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International Journal of Neuropsychopharmacology logoLink to International Journal of Neuropsychopharmacology
. 2023 May 19;26(6):415–425. doi: 10.1093/ijnp/pyad020

Characterization of 2 Novel Phosphodiesterase 2 Inhibitors Hcyb1 and PF-05180999 on Depression- and Anxiety-Like Behavior

Yuqing Yan 1,#, Yuhan Zhao 2,#, Yue Lu 3, Abhinav P Acharya 4, Wei Wang 5, Chang-Guo Zhan 6, Jianghong Ye 7, Fu Du 8, Xiongwei Zhu 9, Ying Xu 10,11,✉
PMCID: PMC10289143  PMID: 37208298

Abstract

Background

Phosphodiesterase 2A (PDE2A) represents a novel target for new therapies addressing psychiatric disorders. To date, the development of PDE2A inhibitors suitable for human clinical evaluation has been hampered by the poor brain accessibility and metabolic stability of the available compounds.

Methods

Corticosterone (CORT)-induced neuronal cell lesion and restraint stress mouse model were used to measure the neuroprotective effect in cells and antidepressant-like behavior in mice.

Results

The cell-based assay showed that both Hcyb1 and PF were potent in protecting cells against stress hormone CORT insults by stimulating cAMP and cGMP signaling in hippocampal cells (HT-22). Administration of both compounds before treatment of CORT to cells increased cAMP/cGMP, VASP phosphorylation at Ser239 and Ser157, cAMP response element binding protein phosphorylation at Ser133, and brain derived neurotrophic factor BDNF expression. Further in vivo study showed that both Hcyb1 and PF displayed ­antidepressant- and anxiolytic-like effects against restraint stress as indicated by reduced immobility time in the forced swimming and tail suspension tasks as well as increased open arm entries and time spent in open arms and holes visit in elevated plus maze and hole-board tests, respectively. The biochemical study confirmed that these antidepressant- and anxiolytic-like effects of Hcyb1 and PF were related to cAMP and cGMP signaling in the hippocampus.

Conclusions

The results extend the previous studies and validate that PDE2A is a tractable target for drug development in the treatment of emotional disorders such as depression and anxiety.

Keywords: Hcyb1, PF-05180999, PDE2A, cGMP/cAMP, stress, anxiety- and depression-like behavior

Significance Statement.

PDE2A is a novel target for new therapies addressing psychiatric disorders. Previous development of these therapies has been stymied by poor brain penetration and metabolization of the currently available therapies. Hcyb1 and PF were potent in protecting cell viability against stress hormone corticosterone insults demonstrated by increased cAMP/cGMP, VASP, and cAMP response element binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) expression. Additionally, Hcyb1 and PF displayed the desired antidepressant- and anxiety-like effects against stress in mice as indicated by reduced immobility time in the forced swim and tail suspension tasks. These findings extend the previous studies and support PDE2A as a target for amelioration of depression- and anxiety-like behavior. Further chemical findings garnered from this study supports the hypothesis that PDE2A is an excellent target for drug development in the treatment of emotional disorders.

INTRODUCTION

Major depressive disorder is currently ranked as the second-leading cause of disability worldwide and the eleventh-leading cause of global disease burden (Paul et al., 2020). In the United States, the incidence of suffering from depression and anxiety sharply increased from 34.6% to 47.5% by 2020, and even worse during the pandemic, leading to a disease burden exceeding $210 billion over the last 2 decades (Sussman et al., 2019; Greenberg et al., 2021). Despite the variety of therapeutics for treatment of symptoms of depression and anxiety, only approximately one-third of patients respond to the first treatment, leaving the other two-thirds of patients who either never respond to antidepressants (Hetrick et al., 2007) or experience relapse (Al-Harbi 2012). Some studies suggest that patients with depression who take selective serotonin reuptake inhibitors may experience side effects such as definitive suicidal behavior and suicidal ideation (Hetrick et al., 2007; Viswanathan et al., 2020). The challenge in finding effective targets for depression and anxiety (Flint and Kendler, 2014) has led to increased efforts to investigate the post-monoamine receptor signaling pathway underlying this disorder.

It has been suggested that intracellular cyclic nucleotide cAMP/cGMP signaling is inversely involved in an individual’s vulnerability to stress-induced depression and anxiety (Xu et al., 2013). Phosphodiesterases comprise a class of key enzymes that catalyze the hydrolysis of intracellular cAMP and cGMP (Masood et al., 2008; 2009; Delhaye and Bardoni 2021). Among the 11 PDE families, PDE2 is a dual-specificity enzyme hydrolyzing both cAMP and cGMP (Li et al., 2005; Masood et al., 2008, 2009). A recent study suggests that PDE2 is encoded by a single gene PDE2A and highly expressed in the frontal cortex, hippocampus, amygdala, hypothalamus, pituitary, and adrenal cortex, indicating its crucial role in mediating negative feedback of the limbic-hypothalamus-pituitary-adrenal gland axis in stress-related psychiatric disorders (Xu et al., 2011). Our previous studies demonstrated that inhibition of PDE2 by Bay 60-7550 (Bay) ameliorated anxiety- and depression-like behavior and improved memory performance in stressed mice (Xu et al., 2015; Huang et al., 2018b). Considering that PDE2 is not found in the area postrema, the brain region related to nausea and emesis (Ruan et al., 2019), modulation of cAMP and cGMP levels by inhibition of PDE2 implicates a particular advantage over other phosphodiesterases toward treatment of MAJOR depressive disorder. However, the efficacy of Bay is limited because of its low penetration of the blood-brain barrier and poor metabolic stability. It is imperative to develop novel PDE2 inhibitors with high selectivity and potency and good brain penetration for the treatment of emotional disorders.

