Allosteric Modulation of GPCRs of Class A by Cholesterol.

G-protein coupled receptors (GPCRs) are membrane proteins that convey extracellular signals to the cellular milieu. They represent a target for more than 30% of currently marketed drugs. Here we review the effects of membrane cholesterol on the function of GPCRs of Class A. We review both the specific effects of cholesterol mediated via its direct high-affinity binding to the receptor and non-specific effects mediated by cholesterol-induced changes in the properties of the membrane. Cholesterol binds to many GPCRs at both canonical and non-canonical binding sites. It allosterically affects ligand binding to and activation of GPCRs. Additionally, it changes the oligomerization state of GPCRs. In this review, we consider a perspective of the potential for the development of new therapies that are targeted at manipulating the level of membrane cholesterol or modulating cholesterol binding sites on to GPCRs.

Agonist-Specific Conformations of the M2 Muscarinic Acetylcholine Receptor Assessed by Molecular Dynamics.

Binding of muscarinic ligands, both antagonists and agonists, and their effects on the conformation of the M2 acetylcholine receptor were modeled in silico and compared to experimental data. After docking of antagonists to the M2 receptor in an inactive conformation (3UON, 5ZK3, 5ZKB, or 5ZKB) and agonists in an active conformation (4MQS), 100 ns of conventional molecular dynamics (MD) followed by 500 ns of accelerated MD was run. Conventional MD revealed ligand-specific interactions with the receptor. Antagonists stabilized the receptor in an inactive conformation during accelerated MD. The receptor in complex with various agonists attained different conformations specific to individual agonists. The magnitude of the TM6 movement correlated with agonist efficacy at the non-preferential Gs pathway. The shape of the intracellular opening where the receptor interacts with a G-protein was different for the classical agonist carbachol, super-agonist iperoxo, and Gi/o-biased partial agonists JR-6 and JR-7, being compatible with experimentally observed agonist bias at the G-protein level. Moreover, a wash-resistant binding of the unique agonist xanomeline associated with interactions with membrane lipids was formed during accelerated MD. Thus, accelerated MD is suitable for modeling of ligand-specific receptor binding and receptor conformations that is essential for the design of experiments aimed at identification of the secondary binding sites and understanding molecular mechanisms underlying receptor activation.

Changes in Membrane Cholesterol Differentially Influence Preferential and Non-preferential Signaling of the M1 and M3 Muscarinic Acetylcholine Receptors.

We have found earlier that changes in membrane cholesterol content have distinct impact on signaling via the M1, M2, or M3 receptors expressed in CHO cells (CHO-M1 through CHO-M3). Now we investigated whether gradual changes in membrane cholesterol exerts differential effects on coupling of the M1 and M3 muscarinic receptors to preferential signaling pathways through Gq/11 and non-preferential Gs G-proteins signaling. Changes in membrane cholesterol resulted in only marginal alterations of antagonist and agonist affinity of the M1 and M3 receptors, and did not influence precoupling of either subtype. Changes in membrane cholesterol did not influence parameters of carbachol-stimulated GTP-γ(35)S binding in CHO-M1 membranes while reduction as well as augmentation of membrane cholesterol lowered the efficacy but increased the potency of carbachol in CHO-M3 membranes. Gradual increase or decrease in membrane cholesterol concentration dependently attenuated agonist-induced inositolphosphates release while only cholesterol depletion increased basal values in both cell lines. Similarly, membrane cholesterol manipulation modified basal and agonist-stimulated cAMP synthesis via Gs in the same way in both cell lines. These results demonstrate that changes in membrane cholesterol concentration differentially impact preferential and non-preferential M1 and M3 receptor signaling. They point to the activated G-protein/effector protein interaction as the main site of action in alterations of M1 receptor-mediated stimulation of second messenger pathways. On the other hand, modifications in agonist-stimulated GTP-γ(35)S binding in CHO-M3 membranes indicate that in this case changes in ligand-activated receptor/G-protein interaction may also play a role.

Classical and atypical agonists activate M1 muscarinic acetylcholine receptors through common mechanisms.

