Molecular Targets in Streptococcus pyogenes for the Development of Anti-Virulence Agents.

Streptococcus pyogenes, commonly known as Group A Streptococcus (GAS), is a significant human pathogen responsible for a wide range of diseases, from mild pharyngitis to severe conditions such as necrotizing fasciitis and toxic shock syndrome. The increasing antibiotic resistance, especially against macrolide antibiotics, poses a challenge to the effective treatment of these infections. This paper reviews the current state and mechanisms of antibiotic resistance in S. pyogenes. Furthermore, molecular targets for developing anti-virulence agents, which aim to attenuate virulence rather than killing it outright, are explored. This review specifically focuses on virulence regulators, proteins that coordinate the expression of multiple virulence factors in response to environmental signals, playing a crucial role in the pathogen's ability to cause disease. Key regulatory systems, such as RopB, Mga, CovRS, and the c-di-AMP signaling system, are discussed for their roles in modulating virulence gene expression. Additionally, potential molecular target sites for the development of anti-virulence agents are suggested. By concentrating on these regulatory pathways, it is proposed that anti-virulence strategies could enhance the effectiveness of existing antibiotics and reduce the selective pressure that drives the development of resistance.

Protein Kinase A Negatively Regulates the Acetic Acid Stress Response in S. cerevisiae.

Bioethanol fermentation from lignocellulosic hydrolysates is negatively affected by the presence of acetic acid. The budding yeast S. cerevisiae adapts to acetic acid stress partly by activating the transcription factor, Haa1. Haa1 induces the expression of many genes, which are responsible for increased fitness in the presence of acetic acid. Here, we show that protein kinase A (PKA) is a negative regulator of Haa1-dependent gene expression under both basal and acetic acid stress conditions. Deletions of RAS2, encoding a positive regulator of PKA, and PDE2, encoding a negative regulator of PKA, lead to an increased and decreased expression of Haa1-regulated genes, respectively. Importantly, the deletion of HAA1 largely reverses the effects of ras2∆. Additionally, the expression of a dominant, hyperactive RAS2A18V19 mutant allele also reduces the expression of Haa1-regulated genes. We found that both pde2Δ and RAS2A18V19 reduce cell fitness in response to acetic acid stress, while ras2Δ increases cellular adaptation. There are three PKA catalytic subunits in yeast, encoded by TPK1, TPK2, and TPK3. We show that single mutations in TPK1 and TPK3 lead to the increased expression of Haa1-regulated genes, while tpk2Δ reduces their expression. Among tpk double mutations, tpk1Δ tpk3Δ greatly increases the expression of Haa1-regulated genes. We found that acetic acid stress in a tpk1Δ tpk3Δ double mutant induces a flocculation phenotype, which is reversed by haa1Δ. Our findings reveal PKA to be a negative regulator of the acetic acid stress response and may help engineer yeast strains with increased efficiency of bioethanol fermentation.

Gene therapy with phosphodiesterases 2A and 4B ameliorates heart failure and arrhythmias by improving subcellular cAMP compartmentation.

AIMS: Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains. METHODS AND RESULTS: HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia. CONCLUSION: Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.

A mini-review: phosphodiesterases in charge to balance intracellular cAMP during T-cell activation.

