Supplemental material to this article can be found at: http://dmd.aspetjournals.org/content/suppl/2017/03/03/dmd.117.075184.DC1

1521-009X/45/5/497–500$25.00 https://doi.org/10.1124/dmd.117.075184 DRUG METABOLISM AND DISPOSITION Drug Metab Dispos 45:497–500, May 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Short Communication

Marmoset Flavin-Containing 3 in the Liver Is a Major Benzydamine and Sulindac Sulfide s

Received January 20, 2017; accepted March 1, 2017

ABSTRACT Common marmosets (Callithrix jacchus) are potentially primate FMO3 protein was detected by immunoblotting. FMO inhibition assays models for preclinical drug metabolism studies because there are using preheated tissue microsomes indicated that benzydamine similarities in the molecular characteristics of cytochrome P450 N-oxygenation and sulindac sulfide S-oxygenation in the marmo- between this species and humans. However, character- set liver was mainly catalyzed by FMO3, the major hepatic FMO. ization of non–cytochrome P450 enzymes has not been clarified in Marmoset FMO3 protein heterologously expressed in Escherichia coli Downloaded from marmosets. Here, we report characterization of flavin-containing effectively catalyzed benzydamine N-oxygenation and sulindac FMO1–FMO5 identified in marmoset tissues. sulfide S-oxygenation comparable to marmoset liver microsomes. Marmoset FMO forms shared high amino acid sequence identities These results indicate that the FMO3 expressed in marmoset (93%–95%) and phylogenetic closeness with human homologous livers mainly metabolizes benzydamine and sulindac sulfide (typical FMO forms. FMO1 and FMO3 mRNA were abundantly expressed in human FMO substrates), suggesting its importance for FMO-dependent the liver and kidneys among five marmoset tissues examined, where drug metabolism in marmosets. dmd.aspetjournals.org

Introduction In this study, we isolated three marmoset FMO cDNAs based on FMO Flavin-containing monooxygenases (FMOs; EC 1.14.13.8) are a cluster organization and we analyzed them for their sequence family of xenobiotic-metabolizing enzymes involved in the oxygenation identity, tissue expression, and catalytic activities using recombinant of a broad range of chemicals containing nitrogen, sulfur, or phospho- proteins heterologously expressed in Escherichia coli. This work is of at ASPET Journals on October 1, 2021 rous (Krueger and Williams, 2005). In humans, functional importance for understanding the fundamental characteristics and – FMO1–FMO5 (Lawton et al., 1994) and a nonfunctional pseudogene functions of marmoset FMOs and the use of this species as a non FMO6 (Hines et al., 2002) have been identified, and their mRNAs are human primate model in preclinical drug development. expressed in various tissues. FMO3 is considered a major functional FMO enzyme in the human liver and contributes to the metabolism of the anti-inflammatory drugs benzydamine and sulindac sulfide and diet- Materials and Methods derived trimethylamine (Shimizu et al., 2015). Detailed methods are provided separately in the Supplemental Material. FMO The common marmoset (Callithrix jacchus) is a useful non–human cDNAs were isolated by reverse transcription (RT)-polymerase chain reaction primate species for pharmacokinetics studies because it has cytochrome (PCR) with cDNA libraries transcribed from total RNA from marmoset tissues as P450 (EC 1.14.14.1) characteristic features that are significantly similar described (Uehara et al., 2015). The structure of primate FMO gene clusters was determined by BLAT (UCSC Genome Bioinformatics; University of California, to humans (Uno et al., 2016). De novo transcriptome analysis indicate Santa Cruz, CA). Multiple alignment of amino acid sequences and phylogenetic that FMO1-, FMO3-, FMO4-, and FMO5-like genes are expressed in the analysis were performed with the GENETYX system (Software Development, marmoset liver, kidneys, and intestines (Shimizu et al., 2014). FMO3 Tokyo, Japan) and DNASIS Pro (Hitachi Software, Tokyo, Japan), respectively. effectively catalyzes the N-oxygenation of potential proneurotoxin The FMO amino acid sequences used were from GenBank (National Center for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the marmoset liver Biotechnology Information, Bethesda, MD). Pooled liver microsomes from (Uehara et al., 2015). To date, only two FMO forms have been marmosets (n = 5 males) were purchased from Corning Life Sciences (Woburn, identified; however, little information on the molecular characteristics MA). Pooled microsomes of the brain, lungs, liver, kidneys, and small intestine in marmosets is available (Uehara et al., 2015). were prepared from tissue samples of 20 marmosets (10 males and 10 females, aged .2 years) at the Central Institution for Experimental Animals (Kawasaki, Japan) as described (Uehara et al., 2016a) and with approval from the Institutional Animal Care and Use Committee. FMO mRNA distribution in the brain, lungs, This research was supported partly by the Japan Society for the Promotion of liver, kidneys, and small intestine, each pooled from six male and six female adult Science [Grant-in-Aid for Young Scientists B 15K18934 (to S.U.)]. This work marmosets (aged .2 years), was analyzed by real-time RT-PCR as described resulted from “Construction of System for Spread of Primate Model Animals” (Uehara et al., 2016b). Recombinant marmoset FMO1 and FMO3 were under the Strategic Research Program for Brain Sciences of the Japan Agency for heterologously expressed in E. coli using pET30 vectors (Novagen, Madison, Medical Research and Development. WI) as described (Yamazaki et al., 2014). Tissue microsomes (10 mg), S.U., M.S., and Y.U. contributed equally to this work. microsomes from five individual livers, and recombinant FMO3 protein (0.1 https://doi.org/10.1124/dmd.117.075184. pmol) were separated on a 10% SDS-polyacrylamide gel by electrophoresis and s This article has supplemental material available at dmd.aspetjournals.org. were transferred to a polyvinylidene difluoride membrane (Uehara et al., 2016a).

ABBREVIATIONS: FMO, flavin-containing monooxygenase; PCR, polymerase chain reaction; RT, reverse transcription.

497 498 Uehara et al.