We mutated key amino acids of the human variant of the M1 muscarinic receptor that target ligand binding, receptor activation, and receptor-G protein interaction. We compared the effects of these mutations on the action of two atypical M1 functionally preferring agonists (N-desmethylclozapine and xanomeline) and two classical non-selective orthosteric agonists (carbachol and oxotremorine). Mutations of D105 in the orthosteric binding site and mutation of D99 located out of the orthosteric binding site decreased affinity of all tested agonists that was translated as a decrease in potency in accumulation of inositol phosphates and intracellular calcium mobilization. Mutation of D105 decreased the potency of the atypical agonist xanomeline more than that of the classical agonists carbachol and oxotremorine. Mutation of the residues involved in receptor activation (D71) and coupling to G-proteins (R123) completely abolished the functional responses to both classical and atypical agonists. Our data show that both classical and atypical agonists activate hM1 receptors by the same molecular switch that involves D71 in the second transmembrane helix. The principal difference among the studied agonists is rather in the way they interact with D105 in the orthosteric binding site. Furthermore, our data demonstrate a key role of D105 in xanomeline wash-resistant binding and persistent activation of hM1 by wash-resistant xanomeline.

Molecular mechanisms of methoctramine binding and selectivity at muscarinic acetylcholine receptors.

Methoctramine (N,N'-bis[6-[[(2-methoxyphenyl)-methyl]hexyl]-1,8-octane] diamine) is an M(2)-selective competitive antagonist of muscarinic acetylcholine receptors and exhibits allosteric properties at high concentrations. To reveal the molecular mechanisms of methoctramine binding and selectivity we took advantage of reciprocal mutations of the M(2) and M(3) receptors in the second and third extracellular loops that are involved in the binding of allosteric ligands. To this end we performed measurements of kinetics of the radiolabeled antagonists N-methylscopolamine (NMS) in the presence of methoctramine and its precursors, fluorescence energy transfer between green fluorescent protein-fused receptors and an Alexa-555-conjugated precursor of methoctramine, and simulation of molecular dynamics of methoctramine association with the receptor. We confirm the hypothesis that methoctramine high-affinity binding to the M(2) receptors involves simultaneous interaction with both the orthosteric binding site and the allosteric binding site located between the second and third extracellular loops. Methoctramine can bind solely with low affinity to the allosteric binding site on the extracellular domain of NMS-occupied M(2) receptors by interacting primarily with glutamate 175 in the second extracellular loop. In this mode, methoctramine physically prevents dissociation of NMS from the orthosteric binding site. Our results also demonstrate that lysine 523 in the third extracellular loop of the M(3) receptors forms a hydrogen bond with glutamate 219 of the second extracellular loop that hinders methoctramine binding to the allosteric site at this receptor subtype. Impaired interaction with the allosteric binding site manifests as low-affinity binding of methoctramine at the M(3) receptor.

Neuroactive steroids, WIN-compounds and cholesterol share a common binding site on muscarinic acetylcholine receptors.

Endogenous neurosteroids and their synthetic analogues-neuroactive steroids-have been found to bind to muscarinic acetylcholine receptors and allosterically modulate acetylcholine binding and function. Using radioligand binding experiments we investigated their binding mode. We show that neuroactive steroids bind to two binding sites on muscarinic receptors. Their affinity for the high-affinity binding site is about 100 nM. Their affinity for the low-affinity binding site is about 10 µM. The high-affinity binding occurs at the same site as binding of steroid-based WIN-compounds that is different from the common allosteric binding site for alcuronium or gallamine that is located between the second and third extracellular loop of the receptor. This binding site is also different from the allosteric binding site for the structurally related aminosteroid-based myorelaxants pancuronium and rapacuronium. Membrane cholesterol competes with neurosteroids/neuroactive steroids binding to both high- and low-affinity binding site, indicating that both sites are oriented towards the cell membrane..

Current Advances in Allosteric Modulation of Muscarinic Receptors.

Allosteric modulators are ligands that bind to a site on the receptor that is spatially separated from the orthosteric binding site for the endogenous neurotransmitter. Allosteric modulators modulate the binding affinity, potency, and efficacy of orthosteric ligands. Muscarinic acetylcholine receptors are prototypical allosterically-modulated G-protein-coupled receptors. They are a potential therapeutic target for the treatment of psychiatric, neurologic, and internal diseases like schizophrenia, Alzheimer's disease, Huntington disease, type 2 diabetes, or chronic pulmonary obstruction. Here, we reviewed the progress made during the last decade in our understanding of their mechanisms of binding, allosteric modulation, and in vivo actions in order to understand the translational impact of studying this important class of pharmacological agents. We overviewed newly developed allosteric modulators of muscarinic receptors as well as new spin-off ideas like bitopic ligands combining allosteric and orthosteric moieties and photo-switchable ligands based on bitopic agents.

