Azole derivatives as histamine H3 receptor antagonists, part 2: C-C and C-S coupled heterocycles.

With a small series of compounds we demonstrated the variability in the core region of the human histamine H(3) receptor (hH(3)R) antagonist structural blueprint by introducing polar azole groups (oxazole, oxadiazole, thiazole and triazole). Additional variations achieved by coupling different residues to the heterocyclic core structure led to further optimisation of in vitro receptor binding of the novel azole derivatives.

Azole derivatives as histamine H3 receptor antagonists, part I: thiazol-2-yl ethers.

Most human histamine H(3) receptor (hH(3)R) antagonists follow a general structural blueprint, containing a basic moiety linked by a spacer to a substituted core element. In this investigation the acceptance of thiazol-2-yl ether moieties in the core region is proved with some ether derivatives showing hH(3)R binding affinities in the nanomolar concentration range. A diversity of structural motifs is used as substituents to enhance the in vitro hH(3)R binding affinity.

Fluorinated non-imidazole histamine H3 receptor antagonists.

Fluorine substituents have become a widespread and important component in drug design and development. Here, the synthesis of fluorine containing compounds and some corresponding precursor molecules are presented for potential isotope labelling as well as their data obtained with in vitro and in vivo screenings. The compounds vary in the basic centres (piperidine or pyrrolidine) and are fluoro substituted in different positions of the basic alicyclic moiety. Pharmacological evaluation resulted in ligands with high affinities at hH(3) receptor in the nanomolar and subnanomolar concentration range and some with high antagonist in vivo potencies.

BF2.649 [1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride], a nonimidazole inverse agonist/antagonist at the human histamine H3 receptor: Preclinical pharmacology.

Histamine H3 receptor inverse agonists are known to enhance the activity of histaminergic neurons in brain and thereby promote vigilance and cognition. 1-{3-[3-(4-Chlorophenyl)propoxy]propyl}piperidine, hydrochloride (BF2.649) is a novel, potent, and selective nonimidazole inverse agonist at the recombinant human H3 receptor. On the stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate binding to this receptor, BF2.649 behaved as a competitive antagonist with a Ki value of 0.16 nM and as an inverse agonist with an EC50 value of 1.5 nM and an intrinsic activity approximately 50% higher than that of ciproxifan. Its in vitro potency was approximately 6 times lower at the rodent receptor. In mice, the oral bioavailability coefficient, i.e., the ratio of plasma areas under the curve after oral and i.v. administrations, respectively, was 84%. BF2.649 dose dependently enhanced tele-methylhistamine levels in mouse brain, an index of histaminergic neuron activity, with an ED50 value of 1.6 mg/kg p.o., a response that persisted after repeated administrations for 17 days. In rats, the drug enhanced dopamine and acetylcholine levels in microdialysates of the prefrontal cortex. In cats, it markedly enhanced wakefulness at the expense of sleep states and also enhanced fast cortical rhythms of the electroencephalogram, known to be associated with improved vigilance. On the two-trial object recognition test in mice, a promnesiant effect was shown regarding either scopolamine-induced or natural forgetting. These preclinical data suggest that BF2.649 is a valuable drug candidate to be developed in wakefulness or memory deficits and other cognitive disorders.

The gamma-subunit of (Na+,K+)-ATPase: a representative example of human single transmembrane protein with a key regulatory role.

The (Na+,K+)-ATPase is a plasma membrane protein complex composed of at least three subunits (alpha,beta,gamma) that couples the exchange of three cytoplasmic Na+ ions with two extracellular K+ ions, to the hydrolysis of one molecule ofATP in most animal cells. The gamma-subunit is a 66 residue membrane protein associated with the active alpha/beta binary complex. It can be considered as an archetype of single transmembrane proteins (type I) which may play a modulatory role upon association with functional membrane partners. This paper highlights similar associations observed with other ATPases such as the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA1/SERCA 2a), but also with Cl- and/or K+ currents, ionic channels (HERG, KCNQ1) and G-protein coupled receptors (adrenomedullin, CGRP and calcitonin) which are of particular interest in the cardiovascular field. Here is reviewed the assessed or suggested regulatory role of a family of small plasma/SR associated membrane proteins including gamma-subunit, phospholemman, Mat 8, KCNE (type 1, 2 and 3), RAMP (type 1, 2 and 3), sarcolipin and phospholamban, mainly found in muscular and vascular tissues. These proteins are critical in controlling important biological processes which derive from specific associations with a binding partner and particular subcellular localizations.

Biophysical interaction between phospholamban and protein phosphatase 1 regulatory subunit GM.

