A sensitive assay of human blood platelet cyclic nucleotide phosphodiesterase activity by HPLC using fluorescence derivatization and its application to assessment of cyclic nucleotide phosphodiesterase inhibitors

A selective and sensitive HPLC measurement of 3',5'-cyclic nucleotide phosphodiesterase (PDE) activity in human platelets using (3,4-dimethoxyphenyl)glyoxal (DMPG) as a fluorogenic reagent for guanine and its nucleosides and nucleotides is described. cGMP, a substrate for PDE, and GMP, which was produced by the enzyme reaction, are selectively converted by the reaction with DMPG to the fluorescent derivatives. The derivatives were separated by reversed-phase HPLC. Human platelet PDE activity was measured and the inhibitory effects of several compounds were investigated.     

Control of platelet activation by cyclic AMP turnover and cyclic nucleotide phosphodiesterase type-3

Prostaglandin-induced cAMP elevation restrains key signaling pathways in platelet activation including Ca(2+) mobilization and integrin alphaIIbbeta3 affinity regulation. We investigated how cAMP turnover by cyclic nucleotide phosphodiesterases (PDEs) regulates platelet activation. In washed human platelets, inhibition of all PDEs and also specific inhibition of PDE3 but not of PDE5 suppressed thrombin-induced Ca(2+) responses. The effect of general PDE or PDE3 inhibition was accompanied by an increase in cAMP, and potentiated by Gs stimulation with prostaglandin E(1). In platelet-rich plasma, general or PDE3 inhibition blocked platelet aggregation, integrin activation, secretion and thrombin generation. In contrast, inhibition of PDE5 increased the cGMP level, but without significant influence on aggregation, alphaIIbbeta3 activation, secretion or procoagulant activity. Nitroprusside (nitric oxide) potentiated the effect of PDE5 inhibition in elevating cGMP. Nitroprusside inhibited platelet responses, but this was accompanied by elevation of cAMP. Together, these results indicate that cAMP is persistently formed in platelets, independently of agonist-induced Gs stimulation. PDE3 thus functions to keep cAMP at a low equilibrium level and reduce the cAMP-regulated threshold for platelet activation. This crucial role of PDE3, but not of PDE5, extends to all major processes in thrombus formation: assembly of platelets into aggregates, secretion of autocrine products, and procoagulant activity.     

Cilostazol (pletal): a dual inhibitor of cyclic nucleotide phosphodiesterase type 3 and adenosine uptake

Cilostazol (Pletal), a quinolinone derivative, has been approved in the U.S. for the treatment of symptoms of intermittent claudication (IC) since 1999 and for related indications since 1988 in Japan and other Asian countries. The vasodilatory and antiplatelet actions of cilostazol are due mainly to the inhibition of phosphodiesterase 3 (PDE3) and subsequent elevation of intracellular cAMP levels. Recent preclinical studies have demonstrated that cilostazol also possesses the ability to inhibit adenosine uptake, a property that may distinguish it from other PDE3 inhibitors, such as milrinone. Elevation of interstitial and circulating adenosine levels by cilostazol has been found to potentiate the cAMP-elevating effect of PDE3 inhibition in platelets and smooth muscle, thereby augmenting antiplatelet and vasodilatory effects of the drug. In contrast, elevation of interstitial adenosine by cilostazol in the heart has been shown to reduce increases in cAMP caused by the PDE3-inhibitory action of cilostazol, thus attenuating the cardiotonic effects. Cilostazol has also been reported to inhibit smooth muscle cell proliferation in vitro and has been demonstrated in a clinical study to favorably alter plasma lipids: to decrease triglyceride and to increase HDL-cholesterol levels. One, or a combination of several of these effects may contribute to the clinical benefits and safety of this drug in IC and other disease conditions secondary to atherosclerosis. In eight double-blind randomized placebo-controlled trials, cilostazol significantly increased maximal walking distance, or absolute claudication distance on a treadmill. In addition, cilostazol improved quality of life indices as assessed by patient questionnaire. One large randomized, double-blinded, placebo-controlled, multicenter competitor trial demonstrated the superiority of cilostazol over pentoxifylline, the only other drug approved for IC. Cilostazol has been generally well-tolerated, with the most common adverse events being headache, diarrhea, abnormal stools and dizziness. Studies involving off-label use of cilostazol for prevention of coronary thrombosis/restenosis and stroke recurrence have also recently been reported.     

