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PubMed1996

Sigma receptor activation does not mediate fentanyl-induced attenuation of muscarinic coronary contraction.

Our overall goal was to investigate the mechanism by which fentanyl attenuates acetylcholine-induced contraction in porcine coronary artery. We tested the hypothesis that fentanyl attenuates muscarinic coronary contraction via sigma receptor activation. Left coronary artery vascular rings were isolated from porcine hearts and were suspended in organ chambers for isometric tension recording. In untreated coronary vascular rings, acetylcholine administration resulted in dose-dependent contraction. Fentanyl attenuated acetylcholine-induced contraction. The sigma ligands--(+)-pentazocine, (+)-cyclazocine, haloperidol, and 1,3-di-o-tolylguanidine--also inhibited acetylcholine-induced contraction. In contrast, the selective sigma ligand, (+)-3-(3-hydroxyphenyl)-N-(1-propyl) piperidine failed to have an inhibitory effect on acetylcholine-induced contraction. Moreover, metaphit (1-[1(3-isothiocyanatophenyl)cyclohexyl]piperidine), which causes irreversible acylation of sigma receptors, only inhibited acetylcholine-induced contraction when it was present in the organ chamber. We also assessed the effects of inhibiting various points in the signal transduction pathway distal to naloxone-sensitive opioid receptor activation on acetylcholine-induced contraction. Selective (glybenclamide) and nonselective (tetraethylammonium) K(+)-channel inhibition, guanosine triphosphate-binding protein inactivation (pertussis toxin), and Type 1 and Type 2 dopamine receptor inhibition all failed to alter the attenuating effect of fentanyl on acetylcholine-induced contraction. Thus, neither sigma or opioid receptor activation is a prerequisite for fentanyl-induced inhibition of muscarinic coronary contraction.

Acetylcholine↗
PubMed2000

High morpholine degradation rates and formation of cytochrome P450 during growth on different cyclic amines by newly isolated Mycobacterium sp. strain HE5.

Using morpholine as sole source of carbon, nitrogen and energy, strain HE5 (DSM 44238) was isolated from forest soil. The isolated strain was identified as a member of the subgroup of fast-growing Mycobacterium species as revealed by 16S rDNA analysis. An identity of 99.4% was obtained to Mycobacterium gilvum; however, the type strain was unable to utilize morpholine. A maximal growth rate of 0.17 h(-1) was observed at a morpholine concentration of 30 mM, 30 degrees C and pH 7.2. The substrate was tolerated at concentrations up to 100 mM. Besides morpholine, the strain utilized pyrrolidine, piperidine and proposed intermediates in morpholine metabolism such as glycolate, glyoxylate and ethanolamine. Degradation of morpholine, piperidine and pyrrolidine by resting or permeabilized cells was strictly dependent on the presence of oxygen. Addition of the cytochrome-P450-specific inhibitor metyrapone to the growth medium resulted in a significantly decreased growth rate if these cyclic amines were used as a substrate. Carbon monoxide difference spectra of crude extracts from cells grown on these substrates compared to spectra obtained for extracts of succinate-grown cells indicated that cytochrome P450 is specifically expressed during growth on the cyclic amines. These data indicated that a cytochrome-P450-dependent monooxygenase is involved in the degradation of the three cyclic amines.

Amines↗
PubMed2005

Three trifluoromethyl-substituted protoporphyrinogen IX oxidase inhibitors.

The structures of methyl 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoate, C15H9ClF3N3O5, (I), methyl 2-chloro-5-[3-methyl-2,6-dioxo-4-(trifluoromethyl)-1,2,3,6-tetrahydropyrimidin-1-yl]benzoate, C14H10ClF3N2O4, (II), and 2-[4-chloro-2-fluoro-5-(prop-2-ynyloxy)phenyl]-4-(trifluoromethyl)piperidine-2,6-dione, C15H10ClF4NO3, (III), are similar in their dihedral angles and in the distances between the farthest two atoms. There are two independent molecules in the structure of (I). The dihedral angles between the two aromatic rings in each molecule in (I), between the benzene and tetrahydropyrimidine rings in (II), and between the benzene ring and the five-atom planar portion of the piperidine-2,6-dione ring in (III) are 80.78 (11)/89.75 (11), 89.13 (9) and 87.52 (13) degrees , respectively. The distances between the farthest two atoms, viz. O...F in the two molecules of (I), and Cl...F in (II) and (III), are 11.763 (7)/11.953 (6), 10.734 (10) and 10.889 (9) A, respectively. In all three crystal structures, the molecules are linked to generate sheets of molecules via C-H...O interactions.

