Short Communication Expression and Characterization of Functional Dog Flavin- Containing Monooxygenase

Mammalian flavin-containing monooxygenase (FMO) enzymes catalyze oxidation at nucleophilic, heteroatom centers and are important for drug, xenobiotic, and endogenous substrate metabolism. In human liver, human FMO3 (hFMO3) is the most abundant FMO isoform and is known to contribute to the hepatic clearance of a variety of clinical drugs. The purpose of the current study was to express and compare the dog (beagle) FMO3 (dFMO3) to hFMO3. A full-length dFMO3 cDNA was obtained from liver by reverse transcription-polymerase chain reaction. Using a baculovirus expression system in Spodoptera frugiperda insect cells, dFMO3 was expressed to protein levels of 0.50 nmol/mg, as determined by liquid chromatography-fluorescence detection. Expressed dFMO3 displayed Michaelis-Menten kinetics, catalyzing NADPH-dependent N-oxidation of benzydamine, with Km and Vmax values of 18.6 M and 0.63 nmol N-oxide formed/min/nmol of enzyme, respectively. Benzydamine N-oxidation catalyzed by hFMO3 showed values of 42.6 M (Km) and 3.56 nmol/min/nmol of enzyme (Vmax). Human FMO3 was observed to catalyze the S-oxidation of sulindac sulfide, with respective Km and Vmax values of 69.3 M and 35.4 nmol/min/nmol of enzyme. dFMO3 also catalyzed sulindac sulfide S-oxidation with 6.8 nmol/min/nmol of enzyme being the highest velocity observed. Finally, Western blot analysis indicated protein expression levels of dFMO3 in pooled dog liver and lung microsomes to be 27 and 9 pmol/mg, respectively. In summary, dFMO3 appears to be a functional enzyme expressed at appreciable levels in liver, but one with some kinetic properties that are substantially different from its human homolog hFMO3. The flavin-containing monooxygenases (FMOs) are a family of enzymes capable of catalyzing the oxidation of various drugs, xenobiotics, and endogenous substrates containing a soft nucleophile, usually nitrogen or sulfur (Cashman, 2000; Krueger and Williams, 2005). In humans, FMO-dependent drug metabolism can have important clinical implications (Cashman, 2000). Like cytochrome P450s (P450s), the FMOs are microsomal enzymes that require NADPH and O2, and FMOs have shown overlapping substrate specificity with P450s. FMOs also typically convert their xenobiotic substrates into more polar products that are less pharmacologically active and more easily excreted, thereby enhancing their elimination from the body (Cashman, 1995). The mammalian FMO gene family includes five different isoforms (FMO1 through FMO5) (Lawton et al., 1994). In humans, as well as in a variety of preclinical species, tissue distribution patterns of FMO isoforms have been described previously (Cashman and Zhang, 2006; Phillips and Shephard, 2008). FMO3 is the most abundantly expressed isoform in adult human liver, existing at levels similar to the major human liver P450 isoform, CYP3A4 (Haining et al., 1997). FMO3 has been observed to contribute to the metabolic clearance of a variety of drugs, e.g., cimetidine, nicotine, and tamoxifen, as well as the diet-derived substrate trimethylamine (Cashman et al., 1992, 1993; Mani et al., 1993). It has been demonstrated that FMO3 is essential for the N-oxygenation and metabolic clearance of trimethylamine (Dolphin et al., 1997; Lang et al., 1998). This finding led to the discovery that human FMO3 (hFMO3) is also a highly polymorphic gene (Koukouritaki et al., 2005). In general, a total of 29 allelic variants of FMO3 have each been observed to be associated with the human condition known as trimethylaminuria or “fish odor syndrome” (Phillips and Shephard, 2008). Preclinical species, e.g., mouse, rat, dog, monkey, serve as a valuable tool for the drug discovery and drug development process. Data obtained from in vivo metabolism and toxicology studies in these models are essential for scaling and prediction of pharmacokinetic and pharmacodynamic behavior of drug candidates to be potentially administered to humans. It is noteworthy that the accuracy