Flavin-Containing Monooxygenase 3 Polymorphic Variants Significantly Affect Clearance of Tamoxifen and Clomiphene.

Human flavin-containing monooxygenase 3 (hFMO3) is a drug-metabolising enzyme that oxygenates many drugs and xenobiotics in the liver. This enzyme is also known to exhibit single nucleotide polymorphisms (SNPs) that can alter the rates of monooxygenation of therapeutic agents. The purpose of this study was to investigate the effect of the three common polymorphic variants of hFMO3 (V257M, E158K and E308G) on the metabolism and clearance of three structurally similar compounds: tamoxifen (breast cancer medication), clomiphene (infertility medication) and GSK5182 (antidiabetic lead molecule). For GSK5182, none of the three variants showed any significant differences in its metabolism when compared to the wild-type enzyme. In the case of clomiphene, two of the variants, V257M and E308G, exhibited a significant increase in all the kinetic parameters measured with nearly two times faster clearance. Finally, for tamoxifen, a mixed behaviour was observed; E158K variant showed a significantly higher clearance compared to the wild type, whereas V257M mutation had the opposite effect. Overall, the data obtained demonstrate that there is no direct correlation between the SNPs and the metabolism of these three hFMO3 substrates. The metabolic capacity is both variant-dependent and substrate-dependent and therefore when testing new drugs or administering already approved therapies, these differences should be taken into consideration.

Identification of human flavin-containing monooxygenase 3 substrates by a colorimetric screening assay.

Human hepatic flavin-containing monooxygenase 3 is a phase I drug-metabolizing enzyme that is responsible for the oxidation of a variety of drugs and xenobiotics. This work reports on a high throughput rapid colorimetric assay for the screening of substrates or inhibitors of this enzyme. The method is based on the competition of two substrates for access to the active site of hFMO3 whereby the enzymatic product of the first drug converts nitro-5-thiobenzoate (TNB, yellow) to 5,5'-dithiobis (2-nitrobenzoate) (DTNB, colourless). Upon addition of a competing substrate, the amount of detected DNTB is decreased. The assay is validated testing three known substrates of hFMO3, namely benzydamine, tozasertib and tamoxifen. The latter drugs resulted in 41%-55% inhibition. In addition, two other drugs also classified as doping drugs, selegiline and clomiphene, were selected based on their chemical structure similarity to known substrates of hFMO3. These drugs showed 21% and 60% inhibition in the colorimetric assay and therefore were proven to be hFMO3 substrates. LC-MS was used to confirm their N-oxide products. Further characterisation of these newly identified hFMO3 substrates was performed determining their Km and kcat values that resulted to be 314 µM and 1.4 min-1 for selegiline and, 18 µM and 0.1 min-1 for clomiphene. This method paves the way for a rapid automated high throughput screening of nitrogen-containing compounds as substrates/inhibitors of hFMO3.