Table3_Novel Mechanism for an Old Drug: Phenazopyridine is a Kinase Inhibitor Affecting Autophagy and Cellular Differentiation.XLSX

Phenazopyridine is a widely used drug against urinary tract pain. The compound has also been shown to enhance neural differentiation of pluripotent stem cells. However, its mechanism of action is not understood. Based on its chemical structure, we hypothesized that phenazopyridine could be a kinase inhibitor. Phenazopyridine was investigated in the following experimental systems: 1) activity of kinases in pluripotent stem cells; 2) binding to recombinant kinases, and 3) functional impact on pluripotent stem cells. Upon addition to pluripotent stem cells, phenazopyridine induced changes in kinase activities, particularly involving Mitogen-Activated Protein Kinases, Cyclin-Dependent Kinases, and AKT pathway kinases. To identify the primary targets of phenazopyridine, we screened its interactions with 401 human kinases. Dose-inhibition curves showed that three of these kinases interacted with phenazopyridine with sub-micromolar binding affinities: cyclin-G-associated kinase, and the two phosphatidylinositol kinases PI4KB and PIP4K2C, the latter being known for participating in pain induction. Docking revealed that phenazopyridine forms strong H-bonds with the hinge region of the ATP-binding pocket of these kinases. As previous studies suggested increased autophagy upon inhibition of the phosphatidyl-inositol/AKT pathway, we also investigated the impact of phenazopyridine on this pathway and found an upregulation. In conclusion, our study demonstrates for the first time that phenazopyridine is a kinase inhibitor, impacting notably phosphatidylinositol kinases involved in nociception.

非那吡啶（Phenazopyridine）是一款广泛应用于缓解尿路疼痛的临床药物。该化合物还被证实可促进多能干细胞的神经分化。然而，其具体的作用分子机制尚未明确。基于其化学结构特征，本研究提出假说：非那吡啶可能属于激酶抑制剂。
本研究通过以下三类实验体系对非那吡啶展开探究：1）多能干细胞内的激酶活性检测；2）与重组激酶的结合实验；3）对多能干细胞的功能影响分析。
当向多能干细胞中加入非那吡啶后，其可引发激酶活性谱的显著改变，尤其涉及丝裂原活化蛋白激酶（Mitogen-Activated Protein Kinases）、细胞周期蛋白依赖性激酶（Cyclin-Dependent Kinases）以及AKT通路激酶。
为明确非那吡啶的核心作用靶点，本研究对其与401种人类激酶的相互作用进行了筛选。剂量-抑制曲线分析显示，其中三种激酶可与非那吡啶以亚微摩尔级结合亲和力产生互作：周期蛋白G相关激酶（cyclin-G-associated kinase）、以及两种磷脂酰肌醇激酶PI4KB与PIP4K2C，其中PIP4K2C已被证实参与疼痛诱导过程。
分子对接实验结果表明，非那吡啶可与上述激酶ATP结合口袋的铰链区形成稳定的氢键相互作用。
既往研究显示，抑制磷脂酰肌醇/AKT通路可引发细胞自噬水平上调，据此本研究同时检测了非那吡啶对该通路的影响，结果发现通路活性出现上调。
综上，本研究首次证实非那吡啶属于一类激酶抑制剂，可显著作用于参与痛觉传导的磷脂酰肌醇激酶。