Note N-Methyltyramine, a Gastrin-Releasing Factor in Beer, and Structurally Related Compounds As Agonists for Human Trace Amine-Associated Receptor 1

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Note N-Methyltyramine, a Gastrin-releasing Factor in Beer, and Structurally Related Compounds as Agonists for Human Trace Amine-associated Receptor 1

1,2* 1 1 1 3 3 Hiroto Ohta , Yuka Murakami , Youhei Takebe , Kaori Murasaki , Kenji Oshima , Hiroshi Yoshihara 1 and Shigeru Morimura

1Graduate School of Science and Technology, Kumamoto University, 2-39-1 Kurokami Chuo-ku, Kumamoto 860- 8555, Japan 2Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, 4-22-1 Ikeda Nishi-ku, Kumamoto 860-0082, Japan 3Department of Biological and Chemical Systems Engineering, National Institute of Technology, Kumamoto College, 2627 Hirayama-shinmachi, Yatsushiro, Kumamoto 866-8501, Japan

Received September 30, 2019 ; Accepted December 5, 2019

N-Methyltyramine (N-MeTA) is known as a gastrin-releasing factor in beer. In this study, the agonistic actions of N-MeTA as well as tyramine (TA)/β-phenylethylamine (PEA) and their other N-methylated derivatives were examined to elucidate their structure-activity relationships, using a secreted placental alkaline phosphatase (SEAP)-based reporter assay in HEK-293 cells transiently expressing a G protein- coupled receptor, human trace amine-associated receptor 1 (hTAAR1). We detected the agonistic actions of

six test compounds, including N-MeTA (EC50 = 6.78 µM), on hTAAR1. The agonistic actions were reduced depending on the number of N-methyl groups introduced into TA and PEA; the order of potency is PEA > N-methylphenylethylamine > TA ≈ N,N-dimethylphenylethylamine ≥ N-MeTA ≥ N,N-dimethyltyramine. Taken together with our previous study on TA/PEA as agonists for hTAAR1 in the stomach, this finding suggests that hTAAR1 might be the primary target of N-MeTA in the stomach; however, the agonistic potency of N-MeTA is weaker compared to TA and PEA.

Keywords: beer, N-methyltyramine (N-MeTA), trace amine-associated receptor 1 (hTAAR1), gastrin

Introduction receptor (GPCR) family (Gainetdinov et al., 2018). TA and Aromatic biogenic amines such as tyramine (TA) and PEA are abundantly present in fermented foods such as cheese, β-phenylethylamine (PEA) (Fig. 1A) are well-known trace wine, chocolate, and traditional fermented food products amines in the mammalian nervous system. These two amines consumed worldwide (Naila et al., 2010; Kim et al., 2012; modulate various neuronal functions via trace amine-associated Gainetdinov et al., 2018). These amines are also derived from receptors (TAARs), which belong to the G protein-coupled decarboxylation of L-tyrosine and L-phenylalanine during

Abbreviations: TA, tyramine; PEA, β-phenylethylamine; TAARs, trace amine-associated receptors; GPCR, G protein-coupled receptor; hTAAR1, human TAAR 1; N-MeTA, N-methyltyramine; N,N-diMeTA, N,N-dimethyltyramine; N-MePEA, N-methylphenylethylamine; N,N-diMePEA, N,N-dimethylphenylethylamine; SEAP, secreted placental alkaline phosphatase; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; cAMP, cyclic AMP; CRE, cAMP response element; CL, confidence limit.

*To whom correspondence should be addressed. E-mail: [email protected] 314 H. Ohta et al.

Fig. 1. (A) Structures of tyramine (TA), β-phenylethylamine (PEA), and their N-methylated/N,N-dimethylated compounds, and (B) syntheses of N-methylated TA/PEA derivatives. fermentation by specific bacteria, including those belonging to Nacalai Tesque, Inc. (Kyoto, Japan). PEA hydrochloride and the genera Lactobacillus and Enterococcus (Fernández et al., N,N-diMeTA (hordenine) were obtained from Tokyo Chemical 2015; Takebe et al., 2016). We recently reported that food- Industry Co., Ltd. (Tokyo, Japan). N-MePEA was purchased derived TA and PEA are putative agonists for human TAAR 1 from Sigma-Aldrich Corp. (St. Louis, MO, USA). N,N- (hTAAR1) in the stomach, and especially in the pylorus (Ohta diMePEA hydrochloride and N-MeTA hydrochloride were et al., 2017). The pylorus contains G cells, which release synthesized from the corresponding phenethylbromides and a gastrin to stimulate gastric fluid secretion. The physiological large excess of N-methylated amines, as shown in Fig. 1B. The roles of hTAAR1 and its agonists in peripheral tissues are products were extracted with ether or chloroform, purified by gaining attention in not only the medicinal field but also the column chromatography on silica gel (7/3-chloroform/ food/nutrition field (Adriaenssens et al., 2015; Raab et al., methanol), neutralized by hydrochloric acid, and then isolated 2016; Gainetdinov et al., 2018; Batista-Lima et al., 2019). as colorless crystals. The NMR data are as follows. N,N-

