The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) controls numerous physiological functions of the central and peripheral nervous systems, including sleep/wake cycle, food intake, nociception, locomotion, and cardiovascular homeostasis (Darmon et al., 2015).

5-HT receptors (5-HTRs) are classified into seven distinct subfamilies, 5-HT_1-7R. All 5-HTR subtypes belong to G protein-coupled receptor (GPCR) superfamily, except for 5-HT₃R, which is a ligand-gated ion channel (Yun and Rhim, 2011).

The type 2 serotonin receptor subfamily contains 5-HT_2AR, 5-HT_2BR, and 5-HT_2CR (Bonhaus et al., 1995; Leysen, 2004). Among these subtypes, 5-HT_2AR is expressed widely throughout the central nervous system that includes neocortex (mainly prefrontal, parietal, and somatosensory cortex) and the olfactory tubercle. High concentrations of this receptor on the apical dendrites of pyramidal cells in layer V of the cortex play a key role in several diseases such as drug addiction (Krebs and Johansen, 2012), schizophrenia (Vollenweider et al., 1998), obsessive compulsive disorder (Adams et al., 2005; Moreno et al., 2006), depression (Celada et al., 2004; Carhart-Harris et al., 2012), and neuropathic pain (Okamoto et al., 2007).

5-HT_2AR agonists have traditionally been divided into three structural groups: phenethylamines, tryptamines, and ergolines (Nichols, 2012). Phenethylamines have been most extensively characterized (Parker et al., 1998; McLean et al., 2006). They generally show selectivity for 5-HT_2AR; however, they also bind to 5-HT_2CR with high affinity (Nelson et al., 1999).

Tryptamines with tryptophan ring as the basal structural moiety are structurally close to 5-HT, the endogenous transmitter. Tryptamines are more selective and have stronger affinity than ergolines for 5-HT_2ARs. Tryptamine derivatives include ring substituents, N-alkylation, and side chain alkylation (Nichols, 2012).

Ergolines are tetracyclic molecules derived from alkaloids produced by the ergot fungus. Ergolines are considered to be rigidified tryptamines; however, they generally show little subtype selectivity compared to that of phenethylamines and tryptamines (Nelson et al., 1999). Ergolines are structurally complex, and deriving their structural analogs is difficult.

In this study, we conducted radioligand binding study on 5-HT_2AR with 11 tryptamines and 14 phenethylamine derivatives. The affinity for 5-HT_2AR was largely determined by scaffolds, and the details were determined by the size or inclusion of the side branches. The relative efficacies and potencies of 5-HT agonists were in accordance with their affinity for 5-HT_2AR as evident by the activation of ERK signaling and receptor endocytosis. The information obtained here will be useful in developing 5-HT_2AR ligands optimized for therapeutic purposes and avoiding side effects, such as psychedelic actions.

Reagents

5-HT, ketanserin (+)-tartrate, 2-bromo-α-ergocryptine, and rabbit anti-hemagglutinin (HA) were obtained from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Peroxidase-conjugated anti-rabbit antibodies were purchased from Invitrogen (Waltham, MA, USA). TMB (3,3’,5,5’-Tetramethylbenzidine)-ELISA substrate was procured from Thermo Fisher Scientific (Waltham, MA, USA). Methylspiperone (84.2 Ci/mmol) was purchased from PerkinElmer Life Sciences (Waltham, MA, USA). Antibodies to phospho-ERK1/2 and ERK2 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and anti-mouse horse peroxidase (HRP)-conjugated secondary antibodies were obtained from Jackson ImmunoResearch (West Grove, PA, USA). Phenethylamine, tryptamine, and their derivatives were provided by Korean Ministry of Food and Drug Safety (Cheongju, Korea).

Plasmid constructs

5-HT_2AR in pCNS-D2 was provided from Korea Human Gene Bank, Medical Genomics Research center, KRIBB, Daejeon, Korea. 5-HT_2AR was tagged either with FLAG or HA at amino terminal. Wildtype and dominant negative mutants of dynamin2 (K44A-Dyn2) were described previously (Henley et al., 1998; Guo et al., 2015).

