Different Roles of N-Terminal and C-Terminal Domains in Calmodulin for Activation of Bacillus anthracis Edema Factor

Bacillus anthracis adenylyl cyclase toxin edema factor (EF) is one component of the anthrax toxin and is essential for establishing anthrax disease. EF activation by the eukaryotic Ca²⁺-sensor calmodulin (CaM) leads to massive cAMP production resulting in edema. cAMP also inhibits the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, thus reducing production of reactive oxygen species (ROS) used for host defense in activated neutrophils and thereby facilitating bacterial growth. Methionine (Met) residues in CaM, important for interactions between CaM and its binding partners, can be oxidized by ROS. We investigated the impact of site-specific oxidation of Met in CaM on EF activation using thirteen CaM-mutants (CaM-mut) with Met to leucine (Leu) substitutions. EF activation shows high resistance to oxidative modifications in CaM. An intact structure in the C-terminal region of oxidized CaM is sufficient for major EF activation despite altered secondary structure in the N-terminal region associated with Met oxidation. The secondary structures of CaM-mut were determined and described in previous studies from our group. Thus, excess cAMP production and the associated impairment of host defence may be afforded even under oxidative conditions in activated neutrophils.     

The N-terminal Ca2+-independent calmodulin-binding site on the inositol 1,4,5-trisphosphate receptor is responsible for calmodulin inhibition, even though this inhibition requires Ca2+

Calmodulin (CaM) is a ubiquitous Ca(2+)-sensor protein that plays an important role in regulating a large number of Ca(2+) channels, including the inositol 1,4,5-trisphosphate receptor (IP(3)R). CaM binds to the IP(3)R at Ca(2+)-dependent as well as at Ca(2+)-independent interaction sites. In this study, we have investigated the Ca(2+)-independent CaM-binding site for its role in the regulation of the Ca(2+)-dependent bell-shaped activation curve of the IP(3)R. Suramin, a polysulfonated napthylurea, displaced CaM in both the presence and the absence of Ca(2+). Suramin competed with CaM for binding to different peptides representing the previously identified CaM-binding sites on IP(3)R1. By interacting with the N-terminal Ca(2+)-independent CaM-binding site, suramin mimicked the functional effect of CaM and induced an allosteric but competitive inhibition of IP(3) binding. Therefore, suramin also potently inhibited IP(3)-induced Ca(2+) release (IICR) from permeabilized cells predominantly expressing IP(3)R1 (L15 fibroblasts) or IP(3)R3 (Lvec fibroblasts), even though the IP(3)R3 does not contain Ca(2+)-dependent CaM-binding sites. Furthermore, we have found that CaM(1234), a CaM mutated in its four EF hands, inhibited IICR in a Ca(2+)-dependent way with the same potency as CaM. We conclude that CaM inhibits IICR via the N-terminal binding site. The inhibition requires Ca(2+) but CaM itself is not the Ca(2+) sensor for the inhibition of the IP(3)R.     

SB265610 is an allosteric,inverse agonist at the human CXCR2 receptor

Background and purpose:

In several previous studies, the C-X-C chemokine receptor (CXCR)2 antagonist 1-(2-bromo-phenyl)-3-(7-cyano-3H-benzotriazol-4-yl)-urea (SB265610) has been described as binding competitively with the endogenous agonist. This is in contrast to many other chemokine receptor antagonists, where the mechanism of antagonism has been described as allosteric.

Experimental approach:

To determine whether it displays a unique mechanism among the chemokine receptor antagonists, the mode of action of SB265610 was investigated at the CXCR2 receptor using radioligand and [³⁵S]-GTPγS binding approaches in addition to chemotaxis of human neutrophils.

Key results:

In equilibrium saturation binding studies, SB265610 depressed the maximal binding of [¹²⁵I]-interleukin-8 ([¹²⁵I]-IL-8) without affecting the K_d. In contrast, IL-8 was unable to prevent binding of [³H]-SB265610. Kinetic binding experiments demonstrated that this was not an artefact of irreversible or slowly reversible binding. In functional experiments, SB265610 caused a rightward shift of the concentration-response curves to IL-8 and growth-related oncogene α, but also a reduction in maximal response elicited by each agonist. Fitting these data to an operational allosteric ternary complex model suggested that, once bound, SB265610 completely blocks receptor activation. SB265610 also inhibited basal [³⁵S]-GTPγS binding in this preparation.