A related amine compound, the TA metabolite diMePEA hydrochloride: NMR δH(dimethyl sulfoxide-d6): N-methyltyramine (N-MeTA) (Fig. 1A), is found in various 10.86 (1H, NH+), 7.36-7.24 (5H, aromatic), 3.24 (2H, Ph- plants, particularly malted barley, with levels as high as 2 mg/g. CH2-), 3.03 (2H, N-CH2-), 2.78 [6H, N-(CH3)2]. N-MeTA

Thus, an equivalent concentration is present in beer as well hydrochloride: NMR δH(dimethyl sulfoxide-d6): 9.31 (1H, OH), + (Stohs and Hartman, 2015) and Tsutsumi et al. (2010) reported 8.69 (2H, NH2 ), 7.05 (2H, aromatic), 6.73 (2H, aromatic), 3.06 that the N-MeTA content in beer was 2 mg/L. Administration (2H, Ph-CH2-), 2.82 (2H, N-CH2-), 2.56 (3H, N-CH3). of N-MeTA has been shown to induce increases in blood CRE-SEAP reporter assays Prior to the SEAP reporter pressure, enhancement of cardiac contraction, and inhibition of assays, HEK-293 cells were grown in Dulbecco’s modified lipolysis (Stohs and Hartman, 2015). In addition, N-MeTA is Eagle’s medium (DMEM) (Nacalai Tesque, Inc.) supplemented known as a gastrin-releasing factor in beer (Yokoo et al., 1999; with 10 % fetal bovine serum (FBS; Life Technologies,

Stohs and Hartman, 2015). Considering the gastrin-releasing Carlsbad, CA, USA) at 37 ℃ and 5 % CO2. As shown in our activity of N-MeTA together with our previous work (Ohta et previous study (Ohta et al., 2017), the receptor expression al., 2017), we assumed that N-MeTA acts as an hTAAR1 vector pcDNA3-β2N9/hTAAR1 (1 μg) and the reporter vector agonist to stimulate gastrin secretion in the stomach. In this pCRE-SEAP (1 μg) (Clontech, Mountain View, CA, USA) study, the agonistic actions of N-MeTA as well as TA/PEA and were transiently co-transfected into HEK-293 cells (6 × 105 their other N-methylated derivatives [N,N-dimethyltyramine cells/35-mm diameter dish) using GeneJuice® Transfection (N,N-diMeTA), N-methylphenylethylamine (N-MePEA), and Reagent (Novagen, Gibbstown, NJ, USA). The following N,N-dimethylphenylethylamine (N,N-diMePEA)] (Fig. 1A) cyclic AMP (cAMP) response element (CRE)-SEAP reporter were examined to elucidate their structure-activity assays were performed according to the procedure described relationships, using a secreted placental alkaline phosphatase previously (Ohta et al., 2017). TA/PEA and their N-methylated _ _ (SEAP)-based reporter assay system (Ohta et al., 2012) in compounds at concentrations ranging from 10 9–10 4 M were HEK-293 cells transiently expressing hTAAR1 (Ohta et al., added to the transfected cells along with 100 μM 3-isobutyl-1- 2017). methylxanthine (IBMX; Nacalai Tesque, Inc.) in FBS-free DMEM. Dimethyl sulfoxide, used to dissolve and dilute the Materials and Methods compounds, was included in the FBS-free medium at 1 % Test compounds TA hydrochloride was purchased from concentration, without undesirable effects on SEAP activity. N-Methyltyramine and Related Compounds as hTAAR1 Agonists 315

Fig. 2. Agonist response of TA, N-MeTA, and N,N-diMeTA in (A) transiently β2N9/hTAAR1-expressing HEK-293 cells _ _ and (B) HEK-mock cells by CRE-SEAP assay. Each compound at 10 9-10 4 M was added to cells, and the cells were

incubated at 37 ℃ and 5 % CO2 for 1 day. The SEAP activity at the basal level (Ba, SEAP activity with no agonist) was set as 1. HEK cells transiently transfected with the empty pcDNA3 were used as HEK-mock cells for negative control experiments. The adenylate cyclase activator forskolin was used to confirm the negative control experiment. Data represent the mean ± SE for three independent experiments, each performed in duplicate.

