G protein-coupled receptors (GPCRs) are hard to crystallize. However, attempts to predict their structure have boomed as a result of advancements in crystallographic techniques. This trend has allowed computer-aided molecular modeling of GPCRs. We analyzed the performance of four molecular modeling programs in pose evaluation of re-docked antagonists / inverse agonists to 11 original crystal structures of aminergic GPCRs using an induced fit-docking procedure. AutoDock and Glide were used for docking. AutoDock binding energy function, GlideXP, Prime MM-GB/SA, and YASARA binding function were used for pose scoring. Root mean square deviation (RMSD) of the best pose ranged from 0.09 to 1.58 Å, and median RMSD of the top 60 poses ranged from 1.47 to 3.83 Å. However, RMSD of the top pose ranged from 0.13 to 7.33 Å and ranking of the best pose ranged from the 1st to 60th out of 60 poses. Moreover, analysis of ligand-receptor interactions of top poses revealed substantial differences from interactions found in crystallographic structures. Bad ranking of top poses and discrepancies between top docked poses and crystal structures render current simple docking methods unsuitable for predictive modeling of receptor-ligand interactions. Prime MM-GB/SA optimized for 3NY9 by multiple linear regression did not work well at 3NY8 and 3NYA, structures of the same receptor with different ligands. However, 9 of 11 trajectories of molecular dynamics simulations by Desmond of top poses converged with trajectories of crystal structures. Key interactions were properly detected for all structures. This procedure also worked well for cross-docking of tested β2-adrenergic antagonists. Thus, this procedure represents a possible way to predict interactions of antagonists with aminergic GPCRs.

• Klíčová slova
• Induced-fit docking, Ligand-receptor interaction, Molecular dynamics, Pose scoring,
• MeSH
• krystalografie rentgenová MeSH
• ligandy MeSH
• receptory spřažené s G-proteiny chemie   MeSH
• simulace molekulového dockingu metody   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ligandy MeSH
• receptory spřažené s G-proteiny MeSH

Xanomeline is an agonist endowed with functional preference for M1/M4 muscarinic acetylcholine receptors. It also exhibits both reversible and wash-resistant binding to and activation of these receptors. So far the mechanisms of xanomeline selectivity remain unknown. To address this question we employed microfluorometric measurements of intracellular calcium levels and radioligand binding to investigate differences in the short- and long-term effects of xanomeline among muscarinic receptors expressed individually in Chinese hamster ovary cells. 1/One-min exposure of cells to xanomeline markedly increased intracellular calcium at hM1 and hM4, and to a lesser extent at hM2 and hM3 muscarinic receptors for more than 1 hour. 2/Unlike the classic agonists carbachol, oxotremorine, and pilocarpine 10-min exposure to xanomeline did not cause internalization of any receptor subtype. 3/Wash-resistant xanomeline selectively prevented further increase in intracellular calcium by carbachol at hM1 and hM4 receptors. 4/After transient activation xanomeline behaved as a long-term antagonist at hM5 receptors. 5/The antagonist N-methylscopolamine (NMS) reversibly blocked activation of hM1 through hM4 receptors by xanomeline. 6/NMS prevented formation of xanomeline wash-resistant binding and activation at hM2 and hM4 receptors and slowed them at hM1, hM3 and hM5 receptors. Our results show commonalities of xanomeline reversible and wash-resistant binding and short-time activation among the five muscarinic receptor subtypes. However long-term receptor activation takes place in full only at hM1 and hM4 receptors. Moreover xanomeline displays higher efficacy at hM1 and hM4 receptors in primary phasic intracellular calcium release. These findings suggest the existence of particular activation mechanisms specific to these two receptors.

