Recombinant Phosphodiesterase Research Articles

Treatment of Cardiovascular Dysfunction with PDE3-Inhibitors in Moderate and Severe Hypothermia-Effects on Cellular Elimination of Cyclic Adenosine Monophosphate and Cyclic Guanosine Monophosphate.

Introduction: Rewarming from accidental hypothermia is often complicated by hypothermia-induced cardiovascular dysfunction, which could lead to shock. Current guidelines do not recommend any pharmacological treatment at core temperatures below 30°C, due to lack of knowledge. However, previous in vivo studies have shown promising results when using phosphodiesterase 3 (PDE3) inhibitors, which possess the combined effects of supporting cardiac function and alleviating the peripheral vascular resistance through changes in cyclic nucleotide levels. This study therefore aims to investigate whether PDE3 inhibitors milrinone, amrinone, and levosimendan are able to modulate cyclic nucleotide regulation in hypothermic settings. Materials and methods: The effect of PDE3 inhibitors were studied by using recombinant phosphodiesterase enzymes and inverted erythrocyte membranes at six different temperatures—37°C, 34°C, 32°C, 28°C, 24°C, and 20°C- in order to evaluate the degree of enzymatic degradation, as well as measuring cellular efflux of both cAMP and cGMP. The resulting dose-response curves at every temperature were used to calculate IC50 and Ki values. Results: Milrinone IC50 and Ki values for cGMP efflux were significantly lower at 24°C (IC50: 8.62 ± 2.69 µM) and 20°C (IC50: 7.35 ± 3.51 µM), compared to 37°C (IC50: 22.84 ± 1.52 µM). There were no significant changes in IC50 and Ki values for enzymatic breakdown of cAMP and cGMP. Conclusion: Milrinone, amrinone and levosimendan, were all able to suppress enzymatic degradation and inhibit extrusion of cGMP and cAMP below 30°C. Our results show that these drugs have preserved effect on their target molecules during hypothermia, indicating that they could provide an important treatment option for hypothermia-induced cardiac dysfunction.

The neuroprotective and antidepressant-like effects of Hcyb1, a novel selective PDE2 inhibitor.

Depression is currently the most common mood disorder. Regulation of intracellular cyclic adenosine monophosphate (cAMP) and/or cyclic guanosine monophosphate (cGMP) signaling by phosphodiesterase (PDE) inhibition has been paid much attention for treatment of depression. This study aimed to investigate the neuroprotective effects of Hcyb1, a novel PDE2 inhibitor, in HT-22 cells and antidepressant-like effects in mouse models of depression. Hcyb1 was synthesized and its selectivity upon PDE2 was tested. Moreover, HT-22 hippocampal cells were used to determine the effects of Hcyb1 on cell viability, cyclic nucleotide levels, and the downstream molecules related to cAMP/cGMP signaling by neurochemical, enzyme-linked immunosorbent, and immunoblot assays in vitro. The antidepressant-like effects of Hcyb1 were also determined in the forced swimming and tail suspension tests in mice. Hcyb1 had a highly selective inhibition of PDE2A (IC50 =0.57±0.03μmol/L) and over 250-fold selectivity against other recombinant PDE family members. Hcyb1 at concentrations of 10-10 and 10-9 mol/L significantly increased cell viability after treatment for 24hours. At concentrations of 10-9 ~10-7 mol/L, Hcyb1 also increased cGMP levels by 1.7~2.3 folds after 10-minute treatment. Furthermore, Hcyb1 at the concentrations of 10-9 mol/L increased both cGMP and cAMP levels 24hours after treatment. The levels of phosphorylation of CREB and BDNF were also increased by Hcyb1 treatment in HT-22 cells for 24hours. Finally, in the in vivo tests, Hcyb1 (0.5, 1, and 2mg/kg, i.g.) decreased the immobility time in both forced swimming and tail suspension tests, without altering locomotor activity. These results suggest that the novel PDE2 inhibitor Hcyb1 produced neuroprotective and antidepressant-like effects most likely mediated by cAMP/cGMP-CREB-BDNF signaling.

