Investigation of PDE5/PDE6 and PDE5/PDE11 selective potent tadalafil-like PDE5 inhibitors using combination of molecular modeling approaches,molecular fingerprint-based virtual screening protocols and structure-based pharmacophore development

The essential biological function of phosphodiesterase (PDE) type enzymes is to regulate the cytoplasmic levels of intracellular second messengers, 3′,5′-cyclic guanosine monophosphate (cGMP) and/or 3′,5′-cyclic adenosine monophosphate (cAMP). PDE targets have 11 isoenzymes. Of these enzymes, PDE5 has attracted a special attention over the years after its recognition as being the target enzyme in treating erectile dysfunction. Due to the amino acid sequence and the secondary structural similarity of PDE6 and PDE11 with the catalytic domain of PDE5, first-generation PDE5 inhibitors (i.e. sildenafil and vardenafil) are also competitive inhibitors of PDE6 and PDE11. Since the major challenge of designing novel PDE5 inhibitors is to decrease their cross-reactivity with PDE6 and PDE11, in this study, we attempt to identify potent tadalafil-like PDE5 inhibitors that have PDE5/PDE6 and PDE5/PDE11 selectivity. For this aim, the similarity-based virtual screening protocol is applied for the “clean drug-like subset of ZINC database” that contains more than 20 million small compounds. Moreover, molecular dynamics (MD) simulations of selected hits complexed with PDE5 and off-targets were performed in order to get insights for structural and dynamical behaviors of the selected molecules as selective PDE5 inhibitors. Since tadalafil blocks hERG1 K channels in concentration dependent manner, the cardiotoxicity prediction of the hit molecules was also tested. Results of this study can be useful for designing of novel, safe and selective PDE5 inhibitors.     

Cartilage cyclic nucleotide phosphodiesterase: inhibition by anti-inflammatory agents

Soluble 3′,5′-nucleotide phosphodiesterase (PDE) activity is described in chicken epiphyseal and articular cartilage. Kinetic studies of these enzymes demonstrate a high and low K_m for the substrates, adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Epiphyseal and articular PDE activities are inhibited by those anti-inflammatory agents which are potent inhibitors of the enzyme, prostaglandin synthetase (PS). Specificity of this inhibition is indicated by the activity of these agents against the low K_m enzyme. Other anti-inflammatory agents with significantly less potency as PS inhibitors or with no activity against prostaglandin synthetase are found to be either inactive or relatively less potent as inhibitors of cartilage PDE activity. A variety of other anti-inflammatory or anti-rheumatic agents, which are not known to affect prostaglandin synthetase activity, are poor inhibitors of cartilage PDE activity. These data provide insight into the mechanism of action of certain anti-inflammatory agents and into the relationships between prostaglandins and inflammatory reactions.     

cAMP AND cAMP-STIMULATED PROTEIN PHOSPHORYLATION IN ZOOSPORES OF BROWN ALGAE1

Adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP) were detected at concentrations of 8–11 and 10–20 pmol · mg^?1 protein, respectively, in zoospores of a brown alga, Undaria pinnatifida (Harvey) Suringer. Cellular levels of these cyclic nucleotides did not substantially change during dark to light transition. cAMP-stimulated protein phosphorylation was found in soluble cell-free extracts of zoospores of Undaria pinnatifida and Laminaria angustata Kjellman.     

Nitric oxide and downstream second messenger cGMP and cAMP enhance adipogenesis in primary human preadipocytes

