Identification of Phosphodiesterase-7A (PDE7A) as a Novel Target for Reducing Ethanol Consumption in Mice.

BACKGROUND: Ethanol elicits a rapid stimulatory effect and a subsequent, prolonged sedative response, which are potential predictors of EtOH consumption by decreasing adenosine signaling; this phenomenon also reflects the obvious sex difference. cAMP (cyclic Adenosine Monophosphate)-PKA (Protein Kinase A) signaling pathway modulation can influence the stimulatory and sedative effects induced by EtOH in mice. This study's objective is to clarify the role of phosphodiesterase (PDE) in mediating the observed sex differences in EtOH responsiveness between male and female animals. METHODS: EtOH was administered i.p. for 7 days to identify the changes in PDE isoforms in response to EtOH treatment. Additionally, EtOH consumption and preference of male and female C57BL/6J mice were assessed using the drinking-in-the-dark and 2-bottle choice tests. Further, pharmacological inhibition of PDE7A heterozygote knockout mice was performed to investigate its effects on EtOH-induced stimulation and sedation in both male and female mice. Finally, Western blotting analysis was performed to evaluate the alterations in cAMP-PKA/Epac2 pathways. RESULTS: EtOH administration resulted in an immediate upregulation in PDE7A expression in female mice, indicating a strong association between PDE7A and EtOH stimulation. Through the pharmacological inhibition of PDE7A KD mice, we have demonstrated for the first time, to our knowledge, that PDE7A selectively attenuates EtOH responsiveness and consumption exclusively in female mice, whichmay be associated with the cAMP-PKA/Epac2 pathway and downstream phosphorylation of CREB and ERK1/2. CONCLUSIONS: Inhibition or knockdown of PDE7A attenuates EtOH responsivenessand consumption exclusively in female mice, which is associated with alterations in the cAMP-PKA/Epac2 signaling pathways, thereby highlighting its potential as a novel therapeutic target for alcohol use disorder.

Methylation-regulated tumor suppressor gene PDE7B promotes HCC invasion and metastasis through the PI3K/AKT signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) has a high mortality rate, and the mechanisms underlying tumor development and progression remain unclear. However, inactivated tumor suppressor genes might play key roles. DNA methylation is a critical regulatory mechanism for inactivating tumor suppressor genes in HCC. Therefore, this study investigated methylation-related tumor suppressors in HCC to identify potential biomarkers and therapeutic targets. METHODS: We assessed genome-wide DNA methylation in HCC using whole genome bisulfite sequencing (WGBS) and RNA sequencing, respectively, and identified the differential expression of methylation-related genes, and finally screened phosphodiesterase 7B (PDE7B) for the study. The correlation between PDE7B expression and clinical features was then assessed. We then analyzed the changes of PDE7B expression in HCC cells before and after DNA methyltransferase inhibitor treatment by MassArray nucleic acid mass spectrometry. Furthermore, HCC cell lines overexpressing PDE7B were constructed to investigate its effect on HCC cell function. Finally, GO and KEGG were applied for the enrichment analysis of PDE7B-related pathways, and their effects on the expression of pathway proteins and EMT-related factors in HCC cells were preliminarily explored. RESULTS: HCC exhibited a genome-wide hypomethylation pattern. We screened 713 hypomethylated and 362 hypermethylated mCG regions in HCC and adjacent normal tissues. GO analysis showed that the main molecular functions of hypermethylation and hypomethylation were "DNA-binding transcriptional activator activity" and "structural component of ribosomes", respectively, whereas KEGG analysis showed that they were enriched in "bile secretion" and "Ras-associated protein-1 (Rap1) signaling pathway", respectively. PDE7B expression was significantly down-regulated in HCC tissues, and this low expression was negatively correlated with recurrence and prognosis of HCC. In addition, DNA methylation regulates PDE7B expression in HCC. On the contrary, overexpression of PDE7B inhibited tumor proliferation and metastasis in vitro. In addition, PDE7B-related genes were mainly enriched in the PI3K/ATK signaling pathway, and PDE7B overexpression inhibited the progression of PI3K/ATK signaling pathway-related proteins and EMT. CONCLUSION: PDE7B expression in HCC may be regulated by promoter methylation. PDE7B can regulate the EMT process in HCC cells through the PI3K/AKT pathway, which in turn affects HCC metastasis and invasion.