In vivo interaction of prostacyclin with an inhibitor of cyclic nucleotide phosphodiesterase, HL 725.

Prostacyclin (PGI2) inactivates platelets by stimulation of adenylate cyclase, and its effect can be potentiated in vitro by simultaneous inhibition of cyclic AMP phosphodiesterase. The interaction of synthetic PGI2 and the potent phosphodiesterase inhibitor HL 725 was studied in a model of systemic platelet activation by intravenous injection of collagen. Platelet aggregate formation was evaluated by continuous on-line measurement of the circulating platelet count. Collagen injection in rabbits receiving vehicle caused a 30 +/- 3% decrease in the circulating platelet count. Infusion of PGI2 (0.05, 0.1 and 0.75 micrograms kg-1 min-1) dose-dependently inhibited this decrease. HL 725 (0.5, 1 and 3 micrograms kg-1 min-1) caused a slight but significant effect. Combinations of PGI2 and HL 725, respectively, at 0.25 + 1.0 and 0.1 + 0.5 micrograms kg-1 min-1 inhibited platelet aggregate formation to a greater extent than when either substance was used alone and produced a comparable inhibition to PGI2 at 0.75 micrograms kg-1 min-1. Collagen induced an acute fall in the mean arterial blood pressure (MABP) which also was inhibited by PGI2, HL 725 and their combinations. The infusion of a combination of PGI2 and HL 725 before collagen produced a decrease in the MABP which was greater than when either compound was used on its own. Thus, PGI2 and the phosphodiesterase inhibitor HL 725 interact in vivo to inhibit platelet aggregation and lower MABP.     

Comparison of cyclic nucleotide phosphodiesterase isoenzymes in rat and rabbit ventricular myocardium: positive inotropic and phosphodiesterase inhibitory effects of Org 30029, milrinone and rolipram

Summary The species dependent variation in the cardiotonic activity of selective cyclic nucleotide phosphodiesterase (PDE) isoenzyme inhibitors was examined by comparing the inotropic and PDE inhibitory effects of Org 30029 (N-hydroxy-5,6-dimethoxy-benzo[b]thiophene-2-carboximidamide HCI), 3-isobutyl-l-methylxanthine (IBMX), milrinone and rolipram in rat and rabbit ventricular myocardium. The relative activities of PDE isoenzymes in rat and rabbit cardiac ventricle were also examined to assess the role of the different PDE subtypes in modulating contractile force in the two species. In rabbit papillary muscles, IBMX, Org 30029 and milrinone increased contractile force whilst rolipram was inactive. The rank order of potency of the active compounds was Org 30029 > IBMX > milrinone. Only Org 30029 and IBMX produced significant positive inotropic responses in rat papillary muscles, milrinone and rolipram being inactive. However, large positive inotropic responses were obtained in rat papillary muscles when milrinone and rolipram were tested in combination. In rabbit papillary muscles, the positive inotropic action of milrinone was markedly potentiated by rolipram. Four main types of PDE (I, II, III, IV) isoenzymes were resolved, by DEAE-sepharose or Mono-Q ion-exchange chromatography, from both rat and rabbit cardiac ventricular tissue. In rabbit, Ca²⁺/calmodulin dependent PDE (PDE I) and cyclic GMP inhibited PDE (PDE III) were the dominant cAMP activities. In contrast, cyclic GMP stimulated PDE (PDE II), PDE III and cGMP insensitive PDE (PDE IV) represented the main cAMP activities in rat cardiac ventricle. The inhibitory effects of Org 30029, IBMX, milrinone and rolipram on PDE isoenzymes from rat and rabbit cardiac ventricle were essentially similar. Milrinone and rolipram produced selective inhibition of PDE III and PDE IV, respectively. Org 30029 selectively inhibited PDE III and PDE IV whilst IBMX was non-selective and inhibited all PDE isoenzymes. In conclusion, the results show that species-dependent differences in the inotropic action of PDE isoenzyme selective inhibitors may be related to differences in the relative cAMP activity levels of PDE isoenzymes. It seems that in order to induce positive inotropism it is necessary to inhibit a sufficient proportion of low Km cAMP PDEs (such as dual inhibition of PDE III and PDE IV) in a relevant intracellular locale. Send offprint requests to M. Shahid at the above address     

