Use of Protein Kinase-Focused Compound Libraries for the Discovery of New Inositol Phosphate Kinase Inhibitors
Use of Protein Kinase-Focused Compound Libraries for the Discovery of New Inositol Phosphate Kinase Inhibitors
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DOI：
10.1177/2472555218775323
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发表时间：
2018-10-01
期刊：
影响因子：
3.1
通讯作者：
Pearce, Kenneth H.
中科院分区：
文献类型：
--
作者：
Puhl-Rubio, Ana C.;Stashko, Michael A.;Pearce, Kenneth H.
肌醇六磷酸激酶（IP 6 Ks）不仅通过其催化活性（合成InsP（7），一种多功能肌醇焦磷酸信号分子），而且通过蛋白质-蛋白质相互作用调节无数细胞过程。为了进一步研究酶的功能并区分这些不同的机制，需要靶向IP 6 K催化活性的特异性抑制剂。只有一种IP 6 K抑制剂是常用的：N2-（m-（三氟甲基）苄基）N6-（对硝基苄基）嘌呤（TNP）。然而，TNP受到弱效力、不能区分IP 6 K同工酶、脱靶活性和差的药代动力学性质的影响。在此，我们描述了一种新的抑制剂发现策略，基于IP 6 Ks和蛋白激酶的核苷酸结合位点的高度结构保守性;我们使用一组具有已知或计算预测的特征的化合物筛选新型IP 6 K2抑制剂，以靶向蛋白激酶的核苷酸结合。我们开发了一种时间分辨荧光共振能量转移（TR-FRET）分析从三磷酸腺苷（ATP）形成二磷酸腺苷（ADP）。新的命中IP 6 K2的化合物进行了鉴定和验证的剂量-反应曲线和正交试验。这些抑制剂都不影响另一种肌醇焦磷酸激酶PPIP 5 K。我们的筛选策略为未来的开发和优化提供了多种IP 6 K2抑制剂。这种方法将适用于其他肌醇磷酸激酶的抑制剂发现活动。
Inositol hexakisphosphate kinases (IP6Ks) regulate a myriad of cellular processes, not only through their catalytic activity (which synthesizes InsP(7), a multifunctional inositol pyrophosphate signaling molecule) but also through protein-protein interactions. To further study the enzymatic function and distinguish between these different mechanisms, specific inhibitors that target IP6K catalytic activity are required. Only one IP6K inhibitor is commonly used: N2-(m-(trifluoromethyl)benzyl) N6-(p-nitrobenzyl)purine (TNP). TNP is, however, compromised by weak potency, inability to distinguish between IP6K isoenzymes, off-target activities, and poor pharmacokinetic properties. Herein, we describe a new inhibitor discovery strategy, based on the high degree of structural conservation of the nucleotide-binding sites of IP6Ks and protein kinases; we screened for novel IP6K2 inhibitors using a focused set of compounds with features known, or computationally predicted, to target nucleotide binding by protein kinases. We developed a time-resolved fluorescence resonance energy transfer (TR-FRET) assay of adenosine diphosphate (ADP) formation from adenosine triphosphate (ATP). Novel hit compounds for IP6K2 were identified and validated with dose-response curves and an orthogonal assay. None of these inhibitors affected another inositol pyrophosphate kinase, PPIP5K. Our screening strategy offers multiple IP6K2 inhibitors for future development and optimization. This approach will be applicable to inhibitor discovery campaigns for other inositol phosphate kinases.