Langkolide, a 32-Membered Macrolactone Antibiotic Produced by Streptomyces sp. Acta 3062

A new 32-membered macrolactone antibiotic, named langkolide, was isolated from the mycelium of Streptomyces sp. Acta 3062. The langkolide structure was determined by HR-MS and 1D and 2D NMR as a 32-membered macrolactone connected from an overhanging polyketide tail to a naphthoquinone unit mediated by two carbohydrate moieties. The producing strain was isolated from a rhizosphere soil of Clitorea sp. collected at Burau Bay, Langkawi, Malaysia, and was characterized by its morphological and chemotaxonomic features in addition to its 16S rRNA gene sequence. It was identified as a member of the Streptomyces galbus clade. Langkolide exhibited various bioactivities including antimicrobial and antiproliferative activities. Furthermore, langkolide inhibited human recombinant phosphodiesterase 4 with an IC(50) value of 0.48 μM.

Abstract 837: Novel sulindac derivatives that inhibit colon cancer growth by a COX independent mechanism involving PDE5 inhibition and the suppression of nuclear β-catenin levels

Abstract Many colon cancers harbor APC mutations that prevent the proteosomal degradation of oncogenic β-catenin. Previous studies have found that certain nonsteroidal anti-inflammatory drugs (NSAIDs) have the ability to reduce nuclear levels of β-catenin and inhibit tumor cell growth through a cyclooxygenase (COX)-independent mechanism, which might be responsible for their cancer chemopreventive activity. A potential mechanism may involve cGMP phosphodiesterase (PDE) inhibition and the activation of protein kinase G (PKG). In this study we evaluated over 500 novel sulindac derivatives for colon tumor cell growth inhibitory activity. Select compounds were also evaluated for COX and cGMP PDE inhibitory activity to determine which activity is associated with colon tumor cell growth inhibition and ability to suppress nuclear β-catenin levels. A subset of derivatives was identified that displayed reduced COX-1 and COX-2 inhibitory activity, yet increased potency to inhibit the growth of several colon tumor cell lines (HT29, HCT116, SW480, and COLO741) with IC50 values of 2-10 µM compared with IC50 values of 80-110 µM for the parent compound, sulindac sulfide (SS). Certain derivatives also displayed increased potency to inhibit recombinant phosphodiesterase 5 (PDE5), which appears to be the isozyme responsible for the cGMP PDE inhibitory activity of SS. Treatment of colon tumor cells with such compounds also increased the phosphorylation of vasoactive stimulated protein (VASP), a known PKG substrate. Furthermore, nuclear levels of β-catenin decreased within the same time period as PKG activation, which is consistent with previous reports suggesting that PKG can phosphorylate β-catenin to induce proteosomal degradation. Together, these data indicate that sulindac can be chemically modified to eliminate COX inhibitory activity while enhancing its ability to inhibit colon tumor cell growth. We conclude that the suppression of nuclear β-catenin levels by SS is independent of COX inhibition and that novel derivatives can be developed with improved antineoplastic activity while reducing the toxicities associated with COX inhibition. Funding provided by NIH/NCI grants CA131378 and CA148817. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 837. doi:10.1158/1538-7445.AM2011-837

Bioactive Metabolites from the Sponge Suberea sp.

Two new brominated compounds, subereaphenol K (2) and 2-(3,5-dibromo-1-ethoxy-4-oxocyclohexa-2,5-dien-1-yl)acetamide (3), together with subereaphenol B (methyl 2-(2,4-dibromo-3,6-dihydroxyphenyl)acetate; 1) with a revised structure, and five dibromotyrosine-derived metabolites, 4-8, were isolated from the sponge Suberea sp. and characterized by 1D- and 2D-NMR spectroscopic and HR-MS spectrometric data. Compounds 1, 2, 6, and 8 exhibited various weak or moderate bioactivities, including antimicrobial and cytotoxic activities. Furthermore, compounds 1 and 2 inhibited human recombinant phosphodiesterase 4 (PDE4) with IC₅₀ values of 2 μM, whereas compounds 6 and 8 were less active.