Fig. 1. Determination of FMO mRNA (A) and FMO protein (B and C) levels in marmoset tissues. (A) Expression levels of marmoset FMO1– FMO5 mRNAs in five marmoset tissues (each pool of six male and six female marmosets) were measured by real-time RT-PCR. Raw values of target gene expression were normalized with the 18S rRNA level. Each data point represents the average and S.D. of triplicate determinations from three representative experiments. (B and C) Pooled tissue microsomes from marmosets (20 mg/lane) (B) and recombinant marmoset FMO3 protein (0.1 pmol FMO3/lane), individual liver microsomes from marmosets (male, lanes 1–2; female, lanes 3–5) (20 mg/lane) (C) were

analyzed by immunoblotting using anti-human FMO3 antibodies. Protein Downloaded from disulfide isomerase (PDI) expression was assessed as a loading control. dmd.aspetjournals.org

Benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities by 532–556 amino acid residues, respectively (Supplemental Fig. 2), and recombinant FMO proteins and tissue microsomes were measured by high- had functionally important regions (FAD- and NADPH-pyrophosphate at ASPET Journals on October 1, 2021 performance liquid chromatography as described (Yamazaki et al., 2014). For binding sites), and the two characteristic FMO pentapeptides (EGLEP FMO inactivation, liver or kidney microsomes were preheated at 45 C for and FATGY). Marmoset FMO1–FMO5 shared high amino acid 5 minutes without an NADPH-generating system (Taniguchi-Takizawa et al., sequence identities (93%–95%) with human FMO counterparts (Sup- 2015). All other reagents used were of the highest quality commercially available. plemental Table 1) and were phylogenetically more closely clustered with the corresponding primate orthologs than other species (Supple- Results and Discussion mental Fig. 3). Interestingly, in cynomolgus monkeys, FMO6 is a Analysis of common marmoset’s genome sequence showed that functional enzyme that is widely expressed in the kidneys, heart, testes, FMO1–FMO4 and FMO6 genes were localized in the gene cluster; the uterus, and liver (Uno et al., 2013); although we have tried, we failed to FMO5 gene was localized outside this cluster, in marmoset clone FMO6 cDNA from marmoset tissues. 18 (Supplemental Fig. 1). Marmoset FMO genes had one-to-one To investigate the tissue distribution of FMO1–FMO5 mRNAs and orthologous relationships with human FMO genes, even though FMO proteins in marmosets, real-time RT-PCR was performed to measure gene cluster organization is different between marmosets and humans. expression levels of FMO1–FMO5 in pooled brains, lungs, livers, We successfully isolated FMO1–FMO5 cDNA in the marmoset liver by kidneys, and small intestine. FMO3 mRNA was the most abundant in RT-PCR. Marmoset FMO1–FMO5 contained open reading frames of the liver and kidneys, followed by the lungs (Fig. 1A), whereas FMO1

TABLE 1 Kinetic parameters of benzydamine N-oxygenation and sulindac sulfide S-oxygenation by recombinant FMO proteins and liver microsomes from marmosets Kinetic parameters were calculated from fitted curves by nonlinear regression and are presented as means 6 S.E.

Benzydamine N-Oxygenation Sulindac Sulfide S-Oxygenation Enzyme Source a b Km Vmax Vmax/Km Km Vmax Vmax/Km

mM nmol/min per mg protein ml/min per mg protein mM nmol/min per mg protein ml/min per mg protein Liver microsomes 47 6 9 3.8 6 0.2 0.081 23 6 14 2.7 6 0.4 0.12 Marmoset FMO1 40 6 10 0.76 6 0.04 0.019 43 6 15 23 6 2 0.53 Marmoset FMO3 21 6 4276 1 1.3 38 6 13 31 6 2 0.80

Each substrate (10–1000 mM) was incubated with liver microsomes (0.05 mg protein) and recombinant proteins (10 pmol equivalent) at 37C for 10–15 minutes in the presence of an NADPH-generating system. a Vmax values for marmosets are given in nanomoles per minute per nanomole FMO. b Vmax/Km values for marmosets are given in milliliters per minute per nanomole FMO. Cloning of Marmoset FMO cDNAs 499 mRNA was also expressed abundantly in the liver and kidneys but not as had similar enzymatic properties, suggesting the similarity of FMO- much as FMO3 mRNA, the same as results previously reported by de dependent drug metabolism for marmosets and humans. novo transcriptome analysis (Shimizu et al., 2014). Indeed, FMO3 Laboratory of Drug Metabolism and SHOTARO UEHARA protein (approximately 50 kDa) was detected immunologically with Pharmacokinetics, Showa MAKIKO SHIMIZU anti-human FMO3 antibodies in pooled marmoset livers and kidneys Pharmaceutical University, YASUHIRO UNO (Fig. 1B), with a small nonspecific unknown band. No immunoreactive Machida, Tokyo, Japan (S.U., M.S., TAKASHI INOUE bands in these tissue microsomes were seen with commercial anti- H.Y.); Pharmacokinetics and ERIKA SASAKI human FMO1 antibodies (results not shown). Similar to marmosets, Bioanalysis Center, Shin Nippon HIROSHI YAMAZAKI FMO3 is postnatally expressed in the kidneys of rabbits and rats (Ripp Biomedical Laboratories, Ltd., et al., 1999). FMO1 is reportedly expressed postnatally in the liver of Kainan, Wakayama, Japan (Y.U.); dogs (Lattard et al., 2002), rabbits (Shehin-Johnson et al., 1995), rats Department of Applied (Novick et al., 2009), and mice (Itoh et al., 1997). Human FMO1, Developmental Biology (T.I.) and FMO2, and FMO3 are predominantly expressed in the kidneys, lungs, Center of Applied Developmental and liver, respectively, whereas FMO4 and FMO5 are widely expressed Biology (E.S.), Central Institute for in various tissues (Zhang and Cashman, 2006; Uno et al., 2013). In Experimental Animals, Kawasaki, humans, FMO1 is expressed in the fetal liver, but its expression is Japan; and Keio Advanced Research rapidly extinguished after birth (Koukouritaki et al., 2002). Apparent sex Center, Keio University, Minato-ku, differences in FMO3 protein expression levels in the liver (Ripp et al., Downloaded from Tokyo, Japan (E.S.) 1999) were found in mice, but not in marmosets (Fig. 1A). Marmoset FMO2 and FMO5 mRNA were dominantly expressed in the lungs and Authorship Contributions liver, whereas FMO4 mRNA was expressed in the liver, kidneys, and Participated in research design: Uehara, Shimizu, Uno, Yamazaki. small intestine at very low levels, similar to human and cynomolgus Conducted experiments: Uehara, Shimizu. monkey FMO forms (Zhang and Cashman, 2006; Uno et al., 2013). Contributed new reagents or analytic tools: Inoue, Sasaki.

