GW117: A novel serotonin (5‐HT2C) receptor antagonist and melatonin (MT1/MT2) receptor agonist with potential antidepressant‐like activity in rodents

Abstract Aims To evaluate the antidepressant‐like effect of compound GW117 in rodents using in vitro binding and uptake assays as well in vivo behavioral tests. Methods We investigated the target profile of GW117 using [35S]‐GTPγS and [3H]PIP binding. Using the forced swimming test and chronic unpredictable stress in rats, tail suspension test in mice and rats, and learned helplessness model in mice, we further revealed the antidepressant‐like and anxiolytic‐like effects of GW117. Results The current study suggests that GW117 displays serotonin 2C (5‐HT2C) receptor antagonist and melatonin type 1 and 2 (MT1/MT2) receptor agonist properties, as well as evident antidepressant and anxiolytic effects. Conclusion These data suggest that GW117 is probably a potent antidepressant.

side effects of SSRIs, especially anhedonia, apathy, and extrapyramidal motor symptoms, affect the behavioral and emotional efficacy of SSRIs. [6][7][8][9][10][11] These caveats highlight a major unmet need for more efficacious and faster-acting treatments to improve the symptoms of MDD. In addition to antidepressants with different mechanisms, researchers are also interested in drugs that interact with nonselective, multitarget, or multiple ligands which can more effectively, rapidly, and broadly control the core symptoms of depression, as well treat comorbid symptoms such as pain, sexual dysfunction, cognitive impairment, weight gain, and insomnia. 12 Agomelatine, which play the role of "synergism" as a MT 1 /MT 2 receptor agonist and 5-HT 2C receptor antagonist, alleviated depressive symptoms in both rodents and patients, while also showing anxiolytic effects, sleep-promoting, and anti-circadian desynchronization effects. 12 Of particular importance, however, is the fact that currently more than 30% of depressed patients do not respond to firstline treatment, 3 whereas lower doses of ketamine confer a rapid antidepressant effect in these patients. 13,14 But its application is limited because of its psychotropic and addictive properties. 15 In furthermore, recent studies also have suggested a number of pharmacological or non-pharmacological ways to exert antidepressantlike activity. 16,17 Consequently, agomelatine could represent a novel antidepressant with higher therapeutic efficacy. Studies have shown that in contrast to sexual dysfunction caused by SSRIs, treatment with agomelatine does not result in sexual dysfunction, possibly owing to overstimulation of 5-HT 2C receptors. 18 Moreover, this mechanism may explain the remission of anxiety symptoms in MDD. Evidence of other researches suggests that along with 5-HT 2C receptors, gamma aminobutyric acid-ergic neurons within the suprachiasmatic nucleus modulate the action of agomelatine and may also account for its anxiolytic effect. 19,20 Furthermore, studies have postulated that the synergistic effect of melatonin and 5-HT 2C receptors underlies the antidepressant effect of agomelatine. 21,22 Considering the factors given above, we designed and synthesized a string of compounds with new structures and screened the GW117 (Figure 1) a derivate compound of agomelatine with the same targets. Our previous study found that GW117 has lower toxic effects compared with agomelatine. 23 In this research, we did the pharmacological characteristics of the GW117 detailed and believe that the results can provide a reference for progressing in phase II clinical trial in China.
Scintillation liquid was purchased from Perkin-Elmer Life Sciences, and Folin-phenol reagent was purchased from HAWI Science & Technology Co., Ltd. Each group was given suspension of the corresponding drug by gavage at 9 a.m. daily, the volume was 1 ml/100 g in rats or 0.1 ml/10 g in mice, and the control group was given the same volume of 0.5% CMC suspension.

| Hippocampus membrane preparation
Hippocampal membrane preparations were obtained using previously described methods. [24][25][26] Briefly, rats were decapitated and their brains were rapidly removed. Next, the hippocampus was dissected and subsequently homogenized in 40 volumes of ice-cold buffer (50 mM Tris-HCl Buffer, pH 7.4) and then centrifuged at 40,000 × g at 4°C for 10 min. The particles were re-suspended and centrifuged again. In order to remove endogenous monoamines, the final suspension was incubated at 37°C for 20 min and then centrifuged as usual. The final pellets were frozen at −80°C at once for up to 1 week.   Tris-HCl buffer (50 mM, pH 7.4), and dried by suction. Next, the filter paper was removed, dried by baking, and then put it in a scintillation bottle. Finally, 1 ml of scintillation fluid was added and the radioactive intensity measured by liquid scintillation counting.