Conclusions and implications:

Taken together, these data suggest that SB265610 behaves as an allosteric inverse agonist at the CXCR2 receptor, binding at a region distinct from the agonist binding site to prevent receptor activation, possibly by interfering with G protein coupling.     

Changes in soluble calmodulin following activation of dopamine receptors in rat striatal slices.

Various molecular forms of cyclic nucleotide phosphodiesterase (PDE) are present in the striatum of rats. While Ca²⁺ by itself cannot modulate striatal PDE, this ion is essential for the activation of striatal PDE by calmodulin (CaM). Incubation of striatal slices with apomorphine (10^?7 M) for 30 min increased the total CaM content of the supernatant fraction. Also the amount of CaM associated with PDE was increased and the K_m of PDE for cAMP was lowered. A shorter incubation with dopamine or apomorphine (10 min) failed to increase CaM and to lower the K_m of PDE.Haloperidol (10^?7 M), a dopamine receptor antagonist, prevented the change in the kinetic profile of PDE elicited by dopamine (2 × 10^?7M). Transection of the nigra-striatal fibre bundle by itself did not change the kinetic profile of striatal PDE, but in slices prepared from deafferented striata, a 30 min activation of dopamine receptors still elicited a decrease in the K_m of PDE for cAMP. These findings suggest that following a persistent stimulation of dopamine receptors, the CaM content increases in the cytosol because it is mobilized from a pool located in post-synaptic membranes. This mobilization of CaM regulates PDE; thus, regulation of PDE through a translocation of CaM may participate in reducing the functional output of dopamine receptors following persistent stimulation.     

Allosteric modulators of the hERG K channel: Radioligand binding assays reveal allosteric characteristics of dofetilide analogs

Drugs that block the cardiac K⁺ channel encoded by the human ether-à-go-go gene (hERG) have been associated with QT interval prolongation leading to proarrhythmia, and in some cases, sudden cardiac death. Because of special structural features of the hERG K⁺ channel, it has become a promiscuous target that interacts with pharmaceuticals of widely varying chemical structures and a reason for concern in the pharmaceutical industry. The structural diversity suggests that multiple binding sites are available on the channel with possible allosteric interactions between them. In the present study, three reference compounds and nine compounds of a previously disclosed series were evaluated for their allosteric effects on the binding of [³H]astemizole and [³H]dofetilide to the hERG K⁺ channel. LUF6200 was identified as an allosteric inhibitor in dissociation assays with both radioligands, yielding similar EC₅₀ values in the low micromolar range. However, potassium ions increased the binding of the two radioligands in a concentration-dependent manner, and their EC₅₀ values were not significantly different, indicating that potassium ions behaved as allosteric enhancers. Furthermore, addition of potassium ions resulted in a concentration-dependent leftward shift of the LUF6200 response curve, suggesting positive cooperativity and distinct allosteric sites for them. In conclusion, our investigations provide evidence for allosteric modulation of the hERG K⁺ channel, which is discussed in the light of findings on other ion channels.     

A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands

BACKGROUND AND PURPOSE

The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance.

EXPERIMENTAL APPROACH

Using [³H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc.

KEY RESULTS

Kinetic characterization of maraviroc binding to the wild-type CCR5 was consistent with a two-step kinetic mechanism that involved an initial receptor–ligand complex (RA), which transitioned to a more stable complex, R''A, with at least a 13-fold increase in affinity. The dissociation rate from R''A, k₋₂, was 1.2 × 10⁻³ min⁻¹. The maraviroc time-dependent transition was influenced by F85L, W86A, Y108A, I198A and Y251A mutations of CCR5.

CONCLUSIONS AND IMPLICATIONS

The interaction between maraviroc and CCR5 proceeded according to a multi-step kinetic mechanism, whereby initial mass action binding and later reorganizations of the initial maraviroc–receptor complex lead to a complex with longer residence time. Site-directed mutagenesis identified a kinetic fingerprint of residues that affected the binding kinetics, leading to the conclusion that allosteric ligand binding to CCR5 involved the rearrangement of the binding site in a manner specific to each allosteric ligand.     