The operational model of allosteric modulation of pharmacological agonism.

Proper determination of agonist efficacy is indispensable in the evaluation of agonist selectivity and bias to activation of specific signalling pathways. The operational model (OM) of pharmacological agonism is a useful means for achieving this goal. Allosteric ligands bind to receptors at sites that are distinct from those of endogenous agonists that interact with the orthosteric domain on the receptor. An allosteric modulator and an orthosteric agonist bind simultaneously to the receptor to form a ternary complex, where the allosteric modulator affects the binding affinity and operational efficacy of the agonist. Allosteric modulators are an intensively studied group of receptor ligands because of their selectivity and preservation of physiological space-time pattern of the signals they modulate. We analysed the operational model of allosterically-modulated agonism (OMAM) including modulation by allosteric agonists. Similar to OM, several parameters of OMAM are inter-dependent. We derived equations describing mutual relationships among parameters of the functional response and OMAM. We present a workflow for the robust fitting of OMAM to experimental data using derived equations.

Novel M2 -selective, Gi -biased agonists of muscarinic acetylcholine receptors.

BACKGROUND AND PURPOSE: More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. EXPERIMENTAL APPROACH: We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M2 receptors was modelled in silico. KEY RESULTS: Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M2 muscarinic receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. CONCLUSIONS AND IMPLICATIONS: The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

M2, M3, and M4 muscarinic receptors are expressed in the guinea pig gallbladder.

AIM: The identity of muscarinic acetylcholine receptors (mAchR) involved in cholinergic-mediated contraction of the guinea pig gallbladder has been a matter of debate. Different groups have suggested the involvement of M(1), M(2), M(3), or M(4) receptor subtypes in the contraction of this tissue. The objective of this study was to identify the mAchR subtypes expressed in the guinea pig gallbladder by RT-PCR. METHODS: Total RNA prepared from frozen guinea pig gallbladder tissue was amplified by using specific primers for the M(1)-M(4) receptor subtypes. RESULTS: M(2), M(3), and M(4) transcripts were detected in the following rank order: M(4) > M(2) > M(3). We were unable to demonstrate the expression of the M(1) receptor subtype in this tissue. CONCLUSIONS: Our results are in agreement with our previous binding and functional data.

Immediate and delayed consequences of xanomeline wash-resistant binding at the M3 muscarinic receptor.

Xanomeline is thought to be a M1/M4 functionally selective agonist at muscarinic receptors. We have previously demonstrated that it binds in a unique manner at the M1 receptor. In the current study, we examined the ability of xanomeline to bind to the M3 receptor and determined the long-term consequences of this mode of binding in Chinese hamster ovary cells expressing M3 receptors. Xanomeline binds in a reversible and wash-resistant manner at the M3 receptor and elicits a functional response under both conditions. Long-term exposure to xanomeline resulted in changes in the binding profile of [(3)H]NMS and a decrease in cell-surface receptor density. Additionally, pretreatment with xanomeline was associated with antagonism of the functional response to subsequent stimulation by conventional agonists. Our results indicate that xanomeline binds to and activates the M3 muscarinic receptor in a wash-resistant manner, and that this type of binding results in time-dependent receptor regulation.

Divergence of allosteric effects of rapacuronium on binding and function of muscarinic receptors.

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.

Mechanisms of M3 muscarinic receptor regulation by wash-resistant xanomeline binding.

BACKGROUND/AIMS: Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect. METHODS: Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments. RESULTS: The above treatment protocol with xanomeline resulted in similar effects of the binding of [(3)H]NMS and [(3)H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent. CONCLUSION: Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.

Analysis of equilibrium binding of an orthosteric tracer and two allosteric modulators.