Background aimsObesity is correlated with chronic low-grade inflammation. Thus the induction of inflammation could be used to stimulate adipose tissue formation in tissue-engineering approaches. As nitric oxide (NO) is a key regulator of inflammation, we investigated the effect of NO and its downstream signaling molecule guanosine 3′,5′-cyclic monophosphate (cGMP) as well as adenosine 3′,5′-cyclic monophosphate (cAMP) on preadipocytes in vitro.MethodsPreadipocytes were isolated from human subcutaneous adipose tissue, cultured until confluence, and differentiated. The NO donor diethylenetriamine (DETA)/NO (30–150 μm) was added during proliferation and differentiation. Additionally, cGMP/cAMP analogs 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP), 8-(4-chlorophenylthio)-guanosine 3′,5′-cyclic monophosphate (8-pCPT-cGMP) and 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP), and the adenylyl cyclase activator forskolin, specific guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and adenylyl cyclase inhibitor 2′-5′-dideoxyadenosine (ddA), were applied. Proliferation and differentiation were evaluated.ResultsDETA/NO in combination with the standard differentiation procedure significantly enhanced maturation of precursor cells to adipocytes. Proliferation, in contrast, was inhibited in the presence of NO. The application of cGMP and cAMP, respectively, increased pre-adipocyte differentiation to an even higher extent than NO. Inhibitors of the underlying pathways caused a significant decrease in adipogenic conversion.ConclusionsOur results support the application of NO donors during transplantation of preadipocytes in a 3-dimensional setting to accelerate and optimize differentiation. The results suggest that, instead of the rather instable and reactive molecule NO, the application of cGMP and cAMP would be even more effective because these substances have a stronger adipogenic effect on preadipocytes and a longer half-life than NO. Also, by applying inhibitors of the underlying pathways, the induced inflammatory condition could be regulated to the desired level.     

Adenosine 3' , 5'-cyclic monophsphate phosphodiesterase activities in the x-irradiation induced rat small bowel adenocarcinoma.

The adenosine 3′, 5′-cyclic monophosphate phosphodiesterase (PDE) activities were evaluated in X-irradiation induced Holtzman rat small bowell adenocarcinoma and age-matched normal small intestine. Within normal small intestine, PDE activity was optimal at pH 7.4, and highly dependent upon the addition of Mg²⁺ or Mn²⁺. Analyses of the rat small bowel adenocarcinoma revealed significantly elevated PDE activities above the normal small bowel which were found to be relatively constant throughout the length of the ileum and jejunum. These findings suggest that the diminished intracellular adenosine 3′, 5′-cyclic monophosphate levels observed in this lesion (1) may be the consequence of elevated PDE activities.     

In silico investigation of PARP-1 catalytic domains in holo and apo states for the design of high-affinity PARP-1 inhibitors

The rational design of high-affinity inhibitors of poly-ADP-ribose polymerase-1 (PARP-1) is at the heart of modern anti-cancer drug design. While relevance of enzyme to DNA repair processes in cellular environment is firmly established, the structural and functional understanding of the main determinants for high-affinity ligands controlling PARP-1 activity is still lacking. The conserved active site of PARP-1 represents an ideal target for inhibitors and may offer a novel target at the treatment of breast cancer. To fill the gap in the structural knowledge, we report on the combination of molecular dynamics (MD) simulations, principal component analysis (PCA), and conformational analysis that analyzes in great details novel binding mode for a number of inhibitors at the PARP-1. While optimization of the binding affinity for original target is an important goal in the drug design, many of the promising molecules for treatment of the breast cancer are plagued by significant cardiotoxicity. One of the most common side-effects reported for a number of polymerase inhibitors is its off-target interactions with cardiac ion channels and hERG1 channel, in particular. Thus, selected candidate PARP-1 inhibitors were also screened in silico at the central cavities of hERG1 potassium ion channel.     

Detection of a 3′, 5′-cyclic-AMP phosphodiesterase activity in the lichenized fungus Evernia prunastri

Abstract

A 3′, 5′-cyclic-AMP phosphodiesterase (PDE) was detected and measured in the lichen Evernia prunastri. The percentage of hydrolysis of tritiated 3′, 5′-cyclic-adenosine monophosphate ([³H]-cAMP) and 3′, 5′-cyclic-guanosine monophosphate ([³H]-cGMP) by the PDE enzyme into tritiated 5′-adenosine-monophospahte ([³H]-AMP) and tritiated 5′-guanosine-monophospahte ([³H]-GMP) was measured by treating the PDE products with a 5′-nucleotidase enzyme present in snake venom. The lysate fraction (L) (plasma membranes and cell walls) and the supernatant (S) (soluble fraction of the cells) were tested. In both fractions, competition of unlabelled cAMP, but not unlabelled cGMP, was revealed. Specific competitive PDE inhibitors such as IBMX inhibited enzymatic activity. Although it is thought that in this species cAMP is regulated by red/far red light through PDE activity, this is the first report that seems to suggest the presence of a PDE activity specific for cAMP in lichenized fungi. However, this work is at a preliminary stage and despite the high levels of enzymatic activity with cAMP found in both fractions, data are still insufficient to state the absolute specificity for this nucleotide.     