Ca2+-calmodulin can activate and inactivate cardiac ryanodine receptors

Background and purpose:

Ca²⁺-calmodulin (Ca²⁺CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated.

Experimental approach:

We investigated the effects of low concentrations of Ca²⁺CaM (50–100 nmol·L⁻¹) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol·L⁻¹ CaM on sarcoplasmic reticulum (SR) Ca²⁺-release.

Key results:

Ca²⁺CaM activated or inhibited single RyR2, but activation was much more likely at low (50–100 nmol·L⁻¹) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol·L⁻¹ CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca²⁺-release events. Application of caffeine demonstrated that 50 nmol·L⁻¹ CaM reduced SR Ca²⁺ content.

Conclusions and implications:

We describe for the first time how Ca²⁺CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca²⁺-release and the use of caffeine suggests that this is a RyR2-mediated effect.     

Small-Molecule Allosteric Activation of Human Glucokinase in the Absence of Glucose

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Molecular mechanism of positive allosteric modulation of the metabotropic glutamate receptor 2 by JNJ‐46281222

Background and Purpose

Allosteric modulation of the mGlu₂ receptor is a potential strategy for treatment of various neurological and psychiatric disorders. Here, we describe the in vitro characterization of the mGlu₂ positive allosteric modulator (PAM) JNJ‐46281222 and its radiolabelled counterpart [³H]‐JNJ‐46281222. Using this novel tool, we also describe the allosteric effect of orthosteric glutamate binding and the presence of a bound G protein on PAM binding and use computational approaches to further investigate the binding mode.

Experimental Approach

We have used radioligand binding studies, functional assays, site‐directed mutagenesis, homology modelling and molecular dynamics to study the binding of JNJ‐46281222.

Key Results

JNJ‐46281222 is an mGlu₂‐selective, highly potent PAM with nanomolar affinity (K _D = 1.7 nM). Binding of [³H]‐JNJ‐46281222 was increased by the presence of glutamate and greatly reduced by the presence of GTP, indicating the preference for a G protein bound state of the receptor for PAM binding. Its allosteric binding site was visualized and analysed by a computational docking and molecular dynamics study. The simulations revealed amino acid movements in regions expected to be important for activation. The binding mode was supported by [³H]‐JNJ‐46281222 binding experiments on mutant receptors.

Conclusion and Implications

Our results obtained with JNJ‐46281222 in unlabelled and tritiated form further contribute to our understanding of mGlu₂ allosteric modulation. The computational simulations and mutagenesis provide a plausible binding mode with indications of how the ligand permits allosteric activation. This study is therefore of interest for mGlu₂ and class C receptor drug discovery.

Abbreviations

JNJ‐46281222

3‐(Cyclopropylmethyl)‐7‐[(4‐phenyl‐1‐piperidinyl)methyl]‐8‐(trifluoromethyl)‐1,2,4‐triazolo[4,3‐a]pyridine

NAM

negative allosteric modulator

PAM

positive allosteric modulator

VFT

Venus Flytrap domain

     

Real-time characterization of cannabinoid receptor 1 (CB1) allosteric modulators reveals novel mechanism of action

BACKGROUND AND PURPOSE

The cannabinoid receptor type 1 (CB₁) has an allosteric binding site. The drugs {"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593"}}ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(−)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB₁, we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators.

EXPERIMENTAL APPROACH

Studies of CB₁ signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB₁. We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB₁ internalization.

KEY RESULTS

{"type":"entrez-protein","attrs":{"text":"ORG27569","term_id":"1179174593"}}ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB₁-mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization.

CONCLUSIONS AND IMPLICATIONS

Contrary to previous studies characterizing allosteric modulators at CB_1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.     

Influence of neuroleptics on the binding of met-enkephalin, morphine and dihydromorphine to synaptosome-enriched fractions of rat brain.

The influence of three neuroleptics on the binding of tritiated met-enkephalin to synaptosomeenriched fractions of rat brain was studied in the high-affinity binding region. All three drugs tested blocked the binding of met-enkephalin in a dose-dependent fashion with inhibition constants (k_i) of 11.6, 39.5 and 29.5 μM for haloperidol, chlorpromazine and prochlorperazine, respectively. Hill plots suggest that the inhibitory mechanism involves negative allosteric effects between neuroleptic molecules. The effects of these neuroleptics on the binding of tritiated morphine and dihydromorphine were also studied.     