Allosteric ligands bind to receptors at sites that are distinct from those endogenous agonists and orthosteric pharmacological agents interact with. Both an allosteric and orthosteric ligand bind simultaneously to the receptor to form a ternary complex, where each ligand influences binding affinity of the other to the receptor, either positively or negatively. Allosteric modulators are an intensively studied group of receptor ligands because of their potentially greater selectivity over orthosteric ligands, with the possibility of fine tuning of the effects of endogenous neurotransmitters and hormones. The affinity of an unlabelled allosteric ligand is commonly estimated by measuring its effects on binding of a radio-labelled orthosteric tracer. This scenario is complicated by many folds when one studies the kinetics of interactions of two allosteric agents, added simultaneously, on binding of an orthosteric tracer. In this paper, we provide, for the first time, theoretical basis for analysis of such complex interactions. We have expanded our analysis to include the possibility of having two allosteric modulators interact with the same or different sites on the receptor. An added value of our analysis is to provide a tool to distinguish between the two situations. Finally, we also modelled binding of two molecules of one allosteric modulator to one receptor.

Applications and limitations of fitting of the operational model to determine relative efficacies of agonists.

Proper determination of agonist efficacy is essential in the assessment of agonist selectivity and signalling bias. Agonist efficacy is a relative term that is dependent on the system in which it is measured, especially being dependent on receptor expression level. The operational model (OM) of functional receptor agonism is a useful means for the determination of agonist functional efficacy using the maximal response to agonist and ratio of agonist functional potency to its equilibrium dissociation constant (KA) at the active state of the receptor. However, the functional efficacy parameter τ is inter-dependent on two other parameters of OM; agonist's KA and the highest response that could be evoked in the system by any stimulus (EMAX). Thus, fitting of OM to functional response data is a tricky process. In this work we analyse pitfalls of fitting OM to experimental data and propose a rigorous fitting procedure where KA and EMAX are derived from half-efficient concentration of agonist and apparent maximal responses obtained from a series of functional response curves. Subsequently, OM with fixed KA and EMAX is fitted to functional response data to obtain τ. The procedure was verified at M2 and M4 muscarinic receptors fused with the G15 G-protein α-subunit. The procedure, however, is applicable to any receptor-effector system.

A randomized controlled pilot study of the University of Minnesota mentoring excellence training academy: A hybrid learning approach to research mentor training.

INTRODUCTION: Research mentor training is a valuable professional development activity. Options for training customization (by delivery mode, dosage, content) are needed to address the many critical attributes of effective mentoring relationships and to support mentors in different institutional settings. METHODS: We conducted a pilot randomized controlled trial to evaluate a hybrid mentor training approach consisting of an innovative, 90-minute, self-paced, online module (Optimizing the Practice of Mentoring, OPM) followed by workshops based on the Entering Mentoring (EM) curriculum. Mentors (n = 59) were randomized to intervention or control arms; the control condition was receipt of a two-page mentoring tip sheet. Surveys (pre, post, 3-month follow up) and focus groups assessed training impact (self-appraised knowledge, skills, behavior change) and participants' perceptions of the blended training model. RESULTS: The intervention (∼6.5 hours) produced significant improvements in all outcomes, including skills gains on par with those reported previously for the 8-hour EM model. Knowledge gains and intention-to-change mentoring practices were realized after completion of OPM and augmented by the in-person sessions. Mentors valued the synergy of the blended learning format, noting the unique strengths of each modality and specific benefits to completing a foundational online module before in-person engagement. CONCLUSIONS: Findings from this pilot trial support the value of e-learning approaches, both as standalone curricula or as a component of hybrid implementation models, for the professional development of research mentors.

Synthesis and evaluation of xanomeline analogs--probing the wash-resistant phenomenon at the M1 muscarinic acetylcholine receptor.

A series of xanomeline analogs were synthesized and evaluated for binding at the M(1) muscarinic acetylcholine receptor (M(1) receptor). Specifically, compounds that substitute the O-hexyl chain of xanomeline with polar, ionizable, or conformationally restricted moieties were assessed for their ability to bind to the M(1) receptor in a wash-resistant manner (persistent binding). From our screen, several novel ligands that persistently bind to the M(1) receptor with greater affinity than xanomeline were discovered. Results indicate that persistent binding may arise not only from hydrophobic interactions but also from ionic interactions with a secondary M(1) receptor binding site. Herein, a qualitative model that accounts for both binding scenarios is proposed and applied to understand the structural basis to wash-resistant binding and long-acting effects of xanomeline-based compounds.