T-cell activation is a pivotal process of the adaptive immune response with 3',5'-cyclic adenosine monophosphate (cAMP) as a key regulator of T-cell activation and function. It governs crucial control over T-cell differentiation and production of pro-inflammatory cytokines, such as IFN-γ. Intriguingly, levels of intracellular cAMP differ between regulatory (Treg) and conventional T-cells (Tcon). During cell-cell contact, cAMP is transferred via gap junctions between these T-cell subsets to mediate the immunosuppressive function of Treg. Moreover, the activation of T-cells via CD3 and CD28 co-stimulation leads to a transient upregulation of cAMP. Elevated intracellular cAMP levels are balanced precisely by phosphodiesterases (PDEs), a family of enzymes that hydrolyze cyclic nucleotides. Various PDEs play distinct roles in regulating cAMP and cyclic guanosine monophosphate (cGMP) in T-cells. Research on PDEs has gained growing interest due to their therapeutic potential to manipulate T-cell responses. So far, PDE4 is the best-described PDE in T-cells and the first PDE that is currently targeted in clinical practice to treat autoimmune diseases. But also, other PDE families harbor additional therapeutic potential. PDE2A is a dual-substrate phosphodiesterase which is selectively upregulated in Tcon upon activation. In this Mini-Review, we will highlight the impact of cAMP regulation on T-cell activation and function and summarize recent findings on different PDEs regulating intracellular cAMP levels in T-cells.

Insights into the selective mechanism of PDE2/9a inhibitors from silico aspects.

The selective design of competitive enzyme inhibitors is an extremely difficult task but necessary work for certain types of systems, such as the phosphodiesterase (PDE) system addressed in this article. In the PDE family, PDE2A and PDE9 respectively target the central nervous system and heart failure, and share many conserved amino acids at their binding sites. Therefore, gaining a deep understanding of the selective mechanisms of PDE2A/9A is crucial for designing highly selective drugs. In this study, various computer-aided drug design (CADD) methods, including molecular docking, molecular dynamics simulations (MD), and binding free energy calculations, are employed to explore the selective mechanisms of PDE2A/9A. Overall, our research results indicate a selective design strategy for PDE2A, which involves incorporating hydrophobic or aromatic moieties into the molecular structure to better accommodate the hydrophobic pocket of PDE2A. Additionally, it is recommended to introduce functional groups capable of forming connections with selective residues, such as Phe830 and Gln812 for PDE2A, or Ala452 and Tyr424 for PDE9A, to enhance the selectivity of inhibitors targeting PDE2A/9A. This achievement is anticipated to pave the way for the development of innovative and selective small molecules targeting PDE2A/9A.Communicated by Ramaswamy H. Sarma.

Defects in AMPAR trafficking and microglia activation underlie socio-cognitive deficits associated to decreased expression of phosphodiesterase 2 a.

Phosphodiesterase 2 A (PDE2A) is an enzyme involved in the homeostasis of cAMP and cGMP and is the most highly expressed PDE in human brain regions critical for socio-cognitive behavior. In cerebral cortex and hippocampus, PDE2A expression level is upregulated in Fmr1-KO mice, a model of the Fragile X Syndrome (FXS), the most common form of inherited intellectual disability (ID) and autism spectrum disorder (ASD). Indeed, PDE2A translation is negatively modulated by FMRP, whose functional absence causes FXS. While the pharmacological inhibition of PDE2A has been associated to its pro-cognitive role in normal animals and in models of ID and ASD, homozygous PDE2A mutations have been identified in patients affected by ID, ASD and epilepsy. To clarify this apparent paradox about the role of PDE2A in brain development, we characterized here Pde2a+/- mice (homozygote animals being not viable) at the behavioral, cellular, molecular and electrophysiological levels. Pde2a+/- females display a milder form of the disorder with reduced cognitive performance in adulthood, conversely males show severe socio-cognitive deficits throughout their life. In males, these phenotypes are associated with microglia activation, elevated glutathione levels and increased externalization of Glutamate receptor (GluR1) in CA1, producing reduced mGluR-dependent Long-term Depression. Overall, our results reveal molecular targets of the PDE2A-dependent pathway underlying socio-cognitive performance. These results clarify the mechanism of action of pro-cognitive drugs based on PDE2A inactivation, which have been shown to be promising therapeutic approaches for Alzheimer's disease, schizophrenia, FXS as well as other forms of ASD.

The phosphodiesterase 2A controls lymphatic junctional maturation via cGMP-dependent notch signaling.

The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.