BF0801, a novel adenine derivative, inhibits platelet activation via phosphodiesterase inhibition and P2Y12 antagonism

Though antiplatelet drugs are proven beneficial to patients with coronary heart disease and stroke, more effective and safer antiplatelet drugs are still needed. In this study we report the antiplatelet effects and mechanism of BF0801, a novel adenine derivative. BF0801 dramatically inhibited platelet aggregation and ATP release induced by ADP, 2MeSADP, AYPGKF, SFLLRN or convulxin without affecting shape change in vitro . It also potentiated the inhibitory effects of adenosine-based P2Y12 antagonist AR-C69931MX or phosphodiesterase (PDE) inhibitor IBMX on platelet aggregation. The cAMP levels in both resting and forskolin-stimulated platelets were increased by BF0801 suggesting its PDE inhibitor activity, which is further confirmed by the concentration-dependent suppression of BF0801 on the native and recombinant PDE. Similar to AR-C69931MX, BF0801 drastically inhibited 2MeSADP- induced adenylyl cyclase inhibition in platelets indicating its P2Y12 antagonism activity, which is substantiated by the inhibition of BF0801 on the interaction between ADP and P2Y12 receptor expressed in CHO-K1 cells measured by atomic force microscopy. Moreover, we confirmed the antiplatelet effects of BF0801 using platelets from rats intravenously given BF0801. In summary, for the first time we developed a novel adenine derivative bearing dual activities of PDE inhibition and P2Y12 antagonism, which may have therapeutic advantage as a potential antithrombotic drug.

Sulindac sulfide selectively inhibits growth and induces apoptosis of human breast tumor cells by phosphodiesterase 5 inhibition, elevation of cyclic GMP, and activation of protein kinase G

Sulindac displays promising antineoplastic activity, but toxicities from cyclooxygenase inhibition limit its use for chemoprevention. Previous reports suggest that its anticancer properties may be attributed to a cyclooxygenase-independent mechanism, although alternative targets have not been well defined. Here, we show that sulindac sulfide (SS) induces apoptosis and inhibits the growth of human breast tumor cells with IC50 values of 60 to 85 micromol/L. Within the same concentration range, SS inhibited cyclic GMP (cGMP) hydrolysis in tumor cell lysates but did not affect cyclic AMP hydrolysis. SS did not induce apoptosis of normal human mammary epithelial cells (HMEC) nor did it inhibit phosphodiesterase (PDE) activity in HMEC lysates. SS increased intracellular cGMP levels and activated protein kinase G in breast tumor cells but not HMEC. The guanylyl cyclase (GC) activator, NOR-3, and cGMP PDE inhibitors, trequinsin and MY5445, displayed similar growth-inhibitory activity as SS, but the adenylyl cyclase activator, forskolin, and other PDE inhibitors had no effect. Moreover, GC activation increased the sensitivity of tumor cells to SS, whereas GC inhibition reduced sensitivity. By comparing PDE isozyme profiles in breast tumor cells with HMEC and determining the sensitivity of recombinant PDE isozymes to SS, PDE5 was found to be overexpressed in breast tumor cells and selectively inhibited by SS. The mechanism of SS binding to the catalytic domain of PDE5 was revealed by molecular modeling. These data suggest that PDE5 inhibition is responsible for the breast tumor cell growth-inhibitory and apoptosis-inducing activity of SS and may contribute to the chemopreventive properties of sulindac.

Purification of recombinant human phosphodiesterase 7A expressed in Dictyostelium discoideum

Phosphodiesterase plays an important role in regulating inflammatory pathways and T cell function. The development of phosphodiesterase 7 inhibitor may give better efficacy profile over phosphodiesterase 4 inhibitors. However, the recombinant phosphodiesterase 7 is required in large quantity for high-throughput screening of new drugs by in vitro enzymatic assays. In the present study, recombinant human PDE7A1 was expressed in Dictyostelium discoideum under the control of constitutively active actin-15 promoter. The cytosolic localization of the expressed protein was confirmed by immunofluorescence studies. Upto 2 mg of recombinant protein was purified using His-Tag affinity column chromatography followed by ion-exchange Resource Q column purification. The recombinant protein expressed in D. discoideum followed Michaelis–Menten kinetics similar to the protein expressed in mammalian system and showed no major changes in affinity to substrate or inhibitors. Thus, our study clearly demonstrates a robust expression system for successful bulk production of pharmacologically active isoform of human PDE7A1 required for high-throughput assays.

