Cyclic Nucleotide-specific Phosphodiesterases Research Articles

72 - The tyrosine nitration of PKG-1α inhibits its regulatory function in the pulmonary endothelium

Protein tyrosine nitration secondary to peroxynitrite generation is involved in the pathogenesis of vascular diseases. We have previously shown that tyrosine nitration is associated with the development of acute lung injury (ALI). Protein kinase PKG-1a, which integrates cAMP/cGMP signaling and is important for the endothelial barrier maintenance, can be inhibited by nitration at Tyr247. We studied a tyrosine nitration effect on PKG-1a regulatory function in the pulmonary endothelium. Since cyclic nucleotide-specific phosphodiesterases (PDEs) can be modulated by phosphorylation, we explored the functional link between nitration-mediated PKG-1a inhibition and PDE(s) activation. LPS-challenged mice were utilized as ALI model. The peroxynitrite scavenger, MnTMPyP was used to attenuate protein nitration. Cyclic nucleotide levels, PKA/PKG/PDEs activities and PKG nitration were evaluated. PKG-1a-dependent phosphorylation sites on PDE3A were elucidated by mass-spectrometry. LPS-treatment decreased cAMP and increased cGMP levels in mouse lungs. PKA/PKG activities were also decreased. Nitrosative stress played a pivotal role in these changes. We identified an increase in PKG-1a nitration at Tyr247 and PDE3A activity increase. PKG activation in vitro induced an increase in PDE3A serine phosphorylation that correlated with a decrease in its activity. Mass-spectrometry identified a novel PKG-1a-dependent phosphorylation site, Ser654, in PDE3A. We confirmed PKG-1a-PDE3A interaction, and PKG-dependent PDE3A phosphorylation. This phosphorylation suppressed PDE3A activity and elevated intracellular cAMP levels. Ser654Ala-PDE3A mutant overexpression abolished PKG-1a-dependent inhibitory effect and prevented cAMP increase. We designed a peptide aimed to protect PKG-1a from Tyr247 nitration. This peptide attenuated the endothelial barrier disruption in LPS-challenged mice. We have identified a novel mechanism of cyclic nucleotide cross-talk regulated by PKG-dependent inhibitory phosphorylation of PDE3A. In ALI, PKG-1a Tyr247 nitration abolishes PDE3A phosphorylation leading to cAMP signaling impairment and endothelial dysfunction. Prevention of PKG-1a nitration significantly improved the barrier-protective mechanisms in ALI model. These data suggest a therapeutic approach of PKG activity protection in ALI treatment.

Cyclic Nucleotide-Specific Phosphodiesterases as Potential Drug Targets for Anti-Leishmania Therapy.

The available treatments for leishmaniasis are less than optimal due to inadequate efficacy, toxic side effects, and the emergence of resistant strains, clearly endorsing the urgent need for discovery and development of novel drug candidates. Ideally, these should act via an alternative mechanism of action to avoid cross-resistance with the current drugs. As cyclic nucleotide-specific phosphodiesterases (PDEs) of Leishmania major have been postulated as putative drug targets, a series of potential inhibitors of Leishmania PDEs were explored. Several displayed potent and selective in vitro activity against L. infantum intracellular amastigotes. One imidazole derivative, compound 35, was shown to reduce the parasite loads in vivo and to increase the cellular cyclic AMP (cAMP) level at in a dose-dependent manner at just 2× and 5× the 50% inhibitory concentration (IC50), indicating a correlation between antileishmanial activity and increased cellular cAMP levels. Docking studies and molecular dynamics simulations pointed to imidazole 35 exerting its activity through PDE inhibition. This study establishes for the first time that inhibition of cAMP PDEs can potentially be exploited for new antileishmanial chemotherapy.

The ever unfolding story of cAMP signaling in trypanosomatids: vive la difference!