| [ 35 S]-GTPγS and [ 3 H]PIP binding assays
This experiment was mentioned earlier. 27 In short, HEK293 cell membrane stably expressing human 5-HT 2C receptors or rat hippocampal tissue membrane or MT 1 /MT 2 receptors was re-

| Tail suspension test in mice
The tail suspension test (TST) was conducted as described earlier. 28,29 Eighty naive mice were randomly divided into eight treatment groups (n = 10/group). All mice received a single dose of the drug (p.o.). After 60 min of gavage administration, mice were suspended from the top of the apparatus (25 × 25 × 35 cm) using tape about 1 cm from the tail tip. The duration of immobility was recorded in the last 4 min for a total of 6 min. When mice are passively suspended immobile, they are judged to be stationary.

| Forced swimming test in rats
The forced swimming test (FST) was conducted as described earlier. 30 Eighty naive rats were randomized into eight treatment groups (n = 9-12/group). All rats received a single drug administration (p.o.).
The procedure consisted of two sessions, to be specific, a pre-test session and a test session. A cylindrical container (diameter, 20 cm; height, 40 cm; including 30 cm of water and kept at 25℃) was used.
During the pre-test phase, rats were required to perform forced swimming for 15 min. The test session was performed 24 h later, put the rats in the same cylindrical container for 5 min and immobility duration over 5 min recorded. Vehicle, agomelatine, or GW117 was given 1 h before the test. Rats were considered immobile when they entered a floating posture, specifically, immobile, passive, and with their heads above water.

| Locomotor activity in mice and rats
In order to confirm whether GW117 has an antidepressant activity, we used spontaneous activities in mice and rats to determine whether GW117 affects the central system. 64 ICR mice were randomly allocation to five therapy groups (n = 9-12/group): control group, 5 mg/kg GW117, 10 mg/kg GW117, 20 mg/kg GW117, and 40 mg/kg GW117. All rats and mice received one administration (p.o.). After sixty minutes of gavage administration, each mouse or rat was located in the corner of an open field chamber (36 × 29 × 23 cm for mice; 76 × 76 × 46 cm for rats) to adapt to 5 min. The numbers of crossings and rearings were recorded during the subsequent 5 min.

| Chronic unpredictable stress model of rats
In order to further examine the antidepressant effects of GW117, chronic unpredictable stress (CUS) model was utilized; the methods were as described before. 29,31 After 1 week of training, rats underwent sucrose training of 48 h. After training, a sucrose baseline test was performed. Rats were randomly and evenly grouped depending on baseline of sucrose preference: vehicle (non-stress), stress-vehicle (distilled water), and stress-fluoxetine (10 mg/kg), stress-agomelatine (10, 20 or 40 mg/kg), and stress-GW117 (5, 10, 20, or 40 mg/kg). Gastric gavage was given 1 h before the stress (08:00-09:00). Except for the non-stressed group, all rats were stimulated by a series of stressors. stress methods included the following: fast on food and water (24 h), moisture cage (150 g sawdust bedding in 200 ml water), overnight illumination, low-intensity strobe illumination (100 flashes/min), forced swimming (5 min at 10℃), white noise (110 dB), tail pinch (1 cm from tail root, 6 min), 45℃ cage tilt, and restraint (1-2 h). Stressors need to be used continuously and randomly. Non-stressed rats received free food and water, but were required to fast for 14 h before sucrose preference test. After 4 weeks of stress, the sucrose preference test (SPT) (on day 25), open field test (OFT) (on day 28), and novelty-suppressed feeding (NSF) test (on day 29) were carried out. A diagram of chronic unpredictable stress and behavioral testing was performed as shown in Figure 5.