Development of the fluorescent biosensor hCalmodulin (hCaM)L39C-monobromobimane(mBBr)/V91C-mBBr, a novel tool for discovering new calmodulin inhibitors and detecting calcium

A novel, sensible, and specific fluorescent biosensor of human calmodulin (hCaM), namely hCaM L39C-mBBr/V91C-mBBr, was constructed. The biosensor was useful for detecting ligands with opposing fluorescent signals, calcium ions (Ca(2+)) and CaM inhibitors in solution. Thus, the device was successfully applied to analyze the allosteric effect of Ca(2+) on trifluoroperazine (TFP) binding to CaM (Ca(2+)K(d) = 0.24 μM ± 0.03 with a stoichiometry 4.10 ± 0.15; TFPK(d) ～ 5.74-0.53 μM depending on the degree of saturation of Ca(2+), with a stoichiometry of 2:1). In addition, it was suitable for discovering additional xanthones (5, 6, and 8) with anti-CaM properties from the fungus Emericella 25379. The affinity of 1-5, 7, and 8 for the complex (Ca(2+))(4)-CaM was excellent because their experimental K(d)s were in the nM range (4-498 nM). Docking analysis predicted that 1-8 bind to CaM at sites I, III, and IV as does TFP.     

Inhibition of nitric oxide-activated guanylyl cyclase by calmodulin antagonists

Background and purpose:

Nitric oxide (NO) controls numerous physiological processes by activation of its receptor, guanylyl cyclase (sGC), leading to the accumulation of 3′-5′ cyclic guanosine monophosphate (cGMP). Ca²⁺-calmodulin (CaM) regulates both NO synthesis by NO synthase and cGMP hydrolysis by phosphodiesterase-1. We report that, unexpectedly, the CaM antagonists, calmidazolium, phenoxybenzamine and trifluoperazine, also inhibited cGMP accumulation in cerebellar cells evoked by an exogenous NO donor, with IC₅₀ values of 11, 80 and 180 µM respectively. Here we sought to elucidate the underlying mechanism(s).

Experimental approach:

We used cerebellar cell suspensions to determine the influence of CaM antagonists on all steps of the NO-cGMP pathway. Homogenized tissue and purified enzyme were used to test effects of calmidazolium on sGC activity.

Key results:

Inhibition of cGMP accumulation in the cells did not depend on changes in intracellular Ca²⁺ concentration. Degradation of cGMP and inactivation of NO were both inhibited by the CaM antagonists, ruling out increased loss of cGMP or NO as explanations. Instead, calmidazolium directly inhibited purified sGC (IC₅₀= 10 µM). The inhibition was not in competition with NO, nor did it arise from displacement of the haem moiety from sGC. Calmidazolium decreased enzyme V_max and K_m, indicating that it acts in an uncompetitive manner.

Conclusions and implications:

The disruption of every stage of NO signal transduction by common CaM antagonists, unrelated to CaM antagonism, cautions against their utility as pharmacological tools. More positively, the compounds exemplify a novel class of sGC inhibitors that, with improved selectivity, may be therapeutically valuable.     

Mechanism of allosteric activation of SIRT6 revealed by the action of rationally designed activators

The recent discovery of activator compounds binding to an allosteric site on the NAD⁺-dependent protein lysine deacetylase, sirtuin 6 (SIRT6) has attracted interest and presents a pharmaceutical target for aging-related and cancer diseases. However, the mechanism underlying allosteric activation of SIRT6 by the activator MDL-801 remains largely elusive because no major conformational changes are observed upon activator binding. By combining molecular dynamics simulations with biochemical and kinetic analyses of wild-type SIRT6 and its variant M136A, we show that conformational rotation of 2-methyl-4-fluoro-5-bromo substituent on the right phenyl ring (R-ring) of MDL-801, which uncovers previously unseen hydrophobic interactions, contributes to increased activating deacetylation activity of SIRT6. This hypothesis is further supported by the two newly synthesized MDL-801 derivatives through the removal of the 5-Br atom on the R-ring (MDL-801-D1) or the restraint of the rotation of the R-ring (MDL-801-D2). We further propose that the 5-Br atom serves as an allosteric driver that controls the ligand allosteric efficacy. Our study highlights the effect of allosteric enzyme catalytic activity by activator binding and provides a rational approach for enhancing deacetylation activity.KEY WORDS: Allosteric driver, Allosteric sites, Allosteric mechanisms, Drug design, Enzyme catalysis, Protein dynamics     

Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7

BACKGROUND AND PURPOSE

The mGlu₇ receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu₇ monoclonal antibody, MAB1/28, that triggers receptor internalization.

