Hydrolysis Of Cyclic Nucleotides Research Articles

Apicomplexan phosphodiesterases in cyclic nucleotide turnover: conservation, function, and therapeutic potential.

Apicomplexa encompasses a large number of intracellular parasites infecting a wide range of animals. Cyclic nucleotide signaling is crucial for a variety of apicomplexan life stages and cellular processes. The cyclases and kinases that synthesize and respond to cyclic nucleotides (i.e., 3',5'-cyclic guanosine monophosphate and 3',5'-cyclic adenosine monophosphate) are highly conserved and essential throughout the parasite phylum. Growing evidence indicates that phosphodiesterases (PDEs) are also critical for regulating cyclic nucleotide signaling via cyclic nucleotide hydrolysis. Here, we discuss recent advances in apicomplexan PDE biology and opportunities for therapeutic interventions, with special emphasis on the major human apicomplexan parasite genera Plasmodium, Toxoplasma, Cryptosporidium, and Babesia. In particular, we show a highly flexible repertoire of apicomplexan PDEs associated with a wide range of cellular requirements across parasites and lifecycle stages. Despite this phylogenetic diversity, cellular requirements of apicomplexan PDEs for motility, host cell egress, or invasion are conserved. However, the molecular wiring of associated PDEs is extremely malleable suggesting that PDE diversity and redundancy are key for the optimization of cyclic nucleotide turnover to respond to the various environments encountered by each parasite and life stage. Understanding how apicomplexan PDEs are regulated and integrating multiple signaling systems into a unified response represent an untapped avenue for future exploration.

PDE4 Phosphodiesterases in Cardiovascular Diseases: Key Pathophysiological Players and Potential Therapeutic Targets

3′,5′-cyclic adenosine monophosphate (cAMP) is a second messenger critically involved in the control of a myriad of processes with significant implications for vascular and cardiac cell function. The temporal and spatial compartmentalization of cAMP is governed by the activity of phosphodiesterases (PDEs), a superfamily of enzymes responsible for the hydrolysis of cyclic nucleotides. Through the fine-tuning of cAMP signaling, PDE4 enzymes could play an important role in cardiac hypertrophy and arrhythmogenesis, while it decisively influences vascular homeostasis through the control of vascular smooth muscle cell proliferation, migration, differentiation and contraction, as well as regulating endothelial permeability, angiogenesis, monocyte/macrophage activation and cardiomyocyte function. This review summarizes the current knowledge and recent advances in understanding the contribution of the PDE4 subfamily to cardiovascular function and underscores the intricate challenges associated with targeting PDE4 enzymes as a therapeutic strategy for the management of cardiovascular diseases.

Development of Phosphodiesterase-Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity.

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–RD complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.

A phosphodiesterase 11 (Pde11a) knockout mouse expressed functional but reduced Pde11a: Phenotype and impact on adrenocortical function

Phosphodiesterases catalyze the hydrolysis of cyclic nucleotides and maintain physiologic levels of intracellular concentrations of cyclic adenosine and guanosine mono-phosphate (cAMP and cGMP, respectively). Increased cAMP signaling has been associated with adrenocortical tumors and Cushing syndrome. Genetic defects in phosphodiesterase 11A (PDE11A) may lead to increased cAMP signaling and have been found to predispose to the development of adrenocortical, prostate, and testicular tumors. A previously reported Pde11a knockout (Pde11a−/−) mouse line was studied and found to express PDE11A mRNA and protein still, albeit at reduced levels; functional studies in various tissues showed increased cAMP levels and reduced PDE11A activity. Since patients with PDE11A defects and Cushing syndrome have PDE11A haploinsufficiency, it was particularly pertinent to study this hypomorphic mouse line. Indeed, Pde11a−/− mice failed to suppress corticosterone secretion in response to low dose dexamethasone, and in addition exhibited adrenal subcapsular hyperplasia with predominant fetal-like features in the inner adrenal cortex, mimicking other mouse models of increased cAMP signaling in the adrenal cortex. We conclude that a previously reported Pde11a−/− mouse showed continuing expression and function of PDE11A in most tissues. Nevertheless, Pde11a partial inactivation in mice led to an adrenocortical phenotype that was consistent with what we see in patients with PDE11A haploinsufficiency.

An NMR based phosphodiesterase assay.