Pharmacological modulation of phosphodiesterase-7 as a novel strategy for neurodegenerative disorders.

Neurodegenerative illness develops as a result of genetic defects that cause changes at numerous levels, including genomic products and biological processes. It entails the degradation of cyclic nucleotides, cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP). PDE7 modulates intracellular cAMP signalling, which is involved in numerous essential physiological and pathological processes. For the therapy of neurodegenerative illnesses, the normalization of cyclic nucleotide signalling through PDE inhibition remains intriguing. In this article, we shall examine the role of PDEs in neurodegenerative diseases. Alzheimer's disease, Multiple sclerosis, Huntington's disease, Parkinson's disease, Stroke, and Epilepsy are related to alterations in PDE7 expression in the brain. Earlier, animal models of neurological illnesses including Alzheimer's disease, Parkinson's disease, and multiple sclerosis have had significant results to PDE7 inhibitors, i.e., VP3.15; VP1.14. In addition, modulation of CAMP/CREB/GSK/PKA signalling pathways involving PDE7 in neurodegenerative diseases has been addressed. To understand the etiology, treatment options of these disorders mediated by PDE7 and its subtypes can be the focus of future research.

Circ_0091702 relieves lipopolysaccharide (LPS)-induced cell injury by regulating the miR-182/PDE7A axis in sepsis.

Circular RNA plays an important role in the progression of sepsis. Circ_0091702 has been found to be an important regulator of sepsis progression, so its role and mechanism in sepsis progression deserve to be further explored. Lipopolysaccharide (LPS) could suppress cell viability, while enhance cell apoptosis and inflammation to induce cell injury. Circ_0091702 was downregulated in LPS-induced HK2 cells, and its overexpression alleviated LPS-induced cell injury. MiR-182 could be sponged by circ_0091702. Moreover, miR-182 inhibitor could relieve LPS-induced cell injury, and its overexpression also reversed the inhibition of circ_0091702 on LPS-induced cell injury. PDE7A was a target of miR-182, and its expression was reduced in LPS-induced HK2 cells. Additionally, silencing of PDE7A reversed the suppressive effect of circ_0091702 on LPS-induced cell injury. Our data suggested that circ_0091702 sponged miR-182 to regulate PDE7A, thereby alleviating LPS-induced cell injury in sepsis.

Steroidogenic differentiation and PKA signaling are programmed by histone methyltransferase EZH2 in the adrenal cortex.

Adrenal cortex steroids are essential for body homeostasis, and adrenal insufficiency is a life-threatening condition. Adrenal endocrine activity is maintained through recruitment of subcapsular progenitor cells that follow a unidirectional differentiation path from zona glomerulosa to zona fasciculata (zF). Here, we show that this unidirectionality is ensured by the histone methyltransferase EZH2. Indeed, we demonstrate that EZH2 maintains adrenal steroidogenic cell differentiation by preventing expression of GATA4 and WT1 that cause abnormal dedifferentiation to a progenitor-like state in Ezh2 KO adrenals. EZH2 further ensures normal cortical differentiation by programming cells for optimal response to adrenocorticotrophic hormone (ACTH)/PKA signaling. This is achieved by repression of phosphodiesterases PDE1B, 3A, and 7A and of PRKAR1B. Consequently, EZH2 ablation results in blunted zF differentiation and primary glucocorticoid insufficiency. These data demonstrate an all-encompassing role for EZH2 in programming steroidogenic cells for optimal response to differentiation signals and in maintaining their differentiated state.

Synthesis, biological screening, and molecular docking of quinazolinone and quinazolinethione as phosphodiesterase 7 inhibitors.

