Metabolismo di farmaci antitumorali da parte dell'FMO3 umana e delle sue varianti polimorfiche

Flavin-containing monooxygenase 3 (hFMO3) is a human hepatic enzyme involved in Phase I Drug Metabolism. It has been shown that genetic variations of this enzyme are involved in the alteration of the rate of metabolism of different drugs, so understanding these differences is fundamental to predict drug response of different individuals. In this work, the activity of two common polymorphic variants of hFMO3 (E158K and V257M) has been tested against the one of the WT by measuring and comparing kinetic parameters, in order to estimate the presence of statistically significant differences. First thing first, UV-visible spectroscopy experiments have been performed to show the presence of enzymes in their holo form. Subsequently, two anti-cancer drugs, vandetanib (medullary thyroid cancer treatment) and cediranib (ovarian cancer treatment) have been selected for in vitro enzymatic assays. Incubations of the three purified enzymes with increasing concentrations of these two drugs allowed the HPLC quantification of metabolites, making possible the calculation of kinetic parameters. For vandetanib, E158K and V257M show turnover numbers higher and significantly different to that of the WT (13.7 ± 0.7 min-1 and 11.89 ± 1.5 min-1, respectively). For cediranib, only comparison between E158K and the WT have been performed, because V257M did not reach Vmax in the range of concentrations tested. Even in this case, the kcat value was higher for the mutant and statistically different to the WT. In both cases, no significant differences have been observed in terms of Km and kcat/Km. Given the unavailability of the products during the experimental phase, the standards of the products have been obtained through bioconversion with the pure WT enzyme. For this reason, more in depth studies, for example experiments of mass spectrometry, are needed to confirm the N-oxidation of these two compounds in order to make a contribution in the development of personalized medicine.