| Open field test
The OFT device was a chamber (diameter, 122 cm; height, 45 cm) that was grouped equally into 16 sections. 24 h after the last administration, rats were put into the center of the chamber and the numbers of crossings and readings were recorded in 5 min.

| Sucrose preference test
The SPT was performed as previously reported. 32 Rats were trained to administer sucrose water solution after 48 h fasting and water deprivation. Only 1% sucrose water was given for the first 24 h, and then 1% sucrose water and tap water were given for training at the end of 24 h. After training, feeding was performed for 3 days and a baseline measurement of sucrose drinking water was taken.
Next, rats were fasted from food and water for 14 h. Rats were then made to select from two identical bottles for 1 h: one bottle with 1% sucrose solution and the other bottle with water. Sucrose and water intake were measured, and sucrose preference was calculated: SP =sucrose intake ×100%/ (sucrose intake +water intake).
After 25 days of exposure to a stressful environment, the SPT was repeated to evaluate the drug effect.

| Novelty-suppressed feeding test in the CUS model
The NSF test method performed as previously described. 33 In summary, rats were fasted for 24 h and then put it into the corner of the chamber (76 × 76 × 46 cm) with the floor covered with 2 cm thick of sawdust and with 12 food pellets on it; the rats were allowed to explore freely for 5 min. Record the latency to eating pellets of rats.
Eating food in rats refers to chewing or biting, but not by sniffing or playing with pellets.

| Learned helplessness paradigm
In order to demonstrate the antidepressant effect of sub-chronic administration of GW117, ICR mice were acclimatized to the learned helplessness test, as reported previously. 34 Ninety-eight mice were randomly divided into no inescapable shock group (NIS, n = 10) and inescapable shock group (IS, n = 88). For 4 consecutive days, mice were putted into the shuttle box device (40 × 10 × 13 cm), the device was splited into two compartments, and mice could free access. In the IS group, each mouse received 360 inescapable shocks (current

| Statistical analysis
Differences between groups were determined by one-way ANOVA and Dunnett's test. In all tests, differences with p < 0.05 were regard as significant difference. All the data are based on the mean ± S.E.M. and use GraphPad Prism 8 for analysis (GraphPad Software Inc.,).

Competition binding experiments with [ 3 H]-LSD and [ 3 H]-melatonin
showed that GW117 has high affinity for 5-HT 2C receptors, MT 1 receptors, and MT 2 receptors. The K i value of GW117 was 0.32 ± 0.08, 0.12 ± 0.06, and 54 ± 7.31 nM, respectively (Table 1). Furthermore, the affinity of GW117 for the three targets was similar to that of agomelatine ( Figure 2 and Table 1). Encouragingly, our agomelatine results are consistent with those reported in the literature. 35

| Effect of GW117 on open field behavior in CUS rats
As illustrated in Figure 5A

| Effect of GW117 on sucrose preference in CUS rats
As showed in Figure 5C, chronic administration of GW117 (5, 10, 20, and 40 mg/kg) markedly increased sucrose preference in stressed

| Effect of GW117 on latency to begin eating in CUS rats
As illustrated in Figure 5D, chronic treatment with GW117(5, 10,

| Antidepressant effect of GW117 on the learned helplessness paradigm in mice
The results are shown in Figure 6, after 4 consecutive days of inescapable shock training, it was found that mice in the IS group continued to show the deficit of avoidance behavior between the second day ( Figure 6A and B) and fourth days ( Figure 6C and D) after ines- tine has no effect on the number of failures and the escape.

