Cyclic Nucleotide Phosphodiesterase Research Articles

Resonant-Mie-scattering of aggregates of phosphomolybdate and papaverine for measuring activities and screening inhibitors of cyclic nucleotide phosphodiesterase isozymes

A resonant-light-scattering (RLS) method was proposed to quantify phosphate for screening inhibitors of isozymes of cyclic nucleotide phosphodiesterase (PDE). In acidified mixtures of phosphate, papaverine and molybdate, there were aggregates exhibiting micrometre sizes, no absorbance peaks over 360nm but strong RLS peaks at 392nm; Mie scattering thus accounted for the RLS signals. When papaverine was added before molybdate to acidified samples of phosphate, RLS signals at 392nm were stable from 5 to 25min since the addition of molybdate; after optimization, phosphate from 0.40 to 3.60μM was quantifiable. This RLS method tolerated 60mgL−1 proteins besides common PDE inhibitors and dimethyl sulfoxide in acidified samples of phosphate; the integration of this RLS method with the coupled action of a phosphomonoesterase on PDE product was thus rational to measure PDE activities without the removal of proteins in samples. By quantifying activities of a truncated mutant of human PDE4B2 via this RLS method, Michaelis–Menten constant, inhibition constants of rolipram, papaverine and theophylline varied over three magnitudes and were consistent with those estimated by an improved malachite green assay of phosphate, respectively. Hence, this novel RLS method was promising for screening inhibitors of PDE isozymes.

A survey of rare coding variants in candidate genes in schizophrenia by deep sequencing

The genetic architecture of schizophrenia is likely contributed by both common and rare variants.1 Recent genome-wide studies have revealed that common variants in the major histocompatibility complex (MHC) region, TCF4 and other genes are associated with schizophrenia.1 In addition, rare copy-number variation (CNV) regions in broad regions like 1q21.1, 15q13.3, 15q11.2, 22q111 as well as individual genes such as Neurexin2, 3 have been identified. Unbiased exome or whole genome scanning procedures have the potential to identify novel loci while likely requiring large sample sets to reach a genome-wide significance level. It is possible that the previously identified genes/regions from high-throughput single-nucleotide polymorphisms (SNP) chip genome-wide scanning techniques, in contrast to some ‘classical' candidate genes,4 may harbor rare coding variants that have a role in disease risk. We selected a total of 101 genes from within the 1q21.1, 15q13.3, 22q11 and 15q11.2 regions and a number of other candidate genes, with either a priori knowledge for association with schizophrenia, for example, TCF4/CCDC68, NRXN1, or interesting for drug-discovery efforts, for example, cyclic nucleotide phosphodiesterase genes, and surveyed rare variants in their coding regions through deep sequencing.

Nfasc155H and MAG are Specifically Susceptible to Detergent Extraction in the Absence of the Myelin Sphingolipid Sulfatide

Mice incapable of synthesizing the myelin lipid sulfatide form paranodes that deteriorate with age. Similar instability also occurs in mice that lack contactin, contactin-associated protein or neurofascin155 (Nfasc155), the proteins that cluster in the paranode and form the junctional complex that mediates myelin-axon adhesion. In contrast to these proteins, sulfatide has not been shown to be enriched in the paranode nor has a sulfatide paranodal binding partner been identified; thus, it remains unclear how the absence of sulfatide results in compromised paranode integrity. Using an in situ extraction procedure, it has been reported that the absence of the myelin sphingolipids, galactocerebroside and sulfatide, increased the susceptibility of Nfasc155 to detergent extraction. Here, employing a similar approach, we demonstrate that in the presence of galactocerebroside but in the absence of sulfatide Nfasc155 is susceptible to detergent extraction. Furthermore, we use this in situ approach to show that stable association of myelin-associated glycoprotein (MAG) with the myelin membrane is sulfatide dependent while the membrane associations of myelin/oligodendrocyte glycoprotein, myelin basic protein and cyclic nucleotide phosphodiesterase are sulfatide independent. These findings indicate that myelin proteins maintain their membrane associations by different mechanisms. Moreover, the myelin proteins that cluster in the paranode and require sulfatide mediate myelin-axon adhesion. Additionally, the apparent dependency on sulfatide for maintaining Nfasc155 and MAG associations is intriguing since the fatty acid composition of sulfatide is altered and paranodal ultrastructure is compromised in multiple sclerosis. Thus, our findings present a potential link between sulfatide perturbation and myelin deterioration in multiple sclerosis.