Functional and biochemical evidence for diazepam as a cyclic nucleotide phosphodiesterase type 4 inhibitor

1. The responses of the electrically-driven right ventricle strip of the guinea-pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors.
2. Diazepam, at concentrations ranging from 1 μM to 100 μM, was devoid of effect on the contractile force in this preparation.
3. Conversely, diazepam (5 μM–100 μM) produced a consistent positive inotropic response in the presence of a concentration (1 μM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram.
4. This effect of diazepam was not γ-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 μM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and β-carboline-3-carboxylic acid methyl ester (βCCMe), or by the inverse agonists, β-carboline-3-carboxylic acid N-methylamide (βCCMa) and methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM, 0.1 μM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 μM), did not influence the effect of diazepam.
5. Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea-pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non-competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4.
6. Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea-pig eosinophils, where only type 4 PDE is present.
7. The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally-related benzodiazepines, that also displaced [³H]-rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site.
8. The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors.

     

Identification of Leu276 of the S1P1 receptor and Phe263 of the S1P3 receptor in interaction with receptor specific agonists by molecular modeling, site-directed mutagenesis, and affinity studies

Sphingosine-1-phosphate (S1P) receptor agonists are novel immunosuppressive agents. The selectivity of S1P1 against S1P3 is strongly correlated with lymphocyte sequestration and minimum acute toxicity and bradycardia. This study describes molecular modeling, site-directed mutagenesis, and affinity studies exploring the molecular basis for selectivity between S1P1 and S1P3 receptors. Computational models of human S1P1 and S1P3 receptors bound with two nonselective agonists or two S1P1-selective agonists were developed based on the X-ray crystal structure of bovine rhodopsin. The models predict that S1P1 Leu276 and S1P3 Phe263 contribute to the S1P1/S1P3 selectivity of the two S1P1-selective agonists. These residues were subjected to site-directed mutagenesis. The wild-type and mutant S1P receptors were expressed in Chinese hamster ovary cells and examined for their abilities to bind to and be activated by agonists in vitro. The results indicate that the mutations have minimal effects on the activities of the two nonselective agonists, although they have dramatic effects on the S1P1-selective agonists. These studies provide a fundamental understanding of how these two receptor-selective agonists bind to the S1P1 and S1P3 receptors, which should aid development of more selective S1P1 receptor agonists with immunosuppressive properties and improved safety profiles.     

Identification and selective inhibition of four distinct soluble forms of cyclic nucleotide phosphodiesterase activity from kidney

Homogenization of rat kidney under isotonic conditions and in the presence of protease inhibitors showed that some 92% of the cyclic AMP phosphodiesterase activity and some 83% of the cyclic GMP phosphodiesterase activity was released into the soluble fraction. Analysis of soluble phosphodiesterase activity by FPLC on a Mono-Q column resolved four distinct fractions expressing cyclic nucleotide phosphodiesterase activity. Lineweaver-Burk plots for the hydrolysis of both cyclic GMP and cyclic AMP yielded linear results. The first two peaks (KPDE-MQ-II, KPDE-MQ-III) showed higher activities towards cyclic GMP than cyclic AMP with the ratio of their Vmax values for the hydrolysis of cyclic AMP/cyclic GMP being 0.66 and 0.16, respectively. For the second two peaks (KPDE-MQ-IV, KPDE-MQ-V) the Vmax ratios for the hydrolysis of cyclic AMP/cyclic GMP were 6.4 and 16.7, respectively. All enzymes exhibited similar low Km values for both cyclic AMP and cyclic GMP but had very different Vmax values. KPDE-MQ-II was activated by Ca2+/calmodulin. The cyclic AMP phosphodiesterase activity of KPDE-MQ-III was augmented by the presence of low concentrations of cyclic GMP. Thermal denaturation studies showed that the phosphodiesterase activity of each fraction decayed as a single exponential indicating that each phosphodiesterase fraction contained but a single phosphodiesterase activity. The inhibitors IBMX, zaprinast, milrinone, amrinone, buquineran, carbazeran, ICI 118233, ICI 63197 exerted selective effects on the activities of these enzymes. We compared the action of these compounds on cyclic GMP phosphodiesterases from bovine retina. Over the concentration ranges used, the bovine retinal enzyme was only inhibited by IBMX, zaprinast and carbazeran. The cytosolic isoenzymes of cyclic AMP phosphodiesterases play a much more important role in metabolizing cyclic AMP in kidney compared with liver, where the activity of membrane-bound isoenzymes predominate.     