These results suggested that tissue distribution of FMO3 and FMO1 was Performed data analysis: Uehara, Shimizu, Uno, Yamazaki. dmd.aspetjournals.org partially different between marmosets and humans. Wrote or contributed to the writing of the manuscript: Uehara, Shimizu, Uno, To assess the importance of FMO forms in drug oxidation in the Yamazaki. marmoset liver, drug oxygenation activities by liver microsomes preheated for FMO inhibition were measured. Preheat-sensitive benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities in marmo- References set liver and kidney microsomes was found (Supplemental Table 2). Hines RN, Hopp KA, Franco J, Saeian K, and Begun FP (2002) Alternative processing of the human FMO6 gene renders transcripts incapable of encoding a functional flavin-containing Recombinant FMO3-mediated benzydamine N-oxygenation activity monooxygenase. Mol Pharmacol 62:320–325. at ASPET Journals on October 1, 2021 and its Vmax/Km value were higher than those for recombinant FMO1, Itoh K, Nakamura K, Kimura T, Itoh S, and Kamataki T (1997) Molecular cloning of mouse liver suggesting that FMO3 abundantly expressed in the liver plays a flavin containing monooxygenase (FMO1) cDNA and characterization of the expression prod- uct: metabolism of the neurotoxin, 1,2,3,4-tetrahydroisoquinoline (TIQ). J Toxicol Sci 22:45–56. role in the N-oxygenation of benzydamine in the marmoset liver Koukouritaki SB, Simpson P, Yeung CK, Rettie AE, and Hines RN (2002) Human hepatic flavin- (Table 1), similar to the human liver (Taniguchi-Takizawa et al., containing monooxygenases 1 (FMO1) and 3 (FMO3) developmental expression. Pediatr Res 51:236–243. 2015). Kinetic analyses also indicated that marmoset liver micro- Krueger SK and Williams DE (2005) Mammalian flavin-containing monooxygenases: structure/ function, genetic polymorphisms and role in drug metabolism. Pharmacol Ther 106:357–387. somes effectively catalyzed sulindac sulfide S-oxygenation (Vmax/Km, Lattard V, Longin-Sauvageon C, Lachuer J, Delatour P, and Benoit E (2002) Cloning, sequencing, 0.12 ml/min per milligram protein) (Table 1), compared with those of and tissue-dependent expression of flavin-containing monooxygenase (FMO) 1 and FMO3 in the dog. Drug Metab Dispos 30:119–128. humans (Vmax/Km, 0.02 ml/min per milligram protein), as previously Lawton MP, Cashman JR, Cresteil T, Dolphin CT, Elfarra AA, Hines RN, Hodgson E, Kimura T, reported (Yamazaki et al., 2014). Marmoset FMO3 was catalytically Ozols J, Phillips IR, et al. (1994) A nomenclature for the mammalian flavin-containing mono- efficient (Vmax/Km, 0.80 ml/min per nanomole) for sulindac sulfide oxygenase gene family based on amino acid sequence identities. Arch Biochem Biophys 308: m 254–257. S-oxygenation and showed a low Km value (38 M), comparable to Novick RM, Mitzey AM, Brownfield MS, and Elfarra AA (2009) Differential localization of marmoset liver microsomes (23 mM), similar to human FMO3 flavin-containing monooxygenase (FMO) isoforms 1, 3, and 4 in rat liver and kidney and m evidence for expression of FMO4 in mouse, rat, and human liver and kidney microsomes. J (Yamazaki et al., 2014). Marmoset FMO1 showed low Km (43 M) Pharmacol Exp Ther 329:1148–1155. and high Vmax/Km (0.53 ml/min per nanomole), compared with human Ripp SL, Itagaki K, Philpot RM, and Elfarra AA (1999) Species and sex differences in expression m of flavin-containing monooxygenase form 3 in liver and kidney microsomes. Drug Metab FMO1 (Km,280 M; Vmax/Km, 0.01 ml/min per nanomole). Considering Dispos 27:46–52. this together with tissue distribution, FMO1 might account for the Shehin-Johnson SE, Williams DE, Larsen-Su S, Stresser DM, and Hines RN (1995) Tissue-specific potential species differences in sulindac sulfide S-oxygenation rates expression of flavin-containing monooxygenase (FMO) forms 1 and 2 in the rabbit. JPharmacol Exp Ther 272:1293–1299. between marmoset and human livers. These results indicated that Shimizu M, Iwano S, Uno Y, Uehara S, Inoue T, Murayama N, Onodera J, Sasaki E, and Yamazaki benzydamine N-oxygenation and sulindac sulfide S-oxygenation in the H (2014) Qualitative de novo analysis of full length cDNA and quantitative analysis of gene expression for common marmoset (Callithrix jacchus) transcriptomes using parallel long-read marmoset liver were mainly catalyzed by FMO3. technology and short-read sequencing. PLoS One 9:e100936. In summary, marmoset FMO1–FMO5 had amino acid sequences that Shimizu M, Shiraishi A, Sato A, Nagashima S, and Yamazaki H (2015) Potential for drug inter- actions mediated by polymorphic flavin-containing monooxygenase 3 in human livers. Drug were highly identical (.93%) to human FMO1–FMO5, as well as a Metab Pharmacokinet 30:70–74. phylogenetically close relationship with human FMO1–FMO5. In Taniguchi-Takizawa T, Shimizu M, Kume T, and Yamazaki H (2015) Benzydamine N-oxygenation as an index for flavin-containing monooxygenase activity and benzydamine contrast with humans, FMO3 and FMO1 mRNA was abundant in the N-demethylation by cytochrome P450 enzymes in liver microsomes from rats, dogs, monkeys, marmoset liver and kidneys among five FMO forms. Recombinant and humans. Drug Metab Pharmacokinet 30:64–69. Uehara S, Uno Y, Inoue T, Murayama N, Shimizu M, Sasaki E, and Yamazaki H (2015) Activation marmoset FMO3 enzymes heterologously expressed in E. coli effec- and deactivation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by cytochrome P450 enzymes tively metabolized typical human FMO substrates, benzydamine and and flavin-containing monooxygenases in common marmosets (Callithrix jacchus). Drug Metab – Dispos 43:735–742. sulindac sulfide. These results indicated that marmoset FMO1 FMO5 Uehara S, Uno Y, Ishii S, Inoue T, Sasaki E, and Yamazaki H (2016a) Marmoset cytochrome P450 has sequences that are similar to those of humans but are partially 4A11, a novel arachidonic acid and lauric acid v-hydroxylase expressed in liver and kidney tissues. Xenobiotica DOI: 10.1080/00498254.2016.1206673 [published ahead of print]. different from humans in terms of tissue expression. Importantly, Uehara S, Uno Y, Suzuki T, Inoue T, Utoh M, Sasaki E, and Yamazaki H (2016b) Strong induction marmoset and human FMO3, a major hepatic FMO in both species, of cytochrome P450 1A/3A, but not P450 2B, in cultured hepatocytes from common marmosets 500 Uehara et al.

