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

Abstract

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.

Keywords: autophagy; differentiation; kinase; phenazopyridine; phosphatidylinositol kinase.

FIGURE 1

phenazopyridine regulates the human kinome. (A) Chemical structure of phenazopyridine. (B) The impact of phenazopyridine on kinases and their respective pathways was analysed in PSC by the Tyrosine or Serine/Threonine kinase assays (Pamgene). Substrates of336 kinases were exposed to the cellular content of PSC exposed to the drug. Phosphorylation of these substrates, as an indication of kinase activity, was measured by fluorescent antibodies at different time points. The fold change between phenazopyridine and DMSO control conditions was calculated. Kinases altered by phenazopyridine with a significant regulation of fold change (p < 0,05) were then hierarchically clustered. (C) Functional clustering into networks of kinases upregulated in the presence of phenazopyridine, according to an analysis with the STRING database. (D) Functional clustering into networks of kinases down-regulated in the presence of phenazopyridine.

FIGURE 2

phenazopyridine induces differentiation of PSC with no effects on proliferation. (A) The CGR8 PSC line was exposed to phenazopyridine at 10 µM or DMSO control conditions. After two days, cells were lysed for the quantification of intracellular ATP. (B) CGR8_{EF1αS,RLuc-Tα1,FLuc} cells were exposed to phenazopyridine at various concentrations or DMSO control conditions. After three days, cells were lysed for the quantification of F-luminescence (FLuc) and R-luminescence (RLuc) in the presence of the respective substrates. The left panel shows the ratio of luminescence intensity between FLuc and RLuc for different concentrations of phenazopyridine. The right panel shows cell viability for various phenazopyridine concentrations, assessed by analysis of the fluorescence in the presence of propidium iodide.

FIGURE 3

In silico confirmation of phenazopyridine binding to the three selected kinases. Binding mode and interaction diagrams of phenazopyridine within (A) human cyclin-G-associated kinase (GAK; PDB identifier: 4Y8D), (B) phosphatidylinositol 4-kinase β (PI4KB; PDB identifier: 6GL3), and (C) phosphatidylinositol-5-phosphate four kinase type 2 gamma (PIP4K2C; PDB identifier: 2GK9). The atoms of phenazopyridine are shown as sticks with yellow carbons and the interacting residues of each protein as sticks with orange (A), blue (B), and brown carbons (C), respectively. For the holo crystal structures (A) and (B), the carbon atoms of the respective co-crystallized ligands are displayed with white sticks. H-bonds are shown as dashed yellow lines, and their lengths are indicated in Ångströms. Binding site residues directly interacting with phenazopyridine and the catalytic Lys are labelled as well.

FIGURE 4

Analysis of PSC autophagy in the presence of phenazopyridine. PSC or HeLa cells were exposed for three days to 10 µM of phenazopyridine or PIP4K2C inhibitors, prior to analysis and quantification of LC3B positive autophagosomes by immunofluorescent staining.