Inhibitors of cyclic nucleotide phosphodiesterase 3 and 5 as therapeutic agents in heart failure

Cyclic nucleotide phosphodiesterases (PDE) 3 and 5 regulate cAMP and cGMP signalling in cardiac and smooth muscle myocytes. Important advances in the understanding of the roles of these enzymes have recently been made. PDE3 inhibitors have inotropic and vasodilatory properties, and although they acutely improve haemodynamics in patients with heart failure, they do not improve long-term morbidity and mortality. Although combination therapy with β-adrenergic receptor antagonists or selective inhibition of specific PDE3 isoforms might result in a more favourable long-term outcome, more clinical data are needed to test this proposition. The role of PDE5 inhibitors in the treatment of cardiac disease is evolving. PDE5 inhibitors cause pulmonary and systemic vasodilation. How these drugs will compare with other vasodilators in terms of long-term outcomes in patients with heart failure is unknown. Recent studies also suggest that PDE5 inhibitors may have antihypertropic effects, exerted through increased myocardial cGMP signalling, that could be of additional benefit in patients with heart failure.     

Inhibitors of cyclic nucleotide phosphodiesterase 3 and 5 as therapeutic agents in heart failure

Cyclic nucleotide phosphodiesterases (PDE) 3 and 5 regulate cAMP and cGMP signalling in cardiac and smooth muscle myocytes. Important advances in the understanding of the roles of these enzymes have recently been made. PDE3 inhibitors have inotropic and vasodilatory properties, and although they acutely improve haemodynamics in patients with heart failure, they do not improve long-term morbidity and mortality. Although combination therapy with beta-adrenergic receptor antagonists or selective inhibition of specific PDE3 isoforms might result in a more favourable long-term outcome, more clinical data are needed to test this proposition. The role of PDE5 inhibitors in the treatment of cardiac disease is evolving. PDE5 inhibitors cause pulmonary and systemic vasodilation. How these drugs will compare with other vasodilators in terms of long-term outcomes in patients with heart failure is unknown. Recent studies also suggest that PDE5 inhibitors may have antihypertropic effects, exerted through increased myocardial cGMP signalling, that could be of additional benefit in patients with heart failure.     

Inhibitors of cyclic nucleotide phosphodiesterase PDE3 as adjunct therapy for dilated cardiomyopathy

PDE3 cyclic nucleotide phosphodiesterases are important in cyclic AMP (cAMP) and possibly cyclic GMP-mediated signalling in cardiac and vascular smooth muscle myocytes. Drugs that inhibit these enzymes have inotropic and vasodilatory actions that have proven useful in the short-term treatment of contractile failure and pulmonary hypertension in dilated cardiomyopathy (both ischaemic and idiopathic). With long-term usage, however, these drugs appear to increase mortality in treated patients through an as yet undetermined mechanism that is in some way attributable to an increase in intracellular cAMP content in cardiac myocytes. Several recent clinical trials have raised the possibility that these drugs may be used to advantage in dilated cardiomyopathy when they are administered in combination with beta-adrenoceptor antagonists, which act to lower intracellular cAMP content. In this review, the relevant basic and clinical data are examined and the possible justification for the combination of two therapies with seemingly opposite effects on intracellular cAMP content is considered.     

Inhibitors of cyclic nucleotide phosphodiesterase PDE3 as adjunct therapy for dilated cardiomyopathy

PDE3 cyclic nucleotide phosphodiesterases are important in cyclic AMP (cAMP) and possibly cyclic GMP-mediated signalling in cardiac and vascular smooth muscle myocytes. Drugs that inhibit these enzymes have inotropic and vasodilatory actions that have proven useful in the short-term treatment of contractile failure and pulmonary hypertension in dilated cardiomyopathy (both ischaemic and idiopathic). With long-term usage, however, these drugs appear to increase mortality in treated patients through an as yet undetermined mechanism that is in some way attributable to an increase in intracellular cAMP content in cardiac myocytes. Several recent clinical trials have raised the possibility that these drugs may be used to advantage in dilated cardiomyopathy when they are administered in combination with β-adrenoceptor antagonists, which act to lower intracellular cAMP content. In this review, the relevant basic and clinical data are examined and the possible justification for the combination of two therapies with seemingly opposite effects on intracellular cAMP content is considered.     