The Dlt and LiaFSR systems derepress SpeB production independently in the Δpde2 mutant of Streptococcus pyogenes.

The second messenger molecule, c-di-AMP, plays a critical role in pathogenesis and virulence in S. pyogenes. We previously reported that deleting the c-di-AMP phosphodiesterase gene pde2 severely suppresses SpeB production at the transcriptional level. We performed transposon mutagenesis to gain insight into the mechanism of how Pde2 is involved in SpeB regulation. We identified one of the genes of the dlt operon, dltX, as a suppressor of the SpeB-null phenotype of the Δpde2 mutant. The dlt operon consists of five genes, dltX, dltA, dltB, dltC, and dltD in many Gram-positive bacteria, and its function is to incorporate D-alanine into lipoteichoic acids. DltX, a small membrane protein, is a newly identified member of the operon. The in-frame deletion of dltX or insertional inactivation of dltA in the Δpde2 mutant restored SpeB production, indicating that D-alanylation is crucial for the suppressor phenotype. These mutations did not affect the growth in lab media but showed increased negative cell surface charge and enhanced sensitivity to polymyxin B. Considering that dlt mutations change cell surface charge and sensitivity to cationic antimicrobial peptides, we examined the LiaFSR system that senses and responds to cell envelope stress. The ΔliaR mutation in the Δpde2 mutant also derepressed SpeB production, like the ΔdltX mutation. LiaFSR controls speB expression by regulating the expression of the transcriptional regulator SpxA2. However, the Dlt system did not regulate spxA2 expression. The SpeB phenotype of the Δpde2ΔdltX mutant in higher salt media differed from that of the Δpde2ΔliaR mutant, suggesting a unique pathway for the Dlt system in SpeB production, possibly related to ion transport or turgor pressure regulation.

Unbiased discovery of natural sequence variants that influence fungal virulence.

Isolates of Cryptococcus neoformans, a fungal pathogen that kills over 112,000 people each year, differ from a 19-Mb reference genome at a few thousand up to almost a million DNA sequence positions. We used bulked segregant analysis and association analysis, genetic methods that require no prior knowledge of sequence function, to address the key question of which naturally occurring sequence variants influence fungal virulence. We identified a region containing such variants, prioritized them, and engineered strains to test our findings in a mouse model of infection. At one locus, we identified a 4-nt variant in the PDE2 gene that occurs in common laboratory strains and severely truncates the encoded phosphodiesterase. The resulting loss of phosphodiesterase activity significantly impacts virulence. Our studies demonstrate a powerful and unbiased strategy for identifying key genomic regions in the absence of prior information and provide significant sequence and strain resources to the community.

Phosphodiesterase 2 and Its Isoform a as Therapeutic Targets in the Central Nervous System Disorders.

Cyclic adenosine monophosphates (cAMP) and cyclic guanosine monophosphate (cGMP) are two essential second messengers, which are hydrolyzed by phosphodiesterase's (PDEs), such as PDE-2. Pharmacological inhibition of PDE-2 (PDE2A) in the central nervous system improves cAMP and cGMP signaling, which controls downstream proteins related to neuropsychiatric, neurodegenerative, and neurodevelopmental disorders. Considering that there are no specific treatments for these disorders, PDE-2 inhibitors' development has gained more attention in the recent decade. There is high demand for developing new-generation drugs targeting PDE2 for treating diseases in the central nervous and peripheral systems. This review summarizes the relationship between PDE-2 with neuropsychiatric, neurodegenerative, and neurodevelopmental disorders as well as its possible treatment, mainly involving inhibitors of PDE2.

Deciphering the regulatory mechanisms of the cAMP/protein kinase A pathway and their roles in the pathogenicity of Candida auris.

