3',5'-cyclic nucleotide phosphodiesterase from human brain

3':5'-Cyclic nucleotide phosphodiesterase was isolated from human brain and characterized. After the first stage of purification on phenyl-Sepharose, the enzyme activity was stimulated by Ca2+ and micromolar concentrations of cGMP. High pressure liquid chromatography on a DEAE-TSK-3SW column permitted to identify three ranges of enzymatic activity designated as PDE I, PDE II and PDE III. Neither of the three enzymes possessed a high selectivity for cAMP and cGMP substrates. The catalytic activity of PDE I and PDE II increased in the presence of Ca2+-calmodulin (up to 6-fold); the degradation of cAMP was decreased by cGMP. The Ca2+-calmodulin stimulated PDE I and PDE II activity was decreased by W-7. PDE I and PDE II can thus be classified as Ca2+-calmodulin-dependent phosphodiesterases. With cAMP as substrate, the PDE III activity increased in the presence of micromolar concentrations of cGMP (up to 10-fold), Ca2+ and endogenous calmodulin (up to 2-3-fold). No additivity in the effects of saturating concentrations of these compounds on PDE III was observed. Ca2+ did not influence the rate of cGMP hydrolysis catalyzed by PDE III. In comparison with PDE I and PDE II, the inhibition of PDE III was observed at higher concentrations of W-7 and was not limited by the basal level of the enzyme. These results do not provide any evidence in favour of the existence of several forms of the enzyme in the PDE III fraction. The double regulation of PDE III creates some difficulties for its classification.     

Adenosine-3':5'-monophosphate phosphodiesterase from Rhizobium

The phosphodiesterase (PDE) activity of adenosine-3':5'-monophosphate was detected in the cells of tubercular bacteria of Rhizobium lupini and Rhizobium japonicum. The specific activity of three Rhizobium forms, e.g. bacteroids from lupine root tubercles, free-nitrogen-fixing culture and vegetative cells grown on a mannitol--yeast agar, were compared. In the bacteroids PDE is represented both by soluble and membrane-bound forms. The optimal enzyme activity is revealed in an alkaline medium, whereas the curve of PDE activity dependence on pH has a broad maximum. PDE is inhibited by methylxanthines, the inhibiting effect being stronger than that of theophylline.     

Hepatic adenosine-3',5'-monophosphate metabolism in newborn rats irradiated during the period of organogenesis]

The adenylate cylcase (AC) and phosphodiesterase (PDE) activities, as well as the 3'5'-AMP (cAMP) fund were studied in the liver of newborn rats, intact, and after irradiation on the 9th day of the embryonic development. A decrease of AC and PDE activities was noted with a dose of 50 r. The stationary level of cAMP in the tissue remained unchanged. The adrenaline-stimulated AC activity only tended to decrease in case of irradiation. As suggested, during critical periods of development, in the presence of the hormone inductor in the liver of irradiated rats, conditions can be created for an increase of the cAMP pool.     

Regulation of cyclic adenosine 3':5'-monophosphate phosphodiesterases: altered pattern in transformed myoblasts

Rat skeletal myoblasts, L6 and L8, have two major forms of phosphodiesterases, PDE II and PDE III. Only the former is activated by treatment with proteases. When the myoblasts are exposed to cAMP for 10-16 h, the activity of PDE III increases considerably. This increase is accompanied by a loss of activatability of PDE II by proteases. Leupeptin prevents the increase in the levels of PDE III suggesting that a protease in vivo may be responsible for the formation of PDE III from PDE II. Spontaneously or Rous sarcoma virus-transformed myoblasts, however, show altered regulation of the two forms of PDE. In the presence of cAMP in the medium, unlike the nontransformed cells, the levels of PDE III do not increase but the activity of PDE II rises. Simultaneously, PDE II becomes refractory to activation by proteases. The altered mode of PDE regulation in transformed cells is dominant in hybrids between normal and transformed myoblasts, which suggests that altered regulation is due to an "acquisition" of some new property by transformed cells.     