Increase in Cellular Cyclic AMP Concentrations Reverses the Profibrogenic Phenotype of Cardiac Myofibroblasts: A Novel Therapeutic Approach for Cardiac Fibrosis

Tissue fibrosis is characterized by excessive production, deposition, and contraction of the extracellular matrix (ECM). The second messenger cAMP has antifibrotic effects in fibroblasts from several tissues, including cardiac fibroblasts (CFs). Increased cellular cAMP levels can prevent the transformation of CFs into profibrogenic myofibroblasts, a critical step that precedes increased ECM deposition and tissue fibrosis. Here we tested two hypotheses: 1) myofibroblasts have a decreased ability to accumulate cAMP in response to G protein-coupled receptor (GPCR) agonists, and 2) increasing cAMP will not only prevent, but also reverse, the myofibroblast phenotype. We found that myofibroblasts produce less cAMP in response to GPCR agonists or forskolin and have decreased expression of several adenylyl cyclase (AC) isoforms and increased expression of multiple cyclic nucleotide phosphodiesterases (PDEs). Furthermore, we found that forskolin-promoted increases in cAMP or N(6)-phenyladenosine-cAMP, a protein kinase A-selective analog, reverse the myofibroblast phenotype, as assessed by the expression of collagen Iα1, α-smooth muscle actin, plasminogen activator inhibitor-1, and cellular contractile abilities, all hallmarks of a fibrogenic state. These results indicate that: 1) altered expression of AC and PDE isoforms yield a decrease in cAMP concentrations of cardiac myofibroblasts (relative to CFs) that likely contributes to their profibrotic state, and 2) approaches to increase cAMP concentrations not only prevent fibroblast-to-myofibroblast transformation but also can reverse the profibrotic myofibroblastic phenotype. We conclude that therapeutic strategies designed to enhance cellular cAMP concentrations in CFs may provide a means to reverse excessive scar formation following injury and to treat cardiac fibrosis.

Cyclic AMP regulates the migration and invasion potential of human pancreatic cancer cells

Aggressive dissemination and metastasis of pancreatic ductal adenocarcinoma (PDAC) results in poor prognosis and marked lethality. Rho monomeric G protein levels are increased in pancreatic cancer tissue. As the mechanisms underlying PDAC malignancy are little understood, we investigated the role for cAMP in regulating monomeric G protein regulated invasion and migration of pancreatic cancer cells. Treatment of PDAC cells with cAMP elevating agents that activate adenylyl cyclases, forskolin, protein kinase A (PKA), 6-Bnz-cAMP, or the cyclic nucleotide phosphodiesterase inhibitor cilostamide significantly decreased migration and Matrigel invasion of PDAC cell lines. Inhibition was dose-dependent and not significantly different between forskolin or cilostamide treatment. cAMP elevating drugs not only blocked basal migration, but similarly abrogated transforming-growth factor-β-directed PDAC cell migration and invasion. The inhibitory effects of cAMP were prevented by the pharmacological blockade of PKA. Drugs that increase cellular cAMP levels decreased levels of active RhoA or RhoC, with a concomitant increase in phosphorylated RhoA. Diminished Rho signaling was correlated with the appearance of thickened cortical actin bands along the perimeter of non-motile forskolin or cilostamide-treated cells. Decreased migration did not reflect alterations in cell growth or programmed cell death. Collectively these data support the notion that increased levels of cAMP specifically hinder PDAC cell motility through F-actin remodeling.

Cyclic nucleotide phosphodiesterase 3A1 protects the heart against ischemia-reperfusion injury