EXPERIMENTAL APPROACH

MAB1/28''s activity was investigated using Western blot and direct immunofluorescence on live cells, in vitro pharmacology by functional cAMP and [³⁵S]-GTPγ binding assays, the kinetics of IgG-induced internalization by image analysis, and the activation of the ERK1/2 by elisa.

KEY RESULTS

mGlu₇/mGlu₆ chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu₇. MAB1/28 potently antagonized both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation. The potency of the antagonistic actions was similar to the potency in triggering receptor internalization. The internalization mechanism occurred via a pertussis toxin-insensitive pathway and did not require Gα_i protein activation. MAB1/28 activated ERK1/2 with potency similar to that for receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu₇ dimers.

CONCLUSIONS AND IMPLICATIONS

We obtained evidence for an allosteric-biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu₇ function and selective activation of its intracellular trafficking.     

Analysis of the actions of the novel dopamine receptor-directed compounds (S)-OSU6162 and ACR16 at the D2 dopamine receptor

BACKGROUND AND PURPOSE

The two phenylpiperidines, OSU6162 and ACR16, have been proposed as novel drugs for the treatment of brain disorders, including schizophrenia and Huntington''s disease, because of their putative dopamine stabilizing effects. Here we evaluated the activities of these compounds in a range of assays for the D₂ dopamine receptor in vitro.

EXPERIMENTAL APPROACH

The affinities of these compounds for the D₂ dopamine receptor were evaluated in competition with [³H]spiperone and [³H]NPA. Agonist activity of these compounds was evaluated in terms of their ability to stimulate [³⁵S]GTPγS binding.

KEY RESULTS

Both compounds had low affinities for inhibition of [³H]spiperone binding (pK_i vs. [³H]spiperone, ACR16: <5, OSU6162: 5.36). Neither compound was able to stimulate [³⁵S]GTPγS binding when assayed in the presence of Na⁺ ions, but if the Na⁺ ions were removed, both compounds were low-affinity, partial agonists (E_max relative to dopamine: ACR16: 10.2%, OSU6162:54.3%). Schild analysis of the effects of OSU6162 to inhibit dopamine-stimulated [³⁵S]GTPγS binding indicated Schild slopes of ∼0.9, suggesting little deviation from competitive inhibition. OSU6162 was, however, able to accelerate [³H]NPA dissociation from D₂ dopamine receptors, indicating some allosteric effects of this compound.

CONCLUSIONS AND IMPLICATIONS

The two phenylpiperidines were low-affinity, low-efficacy partial agonists at the D₂ dopamine receptor in vitro, possibly exhibiting some allosteric effects. Comparing their in vitro and in vivo effects, the in vitro affinities were a reasonable guide to potencies in vivo. However, the lack of in vitro–in vivo correlation for agonist efficacy needs to be further addressed.     

Allosteric interactions at adenosine A1 and A3 receptors: new insights into the role of small molecules and receptor dimerization

The purine nucleoside adenosine is present in all cells in tightly regulated concentrations. It is released under a variety of physiological and pathophysiological conditions to facilitate protection and regeneration of tissues. Adenosine acts via specific GPCRs to either stimulate cyclic AMP formation, as exemplified by G_s-protein-coupled adenosine receptors (A_2A and A_2B), or inhibit AC activity, in the case of G_i/o-coupled adenosine receptors (A₁ and A₃). Recent advances in our understanding of GPCR structure have provided insights into the conformational changes that occur during receptor activation following binding of agonists to orthosteric (i.e. at the same binding site as an endogenous modulator) and allosteric regulators to allosteric sites (i.e. at a site that is topographically distinct from the endogenous modulator). Binding of drugs to allosteric sites may lead to changes in affinity or efficacy, and affords considerable potential for increased selectivity in new drug development. Herein, we provide an overview of the properties of selective allosteric regulators of the adenosine A₁ and A₃ receptors, focusing on the impact of receptor dimerization, mechanistic approaches to single-cell ligand-binding kinetics and the effects of A₁- and A₃-receptor allosteric modulators on in vivo pharmacology.Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5     