Endogenous inhibitors of cyclic adenosine 3',5'-monophosphate-phosphodiesterase in rat epididymis

Stabilization of cyclic adenosine 3',5'-monophosphate (cAMP)-phosphodiesterase (PDE) in 50% glycerol made possible the removal of endogenous inhibitors from tissue extracts by dialysis and the storage of the extracts at -20 degrees C without loss of PDE activity. Dialysates of heat-inactivated epididymal extracts were fractionated by liquid chromatography, and 4 fractions-F2, F5, F7, and F12-were found to contain endogenous inhibitors of PDE. The masses of the fractions required to inhibit low-Km PDE activity by ca. 50% in 430-microliter incubation mixtures were F2, 89 micrograms; F5, 23 micrograms; F7, 275 micrograms; and F12, 1.2 mg. The mechanisms of inhibition of low-Km PDE by the endogenous inhibitors were investigated by kinetic analysis of enzyme-inhibitor interaction. F2 and F12 inhibited PDE competitively; F5 and F7 decreased both apparent Km and Vmax, suggesting an uncompetitive mechanism of inhibition. The high potency of F5 in low concentration suggests that it may be a physiological modulator of low-Km cAMP-PDE activity.     

Effects of ethanol on gastric mucosal adenosine 3', 5' monophosphate (cAMP)

The effects of ethanol on the gastric mucosal adenosine 3′, 5′-monophosphate (cAMP) system were evaluated. The activity of adenylate cyclase (AC), phosphodiesterase (PDE), and tissue content of cAMP were determined in the presence of ethanol. NaF stimulated AC in rat gastric mucosa was inhibited in vitro and in vivo by 20% ethanol. Basal AC activity was so low (0.05 ± 0.10 pmoles cAMP formed/min/mg protein) that consistent results without NaF could not be obtained. The PDE activity (172 ± 11 pmoles cAMP consumed/min/mg protein) was approximately 350 fold greater than the basal AC activity. All levels of ethanol tested (2.0–20.0%) significantly inhibited (p<0.05) PDE in vitro. Gastric mucosal levels of cAMP are not measurably altered by ethanol in vivo (5–20%).     

Inhibition of rat heart and brain cyclic adenosine-3',5'-monophosphate phosphodiesterase in vitro under the influence of neurohormone C]

The work was devoted to one of the new neurohormones (neurohormone "C") obtained from the bovine hypothalamus which produced a pronounced relaxation of the coronary vessels. A study of the effect of "C" on cyclic 3',5'-adenosine monophosphate (cAMP) phosphodiesterase (PDE) showed "C" to be a potent inhibitor of PDE in the crude PDE-preparations of the rat heart and brain. Experiments were carried out on the 2000 g supernatant obtained from the homogenized tissues. It is supposed that the relaxant effect of "C" on the coronary vessels is due to its inhibitory action on PDE mediated by the accumulated cAMP.     

Synthesis and phosphodiesterase 5 inhibitory activity of novel pyrido[1,2-e]purin-4(3H)-one derivatives

Synthesis and primary SAR of a novel series of 2-phenylpyrido[1,2-e]purin-4(3H)-one derivatives with piperazinyl sulfonamide substituents were described herein. As potential PDE5 inhibitors for erectile dysfunction (ED) treatment, representative compounds exhibit improved selectivity versus PDE1 and PDE6. Meanwhile, compound 3e demonstrated functional efficacy on rabbit corpus cavernosum strip in vitro.     

Isolation and characterization of PDE10A, a novel human 3', 5'-cyclic nucleotide phosphodiesterase.