The novel and selective PDE2A inhibitor, Hcyb1 (Figure 1), was designed with our collaborators based on PDE-focused library screening by a high-throughput fluorescence polarization phosphodiesterase assay (Liu et al., 2018). The present study assessed the pharmacological profile of Hcyb1 and the related cAMP/cGMP-pVASP-CREB-BDNF signaling pathway in vitro and in vivo compared with the Pfizer compound PF-05180999 (PF; Figure 1).

Figure 1.

Figure 1.

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The chemical structure of Phosphodiesterase 2A inhibitors Hcyb1 and PF-05180999 (PF).

MATERIALS AND METHODS

Drugs and Reagents

In cell-based assays, Hcyb1, PF, Bay, and corticosterone were dissolved in 100% dimethyl sulfoxide (DMSO) then rapidly diluted with stirring into 5% Kolliphor H15 (Solutol H15) in the cell culture media (or phosphate-buffered saline for in vivo test) to the final dosing concentration. The final concentration of DMSO did not exceed 0.1%. Cells were exposed to 100 μM corticosterone for 24 hours, and then the parameters reflecting cell lesion were examined. PDE2 inhibitors were pretreated to these cells 30 minutes before corticosterone treatment (Huang et al., 2019). Two lead compounds, Hcyb1 and PF, were dissolved in 100% DMSO at a stock concentration of 10 mM and then diluted into culture medium at a final concentration in which the DMSO concentration was <0.1%. The levels of cGMP, cAMP, pVASP, pCREB, and BDNF were measured by enzyme-linked immunosorbent assay (ELISA) or immunoblot assays.

In the behavioral tests, mice were assigned to 1 of 10 treatment groups: control (vehicle-treated only); Hcyb1 (0.3, 1, and 3 mg/kg i.p. injection); PF (0.3, 1, and 3 mg/kg i.p.); Bay (3 mg/kg i.p.); or diazepam (0.5 mg/kg i.p.). The dosing volume was 10 mL/kg. The working solution was prepared in the morning of the experiment. Drugs were administered 30 minutes before restraint stress procedure for 2 days. The behavioral tests were conducted 30 minutes after last restraint stress.

HT-22 Cell Cultures

HT-22 cells were generous gifts from Dr David Schubert at the Salk Institute. Cell cultures were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and cultured at 37°C in 5% CO2. Cells were plated at 1 × 105 cells/mL for all the tests. For the assays, HT-22 cells were treated with corticosterone (100 μM) to replicate stress.

Measurement of Cell Viability by CCK8 Assay in HT-22 Cells

Cell viability was performed by CCK8 assay according to the manufacturer’s instructions (Cat. No. CK04-11, Dojindo, Japan). In brief, the cells were first seeded onto a 96-well plate. After a period of 24 hours, drugs were administered at various concentrations from 0.01 to approximately 10 μM. The 10-µL/well CCK8 reaction solution was added to each well in 24 hours. The plate was incubated at 37°C for 30 minutes, and the optical density (OD) value was read at an absorbance of 450 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

Measurement of Cell Death by Lactate Dehydrogenase (LDH) Assay in HT-22 Cells

In the LDH assay, the cell injury was tested by measuring release of LDH from damaged cells (Wang et al., 2008). LDH assay was performed according to the manufacturer’s instructions (Cat. No. C20301, Invitrogen, Waltham, MA, USA). In brief, cells were plated at 1 × 105 cells/mL and incubated overnight at 37°C. The 10 µL of ultrapure water was added to 1 set of triplicate wells (spontaneous LDH) the next day. However, nothing was added to 1 set of triplicate wells (maximum LDH), while 10 µL of drug was added to the other well to pertinent triplicate wells (chemical-treated LDH activity). After 24 hours, 10 µL of 10 × Lysis buffer was added to the maximum LDH activity wells then mixed by gentle tapping. Then 7.50 µL of medium for each sample was added for triplicate. A total of 50 µL of reaction mixture was then added to each well, and the plate was incubated for 30 minutes, protected from light. After 50 µL of stop solution was added, the absorbance was read at 490 nm.