Cell culture

Human embryonic kidney (HEK-293) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in a humid environment containing 5% CO2, using a minimal essential medium containing 8% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin. Cells were transfected using polyethylenimine (Polysciencies Inc., Warrington, PA, USA).

5-HT_2AR binding assay

HEK-293 cells expressing 5-HT_2AR were sub-seeded in a 24-well plate coated with poly-L-lysine. Radioligand binding assay was performed by incubating cells with 1 nM [³H]-methylspiperone at 4°C for 90 min. Cells were washed three times with ice-cold serum-free media and then lysed with phosphate-buffered saline (PBS) containing 1% sodium dodecyl sulfate (SDS). Radioactivity was determined by Wallac 1450 MicroBeta^® TriLux liquid scintillation counter (PerkinElmer Life Sciences). The binding of [³H]-methylspiperone in the presence of 10 μM ketanserin was defined as non-specific. Preparation of dose–response curves and statistical analysis were performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). Ki values were converted from IC₅₀ values according to the following Cheng-Prusoff equation (Cheng and Prusoff, 1973), Ki=IC₅₀/(1+[A]/Kd) where [A] and Kd represent the concentration of the compound used and dissociation constant of [³H]-methylspiperone for 5-HT_2AR, respectively.

ERK measurement

Transfected cells were cultured in 6-well plates, and were starved overnight in a serum-free culture medium containing 0.1% bovine serum albumin (BSA). Cells were treated with 5-HT dissolved in serum-free culture medium, and sodium dodecyl sulfate (SDS) sample buffer was directly added to culture wells. After incubating for 20 min at 65°C, samples were sonicated to shear genomic DNA. Proteins were separated by SDS-polyacrylamide gel electrophoresis (10% running gel, 5% stacking gel) and electroblotted onto polyvinylidene difluoride or nitrocellulose membranes. The membranes were incubated for 1 h at 22°C in TBS-Tween 20 (TBS-T) containing 5% nonfat dry milk or 4% BSA, followed by 1 h of incubation with antibody to phospho-ERK (1:1,000 dilution) and 1 h with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000 dilution) in 2% nonfat dry milk. Blots were visualized with chemiluminescent western blotting kit. The same samples were processed to detect ERK. Signals were quantified using Multi Gauge version 3.1 (FUJIFILM Corporation, Tokyo, Japan).

Receptor endocytosis assay

Endocytosis of 5-HT_2AR was determined by enzyme-linked immunosorbent assay (ELISA). HEK-293 cells were transfected with either HA-tagged 5-HT_2AR. Cells were treated with 1 μM 5-HT for a designated time period and washed with PBS three times. Cells were fixed with 4% paraformaldehyde for 15-20 min on ice and washed with PBS three times and then treated with 1% BSA for 1 h, followed by incubation with anti-HA or ant-FLAG antibodies (1:2,000 dilution in 1% BSA) for 1 h. Cells were then washed with PBS three times and treated with of HRP-conjugated anti-rabbit secondary antibody (1:2,000 dilution in 1% BSA) for 1 h at 20°C. After washing with PBS three times, cells were treated with TMB substrate solution for at least 10 min, and the reaction was stopped by adding sulfuric or phosphoric acid. Optical density was monitored at 450 nm.

Statistical analysis

Values are expressed as the mean ± standard deviation. Statistical significance of the data was analyzed using a one-way analysis of variance with Tukey’s post-hoc test using GraphPad Prism 5.0. A p-value<0.05 was considered significant.

Characterization of phenethylamine derivatives in 5-HT_2AR binding

Phenethylamine is a primary amine in which the amino group is attached to a benzene ring through a two-carbon or ethyl group (Table 1, upper panel). Radioligand binding assay on 5-HT_2AR was conducted with 14 phenethylamine derivatives using ketanserin as a positive control (Glennon et al., 2002). The structure-activity relationship (SAR) was determined by classifying them according to the substitutions in seven positions (R¹–R⁷, Table 1).

Table 1 Binding affinity of phenethylamine derivatives for 5-HT_2AR.