Crystallography, X-Ray↗
PubMed2005

Hydrogen-bonding and C-H...pi interactions in 7-hydroxy-3-methoxy-4-methyl-5,6,7,8-tetrahydropyrido[1,2-c]pyrimidin-1(9H)-one.

In the title compound, C10H14N2O3, a pyrimidine ring is fused with a piperidine ring. The pyrimidine ring is planar, whereas the piperidine ring adopts a half-chair conformation. The molecules of the title compound are connected via O-H...O intermolecular hydrogen bonds into infinite zigzag chains. The pyrimidine ring is involved in three C-H...pi interactions, which link the hydrogen-bonded chains into a three-dimensional framework.

Antineoplastic Agents↗
PubMed2005

1-(4-Chlorophenyl)-2-methyl-4-nitro-5-(1-piperidyl)-1H-imidazole.

The only specific interactions that influence the crystal packing of the title compound, C15H17ClN4O2, are weak C-H...N and C-H...Cl hydrogen bonds, even though there is a possibility of, for example, pi-pi stacking or halogen bonding. The dihedral angle between the mean planes of the imidazole and benzene rings is 59.82 (5) degrees . The length of the C-N bond connecting the imidazole and piperidine fragments is correlated with the degree of pyramidalization of the piperidine N atom.

Crystallization↗
PubMed2006

Donepezilium oxalate trihydrate, a therapeutic agent for Alzheimer's disease.

Donepezil, a cholinesterase inhibitor with good central nervous system penetration, has been crystallized as a tertiary amine salt with a disordered oxalate anion to give the title compound, (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-oxoindan-2-yl)methyl]piperidinium hydrogen oxalate trihydrate, C24H30NO3+.C2HO4-.3H2O. The indanone and piperidine ring planes are inclined at an angle of 33.4 (1) degrees. A comparison is made with the piperidinium cation bound in acetylcholinesterase in the solid state. The methylene units bridging the indanone-piperidine-benzyl groups determine the molecular shape and conformational features. The structure is stabilized mainly by O-H...O and N-H...O hydrogen bonds, with water molecules mediating interactions between oxalate anions and donepezilium cations.

Alzheimer Disease↗
PubMed1990

N-methyl-D-aspartate recognition site ligands modulate activity at the coupled glycine recognition site.

In synaptic plasma membranes from rat forebrain, the potencies of glycine recognition site agonists and antagonists for modulating [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding and for displacing strychnine-insensitive [3H]glycine binding are altered in the presence of N-methyl-D-aspartate (NMDA) recognition site ligands. The NMDA competitive antagonist, cis-4-phosphonomethyl-2-piperidine carboxylate (CGS 19755), reduces [3H]glycine binding, and the reduction can be fully reversed by the NMDA recognition site agonist, L-glutamate. Scatchard analysis of [3H]glycine binding shows that in the presence of CGS 19755 there is no change in Bmax (8.81 vs. 8.79 pmol/mg of protein), but rather a decrease in the affinity of glycine (KD of 0.202 microM vs. 0.129 microM). Similar decreases in affinity are observed for the glycine site agonists, D-serine and 1-aminocyclopropane-1-carboxylate, in the presence of CGS 19755. In contrast, the affinity of glycine antagonists, 1-hydroxy-3-amino-2-pyrrolidone and 1-aminocyclobutane-1-carboxylate, at this [3H]glycine recognition site increases in the presence of CGS 19755. The functional consequence of this change in affinity was addressed using the modulation of [3H]TCP binding. In the presence of L-glutamate, the potency of glycine agonists for the stimulation of [3H]TCP binding increases, whereas the potency of glycine antagonists decreases. These data are consistent with NMDA recognition site ligands, through their interactions at the NMDA recognition site, modulating activity at the associated glycine recognition site.

Animals↗
PubMed1974

Relationships between chemical structure and affinity for postganglionic acetylcholine receptors of the guinea-pig ileum.