Measurement of cAMP and cGMP in HT-22 Cells and Hippocampal Tissues of Mice

The cGMP (cyclic GMP Complete Enzyme Immunoassay Kit, ENZO Life Science) and cAMP (cyclic AMP Complete Enzyme Immunoassay Kit, ENZO Life Science) ELISA kits were used for measuring cGMP and cAMP levels in the HT-22 cells and the hippocampus of mice. Briefly, cells or tissues were collected in labeled and preweighed 1.5 mL U-bottom polycarbonate tubes (Micronic, Aston, PA, USA). A total of 50 μM of the conjugate and antibody solution were added to 100 μL of tissue-derived or cell-derived supernatant in a 96-well plate. The mixture was prepared for shaking incubation (500 rpm) at room temperature for 2 hours. After washing 3 times, 200 μL para-Nitrophenylphosphate (pNpp) substrate solution was added to each well, and the plate was incubated for another 1 hour. After stop solution was added to each well, the sample absorbance was measured at 405 nm by microplate reader (SpectraMax, San Jose, CA, USA).

Animals and Housing

Male C57BL/6 mice (ENVIGO RMS INC, Indianapolis, IN, USA) weighing 20–28 g (2.5 months of age) were used. Rodent chow and tap water were freely available. Mice were housed 3 per cage under standard colony conditions, with a 12-hour-light/-dark cycle (lights on at 6:00 am). Experimental procedures were performed between 9:00 am and 5:00 pm. All experiments were carried out according to the NIH Guide for the Care and Use of Laboratory Animals (revised 2014) and approved by the Animal Care and Use Committee of University at Buffalo, the State University of New York, and Rutgers, the State University of New Jersey.

Subacute Restraint Stress Procedure

Subacute restraint stress was performed as previously described with minor modifications (Huang et al., 2018a; Zhu et al., 2020). Mice were subjected to restraint stress in ventilated 50-mL conical tubes (with no access to food or water) 4 hours daily for 2 days. The control and stressed mice were treated with vehicle, different doses of Hcyb1 and PF, or the positive drugs 30 minutes prior to stress procedure for 2 days. The behavioral tests, including forced swimming (FST), tail suspension (TST), elevated plus-maze (EPM), and hole-board (HB), were performed 30 minutes after the stress procedure on the second day to rule out false- negative or false-positive behaviors.

Tail Suspension Test

To assess antidepressant-like effects of PDE2 inhibitors, TST was carried out as previously described (Panconi et al., 1993) with minor modification. Briefly, individual mice were acoustically and visually isolated and suspended approximately 50 cm above the floor with adhesive tape placed on the mice approximately 1 cm from the tail tip. Mice were considered immobile when they were passively suspended and remained completely motionless. Each animal was suspended for 6 minutes, and total immobility time was recorded during the last 4 minutes of the test.

Forced Swimming Test

To assess antidepressant-like effects of PDE2 inhibitors, FST was carried out as previously described (Panconi et al., 1993). Briefly, mice were forced to swim in a cylinder (20 cm high × 14 cm diameter) containing fresh water (25 ± 1°C) to a height of 10 cm. The immobility time was recorded as the time the mice spent floating in the water without struggling or only making movements necessary to keep their heads above the water. Each animal was forced to swim for 6 minutes, and the total immobility time was recorded during the last 4 minutes of the test.

Elevated Plus-Maze Test

This test is widely accepted for anxiety-like behavior as previously described in detail (Xu et al., 2011). Briefly, the EPM test (San Diego Instruments, San Diego, CA, USA) consisted of 2 open arms (30 cm × 5 cm) and 2 closed arms (30 × 5 × 15 cm) that extended from a central platform (5 cm × 5 cm). The entire maze was elevated 40 cm above the floor. During the 5 minutes of free exploration, the number of entries (with the first 2 paws in) and time spent in both open arms and closed arms was recorded for 5 minutes by a camera and analyzed by the EthoVision XT system (Noldus Informative Technology, Inc., Leesburg, Virginia, USA). The percentages of entries onto open arms and time spent on open arms were calculated as open-arm entry (%) = number of open-arm entries/total number of arm entries × 100%; open-arm time (%) = time (s) spent in open arm/total time (s) in all arms × 100%. To eliminate any effect of olfactory cues from previous trials, the maze was cleaned with 75 % ethanol solution and dried thoroughly after each test.

Hole-Board Test

This test is widely used for assessing exploratory behavior of mice as previously described (Juilfs et al., 1999; Zhang et al., 2017). The apparatus consisted of a box of 60 cm×30 cm with 16 evenly spaced holes with built-in infrared sensors used to detect the number of head-dips into the holes and the time of head-dipping. Each mouse was placed in the center of the HB and allowed to freely explore the apparatus for 5 minutes. The number of head-dips and total time spent in head-dipping were recorded.

Locomotor Activity (LA)

LA was performed using an open-field arena as previously described (Liu et al., 2018) with minor modification. The floor of the chamber was divided into 20 squares. Mice were individually placed in the center of the arena and allowed to freely explore the environment for 20 minutes. The locomotor counts were calculated by counting the number of times that all 4 paws crossed the line in the following 10 minutes. After completion of behavioral tests, mice were killed, and the hippocampus was dissected out and stored at −80ºC for later analysis.

