Effect of Human Flavin-Containing Monooxygenase 3 Polymorphism on the Metabolism of Aurora Kinase Inhibitors

Abstract

Aurora kinases were recently identified as a potential target in anticancer therapy and, amongst their available inhibitors, Tozasertib (VX-680) and Danusertib (PHA-739358) have been indicated as possible substrates of human flavin-containing monooxygenase 3 (hFMO3). Here we report the in vitro rate of oxidation of these drugs by wild-type hFMO3 and its polymorphic variant V257M. The conversion of Tozasertib and Danusertib to their corresponding metabolites, identified by LC-MS, by the purified wild-type and V257M hFMO3 show significant differences. In the case of Tozasertib, the V257M variant shows a catalytic efficiency, expressed as kcat/Km, similar to the wild-type: 0.39 ± 0.06 min−1μM−1 for V257M compared to 0.33 ± 0.04 min−1μM−1 for the wild type. On the other hand, in the case of Danusertib, V257M shows a 3.4× decrease in catalytic efficiency with kcat/Km values of 0.05 ± 0.01 min−1μM−1 for V257M and 0.17 ± 0.03 min−1μM−1 for the wild type. These data reveal how a simple V257M substitution ascribed to a single nucleotide polymorphism affects the N-oxidation of relevant anticancer drugs, with important outcome in their therapeutic effects. These findings demonstrate that codon 257 is important for activity of the hFMO3 gene and the codon change V to M has an effect on the catalytic efficiency of this enzyme.

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

“…[10] b ref [9] c ref [6] Figure legends Figure 1. Scheme depicting the reactions involved in the colorimetric inhibition assay.…”

“…Aurora A was found to be overexpressed in many human tumors including breast, colorectal, and ovarian cancers with Aurora B also overexpressed in various types of cancer. Recently our group has demonstrated the conversion of tozasertib to its N--oxide using purified recombinant hFMO3 enzyme [10]. Different concentrations of this drug, dissolved in methanol, were tested in the colorimetric assay and the inhibition data obtained are shown in Figure 3B.…”

Human flavin-containing monooxygenase 3: Structural mapping of gene polymorphisms and insights into molecular basis of drug binding

“…The five ligands used for molecular docking were prepared in DS Visualizer and optimized using Clean Geometry function (Fig. 5A) and include benzydamine (Castrignanò et al, 2010), a nonsteroidal antiinflammatory drug; methimazole, a drug used to treat hyperthyroidism; sulindac sulfide (Castrigna nò et al, 2012), a non-steroidal anti-inflammatory drug that reduces tumour burden in human colorectal cancer; tozasertib (Catucci et al, 2013), a pan-Aurora kinase inhibitor for cancer treatment and, trimethylyamine, an organic compound found in dietary food with a "fishy" odour which if not metabolised by hFMO3 causes the fish odour syndrome (Dolphin et al, 1997). The best binding modes of each of these drugs with the highest binding energies were selected and optimized by energy minimization for further analysis.…”

Bioelectrochemical profiling of two common polymorphic variants of human FMO3 in presence of graphene oxide

“…Among these enzymes, human flavin-containing monooxygenase isoform 3 (hFMO3) is able to catalyse the oxygenation of a variety of xenobiotics with varied chemical structures, including many therapeutic drugs [1]. This enzyme is microsomal, FAD-and NADPH-dependent and, is able to activate molecular oxygen to catalyse substrate oxygenation [1][2][3]4]. Recently, in addition to xenobiotic metabolism, FMO3 has also been implicated in the development of atherosclerosis, cholesterol imbalance and to glucose and lipid metabolism [5][6][7].…”

“…These two hFMO3 variants were expressed in a recombinant system and immobilised on didodecyldimethylammonium bromide (DDAB)/GO glassy carbon electrodes and their activity tested with three hFMO3 marker drugs: benzydamine, a non-steroidal anti-inflammatory drug that is metabolised to its N-oxide by hFMO3 [15][16][17]21,25], tamoxifen, a breast cancer drug with antiestrogenic effect, also Noxygenated by hFMO3 [15][16][17]21,26,27],and sulindac sulfide, a nonsteroidal antiinflammatory drug [11] that is selectively re-oxidised to sulindac by hFMO3 S-oxygenation [2,25].…”