1 Some phenylacetyl, diphenylacetyl, benziloyl and (+/-)-cyclohexylphenylglycolloyl esters have been made with 2- and 3-hydroxymethylpyrrolidines, 3-hydroxymethyl-N-methylpiperidine, piperidin-3-ols, piperidin-4-ols, 2,2,6,6-tetramethyl-N-methylpiperidin-4-ol, tropine, pseudotropine and quinuclidin-3-ol, and the affinity of these compounds and of their metho- and etho- derivatives has been measured for postganglionic acetylcholine receptors of the guinea-pig isolated ileum.2 Some of the compounds were very active indeed; the benziloyl esters of N-methylpiperidin-4-ol methiodide, tropine methiodide, and quinculidin-3-ol, and the (+/-)-cyclohexylphenylglycolloyl esters of N-methylpiperidin-4-ol and its methiodide had affinity constants greater than 10(10).3 The effects of inserting an additional methylene group onto the nitrogen were extremely variable, ranging from a decrease in log K of 1.64 units to an increase of 0.97 units. The effects of replacing hydrogen by phenyl in the acid portion ranged from an increase of 1.04 units to an increase of 3.06 units and of replacing hydrogen by hydroxyl from a decrease of 0.09 units to an increase of 1.94 units.4 The extent of the variation in the effects of a particular change in structure on affinity does not appear to be any different in these relatively rigid compounds from that observed with the same changes in open-chain aminoalcohols.5 Reasons for the variable effects of groups on affinity are discussed. If differences in effects on preferred conformations of these particular compounds in solution are of secondary importance, the effect of a group on affinity will be the net result of what it could contribute to binding, offset by the disturbance it causes to existing binding. The maximum effect observed in a large number of comparisons may indicate the contribution in the absence of disturbance and for groups containing only carbon and hydrogen it appears to be related to size, assessed from the increments in apparent molal volume at infinite dilution. The variation in the effects of these groups also appears to be related to size. Changes involving groups containing oxygen can produce bigger contributions to binding, and a bigger variation in contribution, than would be expected from their size.6 It is difficult to predict the extent to which groups may fail to produce their maximum effects. Variation is greatest with groups which could produce the biggest changes and so are of the greatest interest.7 The relevance of the results to the successful prediction of biological activity is discussed.

Acetylcholine↗
PubMed1988

The affinity of some acetylenic analogues of 4-DAMP methobromide for muscarinic receptors in guinea-pig ileum and atria.

1. The replacement of 4-hydroxy-N-methyl piperidine (HO NMe) in 4-diphenylacetoxy-N-methyl piperidine (4-DAMP) metho-bromide by 4-hydroxy-but-2-ynylamines (HOCH2C=CCH2NR2) reduces the affinity for muscarine-sensitive acetylcholine receptors in guinea-pig ileum and atria. It does not abolish selectivity. The tertiary amines are more active and more selective than the corresponding quaternary salts. 2. Analogous derivatives of 4-hydroxy-but-2-ynylamines which lack the ester group (i.e. substituted 4-hydroxymethyl-propynyl amines) are less active and less selective. The quaternary compounds are more active than the tertiary bases. 3. The diphenylcarbamyl ester of 4-hydroxy-N-methylpiperidine methobromide has less than one-thousandth of the activity of the diphenylacetyl ester (4-DAMP methobromide) and is not selective. 4. Although 4-diphenylacetoxy-butynyl dimethylamine is only about one-hundredth as active as 4-DAMP methobromide it appears to have comparable selectivity. It is an interesting compound because it is a tertiary amine and should cross membranes.

Animals↗
PubMed1990

Effects of chain-length and unsaturation on affinity and selectivity at muscarinic receptors.