Role of phosphodiesterase 2 in growth and invasion of human malignant melanoma cells

Cyclic nucleotide phosphodiesterases (PDEs) regulate the intracellular concentrations and effects of adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The role of PDEs in malignant tumor cells is still uncertain. The role of PDEs, especially PDE2, in human malignant melanoma PMP cell line was examined in this study. In PMP cells, 8-bromo-cAMP, a cAMP analog, inhibited cell growth and invasion. However, 8-bromo-cGMP, a cGMP analog, had little or no effect. PDE2 and PDE4, but not PDE3, were expressed in PMP cells. Growth and invasion of PMP cells were inhibited by erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), a specific PDE2 inhibitor, but not by rolipram, a specific PDE4 inhibitor. Moreover, cell growth and invasion were inhibited by transfection of small interfering RNAs (siRNAs) specific for PDE2A and a catalytically-dead mutant of PDE2A. After treating cells with EHNA or rolipram, intracellular cAMP concentrations were increased. Growth and invasion were stimulated by PKA14-22, a PKA inhibitor, and inhibited by N⁶-benzoyl-c AMP, a PKA specific cAMP analog, whereas 8-(4-chlorophenylthio)-2′-O-methyl-cAMP, an Epac specific cAMP analog, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell line. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines.     

Pharmacophore elucidation and molecular docking studies on phosphodiesterase-5 inhibitors

cGMP-binding cGMP-specific PDE, PDE5 plays a key role in the hydrolysis of cyclic guanidine monophosphate. Because cGMP mediates vascular functions, a PDE5 inhibitor that elevates cGMP level is an attractive means for vasodilatation and treatment of erectile dysfunction. In this paper we report the elucidation of the common pharmacophore hypothesis of different classes of PDE5 inhibitors. Using LigandScout program, pharmacophore modelling studies were performed on prior reported potent PDE5 inhibitors with a variety of scaffolds in order to identify one common set of critical chemical features of these PDE5 inhibitors 1-52. The best pharmacophore model, model-1, characterized by four chemical features: one aromatic ring, one hydrophobe, one hydrogen acceptors and one hydrogen donor. Using Dock6 program, docking studies were performed in order to investigate the mode of binding of these compounds. The molecular docking study allowed confirming the preferential binding mode of different classes of PDE5 inhibitors inside the active site. The obtained binding mode was as same as that of vardenafil, X-ray ligand with different orientation with varied PDE5 inhibitors׳ scaffold.     

Cyclic nucleotide esterification: relationship to phosphate ring geometry and growth regulation in synchronized human lymphocytes.

Infrared spectra of neutral aqueous solutions of nucleoside 3′,5′-cyclic monophosphates indicate an increase in the antisymmetric phosphoryl stretching frequency to 1236 cm^?1 from 1215 cm^?1 in trimethylene cyclic phosphates. A further increase to 1242 cm^?1 accompanies esterification of the 2′-ribose hydroxyl. The O²′-esterified and 2′-deoxy cyclic nucleotides examined display both reduced kinase binding and altered phosphoryl stretching frequencies, suggesting that modification of the phosphate ring represents a common feature in decreased kinase activation. Reversible inhibition of mitosis in thymidine-synchronized human lymphocytes by 2 mmN⁶,O²′-dibutyryladenosine 3′,5′-cyclic monophosphate and N⁶-monobutyryladenosine 3′,5′-cyclic monophosphate was observed. However, adenosine 3′,5′-cyclic monophosphate, O²′-monobutyryladenosine 3′,5′-cyclic monophosphate, butyric acid, and ethyl butyrate had no effect on mitosis when present at 2 mm concentrations during S and G2. These results are consistent with hydrolysis of O²′-monobutyryladenosine 3′,5′-cyclic monophosphate and adenosine 3′,5′-cyclic monophosphate by esterase and phosphodiesterase enzymes and suggest that modification of the N⁶ amino group is necessary for the antimitotic activity of N⁶,O²′-dibutyryladenosine 3′, 5′-cyclic monophosphate.     

