PKD3 (IC50 = 109.4 nM); PKD2 (IC50 = 133.4 nM); PKD1 (IC50 = 154.6 nM); v-Fyn (IC50 = 0.6 μM); c-Abl (IC50 = 0.6 μM)

1-NA-PP1和IKK-16是新型泛pkd抑制剂。[1]
1-NA-PP1是一种atp竞争性抑制剂，对密切相关的激酶具有高选择性。[1]
1-NA-PP1在前列腺癌细胞中具有细胞活性并引起靶抑制。[1]
1-NA-PP1通过诱导G2/M阻滞有效阻断前列腺癌细胞增殖。[1]
1- na - pp1诱导的生长停滞是通过靶向抑制PKD介导的。[1]
1-NA-PP1能有效抑制前列腺肿瘤细胞的迁移和侵袭。[1]
在完整细胞中，PKD1的守门人突变体对1-NA-PP1抑制的敏感性提高了12倍。[1]

系统给药1-NA-PP1容易穿过血脑屏障，抑制pkcε介导的磷酸化。1-NA-PP1可逆地降低了AS-PKCε小鼠的乙醇消耗量，但对缺乏AS-PKCε突变的野生型小鼠没有作用。这些结果支持了PKCε催化活性抑制剂作为减少乙醇消耗的策略的开发，并且它们表明as - PKCε小鼠是研究PKCε在行为中的作用的有用工具。[3]

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我们培育了一种新的AS-PKCε小鼠系，其ATP结合位点发生点突变，使其对纳米摩尔浓度的PP1类似物1-NA-PP1的抑制高度敏感。系统给药的1-NA-PP1穿过血脑屏障，在脑内达到足够高的浓度来抑制AS-PKCε。1-NA-PP1延长了乙醇的共济失调和催眠作用，减少了AS-PKCε小鼠的乙醇消耗。在缺乏AS-PKCε突变的野生型小鼠中未观察到1-NA-PP1的这些作用。这些结果表明，抑制PKCε催化活性的化合物可能有助于减少乙醇的消耗。 [3]

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1-NA-PP1降低AS-PKCε小鼠的乙醇消耗[3]
为了确定1-NA-PP1是否会改变乙醇的消耗，我们对AS-PKCε小鼠进行了连续的两瓶选择饮用程序，其中乙醇浓度从3%上升到6%，最后在8天内上升到10%。小鼠习惯了载体注射并连续三次饮用10%乙醇达到稳定水平后[F(2,34) = 1.474, P = 0.2433；图4A]，它们使用受试者内设计给药1-NA-PP1，其中所有动物在不同日期接受载体或1-NA-PP1。1-NA-PP1浓度为20或30mg/kg时，前24 h乙醇消耗量降低[F(2,34) = 10.69；P = 0.0003；图4 b)。这种效应是可逆的，因为乙醇消耗量在用载体或1-NA-PP1处理48小时后相似[F(2,34) = 3.058；P = 0.0601；图4 c)。1-NA-PP1未显著改变乙醇偏好[F(2,34) = 0.9508；P = 0.3965；图4 d)。虽然在30mg/kg时有减少水消耗的趋势，但这种影响在统计学上并不显著[F(2,34) = 1.722；P = 0.1940；图4 e]。 [3]

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1-NA-PP1延长AS-PKCε小鼠乙醇中毒[3]
我们之前发现Prkce - / -小鼠由于对乙醇的急性功能耐受性受损而表现出长期的乙醇中毒迹象（Hodge等人，1999年，Wallace等人，2007年）。因此，为了确定抑制PKCε是否会改变乙醇中毒，并测试口服1-NA-PP1是否有效产生表型，我们给AS-PKCε小鼠喂食1-NA-PP1或对照食物和水11天。平均而言，1-NA-PP1组小鼠每天消耗3.00±0.14g 1-NA-PP1食物颗粒，低于对照组(3.65±0.16g/d；P = 0.02)。1-NA-PP1组小鼠的饮水量（2.00±0.01ml）也少于对照组（3.5±0.25ml）；P < 0.0001)。然而，尽管在食物和水的摄取量上存在差异，但1- na - pp1喂养的动物（25.5±0.18g）和对照喂养的动物（25.8±0.23g）的体重相似。

体外放射学PKD1筛选试验[1]
采用体外放射激酶法筛选80个化合物库，在1µM浓度下检测PKD1抑制活性。用1.2µM的HDAC5肽作为底物进行反应。在含有50µL含有50 mM Tris-HCl、pH 7.5、4 mM MgCl2和10 mM β-巯基乙醇的激酶缓冲液中，用1µCi [γ-32P] ATP、25µM ATP、50 ng纯化的重组PKD1进行激酶反应，检测HDAC5的磷酸化。反应在30℃下孵育10分钟，取25µL的反应液滴在Whatman P81滤纸上。滤纸在0.5%磷酸中洗涤3次，风干后用Beckman LS6500多功能闪烁计数器计数。使用GraphPad Prism软件5.0绘制PKD1抑制百分比图。