New biscoumarin derivatives-cytotoxicity and enzyme inhibitory activities

Biscoumarin derivatives 1–27 were tested for their inhibition of snake venom and human nucleotide pyrophosphatase phosphodiesterase-1 enzymes. Lineweaver–Burk and Dixon plots and their secondary replots showed that these compounds are pure non-competitive inhibitors of both the enzymes. Ki and IC50 values of biscoumarins were found to be in the range of 50 to 1000 and 164 to >1000μM, respectively, against human recombinant phosphodiesterase 1 enzyme and 8.0 to 1150 and 9.44 to >1000μM, respectively, against snake venom phosphodiesterase. Compounds 1, 3, 4, 6, 7, 17, 26, and 30 were found to be non-competitive and non-cytotoxic upto a concentration of 200μg/mL as evident by less than 10% cell death after 3h of incubation.

Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity

The inhibitory potential of novel anti-platelet aggregatory cilostamide analogues on phosphodiesterase (PDE) isozyme activities was investigated with recombinant PDE isozymes expressed in a baculovirus/Sf9 expression system. The recombinant enzymes (PDE1–PDE5 and PDE7) showed K m values and sensitivities to selective inhibitors similar to those reported previously for native enzymes purified from tissues. The cyclooctylurea derivative OPC-33540 (6-[3-[3-cyclooctyl-3-[(1 R∗,2 R∗)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1 H)-quinolinone) inhibited recombinant PDE3A ( ic 50 = 0.32 nM) more potently and selectively than the classical PDE3 inhibitors cilostamide, cilostazol, milrinone, and amrinone. The cyclopropylurea derivative OPC-33509 [(-)-6-[3-[3-cyclopropyl-3-[(1 R,2 R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1 H)-quinolinone] was less potent ( ic 50 = 0.10 μM) than OPC-33540, demonstrating that the cyclooctyl moiety was important for a potent inhibitory effect. In platelets, OPC-33540 potentiated cyclic AMP accumulation concentration-dependently in both the absence and the presence of 3 nM prostaglandin E 1 (PGE 1) (doubling concentrations: 32.5 and 6.2 nM, respectively). OPC-33540 inhibited thrombin-induced platelet aggregation potently ( ic 50 = 27.8 nM). The anti-platelet aggregation effect also was stimulated in the presence of 3 nM PGE 1 ( ic 50 = 6.0 nM). There was a good correlation between the ic 50 values of PDE3 inhibitors in this study for recombinant PDE3A activity and their ic 50 values for thrombin-induced platelet aggregation ( r = 0.998). These data demonstrated that OPC-33540 is a highly selective and potent PDE3 inhibitor and a useful probe for identification of the intracellular functions of PDE3.

Role of conserved histidines in catalytic activity and inhibitor binding of human recombinant phosphodiesterase 4A.

To identify critical amino acids within the central conserved region of recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE4A), we engineered the expression of point mutants in a fully active rhPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were changed independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to bind prototypical PDE4 inhibitors [3H]-(R)-rolipram or [3H]RP 73401 was also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests that these changes resulted from non-localized perturbations in the structure of the enzyme. More interesting results were obtained when histidine residues at positions 505 and 506 were changed independently to aspar agines. The K(m) value for cAMP increased 3-fold in H505N (K(m) = 11 +/- 3 microM) and 11-fold in H506N (K(m) = 44 +/- 6 microM) compared with the wild-type protein (K(m) = 4 +/- 1 microM). These mutant proteins bound [3H]-(R)-rolipram and [3H]RP 73401 with K(d) values of 1.8 +/- 0.4 and 0.3 +/- 0.1 nM, respectively, for H505N, and 3.9 +/- 0.9 and 0.5 +/- 0.1 nM, respectively, for H506N. These values are nearly identical to those obtained with the wild-type rhPDE4A/Met201-886. In contrast, the IC50 values for cAMP competition with either [3H]-(R)-rolipram or [3H]RP 73401 binding increased approximately 2-fold in H505N and approximately 13-fold in H506N compared with the wild type protein. These increases are virtually identical to the changes in the K(m) value for cAMP in these mutants. We conclude that His506 and, perhaps, His505 are involved in binding of cAMP to PDE4A/Met201-886 but not in binding of PDE4-selective inhibitors.