Kinetoplastids are unicellular, eukaryotic, flagellated protozoans containing the eponymous kinetoplast. Within this order, the family of trypanosomatids are responsible for some of the most serious human diseases, including Chagas disease (Trypanosoma cruzi), sleeping sickness (Trypanosoma brucei spp.), and leishmaniasis (Leishmania spp). Although cAMP is produced during the life cycle stages of these parasites, its signaling pathways are very different from those of mammals. The absence of G-protein-coupled receptors, the presence of structurally different adenylyl cyclases, the paucity of known cAMP effector proteins and the stringent need for regulation of cAMP in the small kinetoplastid cells all suggest a significantly different biochemical pathway and likely cell biology. However, each of the main kinetoplastid parasites express four class 1-type cyclic nucleotide-specific phosphodiesterases (PDEA-D), which have highly similar catalytic domains to that of human PDEs. To date, only TbrPDEB, expressed as two slightly different isoforms TbrPDEB1 and B2, has been found to be essential when ablated. Although the genomes contain reasonably well conserved genes for catalytic and regulatory domains of protein kinase A, these have been shown to have varied structural and functional roles in the different species. Recent discovery of a role of cAMP/AMP metabolism in a quorum-sensing signaling pathway in T. brucei, and the identification of downstream cAMP Response Proteins (CARPs) whose expression levels correlate with sensitivity to PDE inhibitors, suggests a complex signaling cascade. The interplay between the roles of these novel CARPs and the quorum-sensing signaling pathway on cell division and differentiation makes for intriguing cell biology and a new paradigm in cAMP signal transduction, as well as potential targets for trypanosomatid-specific cAMP pathway-based therapeutics.

Pharmacological Validation of Trypanosoma brucei Phosphodiesterases as Novel Drug Targets

The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug targets.

Quantitative comparison of phosphodiesterase mRNA distribution in human brain and peripheral tissues

Cyclic nucleotide-specific phosphodiesterases (PDEs) play a critical role in signal transduction by regulating the level of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) in cells. The gene expression pattern of a PDE provides important information regarding its role in physiological and pathological processes. In this study, we have established the mRNA expression profile all PDE isoenzymes (PDE1A/B/C, 2A, 3A/B, 4A/B/C/D, 5A, 6A/B/C, 7A/B, 8A/B, 9A, 10A, 11A) in a human cDNA collection consisting of 10 brain regions (parietal, frontal, temporal cortex, hippocampus, striatum, thalamus, hypothalamus, substantia nigra, nucleus accumbens, cerebellum), spinal cord, dorsal root ganglia and 12 peripheral tissues (skeletal muscle, heart, thyroid, adrenal gland, pancreas, bladder, kidney, liver, lung, small intestine, spleen, and stomach). Using quantitative real-time polymerase chain reaction and parallel analysis of a carefully selected group of reference genes, we have determined the relative expression of each PDE isoenzyme across the 24 selected tissues, and also compared the expression of selected PDEs to each other within a given tissue type. Several PDEs show strikingly selective expression (e.g. PDE10A and PDE1B mRNA levels in the caudate nucleus are 20-fold higher than in most other tissues; PDE1C and PDE3A are highly expressed in the heart and PDE8B is expressed very strongly in the thyroid gland). This comprehensive approach provides a coherent and quantitative view of the mRNA expression of the PDE gene family and enables an integration of data obtained with other non-quantitative methods.

Gene Conversion Transfers the GAF-A Domain of Phosphodiesterase TbrPDEB1 to One Allele of TbrPDEB2 of Trypanosoma brucei