• MeSH
• AMP cyklický metabolismus   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• časové faktory MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• extracelulární prostor účinky léků   metabolismus   MeSH
• intracelulární prostor účinky léků   metabolismus   MeSH
• kinetika MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin farmakologie   MeSH
• pyridiny farmakologie   MeSH
• receptor muskarinový M1 agonisté   antagonisté a inhibitory   metabolismus   MeSH
• receptor muskarinový M4 agonisté   antagonisté a inhibitory   metabolismus   MeSH
• thiadiazoly farmakologie   MeSH
• vápník metabolismus   MeSH
• vazebná místa účinky léků   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AMP cyklický MeSH
• N-methylskopolamin MeSH
• pyridiny MeSH
• receptor muskarinový M1 MeSH
• receptor muskarinový M4 MeSH
• thiadiazoly MeSH
• vápník MeSH
• xanomeline MeSH Prohlížeč

An allosteric modulator is a ligand that binds to an allosteric site on the receptor and changes receptor conformation to produce increase (positive cooperativity) or decrease (negative cooperativity) in the binding or action of an orthosteric agonist (e.g., acetylcholine). Since the identification of gallamine as the first allosteric modulator of muscarinic receptors in 1976, this unique mode of receptor modulation has been intensively studied by many groups. This review summarizes over 30 years of research on the molecular mechanisms of allosteric interactions of drugs with the receptor and for new allosteric modulators of muscarinic receptors with potential therapeutic use. Identification of positive modulators of acetylcholine binding and function that enhance neurotransmission and the discovery of highly selective allosteric modulators are mile-stones on the way to novel therapeutic agents for the treatment of schizophrenia, Alzheimer's disease and other disorders involving impaired cognitive function.

• Klíčová slova
• Alzheimer’s disease, allosteric modulation, muscarinic acetylcholine receptors, schizophrenia,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

BACKGROUND: Many neuromuscular blockers act as negative allosteric modulators of muscarinic acetylcholine receptors by decreasing affinity and potency of acetylcholine. The neuromuscular blocker rapacuronium has been shown to have facilitatory effects at muscarinic receptors leading to bronchospasm. We examined the influence of rapacuronium on acetylcholine (ACh) binding to and activation of individual subtypes of muscarinic receptors expressed in Chinese hamster ovary cells to determine its receptor selectivity. RESULTS: At equilibrium rapacuronium bound to all subtypes of muscarinic receptors with micromolar affinity (2.7-17 microM) and displayed negative cooperativity with both high- and low-affinity ACh binding states. Rapacuronium accelerated [3H]ACh association with and dissociation from odd-numbered receptor subtypes. With respect to [35S]GTPgammaS binding rapacuronium alone behaved as an inverse agonist at all subtypes. Rapacuronium concentration-dependently decreased the potency of ACh-induced [35S]GTPgammaS binding at M2 and M4 receptors. In contrast, 0.1 microM rapacuronium significantly increased ACh potency at M1, M3, and M5 receptors. Kinetic measurements at M3 receptors showed acceleration of the rate of ACh-induced [35S]GTPgammaS binding by rapacuronium. CONCLUSIONS: Our data demonstrate a novel dichotomy in rapacuronium effects at odd-numbered muscarinic receptors. Rapacuronium accelerates the rate of ACh binding but decreases its affinity under equilibrium conditions. This results in potentiation of receptor activation at low concentrations of rapacuronium (1 microM) but not at high concentrations (10 microM). These observations highlight the relevance and necessity of performing physiological tests under non-equilibrium conditions in evaluating the functional effects of allosteric modulators at muscarinic receptors. They also provide molecular basis for potentiating M3 receptor-mediated bronchoconstriction.

• MeSH
• acetylcholin metabolismus   MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• alosterická regulace účinky léků   MeSH
• alosterické místo účinky léků   MeSH
• CHO buňky MeSH
• Cricetulus MeSH
• guanosin 5'-O-(3-thiotrifosfát) metabolismus   MeSH
• kompetitivní vazba účinky léků   MeSH
• křečci praví MeSH
• N-methylskopolamin metabolismus   MeSH
• nervosvalové látky nedepolarizující farmakologie   MeSH
• radioligandová zkouška metody   MeSH
• receptory muskarinové účinky léků   fyziologie   MeSH
• vekuronium analogy a deriváty   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• acetylcholin MeSH
• agonisté muskarinových receptorů MeSH
• guanosin 5'-O-(3-thiotrifosfát) MeSH
• N-methylskopolamin MeSH
• nervosvalové látky nedepolarizující MeSH
• rapacuronium MeSH Prohlížeč
• receptory muskarinové MeSH
• vekuronium MeSH