A novel insulin secretagogue is a phosphodiesterase inhibitor.

The arylpiperazine L-686,398 was described as an oral hypoglycemic agent and is shown to be an insulin secretagogue in vitro. The characteristics of its activity were similar to those of the incretin glucagon-like peptide I (GLP-I). We demonstrate that both the peptide and L-686,398 increase the accumulation of cAMP in isolated ob/ob mouse pancreatic islet cells, but by different mechanisms. Although GLP-I activates adenylate cyclase, the arylpiperazine has no effect on this enzyme or on the binding of 125I-labeled GLP-I to its receptor on RINm5F rat insulinoma cell membranes. However, L-686,398 inhibits the total cAMP phosphodiesterase (PDE) activity in homogenates of ob/ob mouse pancreatic islets with an EC50 of approximately 50 mumol/l. To determine the mechanism of PDE inhibition by the arylpiperazine and to examine its specificity, we studied the kinetics of arylpiperazine inhibition of two recombinant PDEs. The arylpiperazine is a competitive inhibitor of both a human heart type III PDE and a rat type IV-D PDE. Inhibition of the type III and IV isozymes are characterized by Ki values of 27 and 5 mumol/l, respectively. Although not extremely potent, the arylpiperazine does exhibit modest selectivity between these PDEs. The observation that L-686,398 acts as a PDE inhibitor suggests that exploration for beta-cell-specific PDE isoforms may reveal novel PDEs as targets for the development of therapeutically useful glucose-dependent insulin secretagogues.

Regulation of transducin GTPase activity in bovine rod outer segments.

The photoreceptor G-protein, transducin, belongs to the class of heterotrimeric GTP-binding proteins that transfer information from activated seven-span membrane receptors to effector enzymes or ion channels. Like other G-proteins, transducin acts as a molecular clock. It is activated by photoexcited rhodopsin which catalyzes the exchange of transducin-bound GDP for GTP and then stays active until bound GTP is hydrolyzed by an intrinsic GTPase activity. Our previous study on the components of the amphibian phototransduction cascade (Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417) has shown that transducin GTPase can be significantly accelerated by the target enzyme, cGMP phosphodiesterase (PDE), and more specifically its gamma-subunit (PDE gamma). Here we report that an analogous mechanism is present in bovine photoreceptors. Addition of recombinant PDE gamma to the test photoreceptor membranes which retain transducin but are depleted of endogenous PDE causes a significant acceleration of transducin GTPase activity. A similar effect was observed with the PDE holoenzyme, but not with the complex of PDE alpha- and beta-subunits prepared by a limited proteolysis of PDE with trypsin. The activating effect of PDE gamma is increased as test membrane concentration increases, exceeding 20-fold at rhodopsin concentrations over 80 microM and approaching the rate of the photoresponse turnoff. This suggests either that photoreceptor membranes contain a further factor which is essential for PDE-dependent regulation of transducin-bound GTP hydrolysis or that components of the phototransduction cascade interact in a cooperative manner. We also report that the GTPase-activating epitope is located within the C-terminal third of PDE gamma: the peptide corresponding to the 25 C-terminal amino acid residues of PDE gamma can accelerate transducin GTPase almost as well as the full-length PDE gamma. A part of the GTPase activating epitope is located within the 3 C-terminal amino acid residues: the truncation PDE gamma mutant lacking these residues accelerates transducin GTPase considerably less than the whole length PDE gamma.