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体外放射PKC和CAMKIIα激酶测定[1]
PKC激酶检测是通过将1µCi [γ-32P]ATP、20µM ATP、50 ng纯化PKCα或PKCδ和5µg髓鞘碱性蛋白4 - 14、0.25 mg/mL牛血清白蛋白、0.1 mg/mL磷脂酰胆碱/磷脂酰丝氨酸（80/20%）（1µM）、1µM二丁酸磷在50µL含有50 mM Tris-HCl、pH 7.5、4 mM MgCl2和10 mM β-巯基乙醇的激酶缓冲液中共孵育进行的。CAMK实验中，50 ng CAMKIIα和2µg syntide-2底物在50µL激酶缓冲液中与0.1 mM MgCl2、1µCi [γ-32P] ATP、70µM ATP孵育。0.5 mM CaCl2和30 ng/µL钙调素在冰上预孵育15 min后加入激酶反应。反应在30℃下孵育10 min，取25µL的反应液滴在Whatman P81滤纸上。滤纸在0.5%磷酸中洗涤3次，风干后用Beckman LS6500多功能闪烁计数器计数。

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体外激酶测定[2]
我们在低ATP浓度（10 nM）的0.2µCiµl-1 [γ-32P]ATP存在下进行了体外激酶实验（Cdc28除外），因此IC50值代表了抑制常数（Ki）的粗略测量。抑制剂IC50值的测定方法如上所述。
纯化的Cdc28-His6 （1 nM）和MBP-Clb2 （3 nM）在25µl反应混合物中23°C孵育10分钟，反应混合物中含有5µg组蛋白H1, 1µCi的[γ-32P]ATP(1µCi / 10µM和1µCi / 1 mM)，以及不同浓度的化合物9激酶缓冲液（25 mM hepe - naoh pH 7.4, 10 mM NaCl， 10 mM MgCl2和1 mM二硫代索糖醇）。反应产物用15% SDS-PAGE进行分析，然后进行放射自显影。为了测定Cdc28的动力学常数，不同浓度的[γ-32P]ATP（1µCi / 100µM）孵育和分析如上所述。

MTT试验[1]
PC3细胞接种于96孔板（3000个细胞/孔），贴壁过夜。然后将细胞在含有0.7-100µM抑制剂的培养基中孵育72 h，以2mg /mL浓度在PBS中制备3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化甲基噻唑基四唑（MTT）溶液，通过0.2µM过滤器过滤灭菌，并用铝箔包裹以遮光。每孔加入50µL MTT溶液，37℃孵育4 h。然后去除培养基，每孔加入200µL DMSO。振荡混合5 min，在570 nm处测定光密度。

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细胞增殖试验和细胞周期分析[1]
通过台盼蓝染色计数活细胞数（如前所述）来测量PC3细胞的增殖。按照描述进行细胞周期分析。简单地说，用指定的化合物在30µM下处理PC3细胞72 h，然后在70%的冷冻乙醇中固定过夜，然后用碘化丙啶标记。标记的细胞使用FACSCalibur流式细胞仪 进行分析。

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创面愈合试验[1]
PC3或DU145细胞在6孔板中培养融合。迁移是通过用移液管尖端刮擦单层开始的，形成一个“伤口”。在培养基中加入指定浓度的化合物，并立即在10倍物镜的倒置相差显微镜下对伤口进行成像。24小时后，拍摄最终图像。测量创面间隙，计算创面愈合率。平均伤口愈合百分比是根据至少6次伤口间隙测量来确定的。

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基质侵袭试验[1]
将DU145细胞（4.0×104 cells/ml）在含有0.1%胎牛血清（FBS）的RPMI中接种到BioCoat对照植入物（孔径8µm）或BioCoat Matrigel侵袭植入物（带有Matrigel涂层过滤器）的顶室中。为了刺激侵入，植入物下腔的介质中含有20%的FBS。上、下腔均加入浓度为30µM的抑制剂，细胞孵育22 h。孵育后，用棉签去除无创细胞，将有创细胞用100%甲醇固定，并用0.4%苏木精染色。染色后，在200倍放大镜下计数。侵染率由侵染基质的细胞数相对于通过对照插入的细胞数在5个区域内的细胞计数来确定。

