AUTHOR=Gerstmeier Jana , Seegers Julia , Witt Finja , Waltenberger Birgit , Temml Veronika , Rollinger Judith M. , Stuppner Hermann , Koeberle Andreas , Schuster Daniela , Werz Oliver 

TITLE=Ginkgolic Acid is a Multi-Target Inhibitor of Key Enzymes in Pro-Inflammatory Lipid Mediator Biosynthesis

JOURNAL=Frontiers in Pharmacology

VOLUME=10

YEAR=2019

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2019.00797

DOI=10.3389/fphar.2019.00797

ISSN=1663-9812

ABSTRACT=<p><bold>Introduction:</bold> Lipid mediators (LMs) comprise bioactive metabolites of polyunsaturated fatty acids, including pro-inflammatory prostaglandins (PGs), thromboxanes (TXs), and leukotrienes (LTs), as well as specialized pro-resolving mediators (SPMs). They are essentially biosynthesized <italic>via</italic> cyclooxygenase (COX) and lipoxygenase (LO) pathways in complex networks and regulate the progression as well as the resolution of inflammatory disorders including inflammation-triggered cancer. Ginkgolic acid (GA) is a phenolic acid contained in <italic>Ginkgo biloba</italic> L. with neuroprotective, antimicrobial, and antitumoral properties. Although LMs regulate microbial infections and tumor progression, whether GA affects LM biosynthesis is unknown and was investigated here in detail.</p><p><bold>Methods:</bold> Pharmacophore-based virtual screening was performed along with docking simulations. Activity assays were conducted for isolated human recombinant 5-LO, cytosolic phospholipase (PLA)<sub>2</sub>α, COX-2, and ovine COX-1. The activity of human mPGES-1 and thromboxane A<sub>2</sub> synthase (TXAS) was determined in crude cellular fractions. Cellular LM formation was studied using human monocytes, neutrophils, platelets, and M1- and M2-like macrophages. LMs were identified after (ultra)high-performance liquid chromatography by UV detection or ESI-tandem mass spectrometry.</p><p><bold>Results:</bold> GA was identified as virtual hit in an mPGES-1 pharmacophore-based virtual screening. Cell-free assays revealed potent suppression of mPGES-1 activity (IC<sub>50</sub> = 0.7 µM) that is fully reversible and essentially independent of the substrate concentration. Moreover, cell-free assays revealed COX-1 and TXAS as additional targets of GA with lower affinity (IC<sub>50</sub> = 8.1 and 5.2 µM). Notably, 5-LO, the key enzyme in LT biosynthesis, was potently inhibited by GA (IC<sub>50</sub> = 0.2 µM) in a reversible and substrate-independent manner. Docking simulations support the molecular interaction of GA with mPGES-1 and 5-LO and suggest concrete binding sites. Interestingly, interference of GA with mPGES-1, COX-1, TXAS, and 5-LO was evident also in intact cells with IC<sub>50</sub> values of 2.1–3.8 µM; no radical scavenging or cytotoxic properties were obvious. Analysis of LM profiles from bacteria-stimulated human M1- and M2-like macrophages confirmed the multi-target features of GA and revealed LM redirection towards the formation of 12-/15-LO products including SPM.</p><p><bold>Conclusions:</bold> We reveal GA as potent multi-target inhibitor of key enzymes in the biosynthesis of pro-inflammatory LMs that contribute to the complex pharmacological and toxicological properties of GA.</p>