and cynomolgus monkeys by typical human P450 inducing agents. Drug Metab Lett DOI: Zhang J and Cashman JR (2006) Quantitative analysis of FMO gene mRNA levels in human 10.2174/1872312810666161114144412 [published ahead of print]. tissues. Drug Metab Dispos 34:19–26. Uno Y, Shimizu M, and Yamazaki H (2013) Molecular and functional characterization of flavin- containing monooxygenases in cynomolgus macaque. Biochem Pharmacol 85:1837–1847. Uno Y, Uehara S, and Yamazaki H (2016) Utility of non-human primates in drug development: Address correspondence to: Dr. Hiroshi Yamazaki, Laboratory of Drug Metab- comparison of non-human primate and human drug-metabolizing cytochrome P450 enzymes. olism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165 Higashi- Biochem Pharmacol 121:1–7. tamagawa Gakuen, Machida, Tokyo 194-8543, Japan. E-mail: hyamazak@ac. Yamazaki M, Shimizu M, Uno Y, and Yamazaki H (2014) Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and shoyaku.ac.jp minipigs. Biochem Pharmacol 90:159–165. Downloaded from dmd.aspetjournals.org at ASPET Journals on October 1, 2021 DMD # 75184

Supplemental Data

Marmoset flavin-containing monooxygenase 3 in liver is a major benzydamine and sulindac sulfide oxygenase

Shotaro Uehara, Makiko Shimizu, Yasuhiro Uno, Takashi Inoue, Erika Sasaki, and Hiroshi

Yamazaki

Drug Metabolism and Disposition

Supplemental Methods

FMO cDNA cloning

FMO cDNAs were isolated by reverse transcription (RT)-polymerase chain reaction (PCR) with cDNA libraries transcribed from total RNA from marmoset tissues (FMO2, lungs; FMO4 and FMO5, livers) as described previously (Uehara et al., 2015). Briefly, the first strand cDNA libraries were synthesized with total RNA from marmoset tissues, oligo (dT) (Invitrogen,

Carlsbad, CA), and SuperScript III RT reverse transcriptase (Invitrogen) following the instructions of the manufacturer. PCR was carried out using KOD-Plus-Neo DNA polymerase

(Toyobo, Osaka, Japan) with conditions consisting of an initial denaturation step at 98 °C for

2 min; 30 cycles of denaturation at 98 °C for 15 s, annealing at 60 °C for 30 s, and extension at 68 °C for 3 min; and a final extension at 68 °C for 7 min. The PCR primers used were 5-

CAGCACACAACCCAGGAAGA-3 and 5-CCCTCTCACTTGTTCTACCTAAC-3 for

FMO2, 5-GCCCAAACCTCCTACTCCTC-3 and 5-CAGCCCTCAGAACCAATCAG-3 for

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FMO4, and 5-GCCACACGTGAAGATCTCTG-3 and 5-

TGGGCAATATGAAGCTGAGTG-3 for FMO5. Amplified DNA fragments were cloned into pGEM-T easy vectors (Promega, Madison, WI) and then sequenced with an ABI PRISM 3730

DNA Analyzer (Applied Biosystems, Foster City, CA).

Sequence analysis

The structure of primate FMO gene clusters was determined by BLAT (UCSC Genome

Bioinformatics, University of California, Santa Cruz, CA). Multiple alignment of amino acid sequences and phylogenetic analysis were performed by Genetyx system (Software

Development, Tokyo, Japan) and DNASIS Pro (Hitachi Software, Tokyo, Japan), respectively.

FMO amino acid sequences used were from GenBank (National Center for Biotechnology

Information, Bethesda, MD): human FMO1 (NP_001269621), FMO2 (NP_001451), FMO3

(NP_008825), FMO4 (NP_002013), and FMO5 (NP_001452); chimpanzee FMO2

(NP_001009008) and FMO3 (NP_001009092); orangutan FMO1 (NP_001126721), FMO2

(NP_001124835), FMO3 (NP_001124820), and FMO4 (NP_001127523); cynomolgus monkey FMO1 (AHH29283), FMO2 (AHH29286), FMO3 (AHH29287), FMO4

(NP_001306349), FMO5 (AHH29291), and FMO6 (AHH29292); rhesus monkey FMO1

(NP_001305113), FMO2 (NP_001036242), and FMO3 (NP_001028065); dog FMO1

(NP_001003061) and FMO3 (NP_001003060); pig FMO1 (NP_999229); rabbit FMO1

(NP_001075754), FMO2 (NP_001075753), FMO3 (NP_001075715), FMO4

(NP_001076253), and FMO5 (NP_001075714); guinea pig FMO5 (NP_001166418); rat

FMO1 (NP_036924), FMO2 (NP_653338), FMO3 (NP_445885), FMO4 (NP_653147),

FMO5 (NP_653340), and FMO9 (NP_001102936); mouse FMO1 (NP_034361), FMO2

(NP_061369), FMO3 (NP_032056), FMO4 (NP_659127), FMO5 (NP_001155237), FMO6

(NP_001171509), FMO9 (NP_766432), FMO12 (NP_001157784), and FMO13 2

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(NP_001157778); Caenorhabditis elegans FMO (NP_001300132).

