It is well recognized that patients administered a particular drug will exhibit significant inter-individual variability in their response to treatment. Currently, Personalized medicine which defines the specificity and dosage of drugs according to effectiveness and safety for each patient is a very fast growing area of research. Dosage strongly depends on the rate of metabolism that is primarily regulated by the activity of cytochromes P450 and flavin-containing monooxygenase 3 (hFMO3). Human FMO3 is a microsomal enzyme capable of metabolising drugs containing a soft-nucleophile, usually sulphur or nitrogen. This enzyme has three common polymorphic variants, one of which is V257M (rs1736557). In this work, the wild type enzyme (WT) and the engineered V257M polymorphic variant were heterologously expressed in bacteria and purified with a yield of 12 mg/L of culture. The activity of the purified enzymes was subsequently measured towards several drug and/or drug candidates. Initially benzydamine, a nonsteroidal anti-inflammatory drug (NSAID) and a marker substrate of hFMO3, and sulindac sulphide, another NSAID used in the treatment of chronic inflammatory conditions, were tested. For benzydamine the results obtained with the purified enzymes showed no differences between the WT and the V257M polymorphic variant in the N-oxygenation of this drug. In the case of sulindac sulphide, the variant showed a decrease in its catalytic efficiency when compared to the WT enzyme. Subsequently two Aurora kinase inhibitors, Tozasertib (VX-680) and Danusertib (PHA-739358), which are anti-cancer drug candidates, were tested. The conversion of Tozasertib and Danusertib to their corresponding metabolites by the purified WT and V257M hFMO3 showed significant differences. In the case of Tozasertib, the V257M variant showed a catalytic efficiency, expressed as kcat/KM, similar to the WT (0.33-0.36 min−1μM−1). On the other hand, in the case of Danusertib, V257M showed a 3.4 fold decrease in catalytic efficiency (kcat/KM = 0.05 versus 0.17 min−1μM−1 for V257M and WT, respectively). These data reveal how a simple V257M substitution ascribed to a single nucleotide polymorphism may affect drug metabolism and efficacy. Supported by Progetto di Ateneo-2012 grant.
Human flavin-containing monoxygenase 3 polymorphism and its effect on drug metabolism
BORTOLUSSI, STEFANIA;CASTRIGNANO', SILVIA;CATUCCI, GIANLUCA;GILARDI, Gianfranco;SADEGHI, JILA
2013-01-01
Abstract
It is well recognized that patients administered a particular drug will exhibit significant inter-individual variability in their response to treatment. Currently, Personalized medicine which defines the specificity and dosage of drugs according to effectiveness and safety for each patient is a very fast growing area of research. Dosage strongly depends on the rate of metabolism that is primarily regulated by the activity of cytochromes P450 and flavin-containing monooxygenase 3 (hFMO3). Human FMO3 is a microsomal enzyme capable of metabolising drugs containing a soft-nucleophile, usually sulphur or nitrogen. This enzyme has three common polymorphic variants, one of which is V257M (rs1736557). In this work, the wild type enzyme (WT) and the engineered V257M polymorphic variant were heterologously expressed in bacteria and purified with a yield of 12 mg/L of culture. The activity of the purified enzymes was subsequently measured towards several drug and/or drug candidates. Initially benzydamine, a nonsteroidal anti-inflammatory drug (NSAID) and a marker substrate of hFMO3, and sulindac sulphide, another NSAID used in the treatment of chronic inflammatory conditions, were tested. For benzydamine the results obtained with the purified enzymes showed no differences between the WT and the V257M polymorphic variant in the N-oxygenation of this drug. In the case of sulindac sulphide, the variant showed a decrease in its catalytic efficiency when compared to the WT enzyme. Subsequently two Aurora kinase inhibitors, Tozasertib (VX-680) and Danusertib (PHA-739358), which are anti-cancer drug candidates, were tested. The conversion of Tozasertib and Danusertib to their corresponding metabolites by the purified WT and V257M hFMO3 showed significant differences. In the case of Tozasertib, the V257M variant showed a catalytic efficiency, expressed as kcat/KM, similar to the WT (0.33-0.36 min−1μM−1). On the other hand, in the case of Danusertib, V257M showed a 3.4 fold decrease in catalytic efficiency (kcat/KM = 0.05 versus 0.17 min−1μM−1 for V257M and WT, respectively). These data reveal how a simple V257M substitution ascribed to a single nucleotide polymorphism may affect drug metabolism and efficacy. Supported by Progetto di Ateneo-2012 grant.
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