1. Lengthening the chain in diphenylacetylcholine decreases affinity for muscarinic cholinoceptors in guinea-pig ileum. Diphenylacetoxypropyldimethylamine and its quaternary trimethylammonium salt are roughly equiactive: the dimethylamine and the piperidine have some selectivity for ileum compared with atria, but are not as active nor as selective as 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) methobromide (MeBr). With the weaker diphenylacetoxybutyl compounds the base is more active than the quaternary salt. 2. The diphenylacetoxybutyl-, cis-butenyl and trans-butenyl compounds have similar affinities. The quaternary salts are less active than the tertiary bases, but they are less selective than the butynyl analogues studied in earlier work. 3. 1,1-Diphenyl-1-hydroxy-2,4-hexadiynyl dimethylamine and its trimethylammonium salt are inactive in concentrations below 100 microM, as are the (+)-camphor-sulphonyl ester of 4-hydroxy-N-methyl piperidine and its methiodide. The (+/-)-phenylcyclopentylacetyl ester of 4-hydroxy-N-methylpiperidine methobromide is more active than its cyclohexyl analogue and than 4-DAMP MeBr but it is less selective than 4-DAMP MeBr. 4. The high selectivity of p-fluoro-hexahydrosila-diphenidol is confirmed but this compound has relatively low affinity (for ileum log K = 7.8). 5. The results indicate steric constraints to binding at muscarinic receptors which could be used to check molecular modelling of the receptor based on its known amino acid sequence. The group binding the charged nitrogen is probably at the mouth of a cavity which can accommodate two large rings (as in 4-DAMP MeBr) but with a depth less than about 7 A so that the rod-like hexadiynes cannot fit. Differences between types of receptor may only involve small changes in geometry secondary to differences in amino acids not directly involved in binding and the production of selectivity depends upon finding substituents which interfere with binding more at one type of receptor than at another.

Animals↗
PubMed1995

The pharmacology of GR203040, a novel, potent and selective non-peptide tachykinin NK1 receptor antagonist.

1. The in vitro and in vivo pharmacology of GR203040 ((2S, 3S)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), a novel, highly potent and selective non-peptide tachykinin NK1 receptor antagonist, was investigated in the present study. 2. GR203040 potently inhibited [3H]-substance P binding to human NK1 receptors expressed in Chinese hamster ovary (CHO) and U373 MG astrocytoma cells, and NK1 receptors in ferret and gerbil cortex (pKi values of 10.3, 10.5, 10.1 and 10.1 respectively). GR203040 had lower affinity at rat NK1 receptors (pKi = 8.6) and little affinity for human NK2 receptors (pKi < 5.0) in CHO cells and NK3 receptors in guinea-pig cortex (pKi < 6.0). With the exception of the histamine H1 receptor (pIC50 = 7.5). GR203040 had little affinity (pIC50 < 6.0) at all non-NK1 receptors and ion channels examined. Furthermore, GR203040 produced only weak inhibition of Na+ currents in SH-SY5Y neuroblastoma and superior cervical ganglion cells (pIC50 values < 4.0). GR203040 produced only weak antagonism of Ca(2+)-evoked contractions of rat isolated portal vein (pKn = 4.1). The enantiomer of GR203040, GR205608 (2R, 3R)-2-methoxy-5-tetrazol-1-yl-benzyl-(2-phenyl-piperidin-3-y l)-amine), had 10,000 fold lower affinity at the human NK1 receptor expressed in CHO cells (pKi = 6.3). 3. In gerbil ex vivo binding experiments, GR203040 produced a dose-dependent inhibition of the binding of [3H]-substance P to cerebral cortical membranes (ED50 = 15 micrograms kg-1 s.c. and 0.42 mg kg-1 p.o.). At 10 micrograms kg-1 s.c., the inhibition of [3H]-substance P binding was maintained for > 6 h. In the rat, GR203040 was less potent (ED50 = 15.4 mg kg-1 s.c.) probably reflecting, at least in part, its lower affinity at the rat NK1 receptor. 4. In guinea-pig isolated ileum and dog isolated middle cerebral and basilar arteries, GR203040 produced a rightward displacement of the concentration-effect curves to substance P methyl ester (SPOMe) with suppression of the maximum agonist response (apparent pKB values of 11.9, 11.2 and 11.1 respectively). 5. In anaesthetized rabbits, GR203040 antagonized reductions in carotid arterial vascular resistance evoked by SPOMe, injected via the lingual artery (DR10 (i.e. the dose producing a dose-ratio of 10) = 1.1 micrograms kg-1, i.v.). At a dose 20 fold greater than its DR10 value (i.e. 22 micrograms kg-1, i.v.), significant antagonism was evident more than 2 h after GR203040 administration. 6. In anaesthetized rats, GR203040 (3 and 10 mg kg-1, i.v.) produced a dose-dependent inhibition of plasma protein extravasation in dura mater, conjunctiva, eyelid and lip in response to electrical stimulation of the trigeminal ganglion. 7. It is concluded that GR203040 is one of the most potent and selective NK1 receptor antagonists yet described, and as such, has considerable potential as a pharmacological tool to characterize the physiological and pathological roles of substance P and NK1 receptors. GR203040 may also have potential as a novel therapeutic agent for the treatment of conditions such as migraine, emesis and pain.