Identification of tyrosine 189 and asparagine 358 of the cholecystokinin 2 receptor in direct interaction with the crucial C-terminal amide of cholecystokinin by molecular modeling,site-directed mutagenesis,and structure/affinity studies

The cholecystokinin (CCK) receptors CCK1R and CCK2R exert important central and peripheral functions by binding the neuropeptide cholecystokinin. Because these receptors are potential therapeutic targets, great interest has been devoted to the identification of efficient ligands that selectively activate or inhibit these receptors. A complete mapping of the CCK binding site in these receptors would help to design new CCK ligands and to optimize their properties. In this view, a molecular model of the CCK2R occupied by CCK was built to identify CCK2R residues that interact with CCK functional groups. No such study has yet been reported for the CCK2R. Docking of CCK in the receptor was performed by taking into account our previous mutagenesis data and by using, as constraint, the direct interaction that we demonstrated between His207 in the CCK2R and Asp8 of CCK (Mol Pharmacol 54:364-371, 1998; J Biol Chem 274:23191-23197, 1999). Two residues that had not been revealed in our previous mutagenesis studies, Tyr189 (Y4.60) and Asn358 (N6.55), were identified in interaction via hydrogen bonds with the C-terminal amide of CCK, a crucial functional group of the peptide. Mutagenesis of Tyr189 (Y4.60) and Asn358 (N6.55) as well as structure-affinity studies with modified CCK analogs validated these interactions and the involvement of both residues in the CCK binding site. These results indicate that the present molecular model is an important tool to identify direct contact points between CCK and the CCK2R and to rapidly progress in mapping of the CCK2R binding site. Moreover, comparison of the present CCK2R.CCK molecular model with that of CCK1R.CCK, which we have previously published and validated, clearly argues that the positioning of CCK in these receptors is different.     

Ligand recognition by drug-activated nuclear receptors PXR and CAR: structural, site-directed mutagenesis and molecular modeling studies

Pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are the principal regulators of drug/xenobiotic disposition and toxicity. These nuclear receptors display considerable cross-regulation of their target genes, and species-specific, yet promiscuous activation by a large number of structurally dissimilar ligands. Activation of PXR and/or CAR will frequently result in enhanced drug metabolism, disturbances in homeostasis of endogenous substances, and increased toxicity. Thus, understanding, measurement and prediction of ligand-elicited activation of PXR and CAR receptors is of utmost importance for the drug development process. In this mini-review, we will review the recent elucidation of structural properties of PXR and CAR, the molecular determinants of their ligand and species specificities and progress made in in silico models for identification of PXR and CAR activators.     

Human bronchial cyclic nucleotide phosphodiesterase isoenzymes: biochemical and pharmacological analysis using selective inhibitors.

1 The aims of the present study were to characterize the cyclic nucleotide phosphodiesterase (PDE) isoenzyme activities present in human bronchi and to examine the ability of selective isoenzyme inhibitors to relax histamine and methacholine precontracted preparations of human bronchi. 2 Three separations of pooled human bronchial tissue samples were performed. Ion-exchange chromatography showed that the soluble fraction of human bronchial preparations contains PDE I, II, III, IV and V isoenzyme activities. Multiple forms of PDE I and PDE IV were observed and PDE IV was the main cyclic AMP hydrolytic activity. 3 3-Isobutyl-l-methylxanthine (IBMX) non-selectively inhibited all separated isoenzyme activities. Zaprinast selectively inhibited PDE V, but also effectively inhibited one of the two PDE I isoforms identified. The PDE IV selective inhibitors rolipram and RO-201724, inhibited the PDE IV activities as did the dual PDE III/IV inhibitor, Org 30029. Org 9935, a PDE III selective inhibitor, potently attenuated part of the PDE IV activity peak in one of three separations performed, indicating that some PDE III activity may co-elute with PDE IV under the experimental conditions employed. 4 PDE IV-selective (rolipram), PDE III-selective (Org 9935) and dual PDE III/IV (Org 30029) inhibitors were effective relaxants of human bronchial smooth muscle. The PDE V/PDE I inhibitor, zaprinast was relatively ineffective. 5 The present study demonstrates in human bronchi, as in animal airways smooth muscle, that inhibitors of PDE III, PDEIV and dual PDE III/IV have potentially useful bronchodilator activity and are worthy of further consideration as anti-asthma drugs.     