Compd	R1	R2	R3	R4	R5=R6=R7	Ki (nM)
1	-CH2CH2CH3	-H	-H	-H	-H		3.473
2	-NO2	-H	-H	-H	-H		25.17
3	-OCH2C(CH3)=CH2	-H	-H	-H	-H		71.92
4	-Cl	-H	-H	-H	-H		13.04
5	-CH3	-OCH3	-H	-H	-H		1.907
6	-CH3	-H	-Ar	-OCH3	-H		2.881
7	-CH2CH3	-H	-Ar	-OCH3	-H		1.524
8	-Cl	-H	-Ar	-OCH3	-H		0.817
9	-Br	-H	-Ar	-OCH3	-H		1.411
10	-Cl	-H	-Ar	-OH	-H		1.099
11	-Cl	-H	-Ar	-F	-H		10.84
12	-Br	-H	-Ar	-F	-H		3.629
13	-I	-H	-Ar	-F	-H		10.00
14	-Cl	-H	-Ar	-H	-OCH3		515.1
Ketanserin							22.21

1. 2C-P HCl: 2,5-dimethoxy-4-propyl-benzeneethanamine, HCl.

2. 2C-N HCl: 2,5-Dimethoxy-4-nitrophenethylamine, HCl.

3. Methallylescaline HCl.

4. 2C-C HCl: 4-Chloro-2,5-Dimethoxyphenethylamine, HCl.

5. BOD HCl: 4-methyl-2,5,beta-trimethoxyphenethylamine, HCl.

6. 25D-NBOMe HCl: 2-(2,5-dimethoxy-4-methylphenyl)-N-(2-methoxybenzyl)ethanamine, HCl.

7. 25E-NBOMe HCl: 2-(4-ethyl-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethan-1-amine, HCl.

8. 25C-NBOMe HCl: 2-(4-chloro-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine, HCl.

9. 25B-NBOMe HCl: 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine, HCl.

10. 25C-NBOH HCl: 2-[[[2-(4-chloro-2,5-dimethoxyphenyl)ethyl]amino]methyl]-phenol, HCl.

11. 25C-NBF HCl: 4-chloro-N-[(2-fluorophenyl)methyl]-2,5-dimethoxy-benzeneethanamine, HCl.

12. 25B-NBF HCl: 4-bromo-N-[(2-fluorophenyl)methyl]-2,5-dimethoxy-benzeneethanamine, HCl.

13. 25I-NBF HCl: N-(2-fluorobenzyl)-2-(4-iodo-2,5-dimethoxyphenyl)ethanamine, HCl.

14. 30C-NBOMe HCl: 2-(4-chloro-2,5-dimethoxyphenyl)-N-(3,4,5-trimethoxybenzyl)ethanamine, HCl.

The affinity for 5-HT_2AR was maintained at similar range when R¹ attached to the para position of the phenyl ring contained an alkyl or a halogen group (1,4, 6-9, 11-13). In contrast, the affinity of the compounds decreased when alkoxy or nitro group was placed at R¹ (2, 3). A methoxy group at R² did not exert noticeable impact on the binding affinity to 5-HT_2AR (1 vs 5). An aromatic group at R³ increased the binding affinity of phenethylamine to 5-HT_2AR if the aromatic ring at R³ contained oxygen-containing group at the ortho position (R⁴) without exception but at less extent with fluoride at R⁴ (6–10 vs 1–4 or 11–13). The presence of methoxy groups at R⁵–R⁷ that correspond to meta and para positions, exerted negative effects on their affinity to 5-HT_2AR (14).

Characterization of tryptamine derivatives in 5-HT_2AR binding

Tryptamine is composed of an indole (benzene ring plus pyrrole ring) and a 2-aminoethyl group that is attached to the third carbon of pyrrole ring. For SAR analysis, IC₅₀ values of 11 compounds, which were determined from our study, were combined with the IC₅₀ values of 25 other compounds that were cited from previous studies (Table 2). SAR analysis was focused on three parts of tryptamine—indole ring, side chain alkylation, and N-alkylation. In addition, cyclic amino groups, such as pyrrolidinyl, piperidyl, and 2,5-dimethylpyrol, at N-alkylation site (34–36) were included.

Table 2 Binding affinity of tryptamine derivatives for 5-HT_2AR.