Identification of a binding site on retinal transducin α for the phosphodiesterase inhibitory γ subunit

Transducin alpha (T alpha) activates retinal rod cyclic GMP phosphodiesterase (PDE) by interacting with and removing the inhibitory PDE gamma subunit. A T alpha-PDE gamma complex can be isolated in vitro, and our previous work [Morrison, Rider and Takemoto (1987) FEBS Lett. 222, 266-270; Morrison, Cunnick, Oppert and Takemoto (1989) J. Biol. Chem. 264, 11671-11681] has identified a region of PDE gamma, residues 24-45, that binds to T alpha. The C-terminal region of PDE gamma is the site that interacts with PDE alpha/beta and inhibits catalytic function. The site on T alpha that binds to the PDE gamma 24-45 region has not been identified. Synthetic peptides (15-mers) which span the bovine T alpha sequence were tested for binding to purified recombinant PDE gamma using a solid-phase assay. The peptides were also tested for ability to activate a PDE complex. We have identified a region, residues 250-275 of T alpha, which shows a high affinity of PDE gamma and for the PDE gamma (24-45) binding peptide. The peptide did not bind to the C-terminal residues 50-87 of PDE gamma. Likewise, a region of T alpha, 1-25 did not exhibit high-affinity binding to PDE gamma or to the 24-45 PDE gamma peptide. Specific binding of the 250-275 peptide to PDE gamma was confirmed by its ability to compete with T alpha binding to PDE gamma, although a higher concentration was required (10x). The T alpha-(250-275) peptide activated a fully inhibited PDE alpha beta gamma 2 complex in a dose-dependent manner. These results suggest that a region on T alpha that recognizes the PDE gamma-binding site is found within residues 250-275 of T alpha.

Interaction between the retinal cyclic GMP phosphodiesterase inhibitor and transducin. Kinetics and affinity studies

In the retinal cyclic GMP phosphodiesterase (PDE), catalysis by the alpha beta-heterodimer is inhibited in the dark by two identical gamma-subunits and stimulated in the light by the GTP-bearing alpha-subunit of the heterotrimeric G-protein transducin (T beta gamma-T alpha GDP). Two T alpha GTP molecules, dissociated from T beta gamma, bind to and displace the PDE gamma subunits from their inhibitory sites on PDE alpha beta. With GTP gamma S in lieu of GTP, this association becomes persistent. Under physiological conditions, the PDE alpha beta (gamma T alpha)2 active complex stays on the membrane. But in low-salt buffers, it becomes soluble and dissociates into a partially active PDE alpha beta catalytic moiety and two PDE gamma-T alpha GTP gamma S complexes. This indicates that T alpha binds preferentially to PDE gamma. We have studied the interaction of recombinant bovine PDE gamma with purified T alpha in solution or with retinal rod outer segments (ROS) containing both T beta gamma-T alpha GDP and PDE alpha beta gamma 2. When added to dark ROS, recombinant PDE gamma did not bind to inactive PDE alpha beta gamma 2 but extracted T alpha GDP from membrane-bound holo-transducin to form a soluble PDE gamma-T alpha GDP complex. PDE gamma also bound to purified T alpha GDP in solution. The kinetics and affinity of the interaction between PDE gamma and T alpha GDP or T alpha GTP gamma S were determined by monitoring changes in the proteins' tryptophan fluorescence. The Kd's for the binding of recombinant PDE gamma to soluble T alpha GTP gamma S and T alpha GDP are < or = 0.1 and 3 nM, respectively. PDE gamma-T alpha GDP falls apart in 3 s. This slow dissociation means that, in situ, T alpha-PDE gamma cannot physically leave the active PDE alpha beta, since after GTP hydrolysis, an isolated T alpha-PDE gamma complex would dissociate too slowly to allow a fast PDE reinhibition by the liberated PDE gamma. When recombinant PDE gamma was added to PDE that had been persistently activated by T alpha GTP gamma S, reinhibition occurred and T alpha GTP gamma S, complexed to the native PDE gamma, was released, indicating that both had hitherto stayed bound to PDE alpha beta. The mutation W70F does not prevent recombinant PDE gamma from inhibiting PDE alpha beta but diminishes its affinity for T alpha GTP and T alpha GDP 100-fold.(ABSTRACT TRUNCATED AT 400 WORDS)