We propose a phosphodiesterase assay based on 1D 1H NMR to monitor the hydrolysis of cyclic nucleotides directly, without requiring tags or the addition of exogenous reagents. The method is suitable to measure phosphodiesterase KM and kcat parameters and to identify phosphodiesterase inhibitors.

Phosphodiesterase inhibitors: Potential role in the respiratory distress of neonates.

Phosphodiesterases (PDEs) are a superfamily of enzymes that catalyze the hydrolysis of phosphodiester bonds of 3',5' cyclic adenosine and guanosine monophosphate (cAMP and cGMP). PDEs control hydrolysis of cyclic nucleotides in many cells and tissues. Inhibition of PDEs by selective or nonselective PDE inhibitors represents an effective targeted strategy for the treatment of various diseases including respiratory disorders. Recent data have demonstrated that PDE inhibitors can also be of benefit in respiratory distress in neonates. This article outlines the pharmacological properties of nonselective and selective PDE inhibitors and provides up-to-date information regarding their use in experimental models of neonatal respiratory distress as well as in clinical studies.

Roles of PDE1 in Pathological Cardiac Remodeling and Dysfunction

Pathological cardiac hypertrophy and dysfunction is a response to various stress stimuli and can result in reduced cardiac output and heart failure. Cyclic nucleotide signaling regulates several cardiac functions including contractility, remodeling, and fibrosis. Cyclic nucleotide phosphodiesterases (PDEs), by catalyzing the hydrolysis of cyclic nucleotides, are critical in the homeostasis of intracellular cyclic nucleotide signaling and hold great therapeutic potential as drug targets. Recent studies have revealed that the inhibition of the PDE family member PDE1 plays a protective role in pathological cardiac remodeling and dysfunction by the modulation of distinct cyclic nucleotide signaling pathways. This review summarizes recent key findings regarding the roles of PDE1 in the cardiac system that can lead to a better understanding of its therapeutic potential.

Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta.

RhoPDE is a type I rhodopsin/phosphodiesterase gene fusion product from the choanoflagellate Salpingoeca rosetta. The gene was discovered around the time that a similar type I rhodopsin/guanylyl cyclase fusion protein, RhoGC, was shown to control phototaxis of an aquatic fungus through a cGMP signaling pathway. RhoPDE has potential as an optogenetic tool catalyzing the hydrolysis of cyclic nucleotides. Here we provide an expression and purification system for RhoPDE, as well as a crystal structure of the C-terminal phosphodiesterase catalytic domain. We show that RhoPDE contains an even number of transmembrane segments, with N- and C-termini both located on the cytoplasmic surface of the cell membrane. The purified protein exhibits an absorption maximum at 490 nm in the dark state, which shifts to 380 nm upon exposure to light. The protein acts as a cGMP-selective phosphodiesterase. However, the activity does not appear to be modulated by light. The protein is also active with cAMP as a substrate, but with a roughly 5-7-fold lower kcat. A truncation consisting solely of the phosphodiesterase domain is also active with a kcat for cGMP roughly 6-9-fold lower than that of the full-length protein. The isolated PDE domain was crystallized, and the X-ray structure showed the protein to be a dimer similar to human PDE9. We anticipate that the purification system introduced here will enable further structural and biochemical experiments to improve our understanding of the function and mechanism of this unique fusion protein.

Role of Cyclic Nucleotide Phosphodiesterases During Meiotic Resumption From Diplotene Arrest in Mammalian Oocytes.

Cyclic nucleotide phosphodiesterases (PDEs) are group of enzymes that hydrolyze cyclic nucleotides in wide variety of cell types including encircling granulosa cells as well as associated oocytes. One group of PDEs are located in encircling granulosa cells and another group get expressed in the oocyte, while few other PDEs are expressed in both compartments. The PDE1A, PDE4D, PDE5A, PDE8A, and PDE8B are granulosa cell specific PDEs that hydrolyze adenosine 3',5'-cyclic monophosphate (cAMP) as well as guanosine 3',5'-cyclic monophosphate (cGMP) with different affinities. PDE3A, PDE8A as well as PDE9A are expressed in oocyte and specifically responsible for the cyclic nucleotide hydrolysis in the oocyte itself. Few other PDEs such as PDE7B, PDE10A, and PDE11A are either detected in granulosa cells or oocytes. Activation of these PDEs either in encircling granulosa cells or in oocyte directly or indirectly reduces intraoocyte cAMP level. Reduction of intraoocyte cAMP level modulates phosphorylation status of cyclin-dependent kinase 1 (Cdk1) and triggers cyclin B1 degradation that destabilizes maturation promoting factor (MPF) and/or increases Cdk1 activity. The destabilized MPF and/or increased Cdk1 activity leads to resumption of meiosis, which initiates the achievement of meiotic competency in preovulatory follicles of several mammalian species. Use of specific PDEs inhibitors block cyclic nucleotides hydrolysis that results in increase of intraoocyte cyclic nucleotides level, which leads to maintenance of meiotic arrest at diplotene stage in vivo as well as in vitro. Thus, cyclic nucleotide PDEs play important role in the achievement of meiotic competency by reducing intraoocyte cyclic nucleotides level in mammalian oocytes. J. Cell. Biochem. 118: 446-452, 2017. © 2016 Wiley Periodicals, Inc.