Animals↗
PubMed1996

Study of SR 142801, a new potent non-peptide NK3 receptor antagonist on cardiovascular responses in conscious guinea-pig.

1. The cardiovascular responses to intravenous (i.v.) injection of natural tachykinins, substance P (SP), neurokinin A (NKA), neurokinin B (NKB) and selective tachykinin (NK) receptor agonists, [Sar9, Met(O2)11]SP, [beta Ala8]NKA(4-10), [MePhe7]NKB and senktide were assessed in conscious, freely moving, guinea-pigs. 2. SP and [Sar9, Met(O2)11]SP (1-1000 pmol kg-1) induced dose-dependent decreases in mean arterial blood pressure (MAP) accompanied by increases in heart rate (HR). NKA evoked only weak hypotensive effects at high doses (3000 pmol kg-1) whereas [beta Ala8]NKA(4-10) (1-3000 pmol kg-1) had no effects. By contrast, NKB [MePhe7]NKB (1-10,000 pmol kg-1) and senktide (1-1000 pmol kg-1), produced dose-related hypertensive effects with the following rank order of potency: senktide > [MePhe7]NKB > NKB. Bradycardia occurred simultaneously with the increases in arterial pressure. 3. The pressor response to intravenous injection of senktide (300 pmol kg-1) was partially reduced by pretreatment with prazosin (0.71 mumol kg-1), or clonidine (0.38 mumol kg-1) and was completely inhibited by the combination of the two compounds. Atropine (1.5 mumol kg-1) suppressed the decrease in HR induced by senktide without altering the blood pressure response. These findings suggest that the blood pressure response to senktide is an indirect effect mediated by noradrenaline released from sympathetic nerve endings, whereas the bradycardia is of vagal reflex origin. 4. SR 142801, ((S)-(N)-(1-(3-(1-benzoyl-3-(3,4-dichlorophenyl) piperidin-3-yl) propyl)-4-phenyl-piperidin-4-yl)-N-methylacetamide), a potent and specific non-peptide NK3 receptor antagonist dose-dependently (0.46-4.6 mumol kg-1, i.v.; 4.6-46 mumol kg-1, p.o.) inhibited the cardiovascular effects of senktide and displayed a long-lasting inhibitory effect after oral administration. By contrast, SR 142806 (4.6 mumol kg-1, i.v.), the (R)-enantiomer of SR 142801 had no effect on the responses to senktide. SR 142801 at a high dose (15 mumol kg-1, i.v.) was inactive toward the [Sar9, Met(O2)11]SP-induced hypotension. 5. SR 142801 did not modify MAP in conscious guinea-pigs both after i.v. (4.6 and 15 mumol kg-1) and oral (46 and 150 mumol kg-1) administration, showing a lack of agonistic properties. However, a slight reduction in HR was observed only after i.v. injection. 6. In conclusion, these results show evident differences in the functional role of tachykinin receptors in the peripheral control of the cardiovascular system. Furthermore, a clear pressor effect of senktide, which was selectively blocked by SR 142801, was observed in conscious guinea-pigs. Hence, this antagonist appears suitable for investigating the functional role of NK3 receptors.

Analysis of Variance↗
PubMed2006

The discovery of tropane-derived CCR5 receptor antagonists.

The development of compound 1, a piperidine-based CCR5 receptor antagonist with Type I CYP2D6 inhibition, into the tropane-derived analogue 5, is described. This compound, which is devoid of CYP2D6 liabilities, is a highly potent ligand for the CCR5 receptor and has broad-spectrum activity against a range of clinically relevant HIV isolates. The identification of human ether a-go-go-related gene channel inhibition within this series is described and the potential for QTc interval prolongation discussed. Furthermore, structure activity relationship (SAR) around the piperidine moiety is also described.