Real-time reverse transcription-PCR

FMO mRNA distribution in brains, lungs, livers, kidneys, and small intestines, each pooled from 6 male and 6 female adult marmosets (>2 years old) was analyzed by real-time RT-PCR as described previously (Uehara et al., 2016b). Briefly, RT sample was prepared from tissue total RNA as described above using random hexamers. PCR amplification was performed with

SYBR Green-based detection system using primers; 5-GGGTTCCATGATACCCACAG-3 and 5-TGCAATAGCACAAGCCAAAC-3 for FMO1, 5-AGCCGCCAATACAAACATCC-

3 and 5-AGATACGGCTCATGACCCAG-3 for FMO2, 5-

CTCCTTCATTGGAGCAAAGC-3 and 5-TCCTGACAACTCGTGTCTGC-3 for FMO3,

5-AGACAGAGGGCAAGCAGAAT-3 and 5-TCCTCCAGTGTTCCCAAGAC-3 for

FMO4, and 5-CATTGCAGAGCTCCAAGGAC-3 and 5-CCCTGAATGGTATGGCGTTG-

3 for FMO5. Real-time PCR was performed using Power SYBR Green PCR Master Mix

(Applied Biosystems) and 400 nM gene-specific primers with an ABI PRISM 7300 sequence detection system (Applied Biosystems) according to the manufacturer’s protocols. The relative expression level of each FMO mRNA was determined by normalizing to the level of 18S rRNA.

Immunoblotting

Anti-human FMO3 antibodies (ab126790) and anti-6×histidine antibodies (F008) were

purchased from Abcam (Cambridge, MA) and BioDynamics Laboratory (Tokyo, Japan),

respectively. Anti-human protein disulfide isomerase (PDI) antibodies (H-160), goat anti-rabbit

IgG-horseradish peroxidase, and goat anti-mouse IgG-horseradish peroxidase were purchased

from Santa Cruz Biotechnology (Santa Cruz, CA). According to methods previously reported

(Uehara et al., 2016a), recombinant FMO3 proteins (0.1 pmol) or tissue microsomes (10 μg)

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The protein bands were visualized by an ECL Prime Western Blotting Detection System (GE

Healthcare, Buckinghamshire, UK).

Enzyme assay

Recombinant marmoset FMO1 and FMO3 proteins were heterologously expressed in

Escherichia coli using pET30 vectors (Novagen, Madison, WI) and purified using HisPur Ni-

NTA Purification Kit (Thermo Fisher Scientific, Waltham, MA) as described previously

(Uehara et al., 2015; Yamazaki et al., 2014). Protein concentration was quantified by immunoblotting using anti-his-tag antibodies as described previously (Yamazaki et al., 2014).

Benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities by recombinant

FMO proteins and tissue microsomes were measured by HPLC as described previously

(Yamazaki et al., 2014). Briefly, the reaction mixture consisted of 5-25 pmol/mL recombinant protein or 0.1 mg/mL microsomes, 5 μM benzydamine or 20-300 μM sulindac sulfide, an

NADPH-generating system (0.25 mM NADP+, 2.5 mM glucose 6-phosphate, and 0.25 units/mL glucose 6-phosphate dehydrogenase), and 100 mM potassium phosphate buffer (pH

8.4) in a total volume of 0.20 mL. After incubation at 37°C for 10-15 min, metabolite concentrations were quantified by HPLC. Kinetic parameters were estimated from the fitted curves employing the Michaelis-Menten equation using the KaleidaGraph program (Synergy

Software, Reading, PA).

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References

Uehara S, Uno Y, Inoue T, Murayama N, Shimizu M, Sasaki E, and Yamazaki H (2015) Activation and deactivation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine by cytochrome P450 enzymes and flavin-containing monooxygenases in common marmosets (Callithrix jacchus). Drug Metab Dispos 43:735-742.

Uehara S, Uno Y, Ishii S, Inoue T, Sasaki E, and Yamazaki H (2016a) Marmoset cytochrome P450 4A11, a novel arachidonic acid and lauric acid omega-hydroxylase expressed in liver and kidney tissues. Xenobiotica, in press (http://dx.doi.org/10.1080/00498254.2016.1206673).

Uehara S, Uno Y, Suzuki T, Inoue T, Utoh M, Sasaki E, and Yamazaki H (2016b) Strong induction of cytochrome P450 1A/3A, but not P450 2B, in cultured hepatocytes from common marmosets and cynomolgus monkeys by typical human P450 inducing agents. Drug Metab Lett, in press (http://dx.doi.org/10.2174/1872312810666161114144412).

Yamazaki M, Shimizu M, Uno Y, and Yamazaki H (2014) Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and minipigs. Biochem Pharmacol 90:159-165.

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Supplemental Table 1

Identities of amino acid sequences between marmoset and human FMO forms.

Marmoset Human Identity (%)

FMO form FMO form cDNA Amino acid

FMO1 FMO1 96 95

FMO2 FMO2 96 95

FMO3 FMO3 94 93

FMO4 FMO4 95 94

FMO5 FMO5 95 93

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Supplemental Table 2

Benzydamine N-oxygenation activities by marmoset liver and kidney microsomes and recombinant FMO proteins

Enzyme source Benzydamine N- Sulindac sulfide S-

oxygenation oxygenation

nmol/min/mg protein nmol/min/mg protein

Liver microsomes 1.7 0.29

Preheated liver 0.085 0.16

microsomes

Kidney microsomes 0.48 0.11

Preheated kidney 0.010 0.057

microsomes

nmol/min/nmol FMO nmol/min/nmol FMO

Marmoset FMO1 0.55 2.40

Marmoset FMO3 4.64 3.60

For FMO inactivation, microsomes from marmoset livers and kidneys were preheated at 45°C for 5 min without NADPH-generating system. The substrates (5 µM) were incubated with recombinant FMOs and microsomes from marmoset livers and kidneys for 10 min in the presence of an NADPH-generating system at 37°C.