A gene encoding a novel human 3', 5'-cyclic nucleotide phosphodiesterase (PDE) was identified and characterized. PDE10A1 encodes a protein that is 779 amino acids in length. An incomplete cDNA for a second 5'-splice variant, PDE10A2, was isolated. The proteins encoded by the two variants share 766 amino acids in common. This common region includes an amino-terminal domain with partial homology to the cGMP-binding domains of PDE2, PDE5 and PDE6 as well as a carboxy-terminal region with homology to the catalytic regions of mammalian PDEs. Northern analysis revealed that PDE10A is widely expressed. The PDE10A gene was mapped to three yeast artificial chromosomes (YACs) that contain human DNA from chromosome 6q26-27. A recombinant protein corresponding to the 766 amino acid region common to PDE10A1 and PDE10A2 was expressed in yeast. It hydrolyzed both cAMP and cGMP. Inhibitors that are selective for other PDE families are poor inhibitors of PDE10A; however, PDE10A is inhibited by the non-specific PDE inhibitor, IBMX.     

Discovery of a new series of [1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors

The synthesis, preliminary evaluation and structure–activity relationship (SAR) of a series of 1-aryl-4-methyl[1,2,4]triazolo[4,3-a]quinoxalines as dual phosphodiesterase 2/phosphodiesterase 10 (PDE2/PDE10) inhibitors are described. From this investigation compound 31 was identified, showing good combined potency, acceptable brain uptake and high selectivity for both PDE2 and PDE10 enzymes. Compound 31 was subjected to a microdosing experiment in rats, showing preferential distribution in brain areas where both PDE2 and PDE10 are highly expressed. These promising results may drive the further development of highly potent combined PDE2/PDE10 inhibitors, or even of selective inhibitors of PDE2 and/or PDE10.     

Synthesis and phosphodiesterase 5 inhibitory activity of new 5-phenyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one derivatives containing an N-acylamido group phenyl ring.

New sildenafil analogues with an N-acylamido group at the 5'-position of the phenyl ring, 6a--e, were prepared from the readily available starting compound 2 in four straightforward steps. Enzyme assays demonstrated that all the target compounds 6a-e showed higher PDE5 inhibitory activities than sildenafil. It was observed that the PDE5 inhibitory activity was enhanced as the chain length of R group increased, but introduction of the branched alkyl groups such as isopropyl (6d) and cyclohexyl (6e) resulted in the drop of potency compared with 6c. In particular the N-butyrylamido derivative 6c exhibited the highest PDE5 inhibitory activity, and was about 6-fold more potent than sildenafil. However, all the compounds exhibited somewhat weak selectivity (1--3-fold) over PDE6, indicating that the compounds 6a--e have intrinsically lower selectivity than sildenafil.     

Properties of a cyclic 3'5'-nucleotide phosphodiesterase from Vigna mungo

Cyclic AMP phosphodiesterase (PDE) partially purified from roots of Vigna mungo exhibited optimum activity at pH 5.5 to 6.0 and maximum enzyme activity at 50 degrees C. Levels of PDE activity in roots remained relatively constant from the first to the eleventh day after germination; on the twelfth day there was a 400% increase in PDE activity. The enzyme was stable for at least 48 hours at 28 degrees C, retaining 92% of its original activity. Plant growth hormones including gibberellic acid, indoleacetic acid and kinetin at 1.0 and 10.0 microM concentrations did not have any significant effect on enzyme activity. Nucleotides tested including cyclic 2'3' AMP, cyclic 2'3' GMP completely abolished enzyme activity at 1.0mM while cyclic 3'5' GMP, cyclic 3'5' GMP, 2'deoxy 5' ATP, 2'deoxy 5'GTP and 5'ADP were also inhibitory to the enzyme. The enzyme was stimulated by Mg2+, Fe2+ and NH4+ while Cu2+ and Fe3+ were inhibitory. Theophylline, caffeine, phosphate, pyrophosphate and EDTA were inhibitory to the enzyme.     