Cyclic nucleotide phosphodiesterase 1 and vascular aging.

VSMCs (vascular smooth muscle cells) play critical roles in arterial remodelling with aging, hypertension and atherosclerosis. VSMCs exist in diverse phenotypes and exhibit phenotypic plasticity, e.g. changing from a quiescent/contractile phenotype to an active myofibroblast-like, often called ‘synthetic’, phenotype. Synthetic VSMCs are able to proliferate, migrate and secrete ECM (extracellular matrix) proteinases and ECM proteins. In addition, they produce pro-inflammatory molecules, providing an inflammatory microenvironment for leucocyte penetration, accumulation and activation. The aging VSMCs have also shown changes in cellular phenotype, responsiveness to contracting and relaxing mediators, replicating potential, matrix synthesis, inflammatory mediators and intracellular signalling. VSMC dysfunction plays a key role in age-associated vascular remodelling. Cyclic nucleotide PDEs (phosphodiesterases), by catalysing cyclic nucleotide hydrolysis, play a critical role in regulating the amplitude, duration and compartmentalization of cyclic nucleotide signalling. Abnormal alterations of PDEs and subsequent changes in cyclic nucleotide homoeostasis have been implicated in a number of different diseases. In the study published in the latest issue of Clinical Science, Bautista Niño and colleagues have shown that, in cultured senescent human VSMCs, PDE1A and PDE1C mRNA levels are significantly up-regulated and inhibition of PDE1 activity with vinpocetine reduced cellular senescent makers in senescent VSMCs. Moreover, in the premature aging mice with genomic instability (Ercc1d/−), impaired aortic ring relaxation in response to SNP (sodium nitroprusside), an NO (nitric oxide) donor, was also largely improved by vinpocetine. More interestingly, using data from human GWAS (genome-wide association studies), it has been found that PDE1A single nucleotide polymorphisms is significantly associated with diastolic blood pressure and carotid intima–media thickening, two hallmarks of human vascular dysfunction in aging. These findings establish a strong relationship between PDE1 expression regulation and vascular abnormalities in aging.

Abstract P6-01-22: PDE5 as a novel biomarker and a potential therapeutic target for breast cancer

