title: Identification of human flavin-containing monooxygenase 3 substrates by a colorimetric screening assay.
abstract: 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 Kmand kcatvalues that resulted to be 314 μM and 1.4 min-1for selegiline and, 18 μM and 0.1 min-1for clomiphene. This method paves the way for a rapid automated high throughput screening of nitrogen-containing compounds as substrates/inhibitors of hFMO3.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Catucci
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Polignano
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Debora
authors.initials: D
authors.lastname: Cusumano
authors.affiliation: Department of Molecular Biotechnology and Health Sciences, University of Torino, 10125 Turin, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Medana
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy. Electronic address: sheila.sadeghi@unito.it.
authors.forename: Sheila J
authors.initials: SJ
authors.lastname: Sadeghi
mesh_terms: D001565:Benzoates / Q000737:chemistry*; D001591:Benzydamine / Q000737:chemistry*; D003124:Colorimetry / Q000379:methods; D004228:Dithionitrobenzoic Acid / Q000737:chemistry*; D006801:Humans; D010105:Oxygenases / Q000737:chemistry*; D010879:Piperazines / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D013629:Tamoxifen / Q000737:chemistry*; D003521:Cyclopropanes; D011720:Pyrazoles; D011743:Pyrimidines
chemical_list: D001565:Benzoates; D001591:Benzydamine; D004228:Dithionitrobenzoic Acid; D010105:Oxygenases; D010879:Piperazines; D011092:Polyethylene Glycols; D013629:Tamoxifen; C489064:thiobenzoate chromophore; C484810:tozasertib; D003521:Cyclopropanes; D011720:Pyrazoles; D011743:Pyrimidines; C020066:dimethylaniline monooxygenase (N-oxide forming)
keywords: Doping drugs; FMO; Flavoprotein; HTS; Mass spectrometry; Tamoxifen