Protein Extracts and Immunoblot Assay

The cells were digested with lysis buffer: 50 mmol/L Tris HCl, pH 7.2, containing 1% sodium deoxycholate, 1% nonyl phenoxypolyethoxylethanol (NP-40), .15 mmol/L NaCl, and 0.1% sodium dodecyl sulfate (SDS) (Roche Applied Science, Mannheim, Germany). Protein extracts were obtained by homogenizing hippocampal tissues or HT-22 cells. The Bicinchoninic acid assay (BCA assay) was then used to obtain the protein concentration (Sigma-Aldrich, St. Louis, MO, USA). Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a PVDF membrane (0.2 μm, EMD Millipore., Billerica, MA, USA). The membranes were blocked for 2–3 hours with 5% nonfat dry milk at room temperature and then incubated at 4°C overnight with the following antibodies: rabbit anti-pCREB (ser 133, 1:1000, Cell Signaling Technology, #9198), anti-CREB (1:1000, Cell Signaling Technology #9197), anti-BDNF (1:500, Cell Signaling Technology, #47808), anti-pVASP (s157, 1:500, Cell Signaling Technology, #47808), anti-pVASP (s239, 1:500, Invitrogen, #PA5-117182), anti-VASP (1:500, Invitrogen, #PA5-82211), and rabbit anti-β-actin (1:1000, Cell Signaling Technology, #4970). After 3 washes in TBS-triton X-100 buffer, the membranes were incubated for 1 hour with horseradish peroxidase–labelled anti-rabbit IgG (1:5000, Bioworld Technology, St. Louis Park, MN, USA). Densitometric measurements were made using the ECL Western detection system (Millipore Co., Ltd.) and the Quantity One imaging program (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Data Analysis

All data are expressed as means ± SEM. Statistical significance was assessed by 1-way ANOVA followed by a Dunnett’s t test. The criterion for significance was P < .05.

RESULTS

Neuroprotective Effects of Hcyb1 and PF in HT-22 Cells With or Without Corticosterone Exposure

The structures of 2 PDE2 inhibitors Hcyb1 and PF are shown in Figure 1. As shown in Figure 2, treatment of naïve HT-22 cells with Hcyb1 at concentrations from 0.01 to 1 μM for 24 hours did not show significant changes in cell viability in CCK8 assay, though there was a tend to increase cell viability at a concentration of 1 μM that may suggest 1 μM Hcyb1 protected cells. This result was consistent with the LDH assay that showed 1 μM Hcyb1 decreased cell death compared with vehicle-treated groups (Figure 2A–B; P < .05). Treatment of PF at a concentration of 1 μM showed significant protective effects on these naïve cells in both CCK8 and LDH assays, as shown by increased cell viability and decreased cell death (P < .05). However, increasing concentrations of both Hcyb1 or PF seemed to increase toxicity, as reflected by the decreased tendency of cell viability in CCK8 assay. When these HT-22 cells were exposed to stress hormone, for example, 100 μM corticosterone, pretreatment of both Hcyb1 and PF showed significant neuroprotective effects, as shown by decreased OD values in LDH assay concentration–dependent and increased cell viability in CCK8 assay (F(9,50) = 3.363, P < .05). As a control group, the PDE2 inhibitor Bay also showed its neuroprotective effects at a concentration of 1 μM in CCK8 and LDH assays in both naïve and corticosterone-treated cells, indicating that PDE2 inhibitors have neuroprotective effects (Figure 2C–D; P < .01, P < .05, respectively).

Figure 2.

Figure 2.

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The effects of Hcyb1 and PF on cell viability in HT-22 cells with or without corticosterone exposure. (A and B) HT-22 cells were treated with different concentrations of Hcyb1 and PF for 24 hours. Cell viability and cell death were assessed by CCK8 and LDH assays. (C and D) HT-22 cells were pretreated with various concentrations of Hcyb1 and PF for 30 minutes, and then corticosterone (100 μM) was added to cells for additional 24 hours before measurement of cell viability and cell death. Values are expressed as mean ± SEM (n = 6). **P < .01 vs vehicle-treated control group. #P < .05, ##P < .01 vs vehicle-treated corticosterone group. Bay, Bay 60-7550.

Effects of Hcyb1 and PF on cGMP and cAMP Levels in Corticosterone-Treated HT-22 Cells

The effects of Hcyb1 and PF on accumulation of cGMP and cAMP in corticosterone-treated HT-22 cells are shown in Figure 3. When HT-22 cells were exposed to 100 μM corticosterone for 24 hours, a significant cGMP and cAMP reduction was observed compared with control groups (Figure 3A–D; P < .01). However, this corticosterone-induced cGMP reduction was reversed by pretreatment of cells with either Hcyb1 or PF at concentrations of 0.1 and 1 μM (Figure 3A and C; P < .05, P < .01). Similar to the cGMP assay, corticosterone-induced cAMP reduction was reversed by Hcyb1 at 1 μM and PF at 0.1 and 1 μM (Figure 3B and D; P < .05, P < .01, respectively). These effects were also observed in Bay-treated groups (P < .01, P < .05).

Figure 3.

Figure 3.

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The effects of Hcyb1 and PF on cGMP and cAMP levels in corticosterone-treated HT-22 cells. HT-22 cells were treated with different concentrations of Hcyb1 and PF for 30 minutes, and then corticosterone (100 μM) was added for additional 24 hours before measurement of cGMP (A, C) and cAMP (B, D) levels by ELISA assay. Values are expressed as mean ± SEM (n = 6). **P < .01 vs vehicle-treated control group. #P < .05, ##P < .01 vs vehicle-treated corticosterone group.

