title: In vitro drug metabolism by C-terminally truncated human flavin-containing monooxygenase 3.
abstract: Human flavin-containing monooxygenase 3 (hFMO3) is a microsomal drug-metabolizing monooxygenase that catalyzes the NADPH-dependent oxygenation of a wide range of drugs and xenobiotics which contain a soft-nucleophiles, usually sulfur or nitrogen. As the release from the microsomal membranes can facilitate the in vitro experimental determination of drug metabolism by hFMO3, in this work we identified and eliminated the membrane anchoring sequence without affecting the activity of the enzyme and producing a soluble active enzyme. The truncated hFMO3 carrying a C-terminal deletion of 17 amino acids (tr-hFMO3) was expressed and purified from the cytosolic fraction. The tr-hFMO3 proves to be detached from the membrane, properly folded and fully active towards well-known marker substrates such as benzydamine and sulindac sulfide with measured apparent K(m) values of 45 ± 8 μM and 25 ± 4 μM, respectively. Its activity was further tested with newly discovered Aurora kinase inhibitors, Tozasertib and Danusertib, and compared to those of the wild type enzyme. The use of this soluble form of the hFMO3 enzyme as opposed to the usual microsomal preparations is advantageous for in vitro drug metabolism studies that are a requirement in the early phases of drug development by pharmaceutical industry.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Catucci
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
authors.forename: Lars
authors.initials: L
authors.lastname: Jeuken
authors.forename: Sheila J
authors.initials: SJ
authors.lastname: Sadeghi
mesh_terms: D001549:Benzamides / Q000737:chemistry / Q000378:metabolism; D001591:Benzydamine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002474:Cell-Free System; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D010879:Piperazines / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011720:Pyrazoles / Q000737:chemistry / Q000378:metabolism; D013467:Sulindac / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D003521:Cyclopropanes; D011743:Pyrimidines
chemical_list: D001549:Benzamides; D001591:Benzydamine; D010105:Oxygenases; D010879:Piperazines; D011720:Pyrazoles; D013467:Sulindac; C484810:tozasertib; D003521:Cyclopropanes; D011743:Pyrimidines; C025462:sulindac sulfide; C020066:dimethylaniline monooxygenase (N-oxide forming); C523797:danusertib