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

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.