Effects of Hcyb1 and PF on pVASPser239 and pVASPser157 Expression in Corticosterone-Treated HT-22 Cells

As the readout for cGMP-mediated signaling showed, VASP phosphorylation at serine 239 (pVASPser239) can be activated by PKG (Smolenski et al., 1998). However, both PKA and PKG can stimulate VASP phosphorylation at serine157 (pVASPser157) (Butt et al., 1994). We examined whether the protective effects of PDE2 inhibitors against corticosterone insults are pVASPser239 or pVASPser157 dependent. As shown in Figure 4, VASP phosphorylation was reduced at serine 239 and serine 157 after treatment of HT-22 cells with 100 μM corticosterone for 24 hours (P < .01). In contrast, Hcyb1 at 1 μM significantly increased pVASP at serine 239 and 157 (Figure 4A, C, and E; P < .01, P < .05). PF at 0.1 and 1 μM significantly increased pVASP at serine 239 (Figure 4B and D; P < .05, P < .01), while 1 μM PF increased expression of pVASP at serine 157 (Figure 4B and F; P < .05). The positive drug Bay also showed a significant increase in VASP phosphorylation at both serine 239 and serine 157 (Figure 4A–F; P < .05, P < .01). These results suggest that cAMP and cGMP signaling was activated after treatment with these PDE2 inhibitors.

Figure 4.

Figure 4.

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Hcyb1 and PF increased the levels of pVASPser239 (A–D) and pVASPser157 (A, B and E, F) in corticosterone-treated HT-22 cells. Values are expressed as mean + SEM (n = 6). **P < .01, vs vehicle-treated control group. #P < .05, ##P < .01 vs vehicle-treated corticosterone group.

Effects of Hcyb1 and PF on pCREBser133 to CREB Ratio and BDNF Expression in Corticosterone-Treated HT-22 Cells

There are at least 3 potential phosphorylation sites, for example, serine 133, 142, and 271, in the human CREB protein related to stress (Sakamoto et al., 2011). The present study examined the phosphorylation site of CREB at serine 133, which is the main site of regulation upon stress. cAMP and cGMP upregulation results in their downstream neuroprotective proteins expression, such as CREB phosphorylation at serine 133. As shown in Figure 5, the ratio of pCREBser133/CREB decreased when HT-22 cells were exposed to 100 μM corticosterone for 24 hours (P < .01). Pretreatment of Hcyb1 at a concentration of 1 μM significantly increased the ratio of pCREBser133/CREB (Figure 5A; P < .01). This concentration of Hcyb1 also reversed corticosterone-induced BDNF reduction (Figure 5C; P < .05). In contrast, PF at concentrations of 0.1 and 1 μM significantly prevented corticosterone-induced reduction of pCREBser133/CREB (Figure 5B; P < .05, P < .01). These concentrations also reversed BDNF reduction induced by 100 μM corticosterone (Figure 5D; P < .05, P < .01). Similarly, as the positive control PDE2 inhibitor, Bay was also effective on reversion of corticosterone-induced reduction of pCREBser133/CREB and BDNF expression (Figure 5A–B; P < .01; Figure 5C–D; P < .05).

Figure 5.

Figure 5.

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The effects of Hcyb1 (A and C) and PF (BandD) on the ratio of pCREBser133 to CREB and BDNF expression in corticosterone-treated HT-22 cells. Values are expressed as mean ± SEM (n = 6). **P < .01, vs vehicle-treated control group. #P < .05, ##P < .01 vs vehicle-treated corticosterone group.

Antidepressant- and Anxiolytic-Like Effects of Hcyb1 and PF in FST and TST in Stressed Mice

Our pilot study found a significant increase in corticosterone levels when mice were subjected to 2 days restraint stress (data not shown). The antidepressant-like properties of treatment with Hcyb1 or PF at doses of 0.1, 1, and 3 mg/kg (i.p.) for 2 days were evaluated by the FST and TST in mice exposed to restraint stress (4 h/d for 2 days). This restraint stress induced a significant increase in immobility time both in the FST and TST (Figure 6A–B; P < .01). However, treatment of Hcyb1 at a dose of 3 mg/kg 30 minutes before the restraint stress for 2 days significantly decreased immobility time in the FST (Figure 6A; P < .01) but not in the TST (Figure 6B). However, PF at doses of 1 and 3 mg/kg for 2 days significantly decreased immobility time both in the FST and TST (P < .05, P < .01). Treatment with Bay at a dose of 3 mg/kg for 2 days decreased immobility time in the FST but not in the TST (Figure 6A–B ; P < .05). Particularly, none of doses of Hcyb1 and PF that induced change in immobility time affected LA, indicating that the stimulation or inhibition of central nervous system (CNS) was not involved in the mechanisms that affected depression-like behavior of these PDE2 inhibitors (Figure 6C).

Figure 6.

Figure 6.