Role of membrane cholesterol in differential sensitivity of muscarinic receptor subtypes to persistently bound xanomeline.

Xanomeline (3-(Hexyloxy)-4-(1-methyl-1,2,5,6-tetrahydropyridin-3-yl)-1,2,5-thiadiazole) is a muscarinic agonist that is considered to be functionally selective for the M1/M4 receptor subtypes. Part of xanomeline binding is resistant to washing. Wash-resistant xanomeline activates muscarinic receptors persistently, except for the M5 subtype. Mutation of leucine 6.46 to isoleucine at M1 or M4 receptors abolished persistent activation by wash-resistant xanomeline. Reciprocal mutation of isoleucine 6.46 to leucine at the M5 receptor made it sensitive to activation by wash-resistant xanomeline. Lowering of membrane cholesterol made M1 and M4 mutants and M5 wild type receptors sensitive to activation by wash-resistant xanomeline. Molecular docking revealed a cholesterol binding site in the groove between transmembrane helices 6 and 7. Molecular dynamics showed that interaction of cholesterol with this binding site attenuates receptor activation. We hypothesize that differences in cholesterol binding to this site between muscarinic receptor subtypes may constitute the basis for xanomeline apparent functional selectivity and may have notable therapeutic implications. Differences in receptor-membrane interactions, rather than in agonist-receptor interactions, represent a novel possibility to achieve pharmacological selectivity. Our findings may be applicable to other G protein coupled receptors.

Long-term changes in the muscarinic M1 receptor induced by instantaneous formation of wash-resistant xanomeline-receptor complex.

Unlike other M1 muscarinic acetylcholine receptor agonists, xanomeline demonstrates a unique mode of binding to the receptor. It not only binds reversibly to the receptor's conventional orthosteric site but also binds persistently at a secondary binding domain(s) on the M1 receptor. This results in persistent activation of the receptor even after extensive washout, and allosteric modulation of the orthosteric site. In the current study, we investigated how the effects of very brief exposure (1 min) of intact Chinese hamster ovary cells expressing M1 receptors to xanomeline followed by washout change with time. Pretreatment with xanomeline for 1 min resulted in a concentration-dependent wash-resistant inhibition of [3H]N-methylscopolamine (NMS) binding, with a lower potency than that observed in the continuous presence of xanomeline in the binding assay medium. This effect was associated with wash-resistant receptor activation. Incubation of pretreated and washed cells in control medium for 24 h transformed the monophasic xanomeline wash-resistant binding curve to one that exhibits two distinct potencies. This was the result of the appearance of a new very high-potency binding component without a change in the low-potency state. The delayed effects of persistently bound xanomeline are mainly due to reduction of the maximal binding of [3H]NMS without a change in its affinity. These treatment conditions also reversed persistent receptor activation by xanomeline. Our results imply that brief exposure to xanomeline followed by washing and prolonged waiting may result in delayed receptor desensitization accompanied by internalization or down-regulation.

Binding of N-methylscopolamine to the extracellular domain of muscarinic acetylcholine receptors.

Interaction of orthosteric ligands with extracellular domain was described at several aminergic G protein-coupled receptors, including muscarinic acetylcholine receptors. The orthosteric antagonists quinuclidinyl benzilate (QNB) and N-methylscopolamine (NMS) bind to the binding pocket of the muscarinic acetylcholine receptor formed by transmembrane α-helices. We show that high concentrations of either QNB or NMS slow down dissociation of their radiolabeled species from all five subtypes of muscarinic acetylcholine receptors, suggesting allosteric binding. The affinity of NMS at the allosteric site is in the micromolar range for all receptor subtypes. Using molecular modelling of the M2 receptor we found that E172 and E175 in the second extracellular loop and N419 in the third extracellular loop are involved in allosteric binding of NMS. Mutation of these amino acids to alanine decreased affinity of NMS for the allosteric binding site confirming results of molecular modelling. The allosteric binding site of NMS overlaps with the binding site of some allosteric, ectopic and bitopic ligands. Understanding of interactions of NMS at the allosteric binding site is essential for correct analysis of binding and action of these ligands.