Compd	Aromatic ring substitution				Side chain Alkylation		N-Alkylation		Ki (nM)
	R1	R2	R3		R4		R5	R6
1	-CH3	-H	-H		-H		-H	-H	685.70
2A	-H	-H	-H		-CH3		-H	-H	130
3	-H	-H	-OCH3		-CH3		-H	-H	3.121
4	-H	-H	-Cl		-CH3		-H	-H	23.97
5	-H	-H	-Br		-CH3		-H	-H	58.78
6	-H	-H	-H		-CH2(CH2)2CH3		-H	-H	123.70
7A	-H	-H	-H		-CH3		-H	-CH3	46
8C	-H	-H	-OCH3		-H		-H	-CH(CH3)2	50
9	-H	-H	-H		-H		-CH3	-CH3	88.49
10A	-H	-OH	-H		-H		-CH3	-CH3	6
11B	-H	-H	-OH		-H		-CH3	-CH3	3.5
12A	-H	-H	-OCH3		-H		-CH3	-CH3	14
13	-H	-OH	-H		-H		-CH3	-CH2CH3	64.8
14C	-H	-H	-H		-H		-CH3	-CH(CH3)2	38
15C	-H	-H	-OCH3		-H		-CH3	-CH(CH3)2	17
16C	-H	-H	-CH3		-H		-CH3	-CH(CH3)2	28
17C	-H	-OH	-H		-H		-CH3	-CH(CH3)2	13
18	-H	-OAc	-H		-H		-CH2CH3	-CH2CH3	595.30
19C	-H	-OH	-H		-H		-CH2CH3	-CH2CH3	14
20	-H	-H	-OCH3		-H		-CH2CH3	-CH2CH2CH3	122.30
21B	-H	-H	-OH		-H		-CH(CH3)2	-CH(CH3)2	5.6
22D	-H	-H	-H		-H		-CH2CH=CH2	-CH2CH=CH2	701
23D	-H	-OAC	-H		-H		-CH2CH=CH2	-CH2CH=CH2	565
24D	-H	-OH	-H		-H		-CH2CH=CH2	-CH2CH=CH2	652
25	-H	-H	-OCH3		-H		-CH2CH=CH2	-CH2CH=CH2	258.41
26D	-CH3	-H	-OCH3		-H		-CH2CH=CH2	-CH2CH=CH2	1,153
27D	-H	-H	F		-H		-CH2CH=CH2	-CH2CH=CH2	247
28D	-H	-H	Br		-H		-CH2CH=CH2	-CH2CH=CH2	477
29D	-CH3	-H	F		-H		-CH2CH=CH2	-CH2CH=CH2	655
30D	-CH2CH3	-H	-H		-H		-CH2CH=CH2	-CH2CH=CH2	1,515
31A	-H	-OH	-H		-H		-CH2CH(CH3)2	-CH2CH(CH3)2	260
32A	-H	-OH	-H		-H		-CH2CH(CH3)CH2CH3	-CH2CH(CH3)CH2CH3	39
33	-H	-H	-H		-H		-CH2(CH2)2CH3	-CH2(CH2)2CH3	822.90
34A	-H	-H	-H		-H		Pyrrolidinyl		110
35A	-H	-H	-H		-H		Piperidyl		760
36C	-H	-H	-H		-H		2,5-dimethylpyrol		2,430
Ketanserin									22.21

^AThese were adapted from (Nichols, 2012). ^BThis was adapted from (Klein et al., 2011). ^CThese were adapted from (McKenna et al., 1990). ^DThese were adapted from (Klein et al., 2018).

1. 2-MT.

3. 5-MeO-AMT: 5-methoxy-α-methyltryptamine.

4. 5-CAMT HCl: 5-Chloro-3-(2-aminoethyl)indole Hydrochloride.

5. 5-BAMT HCl: 5-Bromo-3-(2-aminoethyl)indole Hydrochloride.

6. ABT HCl.

9. DMT fumarate: N,N-dimethyl-1H-indole-3-ethanamine, (2E)-2-butenedioate.

13. 4-OH-MET fumarate: 4-hydroxy-N-methyl-N-ethyltryptamine, metocin, or methylcybin.

18. 4-AcO-DET fumarate (1 : 0.83): 4-Acetoxy-N,N-diethyltryptamine.

20. 5-MeO-EPT: 5- methoxy -N-ethyl-N-propyl tryptamine.

