Identification of food derived phenolics as modulators of proteolytic enzymes in the digestive tract: in vitro and in silico approaches

The interaction of food components with digestive enzymes can modify the digestion process and affect human health. Phenols/polyphenols (PPs) have a role in regulating metabolism and a beneficial impact on some chronic diseases. On a different tune, PPs are often referred to as anti-nutritional factors, having been reported to inhibit proteolytic digestive enzymes. However, reports on this particular topic are often conflicting, stressing the need to evaluate the effects of PPs through standardized approaches. In this study, the effects on proteolyisis of a selected collection of food derived PPS, characterised by different chemotypes and used either as glicosides or aglycones, was tested “in vitro” on pepsin, trypsin, and chymotrypsin acting on albumin, gluten, and hemoglobin as substrates. Results show that PPs may affect proteolytic activity in opposite ways, depending on the substrate/enzyme system. In a second part of the study, “in silico” approaches were used to investigate the binding of a restricted set of PPs to a model substrate (ovalbumin) and a model enzyme (chymotrypsin). After extensive literature search and pocket scanning, molecular docking and molecular dynamics simulations were used to pinpoint the capability of PPs to interact in a stable way with either protein. The combined approaches indicate that the most potent proteolytic inhibitors were PPs interacting with the enzyme binding site, whereas PPs acting as enhancers of proteolysis were more likely to interact with sites on the substrate protein. Concluding, our results indicate that peculiar structural features of PPs have a role in eliciting their specific effects, in terms of 3D- structure, substitution pattern and lipophylicity, suggesting a possible modulatory role of PPs in the formulation of functional foods. This investigation is partially supported by National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.3 - Call for tender No. 341 of 15/03/2022 of Italian Ministry of University and Research funded by the European Union – NextGenerationEU, in the frame of the project: Research and innovation network on food and nutrition Sustainability, Safety and Security (ONFoods) and 1H-HUB.