Phosphodiesterase 3A (PDE3A) is a major regulator of cAMP in cardiomyocytes. PDE3 inhibitors are used for acute treatment of congestive heart failure, but are associated with increased incidence of arrhythmias and sudden death with long-term use. We previously reported that chronic PDE3A downregulation or inhibition induced myocyte apoptosis in vitro. However, the cardiac protective effect of PDE3A has not been demonstrated in vivo in disease models. In this study, we examined the role of PDE3A in regulating myocardial function and survival in vivo using genetically engineered transgenic mice with myocardial overexpression of the PDE3A1 isozyme (TG). TG mice have reduced cardiac function characterized by reduced heart rate and ejection fraction (52.5±7.8% vs. 83.9±4.7%) as well as compensatory expansion of left ventricular diameter (4.19±0.19mm vs. 3.10±0.18mm). However, there was no maladaptive increase of fibrosis and apoptosis in TG hearts compared to wild type (WT) hearts, and the survival rates also remained the same. The diminution of cardiac contractile function is very likely attributed to a decrease in beta-adrenergic receptor (β-AR) response in TG mice. Importantly, the myocardial infarct size (4.0±1.8% vs. 24.6±3.8%) and apoptotic cell number (1.3±1.0% vs. 5.6±1.5%) induced by ischemia/reperfusion (I/R) injury were significantly attenuated in TG mice. This was associated with decreased expression of inducible cAMP early repressor (ICER) and increased expression of anti-apoptotic protein BCL-2. To further verify the anti-apoptotic effects of PDE3A1, we performed in vitro apoptosis study in isolated adult TG and WT cardiomyocytes. We found that the apoptotic rates stimulated by hypoxia/reoxygenation or H2O2 were indeed significantly reduced in TG myocytes, and the differences between TG and WT myocytes were completely reversed in the presence of the PDE3 inhibitor milrinone. These together indicate that PDE3A1 negatively regulates β-AR signaling and protects against I/R injury by inhibiting cardiomyocyte apoptosis.

Inhibition of phosphodiesterase 4 promotes differentiation and fusion of bewo choriocarcinoma cells

Inhibition of phosphodiesterase 4 promotes differentiation and fusion of bewo choriocarcinoma cells

Quercetin acutely relaxes airway smooth muscle and potentiates β-agonist-induced relaxation via dual phosphodiesterase inhibition of PLCβ and PDE4

Asthma is a disease of the airways with symptoms including exaggerated airway narrowing and airway inflammation. Early asthma therapies used methylxanthines to relieve symptoms, in part, by inhibiting cyclic nucleotide phosphodiesterases (PDEs), the enzyme responsible for degrading cAMP. The classification of tissue-specific PDE subtypes and the clinical introduction of PDE-selective inhibitors for chronic obstructive pulmonary disease (i.e., roflumilast) have reopened the possibility of using PDE inhibition in the treatment of asthma. Quercetin is a naturally derived PDE4-selective inhibitor found in fruits, vegetables, and tea. We hypothesized that quercetin relaxes airway smooth muscle via cAMP-mediated pathways and augments β-agonist relaxation. Tracheal rings from male A/J mice were mounted in myographs and contracted with acetylcholine (ACh). Addition of quercetin (100 nM-1 mM) acutely and concentration-dependently relaxed airway rings precontracted with ACh. In separate studies, pretreatment with quercetin (100 μM) prevented force generation upon exposure to ACh. In additional studies, quercetin (50 μM) significantly potentiated isoproterenol-induced relaxations. In in vitro assays, quercetin directly attenuated phospholipase C activity, decreased inositol phosphate synthesis, and decreased intracellular calcium responses to Gq-coupled agonists (histamine or bradykinin). Finally, nebulization of quercetin (100 μM) in an in vivo model of airway responsiveness significantly attenuated methacholine-induced increases in airway resistance. These novel data show that the natural PDE4-selective inhibitor quercetin may provide therapeutic relief of asthma symptoms and decrease reliance on short-acting β-agonists.

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

Correction to: Journal of Human Genetics (2011) 56, 676–681; doi:10.1038/jhg.2011.80; published online 4 August 2011 The authors of the above article noticed errors in publication of this paper (in September 2011 issue). In the list of authors, incorrect middle initial was applied to Dr Kipps. The correct name should have read Thomas J Kipps and is now shown correctly above.

The role and significance of negative regulatory factors in the learning and memory

Background The exact mechanism of learning and memory is still unclear.The existing researches mainly focused on learning and memory regulatory factors,especially on positive regulatory factors.In recent years,the roles of learning and memory negative regulatory factors in correct forming and maintaining of the learning and memory have attracted most attentions.Objective Summary of the role of and significance learning and memory negative regulatory factors in the mechanism of learning and memory formation and maintainance.Content The negative regulatory factors of learning and memory mainly include protein phosphatase,cyclic nucleotide phosphodiesterases,transcriptional regulation factor,histone deacetylases,hippyragranin(HGN) gene and so on.They regulate learning and memory in multiple levels including the cell membrane receptors,cytoplasmic signal transduction cascade composition,transcriptional regulation of gene expression in the nucleus and cell growth and development.Trend In-depth study of learning and memory negative regulatory factors and the balance between learning and memory positive/negative regulation contribute to our understanding of the mechanism of learning and memory formation and maintainance,and searching for effective interventions. Key words: Learning and memory; Regulation; Negative