N-Substituted isatoic anhydrides were used as starting materials for the synthesis of compounds 5-16 through alkali hydrolysis, Schiff base reactions, and oxidation. Compounds 18-23 were obtained by thionation of their oxo isosteres using Lawesson's reagent. Cyclocondesation of anthranilic acid with thiourea afforded compounds 25-27, which were S-alkylated to afford compounds 28-30, which were thionated using Lawesson's reagent to afford 31-33. The compounds were tested for their in vitro inhibitory activity against the phosphodiesterase 7A (PDE7A) enzyme compared with the selective PDE7 inhibitor BRL50481. All the compounds showed the inhibitory activity on the enzyme at micromolar levels. Compounds 9 and 25 showed the highest inhibitory activity on the enzyme: IC50 = 0.096 and 0.074 µM, respectively, comparable to BRL50481 (IC50 = 0.072 µM). The binding mode and binding affinity of the target compounds at the enzyme PDE7A-binding site were studied through molecular docking. Compounds 9 and 25 showed good recognition at the enzyme-binding site and were capable of binding in an inhibitory mode similar to the reference compound BRL50481, forming the necessary interactions with the key amino acids. Docking studies and enzyme assay were in agreement.

Role of Phosphodiesterase 7 (PDE7) in T Cell Activity. Effects of Selective PDE7 Inhibitors and Dual PDE4/7 Inhibitors on T Cell Functions.

Phosphodiesterase 7 (PDE7), a cAMP-specific PDE family, insensitive to rolipram, is present in many immune cells, including T lymphocytes. Two genes of PDE7 have been identified: PDE7A and PDE7B with three or four splice variants, respectively. Both PDE7A and PDE7B are expressed in T cells, and the predominant splice variant in these cells is PDE7A1. PDE7 is one of several PDE families that terminates biological functions of cAMP-a major regulating intracellular factor. However, the precise role of PDE7 in T cell activation and function is still ambiguous. Some authors reported its crucial role in T cell activation, while according to other studies PDE7 activity was not pivotal to T cells. Several studies showed that inhibition of PDE7 by its selective or dual PDE4/7 inhibitors suppresses T cell activity, and consequently T-mediated immune response. Taken together, it seems quite likely that simultaneous inhibition of PDE4 and PDE7 by dual PDE4/7 inhibitors or a combination of selective PDE4 and PDE7 remains the most interesting therapeutic target for the treatment of some immune-related disorders, such as autoimmune diseases, or selected respiratory diseases. An interesting direction of future studies could also be using a combination of selective PDE7 and PDE3 inhibitors.

A Novel, Pan-PDE Inhibitor Exerts Anti-Fibrotic Effects in Human Lung Fibroblasts via Inhibition of TGF-ß Signaling and Activation of cAMP/PKA Signaling.

Phosphodiesterase (PDE) inhibitors are currently a widespread and extensively studied group of anti-inflammatory and anti-fibrotic compounds which may find use in the treatment of numerous lung diseases, including asthma and chronic obstructive pulmonary disease. Several PDE inhibitors are currently in clinical development, and some of them, e.g., roflumilast, are already recommended for clinical use. Due to numerous reports indicating that elevated intracellular cAMP levels may contribute to the alleviation of inflammation and airway fibrosis, new and effective PDE inhibitors are constantly being sought. Recently, a group of 7,8-disubstituted purine-2,6-dione derivatives, representing a novel and prominent pan-PDE inhibitors has been synthesized. Some of them were reported to modulate transient receptor potential ankyrin 1 (TRPA1) ion channels as well. In this study, we investigated the effect of selected derivatives (832-a pan-PDE inhibitor, 869-a TRPA1 modulator, and 145-a pan-PDE inhibitor and a weak TRPA1 modulator) on cellular responses related to airway remodeling using MRC-5 human lung fibroblasts. Compound 145 exerted the most considerable effect in limiting fibroblast to myofibroblasts transition (FMT) as well as proliferation, migration, and contraction. The effect of this compound appeared to depend mainly on its strong PDE inhibitory properties, and not on its effects on TRPA1 modulation. The strong anti-remodeling effects of 145 required activation of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) pathway leading to inhibition of transforming growth factor type ß1 (TGF-ß1) and Smad-dependent signaling in MRC-5 cells. These data suggest that the TGF-ß pathway is a major target for PDE inhibitors leading to inhibitory effects on cell responses involved in airway remodeling. These potent, pan-PDE inhibitors from the group of 7,8-disubstituted purine-2,6-dione derivatives, thus represent promising anti-remodeling drug candidates for further research.