We investigated the influence of membrane cholesterol content on preferential and non-preferential signaling through the M(2) muscarinic acetylcholine receptor expressed in CHO cells. Cholesterol depletion by 39% significantly decreased the affinity of M(2) receptors for [(3)H]-N-methylscopolamine ([(3)H]-NMS) binding and increased B(max) in intact cells and membranes. Membranes displayed two-affinity agonist binding sites for carbachol and cholesterol depletion doubled the fraction of high-affinity binding sites. In intact cells it also reduced the rate of agonist-induced receptor internalization and changed the profile of agonist binding from a single site to two affinity states. Cholesterol enrichment by 137% had no effects on carbachol E(max) of cAMP synthesis inhibition and on cAMP synthesis stimulation and inositolphosphates (IP) accumulation at higher agonist concentrations (non-preferred pathways). On the other hand, cholesterol depletion significantly increased E(max) of cAMP synthesis inhibition or stimulation without change in potency, and decreased E(max) of IP accumulation. Noteworthy, modifications of membrane cholesterol had no effect on membrane permeability, oxidative activity, protein content, or relative expression of G(s), G(i/o), and G(q/11) alpha subunits. These results demonstrate distinct changes of M(2) receptor signaling through both preferential and non-preferential G-proteins consequent to membrane cholesterol depletion that occur at the level of receptor/G-protein/effector protein interactions in the cell membrane. The significant decrease of IP accumulation by cholesterol depletion was also observed in cells expressing M(3) receptors and by both cholesterol depletion and enrichment in cells expressing M(1) receptors indicating relevance of reduced G(q/11) signaling for the pathogenesis of Alzheimer's disease.

• MeSH
• acetylcholin analogy a deriváty   MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• beta-cyklodextriny farmakologie   MeSH
• buněčná membrána účinky léků   metabolismus   MeSH
• CHO buňky MeSH
• cholesterol metabolismus   MeSH
• Cricetulus MeSH
• karbachol analogy a deriváty   metabolismus   farmakologie   MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin metabolismus   farmakologie   MeSH
• proteiny vázající GTP metabolismus   MeSH
• receptor muskarinový M2 metabolismus   MeSH
• systémy druhého messengeru * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• acetylcholin MeSH
• antagonisté muskarinových receptorů MeSH
• beta-cyklodextriny MeSH
• cholesterol MeSH
• karbachol MeSH
• methyl-beta-cyclodextrin MeSH Prohlížeč
• N-methylskopolamin MeSH
• proteiny vázající GTP MeSH
• receptor muskarinový M2 MeSH

Allosteric enhancement of the affinity of muscarinic receptors for their ligands offers a new way to influence cholinergic neurotransmission. The structure of the allosteric binding domain(s) and the features of agonists, antagonists and modulators which determine the occurrence of either positive or negative cooperativity require clarification. We tested interactions between allosteric modulators alcuronium, strychnine and brucine and eight antagonists at muscarinic receptors expressed in CHO cells. In experiments with unlabeled antagonists, all three modulators enhanced the affinity for 4-diphenylacetoxy-N-dimethylpiperidinium (4-DAMP) at the M2 receptors, and strychnine did so also at the M4 receptors. Positive interactions were also observed between alcuronium and L-hyoscyamine (M2) and scopolamine (M2), between strychnine and butylscopolamine (M4), L-hyoscyamine (M2 and M4) and scopolamine (M4), and between brucine and scopolamine (M2). Positive effects of alcuronium, strychnine and brucine on the affinity of the M2 receptors for 4-DAMP have been confirmed by direct measurements of the binding of [3H]-4-DAMP. A comparison of molecular models of several antagonists which are esters revealed that antagonists in which the distance between the N and the carboxyl C atoms corresponds to five chemical bonds are more likely to display positive cooperativity with alcuronium at the M2 receptors than the antagonists in which the N-carboxyl C distance corresponds to four chemical bonds.