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Supplemental Figure 1

FMO gene cluster in marmosets and humans.

Marmoset FMO1-6 genes were localized on marmoset chromosome 18 using BLAT. FMO1- 4 and FMO6 genes were localized in FMO gene cluster between PRRC2C and MROH9 genes, whereas FMO5 gene was localized outside this cluster. Sizes of the genes and the distance between the genes are not proportionate to actual measurement.

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Supplemental Figure 2

Multiple alignment of FMO amino acid sequences from marmosets and humans.

Amino acid sequences of marmoset (cj) FMO1-5 were aligned with those of human (h) FMO 1-5. FAD- and NADPH-pyrophosphate-binding sites and the two characteristic FMO pentapeptides (EGLEP and FATGY) are shown by solid and double solid lines, respectively. Amino acids completely and relatively conserved are shown by asterisks and dots, respectively.

FAD-binding site EGLEP cjFMO1 1:MGKR-VAIVG AGVSGLASIK CCLEEGLEPT CFERSDDLGG LWRFTEHVEE GRASLYESVV SNSCKEMSCY SDFPFPEDYP NYVPNSQFLE YLKMYANHFN 99 cjFMO2 1:MAKK-VAVIG AGVSGLISLK CCVDEGLEPT CFERTEDIGG VWRFKENVED GRASIYQSVI TNTSKEMSCF SDFPMPEDFP NFLHNSKLLE YFRMFAKKFD 99 cjFMO3 1:MGKK-VAIIG AGVSGLASIR SCLEEGLEPT CFEKSNDIGG LWKFSDHAEE GRASIYKSVF TNSSKEMMCF PDFPYPDDFP NFMHNSKIQE YIIAFAKEKN 99 cjFMO4 1:MAKK-VAVIG AGVSGLSSIK CCLDEDLEPT CFERSNDIGG LWKFTESSKD GMTRVYKSLV TNVCKEMSCY SDFPFREDYP NFMNHEKFWG YLHEFAEHFD 99 cjFMO5 1:MTKKRIAVIG GGVSGLSSIK CCLEEGLEPV CFERTDDIGG LWRFQENPEE GRASIYKSVI INTSKEMMCF SDYPIPDHYP NFMHNTQVLE YFRMYAKEFG 100 hFMO1 1:MAKR-VAIVG AGVSGLASIK CCLEEGLEPT CFERSDDLGG LWRFTEHVEE GRASLYKSVV SNSCKEMSCY SDFPFPEDYP NYVPNSQFLE YLKMYANHFD 99 hFMO2 1:MAKK-VAVIG AGVSGLISLK CCVDEGLEPT CFERTEDIGG VWRFKENVED GRASIYQSVV TNTSKEMSCF SDFPMPEDFP NFLHNSKLLE YFRIFAKKFD 99 hFMO3 1:MGKK-VAIIG AGVSGLASIR SCLEEGLEPT CFEKSNDIGG LWKFSDHAEE GRASIYKSVF SNSSKEMMCF PDFPFPDDFP NFMHNSKIQE YIIAFAKEKN 99 hFMO4 1:MAKK-VAVIG AGVSGLSSIK CCVDEDLEPT CFERSDDIGG LWKFTESSKD GMTRVYKSLV TNVCKEMSCY SDFPFHEDYP NFMNHEKFWD YLQEFAEHFD 99 hFMO5 1:MTKKRIAVIG GGVSGLSSIK CCVEEGLEPV CFERTDDIGG LWRFQENPEE GRASIYKSVI INTSKEMMCF SDYPIPDHYP NFMHNAQVLE YFRMYAKEFD 100 * *. .*..* .***** *.. .*..*.***. ***.. *.** .*.* . .. *....*.*. * .***.*. .*.* ....* *...... * .*. .. NADPH-binding site cjFMO1 100:LLKHIQFKTK VCSVTKCSDF TVSGQWEVVT LHKEKQESAI FDAVMVCTGF LTNPYLPLDS FPGINAFKGQ YFHSRQYKHP DIFKDKRVLV IGMGNSGTDI 199 cjFMO2 100:LLKYIQFQTT VLSVRKCPDF SSSGQWKVVT QSNGKEQSAV FDAVMVCSGH HILPHIPLKS FPGIERFKGQ YFHSRQYKHP DGFEGNRILV IGMGNSGSDI 199 cjFMO3 100:LLKYIQFKTF VSHVNKRPDF AMTGQWDVIT ERDGQKESTV FDAVMVCSGH HVYPNLPKES FPGLEHFKGK CFHSRDYKEP GVFKGKRVLV VGLGNSGCDI 199 cjFMO4 100:LLKYIQFKTT VCSITKRPDF FTTGQWDVVT ETEGKQNRAV FDAVMVCTGH FLNPHLPLEA FPGIHKFKGQ ILHSQEYKIP EVFQGKRVLV IGLGNTGGDV 199 cjFMO5 101:LLKYIRFKTT VCTVKKRPDF ATSGQWEVVT ESEGKKETDV FDAVMVCTGH HTNAHLPLDS FPGIEKFNGQ YFHSRDYKNP EGFTGKRVII IGIGNSGGDL 200 hFMO1 100:LLKHIQFKTK VCSVTKCSDS AVSGQWEVVT MHEEKQESAI FDAVMVCTGF LTNPYLPLDS FPGINAFKGQ YFHSRQYKHP DIFKDKRVLV IGMGNSGTDI 199 hFMO2 100:LLKYIQFQTT VLSVRKCPDF SSSGQWKVVT QSNGKEQSAV FDAVMVCSGH HILPHIPLKS FPGMERFKGQ YFHSRQYKHP DGFEGKRILV IGMGNSGSDI 199 hFMO3 100:LLKYIQFKTF VSSVNKHPDF ATTGQWDVTT ERDGKKESAV FDAVMVCSGH HVYPNLPKES FPGLNHFKGK CFHSRDYKEP GVFNGKRVLV VGLGNSGCDI 199 hFMO4 100:LLKYIQFKTT VCSITKRPDF SETGQWDVVT ETEGKQNRAV FDAVMVCTGH FLNPHLPLEA FPGIHKFKGQ ILHSQEYKIP EGFQGKRVLV IGLGNTGGDI 199 hFMO5 101:LLKYIRFKTT VCSVKKQPDF ATSGQWEVVT ESEGKKEMNV FDGVMVCTGH HTNAHLPLES FPGIEKFKGQ YFHSRDYKNP EGFTGKRVII IGIGNSGGDL 200 ***.*.*.*. *... * .*. .*** *.* ...... **.****.*...... *. . ***. *.*. ..**. ** * * ..*... .* **.* *.