Forskolin and 3-Isobutyl-l-Methylxanthine Increase Basal and Sodium Nitroprusside-Elevated Cyclic GMP Levels in Adult Guinea-Pig Cerebellar Slices

Abstract: In this study, the interaction between 3′,5′-cyclic adenosine monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate (cGMP) in [³H]adenine-or [³H]-guanine-prelabelled adult guinea-pig cerebellar slices was investigated. Basal levels of [³H]cGMP were enhanced by forskolin, although no plateau was reached over the concentration range tested (0.1-100 μM). However, forskolin elicited a concentration-dependent, saturable potentiation of sodium nitroprusside (SNP)-stimulated [³H]cGMP accumulation (forskolin EC₅₀ value of 0.98 β 0.23 μM; 10 μM forskolin produced a 1.8 β 0.3-fold potentiation of the SNP response at 2.5 min). The forskolin potentiation was observed at all concentrations of SNP tested (0.001-10 mM). forskolin also elicited a large stimulation of [³H]-cAMP in [³H]adenine-prelabelled guinea-pig cerebellar slices; however, 1,9-dideoxyforskolin failed to elicit either a [³H]cAMP response or a potentiation of the SNP-induced [³H]cGMP response at concentrations up to 100 μM. Pretreatment with oxyhaemoglobin (50 μM) inhibited the response to SNP (1 mM) and forskolin (10 μM), as well as the response evoked by the combination of SNP and forskolih. A^G-Nitro-l -arginine (100 μM) inhibited the response to forskolin alone, but did not change the response to SNP or the potentiation induced by forskolin on SNP-induced [³H]cGMP levels. The protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7; 100 μM), staurosporine (10 μM), polymyxin B (100 μM), and Ro 31-8220 (10 μM) had no effect on the [³H]cGMP response to either SNP or the combination of SNP plus forskolin. N⁶,2′-Dibutyryl cAMP, at concentrations up to 10 mM, was also without effect on [³H]cGMP levels induced by SNP. 3-lso-butyl-1-methylxanthine reproduced the effect of forskolin on SNP-induced [³H]cGMP levels, but a less-than-additive effect was observed when the response to SNP was studied in the presence of forskolin and 3-isobutyl-1-methylxanthine. Taken together, these results infer that crosstalk between cyclic nucleotides takes place in guinea-pig cerebellar slices, and that cAMP may regulate cGMP-mediated responses in this tissue.     

CHANGE IN THE ACTIVITY OF LIPASE IN SEA URCHIN EGGS AND EMBRYOS DURING EARLY DEVELOPMENT*

During the early development of the sea urchin, Anthocidaris crassispina, the activity of lipase was maintained at the same level as in unfertilized eggs until the mesenchymal blastula stage (20 hr culture at 20°C) and then increased gradually after gastrulation. The activity in the embryos kept in SO^2?₄-free artificial sea water changed in a similar manner to that in those kept in normal sea water, during the development until 36 hr of fertilization. At 48 hr, the activity in the embryos, which had developed to the permanent blastulae in SO^2?₄-free sea water, was markedly lower than in normal plutei and was similar to that in unfertilized eggs. The lipase activity in fertilized eggs 30 min after fertilization, which was almost the same as that in unfertilized eggs was found mainly to be localized in the precipitate fraction obtained by the centrifugation at 12,000 x g for 20 min, whereas the activity in unfertilized eggs was found in the precipitate by the centrifugation at 105,000 x g for 60 min. Ca²⁺, adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) had no effect on the lipase activity.     