Administration of 1-NA-PP1 [3]
For ethanol, saccharin, and quinine consumption studies, we dissolved 1-NA-PP1 in 100% DMSO at 20 or 30mg/ml and then diluted it 20-fold in deionized water containing 10% Tween-80 with sonication. For studies using oral administration, we prepared 1-NA-PP1 as a 100mM stock solution in 100% DMSO by gentle heating and sonication. This stock was diluted to 500µM in water containing 1% cremophor-RH40 and 2g/L sucralose to increase palatability. Control animals received an equivalent amount of DMSO vehicle in cremophor-sucralose-water. 1-NA-PP1 food pellets (1g/kg) were obtained from Research Diets (New Brunswick, NJ). Control food pellets contained an equivalent amount of vehicle (DMSO). To determine the effects of 1-NA-PP1 on protein phosphorylation, we dissolved 1-NA-PP1 in vehicle containing 5% DMSO and 20% Cremophor EL

To determine the abundance and half-life of 1-NA-PP1 in plasma and brain, a pharmacokinetic study was performed following intraperitoneal administration of 30mg/kg 1-NA-PP1 in 5% DMSO and 10% Tween-80 to wild type C57BL/6J mice (Fig. 2A). Plasma levels of 1NA-PP1 reached 7.3 ± 0.43µM thirty minutes after injection and declined biphasically (R2= 0.94) with half-lives of 0.47 and 11.62 hours (Fig. 2A). Brain levels reached 2167 ± 85 ng/g (~6.8 ± 0.27µM) one hour after injection and declined in a single-phase (R2 = 0.93) with a half-life of 0.57 hours (Fig. 2B). These results indicate that 1-NA-PP1 enters the brain rapidly and efficiently after intraperitoneal administration and achieves concentrations predicted to inhibit AS-PKCε (Ki = 18.7nM) based on in vitro studies (Qi et al., 2007). [3]

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Plasma and brain concentrations of 1-NA-PP1 were also determined following repeated oral administration. Wild type C57BL/6N mice were provided food pellets containing 1g/kg 1-NA-PP1 and water containing 500µM 1-NA-PP1 in 1% Cremophor-RH40 and 0.2% sucralose. Control animals were fed food and water containing the corresponding vehicles. Mice were sacrificed after 3 days and the concentration of 1-NA-PP1 was determined by LC-MS/MS. Oral administration of 1-NA-PP1 yielded a plasma concentration of 117 ± 23nM (n=5) and brain concentration of 140 ± 54ng/g protein (~ 441 ± 172nM; n=5). These results indicate that repeated administration of 1-NA-PP1 in food and water leads to levels of 1-NA-PP1 in the brain and plasma predicted to inhibit AS-PKCε (Qi et al., 2007). [3]

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To determine whether systemic administration of 1-NA-PP1 inhibits AS-PKCε-mediated phosphorylation in the brain, we examined phosphorylation of the GABAA receptor γ2 subunit since we previously found that PKCε phosphorylates this subunit at S327 (Qi et al., 2007). We administered 1-NA-PP1 by intraperitoneal injection rather than orally in this experiment to better control the dosage relative to the timing of tissue collection. AS-PKCε mice were administered 25mg/kg 1-NA-PP1 or vehicle and sacrificed 1 hour later. Although we used a different vehicle (5%DMSO/20% Cremophor-EL) to dissolve 1-NA-PP1 for this experiment, pharmacokinetic analyses after intraperitoneal injection of 30mg/kg 1-NA-PP1 in this vehicle revealed plasma (6.47 ± 0.25µM; n = 2) and brain concentrations (2055 ± 455ng/g; ~4.43 ± 2.03µM; n = 2) similar to those observed for 1-NA-PP1 dissolved in 5%DMSO/10% Tween-80. Compared with vehicle-injected mice, there was a 33% reduction in γ2-S(P)327 phosphoimmunoreactivity in the striatum of 1-NA-PP1-treated mice (Fig. 3). [3]

[1]. A chemical switch for inhibitor-sensitive alleles of any protein kinase. Nature. 2000 Sep 21;407(6802):395-401.

[2]. New pyrazolopyrimidine inhibitors of protein kinase d as potent anticancer agents for prostate cancer cells. PLoS One. 2013 Sep 23;8(9):e75601.

[3]. Selective chemical genetic inhibition of protein kinase C epsilon reduces ethanol consumption in mice. Neuropharmacology. 2016 Aug;107:40-48.

1-NA-PP1 is a pyrazolopyrimidine. It has a role as a tyrosine kinase inhibitor.

