Targeting Penicillium expansum GMC Oxidoreductase with High Affinity Small Molecules for Reducing Patulin Production

SIMPLE SUMMARY: With the urgent necessity of potential treatments for limiting mycotoxin production and postharvest fungal rots, we propose a combined in silico/in vitro/in vivo strategy for the rapid and effective identification of bioactive small molecules, chosen among a chemical library hosting approved drugs and phytochemicals, to be used after harvest. The molecular target of our analysis was the GMC oxidoreductase from Penicillium expansum involved in the biosynthesis of patulin, a mycotoxin that can contaminate many foods, especially fruits and fruit-based products. The employed in silico/in vitro/in vivo assays described in our study proved the effectiveness of our strategy and in particular of two small molecules, 6-hydroxycoumarin (structurally related to umbelliferon, an already characterized patulin synthase inhibitor) and meticrane (an already approved drug) in reducing patulin accumulation. Our findings highly recommend the mentioned ligands to be subjected to further analysis for being used in the next future in place of other more toxic compounds, in postharvest treatments based on dipping or drenching methods. ABSTRACT: Flavine adenine dinucleotide (FAD) dependent glucose methanol choline oxidoreductase (GMC oxidoreductase) is the terminal key enzyme of the patulin biosynthetic pathway. GMC oxidoreductase catalyzes the oxidative ring closure of (E)-ascladiol to patulin. Currently, no protein involved in the patulin biosynthesis in Penicillium expansum has been experimentally characterized or solved by X-ray diffraction. Consequently, nothing is known about P. expansum GMC oxidoreductase substrate-binding site and mode of action. In the present investigation, a 3D comparative model for P. expansum GMC oxidoreductase has been described. Furthermore, a multistep computational approach was used to identify P. expansum GMC oxidoreductase residues involved in the FAD binding and in substrate recognition. Notably, the obtained 3D comparative model of P. expansum GMC oxidoreductase was used for performing a virtual screening of a chemical/drug library, which allowed to predict new GMC oxidoreductase high affinity ligands to be tested in in vitro/in vivo assays. In vitro assays performed in presence of 6-hydroxycoumarin and meticrane, among the highly affinity predicted binders, confirmed a dose-dependent inhibition (17–81%) of patulin production by 6-hydroxycoumarin (10 µM–1 mM concentration range), whereas the approved drug meticrane inhibited patulin production by 43% already at 10 µM. Furthermore, 6-hydroxycoumarin and meticrane caused a 60 and 41% reduction of patulin production, respectively, in vivo on apples at 100 µg/wound.