| DISCUSS ION
In this research, we comprehensively evaluated the pharmacodynamics and pharmacology of GW117 and proved that GW117 is a Nowadays, immobility and swimming are seen as the expression of different coping strategies. Some studies suggest that drugs tend to improve depressive symptoms in humans and animals, such as anhedonia,. [36][37][38] Since the stimulatory effect of antidepressants on the CNS (cen- weeks of the stresses inhibited locomotor activity, decreased sucrose preference, and prolonged feeding latency in rats (all indicators of core symptom in major depression). Chronic administration of GW117 rehabilitation these symptoms to a normal state indicates that GW117 has an antidepressant effect, consistent with the acute effect. In addition, study has shown that the CUS model can also lead to anxiety behavior. 39 The NSF test is commonly used to test the anxiolytic effects of drugs, and the model is thought to be sensitive to treatment with antidepressants. 33 In our study, chronic administration of GW117 significantly shortened the feeding latency of the rats, compared with agomelatine (10 mg/kg) that was not effective in this test. To date, many laboratories have demonstrated that the learned helplessness model exhibits behavioral outcomes such as withdrawal and passive behaviors consistent with those exhibited by patients with major depressive disorder. 39 We used this paradigm to investigate the antidepressant effect of GW117 and the results suggest that sub-chronic treatment with GW117 has an antidepressant effect; the number of failures and escape latency was significantly reduced compared with the control mice. These findings were similar to those obtained using the first-line SSRI antidepressant fluoxetine.
It should be noted that dose ranges for antidepressant-like and anxiolytic effects of GW117 in rats and mice are between 5 and 40 mg/kg. Together with our present results, they provide support for the hypothesis that the antidepressant effects of GW117 may require combined action at both melatonin (MT 1 /MT 2 ) and 5-HT 2C receptors. In our subsequent study, we hope to elucidate the relationship between the modulation of depression by GW117 and the behavioral effects under stressful conditions. Current antidepressants, including selective serotonin reuptake inhibitors (SSRIs), tricyclics, and monoamine oxidase inhibitors, have major limitations. 40,41 These agents must be continuously administered for a minimum of 2-4 weeks to produce therapeutic effects and only 30-40% of patients respond to first-line treatment 42,43 . Faster-onset antidepressant treatments are greatly needed to improve the treatment of depression. 41 Recent studies in rodents suggest that acute ketamine treatment induces rapid-onset antidepressant effects through rapid activation of extracellular signal-regulated kinase (ERK) and protein kinase B/Akt, which activate the mammalian target of rapamycin (mTOR) pathway. [41][42][43] Early attempts of ketamine in the neurobiology of psychosis and schizophrenia were found to have a rapid antidepressant effect, and it was revealed that this rapid antidepressant-like effect was associated with N-methyl-D-aspartate (NMDA) receptors. 40 John Krystal, Rob Berman, Dennis Charney, and colleagues at Yale University conducted a small, double-blind, placebo-controlled trial to test the antidepressant effects of ketamine. 42 In this trial, a single dose of ketamine was observed to produce a rapid antidepressant response with a mean onset time of four hours lasting at least three days. 43 Transient psychotomimetic and dissociative effects occurred after approximately one to two hours of treatment. But its use is limited due to the side effects of ketamine and the potential for drug abuse. 41 Other work has shown that selective 5-HT 2C antagonists are putative fast-onset antidepressants. Five days of treatment with 5-HT 2C antagonists induced antidepressant behavioral, molecular, and morphological effects that are comparable to those of current antidepressants and the fast-acting agent ketamine. [40][41][42][43] Recently, other work has shown that acute ketamine treatment deactivates eukaryotic elongation factor 2 (eEF2) kinase, resulting in suppression of brain-derived neurotrophic factor (BDNF) translation, which is required for onset of antidepressant behavioral effects. 41 Therefore, GW117, a melatonergic agonist and selective 5-HT 2C antagonist, maybe have a potential to induce faster therapeutic onset than SSRIs. In the following study, we will assess whether GW117 can induce faster-onset antidepressant effects than current antidepressants using chronic models of antidepressant action.

| CON CLUS IONS
In conclusion, our present results demonstrate that GW117 is a novel compound that acts as both a 5-HT 2C receptor antagonist and a MT 1 /MT 2 receptor agonist, and is likely a potent antidepressant in multiple animal models of depression. Our study offers new insights into the advance of antidepressants.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science

Dr Jin has consulted for and received research funding from
Guangwei Pharmaceutical Technology Co., Ltd., (Beijing, China). The remaining authors have nothing to disclose.

AUTH O R CO NTR I B UTI O N S
ZJ designed the study, performed the behavioral tests, analyzed the data, and wrote the manuscript. NG and WG synthesized the novel compounds. TM contributed to the behavioral tests. XL and WZ contributed to the study design, data analysis, and manuscript revision.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.