The emergence of multidrug-resistant fungal pathogens is a significant concern for global public health. Candida auris poses a considerable threat as a multidrug-resistant fungal pathogen. Our recent study revealed that the adenylyl cyclase Cyr1 and protein kinase A (PKA) pathways play distinct and redundant roles in drug resistance and pathogenicity of C. auris. However, the upstream and negative feedback regulatory mechanisms of C. auris are not yet fully understood. In this study, we discovered that the small GTPase Ras1, along with its nucleotide exchange factor Cdc25 and GTPase-activating protein Ira2, plays a major role in regulating cAMP/PKA-dependent traits, while G-protein-coupled receptor Gpr1 and heterotrimeric G-protein α subunit Gpa2 play a minor role. Pde2 plays a major role in negative feedback regulation of the cAMP/PKA pathway, while Pde1 plays a minor role. Hyperactivation of the Ras/cAMP/PKA pathway by deleting PDE2 or BCY1 renders C. auris cells thermosensitive and susceptible to nutrient deficiency, which leads to attenuated virulence. Our study demonstrates the distinct contributions of hyperactivation of the Ras/cAMP/PKA signaling pathway to C. auris pathogenesis and suggests potential therapeutic targets for C. auris-mediated candidiasis. IMPORTANCE Candida auris is a major concern as a multidrug-resistant fungal pathogen. While our previous studies highlighted the crucial roles of the cAMP/protein kinase A (PKA) pathway in regulating drug resistance, stress responses, morphogenesis, ploidy change, biofilm formation, and pathogenicity in this pathogen, their regulatory mechanism remains unclear. In our study, we provided evidence that the cAMP/PKA signaling pathway in C. auris is primarily governed by the small GTPase RAS rather than a G-protein-coupled receptor. Additionally, we discovered that the negative feedback regulation of cAMP, controlled by phosphodiesterases, is vital for C. auris virulence by promoting resistance to high temperatures and nutrient deficiencies. These findings underscore the diverse pathobiological significance of the Ras/cAMP/PKA signaling pathway in C. auris, shedding light on potential therapeutic targets and strategies for combating this multidrug-resistant fungal pathogen.

A homozygous founder variant in PDE2A causes paroxysmal dyskinesia with intellectual disability.

Intellectual developmental disorder with paroxysmal dyskinesia or seizures (IDDPADS, OMIM#619150) is an ultra-rare childhood-onset autosomal recessive movement disorder manifesting paroxysmal dyskinesia, global developmental delay, impaired cognition, progressive psychomotor deterioration and/or drug-refractory seizures. We investigated three consanguineous Pakistani families with six affected individuals presenting overlapping phenotypes partially consistent with the reported characteristics of IDDPADS. Whole exome sequencing identified a novel missense variant in Phosphodiesterase 2A (PDE2A): NM_002599.4: c.1514T > C p.(Phe505Ser) that segregated with the disease status of individuals in these families. Retrospectively, we performed haplotype analysis that revealed a 3.16 Mb shared haplotype at 11q13.4 among three families suggesting a founder effect in this region. Moreover, we also observed abnormal mitochondrial morphology in patient fibroblasts compared to controls. Belonging to diverse age groups (13 years-60 years), patients presented paroxysmal dyskinesia, developmental delay, cognitive abnormalities, speech impairment, and drug-refractory seizures with variable onset of disease (as early as 3 months of age to 7 years). Together with the previous reports, we observed that intellectual disability, progressive psychomotor deterioration, and drug-refractory seizures are consistent outcomes of the disease. However, permanent choreodystonia showed variability. We also noticed that the later onset of paroxysmal dyskinesia manifests severe attacks in terms of duration. Being the first report from Pakistan, we add to the clinical and mutation spectrum of PDE2A-related recessive disease raising the total number of patients from six to 12 and variants from five to six. Together, with our findings, the role of PDE2A is strengthened in critical physio-neurological processes.

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

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.

Essential role of the mitochondrial Na+/Ca2+ exchanger NCLX in mediating PDE2-dependent neuronal survival and learning.