Abstract Background: Phosphodiesterases are enzymes responsible for regulating second messenger signaling by hydrolyzing 3’-5’ cyclic guanosine monophosphate (cGMP), that activates specific pathways resulting in protein phosphorylation, ion fluxes, or cyclic nucleotide hydrolysis to affect gene expression or other aspects of cellular activity. Previous studies have reported increased PDE5 expression in multiple human carcinomas, including bladder, colon, lung and breast cancers, suggesting a role for PDE5 in tumorigenesis. In addition, several in vitro observations have shown antiproliferative and proapoptotic effects of sildenafil and other PDE5 inhibitors in cancer cell lines. However, very little is known about PDE5 expression in human breast tumours and its potential role in breast cancer initiation and progression. We therefore propose to determine whether PDE5 expression may be predictor of outcome in breast cancer patients, and examine PDE5 impact on breast cancer phenotype in vitro. Methods: We employed MCF-10A normal breast epithelial cells, estrogen receptor (ER) α-positive (MCF-7/ZR-75/T-47D) and ERα-negative (BT-20/MDA-MB-468/SKBR-3/MDA-MB-435) breast cancer cells. We used RT-PCR, immunoblotting and immunofluorescence analyses for evaluating PDE5 expression. To examine PDE5 impact on breast cancer phenotype, MCF-7 cells were engineered to stably express PDE5 and four clones were selected. Cell proliferation was assessed by MTT and anchorage-independent assays, motility and invasion by wound-healing, transmigration and matrigel-based invasion assays. Retrospective analysis using 1959 breast cancer patients of the Metabric Project was performed to evaluate relationship between PDE5 expression and overall survival by Cox proportional hazard regression. Results: PDE5 mRNA and protein were constitutively expressed at high levels in all the examined tumor cell lines compared to normal breast cells, except for the less motile and non-invasive MCF-7 cells. Interestingly, higher PDE5 expression was found in more aggressive ER-negative cells. Stable overexpression of PDE5 did not affect proliferation of MCF-7 cells, while it significantly increased motility and invasion of all the stable PDE5-transfected clones tested. Patients having high PDE5 expression had a statistically significant poorer survival compared to patients with low PDE5 expression (p=0.014, HR= 1.2). A more relevant discrimination was achieved in lymph node-negative patients (p=0.0015, HR= 1.6), suggesting that assessing PDE5 levels may be helpful to identify a subgroup of early-stage breast cancer patients who are most likely at the highest risk of progression. Conclusions: PDE5 expression may enhance cancer cell invasive potential, thereby representing prospectively a potential molecular candidate as prognostic marker and target for breast cancer therapy. Citation Format: Ines Barone, Antonella Campana, Cinzia Giordano, Marilena Lanzino, Daniela Bonofiglio, Balazs Gyorffy, Stefania Catalano, Sebastiano Andò. PDE5 as a novel biomarker and a potential therapeutic target for breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-01-22.

PDE5 expression in human thyroid tumors and effects of PDE5 inhibitors on growth and migration of cancer cells.

Recent studies have revealed in normal thyroid tissue the presence of the transcript of several phosphodiesterases (PDEs), enzymes responsible for the hydrolysis of cyclic nucleotides. In this work, we analyzed the expression of PDE5 in a series of human papillary thyroid carcinomas (PTCs) presenting or not BRAF V600E mutation and classified according to ATA risk criteria. Furthermore, we tested the effects of two PDE5 inhibitors (sildenafil, tadalafil) against human thyroid cancer cells. PDE5 gene and protein expression were analyzed in two different cohorts of PTCs by real-time PCR using a TaqMan micro-fluid card system and Western blot. MTT and migration assay were used to evaluate the effects of PDE5 inhibitors on proliferation and migration of TPC-1, BCPAP, and 8505C cells. In a first series of 36 PTCs, we found higher expression levels of PDE5A in tumors versus non-tumor (normal) tissues. PTCs with BRAF mutation showed higher levels of mRNA compared with those without mutation. No significant differences were detected between subgroups with low and intermediate ATA risk. Upregulation of PDE5 was also detected in tumor tissue proteins. Similar results were obtained analyzing the second cohort of 50 PTCs. Moreover, all tumor tissues with high PDE5 levels showed reduction of Thyroglobulin, TSH receptor, Thyroperoxidase, and NIS transcripts. In thyroid cancer cells in vitro, sildenafil and tadalafil determined a reduction of proliferation and cellular migration. Our findings demonstrate for the first time an overexpression of PDE5 in PTCs, and the ability of PDE5 inhibitors to block the proliferation of thyroid cancer cells in culture, therefore, suggesting that specific inhibition of PDE5 may be proposed for the treatment of these tumors.

Phosphodiesterase 10, a novel target in colon cancer.

