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Development of an in vitro screening assay for PIP5K1α lipid kinase and identification of potent inhibitors.
The FEBS Journal ( IF 5.5 ) Pub Date : 2019-12-26 , DOI: 10.1111/febs.15194 Katja Strätker 1 , Samer Haidar 1, 2 , Ángel Amesty 3 , Ehab El-Awaad 1, 4 , Claudia Götz 5 , Ana Estévez-Braun 3 , Joachim Jose 1
The FEBS Journal ( IF 5.5 ) Pub Date : 2019-12-26 , DOI: 10.1111/febs.15194 Katja Strätker 1 , Samer Haidar 1, 2 , Ángel Amesty 3 , Ehab El-Awaad 1, 4 , Claudia Götz 5 , Ana Estévez-Braun 3 , Joachim Jose 1
Affiliation
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The human phosphatidylinositol 4‐phosphate 5‐kinase type I α (hPIP5K1α) participates in the phosphoinositide‐3‐kinase/protein kinase B/mammalian target of rapamycin signaling pathway. Despite the evidence that hPIP5K1α plays a role in the development of prostate cancer (PCa), only one inhibitor is known to date. With the aim of identifying new inhibitors, a nonradiometric assay for measurement of the hPIP5K1α enzyme activity was developed. The assay is based on the separation of the fluorescently labeled substrate phosphatidylinositol‐4‐phosphate (PI(4)P) and the resulting product phosphatidylinositol‐4,5‐bisphosphate (PIP2) by capillary electrophoresis (CE). Furthermore, an inactive mutant K261A of hPIP5K1α was generated by site‐directed mutagenesis and used as a control. Michaelis–Menten analysis revealed a K m value of 21.6 µm and V max of 0.65 pmol·min−1 for the cosubstrate ATP. The average Z ’ value was determined to be 0.86, indicating a high reliability of the assay. An in silico screening of an in‐house compound library was performed employing the crystal structure of zebrafish PIP5K1α. By applying this strategy, three compounds with a 2‐amino‐3‐cyano‐4H‐pyranobenzoquinone scaffold were identified and tested using the CE‐based assay. These compounds inhibited hPIP5K1α to > 90% at a concentration of 50 µm . Subsequently, the inhibitory activity of all compounds with a pyranobenzoquinone scaffold (29) was tested on hPIP5K1α. Compound 4‐(2‐amino‐3‐cyano‐6‐hydroxy‐5,8‐dioxo‐7‐undecyl‐5,8‐dihydro‐4H‐chromen‐4‐yl)benzoic acid appeared to be the most potent inhibitor of hPIP5K1α identified so far with an IC50 value of 1.55 µm , exhibiting a substrate‐competitive mode of action. The effects of this compound on cell viability and the induction of apoptosis were investigated in LNCaP, DU145, and PC3 PCa cells.
中文翻译：
PIP5K1α脂质激酶体外筛选测定方法的开发和有效抑制剂的鉴定。
I型人磷脂酰肌醇4磷酸5激酶（hPIP5K1α）参与了磷酸肌醇3激酶/蛋白激酶B /雷帕霉素信号通路的哺乳动物靶标。尽管有证据表明hPIP5K1α在前列腺癌（PCa）的发生中起作用，但迄今为止仅已知一种抑制剂。为了鉴定新的抑制剂，开发了用于测量hPIP5K1α酶活性的非放射线测定法。该测定基于通过毛细管电泳（CE）分离荧光标记的底物磷脂酰肌醇-4-磷酸（PI（4）P）和所得产物磷脂酰肌醇-4,5-二磷酸（PIP 2）。此外，通过定点诱变产生了hPIP5K1α的无活性突变体K261A，并将其用作对照。Michaelis–Menten分析显示共底物ATP的K m值为21.6 µm，V max为0.65 pmol·min -1。确定的平均Z '值为0.86，表明该测定法的高度可靠性。一个在硅片筛选一个内部化合物文库进行采用斑马鱼PIP5K1α的晶体结构。通过采用该策略，鉴定了三种具有2-氨基-3-氰基-4 H-吡喃苯并醌支架的化合物，并使用基于CE的检测方法进行了测试。这些化合物抑制hPIP5K1α至> 90％的在50μ的浓度米。随后，在hPIP5K1α上测试了所有具有吡喃苯并醌支架（29）的化合物的抑制活性。化合物4-（2-氨基-3-氰基-6-羟基-5,8-二氧代-7-十一烷基-5,8-二氢-4- ħ -苯并吡喃-4-基）苯甲酸似乎是最有效的抑制剂到目前为止，鉴定出的hPIP5K1α的IC 50值为1.55 µm，表现出底物竞争性作用模式。在LNCaP，DU145和PC3 PCa细胞中研究了该化合物对细胞活力和凋亡诱导的影响。
更新日期：2019-12-26
中文翻译：
PIP5K1α脂质激酶体外筛选测定方法的开发和有效抑制剂的鉴定。
I型人磷脂酰肌醇4磷酸5激酶（hPIP5K1α）参与了磷酸肌醇3激酶/蛋白激酶B /雷帕霉素信号通路的哺乳动物靶标。尽管有证据表明hPIP5K1α在前列腺癌（PCa）的发生中起作用，但迄今为止仅已知一种抑制剂。为了鉴定新的抑制剂，开发了用于测量hPIP5K1α酶活性的非放射线测定法。该测定基于通过毛细管电泳（CE）分离荧光标记的底物磷脂酰肌醇-4-磷酸（PI（4）P）和所得产物磷脂酰肌醇-4,5-二磷酸（PIP 2）。此外，通过定点诱变产生了hPIP5K1α的无活性突变体K261A，并将其用作对照。Michaelis–Menten分析显示共底物ATP的K m值为21.6 µm，V max为0.65 pmol·min -1。确定的平均Z '值为0.86，表明该测定法的高度可靠性。一个在硅片筛选一个内部化合物文库进行采用斑马鱼PIP5K1α的晶体结构。通过采用该策略，鉴定了三种具有2-氨基-3-氰基-4 H-吡喃苯并醌支架的化合物，并使用基于CE的检测方法进行了测试。这些化合物抑制hPIP5K1α至> 90％的在50μ的浓度米。随后，在hPIP5K1α上测试了所有具有吡喃苯并醌支架（29）的化合物的抑制活性。化合物4-（2-氨基-3-氰基-6-羟基-5,8-二氧代-7-十一烷基-5,8-二氢-4- ħ -苯并吡喃-4-基）苯甲酸似乎是最有效的抑制剂到目前为止，鉴定出的hPIP5K1α的IC 50值为1.55 µm，表现出底物竞争性作用模式。在LNCaP，DU145和PC3 PCa细胞中研究了该化合物对细胞活力和凋亡诱导的影响。
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