New mechanisms of phenytoin in calcium homeostasis: competitive inhibition of CD38 in hippocampal cells

Abstract

Purpose: Phenytoin is a major anticonvulsant drug that is effective to improve arrhythmia and neuropathic pain. According to early works, phenytoin affected cell membrane depolarization by sodium channel blocking, guanylyl and adenylyl cyclase suppression that cause to intracellular Na⁺ and Ca²⁺ downregulation. This study was aimed to clarify some ambiguities in pathophysiological action of phenytoin by in vitro and molecular docking analyses.

Methods: In this study intracellular free Ca²⁺ of primary culture of embryonic mouse hippocampus evaluated via Fura 2 as fluorescent probe. The effects of phenytoin on ADP ribosyl cyclase activity was assessed by recently developed fluorometric assay. Molecular docking simulation was also implemented to investigate the possible interaction between phenytoin and CD38.

Results: Our results confirmed phenytoin competitively inhibits cyclase activity of CD38 (IC₅₀ = 8.1 μM) and reduces cADPR content. cADPR is a Ca²⁺-mobilising second messenger which binds to L-type calcium channel and ryanodine receptors in cell and ER membrane and increases cytosolic free Ca²⁺. Ca²⁺ content of cells decreased significantly in the presence of phenytoin in a dose dependent manner (IC₅₀ = 12.74 µM). Based on molecular docking analysis, phenytoin binds to deeper site of CD38 active site, mainly via hydrophobic interactions and consequently inhibits proper contact of substrate with catalytic residues specially Glu 226, Trp 186, Thr221.

Conclusion: Taken together, one of the anticonvulsant mechanisms of phenytoin is Ca²⁺ inhibition from CD38 pathway, therefore could be used in disorders that accompanied by CD38 over production or activation such as heart disease, depression, brain sepsis, airway disease, oxidative stress and inflammation. Graphical abstract ᅟ.

Keywords: CD38; Calcium homeostasis; Competitive inhibition; Membrane depolarization; Phenytoin; Sodium blocker.

Graphical abstract

ᅟ

Fig. 1

PHT effects on calcium homeostasis in hippocampal cells. (a) PHT decreased intracellular free Ca²⁺ according to concentration. The data were expressed as mean ± SD of three independent experiments. Asteric (*) symbol indicates significant changes (p < 0.05) than [Ca²⁺]i in the presence of NAD⁺ (b) Cyclase activity of CD38 suppressed by different concentrations of PHT. Enzymatic activity decreased to half of maximum in the presence of 8.1 μM inhibitor. The data were expressed as mean ± SD of three independent experiments

Fig. 2

Cyclase activity related to CD38 that extracted from hippocampal cells in a range of substrate (0–800 μM) resulted to Michaelis-Menten plot in the presence of three different concentration of PHT (0, 5 and 10 μM). CD38 activity decreased in the presence of PHT in a concentration dependent manner. Lineweaver-Burk plot showing the effect of changing inhibitor concentration on 1/-Km (x-intercept) of CD38 conversion of NGD+ to cGDPR. PHT increases K_m without changing V_max value in the reaction, consistent with competitive enzyme inhibition. Whole experiment is repeated independently at least three times and results showed mean ± SD

Fig. 3

PHT molecule in the CD38 active site. a Interaction of PHT with helix and ribbon related to catalytic site of CD38. b Surface view of the active site cavity of the crystal structure of CD38 with PHT bound inside in two resolutions

Fig. 4

PHT and NAD⁺ located in the CD38 active site. a Hydrophobic surface model showed PHT placed in dipper site of catalytic cavity rather than NAD⁺ that was suppressed to proper placement in active site. PHT was showed by pink color and NAD was visualized as a purple molecule in CD38 active site b The carton form of one subunit of CD38 shows substrate binding residues interact with PHT (pink molecule). c PHT is located close to the main residues (Glu 226, Glu 146, Asp 155, Trp 189 and Thr125) in the active site of CD38 that have important roles in the substrate binding and catalytic activity. d Different interactions between PHT and the active site’s residues, especially Glu 226 as a main catalytic residue