Fig. 3. Agonist response of PEA, N-MePEA, and N,N-diMePEA in (A) transiently β2N9/hTAAR1-expressing HEK- 293 cells and (B) HEK-mock cells by CRE-SEAP assay. Cell cultures and CRE-SEAP assays were performed under identical conditions as described in Fig. 2. The basal SEAP activity (Ba) was set as 1. Data represent the mean ± SE for three independent experiments, each performed in duplicate.

Results and Discussion conditions, PEA, N-MePEA, and N,N-diMePEA exhibited a

According to the CRE-SEAP assays, TA, N-MeTA, and typical sigmoidal hTAAR1 agonist curve (Fig. 3A), with EC50 _ _ N,N-diMeTA at concentrations of 10 9–10 4 M showed a dose- values of 0.56 µM (95 % CL, 0.40–0.79 µM), 1.63 µM (95 % dependent increase in SEAP activity in transiently hTAAR1- CL, 1.19–2.26 µM), and 4.31 µM (95 % CL, 3.21–5.93 µM), expressing HEK-293 cells (Fig. 2A), with EC50 values of respectively. The relationship between mono/di N-methylation 4.02 µM [95 % confidence limit (CL), 2.85–6.00 µM), 6.78 µM of PEA and agonist potency for hTAAR1 was similar to that of (95 % CL, 4.85–10.31 µM), and 10.60 µM (95 % CL, 7.65– TA; and the order of potency for the three PEA-related 16.78 µM), respectively. The order of potency was TA ≥ compounds was PEA > N-MePEA > N,N-diMePEA. These N-MeTA ≥ N,N-diMeTA. With the same experimental results indicate that the more methyl groups TA and PEA have 316 H. Ohta et al. at the amino group, the less agonistic potency the TA and PEA thank Editage (www.editage.jp) for English language editing. derivatives show. Overall, the potency of the six compounds tested can be arranged as: PEA > N-MePEA > TA ≈ N,N- References diMePEA ≥ N-MeTA ≥ N,N-diMeTA. As N-MeTA and N,N- Adriaenssens, A., Lam, B.Y.H., Billing, L., Skeffington, K., Sewing, S., diMeTA were the least potent agonists, they did not attain the Reimann, F., and Gribble, F. (2015). A transcriptome-led exploration _ same maximal SEAP activity at 10 4 M as TA or the PEA of molecular mechanisms regulating somatostatin-producing D-cells compounds (Figs. 2A and 3A). Nonetheless, the respective in the gastric epithelium. Endocrinology, 156, 3924–3936. contents of N-MeTA and N,N-diMeTA (hordenine) in different Batista-Lima, F.J., Rodrigues, F.M.D.S., Gadelha, K.K.L., de Oliveira, beer samples are 0.59-4.61 mg/L (3.9-30.5 µM) and 1.05- D.M.N., Carvalho, E.F., Oliveira, T.L., Nóbrega, F.C., Brito, T.S., 6.32 mg/L (6.35-38.25 µM) (Sommer et al., 2019), which are and Magalhães, P.J.C. (2019). Dual excitatory and smooth muscle- comparable with each EC50 value. Therefore, upon direct relaxant effect of β-phenylethylamine on gastric fundus strips in rats. interaction between the ingested beer and hTAAR1 expressed Clin. Exp. Pharmacol. Physiol., 46, 40–47. in the stomach (Ohta et al., 2017), the agonist concentrations in Fernández, M., Hudson, J.A., Korpela, R., and de los Reyes-Gavilán, the beer would be sufficient to activate hTAAR1. C.G. (2015). Impact on human health of microorganisms present in Lindemann et al. (2005) reported that mono N-methyl fermented dairy products: an overview. Biomed. Res. Int., 2015, substitution of TA and PEA had an obscure effect on agonist 412714. activity in cAMP assays with HEK-293 cells stably expressing Gainetdinov, R.R., Hoener, M.C., and Berry, M.D. (2018). Trace hTAAR1/rat TAAR1 chimera receptors. However, Wainscott amines and their receptors. Pharmacol. Rev., 70, 549–620. et al. (2007) showed that N-methyl and N,N-dimethyl Kim, B., Byun, B.Y., and Mah, J.-H. (2012). Biogenic amine formation substitution of PEA induced a 2-3-fold and 14-fold decrease in and bacterial contribution in Natto products. Food Chem., 135, cAMP levels of hTAAR1-expressing GαsAV12-664 cells 2005–2011. (Syrian hamster fibroblast cell line stably transfected with rat Koda, H., Yokoo, Y., Matsumoto, N., Suwa, Y., Fukazawa, H., Ishida,

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