Anti-HIV Agents↗
PubMed2004

Inhibition of epidermal growth factor receptor activity by two pyrimidopyrimidine derivatives.

Overexpression of the epidermal growth factor receptors (EGFRs) and human epidermal growth factor receptor 2 occurs frequently in human cancers and is associated with aggressive tumor behavior and poor patient prognosis. We have investigated the effects in vitro and in vivo of a new class of inhibitor molecules on the growth of several human cancer cell lines. BIBX1382 [N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine] and BIBU1361 [(3-chloro-4-fluoro-phenyl)-[6-(4-diethylaminomethyl-piperidin-1yl)-pyrimido[5,4-d]pyrimidin-4-yl]-amine] are two new selective EGFR kinase inhibitors that do not block the activity of other tyrosine kinases. BIBU1361 blocked epidermal growth factor-induced phosphorylation of EGFR and also prevented downstream responses such as mitogen-activated protein kinase kinase (MAPK/extracellular signal-regulated kinase kinase) and MAPK activation in cells. In accordance with these observations thymidine incorporation into EGFR-expressing KB cells was selectively and potently inhibited by BIBX1382 and BIBU1361 with half-maximally effective doses in the nanomolar range. Oral administration of these compounds inhibited the growth of established human xenografts in athymic mice, including vulval and head and neck squamous cell carcinomas. Tumor growth inhibition by BIBX1382 coincided with reduced pEGFR and Ki-67 levels in vivo, which is in accordance with the expected effect of EGFR inhibitors. Collectively, these results show that the structural class of pyrimidopyrimidines, exemplified here by BIBX1382 and BIBU1361, represents an interesting scaffold for the design of EGFR inhibitors.

Animals↗
PubMed2004

Positive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators have different impact on synaptic transmission in the thalamus and hippocampus.

Earlier studies showed that positive modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors enhance synaptic responses and facilitate synaptic plasticity. Those studies focused mainly on hippocampal functions. However, AMPA receptors have regionally distinct subunit compositions and thus potencies and efficacies of modulators may vary across the brain. The present study compared the effects of CX546 [1-(1,4-benzodioxan-6-ylcarbonyl) piperidine], a benzamide-type modulator, on synaptic transmission in neurons of the reticular thalamic nucleus (RTN), which regulates the firing mode of relay cells in other thalamic nuclei, and on hippocampal CA1 pyramidal cells. CX546 greatly prolonged synaptic responses in CA1 pyramidal cells, but at the same concentration it had only weak modulatory effects in RTN neurons. Effects on miniature excitatory postsynaptic currents (EPSCs) were similar to those on EPSCs in both regions, suggesting that variations in neuronal morphology and transmitter release kinetics do not account for the differences. Relay cells in the ventrobasal thalamus also exhibited weak modulatory effects that were comparable with those in RTN neurons. Regionally different effects on response duration were also observed with CX516 [BDP-12, 1-(quinoxalin-6-ylcarbonyl)piperidine], a second benzamide drug. In contrast, 100 microM cyclothiazide produced comparable synaptic enhancements in hippocampus and RTN. The regional selectivity of benzamide drugs (ampakines) may be explained, at least in part, by a lower potency at thalamic AMPA receptors, perhaps due to the prevalence of the subunits GluR3 and 4. Although regional preferences of the ampakines were modest in their extent, they may be sufficient to be of relevance when considering future therapeutic applications of such compounds.

Animals↗
PubMed2005

Allosteric modulation of the cannabinoid CB1 receptor.