503 Background: Elevation of intracellular cGMP is known to inhibit tumor proliferation and induce apoptosis, although the phosphodiesterase (PDE) isozymes that regulate cGMP levels in tumor cells have not been well studied. We report first evidence that PDE10 is elevated in colon tumors compared with normal colon and suggest that PDE10 inhibitors can be used for the treatment or prevention of colon cancer. Methods: PDE10 protein and mRNA levels were measured in human colon tumor cells (HT29, HCT116, SW480, Caco2), normal colonocytes (NCM460), human clinical samples, and ApcMin/+ mouse model. Two chemically distinct PDE10 selective inhibitors, PQ-10 and Pf-2545920, were tested against the cell lines. The NCI-60 panel of human tumor cell lines was also screened against Pf-2545920 to identify potential differences in sensitivity among histologically diverse tumor types. We also performed siRNA knockdown studies in colonocytes and tumor cell lines. To determine the effect of the PDE10 siRNA knockdown on cyclic nucleotide hydrolysis, whole cell lysates from transfected cells were assayed for PDE activity using cGMP or cAMP as substrates. Results: PDE10 levels were low in normal colonocytes (NCM460) and elevated in tumor cell lines. Similarly, PDE10 was elevated human clinical specimens and the ApcMin+/ mouse model compared with normal mucosa. PDE10 inhibitors and siRNA selectively inhibited colonic tumor growth while stable knockdown inhibited colony formation and increased doubling time. Pf-2545920 also supressed growth of all cell lines within the NCI-60 panel. In comparison with lysates from vector control cells, transfection with PDE10 siRNA reduced cGMP hydrolysis by ~35% in both HCT116 and HT29 cell lines, but did not affect cGMP hydrolysis in colonocytes; siRNA did not significantly affect cAMP degradation in all 3 cell lines. Conclusions: PDE10 plays a role in colon tumorgenesis whereby inhibitors can selectively suppress tumor cell growth. The mechanism by which PDE10 inhibition affects growth appears to involve activation of cGMP/PKG signalling. PDE10 represents a novel anticancer target for the treament and prevention of colon cancer.

Phosphodiesterase 5 is main regulator of brown adipocyte differentiation

Background Obesity, a major threat to global human health, has been shown to be associated with devastating diseases such as stroke, hypertension, cancer and type 2 diabetes. In order to fight the present obesity pandemic it is crucial to better understand regulation of adipocyte differentiation. Phosphodiesterases (PDEs) catalyze hydrolysis of cyclic nucleotides (cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)) to the corresponding 5′ nucleotide monophosphates. To date, eleven different PDEs (PDE1–11) have been characterized, and they differ in their selectivity for cyclic nucleotides, sensitivity to inhibitors and activators, physiological roles, and tissue distribution. The nonselective phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) has been shown to be essential for successful in vitro differentiation of adipocytes.

Identifying the Structural Features of Pyrazolo[4,3-c]Quinoline-3-ones as Inhibitors of Phosphodiesterase 4: An Exploratory CoMFA and CoMSIA Study

Phosphodiesterases (PDEs) are responsible for the hydrolysis of cyclic nucleotides (cAMP and c-GMP). Cyclic nucleotides are important intracellular secondary messengers in cell function, relaying the signals from hormones at specific cell-surface receptors. An increase of cAMP due to the stimulation of adenylyl cyclase or the inhibition of PDEs affects the activity of immune system and inflammatory cells. Thus, PDE4, a cAMP specific PDE, received much attention as a target for the treatment of the diseases like asthma and Chronic Obstructive Pulmonary Disease (COPD). Pyrazolo[4,3-c]quinoline-3-one nucleus has attracted considerable attention recently as PDE4 receptor antagonists which have shown remarkable therapeutic potential in the treatment of asthma and Chronic Obstructive Pulmonary Disease (COPD). In the present study, three dimensional quantitative structure activity relationship (3D QSAR) approach using CoMFA and CoMSIA was applied to a series of 2, 5-dihydropyrazolo [4, 3-c] quinoline-3-ones as PDE4 receptor antagonists. For the purpose, 22 compounds from the series were used to develop and validate models. The robustness of the model was confirmed with the help of leave one out cross-validation method, while the predictive ability of models was tested using a test set containing three molecules. Novel compounds were designed on the basis of results of CoMFA and CoMSIA studies. Designed compounds were evaluated by Docking and Lipinski filters. 3D-QSAR models with high squared correlation coefficient of up to 0.9590 for CoMFA and 0.9740 for CoMSIA were established. Robustness of the models is demonstrated by R2 cv values of up to 0.8600 and 0.8230 for CoMFA and CoMSIA, respectively. Predictive ability of the models is reflected by R2 pred values of 0.865 and 0.926 for CoMFA and CoMSIA respectively. Predicted activity of the designed molecules correlated well with the docking scores and the molecules also passed the Lipinski filters. Developed models highlighted the importance of steric, electrostatic and hydrophobic properties of the molecules for PDE 4 receptor affinity. The designed compounds may serve as lead for the development of newer PDE4 inhibitors based on the 2, 5-dihydropyrazolo [4, 3-c] quinoline-3-one scaffold.

