# Structural Insights into the Interaction of Human ALOX15 with the Natural Antioxidant Nordihydroguaiaretic Acid: Functional Inhibitor Studies and Molecular Dynamics Simulations

## TL;DR

This study reveals how a natural antioxidant interacts with a human enzyme involved in inflammation and disease, identifying a key amino acid important for their binding.

## Contribution

The study identifies Gln595 as a critical residue in the interaction between NDGA and ALOX15 using computational and experimental approaches.

## Key findings

- NDGA binds in the substrate pocket of human ALOX15, with Gln595 playing a major role in the interaction.
- Mutagenesis of Gln595 altered the stability and ligand binding behavior of ALOX15–NDGA complexes.
- Experimental IC50 values confirmed that NDGA strongly inhibits wildtype ALOX15 and specific mutants but not others.

## Abstract

Mammalian arachidonic acid lipoxygenases (ALOXs) are lipid-peroxidizing enzymes, which have been implicated in inflammatory, hyperproliferative and neurodegenerative diseases. Nordihydroguaiaretic acid (NDGA) is a naturally occurring antioxidant and a potent lipoxygenase inhibitor. Unfortunately, the molecular basis of the NDGA–ALOX interaction remains unexplored. Here, we show by in silico docking studies and by molecular dynamics simulations that NDGA binds in the substrate binding pocket of human ALOX15 and that Gln595 plays a major role in this interaction. In silico mutagenesis studies (Glu595Ala, Glu595Leu, Glu595Glu, Glu595Ile) modified the stability of the ALOX15–NDGA complex and altered the ligand binding behavior of the enzyme. To validate the in silico findings, we expressed human ALOX15 and the enzyme mutants as recombinant proteins, characterized their functional properties and quantified the IC50 values for NDGA-induced inhibition. Consistent with our in silico predictions, the experimental IC50 values demonstrated that NDGA strongly inhibited wildtype ALOX15 and its Gln595Glu and Gln595Ile mutants. In contrast, the IC50 values for the Gln595Ala and Gln595Leu mutants were more than one order of magnitude higher. These findings highlight the role of Gln595 for the NDGA–ALOX15 interaction and may facilitate the future development of isoform-specific ALOX15 inhibitors.