Rôle des phosphodiestérases des nucléotides cycliques de types 3 et 4 dans le couplage excitation-contraction et les arythmies cardiaques

Cyclic nucleotide phosphodiesterases (PDE) represent a superfamily of enzymes specialised in the degradation of cAMP and cGMP. In heart, PDE3 and PDE4 are the two major families involved in the regulation of cAMP levels and the control of inotropism. Both families are encoded by several genes, and the recent analysis of the cardiac phenotype of mice lacking these different genes provided new insights into the way they regulate excitation-contraction coupling (ECC). In particular, these studies emphasize the local character of ECC regulation by PDE, as well as the role of these PDE in maintaining calcium homeostasis and preventing cardiac arrhythmias.

Role of expression of phosphodiesterase 4B in spinal cord in inflammatory responses in a rat model of neuropathic pain: relationship with extracellular signal-regulated kinase

Objective To evaluate the role of expression of phosphodiesterase 4B (PDE4B) in the spinal cord in inflammatory responses in a rat model of neuropathic pain and the relationship with extracellular signal-regulated kinase (ERK).Methods Sixty healthy male Sprague-Dawley rats,weighing 180-220 g,in which intrathecal catheters were implanted at L5,6 interspace,were used.The location of catheters was confirmed 6 days later.The rats were randomly divided into 5 groups (n =12 each):sham operation group (group Sham),normal saline (NS) group,vehicle group (group Ⅴ),mismatch siRNA group (group siR-M),and PDE4B-siRNA group (group siR-B).Neuropathic pain was induced by ligation of L5 spinal nerve (SNL).In Sham group,the L5 spinal nerve was only exposed,but not ligated.Immediately after ligation and on 1,3,5,and 7 days after ligation,10 μl NS,10 μl NS,LipofectaminTM RNAiMAX,PDE4B-siRNA (2 μg/10 μl) encapsulated in mismatch siRNA and PDE4B-siRNA (2 μg/10 μl) encapsulated in LipofectaminTM RNAiMAX were injected intrathecally in Sham,NS,V,siR-M,and siR-B groups,respectively.The mechanical pain threshold was measured at 1 day before and 2,4,6 and 8 days after operation.After behavioral testing on 8th day after operation,the rats were sacrificed and the lumbar segment of the spinal cord was removed for determination of PDE4B protein,ERK and phosphor-ERK (p-ERK)expression,and TNF-α,IL-1β and IL-6 levels.Results Compared with Sham group,the mechanical pain threshold was significantly decreased at 2,4,6 and 8 days after operation in NS,V,siR-M and siR-B groups (P ＜0.05),and no significant change was found in the mechanical pain threshold at 2,4,6 and 8 days after operation (P ＞ 0.05) and the expression of p-ERK and PDE4B protein,and levels of TNF-α,IL-1β and IL-6 were increased at 2,4,6 and 8 days after operation in V and siR-M groups (P ＜ 0.05).Compared with NS group,the mechanical pain threshold was significantly increased,and the expression of p-ERK and PDE4B protein and levels of TNF-α,IL-1β and IL-6 were decreased at 2,4,6 and 8 days after operation (P ＜ 0.05),and no significant change was found in the parameters mentioned above in V and siR-M groups (P ＞ 0.05).Conclusion Up-regulation of the expression of PDE4B protein in the spinal cord is involved in the development of neuropathic pain in rats,which may be related to promoted phosphorylation of ERK in the spinal cord and enhanced inflammatory responses. Key words: Cyclic nucleotide phosphodiesterases, type 4; Spinal cord; Neuralgia; Inflammation; Cyclic nucleotide-regulated protein kinases

Phosphodiesterase-8A binds to and regulates Raf-1 kinase

V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A(-/-)) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

PDE3 and PDE4 Isozyme-Selective Inhibitors Are Both Required for Synergistic Activation of Brown Adipose Tissue