Effect of O2-monobutyryl guanosine 3', 5'-cyclic monophosphate on hepatic metabolism of free fatty acids.

Livers from fed male rats were perfused $\text{in vitro}$ with O^2′-monobutyryl guanosine 3′,5′-cyclic monophosphate. The output of triglyceride was reduced, while output of ketone bodies and glucose was stimulated by 10^?4M monobutyryl guanosine 3′,5′-cyclic monophosphate. No effect was observed with 10^?5 M nucleotide. Monobutyryl guanosine 3′,5′-cyclic monophosphate did not affect uptake of free fatty acids. In these respects, monobutyryl guanosine 3′,5′-cyclic monophosphate mimics the effects of dibutyryl adenosine 3′,5′-cyclic monophosphate, although the guanylic nucleotide seems to be less potent than the adenosine 3′,5′-cyclic monophosphate derivative.     

Phosphodiesterase 5 inhibitors prevent 3,4-methylenedioxymethamphetamine-induced 5-HT deficits in the rat

Phosphodiesterase 5 (PDE5) inhibitors are often used in combination with club drugs such as 3,4‐methylenedioxymethamphetamine (MDMA or ecstasy). We investigated the consequences of such combination in the serotonergic system of the rat. Oral administration of sildenafil citrate (1.5 or 8 mg/kg) increased brain cGMP levels and protected in a dose‐dependent manner against 5‐hydroxytryptamine depletions caused by MDMA (3 × 5 mg/kg, i.p., every 2 h) in the striatum, frontal cortex and hippocampus without altering the acute hyperthermic response to MDMA. Intrastriatal administration of the protein kinase G (PKG) inhibitor, KT5823 [(9S, 10R, 12R)‐2,3,9,10,11,12‐Hexahydro‐10‐methoxy‐2,9‐dimethyl‐1‐oxo‐9,12‐epoxy‐1H‐diindolo[1,2,3‐fg:3′,2′,1′‐kl]pyrrolo[3,4‐i][1,6]benzodiazocine‐10‐carboxylic acid, methyl ester)], suppressed sildenafil‐mediated protection. By contrast, the cell permeable cGMP analogue, 8‐bromoguanosine cyclic 3′,5′‐monophosphate, mimicked sildenafil effects further suggesting the involvement of the PKG pathway in mediating sildenafil protection. Because mitochondrial ATP‐sensitive K⁺ channels are a target for PKG, we next administered the specific mitochondrial ATP‐sensitive K⁺ channel blocker, 5‐hydroxydecanoic acid, 30 min before sildenafil. 5‐hydroxydecanoic acid completely reversed the protection afforded by sildenafil, thereby implicating the involvement of mitochondrial ATP‐sensitive K⁺ channels. Sildenafil also increased Akt phosphorylation, and so the possible involvement of the Akt/endothelial nitric oxide synthase (eNOS)/sGC signalling pathway was analysed. Neither the phosphatidylinositol 3‐kinase inhibitor, wortmannin, nor the selective eNOS inhibitor, l ‐N5‐(1‐iminoethyl)‐l ‐ornithine dihydrochloride, reversed the protection afforded by sildenafil, suggesting that Akt/eNOS/sGC cascade does not participate in the protective mechanisms. Our data also show that the protective effect of sildenafil can be extended to vardenafil, another PDE5 inhibitor. In conclusion, sildenafil protects against MDMA‐induced long‐term reduction of indoles by a mechanism involving increased production of cGMP and subsequent activation of PKG and mitochondrial ATP‐sensitive K⁺ channel opening.     

Identification of novel PDEδ interacting proteins

Prenylation is a post-translational modification that increases the affinity of proteins for membranes and mediates protein-protein interactions. The retinal rod rhodopsin-sensitive cGMP 3′,5′-cyclic phosphodiesterase subunit delta (PDEδ) is a prenyl binding protein that is essential for the shuttling of small GTPases between different membrane compartments and, thus, for their proper functioning. Although the prenylome comprises up to 2% of the mammalian proteome, only few prenylated proteins are known to interact with PDEδ. A proteome-wide approach was employed to map the PDEδ interactome among the prenylome and revealed RAB23, CDC42 and CNP as novel PDEδ interacting proteins. Moreover, PDEδ associates with the lamin A mutant progerin in a prenyl-dependent manner. These findings shed new light on the role of PDEδ in binding (and regulating) prenylated proteins in cells.     