Effects of Specific Inhibitor of Phosphodiesterase 7 at the Late Stage of Long-Term Potentiation in Murine Hippocampal Slices.

Second messengers cAMP and cGMP play an important role in synaptic plasticity and memory consolidation. The inhibitors of phosphodiesterases, enzymes hydrolyzing these cyclic nucleotides, are actively studied as potential drugs for the treatment of various cognitive disorders and depression. We studied the effects of a new inhibitor of phosphodiesterase 7 AGF2.20 on the formation of long-term potentiation in hippocampal slices. Administration of AGF2.20 (10 nM) in 90 min after weak tetanization prevented a decrease in the amplitude of excitatory post-synaptic potentials and stabilized long-term potentiation. These data attest to the involvement of phosphodiesterase 7 in the development of synaptic plasticity in the hippocampus. The inhibitor AGF2.20 is considered for the further analysis as a promising substance for the treatment of cognitive impairments.

Phosphodiesterase7 Inhibition Activates Adult Neurogenesis in Hippocampus and Subventricular Zone In Vitro and In Vivo.

The phosphodiesterase 7 (PDE7) enzyme is one of the enzymes responsible for controlling intracellular levels of cyclic adenosine 3',5'-monophosphate in the immune and central nervous system. We have previously shown that inhibitors of this enzyme are potent neuroprotective and anti-inflammatory agents. In addition, we also demonstrated that PDE7 inhibition induces endogenous neuroregenerative processes toward a dopaminergic phenotype. Here, we show that PDE7 inhibition controls stem cell expansion in the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the subventricular zone (SVZ) in the adult rat brain. Neurospheres cultures obtained from SGZ and SVZ of adult rats treated with PDE7 inhibitors presented an increased proliferation and neuronal differentiation compared to control cultures. PDE7 inhibitors treatment of neurospheres cultures also resulted in an increase of the levels of phosphorylated cAMP response element binding protein, suggesting that their effects were indeed mediated through the activation of the cAMP/PKA signaling pathway. In addition, adult rats orally treated with S14, a specific inhibitor of PDE7, presented elevated numbers of proliferating progenitor cells, and migrating precursors in the SGZ and the SVZ. Moreover, long-term treatment with this PDE7 inhibitor shows a significant increase in newly generated neurons in the olfactory bulb and the hippocampus. Also a better performance in memory tests was observed in S14 treated rats, suggesting a functional relevance for the S14-induced increase in SGZ neurogenesis. Taken together, our results indicate for the first time that inhibition of PDE7 directly regulates proliferation, migration and differentiation of neural stem cells, improving spatial learning and memory tasks. Stem Cells 2017;35:458-472.

Discovery and SAR study of 2-(4-pyridylamino)thieno[3,2-d]pyrimidin-4(3H)-ones as soluble and highly potent PDE7 inhibitors.

The discovery and SAR study of a new series of soluble and highly potent phosphodiesterase (PDE) 7 inhibitors are described herein. We explored a new lead compound with improved solubility, which led to the discovery of a 2-(4-pyridylamino)thieno[3,2-d]pyrimidin-4(3H)-one series. The introduction of 3-piperidines at the 7-position resulted in the significant enhancement of PDE7 activity. In particular, compound 32 also showed strong PDE7 inhibitory activity; good selectivity against PDE3, 4, and 5; and good aqueous solubility.

2-(Isopropylamino)thieno[3,2-d]pyrimidin-4(3H)-one derivatives as selective phosphodiesterase 7 inhibitors with potent in vivo efficacy.

A new series of thienopyrimidinones is synthesized and evaluated as selective phosphodiesterase 7 (PDE7) inhibitors for the treatment of inflammatory diseases. The modification of the substituents on thienopyrimidinone revealed that an isopropylamino group at the 2-position was favorable for aqueous solubility. The introduction of 3-pyrrolidines at the 7-position resulted in good solubility, highly potent activity, and good PDE7 selectivity. Among the synthesized compounds, compound 46 exhibited the greatest inhibition of ear edema in a phorbol ester-induced mouse model.