BackgroundChromosome 9 of Trypanosoma brucei contains two closely spaced, very similar open reading frames for cyclic nucleotide specific phosphodiesterases TbrPDEB1 and TbrPDEB2. They are separated by 2379 bp, and both code for phosphodiesterases with two GAF domains in their N-terminal moieties and a catalytic domain at the C-terminus.Methods and FindingsThe current study reveals that in the Lister427 strain of T. brucei, these two genes have undergone gene conversion, replacing the coding region for the GAF-A domain of TbrPDEB2 by the corresponding region of the upstream gene TbrPDEB1. As a consequence, these strains express two slightly different versions of TbrPDEB2. TbrPDEB2a represents the wild-type phosphodiesterase, while TbrPDEB2b represents the product of the converted gene. Earlier work on the subcellular localization of TbrPDEB1 and TbrPDEB2 had demonstrated that TbrPDEB1 is predominantly located in the flagellum, whereas TbrPDEB2 partially locates to the flagellum but largely remains in the cell body. The current findings raised the question of whether this dual localization of TbrPDEB2 may reflect the two alleles. To resolve this, TbrPDEB2 of strain STIB247 that is homozygous for TbrPDEB2a was tagged in situ, and its intracellular localization was analyzed.ConclusionsThe results obtained were very similar to those found earlier with Lister427, indicating that the dual localization of TbrPDEB2 reflects its true function and is not simply due to the presence of the two different alleles. Notably, the gene conversion event is unique for the Lister427 strain and all its derivatives. Based on this finding, a convenient PCR test has been developed that allows the stringent discrimination between Lister-derived strains that are common in many laboratories and other isolates. The technique is likely very useful to resolve questions about potential mix-ups of precious field isolates with the ubiquitous Lister strain.

Cyclic nucleotide-specific phosphodiesterases of Plasmodium falciparum: PfPDEα, a non-essential cGMP-specific PDE that is an integral membrane protein

Cyclic nucleotide-specific phosphodiesterases (PDEs) have come into focus as interesting potential targets for PDE inhibitor-based anti-parasitic drugs. Genomes of the various agents of human malaria, most notably Plasmodium falciparum, all contain four genes for class 1 PDEs. The catalytic domains of these enzymes are closely related to those of the 11 human PDE families. This presents the possibility that the available vast expertise in developing drugs against human PDEs might now also be applied to developing compounds that are active against malarial PDEs. The current study identifies four Plasmodium genes that code for PfPDEα, PfPDEβ, PfPDEγ and PfPDEδ, respectively. It further demonstrates that the PfPDEα polypeptide exists in two versions (PfPDEαA and PfPDEαB) that are generated by alternative splicing of the primary transcript. All malarial PDEs contain several transmembrane helices in their N-terminal regions, indicating that they are integral membrane proteins. In agreement with this prediction, essentially all PDE activity is associated with the cell membranes. PfPDEα was characterized as a cGMP-specific PDE that is not sensitive to a number of standard PDE inhibitors. Genetic ablation of the PfPDE1 gene produced no major phenotype in erythrocyte cultures.

TheTrypanosoma bruceicAMP phosphodiesterases TbrPDEBl and TbrPDEB2: flagellar enzymes that are essential for parasite virulence

Cyclic nucleotide specific phosphodiesterases (PDEs) are pivotal regulators of cellular signaling. They are also important drug targets. Besides catalytic activity and substrate specificity, their subcellular localization and interaction with other cell components are also functionally important. In contrast to the mammalian PDEs, the significance of PDEs in protozoal pathogens remains mostly unknown. The genome of Trypanosoma brucei, the causative agent of human sleeping sickness, codes for five different PDEs. Two of these, TbrPDEB1 and TbrPDEB2, are closely similar, cAMP-specific PDEs containing two GAF-domains in their N-terminal regions. Despite their similarity, these two PDEs exhibit different subcellular localizations. TbrPDEB1 is located in the flagellum, whereas TbrPDEB2 is distributed between flagellum and cytoplasm. RNAi against the two mRNAs revealed that the two enzymes can complement each other but that a simultaneous ablation of both leads to cell death in bloodstream form trypanosomes. RNAi against TbrPDEB1 and TbrPDEB2 also functions in vivo where it completely prevents infection and eliminates ongoing infections. Our data demonstrate that TbrPDEB1 and TbrPDEB2 are essential for virulence, making them valuable potential targets for new PDE-inhibitor based trypanocidal drugs. Furthermore, they are compatible with the notion that the flagellum of T. brucei is an important site of cAMP signaling.

Cyclic nucleotide specific phosphodiesterases of Leishmania major

BackgroundLeishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major.ResultsThis paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation.ConclusionThe genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.