25. 5-MeO-DALT: N-allyl-N-[2-(5-methoxy-1H-indol-3-yl)ethyl] prop-2-en-1- amine.

33. DBT HCl: 1H-Indole-3-ethanamine, N,N-dibutyl-N,N-Dibutyltryptamin.

The presence of alkyl group at R¹ exerted negative effects on the binding affinity (1, 26, 29, and 30). The presence of hydroxyl group at R² exerted favorable effects on the binding affinity without exception (9 vs 10, 14 vs 17, 22 vs 24, and 31 vs 33). The presence of any substitution at R³ exerted positive influences on the binding affinity for 5-HT_2AR regardless of the nature of substituents being electron-donating (-CH₃, -OH, and -OMe; 2 vs 3, 9 vs 11 and 12, and 14 vs 15 and 16) or electron-withdrawing (-Cl and -Br; 2 vs 4 and 5, and 22 vs 27 and 28). In various formats of side chains, the presence of allyl groups at R⁵ and R⁶ exerted strong inhibitory effects on their affinity for 5-HT_2AR (22–30). In general, less bulky alkyl groups at R⁵ and R⁶ exerted more favorable influences on the affinity of compounds in the format of either no substitutions at R¹–R³ (9, 14, 22, and 33) or OH group at R² (10, 13, 17, 19, 24, and 31).

Functional characterization of 5-HT_2AR agonists

The functionality of 5-HT_2AR ligands was tested by evaluating their effects on 5-HT_2AR-mediated ERK activation. Three compounds, including 5-HT (Ki, 11.55 nM) as a positive control, 25C-NBOMe (Ki, 0.817 nM), and methallyescaline (Ki, 71.92 nM) were selected for ERK assay. As shown in Fig. 1, the compounds induced ERK activation in a dose-dependent manner. Their effects on ERK activation were in agreements with their affinity towards 5-HT_2AR, that is, 5-HT and 25C-NBOMe, having high affinity for the receptor, induced ERK activation at the concentration ranges of their Ki values. In contrast, methallyescaline, which had relatively weak affinity for the receptor, failed to induce ERK activation at doses lower than its Ki value.

Figure 1. Effects of 5-HT_2AR agonists on ERK activation. HEK-293 cells were transfected with 5-HT_2AR cDNA in pCNS-D2. Cells were treated with increasing concentrations of 5-HT, 25C-NBOMe, and methallyescaline for 5 min. 5-HT was dissolved in serum-free media; 25C-NBOMe and methallyescaline were dissolved in DMSO. *p<0.05, **p<0.01 compared with the vehicle-treated group (n=3).

Endocytic properties of 5-HT_2AR agonists

Effects of 5-HT agonists on endocytosis of 5-HT_2AR were determined. One compound was selected from each family (25C-NBOME from phenethylamine, 5-BAMT from tryptamine family, and 2-bromo-α-ergocryptine from ergoline family) to examine whether the structural characteristics of the compounds were related to their capacity to induce receptor endocytosis. As shown in Fig. 2A, 5-HT induced endocytosis of 5-HT_2AR in a time-dependent manner. All the compounds tested showed similar extent of endocytosis (Fig. 2B), suggesting that the structural features of agonists do not play critical roles in determining their endocytic activities.

Figure 2. Effects of 5-HT_2AR agonists on receptor endocytosis. (A) HEK-293 cells were transfected with HA-tagged 5-HT_2AR cDNA in pRC/CMV. Cells were treated with 1 μM 5-HT for 0-30 min. Receptor endocytosis was studied as described in Materials and Methods part. (B) HEK-293 cells were transfected with HA-tagged 5-HT_2AR cDNA in pRC/CMV. Cells were treated with 1 μM of 5-HT, 25C-NBOME, 5-BMAT, or 2-bromo-α-ergocryptine for 15 min.