Triazole‐Pyridine Dicarbonitrile Targets Phosphodiesterase 4 to Induce Cytotoxicity in Lung Carcinoma Cells

Phosphodiesterase 4 (PDE4) is a key enzyme involved in the hydrolysis of cyclic adenosine monophosphate (cAMP) and widely expressed in several types of cancers. The inhibition of PDE4 results in an increased concentration of intracellular cAMP levels that imparts the anti‐inflammatory response in the target cells. In the present report, two series of triazolo‐pyridine dicarbonitriles and substituted dihydropyridine dicarbonitriles were synthesized using green protocol (TBAB in refluxed water). We next evaluated the title compounds for their cytotoxicity towards lung cancer (A549) cells and identified 7′‐[4‐(methylsulfonyl)phenyl]‐5′‐oxo‐1′,5′‐dihydrospiro[cyclohexane‐1,2′‐[1,2,4]triazolo[1,5‐a]pyridine]‐6′,8′‐dicarbonitrile ( 5h ) and 7′‐(1‐methyl‐1H‐imidazol‐2‐yl)‐5′‐oxo‐1′,5′‐dihydrospiro[cyclohexane‐1,2′‐[1,2,4]triazolo[1,5‐a]pyridine]‐6′,8′‐dicarbonitrile ( 5j ) as lead analogs with the IC₅₀ values of 15.2 and 24.1 μm , respectively. Furthermore, all the new compounds were tested for PDE4 inhibitory activity and 5j showed relatively good inhibitory activity towards PDE4 with inhibition of 50.9 % at 10 μm . In silico analysis demonstrated the favorable interaction of the title compounds with the target enzyme. Taken together, the present study introduces a new scaffold for the development of novel PDE4 inhibitors to fight against inflammatory diseases.     

A new nonhydrolyzable reactive cGMP analogue, (Rp)-guanosine-3',5'-cyclic-S-(4-bromo-2,3-dioxobutyl)monophosphorothioate, which targets the cGMP binding site of human platelet PDE3A

The amino acids involved in substrate (cAMP) binding to human platelet cGMP-inhibited cAMP phosphodiesterase (PDE3A) are identified. Less is known about the inhibitor (cGMP) binding site. We have now synthesized a nonhydrolyzable reactive cGMP analog, Rp-guanosine-3′,5′-cyclic-S-(4-bromo-2, 3-dioxobutyl)monophosphorothioate (Rp-cGMPS-BDB). Rp-cGMPS-BDB irreversibly inactivates PDE3A (K_I = 43.4 ± 7.2 μM and k_cart = 0.007 ± 0.0006 min⁻¹). The effectiveness of protectants in decreasing the rate of inactivation by Rp-cGMPS-BDB is: Rp-cGMPS (K_d = 72 μM) > Sp-cGMPS (124), Sp-cAMPS (182) > GMP (1517), Rp-cAMPS (3762), AMP (4370 μM). NAD⁺, neither a substrate nor an inhibitor of PDE3A, does not protect. Nonhydrolyzable cGMP analogs exhibit greater affinity than the cAMP analogs. These results indicate that Rp-cGMPS-BDB targets favorably the cGMP binding site consistent with a docking model of PDE3A-Rp-cGMPS-BDB active site. We conclude that Rp-cGMPS-BDB is an effective active site-directed affinity label for PDE3A with potential for other cGMP-dependent enzymes.     

Isolation and characterization of cDNAs encoding PDE5A,a human cGMP-binding,cGMP-specific 3′,5′-cyclic nucleotide phosphodiesterase

Human cGMP-binding, cGMP-specific 3′,5′-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100 012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5′-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25–27.     

