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.

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

CATUCCI, GIANLUCA;POLIGNANO, ISABELLE LAURENCE;MEDANA, Claudio;GILARDI, Gianfranco;SADEGHI, JILA
2017

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 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.
ANALYTICAL BIOCHEMISTRY
522
46
52
http://www.elsevier.com/inca/publications/store/6/2/2/7/8/1/index.htt
Doping drugs; FMO; Flavoprotein; HTS; Mass spectrometry; Tamoxifen
Catucci, Gianluca; Polignano, Isabelle; Cusumano, Debora; Medana, Claudio; Gilardi, Gianfranco; Sadeghi, Jila;
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2318/1623744
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