Characterization of the First Potent and Selective PDE9 Inhibitor Using a cGMP Reporter Cell Line

We report here the in vitro characterization of 1-(2-chlorophenyl)-6-[(2R)-3,3,3-trifluoro-2-methylpropyl]-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidine-4-one (BAY 73-6691), the first potent and selective inhibitor of phosphodiesterase 9 (PDE9), which is currently under preclinical development for the treatment of Alzheimer's disease. This compound selectively inhibits human (IC50 = 55 nM) and murine (IC50 = 100 nM) PDE9 activity in vitro and shows only moderate activity against other cyclic nucleotide-specific phosphodiesterases. We also report the generation and characterization of a stably transfected PDE9 Chinese hamster ovary cell line, additionally expressing soluble guanylate cyclase (sGC), the olfactory cyclic nucleotide-gated cation channel CNGA2 and the photoprotein aequorin. In this cell line, intracellular cGMP levels can be monitored in real-time via aequorin luminescence induced by Ca2+ influx through CNGA2, acting as the intracellular cGMP sensor. This simple and sensitive assay system was used for the characterization of the cellular activity of the new PDE9 inhibitor. BAY 73-6691 alone did not significantly increase basal cGMP levels in this experimental setting. However, in combination with submaximal stimulating concentrations of the sGC activator 4-[((4-carboxybutyl)[2-[(4-phenethyl-benzyl)oxy]phenethyl]amino)methyl] benzoic acid (BAY 58-2667), the compound induced concentration-dependent luminescence signals and intracellular cGMP accumulation. The PDE9 inhibitor significantly potentiated the cGMP signals generated by sGC activating compounds such as BAY 58-2667 or 5-cyclopropyl-2-[1-(2-fluorobenzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]pyrimidin-4-ylamine (BAY 41-2272) and induced leftward shifts of the corresponding concentration-response curves. Using our newly generated PDE9 reporter cell line, we could show that BAY 73-6691 is able to efficiently penetrate cells and to inhibit intracellular PDE9 activity.

TbPDE1, a novel class I phosphodiesterase of Trypanosoma brucei

Cyclic nucleotide specific phosphodiesterases (PDEs) are important components of all cAMP signalling networks. In humans, 11 different PDE families have been identified to date, all of which belong to the class I PDEs. Pharmacologically, they have become of great interest as targets for the development of drugs for a large variety of clinical conditions. PDEs in parasitic protozoa have not yet been extensively investigated, despite their potential as antiparasitic drug targets. The current study presents the identification and characterization of a novel class I PDE from the parasitic protozoon Trypanosoma brucei, the causative agent of human sleeping sickness. This enzyme, TbPDE1, is encoded by a single-copy gene located on chromosome 10, and it functionally complements PDE-deficient strains of Saccharomyces cerevisiae. Its C-terminal catalytic domain shares about 30% amino acid identity, including all functionally important residues, with the catalytic domains of human PDEs. A fragment of TbPDE1 containing the catalytic domain could be expressed in active form in Escherichia coli. The recombinant enzyme is specific for cAMP, but exhibits a remarkably high Km of > 600 microm for this substrate.