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The emergence of protein kinase D (PKD) as a potential therapeutic target for several diseases including cancer has triggered the search for potent, selective, and cell-permeable small molecule inhibitors. In this study, we describe the identification, in vitro characterization, structure-activity analysis, and biological evaluation of a novel PKD inhibitory scaffold exemplified by 1-naphthyl PP1 (1-NA-PP1). 1-NA-PP1 and IKK-16 were identified as pan-PKD inhibitors in a small-scale targeted kinase inhibitor library assay. Both screening hits inhibited PKD isoforms at about 100 nM and were ATP-competitive inhibitors. Analysis of several related kinases indicated that 1-NA-PP1 was highly selective for PKD as compared to IKK-16. SAR analysis showed that 1-NA-PP1 was considerably more potent and showed distinct substituent effects at the pyrazolopyrimidine core. 1-NA-PP1 was cell-active, and potently blocked prostate cancer cell proliferation by inducing G2/M arrest. It also potently blocked the migration and invasion of prostate cancer cells, demonstrating promising anticancer activities on multiple fronts. Overexpression of PKD1 or PKD3 almost completely reversed the growth arrest and the inhibition of tumor cell invasion caused by 1-NA-PP1, indicating that its anti-proliferative and anti-invasive activities were mediated through the inhibition of PKD. Interestingly, a 12-fold increase in sensitivity to 1-NA-PP1 could be achieved by engineering a gatekeeper mutation in the active site of PKD1, suggesting that 1-NA-PP1 could be paired with the analog-sensitive PKD1(M659G) for dissecting PKD-specific functions and signaling pathways in various biological systems.[1]

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Protein kinases have proved to be largely resistant to the design of highly specific inhibitors, even with the aid of combinatorial chemistry. The lack of these reagents has complicated efforts to assign specific signalling roles to individual kinases. Here we describe a chemical genetic strategy for sensitizing protein kinases to cell-permeable molecules that do not inhibit wild-type kinases. From two inhibitor scaffolds, we have identified potent and selective inhibitors for sensitized kinases from five distinct subfamilies. Tyrosine and serine/threonine kinases are equally amenable to this approach. We have analysed a budding yeast strain carrying an inhibitor-sensitive form of the cyclin-dependent kinase Cdc28 (CDK1) in place of the wild-type protein. Specific inhibition of Cdc28 in vivo caused a pre-mitotic cell-cycle arrest that is distinct from the G1 arrest typically observed in temperature-sensitive cdc28 mutants. The mutation that confers inhibitor-sensitivity is easily identifiable from primary sequence alignments. Thus, this approach can be used to systematically generate conditional alleles of protein kinases, allowing for rapid functional characterization of members of this important gene family.[2]

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Reducing expression or inhibiting translocation of protein kinase C epsilon (PKCε) prolongs ethanol intoxication and decreases ethanol consumption in mice. However, we do not know if this phenotype is due to reduced PKCε kinase activity or to impairment of kinase-independent functions. In this study, we used a chemical-genetic strategy to determine whether a potent and highly selective inhibitor of PKCε catalytic activity reduces ethanol consumption. We generated ATP analog-specific PKCε (AS-PKCε) knock-in mice harboring a point mutation in the ATP binding site of PKCε that renders the mutant kinase highly sensitive to inhibition by 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine (1-NA-PP1). Systemically administered 1-NA-PP1 readily crossed the blood brain barrier and inhibited PKCε-mediated phosphorylation. 1-NA-PP1 reversibly reduced ethanol consumption by AS-PKCε mice but not by wild type mice lacking the AS-PKCε mutation. These results support the development of inhibitors of PKCε catalytic activity as a strategy to reduce ethanol consumption, and they demonstrate that the AS- PKCε mouse is a useful tool to study the role of PKCε in behavior.[3]

C19H19N5

317.3877

317.164

221243-82-9

1-Naphthyl PP1 hydrochloride;956025-47-1

4877

Light yellow to khaki solid

1.3±0.1 g/cm3

527.8±45.0 °C at 760 mmHg

219-222ºC

273.0±28.7 °C

0.0±1.4 mmHg at 25°C

1.688

3.88

69.62

1

4

2

24

448

0

N1(C2C(=C(N([H])[H])N=C([H])N=2)C(C2=C([H])C([H])=C([H])C3=C([H])C([H])=C([H])C([H])=C23)=N1)C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H]

XSHQBIXMLULFEV-UHFFFAOYSA-N

InChI=1S/C19H19N5/c1-19(2,3)24-18-15(17(20)21-11-22-18)16(23-24)14-10-6-8-12-7-4-5-9-13(12)14/h4-11H,1-3H3,(H2,20,21,22)

1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine

1-Naphthyl PP1; 221243-82-9; 1-NAPHTHYL PP1; 1-NA-PP1; 1-(tert-Butyl)-3-(naphthalen-1-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; 4-Amino-1-tert-butyl-3-(1'-naphthyl)pyrazolo[3,4-d]pyrimidine; 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine; 1-(1,1-dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine; C19H19N5; 1-NA-PP 1

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: 9~12.5 mg/mL (28.4~39.4 mM)

配方 1 中的溶解度: ≥ 1.25 mg/mL (3.94 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 12.5 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: ≥ 1.25 mg/mL (3.94 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 12.5 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；
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