Discovery of triazines as potent, selective and orally active PDE4 inhibitors.

Expanding on HTS hit 4 afforded a series of [1,3,5]triazine derivatives as novel PDE4 inhibitors. The SAR development and optimization process with the emphasis on ligand efficiency and physicochemical properties led to the discovery of compound 44 as a potent, selective and orally active PDE4 inhibitor.

Effects of selective inhibition of PDE4 and PDE7 on airway reactivity and cough in healthy and ovalbumin-sensitized guinea pigs.

Phosphodiesterases (PDEs) are enzymes responsible for degradation of cAMP and cGMP in cells. Thus, PDE inhibitors may have significant clinical benefit in respiratory diseases associated with inflammation. The aim of the present study was to evaluate the effects of selective PDE4 (rolipram, ROL) and PDE7 inhibitors (BRL50481, BRL) on citric acid-induced cough, in vivo and in vitro airway smooth muscle reactivity in both healthy and ovalbumin sensitized guinea pigs. The drugs tested were administered intraperitoneally to male guinea pigs once daily for 7 days - ROL 1 mg/kg, BRL 1 mg/kg, and ROL+BRL 0.5 mg/kg. Double chamber whole body plethysmography was used for the evaluation of citric acid (0.6 M)-induced cough and specific airway resistance. An organ bath method was used for the measurement of tracheal and lung tissue strip contractions evoked by cumulative doses (10(-8)-10(-3) mol/L) of acetylcholine (ACH) and histamine (HIS). In healthy guinea pigs, the only significant relaxation was observed after ROL in ACH-induced contractions in vitro and the effect on cough was negligible. In ovalbumin-sensitized animals, more pronounced in vitro relaxing effects of BRL in HIS-induced contractions and of combination (ROL+BRL) in ACH-induced contractions were observed, with similar results in vivo, and no significant change in the number of cough efforts was observed in any of the groups tested. The results suggest that PDE4 and PDE7 inhibitors have stronger anti-inflammatory effects compared with direct effects on smooth muscle and cough, with a potential benefit of their concomitant administration.

Neuronal lack of PDE7a disrupted working memory, spatial learning, and memory but facilitated cued fear memory in mice.

BACKGROUND: PDEs regulate cAMP levels which is critical for PKA activity-dependent activation of CREB-mediated transcription in learning and memory. Inhibitors of PDEs like PDE4 and Pde7 improve learning and memory in rodents. However, the role of PDE7 in cognition or learning and memory has not been reported yet. METHODS: Therefore, we aimed to explore the cognitive effects of a PDE7 subtype, PDE7a, using combined pharmacological and genetic approaches. RESULTS: PDE7a-nko mice showed deficient working memory, impaired novel object recognition, deficient spatial learning & memory, and contextual fear memory, contrary to enhanced cued fear memory, highlighting the potential opposite role of PDE7a in the hippocampal neurons. Further, pharmacological inhibition of PDE7 by AGF2.20 selectively strengthens cued fear memory in C57BL/6 J mice, decreasing its extinction but did not affect cognitive processes assessed in other behavioral tests. The further biochemical analysis detected deficient cAMP in neural cell culture with genetic excision of the PDE7a gene, as well as in the hippocampus of PDE7a-nko mice in vivo. Importantly, we found overexpression of PKA-R and the reduced level of pPKA-C in the hippocampus of PDE7a-nko mice, suggesting a novel mechanism of the cAMP regulation by PDE7a. Consequently, the decreased phosphorylation of CREB, CAMKII, eif2a, ERK, and AMPK, and reduced total level of NR2A have been found in the brain of PDE7a-nko animals. Notably, genetic excision of PDE7a in neurons was not able to change the expression of NR2B, BDNF, synapsin1, synaptophysin, or snap25. CONCLUSION: Altogether, our current findings demonstrated, for the first time, the role of PDE7a in cognitive processes. Future studies will untangle PDE7a-dependent neurobiological and molecular-cellular mechanisms related to cAMP-associated disorders.