Brown adipose tissue (BAT) is a highly thermogenic organ that converts lipids and glucose into heat. Many of the metabolic and gene transcriptional hallmarks of BAT activation, namely increased lipolysis, uncoupling protein-1 (UCP1) mRNA, and glucose uptake, are regulated by the adrenergic second messenger, cAMP. Cyclic nucleotide phosphodiesterases (PDEs) catalyze the breakdown of cAMP, thereby regulating the magnitude and duration of this signaling molecule. In the absence of adrenergic stimulus, we found that it required a combination of a PDE3 and a PDE4 inhibitor to fully induce UCP1 mRNA and lipolysis in brown adipocytes, whereas neither PDE inhibitor alone had any substantial effect under basal conditions. Under submaximal β-adrenoceptor stimulation of brown adipocytes, a PDE3 inhibitor alone could potentiate induction of UCP1 mRNA, whereas a PDE4 inhibitor alone could augment lipolysis, indicating differential roles for each of these two PDEs. Neither induction of UCP1 nor lipolysis was altered by inhibition of PDE1, PDE2, or PDE8A. Finally, when injected into mice, the combination of PDE3 and PDE4 inhibitors stimulated glucose uptake in BAT under thermoneutral and fasted conditions, a response that was further potentiated by the global ablation of PDE8A. Taken together, these data reveal that multiple PDEs work in concert to regulate three of the important pathways leading to BAT activation, a finding that may provide an improved conceptual basis for the development of therapies for obesity-related diseases.

Mechanisms of Cyclic Nucleotide Phosphodiesterases in Modulating T Cell Responses in Murine Graft-versus-Host Disease

Graft-versus-host disease (GvHD) is a key contributor to the morbidity and mortality after allogeneic hematopoetic stem cell transplantation (HSCT). Regulatory Foxp3+ CD4+ T cells (Treg) suppress conventional T cell activation and can control GvHD. In our previous work, we demonstrate that a basic mechanism of Treg mediated suppression occurs by the transfer of cyclic adenosine monophosphate (cAMP) to responder cells. Whether this mechanism is relevant for Treg mediated suppression of GvHD is currently unknown. To address this question, bone marrow and T cells from C57BL/6 mice were transferred into lethally irradiated BALB/c recipients, and the course of GvHD and survival were monitored. Transplanted recipients developed severe GvHD that was strongly ameliorated by the transfer of donor Treg cells. Towards the underlying mechanisms, in vitro studies revealed that Treg communicated with DCs via gap junctions, resulting in functional inactivation of DC by a metabolic pathway involving cAMP that is modulated by the phosphodiesterase (PDE) 4 inhibitor rolipram. PDE2 or PDE3 inhibitors as well as rolipram suppressed allogeneic T cell activation, indirectly by enhancing Treg mediated suppression of DC activation and directly by inhibiting responder T cell proliferation. In line with this, we observed a cooperative suppression of GvHD upon Treg transfer and additional rolipram treatment. In conclusion, we propose that an important pathway of Treg mediated control of GvHD is based on a cAMP dependent mechanism. These data provide the basis for future concepts to manipulate allogeneic T cell responses to prevent GvHD.

Chemical informatics uncovers a new role for moexipril as a novel inhibitor of cAMP phosphodiesterase-4 (PDE4)

PDE4 is one of eleven known cyclic nucleotide phosphodiesterase families and plays a pivotal role in mediating hydrolytic degradation of the important cyclic nucleotide second messenger, cyclic 3′5′ adenosine monophosphate (cAMP). PDE4 inhibitors are known to have anti-inflammatory properties, but their use in the clinic has been hampered by mechanism-associated side effects that limit maximally tolerated doses. In an attempt to initiate the development of better-tolerated PDE4 inhibitors we have surveyed existing approved drugs for PDE4-inhibitory activity. With this objective, we utilised a high-throughput computational approach that identified moexipril, a well tolerated and safe angiotensin-converting enzyme (ACE) inhibitor, as a PDE4 inhibitor. Experimentally we showed that moexipril and two structurally related analogues acted in the micro molar range to inhibit PDE4 activity. Employing a FRET-based biosensor constructed from the nucleotide binding domain of the type 1 exchange protein activated by cAMP, EPAC1, we demonstrated that moexipril markedly potentiated the ability of forskolin to increase intracellular cAMP levels. Finally, we demonstrated that the PDE4 inhibitory effect of moexipril is functionally able to induce phosphorylation of the small heat shock protein, Hsp20, by cAMP dependent protein kinase A. Our data suggest that moexipril is a bona fide PDE4 inhibitor that may provide the starting point for development of novel PDE4 inhibitors with an improved therapeutic window.

