Bioconversione di composti naturali tramite aromatasi umana

Natural compounds (NCs) are an important target of drug discovery and biocatalysis. Several studies have been focused on the enzymatic functionalization of bioactive compounds from natural sources to increase their pharmaceutical properties. Cytochromes P450 (CYPs) are a class of heme-thiolate monooxygenases that has been exploited for its huge biotechnological application. These enzymes can recognise and metabolise a great variety of exogenous and endogenous compounds; the main reaction carried out by CYPs is the hydroxylation of unactivated C-H sites. Most of cytochromes P450 require a reduction partner that mediates the flow of electrons from the donor to the heme iron. In humans CYPs work along with Cytochrome P450 Reductase (CPR), that transfers electrons from NADPH to the iron centre. Human aromatase (CYP19A1, or hAro) is the last enzyme in steroidogenesis and catalyses a complex reaction: it converts androgens into estrogens through two hydroxylations and a C-C cleavage, forming an aromatic ring. The active site of CYP19A1 is not the biggest among all CYPs, but it can accommodate steroid and steroid-like compounds, so it can have a biotechnological application. With this knowledge, three steroid-like bioactive compounds were selected for their clinical importance to be tested with human aromatase for late-stage functionalization. Two flavonoids, baicalein and glabridin, and a bile acid, lithocholic acid, were chosen for this thesis work. At first, molecular docking simulations of hAro crystal structure and the three NCs were performed to obtain a prediction of their interaction with the aromatase binding pocket. All three molecules appear to strongly interact with human aromatase. The next step was to investigate if these compounds induced a shift of the Soret band through a spectroscopic binding assay with the wild-type enzyme, but no significant shift was observed. A possible reaction of hAro with the NCs was observed with the NADPH consumption assay: baicalein and lithocholic acid were selected after this assay for further experiments. HPLC was chosen to analyse the reaction over time of human aromatase with the two compounds that showed relevant NADPH consumption. By means of HPLC time-course analyses the substrate consumption and potential product increase can be observed over time. Reactions with baicalein did not show any product, but further experiments with this flavonoid revealed that it can act as an in vitro inhibitor of hAro reactions with its original substrate, androstenedione. The data presented in this work shows that wild-type human aromatase can recognise molecular scaffolds similar to steroids. However, inhibition by these compounds can be an issue. The research on enzymatic functionalization still relies on trial and error, so more NCs should be tested with CYP19A1 to assess its relevance in the biotechnological and pharmaceutical field. In conclusion, this approach can be useful to perform screening of natural compounds with human aromatase to identify potential substrates/inhibitors, as well as identifying functionalized products.