of such predictions greatly depends on similarity of metabolic processes between species. This approach makes it particularly important to have a complete characterization of the metabolic pathways of a candidate compound in a given species before determination of safe doses for humans. The dog is the most widely used nonrodent species in preclinical drug safety studies (Gad and Gad, 2003). We suggest that to gain a definitive understanding of the relevance of drug metabolism in dogs to that in humans, a characterization of species differences in FMOs is necessary. The cDNA sequence for dog FMO1 (dFMO1) (Lattard et al., 2002) and a characterization of functional dFMO1 have been reported (Stevens et al., 2003). Although the cDNA sequence of dog FMO3 (dFMO3) has also been published previously, the investigators were unable to demonstrate activity following recombinant expression 1 Current affiliation: Seventh Wave Laboratories, Chesterfield, Missouri. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.027714. ABBREVIATIONS: FMO, flavin-containing monooxygenase; P450, cytochrome P450; dFMO1, dog FMO1; hFMO3, human FMO3; hFMO1, human FMO1; dFMO3, dog FMO3; FAD, flavin adenine dinucleotide; PCR, polymerase chain reaction; Sf-9, Spodoptera frugiperda; HPLC, highperformance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; DLM, dog liver microsomes. 0090-9556/09/3710-1987–1990$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 10 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 27714/3519169 DMD 37:1987–1990, 2009 Printed in U.S.A. 1987 at A PE T Jornals on Jne 6, 2017 dm d.aspurnals.org D ow nladed from of the enzyme (Lattard et al., 2002). Therefore, the objective of this study was to quantify the protein expression levels of dFMO3 in various tissues and perform a brief functional characterization of a recombinant dFMO3, a highly homologous canine form of the major human liver FMO, hFMO3. Materials and Methods Materials. Benzydamine hydrochloride, benzydamine N-oxide, sulindac sulfide, sulindac sulfoxide, clozapine, clozapine N-oxide, flavin adenine dinucleotide (FAD), and NADPH were purchased from Sigma-Aldrich (St. Louis, MO). Human FMO3 Supersomes (1.0 nmol FMO/mg by FAD content), WB-FMO3 (rabbit anti-human FMO3 antiserum), and horseradish peroxidaseconjugated goat anti-rabbit IgG were purchased from BD Gentest (Woburn, MA). Pooled human liver microsomes (mixed gender and age) and pooled microsomes from beagle dog tissues (liver, lung, kidney, and intestine) were purchased from XenoTech, LLC (Lenexa, KS). DNA and Viral Constructions. Total RNA was isolated from liver tissue freshly obtained from a single adult female beagle dog using the RNeasy Mini Kit (QIAGEN, Valencia, CA). The Pfizer Institutional Animal Care and Use Committee reviewed and approved the animal use in these studies. The animal care and use program is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International. First-strand cDNA synthesis was performed using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA) with slight modifications to the manufacturer’s suggested protocol. Oligonucleotide primers including sense (5 -GTAACTATGGGGAAAAGAGTGGC-3 ) and antisense (5 -GGAATGATAATTAGATCAATGCGAGGA-3 ) were designed based on the published dog FMO3 sequence (Lattard et al., 2002) and used in a polymerase chain reaction (PCR) with first-strand cDNA synthesis product and Platinum Pfx DNA polymerase (Invitrogen). The resulting 1600-base pair PCR product was gel-purified by agarose gel electrophoresis and ligated into pGEM-T Easy vector (Promega, Madison, WI). The cDNA was sequenced on both strands using a series of 10 primers and Gene Codes Sequencher version 4.7 (Ann Arbor, MI). Sequence analysis indicated a 1599-base pair sequence identical to the published dog FMO3 sequence (Lattard et al., 2002). The dFMO3 cDNA was digested with NotI restriction endonuclease and ligated into NotI-cleaved pFastBac1 of the Bac-to-Bac Baculovirus Expression System (Invitrogen). Recombinant baculovirus preparation and selection were