Allosteric-site and catalytic-site ligand effects on PDE5 functions are associated with distinct changes in physical form of the enzyme

Native phosphodiesterase-5 (PDE5) homodimer contains distinct non-catalytic cGMP allosteric sites and catalytic sites for cGMP hydrolysis. Purified recombinant PDE5 was activated by pre-incubation with cGMP. Relatively low concentrations of cGMP produced a Native PAGE gel shift of PDE5 from a single band position (lower band) to a band with decreased mobility (upper band); higher concentrations of cGMP produced a band of intermediate mobility (middle band) in addition to the upper band. Two point mutations (G659A and G659P) near the catalytic site that reduced affinity for cGMP substrate retained allosteric cGMP-binding affinity like that of WT PDE5 but displayed cGMP-induced gel shift only to the middle-band position. The upper band could represent a form produced by cGMP binding to the catalytic site, while the middle band could represent a form produced by cGMP binding to the allosteric site. Millimolar cGMP was required for gel shift of PDE5 when added to the pre-incubation before Native PAGE, presumably due to removal of most of the cGMP during electrophoresis, but micromolar cGMP was sufficient for this effect if cGMP was included in the native gel buffer. cGMP-induced gel shift was associated with stimulation of PDE5 catalytic activity, and the rates of onset and reversibility of this effect suggested that it was due to cGMP binding to the allosteric site. Incubation of PDE5 with non-hydrolyzable, catalytic site-specific, substrate analogs such as the inhibitors sildenafil and tadalafil, followed by dilution, did not produce activation of catalytic activity like that obtained with cGMP, although both inhibitors produced a similar gel shift to the upper band as that obtained with cGMP. This implied that occupation of the catalytic site alone can produce a gel shift to the upper band. PDE5 activation or gel shift was reversed by lowering cGMP with dilution followed by at least 1 h of incubation. Such slow reversibility could prolong effects of cGMP on PDE5 in cells after decline of this nucleotide. Reversal was also achieved by Mg⁺⁺ addition to the pre-incubation mixture to promote cGMP degradation, but Mg⁺⁺ addition did not reverse the gel shift caused by sildenafil, which is not hydrolyzed by PDE5. Upon extensive dilution, the effect of tadalafil, a potent PDE5 inhibitor, to enhance catalytic-site affinity for this inhibitor was rapidly reversed. Thus, kinetic effect of binding of a high-affinity PDE5 inhibitor to the catalytic site is more readily reversible than that obtained by cGMP binding to the allosteric site. It is concluded that cGMP or PDE5 inhibitor binding to the catalytic site, or ligand binding to both the catalytic site and allosteric site simultaneously, changes PDE5 to a similar physical form; this form is distinct from that produced by cGMP binding to the allosteric site, which activates the enzyme and reverses more slowly.     

Exploring binding modes of the selected inhibitors to phosphodiesterase delta by all-atom molecular dynamics simulations and free energy calculations

It’s favorable to alter KRas mutation’s location to endomembrane by interfering the binding of PDEδ (the prenyl-binding protein phosphodiesterase delta) to KRas. In the present work, the binding of four inhibitors (Deltarasin, allyl analogue, pyrazolopyridazinone derivative, and Deltazinone 1) to PDEδ is investigated with all-atom Molecular Dynamic (MD) simulations. The binding free energy calculation results reveal that van der Waals (VDW) energy provides the major force for affinity binding. Moreover, the binding energy decomposition indicates that residues R61 and I129 provide important contributions to binding energies in all systems. The conserved hydrogen bonds play crucial roles in anchoring the inhibitors to the exact site for binding. The results for conformational analysis of PDEδ/free and PDEδ/inhibitors systems show that the structures are more stable after the inhibitors’ binding to the PDEδ. It is also found that the most unstable system among four complexes is PDEδ/pyrazolopyridazinone derivative system whose α3-helix formed by the residues P113-Q116 disappears. This study may provide valuable information for the design of high potency PDEδ inhibitors.

Communicated by Ramaswamy H. Sarma