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Hcyb1 and PF decreased the immobility time in forced swimming (A) and tail suspension (B) tests but did not affect locomotor activity at the doses tested (C). Mice were treated with Hcyb1 or PF 30 minutes before being subjected to restraint stress for 2 days. The behavioral tests were conducted 30 minutes after last restraint stress. Results are expressed as mean ± SEM (n = 8). **P < .01 vs vehicle-treated control group. #P < .05, ##P < .01 vs vehicle-treated stressed group.

In the subsequent study, 2 days restraint stress induced reduced percentage of open arm entries, time spent in the open arms, and the holes visit in the EPM and HB tests (Figure 7A–C; P < .01). In contrast, treatment of PDE2 inhibitor Hcyb1 at 3 mg/kg resulted in a tendency to increase the percentage of open arm entries (Figure 7A) and an increase in the percentage of time spent on open arms (Figure 7B, P < .01) in EPM test as well as time spent in dipping (Figure 7C, P < .05) in HB test. While PF at doses of 1 and 3 mg/kg significantly increased the percentage of open arm entries (P < .01), PF at 3 mg/kg induced an increase in the percentage of time spent on open arms (P < .01) in the EPM test and time spent in dipping (P < .01) in HB test. However, the total arm entries did not change after treatment with both compounds, indicating a lack of CNS stimulation or inhibition involvement (Figure 7D). Similar effects were found in the positive drug diazepam at a dose of 0.5 mg/kg (i.p.), which also increased the percentage of open arm entries and time spent on open arms in the EPM test along with increased holes visited in the HB test (Figure 7A–C; P < .05, P < .01).

Figure 7.

Figure 7.

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Hcyb1 and PF increased the percentage of open arm entries (A) and time spent on open arms (B) in the EPM test as well as increased the number of hole visit in the HB test (C) in stressed mice, whereas drugs did not affect total arm entries at the doses tested (D). Mice were treated with Hcyb1 or PF 30 minutes before being subjected to restraint stress for 2 days. The behavioral tests were conducted 30 minutes after last restraint stress. Values are expressed as mean ± SEM (n = 8). **P < .01 vs vehicle-treated group. #P < .05, ##P < .01 vs vehicle-treated stressed group. DZP, diazepam.

Effects of Hcyb1 and PF on cGMP and cAMP Levels in Hippocampus of Stressed Mice

To determine whether Hcyb1 and PF induced antidepressant- and anxiolytic-like effects are involved in the cAMP- and cGMP-dependent pathway, the cAMP and cGMP levels in mouse hippocampus were determined after behavioral tests. The results showed that both the cAMP and cGMP levels in stressed mice were significantly decreased compared with the control group (Figure 8A–B; P < .01). As expected, treatment of Hcyb1 and PF at a dose of 3 mg/kg significantly increased the levels of cGMP and cAMP (Figure 8A and B; P < .05, P < .01). The positive drug, Bay, also showed an increase in cGMP and cAMP level at a dose of 3 mg/kg (Figure 8A–B; P < .05).

Figure 8.

Figure 8.

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Hcyb1 and PF increased the levels of cGMP (A) and cAMP (B) in the hippocampus of stressed mice. Values are expressed as mean ± SEM (n = 8). **P < .01 vs vehicle-treated control group. #P < .05, ##P < .01 vs vehicle-treated stressed group.

Effects of Hcyb1 and PF on pVASPser239 and pVASPser157 in Hippocampus of Stressed Mice

Considering that pVASP at serine 239 and serine 157 are related to cGMP and cAMP signaling, the present study examined levels of pVASPser239 and pVASPser157 in stressed mice with or without treatment of 2 novel PDE2 inhibitors (supplementary Figure 1). The results showed that the 2 days of restraint stress caused significant decreases in levels of pVASPser239 and pVASPser157 (supplementary Figure 1C–F; P < .01). As expected, these decreased levels of pVASPser239 and pVASPser157 were significantly prevented by treatment with increasing doses of 2 compounds, Hcyb1 and PF (F(5,30) = 8.0, P < .01; F(5,30) = 6.374, P < .01). The maximal increases of pVASPser239 were found as treatment with Hcyb1 at dose of 3 mg/kg (supplementary Figure 1 C; P < .01) and PF at doses of 1 and 3 mg/kg (supplementary Figure 1 D; P < .05 and P < .01). pVASPser157 expression arrived in significant increases as treatment with either Hcyb1 or PF at dose of 3 mg/kg (supplementary Figure 1 E–F; P < .05). Similarly, Bay also induced significant increases in these 2 phosphorylation levels of VASP (supplementary Figure 1 E–F, P < .05; C and D, P < .01).

Effects of Hcyb1 and PF on pCREBser133/CREB Ratio and BDNF Expression in Hippocampus of Stressed Mice

To verify the involvement of CREB phosphorylation at serine 133 in stressed mice treated with PDE2 inhibitors, the ratio of pCREBser133/CREB was examined in these mice with or without Hcyb1 and PF treatment. The results showed that significant decreases in the ratio of pCREBser133/CREB in the hippocampus of mice subjected to restraint stress for 2 days (supplementary Figure 2; P < .01). However, the decreased pCREBser133/CREB ratio was reversed by treatment with 3 mg/kg of Hcyb1 (supplementary Figure 2 A; P < .05) and 1 and 3 mg/kg PF (supplementary Figure 2 B; P < .05, P < .01). As a positive control, Bay at 3 mg/kg showed a significant increase in the ratio of pCREBser133/CREB (supplementary Figure 2 A–B; P < .05).