Phosphodiesterase 11A (PDE11A) Expression in Mouse Tissues and Characterization of a PDE11A Mouse Knock-Out Model

Phosphodiesterases catalyze the hydrolysis of cyclic nucleotides and maintain physiologic levels of intracellular concentrations of cAMP and cGMP. Increased cAMP signaling has been associated with genetics disorders that lead to adrenocortical tumors and Cushing syndrome. Genetics defects in the phosphodiesterase 11A (PDE11A) are associated with increased levels of cAMP and bilateral adrenal hyperplasia; they also contribute to the development of adrenocortical, prostate, and testicular tumors in the general population. The aim of the present work is to study the expression of Pde11a in mouse tissues, as well as to understand better the Pde11a defect in a previously published Pde11a–/– knockout (KO) mouse model.1A colony of Pde11a–/– (KO), Pde11a+/– (Het) and wild type (wt) mice was studied in a complete phenotypical groß and microscopy pathological study. Molecular, immunohistochemical and biochemical studies were done to determine the function and expression of Pde11a in the tissues of normal and mutant mice. In normal mice higher PDE11A immunoreactivity(PDE11A-IR) was found in small intestine villi, thyroid follicular cells, prostate and spermatocytes; moderate PDE11A-IR in brain, liver hepatocytes, and exocrine pancreas and little, if any, in the pituitary gland. In mutant mice RNA studies showed a reduced Pde11a expression in steroidogenic tissues (adrenals and testis), lung, brain, and liver while prostate and kidney presented comparable expression with normal mice. PDE11A protein expression and activity assays did not show differences among three groups. Pathological studies showed differences: Adrenal subcapsular hyperplasia and foamy cells were more frequent in Het(37.7% and 30.2% respectively, p<0.05) than in wt group (26.3% and 15.8% respectively); Spinal epidermoid cyst in Het (15%) and wt mice (10%); kidney-pelvis interstitial lymphocytes only in KO mice (10.7%, P<0.05). KO mice showed increased absolute and organ specific weight. This study show a clear profile of Pde11a protein expression in endocrine and other mouse tissues, as well as convincing evidence of only partial Pde11a suppression in the previously published KO mouse. These mice developed adrenal hyperplasia and other abnormalities like humans with heterozygote PDE11A defects and partial inactivation of the enzyme. These data support the involvement of PDE11A in the pathogenesis of adrenocortical hyperplasia, but also provide significant insight into a previously reported mouse model.

Antidepressant-like effect of etazolate, a cyclic nucleotide phosphodiesterase 4 inhibitor—an approach using rodent behavioral antidepressant tests battery

Etazolate, a pyrazolopyridine class derivative is selective inhibitor of type 4 phosphodiesterase (PDE4), an enzyme catalyzes the hydrolysis of cyclic nucleotide viz. cAMP & regulates cAMP signal transduction. Enhancing cAMP signal transduction by inhibition of PDE4 is known to be beneficial in depression disorders. Thus, the present study was designed to investigate thoroughly the antidepressant potential of etazolate using rodent behavioral models of depression. Acute treatment of etazolate (0.25–1mg/kg, i.p.) exhibited antidepressant-like effects in forced swim test (FST) & tail suspension test (TST) in mice without influencing the baseline locomotion in actophotometer test. Interaction studies of etazolate sub-effective dose (0.12mg/kg, i.p.), were carried out with sub-effective dose of conventional antidepressants like fluoxetine (5mg/kg, i.p.), venlafaxine (4mg/kg, i.p.) & desipramine (5mg/kg, i.p.) in FST. Etazolate at sub-effective dose produced synergistic antidepressant-like effect with conventional antidepressants in the mouse FST. In addition, combined treatment of etazolate & conventional antidepressants had no significant effect on baseline locomotion. Moreover, etazolate (0.5 and 1mg/kg, i.p.) increased head twitch scores in mice & antagonized the reserpine-induced hypothermia in rats. Chronic treatment (14 days) with etazolate (0.5 and 1mg/kg, p.o.) & fluoxetine (10mg/kg, p.o.) significantly reversed the behavioral anomalies induced by bilateral olfactory bulbectomy in rats in modified open field exploration. In conclusion, taken together, our results suggested that etazolate exhibited antidepressant-like activity in acute & chronic rodent models of depression & deserves as a therapeutic tool that could help the conventional pharmacotherapy of depression.