cjFMO1 200:AVEASHLAKK VFLSTTGGAW VISRIFDSGY PWDMVFMTRF QNMLRNSLPT PIVTWLMARK INNWLNHANY GLMPDDRTQL KEFVLNDELP GRIITGKVFI 299 cjFMO2 200:AVELSKKAAQ VFISTRHGTW VMSRISEDGY PWDSVFHTRF RSMLRNVLPR TIVKWMIEQQ MNRWFNHENY GLEPQNKYVM KEPVLNDDLP SRLLYGAIKV 299 cjFMO3 200:ATELSHTAEQ VIISSRSGSW VMSRVWDNGY PWDMVLVTRF GTFLKNNLPT AISDWLYMKE MNARFKHENY GLMPLNGALR KEPVFNDDLP ARILCGTVSI 299 cjFMO4 200:AVELSRTAAQ VLLSTRTGTW VLGRSSAWGY PYNMMITRRC YSFIAQVLPS RFLNWIQERK LNKRFNHEDY GLSITKGKQA KFIVNDE-LP SCILCGSITI 298 cjFMO5 201:AVEISHTAKQ VFLSTRRGAW ILNRVGDYGY PADVLLSSRL KYFMSKICGQ SITNTYLERK MNQRFDHEMF GLKPKHRALS QHPTINDDLP NRIISGMVKV 300 hFMO1 200:AVEASHLAEK VFLSTTGGGW VISRIFDSGY PWDMVFMTRF QNMLRNSLPT PIVTWLMERK INNWLNHANY GLIPEDRTQL KEFVLNDELP GRIITGKVFI 299 hFMO2 200:AVELSKNAAQ VFISTRHGTW VMSRISEDGY PWDSVFHTRF RSMLRNVLPR TAVKWMIEQQ MNRWFNHENY GLEPQNKYIM KEPVLNDDVP SRLLCGAIKV 299 hFMO3 200:ATELSRTAEQ VMISSRSGSW VMSRVWDNGY PWDMLLVTRF GTFLKNNLPT AISDWLYVKQ MNARFKHENY GLMPLNGVLR KEPVFNDELP ASILCGIVSV 299 hFMO4 200:AVELSRTAAQ VLLSTRTGTW VLGRSSDWGY PYNMMVTRRC CSFIAQVLPS RFLNWIQERK LNKRFNHEDY GLSITKGKKA KFIVNDE-LP NCILCGAITM 298 hFMO5 201:AVEISQTAKQ VFLSTRRGAW ILNRVGDYGY PADVLFSSRL THFIWKICGQ SLANKYLEKK INQRFDHEMF GLKPKHRALS QHPTLNDDLP NRIISGLVKV 300 *.*.* .* . *..*.. * * . .* . ** *... .*...... * ...*... ** ...... * ... * . FATGY cjFMO1 300:RPSIKEVKEN SVIFNNTSKE EPIDIIVFAT GYTFAFPFLD ESVVKVEDGQ ASLYKYIFPA NLQKSTLAII GLIKPLGSMI PTGETQARWA VRVLKGVNKL 399 cjFMO2 300:KSTVKELTET SAIFEDGTVE ENIDVIVFAT GYTFSFPFLE DSLVKAENNM VSLYKYIFPP HLEKSTLACI GLIQPLGSIF PTAELQARWV TRVFKGLCSL 399 cjFMO3 300:KPNVKEFTET SAIFEDGTIF EGIDCVIFAT GYSYSYPFLD ESIIKSRNNE IILFKGVFPP LLEKSTLAVI GFVQSLGAAI PTADLQSRWA AQVVKGTCTL 399 cjFMO4 299:KTSVTEFTET SAVFEDGTVE ENIDVVIFTT GYTFSFPFFE EPLKSLCTKK IFLYKNVFPL NLERTTLAII GLLSLKGSIL SGTELQARWA TRVFKGLCKI 398 cjFMO5 301:KGNVKEFTET AAIFEDGSRE DDIDAVIFAT GYTFAFPFLD DSVKVEKNKI S-LYKKVFPP NLERPTLAII GLIQPIGAIM PIAELQGRWA TQVFKGLKTL 399 hFMO1 300:RPSIKEVKEN SVIFNNTSKE EPIDIIVFAT GYTFAFPFLD ESVVKVEDGQ ASLYKYIFPA HLQKPTLAII GLIKPLGSMI PTGETQARWA VRVLKGVNKL 399 hFMO2 300:KSTVKELTET SAIFEDGTVE ENIDVIIFAT GYSFSFPFLE DSLVKVENNM VSLYKYIFPA HLDKSTLACI GLIQPLGSIF PTAELQARWV TRVFKGLCSL 399 hFMO3 300:KPNVKEFTET SAIFEDGTIF EGIDCVIFAT GYSFAYPFLD ESIIKSRNNE IILFKGVFPP LLEKSTIAVI GFVQSLGAAI PTVDLQSRWA AQVIKGTCTL 399 hFMO4 299:KTSVIEFTET SAVFEDGTVE ENIDVVIFTT GYTFSFPFFE EPLKSLCTKK IFLYKQVFPL NLERATLAII GLIGLKGSIL SGTELQARWV TRVFKGLCKI 398 hFMO5 301:KGNVKEFTET AAIFEDGSRE DDIDAVIFAT GYSFDFPFLE DSVKVVKNKI S-LYKKVFPP NLERPTLAII GLIQPLGAIM PISELQGRWA TQVFKGLKTL 399 . ..*..*. ...*...... ** ..*.* **.. .**. .. . . *.* .** *.. *.*.* *.....*...... *.**. ..*.**.. .