Endocytosis of receptors decreases their number on the cell surface, which can be perceived as a mechanism of negative feedback to protect cells from agonistic overstimulation (Sibley and Lefkowitz, 1985). Therefore, blockade of receptor endocytosis is expected to increase their number on the plasma membrane and enhance receptor signaling. In accordance with this expectation, inhibition of receptor endocytosis with co-expression of K44A-dyanmin2, a dominant negative (Fig. 3A) increased ERK activation (Fig. 3B). Dynamin is a critical component involved in the clathrin-mediated and caveolar endocytosis. It cuts the neck of nascent vesicles from the cell membrane, and inhibition of this process by K44A-dynamin2 blocks receptor endocytosis (van der Bliek et al., 1993; Kim et al., 2001).

Figure 3. Effects of 5-HT_2AR endocytosis on ERK activation. (A) HEK-293 cells were transfected with 2 μg HA-tagged 5-HT_2AR cDNA in pRC/CMV together with 4 μg WT-dynamin2 or K44A-dynamin2 constructs in pCMV5. Cells were treated with 1 μM 5-HT for 15 min. ***p<0.001 compared to the WT-dynamin2 group (n=3). (B) HEK-293 cells were transfected as mentioned above. Cells were treated with 1 μM 5-HT for 5 min. **p<0.01 compared to the WT-dynamin2 group (n=3).

The SAR assessment revealed that phenethylamine derivatives showed higher binding affinity towards 5-HT_2AR than did tryptamine derivatives.

In the phenethylamine group, i) compounds with alkyl or halogen groups at R¹ had affinity to 5-HT_2AR higher than those with alkoxy or nitro group; ii) compounds with oxygen-containing group at R⁴ showed higher affinity to 5-HT_2AR than did their counterparts; and iii) the presence of methoxy groups on R⁵–R⁷ exerted strong negative effects on the affinity towards 5-HT_2AR.

In the tryptamine group, i) compounds with alkyl groups at R¹ showed lower affinity to 5-HT_2AR than did their counterparts; ii) compounds with hydroxyl group at R² showed high affinity to 5-HT_2AR; iii) substitutions at R³, regardless of their electron-donating (CH₃, OH, and OMe) or electron-withdrawing (Cl and Br) nature, exerted favorable effects on their affinity towards 5-HT_2AR; and iv) allyl groups at R⁵ and R⁶ exerted strong negative effects on their binding affinity towards 5-HT_2AR.

In Table 2, information on the compounds reported in previous publications was collected and analyzed to construct a more precise SAR for tryptamine derivatives. Although ligand affinity for receptors is not significantly affected by the cell types used compared to signal transduction, it should be recognized that these results were obtained in different cellular environments.

Extracellular signal-regulated kinases are key cellular components that control various aspects of cellular functions such as cell proliferation, differentiation, and synaptic plasticity (Brown and Gerfen, 2006; Girault et al., 2007). The regulation of ERK through GPCRs is a complicated process, and various signaling components play different roles in ERK activation depending on the GPCR and cell types involved (Beom et al., 2004). ERK plays important roles in the pathogenesis, symptomatology, and treatment of depression (Wang and Mao, 2019), which are closely related to the functional roles of 5-HT_2AR. For example, ERK levels were altered in the postmortem frontal cortex of patients of mood disorders and schizophrenia (Yuan et al., 2010).

During endocytosis, receptor signaling may diminish because the number of receptors that can bind to agonists decreases as the receptors on the plasma membrane move to the cytoplasmic regions (Ferguson et al., 1996). Follow-up studies, however, suggested that the main functional role of receptor endocytosis is a restoration of receptor responsiveness rather than a decrement in signaling (Yu et al., 1993; Cho et al., 2010). However, the basis of endocytosis-induced reduction in signaling or rehabilitation of the function of desensitized receptors is largely unexplored. Intuitively, inhibition of receptor endocytosis would increase receptor signaling when a significant proportion of receptors is endocytosed.

Overall, in this study, a SAR was established for binding affinities of 5-HT_2AR agonists of the phenethylamine and tryptamine family members. In addition, ERK activation and receptor endocytosis were evaluated for selected compounds. Since 5-HT_2AR is closely related to affective disorders and drug addiction in humans, these findings will provide valuable information for developing therapeutic agents to treat the related diseases.

This research was supported by the Ministry of Food and Drug Safety (19182MFDS403) and the National Research Foundation of Korea (2020R1F1A1072302) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (KRF-2020R1I1A3062151).

The authors have no conflicts of interest to declare.