performed as recommended by the manufacturer. Viral Infection of Sf-9 Cells and Sf-9 Microsome Isolation. Sf-9 insect cells were maintained in SF-900 II Serum Free Media (Invitrogen) at 27°C on an orbital shaker (90 rpm). Cells were transfected with recombinant dFMO3 bacmid DNA using Cellfectin reagent according to the manufacturer’s protocol (Invitrogen). At 72 h post-transfection, the transfection broth containing recombinant baculovirus was harvested and amplified in Sf-9 suspension cultures. The viral titer of the amplified baculovirus stock was determined by using the BaculoELISA assay (Clontech, Mountain View, CA). Cells were infected in shaker flasks at a density of 1.5 10 cells/ml and a multiplicity of infection of 1 in media supplemented with FAD (10 g/ml). Insect cells were harvested 72 h after infection and washed with sucrose buffer (pH 7.5) containing 280 mM sucrose, 25 mM HEPES, 1 mM EDTA, and protease inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). Cells were pelleted, resuspended in sucrose buffer, homogenized on ice with a glass-glass homogenizer, and microsomes were prepared by differential centrifugation. The protein concentration was determined by using BCA Protein Assay (Pierce Biotechnology, Rockford, IL). Characterization of FMO3-Containing Insect Cell Membranes. Expression levels of the recombinant dFMO3 enzyme were assessed by measurement of both holoenzyme and apoprotein. First, the FAD content of microsomes isolated from the dFMO3-baculovirus-infected and mock-infected Sf-9 cells was quantified. Accordingly, heat treatment of the samples was followed by reversed-phase high-performance liquid chromatography (HPLC) with fluorometric detection as described previously (Lang and Rettie, 2000). Given that naive Sf-9 cells contain endogenous FAD, flavin levels for dFMO3 microsomes were adjusted for levels observed in mock-infected Sf-9 cells. In addition, immunoreactive dFMO3 of these Sf-9 microsomes, as well as pooled microsomes (n 8) from dog liver, kidney, intestine (duodenum/jejunum), and lung (XenoTech, LLC) were quantified by Western blot. After separation of protein bands on a 10% SDS polyacrylamide gel (120 V, 2 h), proteins were transferred to a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen). Antibody dilutions included a 1:500 dilution of primary antibody WB-FMO3 (BD Gentest) and a 1:10,000 dilution of the secondary antibody, IRDye 680 Goat Anti-Rabbit IgG (LI-COR Biotechnologies, Lincoln, NE). According to the manufacturer (BD Gentest), WB-FMO3 is a polyclonal anti-peptide antisera antibody that was generated against amino acids 265 to 282 (KHENYGLMPNGVLRKEP) of the human FMO3 protein and does not cross-react with human FMO1. In addition, although 11 of the 21 amino acids in the hFMO3 peptide antigen correspond to the sequence for dFMO1 (Lattard et al., 2002), the Western blot analysis with WB-FMO3, which included 1 g of dFMO1 microsomes (Stevens et al., 2003) in lane 6 of Fig. 1, indicated there is no cross-reactivity with this homolog. Protein-antibody complexes were detected by measuring fluorescent signal on the Odyssey Infrared Imaging System (LI-COR Biotechnologies) and quantified using a standard curve composed of hFMO3 microsome standard (BD Gentest). Enzyme Kinetics. Based on preliminary determinations of the linearity of metabolite formation with protein concentration and incubation time, all incubation conditions were established in 0.1 M tricine (N-[2-hydroxy-1,1bis(hydroxymethyl)ethyl]glycine) buffer and 1 mM NADPH in a total volume of 500 l. Kinetic experiments for benzydamine and clozapine N-oxidation were determined at pH 7.4 and 8.4, respectively. On the other hand, kinetic experiments for sulindac sulfide metabolism were conducted at pH 9.0 to maintain sufficient substrate solubility. All incubations included negative controls that did not contain NADPH. All negative control incubations were quenched at time 0. Background metabolite levels were always subtracted from the level of metabolite produced in corresponding NADPH-containing