We investigated the pharmacology of three novel compounds, Org 27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide), Org 27759 (3-ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phenyl)-ethyl]-amide), and Org 29647 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt), at the cannabinoid CB1 receptor. In equilibrium binding assays, the Org compounds significantly increased the binding of the CB1 receptor agonist [3H]CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol], indicative of a positively cooperative allosteric effect. The same compounds caused a significant, but incomplete, decrease in the specific binding of the CB1 receptor inverse agonist [3H]SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride], indicative of a limited negative binding cooperativity. Analysis of the data according to an allosteric ternary complex model revealed that the estimated affinity of each Org compound was not significantly different when the radioligand was [3H]CP 55,940 or [3H]SR 141716A. However, the estimated cooperatively factor for the interaction between modulator and radioligand was greater than 1 when determined against [3H]CP 55,940 and less than 1 when determined against [3H]SR 141716A. [3H]CP 55,940 dissociation kinetic studies also validated the allosteric nature of the Org compounds, because they all significantly decreased radioligand dissociation. These data suggest that the Org compounds bind allosterically to the CB1 receptor and elicit a conformational change that increases agonist affinity for the orthosteric binding site. In contrast to the binding assays, however, the Org compounds behaved as insurmountable antagonists of receptor function; in the reporter gene assay, the guanosine 5'-O-(3-[35S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the Emax value for CB1 receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB1 receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands.

Allosteric Regulation↗
PubMed2006

Molecular determinants of HERG channel block.

Drug-induced block of cardiac hERG K+ channels causes acquired long QT syndrome. Here, we characterized the molecular mechanism of hERG block by two low-potency drugs (Nifekalant and bepridil) and two high-potency drugs 1-[2-(6-methyl-2pyridyl)ethyl]-4-(4-methylsulfonyl aminobenzoyl)piperidine (E-4031) and dofetilide). Channels were expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage-clamp technique. All four drugs progressively reduced hERG current during a 20-s depolarization to 0 mV after a 10-min pulse-free period, consistent with the preferential block of open channels. Recovery from block in response to pulses to -160 mV was observed for D540K hERG channels but not for wild-type hERG channels, suggesting that all four drugs are trapped in the central cavity by closure of the activation gate. The molecular determinants of hERG channel block were defined by using a site-directed mutagenesis approach. Mutation to alanine of three residues near the pore helix (Thr623, Ser624, and Val625) and four residues in Ser6 (Gly648, Tyr652, Phe656, and Val659) reduced channel sensitivity to block by dofetilide and E-4031, effects identical with those reported previously for two other methanesulfonanilides, (+)- N -[1' -(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H -1-benzopyran-2,4' -piperidin)-6-yl]-methanesulfonamide] monohydrochloride (MK-499) and ibutilide. The effect of nifekalant on mutant channels was similar, except that V659A retained normal sensitivity and I655A channels were less sensitive. Finally, mutation of the three residues near the pore helix and Phe656 in the Ser6 domain reduced channel block by bepridil. We conclude that the binding site is not identical for all drugs that preferentially block hERG in the open state.

DNA Primers↗
PubMed2006

CB1 receptor antagonism increases hippocampal acetylcholine release: site and mechanism of action.

Evidence indicates that blockade of cannabinoid receptors increases acetylcholine (ACh) release in brain cortical regions. Although it is assumed that this type of effect is mediated through CB1 receptor (CB1R) antagonism, several in vitro functional studies recently have suggested non-CB1R involvement. In addition, neither the precise neuroanatomical site nor the exact mechanisms underlying this effect are known. We thoroughly examined these issues using a combination of systemic and local administration of CB1R antagonists, different methods of in vivo microdialysis, CB1R knockout (KO) mice, tissue measurements of ACh, and immunochemistry. First, we showed that systemic injections of the CB1R antagonists N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR-141716A) and N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) dose-dependently increased hippocampal ACh efflux. Likewise, local hippocampal, but not septal, infusions of SR141716A or AM251 increased hippocampal ACh release. It is noteworthy that the stimulatory effects of systemically administered CB1R antagonists on hippocampal ACh release were completely abolished in CB1R KO mice. CB1R KO mice had similar basal but higher stress-enhanced hippocampal ACh levels compared with wild-type controls. It is interesting that dopamine D1 receptor antagonism counteracted the stimulatory effect of CB1R blockade on hippocampal ACh levels. Finally, immunohistochemical methods revealed that a high proportion of CB1R-positive nerve terminals were found in hippocampus and confirmed the colocalization of CB1 receptors with cholinergic and dopaminergic nerve terminals. In conclusion, hippocampal ACh release may specifically be controlled through CB1Rs located on both cholinergic and dopaminergic neuronal projections, and CB1R antagonism increases hippocampal ACh release, probably through both a direct disinhibition of ACh release and an indirect increase in dopaminergic neurotransmission at the D1 receptors.

Acetylcholine↗