The subsequent experiments examined BDNF expression in the hippocampus of stressed mice with or without drug administration. BDNF expression was significantly decreased when mice were exposed to the subacute restraint stress (supplementary Figure 2 C–D; P < .01). This decreased BDNF expression was reversed by treatment with Hcyb1 and PF at 3 mg/kg (supplementary Figure 2 C–D; P < .01), indicating the neuroprotective mechanism may be involved in the antidepressant- and anxiolytic-like effects of PDE2 inhibitors. As the positive drug, Bay reversed stress-related BDNF reduction (supplementary Figure 2 C–; P < .05), while diazepam did not show such effects.

DISCUSSION

PDE2 is a promising pharmacological target for the treatment of CNS diseases (Zhang et al., 2017). The role of PDE2 in cyclic nucleotide signaling and its expression in the forebrain suggest the involvement in mood and emotional disorders such as depression, anxiety, and learning and memory disorder (Zhang et al., 2015). As most current PDE2 inhibitors contain a pharmacophore of pyrazolopyrimidinone, the selective PDE2 inhibitor Hcyb1 was designed by introducing more lipophilic groups with polar functionality to the scaffold pyrazolopyrimidinone to improve the cell membrane penetration (Liu et al., 2018). This compound was evaluated for protective effects in cultured hippocampal cells, for example, HT-22 cells, and antidepressant- and anxiolytic-like effects in stressed mice. Hcyb1 increased cAMP and cGMP levels in corticosterone-treated HT-22 cells, which were parallel to those of PF. Both Hcyb1 and PF exhibited significant antidepressant- and anxiolytic-like effects in stressed mice in the FST, TST, EPM, and HB tests, the well-established mouse models for screening the behavioral effects of novel compounds. However, the fact that the relatively lower doses of PF produced antidepressant- and anxiolytic-like effects in these behavioral tests suggests that the effects of PF could be better than those of Hcyb1 on depression- and anxiety-like behaviors (Figures 6A, 6B, and 7A). To produce a better antidepressant-like effect, the structure modification of Hcyb1 may be necessary. In addition to increased cAMP and cGMP levels, Hcyb1 increased expression of pVASP, pCREB, and BDNF, 3 cAMP- and cGMP-regulated downstream proteins, in both HT-22 cells and mouse hippocampus, which were similar to those of PF. Overall, the present study demonstrated that 2 PDE2A inhibitors, Hcyb1 and PF, produced antidepressant- and anxiolytic-like effects, which are associated with cGMP- and cAMP-dependent neuroprotective effects.

The classic PDE2A inhibitor Bay has been used as a pharmacological tool to provide proof-of-concept for inhibition of PDE2A, which led to reversal of depression-like behavior in several studies (Ding et al., 2014). However, the concentrations and doses of Bay are higher than predicated by its low nanomolar in vitro potency. Recently, a series of PDE2A inhibitors was described (Buijnsters et al., 2014; Tresadern et al., 2020), but none of them have advanced beyond phase I trials due to poor metabolic stability, low brain penetrance, or tolerability issues. In an attempt to overcome such limitations, we designed and optimized small molecules by scaffold-hopping of known PDE2A inhibitors. Hcyb1 is such a compound that possessed superior brain penetration and metabolic stability paralleled to those of most compounds reported in the literature, including PF and Bay (Liu et al., 2018; Huang et al., 2019).

The discovery of the promising novel inhibitors prompted us to advance this work to in vitro cell-based assay to further assess the cAMP- and/or cGMP-dependent neuroprotective effects in hippocampal cell lines. Our previous studies suggested that inhibition of PDE2A by Bay exhibited neuroprotective effects, as evidenced by increasing the cell viability in both the cultured primary hippocampal neurons and HT-22 cells (Xu et al., 2013; Huang et al., 2018b). The present study tried to address the relationship between stress hormone exposure and the neuroprotective effects of our newly synthesized PDE2 inhibitor Hcyb1 and the other compound, PF, on stress-induced dysregulated cAMP signaling. The results showed that Hcyb1 and PF reversed corticosterone-induced cell lesion and decreases in cAMP and cGMP levels in HT-22 cells in a concentration-dependent manner, indicating that these 2 compounds have favorable potency in this cell-based model. Moreover, both of them increased pVASPser239 and pVASPser157, which are similar to the effects of positive drug Bay. pVASPser239 is a marker for cGMP-/PKG-mediated signaling activation (Butt et al., 1994; Smolenski et al., 1998), while pVASPser157 is mainly activated by PKA, though it is also activated by PKG (Butt et al., 1994). The fact that PDE2 can hydrolyze both cGMP and cAMP prompts us to examine the phosphorylation of VASP on both serine 239 and serine 157. We found that Hcyb1 and PF increased pVASPser239 and pVASPser157 and their downstream proteins pCREBser133 and BDNF expression in the hippocampus of mice, which were similar to the results for Bay. Thus, the in vitro and in vivo data suggest that both Hcyb1 and PF may regulate behavioral changes via inhibiting PDE2A and activating cAMP/cGMP-VASP-CREB-BDNF signaling pathway.