Importance of the interaction protein–protein of the CaM–PDE1A and CaM–MLCK complexes in the development of new anti‐CaM drugs

Protein-protein interactions play central roles in physiological and pathological processes. The bases of the mechanisms of drug action are relevant to the discovery of new therapeutic targets. This work focuses on understanding the interactions in protein-protein-ligands complexes, using proteins calmodulin (CaM), human calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A active human (PDE1A), and myosin light chain kinase (MLCK) and ligands αII-spectrin peptide (αII-spec), and two inhibitors of CaM (chlorpromazine (CPZ) and malbrancheamide (MBC)). The interaction was monitored with a fluorescent biosensor of CaM (hCaM M124C-mBBr). The results showed changes in the affinity of CPZ and MBC depending on the CaM-protein complex under analysis. For the Ca(2+) -CaM, Ca(2+) -CaM-PDE1A, and Ca(2+) -CaM-MLCK complexes, CPZ apparent dissociation constants (Kds ) were 1.11, 0.28, and 0.55 μM, respectively; and for MBC Kds were 1.43, 1.10, and 0.61 μM, respectively. In competition experiments the addition of calmodulin binding peptide 1 (αII-spec) to Ca(2+) -hCaM M124C-mBBr quenched the fluorescence (Kd = 2.55 ± 1.75 pM) and the later addition of MBC (up to 16 μM) did not affect the fluorescent signal. Instead, the additions of αII-spec to a preformed Ca(2+) -hCaM M124C-mBBr-MBC complex modified the fluorescent signal. However, MBC was able to displace the PDE1A and MLCK from its complex with Ca(2+) -CaM. In addition, docking studies were performed for all complexes with both ligands showing an excellent correlation with experimental data. These experiments may help to explain why in vivo many CaM drugs target prefer only a subset of the Ca(2+) -CaM regulated proteins and adds to the understanding of molecular interactions between protein complexes and small ligands.

Gene Cloning, Expression, and Characterization of a Cyclic Nucleotide Phosphodiesterase from Arthrobacter sp. CGMCC 3584

Based on thermal asymmetric interlaced polymerase chain reaction, the arpde gene encoding a cyclic nucleotide-specific phosphodiesterase was cloned from Arthrobacter sp. CGMCC 3584 for the first time. The 930-bp region encoded a 309-amino-acid protein with a molecular weight of 33.6 kDa. The recombinant ArPDE was able to hydrolyze 3',5'-cAMP, 3',5'-cGMP, and 2',3'-cAMP. The K m values of ArPDE for 3',5'-cAMP and 3',5'-cGMP were 6.82 and 12.82 mM, respectively. ArPDE was thermostable and displayed optimal activity at 45 °C and pH 7.5. The enzyme did not require any metal cofactors, although its activity was stimulated by 2 mM Co(2+) and inhibited by Zn(2+). Nucleotides, reducing agents, and sulfhydryl reagents had different inhibitory effects on the activity of ArPDE. NaF, the actual compound used to improve the industrial yield of cAMP, exhibited 62 % inhibitions at concentrations of 10 mM.

2′,3′-cyclic nucleotide phosphodiesterase (CNPase) as a target in neurodegenerative diseases

We review the structure, characteristics, post-translational modifications, and functioning of 2′,3′-cyclic nucleotide phosphodiesterase (CNPase) localized in the CNS myelin and Schwann cells, as well as previously unknown properties of this protein, which was identified in brain mitochondria. We summarize the data on the localization of CNPase in the cell, the specificity of its substrates, the characteristics of the N- and C-terminals of the molecule, and its interactions with proteins-partners and nucleic acids. We describe the possible role of CNPase in ageing and neurodegenerative diseases, such as Alzheimer’s disease and multiple sclerosis, with the main emphasis on the role of mitochondria in these processes.

Discovery of Novel Trypanosoma brucei Phosphodiesterase B1 Inhibitors by Virtual Screening against the Unliganded TbrPDEB1 Crystal Structure

Trypanosoma brucei cyclic nucleotide phosphodiesterase B1 (TbrPDEB1) and TbrPDEB2 have recently been validated as new therapeutic targets for human African trypanosomiasis by both genetic and pharmacological means. In this study we report the crystal structure of the catalytic domain of the unliganded TbrPDEB1 and its use for the in silico screening for new TbrPDEB1 inhibitors with novel scaffolds. The TbrPDEB1 crystal structure shows the characteristic folds of human PDE enzymes but also contains the parasite-specific P-pocket found in the structures of Leishmania major PDEB1 and Trypanosoma cruzi PDEC. The unliganded TbrPDEB1 X-ray structure was subjected to a structure-based in silico screening approach that combines molecular docking simulations with a protein-ligand interaction fingerprint (IFP) scoring method. This approach identified six novel TbrPDEB1 inhibitors with IC50 values of 10-80 μM, which may be further optimized as potential selective TbrPDEB inhibitors.