Binding Mode Analysis of Antifouling Compounds Targeting Tyrosinase and Acetylcholinesterase by Saturation Transfer Difference NMR Spectroscopy.

Ecofriendly and sustainable antifouling (AF) compounds are required to replace toxic additives in maritime AF coatings. Our group has developed synthetic AF compounds with anti-settlement activity toward Mytilus galloprovincialis mussel with nontoxic properties against this target organism. Some compounds have shown to be capable of modulating the activity of key enzymes involved in mussel settlement, namely, tyrosinase and acetylcholinesterase (AChE). The saturation transfer difference nuclear magnetic resonance (STD-NMR) technique is a powerful ligand-based approach to disclose the moieties responsible for binding to macromolecules in solution. This work aimed to study the binding mode of two AF compounds, a xanthone and a polyphenol, with tyrosinase and AChE, respectively, by using STD-NMR. The obtained results showed that the tyrosinase inhibitor exhibited an epitope map based on the hydroxylated aromatic ring, whereas the AChE inhibitor established interactions with both the aromatic ring and the aliphatic moiety. Further competition assays with established inhibitors, namely, kojic acid for tyrosinase and eserine for AChE, suggested that the xanthone derivative engages tyrosinase in a competitive manner, whereas the polyphenol interacts with AChE at sites distinct from the catalytic active site. These structural insights will help the rational design for optimized AF agents by targeting tyrosinase and AChE.