• MeSH
• alkuronium farmakologie   MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů chemie   farmakologie   MeSH
• CHO buňky MeSH
• křečci praví MeSH
• N-methylskopolamin metabolismus   MeSH
• piperidiny farmakologie   MeSH
• radioligandová zkouška MeSH
• strychnin analogy a deriváty   farmakologie   MeSH
• tritium MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 4-diphenylacetoxy-1,1-dimethylpiperidinium MeSH Prohlížeč
• alkuronium MeSH
• antagonisté muskarinových receptorů MeSH
• brucine MeSH Prohlížeč
• N-methylskopolamin MeSH
• piperidiny MeSH
• strychnin MeSH
• tritium MeSH

1. Muscarinic M(2) receptors normally inhibit the production of cyclic AMP via G(i) proteins, but a stimulatory component occurs in their effect at high agonist concentrations, believed to be based on the activation of G(s) proteins. We investigated the conditions which determine the occurrence and extent of the stimulatory component in CHO cells stably expressing muscarinic M(2) receptors. 2. Biphasic concentration-response curves (decline followed by return towards control values) were obtained after 10 min incubation with carbachol, oxotremorine-M, acetylcholine, arecoline and arecaidine propargyl ester, but the upward phase was missing with oxotremorine, methylfurmethide, furmethide and pentylthio-TZTP. Shortening the incubation favoured the occurrence of the stimulatory component. Carbachol (1 mM) and oxotremorine-M (1 mM) brought about net stimulation (above 100% of control) of cyclic AMP synthesis during 2 min incubations. The stimulatory components disappeared after the density of receptors had been lowered with oxyphenonium mustard. 3. All agonists stimulated the synthesis of cyclic AMP in cells pretreated with pertussis toxin. 4. Most differences between agonists regarding the stimulatory component of their effect on cyclic AMP synthesis could be explained by differences in their efficacy and the induced receptor internalization. 5. We propose that the G(s)-mediated stimulatory component of the effect of muscarinic M(2) receptors on cyclic AMP synthesis only occurs if the density of activated receptors is high enough to saturate the G(i) proteins and proportionate to the receptors' low affinity for the G(s) proteins. It tends to be abolished by receptor internalization.

• MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• AMP cyklický biosyntéza   MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• časové faktory MeSH
• CHO buňky MeSH
• cholerový toxin farmakologie   MeSH
• cholinergní agonisté farmakologie   MeSH
• karbachol farmakologie   MeSH
• křečci praví MeSH
• lékové interakce MeSH
• lidé MeSH
• N-methylskopolamin farmakologie   MeSH
• oxyfenon farmakologie   MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go metabolismus   MeSH
• proteiny vázající GTP - alfa-podjednotky Gs metabolismus   MeSH
• receptor muskarinový M2 MeSH
• receptory muskarinové účinky léků   metabolismus   MeSH
• transfekce MeSH
• tritium MeSH
• vazebná místa MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• AMP cyklický MeSH
• antagonisté muskarinových receptorů MeSH
• cholerový toxin MeSH
• cholinergní agonisté MeSH
• karbachol MeSH
• N-methylskopolamin MeSH
• oxyfenon MeSH
• proteiny vázající GTP - alfa-podjednotky Gi-Go MeSH
• proteiny vázající GTP - alfa-podjednotky Gs MeSH
• receptor muskarinový M2 MeSH
• receptory muskarinové MeSH
• tritium MeSH