Identification of ginsenoside interaction sites in 5-HT3A receptors

We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.     

Comparative effects of calmodulin inhibitors on calmodulin's hydrophobic sites and on the activation of cyclic nucleotide phosphodiesterase by calmodulin

Experiments were designed to investigate the effect of inhibitors on calmodulin's hydrophobic sites and their consequences on the activation of a target enzyme, cyclic nucleotide phosphodiesterase. Two fluorescent probes, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS) and 9-anthroylcholine (9AC) were used to study the interactions with calmodulin of inhibitors devoid of direct effect on the probes. Contrary to W-7, nicergoline, nicardipine and quercetin, which decreased the fluorescence of the two probes bound to calmodulin, bepridil only decreased 9AC fluorescence but increased the fluorescence intensity at the wavelength of the emission maximum of TNS. In spite of this difference, bepridil as well as W-7 and nicergoline competitively inhibited calmodulin activation of phosphodiesterase. In addition, nicergoline also inhibited phosphodiesterase activity competitively to cyclic GMP. These results show differences in the interactions of inhibitors with calmodulin; these differences are not detected in functional studies of the effect of inhibitors on phosphodiesterase activation.     

Homology modeling and site-directed mutagenesis to identify selective inhibitors of endothelin-converting enzyme-2

Endothelin-converting enzyme-2 (ECE-2), a member of M13 family of zinc metallopeptidases, has previously been shown to process a number of neuropeptides including those derived from prodynorphin, proenkephalin, proSAAS, and amyloid precursor protein. ECE-2, unlike ECE-1, exhibits restricted neuroendocrine distribution and acidic pH optimum; it is consistent with a role in the regulation of neuropeptide levels in vivo. Here, we report the generation of a three-dimensional (3D) molecular model of ECE-2 using the crystal structure of neprilysin (EC 3.4.24.11) as a template. On the basis of the predictions made from the molecular model, we mutated and tested two residues, Trp 148 and Tyr 563, in the catalytic site. The mutation of Tyr 563 was found to significantly affect the catalytic activity and inhibitor binding. The molecular model was used to virtually screen a small molecule library of 13 000 compounds. Among the top-scoring compounds three were found to inhibit ECE-2 with high affinity and exhibited specificity for ECE-2 compared to neprilysin. Thus, the model provides a new useful tool to probe the active site of ECE-2 and design additional selective inhibitors of this enzyme.     

Inhibition of eotaxin-mediated human eosinophil activation and migration by the selective cyclic nucleotide phosphodiesterase type 4 inhibitor rolipram

1. The effect of the selective type 4 phosphodiesterase (PDE 4) inhibitor rolipram on human eosinophil activation and migration mediated by eotaxin was investigated.
2. Studies were performed with human freshly isolated eosinophils from peripheral blood of healthy donors by a magnetic cell separation (MACS) technique to a purity>99%. To test the effect of rolipram, eosinophils were stimulated with recombinant human eotaxin and the cell surface activation markers CD11b and L-selectin were analysed by flow cytometry. Furthermore, eotaxin mediated eosinophil migration was measured in a transendothelial chemotaxis assay.
3. Our results indicate that rolipram inhibited eotaxin-induced CD11b up-regulation up to 60.6±7.6% at the highest tested dose (10 μM), whereas transendothelial chemotaxis was partially inhibited reaching a plateau of approx. 30% at a rolipram concentration of 0.1 μM.
4. We conclude that the selective PDE 4 inhibitor rolipram decreases eotaxin mediated eosinophil activation, an observation that may contribute to elucidate the mechanism by which PDE 4 inhibitors reduce antigen-induced eosinophil infiltration in different animal models of allergic inflammation.