Impaired phosphodiesterase (PDE) function and mitochondrial Ca2+ (i.e., [Ca2+]m) lead to multiple health syndromes by an unknown pathway. Here, we fluorescently monitor robust [Ca2+]m efflux mediated by the mitochondrial Na+/Ca2+ exchanger NCLX in hippocampal neurons sequentially evoked by caffeine and depolarization. Surprisingly, neuronal depolarization-induced Ca2+ transients alone fail to evoke strong [Ca2+]m efflux in wild-type (WT) neurons. However, pre-treatment with the selective PDE2 inhibitor Bay 60-7550 effectively rescues [Ca2+]m efflux similarly to caffeine. Moreover, PDE2 acts by diminishing mitochondrial cAMP, thus promoting NCLX phosphorylation at its PKA site. We find that the protection of neurons against excitotoxic insults, conferred by PDE2 inhibition in WT neurons, is NCLX dependent. Finally, the administration of Bay 60-7550 enhances new object recognition in WT, but not in NCLX knockout (KO), mice. Our results identify a link between PDE and [Ca2+]m signaling that may provide effective therapy for cognitive and ischemic syndromes.

Radiosynthesis and Preclinical Evaluation of an 18F-Labeled Triazolopyridopyrazine-Based Inhibitor for Neuroimaging of the Phosphodiesterase 2A (PDE2A).

The cyclic nucleotide phosphodiesterase 2A is an intracellular enzyme which hydrolyzes the secondary messengers cAMP and cGMP and therefore plays an important role in signaling cascades. A high expression in distinct brain areas as well as in cancer cells makes PDE2A an interesting therapeutic and diagnostic target for neurodegenerative and neuropsychiatric diseases as well as for cancer. Aiming at specific imaging of this enzyme in the brain with positron emission tomography (PET), a new triazolopyridopyrazine-based derivative (11) was identified as a potent PDE2A inhibitor (IC50, PDE2A = 1.99 nM; IC50, PDE10A ~2000 nM) and has been radiofluorinated for biological evaluation. In vitro autoradiographic studies revealed that [18F]11 binds with high affinity and excellent specificity towards PDE2A in the rat brain. For the PDE2A-rich region nucleus caudate and putamen an apparent KD value of 0.24 nM and an apparent Bmax value of 16 pmol/mg protein were estimated. In vivo PET-MR studies in rats showed a moderate brain uptake of [18F]11 with a highest standardized uptake value (SUV) of 0.97. However, no considerable enrichment in PDE2A-specific regions in comparison to a reference region was detectable (SUVcaudate putamen = 0.51 vs. SUVcerebellum = 0.40 at 15 min p.i.). Furthermore, metabolism studies revealed a considerable uptake of radiometabolites of [18F]11 in the brain (66% parent fraction at 30 min p.i.). Altogether, despite the low specificity and the blood−brain barrier crossing of radiometabolites observed in vivo, [18F]11 is a valuable imaging probe for the in vitro investigation of PDE2A in the brain and has potential as a lead compound for further development of a PDE2A-specific PET ligand for neuroimaging.

Motor, epileptic, and developmental phenotypes in genetic disorders affecting G protein coupled receptors-cAMP signaling.