The SAR development of dihydroimidazoisoquinoline derivatives as phosphodiesterase 10A inhibitors for the treatment of schizophrenia.

The identification of potent and orally active dihydroimidazoisoquinolines as PDE 10A inhibitors is reported. The SAR development led to the discovery of compound 35 as a potent, selective, and orally active PDE10A inhibitor. Compound 35 inhibited MK-801-induced hyperactivity at 3mg/kg and displayed a 10-fold separation between the minimal effective doses for inhibition of MK-801-induced hyperactivity and hypolocomotion in rats.

Imidazopyridazinones as novel PDE7 inhibitors: SAR and in vivo studies in Parkinson's disease model.

The synthesis and structure-activity relationship studies of a series of compounds from imidazopyridazinone scaffold as PDE7 inhibitors are disclosed. Potent analogs such as compounds 7 (31nM), 8 (27nM), and 9 (12nM) were identified. The PDE selectivity and pharmacokinetic profile of compounds 7, 8 and 9 are also disclosed. The adequate CNS penetration of compound 7 in mice allowed it to be tested in the MPTP induced PD model and haloperidol induced catalepsy model to probe the differential pharmacology of PDE7 in the striatal pathway.

Cyclic nucleotide phosphodiesterase 7B mRNA: an unfavorable characteristic in chronic lymphocytic leukemia.

A cost- and time-efficient means to define the prognosis of patients with chronic lymphocytic leukemia (CLL) is desirable but does not yet exist. On the basis of the evidence that CLL cells have enhanced expression of the cyclic nucleotide phosphodiesterase isoform 7B (PDE7B), we hypothesized that PDE7B expression might provide such information. We assessed PDE7B mRNA expression using quantitative real-time PCR in peripheral blood mononuclear cells isolated from 85 patients and 30 normal subjects. We compared PDE7B mRNA expression with that of other disease features to determine if its expression correlates with the prognosis of patients with CLL. We found that CLL patients with PDE7B mRNA levels in the top quartile (greater than ninefold elevation relative to normal controls) have a several-year shorter median time-to-treatment (TTT, 36 months) compared to that of patients whose CLL cells express lower levels of PDE7B mRNA (TTT, 77 months, p=0.001). High PDE7B mRNA expression correlates with expression of zeta-chain-associated protein kinase 70 (ZAP-70), unmutated immunoglobulin heavy chain variable (IGHV) region genes and ß2 microglobulin (ß2M), but use of a multivariate Cox model revealed that high PDE7B mRNA expression independently predicts a short TTT, even after adjusting for several other disease characteristics (ZAP-70 or CD38 expression, IGHV mutation status and Rai status). High expression of PDE7B is an unfavorable characteristic in CLL. Assessment of PDE7B mRNA expression thus appears to be a clinically useful biomarker to define the prognosis of patients with CLL.

Genetic variation in phosphodiesterase (PDE) 7B in chronic lymphocytic leukemia: overview of genetic variants of cyclic nucleotide PDEs in human disease.

Expression of cyclic adenosine monophosphate-specific phosphodiesterase 7B (PDE7B) mRNA is increased in patients with chronic lymphocytic leukemia (CLL), thus suggesting that variation may occur in the PDE7B gene in CLL. As genetic variation in other PDE family members has been shown to associate with numerous clinical disorders (reviewed in this manuscript), we sought to identify single-nucleotide polymorphisms (SNPs) in the PDE7B gene promoter and coding region of 93 control subjects and 154 CLL patients. We found that the PDE7B gene has a 5' non-coding region SNP -347C>T that occurs with similar frequency in CLL patients (1.9%) and controls (2.7%). Tested in vitro, -347C>T has less promoter activity than a wild-type construct. The low frequency of this 5' untranslated region variant indicates that it does not explain the higher PDE7B expression in patients with CLL but it has the potential to influence other settings that involve a role for PDE7B.

Synthesis and structure-activity relationship studies of dihydronaphthyridinediones as a novel structural class of potent and selective PDE7 inhibitors.

The synthesis and SAR studies of a series of structurally novel inhibitors of PDE7 are discussed. The best compounds from the series display low nanomolar inhibitory activity and are selective versus other PDE isoenzymes.