The high expression of PDE2A in the forebrain has been found to be related to mood and emotional disorders (W. Reierson et al., 2011; Stephenson et al., 2012). This specific expression makes its inhibitors valuable as the potential therapeutics for the treatment of depression and anxiety. Previous studies showed that the known PDE2A inhibitor Bay could ameliorate depression-like behavior through regulating cAMP- and cGMP-related antioxidant signaling (Xu et al., 2013; Ding et al., 2014; Huang et al., 2018b; Huang et al., 2019). The present study extended the previous results and found that Hcyb1 and PF were effective in the FST, while only PF at mid- and high-dose significantly decreased the immobility time in the TST. The FST and TST are valid mouse models for depression and widely used for screening the efficacy of antidepressants. However, the antidepressant-like effects of some drugs may not be observed after acute treatment in either TST or FST. This would explain why subacute treatment of Hcyb1 for 2 days was insensitive to the TST. The fact that a high dose of Hcyb1 decreased immobility time in the FST suggests that Hcyb1 should be effective for depression-like behavior. Moreover, Hcyb1 and PF increased the percentage of open arm entries and time spent on open arms in the EPM task and hole exploring in the HB test. These findings are in agreement with the results from the positive drug Bay and our previous study that suggest that PDE2 inhibitors are effective in reversing depression- and anxiogenic-like effects induced by stress (Ding et al., 2014). Further study showed that both Hcyb1 and PF did not affect LA at doses that produced antidepressant- and anxiolytic-like effects, demonstrating that these specific effects are not from CNS stimulation and inhibition.

In summary, the results from cell-based assay and behavioral tests support the hypothesis that the lead compounds from novel chemical scaffolds, exemplified by Hcyb1, are potent and a specific inhibitor of PDE2A. Hcyb1 and PF generally resulted in more robust effects over a broader dose/concentration range. These findings offer compelling evidence that the PDE2A inhibitors such as Hcyb1 and PF represent a novel class of drugs for treatment of depression and/or anxiety and could have a major impact on drug discovery research involving PDE2.

Supplementary Material

pyad020_suppl_Supplementary_Figure_S1
Click here for additional data file. (90.8KB, jpeg)
pyad020_suppl_Supplementary_Figure_S2
Click here for additional data file. (769.2KB, jpeg)

Acknowledgements

This work was supported by research grants from the National Institute of Aging (no. R01 AG070873-01A1, 2021-2026) to Dr Ying Xu, and Small Business Innovation Research (SBIR) Program Phase I (no. R43 AG071045-01A1, 2021-2023) to Dr Fu Du.

Contributor Information

Yuqing Yan, Department of Anesthesiology, Rutgers, the State University of New Jersey, Newark, New Jersey, USA.

Yuhan Zhao, Department of Anesthesiology, Rutgers, the State University of New Jersey, Newark, New Jersey, USA.

Yue Lu, Department of Anesthesiology, Rutgers, the State University of New Jersey, Newark, New Jersey, USA.

Abhinav P Acharya, Chemical Engineering School for the Engineering of Matter, Transport, and Energy, Arizona State University, Tempe, Arizona, USA.

Wei Wang, Department of Pharmacology and Toxicology, Arizona Center for Drug Discovery, College of Pharmacy, University of Arizona, Tucson, Arizona, USA.

Chang-Guo Zhan, Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, Kentucky, USA.

Jianghong Ye, Department of Anesthesiology, Rutgers, the State University of New Jersey, Newark, New Jersey, USA.

Fu Du, FD NeuroTechnologies Consulting and Services, Inc., Columbia, Maryland, USA.

Xiongwei Zhu, Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA.

Ying Xu, Department of Anesthesiology, Rutgers, the State University of New Jersey, Newark, New Jersey, USA; Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA.

Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Author Contributions

Yuqing Yan: Experiment design and conducted experiments, cell culture and animal behavior tests, and original manuscript draft and revision. Yuhan Zhao: Cell culture experiments and original manuscript draft and revision. Yue Lu: Animal and data analysis, animal behavior tests. Wei Wang and Chang-Guo Zhan: Experimental design. Fu Du: Funding acquisition and revision. Xiongwei Zhu, Abhinav Acharya, Jianghong Ye: Revision. Ying Xu: Experimental design, original manuscript draft and revision, funding acquisition.

Data Availability

The access of original data generated in the course of the study in the article should contact to the correspondence author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pyad020_suppl_Supplementary_Figure_S1
Click here for additional data file. (90.8KB, jpeg)
pyad020_suppl_Supplementary_Figure_S2
Click here for additional data file. (769.2KB, jpeg)

Data Availability Statement

The access of original data generated in the course of the study in the article should contact to the correspondence author.

Articles from International Journal of Neuropsychopharmacology are provided here courtesy of Oxford University Press

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