Over the last years, a constantly increasing number of genetic diseases associated with epilepsy and movement disorders have been recognized. An emerging group of conditions in this field is represented by genetic disorders affecting G-protein-coupled receptors (GPCRs)-cAMP signaling. This group of postsynaptic disorders includes genes encoding for proteins highly expressed in the central nervous system and involved in GPCR signal transduction and cAMP production (e.g., GNAO1, GNB1, ADCY5, GNAL, PDE2A, PDE10A, and HPCA genes). While the clinical phenotype associated with ADCY5 and GNAL is characterized by movement disorder in the absence of epilepsy, GNAO1, GNB1, PDE2A, PDE10A, and HPCA have a broader clinical phenotype, encompassing movement disorder, epilepsy, and neurodevelopmental disorders. We aimed to provide a comprehensive phenotypical characterization of genetic disorders affecting the cAMP signaling pathway, presenting with both movement disorders and epilepsy. Thus, we reviewed clinical features and genetic data of 203 patients from the literature with GNAO1, GNB1, PDE2A, PDE10A, and HPCA deficiencies. Furthermore, we delineated genotype-phenotype correlation in GNAO1 and GNB1 deficiency. This group of disorders presents with a highly recognizable clinical phenotype combining distinctive motor, epileptic, and neurodevelopmental features. A severe hyperkinetic movement disorder with potential life-threatening exacerbations and high susceptibility to a wide range of triggers is the clinical signature of the whole group of disorders. The existence of a distinctive clinical phenotype prompting diagnostic suspicion and early detection has relevant implications for clinical and therapeutic management. Studies are ongoing to clarify the pathophysiology of these rare postsynaptic disorders and start to design disease-specific treatments.

Protective effects of phosphodiesterase 2 inhibitor against Aß1-42 induced neuronal toxicity.

Our previous study suggested that inhibition of Phosphodiesterase 2 ameliorates memory loss upon exposure to oxidative stress. While whether memory enhancing effects of PDE2 inhibition on Alzheimer's disease mouse model are involved in antioxidant defense and neuronal remodeling, are largely unexplored. The present study addressed whether and how PDE2 inhibitor Bay 60-7550 rescued Aß oligomers (Aßo)-induced neuronal damage and memory impairment. The results suggested that exposure of primary cortical neurons to Aßo induced neuronal cells damage and increased PDE2 expression, which were paralleled to an increase in the oxidative parameter malondialdehyde (MDA) level and cellular apoptosis. However, this Aßo-induced oxidative damage was blocked by pre-treatment with protein kinase A or G (PKA or PKG) inhibitor, suggesting the involvement of cAMP/cGMP signaling. Moreover, microinjection of Aßo into the prefrontal cortex of mice increased the MDA level; while Bay 60-7550 reversed this effect and increased antioxidant and anti-apoptotic factors, i.e. increased trolox-equivalent-antioxidant capacity and Bcl-2/Bax ratio. Bay 60-7550 also rescued Aßo-induced synaptic atrophy and memory deficits, as evidenced by the increased synaptic proteins' levels and spine density in the prefrontal cortex, and improved cognitive behaviors by decreased working memory errors in the eight-arm maze and increased discrimination index in the novel object recognition test. These findings suggest that inhibition of PDE2 contributes to antioxidant defense and neuronal remodeling by regulation of cAMP/cGMP signaling, which provide a theoretical basis for the future use of PDE2 inhibitors as the anti-AD drugs.

Low Expression of Phosphodiesterase 2 (PDE2A) Promotes the Progression by Regulating Mitochondrial Morphology and ATP Content and Predicts Poor Prognosis in Hepatocellular Carcinoma.

Phosphodiesterase 2 (PDE2A) modulates the levels of cAMP/cGMP and was recently found to be involved in mitochondria function regulation, closely related to multiple types of tumor progression. This study aimed to estimate the prognostic significance and biological effects of PDE2A on hepatocellular carcinoma (HCC). We comprehensively analyzed the PDE2A mRNA expression in HCC based on The Cancer Genome Atlas (TCGA) database and investigated the effects of PDE2A on the proliferation and metastatic capacity of HCC cells. PDE2A was downregulated in 25 cancer types, including HCC. Lower PDE2A expression was a protective factor in HCC and was negatively associated with serum AFP levels, tumor status, vascular invasion, histologic grade, and pathologic stage of HCC. Moreover, tumors with low PDE2A expression displayed a decreased immune function. Then, the ROC curve was used to assess the diagnostic ability of PDE2A in HCC (AUC = 0.823 in TCGA and AUC = 0.901 in GSE76427). Patients with low PDE2A expression exhibited worse outcomes compared with those with high PDE2A expression. Additionally, GO functional annotations demonstrated the involvement of PDE2A in the ECM organization, systems development, and ERK-related pathways, indicating that PDE2A might regulate HCC growth and metastasis. The in vitro experiments confirmed that overexpression of PDE2A inhibited proliferation, colony formation, migration, and invasion in two HCC cell lines (HLF and SNU-368), while inhibition of PDE2A has the opposite results. The mechanism of PDE2A's effect on HCC cells is attributed to the change of mitochondrial morphology and ATP content. These data demonstrated that PDE2A closely participated in the regulation of HCC proliferation and metastasis and can be used as a predictive marker candidate and a potential therapeutic target for HCC.

