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.
Highlights
Phenazopyridine [2,6-diamino-3-(phenylazo)pyridine] is used for its efficiency in the treatment of urinary urgencies and can be helpful in the context of urinary tract infection, interstitial cystitis, as well as medical procedures, which result in irritation and pain of the urinary tract
We demonstrate time-dependent alterations of the cellular kinome by the compound and evidence that phenazopyridine binds to several kinases, notably phosphatidylinositol kinases involved in nociception
The hypothesis on the interaction of phenazopyridine with kinases was reinforced by its chemical structure (Figure 1A): a structure-activity relationship analysis
Summary
Phenazopyridine [2,6-diamino-3-(phenylazo)pyridine] is used for its efficiency in the treatment of urinary urgencies and can be helpful in the context of urinary tract infection, interstitial cystitis, as well as medical procedures (endoscopy or surgery), which result in irritation and pain of the urinary tract. Phenazopyridine is generally well supported and sold as an over the counter medication in the Mechanism of Action of Phenazopyridine. A hypothesis of mechanism of action comes from the chemical structure of phenazopyridine: the pyridine-2,6-diamine moiety of phenazopyridine is a typical kinase inhibition motif (Lin et al, 2005). To corroborate this hypothesis which was not supported by any prior study, we performed an inverse virtual screening on a diverse target set including several thousand proteins, including target classes of pharmaceutical interest. The high frequency of kinases in the high ranks of the interaction-score based ranking obtained from docking phenazopyridine into this large target panel confirmed the involvement of kinases
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