Drug to Genome to Drug: Discovery of New Antiplasmodial Compounds

The dominant strategy for discovery of new antimalarial drugs relies on cell-free assays on specific biochemical pathways of Plasmodium falciparum . However, it appears that screening directly on the parasite is a more rewarding approach. The "drug to genome to drug" approach consists of testing a small set of structural analogues of a drug acting on human proteins that have plasmodial orthologues. Both man and plasmodium possess cyclic nucleotide phosphodiesterases (PDEs) that are key players of cell homeostasis. We synthesized and tested 40 analogues of tadalafil, a human PDE5 inhibitor, on P. falciparum in culture and obtained potent inhibitors of parasite growth. We discuss the structure-activity relationships, which support the hypothesis that our compounds kill the parasite via inhibition of plasmodial PDE activity. We also prove that antiplasmodial derivatives inhibit the hydrolysis of cyclic nucleotides of the parasite, validating the cAMP/cGMP pathways as therapeutic targets against Plasmodium falciparum.

Direct Allosteric Regulation between the GAF Domain and Catalytic Domain of Photoreceptor Phosphodiesterase PDE6

Photoreceptor cGMP phosphodiesterase (PDE6) is the central enzyme in the visual transduction cascade. The PDE6 catalytic subunit contains a catalytic domain and regulatory GAF domains. Unlike most GAF domain-containing cyclic nucleotide phosphodiesterases, little is known about direct allosteric communication of PDE6. In this study, we demonstrate for the first time direct, inter-domain allosteric communication between the GAF and catalytic domains in PDE6. The binding affinity of PDE6 for pharmacological inhibitors or for the C-terminal region of the inhibitory gamma subunit (Pgamma), known to directly inhibit PDE6 catalysis, was increased approximately 2-fold by ligands binding to the GAF domain. Binding of the N-terminal half of Pgamma to the GAF domains suffices to induce this allosteric effect. Allosteric communication between GAF and catalytic domains is reciprocal, in that drug binding to the catalytic domain slowed cGMP dissociation from the GAF domain. Although cGMP hydrolysis was not affected by binding of Pgamma1-60, Pgamma lacking its last seven amino acids decreased the Michaelis constant of PDE6 by 2.5-fold. Pgamma1-60 binding to the GAF domain increased vardenafil but not cGMP affinity, indicating that substrate- and inhibitor-binding sites do not totally overlap. In addition, prolonged incubation of PDE6 with vardenafil or sildenafil (but not 3-isobutyl-1-methylxanthine and zaprinast) induced a distinct conformational change in the catalytic domain without affecting the binding properties of the GAF domains. We conclude that although Pgamma-mediated regulation plays the dominant role in visual excitation, the direct, inter-domain allosteric regulation described in this study may play a feedback role in light adaptational processes during phototransduction.

Phosphodiesterase 2A Forms a Complex with the Co-chaperone XAP2 and Regulates Nuclear Translocation of the Aryl Hydrocarbon Receptor

Phosphodiesterase type 2A (PDE2A) hydrolyzes cyclic nucleotides cAMP and cGMP, thus efficiently controlling cNMP-dependent signaling pathways. PDE2A is composed of an amino-terminal region, two regulatory GAF domains, and a catalytic domain. Cyclic nucleotide hydrolysis is known to be activated by cGMP binding to GAF-B; however, other mechanisms may operate to fine-tune local cyclic nucleotide levels. In a yeast two-hybrid screening we identified XAP2, a crucial component of the aryl hydrocarbon receptor (AhR) complex, as a major PDE2A-interacting protein. We mapped the XAP2 binding site to the GAF-B domain of PDE2A. PDE assays with purified proteins showed that XAP2 binding does not change the enzymatic activity of PDE2A. To analyze whether PDE2A could affect the function of XAP2, we studied nuclear translocation of AhR, i.e. the master transcription factor controlling the expression of multiple detoxification genes. Notably, regulation of AhR target gene expression is initiated by tetrachlorodibenzodioxin (TCDD) binding to AhR and by a poorly understood cAMP-dependent pathway followed by the translocation of AhR from the cytosol into the nucleus. Binding of PDE2A to XAP2 inhibited TCDD- and cAMP-induced nuclear translocation of AhR in Hepa1c1c7 hepatocytes. Furthermore, PDE2A attenuated TCDD-induced transcription in reporter gene assays. We conclude that XAP2 targets PDE2A to the AhR complex, thereby restricting AhR mobility, possibly by a local reduction of cAMP levels. Our results provide first insights into the elusive cAMP-dependent regulation of AhR.