Seven esters of alpha-truxillic acid have been synthesized: bis-3-piperidylpropyl ester and its quaternary bis-N-ethyl derivative, bis-N-diethylaminopropyl ester and its quaternary bis-N-methyl derivative, and bis-4-piperidylbutyl ester and its quaternary bis-N-methyl and bis-N-ethyl derivatives. All esters inhibited the specific binding of muscarinic receptor antagonist [methyl-3H]-N-methylscopolamine ([3H]-NMS) to muscarinic receptors in membranes of CHO cell lines stably expressing the human gene for the M1, M2, M3 or M4 subtype of muscarinic receptors. All esters displayed the highest potency at the M2 and the lowest potency at the M3 receptor subtype. In experiments performed on the M2 muscarinic receptor subtype, the affinity between the receptors and the esters was greatly increased when the concentration of ions was diminished. The highest affinities were found for the tertiary bis-3-piperidylpropyl and bis-4-piperidylbutyl aminoesters (equilibrium dissociation constants of 52 and 179 pM, respectively, in the low ionic strength medium). All investigated esters slowed down the dissociation of [3H]-NMS from the M2 muscarinic receptor subtype. [3H]-NMS dissociation from the M1, M3 and M4 muscarinic receptor subtypes was investigated in experiments with the bis-4-piperidylbutyl aminoester and also found to be decelerated. It is concluded that the esters of alpha-truxillic acid act as M2-selective allosteric modulators of muscarinic receptors and that, by their potency, the tertiary bis-3-piperidylpropyl and bis-4-piperidylbutyl aminoesters surpass the other known allosteric modulators of these receptors.

• MeSH
• alosterická regulace MeSH
• antagonisté muskarinových receptorů metabolismus   farmakologie   MeSH
• CHO buňky MeSH
• cyklobutany farmakologie   MeSH
• křečci praví MeSH
• lidé MeSH
• N-methylskopolamin metabolismus   MeSH
• receptory muskarinové klasifikace   účinky léků   MeSH
• tritium MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• antagonisté muskarinových receptorů MeSH
• cyklobutany MeSH
• N-methylskopolamin MeSH
• receptory muskarinové MeSH
• tritium MeSH
• truxillic acid MeSH Prohlížeč

Ligands that bind to the allosteric-binding sites on muscarinic acetylcholine receptors alter the conformation of the classical-binding sites of these receptors and either diminish or increase their affinity for muscarinic agonists and classical antagonists. It is not known whether the resulting conformational change also affects the interaction between the receptors and the G proteins. We have now found that the muscarinic receptor allosteric modulators alcuronium, gallamine, and strychnine (acting in the absence of an agonist) alter the synthesis of cAMP in Chinese hamster ovary (CHO) cells expressing the M2 or the M4 subtype of muscarinic receptors in the same direction as the agonist carbachol. In addition, most of their effects on the production of inositol phosphates in CHO cells expressing the M1 or the M3 muscarinic receptor subtypes are also similar to (although much weaker than) those of carbachol. The agonist-like effects of the allosteric modulators are not observed in CHO cells that have not been transfected with the gene for any of the subtypes of muscarinic receptors. The effects of alcuronium on the formation of cAMP and inositol phosphates are not prevented by the classical muscarinic antagonist quinuclidinyl benzilate. These observations demonstrate for the first time that the G protein-mediated functional responses of muscarinic receptors can be evoked not only from their classical, but also from their allosteric, binding sites. This represents a new mechanism of receptor activation.

• MeSH
• agonisté muskarinových receptorů farmakologie   MeSH
• alkuronium farmakologie   MeSH
• alosterická regulace MeSH
• AMP cyklický metabolismus   MeSH
• antagonisté muskarinových receptorů farmakologie   MeSH
• chinuklidinylbenzilát farmakologie   MeSH
• CHO buňky MeSH
• inositolfosfáty fyziologie   MeSH
• křečci praví MeSH
• periodicita MeSH
• proteiny vázající GTP fyziologie   MeSH
• receptory muskarinové fyziologie   MeSH
• rekombinantní proteiny MeSH
• signální transdukce MeSH
• strychnin farmakologie   MeSH
• transfekce MeSH
• triethojodid gallaminia farmakologie   MeSH
• zvířata MeSH
• Check Tag
• křečci praví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• agonisté muskarinových receptorů MeSH
• alkuronium MeSH
• AMP cyklický MeSH
• antagonisté muskarinových receptorů MeSH
• chinuklidinylbenzilát MeSH
• inositolfosfáty MeSH
• proteiny vázající GTP MeSH
• receptory muskarinové MeSH
• rekombinantní proteiny MeSH
• strychnin MeSH
• triethojodid gallaminia MeSH