Regulation of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA 2a) depends on the phosphorylation state of phospholamban (PLB). When PLB is phosphorylated, its inhibitory effect towards SERCA 2a is relieved, leading to an enhanced myocardial performance. This process is reversed by a sarcoplasmic reticulum (SR)-associated type 1 protein phosphatase (PP1) composed of a catalytic subunit PP1C and a regulatory subunit GM. Human GM and PLB have been produced in an in vitro transcription/translation system and used for co-immunoprecipitation and biosensor experiments. The detected interaction between the two partners suggests that cardiac PPI is targeted to PLB via GM and we believe that this process occurs with the identified transmembrane domains of the two proteins. Thus, the interaction between PLB and GM may represent a specific way to modulate the SR function in human cardiac muscle.

Relationship between biochemical and functional effects of protein phosphatase 1 inhibitors in rabbit cardiac skinned fibers.

Tautomycin (TT) and calyculin A (CyA) are inhibitors of protein phosphatases type 1 and 2 (PP1, PP2). Inhibitors 1 and 2 are specific for PP1, which is the major phosphatase functionally relevant in heart and able to dephosphorylate phospholamban (PLB). TT and CyA maintain PLB in its phosphorylated state, thereby increasing calcium uptake. Rabbit saponin skinned fibers (SF) are used to assess calcium load of the sarcoplasmic reticulum (SR). The present investigation aimed to examine the effects of PP1 inhibitors on SR calcium load assessed by caffeine-induced tension transient (CITT), and to correlate this activity with the PLB phosphorylation state. TT and CyA (100 nm) applied during the uptake phase increased the amplitude of CITT by 10 and 20%, respectively,P<0.05 without effect on the release phase. Both CyA and TT were devoid of calcium sensitizing effect when studied on Triton X-100 SF. After skinning procedure, SF were grinded for biochemical studies. SDS-PAGE electrophoresis and immunoblots using a monoclonal PLB antibody showed that cAMP or Ca2+/calmodulin-dependent protein kinases phosphorylated PLB in an additive fashion. Inhibition of PP1 by inhibitor 1, CyA and TT maintained PLB in its phosphorylated state in a dose-dependent manner. The results of this study in which functional and biochemical experiments in cardiac SF were combined demonstrate that strong correlation exists between the phosphorylation-dephosphorylation cycle of PLB and calcium uptake.

Specific inhibition of cardiac and skeletal muscle sarcoplasmic reticulum Ca2+ pumps by H-89.

The isoquinolinesulfonamide H-89, an inhibitor of cyclic AMP-dependent protein kinases (EC 2.7.1.37, cAPrK), inhibited the Ca2+-ATPase activity of cardiac and skeletal muscle sarcoplasmic reticulum (SR) with concentrations giving half-maximal inhibition of 8.1 +/- 1.3 and 7.2 +/- 0.9 micromol/L, respectively. The effect of H-89 on cardiac SR Ca2+-ATPase (EC 3.6.1.38) was the same irrespective of the presence or absence of inhibitors of cAPrK and furthermore, was not affected by a neutralising monoclonal antibody raised against phospholamban. Thus, the action of H-89 in inhibiting SR Ca2+-ATPase would not appear to be mediated by inhibition of cAPrK to reduce the phosphorylation state of phospholamban. In both cardiac and skeletal muscle SR, the inhibition by H-89 was noncompetitive with respect to ATP at a low concentration of ATP (<1 mmol/L) and of a mixed pattern at high concentrations of ATP. H-89 produced a decrease in affinity of the SR Ca2+ pump to Ca2+ with an increase in the Km for Ca from 0.52 +/- 0.01 to 0.94 +/- 0.03 micromol/L (P < 0.05) in cardiac SR and from 0.39 +/- 0.01 to 0.79 +/- 0.02 micromol/L (P < 0.05) in skeletal muscle SR. These results suggest that H-89 inhibits SR Ca2+-ATPase by a direct action on the SR Ca2+ pump to decrease its affinity to Ca2+. Such an action may contribute to the pharmacological effect of H-89.

Mechanism of action of sarcoplasmic reticulum calcium-uptake activators--discrimination between sarco(endo)plasmic reticulum Ca2+ ATPase and phospholamban interaction.

