points. All tumor experiments were conducted three times and the data presented are from one experiment.

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In vivo Fru-2,6-BP Measurement[1]
C57Bl/6 female mice (20 g) were injected s.c. with 1 × 106 Lewis lung carcinoma cells. When xenografts were measured to have a mass of 150 to 180 mg, mice were randomized and given i.p. injections of vehicle DMSO or 0.07 mg/g 3PO. Four hours after injection, tumors were removed and homogenized in 1 volume of 0.05 mol/L NaOH and subsequently mixed with 1 volume of 0.1 mol/L NaOH. Fru-2,6-BP assays were done as described previously.

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Micro-Positron Emission Tomography[1]
Lewis lung carcinoma xenograft-bearing mice were given i.p. injections of 50 μL DMSO or 0.07 mg/g 3PO in DMSO. After 30 min, mice were injected i.p. with 18F-2-DG (150 μCi, 100 μL in H2O) and subsequently anesthetized after 15 min with 2% isoflurane in oxygen. The mice were then transferred to a R-4 Rodent Scanner micro-positron emission tomography (CTI Concorde Microsystems; n = 3).

[1]. Mol Cancer Ther . 2008 Jan;7(1):110-20.

[2]. Mol Cancer Ther . 2013 Aug;12(8):1461-70.

3PO is a member of the class of pyridines that is pyridine substituted by a 3-oxo-3-(pyridin-4-yl)prop-1-en-1-yl group at position 3. An inhibitor of PFKFB3 kinase, an enzyme with a key role in glycolysis. It has a role as an antineoplastic agent, an angiogenesis inhibitor, an autophagy inducer, an apoptosis inducer and an EC 2.7.1.105 (6-phosphofructo-2-kinase) inhibitor. It is a member of pyridines and an enone.

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6-phosphofructo-1-kinase, a rate-limiting enzyme of glycolysis, is activated in neoplastic cells by fructose-2,6-bisphosphate (Fru-2,6-BP), a product of four 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isozymes (PFKFB1-4). The inducible PFKFB3 isozyme is constitutively expressed by neoplastic cells and required for the high glycolytic rate and anchorage-independent growth of ras-transformed cells. We report herein the computational identification of a small-molecule inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), which suppresses glycolytic flux and is cytostatic to neoplastic cells. 3PO inhibits recombinant PFKFB3 activity, suppresses glucose uptake, and decreases the intracellular concentration of Fru-2,6-BP, lactate, ATP, NAD+, and NADH. 3PO markedly attenuates the proliferation of several human malignant hematopoietic and adenocarcinoma cell lines (IC50, 1.4-24 micromol/L) and is selectively cytostatic to ras-transformed human bronchial epithelial cells relative to normal human bronchial epithelial cells. The PFKFB3 enzyme is an essential molecular target of 3PO because transformed cells are rendered resistant to 3PO by ectopic expression of PFKFB3 and sensitive to 3PO by heterozygotic genomic deletion of PFKFB3. Importantly, i.p. administration of 3PO (0.07 mg/g) to tumor-bearing mice markedly reduces the intracellular concentration of Fru-2,6-BP, glucose uptake, and growth of established tumors in vivo. Taken together, these data support the clinical development of 3PO and other PFKFB3 inhibitors as chemotherapeutic agents.[1]

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In human cancers, loss of PTEN, stabilization of hypoxia inducible factor-1α, and activation of Ras and AKT converge to increase the activity of a key regulator of glycolysis, 6-phosphofructo-2-kinase (PFKFB3). This enzyme synthesizes fructose 2,6-bisphosphate (F26BP), which is an activator of 6-phosphofructo-1-kinase, a key step of glycolysis. Previously, a weak competitive inhibitor of PFKFB3, 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), was found to reduce the glucose metabolism and proliferation of cancer cells. We have synthesized 73 derivatives of 3PO and screened each compound for activity against recombinant PFKFB3. One small molecule, 1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one (PFK15), was selected for further preclinical evaluation of its pharmacokinetic, antimetabolic, and antineoplastic properties in vitro and in vivo. We found that PFK15 causes a rapid induction of apoptosis in transformed cells, has adequate pharmacokinetic properties, suppresses the glucose uptake and growth of Lewis lung carcinomas in syngeneic mice, and yields antitumor effects in three human xenograft models of cancer in athymic mice that are comparable to U.S. Food and Drug Administration-approved chemotherapeutic agents. As a result of this study, a synthetic derivative and formulation of PFK15 has undergone investigational new drug (IND)-enabling toxicology and safety studies. A phase I clinical trial of its efficacy in advanced cancer patients will initiate in 2013 and we anticipate that this new class of antimetabolic agents will yield acceptable therapeutic indices and prove to be synergistic with agents that disrupt neoplastic signaling.[2]

C13H10N2O

210.2313

210.079

C, 74.27; H, 4.79; N, 13.33; O, 7.61

18550-98-6

18550-98-6; 13309-08-5

5720233

Light yellow to yellow solid powder

1.2±0.1 g/cm3

387.8±42.0 °C at 760 mmHg

191.3±34.3 °C

0.0±0.9 mmHg at 25°C

1.637

1.51

42.8Ų

0

3

3

16

257

0

O=C(/C(/[H])=C(\[H])/C1=C([H])N=C([H])C([H])=C1[H])C1C([H])=C([H])N=C([H])C=1[H]

UOWGYMNWMDNSTL-ONEGZZNKSA-N

InChI=1S/C13H10N2O/c16-13(12-5-8-14-9-6-12)4-3-11-2-1-7-15-10-11/h1-10H/b4-3+

(E)-3-pyridin-3-yl-1-pyridin-4-ylprop-2-en-1-one

3PO; 3-PO; 18550-98-6; 13309-08-5; (E)-3PO; 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one; (E)-3-pyridin-3-yl-1-pyridin-4-ylprop-2-en-1-one; 2-Propen-1-one, 3-(3-pyridinyl)-1-(4-pyridinyl)-; CHEMBL3105848;

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: 4~60 mg/mL (199.8~285.4 mM)
Ethanol: ~11 mg/mL (52.3 mM)

配方 1 中的溶解度: ≥ 3 mg/mL (14.27 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 30.0 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 3 mg/mL (14.27 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 30.0 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: ≥ 3 mg/mL (14.27 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 30.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 2% DMSO+40% PEG 300+2% Tween 80+ddH2O: 2mg/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；
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。