Pharmacological characterization of M1 muscarinic acetylcholine receptor-mediated Gq activation in rat cerebral cortical and hippocampal membranes

This study aimed to pharmacologically characterize the response derived from functional activation of Gq proteins coupled with native muscarinic acetylcholine receptors in rat cerebral cortex and hippocampus. Rat cerebral cortical and hippocampal membranes were prepared, and the effects of a range of mAChR agonists and antagonists, allosteric modulators, and muscarinic toxins were determined by an antibody-capture scintillation proximity assay combined with [³⁵S]GTPγS binding, using the anti-Gα_q antibody sc-393. Increased specific [³⁵S]GTPγS binding, elicited by carbachol (CCh), was selectively inhibited by the muscarinic toxin MT7, and was resistant to membrane pretreatment with N-ethylmaleimide, indicating that the response derived exclusively from Gα_q, selectively coupled with the M₁ mAChR. In addition to CCh, many mAChR agonists, including oxotremorine, arecholine, and methacholine, stimulated binding in a concentration-dependent manner with varied potencies and efficacies. The intrinsic activities of partial M₁ mAChR agonists in the present study were generally lower than previously reported in M₁-expressing cells. Xanomeline and N-desmethylclozapine had negligible or minimal agonist properties. CCh-stimulated [³⁵S]GTPγS binding to Gα_q was inhibited by mAChR antagonists, including scopolamine, ipratropium, atropine, 4-DAMP, pirenzepine, and AF-DX 116, with a rank order of potency consistent with previous studies of M₁-expressing cells. There was a highly significant correlation between the potencies of 13 agonists and 19 antagonists in the cerebral cortex and hippocampus. The effects of several allosteric mAChR modulators were also investigated. These data provide a comprehensive pharmacological profile of the G_q-coupled M₁ mAChR subtype natively expressed at physiological levels in rat cerebral cortex and hippocampus.     

Mechanism of action of species-selective P2X7 receptor antagonists

Background and purpose:

AZ11645373 and N-{2-methyl-5-[(1R, 5S)-9-oxa-3,7-diazabicyclo[3.3.1]non-3-ylcarbonyl]phenyl}-2-tricyclo[3.3.1.13,7]dec-1-ylacetamide hydrochloride (compound-22) are recently described P2X₇ receptor antagonists. In this study we have further characterized these compounds to determine their mechanism of action and interaction with other species orthologues.

Experimental approach:

Antagonist effects at recombinant and chimeric P2X₇ receptors were assessed by ethidium accumulation and radioligand-binding studies.

Key results:

AZ11645373 and compound-22 were confirmed as selective non-competitive antagonists of human or rat P2X₇ receptors respectively. Both compounds were weak antagonists of the mouse and guinea-pig P2X₇ receptors and, for each compound, their potency estimates at human and dog P2X₇ receptors were similar. The potency of compound-22 was moderately temperature-dependent while that of AZ11645373 was not. The antagonist effects of both compounds were slowly reversible and were not prevented by decavanadate, suggesting that they were allosteric antagonists. Indeed, the compounds competed for binding sites labelled by an allosteric radio-labelled P2X₇ receptor antagonist. The species selectivity of AZ11645373, but not compound-22, was influenced by the nature of the amino acid at position 95 of the P2X₇ receptor. N²-(3,4-difluorophenyl)-N¹-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride, a positive allosteric modulator of the rat receptor, reduced the potency of compound-22 at the rat receptor but had little effect on the actions of AZ11645373.

Conclusions:

AZ11645373 and compound-22 are allosteric antagonists of human and rat P2X₇ receptors respectively. The differential interaction of the two compounds with the receptor suggests there may be more than one allosteric regulatory site on the P2X₇ receptor at which antagonists can bind and affect receptor function.