African trypanosomiasis and drug development

African sleeping sickness in humans and Nagana in cattle are caused by the protozoan parasite Trypanosoma brucei. The chemotherapy available for these infections is limited and causes serious side effects to their mammalian hosts. Of the four available drugs used in treatment, three of them were developed >50 years ago. With the emergence of drug-resistant strains of T. brucei, it has become more imperative that new drug targets are identified. To this end, a recent study by R. Zoraghi and T. Seebeck identify a potential metabolic window for intervention in trypanosomiasis [1xThe cAMP-specific phosphodiesterase TbPDE2C is an essential enzyme in bloodstream form of Trypanosoma brucei. Zoraghi, R. and Seebeck, T. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 4343–4348Crossref | PubMed | Scopus (54)See all References][1]. Previous studies have identified cyclic nucleotide-specific phosphodiesterases as candidate drug targets. To explore this possibility, a cAMP-specific phosphodiesterase from T. brucei was characterized called TbPDE2C. Chemical inhibitors targeted against this enzyme are capable of inhibiting enzymatic activity and blocking the proliferation of the bloodstream form (human-infective stage) of the parasite in vitro. Ablation of TbPDE2C mRNA expression by RNA interference demonstrated that TbPDE2C is essential for maintaining intracellular cAMP levels, and loss of TbPDE2C led to increased intracellular levels of cAMP. The bloodstream form of the parasite is sensitive to the elevated levels of cAMP. This increased concentration of cAMP leads to disruption of the nuclear membrane and cell division, and ultimately gives rise to death. These in vitro studies identifying TbPDE2C as a potential target against trypanosomiasis should be followed with animal studies of inhibitors against this enzyme. Simultaneously, crystallization of the protein and subsequent analysis of its three dimensional structure should stimulate further drug development.

The cAMP-specific phosphodiesterase TbPDE2C is an essential enzyme in bloodstream form Trypanosoma brucei.

Chemotherapy of human sleeping sickness, a fatal disease caused by the protozoan parasite Trypanosoma brucei, is in a dismal state, and the identification and characterization of new drug targets is an urgent prerequisite for an improvement of the dramatic situation in the field. Over the last several years, inhibitors of cyclic nucleotide-specific phosphodiesterases have proven to be highly successful drug candidates for an assortment of clinical conditions. Their potential as antiparasitic drugs has not been explored so far. This study reports the characterization of a cAMP-specific phosphodiesterase from T. brucei, TbPDE2C. This enzyme is a class I phosphodiesterase, and it is a member of a small enzyme family in T. brucei, TbPDE2. Inhibitors of this enzyme block the proliferation of bloodstream form trypanosomes in culture. RNA interference experiments demonstrated that the TbPDE2 family, and in particular TbPDE2C, are essential for maintaining intracellular cAMP concentrations within a physiological range. Bloodstream form trypanosomes are exquisitely sensitive to elevated concentrations of intracellular cAMP, and a disruption of TbPDE2C function quickly leads to the disruption of nuclear and cellular cell division, and to cell death. TbPDE2C might represent a novel drug target for the development of new and effective trypanocidal drugs.

Identification and characterization of a novel cyclic nucleotide phosphodiesterase gene (PDE9A) that maps to 21q22.3: alternative splicing of mRNA transcripts, genomic structure and sequence.

Cyclic nucleotide-specific phosphodiesterases (PDEs) play an essential role in signal transduction by regulating the intracellular concentration of second messengers (cAMP and cGMP). We have identified and made an initial characterization of a full-length cDNA encoding a novel human cyclic nucleotide phosphodiesterase, PDE9A. At least four different mRNA transcripts (PDE9A1, A2, A3, A4) are produced as a result of alternative splicing of 5' exons, potentially changing the N-terminal amino acid sequences of the encoded proteins. All these predicted proteins would contain a 3',5'-cyclic nucleotide phosphodiesterase signature motif (Prosite no. PDOC00116). Northern blot analysis revealed several mRNA species of approximately 2.4 kb with varying expression patterns and intensities in most tissues examined, except blood. We have also isolated the mouse homolog of the human PDE9A2 mRNA transcript, pde9A2. The human and mouse isoforms have 93 and 83% sequence identity at the amino acid and nucleotide levels, respectively. PDE9A was mapped to 21q22.3, between TFF1 and D21S360. Comparison of the PDE9A1 cDNA with the genomic sequence from the region revealed that the gene is split into 20 exons that extend over 122 kb. Comparison of the physical map of the region and the genomic sequence further refines the mapping, with D21S113 being derived from intron 15. Several genetic disorders map to 21q22.3, including one form of bipolar affective disorder. Since functional disturbances in intraneuronal signal transmission via second messengers play an important role in the pathophysiology of affective disorders, PDE9A is a strong candidate for such a role by position and function.