The Ca2+ uptake by the sarcoplasmic reticulum (SR) can be affected by direct modulation of the Ca2+ pump or by removing the inhibitory effect of dephosphorylated phospholamban. The effect of these mechanisms was assessed using ellagic acid and 1-(3,4-dimethoxyphenyl)-3-dodecanone. Both compounds (30 micromol/l) enhanced SR-Ca2+ uptake in rabbit cardiomyocytes by 65.3 +/- 13% and 44.3 +/- 6.7% for 1-(3,4-dimethoxyphenyl)-3-dodecanone and ellagic acid, respectively (at pCa 6.2). A similar effect was observed in cardiac SR microsomes (59.5 +/- 7.4% and 45.1 +/- 6.7) with 30 micromol/l 1-(3,4-dimethodoxyphenyl)-3-dodecanone and ellagic acid, respectively. 1-(3,4-Dimethoxyphenyl)-3-dodecanone increased Ca2+ storage by cardiac SR microsomes mainly at high [Ca2+] with a 57% increase of Vmax, whereas ellagic acid increased Vmax to a smaller extent (22%) and stimulated Ca2+ uptake at lower [Ca2+] with a leftward-shift of the pCa/ATPase relationship by pCa 0.24. Ellagic acid also differed from 1-(3,4-dimethoxylphenyl)-3-dodecanone in that it produced a Ca2+ sensitizing effect only in cardiac SR microsomes (by pCa 0.3) whereas 1-(3,4-dimethoxyphenyl)-3-dodecanone stimulated the ATPase, at saturating Ca2+, in both cardiac and skeletal muscle SR vesicles. It is suggested that 1-(3,4-dimethoxyphenyl)-3-dodecanone stimulates directly the Ca2+-ATPase activity, in contrast to ellagic acid which enhances the cardiac SR-Ca2+ uptake by interacting with phospholamban, as confirmed by the lack of additive effect between ellagic acid and monoclonal antibodies raised against phospholamban. 1-(3,4-dimethoxyphenyl)-3-dodecanone and ellagic acid constitute attractive pharmacological tools to investigate the functional consequences of enhancing SR Ca2+, uptake by affecting different mechanisms.

2-Amino-6-trifluoromethoxy benzothiazole, a possible antagonist of excitatory amino acid neurotransmission--II. Biochemical properties.

Two models have been chosen to study the effect of 2-amino-6-trifluoromethoxy benzothiazole (PK 26124) on excitatory amino acid neurotransmission: the pool of cyclic guanosine monophosphate (cGMP) in the cerebellum and the release of acetylcholine in the striatum and olfactory tubercles. The release of acetylcholine induced by N-methyl-DL-aspartate in the striatum and olfactory tubercles was antagonized by PK 26124 which was less potent on the release of acetylcholine induced electrically. The increase in levels of cGMP in the cerebellum induced by excitatory amino acids such as glutamate and quisqualate was antagonized by PK 26124, but the drug was inactive against N-methyl-DL-aspartate, L-aspartate, kainate and cysteine sulphinate. In vivo it antagonized the increases of cGMP in the cerebellum elicited by all these excitatory compounds. All these results are compatible with a possible antagonism by PK 26124 of the excitatory amino acid neurotransmission and may explain its anticonvulsant properties.

Biochemical evidence that 2-phenyl-4[(4-piperidinyl) ethyl]quinoline, a quinoline derivative with pure anticonflict properties, is a partial agonist of benzodiazepine receptors.

The atypical profile of 2-phenyl-4[2-(4-piperidinyl) ethyl]quinoline (PK 8165), a quinoline derivative with pure anticonflict properties, seems to be due to the fact that this compound is a partial agonist of benzodiazepine receptors. The drug PK 8165 is a competitive inhibitor of benzodiazepine binding sites with a Hill coefficient near unity. Opposite to 3-methyl-6-(3-trifluoromethylphenyl)2,4-triazolo(4,5-b)pyridazine (CL 218,872) it was unable to discriminate between BZ1 and BZ2 receptors in sections of brain. However, modulation by gamma-aminobutyric acid (GABA) and the effect of photolabelling by flunitrazepam on the affinity of PK 8165 indicated that GABA or photolabelling shifts of PK 8165 were between full agonists and antagonists. By itself PK 8165 was unable to modify the levels of cGMP in the cerebellum, but potentiated the lowering of levels of cGMP by diazepam and did not present antagonistic properties of this effect.

[Methylation of erythrocyte membrane phospholipids: correlation with membrane viscosity. Study of normal and parkinsonian subjects]. / Méthylation des phospholipides de la membrane érythrocytaire: corrélation avec la viscosité membranaire. Etude de sujets normaux et Parkinsoniens.

Plasma membranes from erythrocytes were used to study various parameters: phospholipid methylation, viscosity and fragility. These parameters were analysed in 57 normal subjects (52 +/- 3 years old) and 14 patients with idiopathic Parkinson's disease without previous treatment (71 +/- 2 years old). No significant correlations were observed between the various parameters and sex or age in the normal group. No correlation was observed between fragility and methylation or viscosity. A statistically significant correlation was found between viscosity and phospholipid methylation. In the patients with Parkinson's disease, fragility is identical to the normal group but the viscosity is increased by 25 p. 100. The increase of viscosity is more obvious among the patients with a pure or predominantly akinetic form of the disease. These results favour the previously described correlation between phospholipid methylation and viscosity of the plasma membrane and suggest a modification of these parameters in Parkinson's disease. They also suggest that the increased viscosity observed in Parkinson patients could render the receptors inaccessible to their endogenous ligands.