An integrated in vitro and in silico approach to assess the potential inhibitory actions of auxins on human placental glutathione S-transferase P1-1.

Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the metabolism of a wide variety of xenobiotics and endogenous compounds. Since some GST isozymes are overexpressed in a variety of malignancies and have been shown to play roles in the development of drug resistance, GSTs have become a promising therapeutic target. Although numerous natural and plant-derived compounds have been evaluated as GST inhibitors, the interaction between human GSTP1-1 and auxins-indole-derived plant hormones that are also detectable in human biological systems-has not been previously characterized. The present study aimed to investigate whether selected auxins can modulate the activity of human placental GSTP1-1 (hpGSTP1-1) and to elucidate the mechanistic and structural basis of any interactions. The inhibitory effects of indole-3-acetic acid (IAA), indole-3-propionic acid (IPA), and indole-3-butyric acid (IBA) on hpGSTP1-1 were evaluated using in vitro enzyme kinetic assays. IC50 determination was conducted at various auxin concentrations. Subsequently, through inhibitory kinetic studies, the inhibition types and kinetic parameters were determined. In parallel, molecular docking was performed to provide structural insight into interactions between hpGSTP1-1 and auxins. All three auxins inhibited hpGSTP1-1 activity in a concentration-dependent manner, with IC50 values of 7.9 mM (IAA), 6.5 mM (IPA), and 4.2 mM (IBA). Kinetic analyses revealed competitive inhibition with respect to both substrates. The Ki values from statistical analysis/secondary plots for IAA, IPA, and IBA at [CDNB]f-[GSH]v and at [GSH]f-[CDNB]v were 4.00 ± 0.62 mM/2.38 mM; 3.33 ± 0.23 mM/2.84 mM; 3.33 ± 0.22 mM/2.61 mM and 3.30 ± 0.24 mM/2.38 mM; 3.52 ± 0.24 mM/2.84 mM; 2.14 ± 0.16 mM/2.61 mM, respectively. These inhibitory auxins were thought to be held mainly by the hydrophilic amino acid residues that are located at the glutathione-binding site of the enzyme. This study provides a biochemical and structural characterization of weak, competitive inhibition of hpGSTP1-1 by auxins based on combined in vitro and in silico analyses. While the inhibitory potency is limited and unlikely to be pharmacologically relevant at physiological concentrations, the combined in vitro and in silico findings offer valuable mechanistic and structural insight into auxin-GSTP1-1 interactions.