miR-139/PDE2A-Notch1 feedback circuit represses stemness of gliomas by inhibiting Wnt/ß-catenin signaling.

Rationale: The malignant phenotypes of glioblastomas (GBMs) are primarily attributed to glioma stem cells (GSCs). Our previous study and other reports have suggested that both miR-139 and its host gene PDE2A are putative antitumor genes in various cancers. The aim of this study was to investigate the roles and mechanisms of miR-139/PDE2A in GSC modulation. Methods: Clinical samples were used to determine miR-139/PDE2A expression. Patient-derived glioma stem-like cells (PD-GSCs) were stimulated for immunofluorescent staining, sphere formation assays and orthotopic GBM xenograft models. Bioinformatic analysis and further in vitro experiments demonstrated the downstream molecular mechanisms of miR-139 and PDE2A. OX26/CTX-conjugated PEGylated liposome (OCP) was constructed to deliver miR-139 or PDE2A into glioma tissue specifically. Results: We demonstrated that miR-139 was concomitantly transcribed with its host gene PDE2A. Both PDE2A and miR-139 indicated better prognosis of gliomas and were inversely correlated with GSC stemness. PDE2A or miR-139 overexpression suppressed the stemness of PD-GSCs. FZD3 and ß-catenin, which induced Wnt/ß-catenin signaling activation, were identified as targets of miR-139 and mediated the effects of miR-139 on GSCs. Meanwhile, PDE2A suppressed Wnt/ß-catenin signaling by inhibiting cAMP accumulation and GSK-3ß phosphorylation, thereby modulating the self-renewal of PD-GSCs. Notably, Notch1, which is also a target of miR-139, suppressed PDE2A/miR-139 expression directly via downstream Hes1, indicating that miR-139 promoted its own expression by the miR-139-Notch1/Hes1 feedback circuit. Expectedly, targeted overexpression miR-139 or PDE2A in glioma with OCP system significantly repressed the stemness and decelerated glioma progression. Conclusions: Our findings elaborate on the inhibitory functions of PDE2A and miR-139 on GSC stemness and tumorigenesis, which may provide new prognostic markers and therapeutic targets for GBMs.

Design, Synthesis, and Evaluation of Dihydropyranopyrazole Derivatives as Novel PDE2 Inhibitors for the Treatment of Alzheimer's Disease.

Phosphodiesterase 2 (PDE2) has been regarded as a novel target for the treatment of Alzheimer's disease (AD). In this study, we obtained (R)-LZ77 as a hit compound with moderate PDE2 inhibitory activity (IC50 = 261.3 nM) using a high-throughput virtual screening method based on molecular dynamics. Then, we designed and synthesized 28 dihydropyranopyrazole derivatives as PDE2 inhibitors. Among them, compound (+)-11h was the most potent PDE2 inhibitor, with an IC50 value of 41.5 nM. The molecular docking of PDE2-(+)-11h reveals that the 4-(trifluoromethyl)benzyl)oxyl side chain of the compound enters the H-pocket and forms strong hydrophobic interactions with L770/L809/F862, which improves inhibitory activity. The above results may provide insight for further structural optimization of highly potent PDE2 inhibitors and may lay the foundation for their use in the treatment of AD.