cjFMO1 400:PPPNVMIEEV NSRKENKPGW FGLCYCKALQ ADYITYIDEL LTYINAKPNL FSMLLMDPHL ALTVFFGPCS PYQFRLTGPG KWEGARNAIM TQWDRTFKVT 499 cjFMO2 400:PSERTMVMDM IKRNEKRIDL FGKSQSQTLQ TNYVDYLDEL ASEIGAKPDF CSLLFKDPKL AVRLYFGPCN SYQYRLVGPG QWEGARSAIF TQKQRILKPL 499 cjFMO3 400:PSREDMMNDI NEKMNKKLKW FGKSDT--IC TDYIEYMDEL ASFIGAKPSI PWLFLTDPKL ATEVYFGPCS PYQFRLVGPG KWPGARNAIL TQWERSLKPM 497 cjFMO4 399:PPSQKLMMEA TKTEQLIKRD VIKDSNKDKL D-YIAYMDGI AACIGAKPSI PLLFLKDPRL AWEVFFGPCT PYQYRLMGPG KWDGARNAIL TQWDRTLKPL 497 cjFMO5 400:PSQSEMMAEI SKAQEEMDKR YVESQRHTIQ GDYIDTMEEL ADLVGVRPNL LALAFTDPKL ALHLLLGPCT PIHYRLQGPG KWDEARKAIL TTEDRIRKPL 499 hFMO1 400:PPPSVMIEEI NARKENKPSW FGLCYCKALQ SDYITYIDEL LTYINAKPNL FSMLLTDPHL ALTVFFGPCS PYQFRLTGPG KWEGARNAIM TQWDRTFKVI 499 hFMO2 400:PSERTMMMDI IKRNEKRIDL FGESQSQTLQ TNYVDYLDEL ALEIGAKPDF CSLLFKDPKL AVRLYFGPCN SYQYRLVGPG QWEGARNAIF TQKQRILKPL 499 hFMO3 400:PSMEDMMNDI NEKMEKKRKW FGKSET--IQ TDYIVYMDEL SSFIGAKPNI PWLFLTDPKL AMEVYFGPCS PYQFRLVGPG QWPGARNAIL TQWDRSLKPM 497 hFMO4 399:PPSQKLMMEA TEKEQLIKRG VFKDTSKDKF D-YIAYMDDI AACIGTKPSI PLLFLKDPRL AWEVFFGPCT PYQYRLMGPG KWDGARNAIL TQWDRTLKPL 497 hFMO5 400:PSQSEMMAEI SKAQEEIDKR YVESQRHTIQ GDYIDTMEEL ADLVGVRPNL LSLAFTDPKL ALHLLLGPCT PIHYRVQGPG KWDGARKAIL TTDDRIRKPL 499 *...... *...... * . . **.* * . .*** ....*. *** .* .**.**. *...* .*..

cjFMO1 500:KVRVVQKSPS PFESFLKLFS FLALLVAIFL IFL------532 cjFMO2 500:KTRSLKNSSN FPASILLKIL GLLAVVVAFF CQLHWS------535 cjFMO3 498:QTRVVRSLQK PCFFFHWLKL FAIPILLIAV FLVLT------532 cjFMO4 498:KTRIVPDSSK PASMSHYLKT WGAPVLLASL LLICKSSVFL KLVRDRLQDR MSPYLVSLW- - 556 cjFMO5 500:MGRVVEKSSS MTSKMTLGKF MLAVAFFAII IAYF------533 hFMO1 500:KARVVQESPS PFESFLKVFS FLALLVAIFL IFL------532 hFMO2 500:KTRALKDSSN FSVSFLLKIL GLLAVVVAFF CQLQWS------535 hFMO3 498:QTRVVGRLQK PCFFFHWLKL FAIPILLIAV FLVLT------532 hFMO4 498:KTRIVPDSSK PASMSHYLKA WGAPVLLASL LLICKSSLFL KLVRDKLQDR MSPYLVSLWR G 558 hFMO5 500:MTRVVERSSS MTSTMTIGKF MLALAFFAII IAYF------533 ..*......

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Supplemental Figure 3

A phylogenetic tree of FMO amino acid sequences in various species.

The phylogenetic tree was constructed by the neighbor-joining method from estimated evolutionary distances using FMO amino acid sequences of marmoset (cj), human (h), chimpanzee (chim), orangutan (ora), cynomolgus monkey (mf), rhesus monkey (mm), dog (d), pig (p), rabbit (rab), guinea pig (cp), rat (r), and mouse (m). Marmoset FMO forms are shown in bold. C. elegans FMO was used as an outgroup. The scale bar shows an evolutionary distance of 0.1 amino acid substitutions per position.

chimFMO3 hFMO3 oraFMO3 mfFMO3 mmFMO3 cjFMO3 dFMO3 rabFMO3 mFMO3 rFMO3 mfFMO6 mFMO6 chimFMO2 hFMO2 oraFMO2 mfFMO2 mmFMO2 cjFMO2 mFMO2 rFMO2 rabFMO2 hFMO4 oraFMO4 mfFMO4 cjFMO4 rabFMO4 mFMO4 rFMO4 hFMO1 oraFMO1 mfFMO1 mmFMO1 cjFMO1 dFMO1 pFMO1 rabFMO1 mFMO1 rFMO1 hFMO5 mfFMO5 cjFMO5 cpFMO5 mFMO5 rFMO5 rabFMO5 mFMO9 rFMO9 mFMO12 mFMO13 C. elegans FMO 0.1 10