Identification,synthesis and SAR of amino substituted pyrido[3,2b]pyrazinones as potent and selective PDE5 inhibitors

A new class of potent and selective PDE5 inhibitors is disclosed. Guided by X-ray crystallographic data, optimization of an HTS lead led to the discovery of a series of 2-aryl, (N8)-alkyl substituted-6-aminosubstituted pyrido[3,2b]pyrazinones which show potent inhibition of the PDE5 enzyme. Synthetic details and some structure–activity relationships are also presented.     

Discovery of benzo[d]imidazo[5,1-b]thiazole as a new class of phosphodiesterase 10A inhibitors

The design, synthesis and structure activity relationship studies of a series of compounds from benzo[d]imidazo[5,1-b]thiazole scaffold as phosphodiesterase 10A (PDE10A) inhibitors are discussed. Several potent analogs with heteroaromatic substitutions (9a–d) were identified. The anticipated binding mode of these analogs was confirmed by performing the in silico docking experiments. Later, the heteroaromatics were substituted with saturated heteroalkyl groups which provided a tool compound 9e with excellent PDE10A activity, PDE selectivity, CNS penetrability and with favorable pharmacokinetic profile in rats. Furthermore, the compound 9e was shown to be efficacious in the MK-801 induced psychosis model and in the CAR model of psychosis.     

Synthesis and phosphodiesterase 5 inhibitory activity of new sildenafil analogues containing a phosphonate group in the 5(')-sulfonamide moiety of phenyl ring

Synthesis of new sildenafil analogues containing a phosphonate group in the 5(')-sulfonamide moiety of the phenyl ring, 12a-e, 13a-d, and 14a-d, and evaluation of their in vitro PDE5 inhibitory activity are disclosed. Enzyme assays revealed that maximum 10-fold increase in PDE5 inhibitory activity, compared with sildenafil, was achieved by introducing a phosphonate group in the 5(')-sulfonamide moiety. Docking model of (PDE5: 12d) complex shows that the PDE5-bound conformation of 12d matches completely with that of sildenafil, while 12d is partially overlapped with cGMP with ethyl phosphonate group of 12d superimposed onto the cyclic phosphate group of cGMP.     

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.     

Identification of pyridazino[4,5-b]indolizines as selective PDE4B inhibitors

Substituted pyridazino[4,5-b]indolizines were identified as potent and selective PDE4B inhibitors. We describe the structure–activity relationships generated around an HTS hit that led to a series of compounds with low nanomolar affinity for PDE4B and high selectivity over the PDE4D subtype.     

Synthesis and structure-activity relationships of 4-oxo-1-phenyl-3,4,6,7-tetrahydro-[1,4]diazepino[6,7,1-hi]indoles: novel PDE4 inhibitors

A novel series of benzodiazepine derivatives have been discovered as inhibitors of PDE4 enzymes. We have found that our compounds are selective versus other PDE enzymes, and that the activity can be modulated by specific structural modifications. One compound exhibited a strong eosinophilic infiltration inhibiting action on sensitized Brown-Norway rats (compound 9, 5.1 mg/kg p.o.), moreover this compound is not emetic at 3 mg/kg i.v.     

Synthesis of pyrrolo[2,3-d]pyridazinones as potent, subtype selective PDE4 inhibitors

A series of pyrrolo [2,3-d]pyridazinones was synthesized and tested for their inhibitory activity on PDE4 subtypes A, B and D and selectivity toward Rolipram high affinity binding site (HARBS). New agents with interesting profile were reported; in particular compound 9e showed a good PDE4 subtype selectivity, being 8 times more potent (IC50 = 0.32 microM) for PDE4B (anti-inflammatory) than for PDE4D (IC50 = 2.5 microM), generally considered the subtype responsible for emesis. Moreover the ratio HARBS/PDE4B was particularly favourable for 9e (147), suggesting that the best arranged groups around the pyrrolopyridazinone core are an isopropyl at position-1, an ethoxycarbonyl at position-2, together with an ethyl group at position-6. For compounds 8 and 15a the ability to inhibit TNFalpha production in PBMC was evaluated and the results are consistent with their PDE4 inhibitory activity.