Attività di elettrodi funzionalizzati con P450 2D6 umano ingegnerizzato con un dominio reduttasico flavodossinico e uno spacer peptidico

Cytochrome P450 2D6 (CYP2D6) is an heme containing mono-oxygenase enzyme belonging to cytochromes P450 superfamily which is responsible for the metabolism of drugs and xenobiotics. CYP2D6 is one of the most important drug-metabolizing enzymes: it is involved in the biotransformation of at least 25% of prescribed drugs and is also important for the activation of pro-drugs such as Tamoxifen which is used for treatment and prevention of estrogen receptor positive breast cancer. In this study the possibility to covalently immobilize CYP2D6 on gold electrodes in a direct and oriented manner using a one step procedure was investigated with the aim of building a bio-electrochemical system useful for the characterization of CYP2D6 metabolism and inhibition and the study of drug-drug interactions mechanism related to CYP2D6 catalytic activity. CYP2D6 was engineered by genetic fusion of the primary sequence with D. vulgaris flavodoxin domain (FLD) in order to allow direct electron transfer with electrode surface. Moreover, a peptide linker composed by four glicine and two cysteine residues was placed at the c-terminus of the fusion protein to allow a covalent direct binding with gold electrode surface. CYP2D6-FLD was also engineered both with and without a six histidine tag for purification purposes. Recombinant expression was carried out in Escherichia coli and purification was performed using two chromatographic steps, using ion exchange and affinity chromatography. The catalytic activity of CYP2D6-FLD modified gold electrodes was investigated towards Tamoxifen metabolism by using electrode surface as electron source in the presence of increasing substrate concentration. To this aim, electrocatalysis experiments were performed in an electrochemical cell using a three electrodes setup in the presence of both Tamoxifen and N-desmethyl-Tamoxifen substrates which are hydroxilated by CYP2D6. Products of electrocatalysis were observed and quantified using HPLC in order to calculate the kinetic parameters of the enzymatic reaction. Inhibition assays were performed using Quinidine, a well known and characterized CYP2D6 inhibitor. The results here reported show that it is possible to covalently immobilize CYP2D6 in a successful one step procedure and that, most importantly, the immobilized enzyme is catalytically active and able to perform an enzymatic reaction which could be monitored to detect the enzyme inhibition due to drug-drug interaction.