Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia.

Current antipsychotics provide symptomatic relief for patients suffering from schizophrenia and related psychoses; however, their effectiveness is variable and many patients discontinue treatment due to side effects. Although the etiology of schizophrenia is still unclear, a leading hypothesis implicates an imbalanced dopaminergic system. Muscarinic acetylcholine (ACh) receptors regulate dopamine levels in key areas of the brain involved in psychosis, with the M(4) subtype emerging as a key regulator of dopaminergic hyperactivity. Unfortunately, no selective small molecule tools exist to provide pharmacological validation of this hypothesis. Here, we describe the discovery of a small molecule modulator, LY2033298, that is highly selective for human M(4) receptors by virtue of targeting an allosteric site on this receptor. Pharmacological assays confirmed the selectivity of LY2033298 for the M(4) receptor and revealed the highest degree of positive allosteric enhancement of ACh potency thus far identified. Radioligand binding assays also show this compound to directly potentiate agonist binding while having minimal effects on antagonist binding. Mutational analysis identified a key amino acid (D(432)) in the third extracellular loop of the human M(4) receptor to be critical for selectivity and agonist potentiation by LY2033298. Importantly, LY2033298 was active in animal models predictive of clinical antipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent.

Unusual presentation for small lymphocytic lymphoma: Case report of a man with bilateral ear involvement and review of the literature.

INTRODUCTION: Ear involvement by non-Hodgkin lymphoma is quite rare and can be mistaken for other common lesions encountered in otolaryngology. The literature on this subject is also limited. CASE SUMMARY: A 45-year-old man with bilateral ear nodules that progressed over two years. Biopsy of the right ear revealed a B-cell small lymphocytic lymphoma (SLL). The patient responded to radiotherapy well. He received an additional dose two months after the initial treatment because of a remaining nodularity on the right earlobe. After several months, he presented a new lesion on his nasal tip, for which a biopsy confirmed a lymphoma relapse. The patient was managed with oral prednisone and low-dose radiation with a favourable response. DISCUSSION: This case highlights the importance of including lymphoma in the differential diagnosis of ear lesions from an otolaryngology perspective. A biopsy of any lesion or nodule with an atypical course should be considered for appropriate diagnosis and management.

Differential engagement of polar networks in the glucagon-like peptide 1 receptor by endogenous variants of the glucagon-like peptide 1.

The glucagon-like peptide 1 receptor (GLP-1R) can be activated by a number of endogenous peptide hormones, including extended, processed, glycine extended and carboxy-terminally amidated versions of glucagon-like peptide 1 (GLP-1). While the main focus of the literature has been on the processed, amidated form, GLP-1(7-36)NH2, the other forms of this peptide are likely to be secreted in physiologically relevant amounts under certain circumstances. This study builds on our existing work examining the effect of mutation of conserved transmembrane polar residues within the receptor to understand the nature of binding and pleiotropic signaling in response to these alternatively processed versions of this important incretin hormone. We show that extended and processed peptides differ not only in their binding to the receptor but also in the way the receptor is engaged for activation that leads to differential signaling bias exhibited by these peptides.

Cyanotoxins as the "common suspects" for the Dalmatian pelican (Pelecanus crispus) deaths in a Mediterranean reconstructed reservoir.

Toxic cyanobacterial blooms have been implicated for their negative consequences on many terrestrial and aquatic organisms. Water birds belong to the most common members of the freshwater food chains and are most likely to be affected by the consumption of toxic cyanobacteria as food. However, the contribution of cyanotoxins in bird mortalities is under-studied. The aim of the study was to investigate the likely role of cyanotoxins in a mass mortality event of the Dalmatian pelican (Pelecanus crispus) in the Karla Reservoir, in Greece. Water, scum, tissues and stomach content of dead birds were examined for the presence of microcystins, cylindrospermopsins and saxitoxins by an enzyme-linked immunosorbent assay. High abundances of potential toxic cyanobacterial species and significant concentrations of cyanotoxins were recorded in the reservoir water. All examined tissues and stomach content of the Dalmatian pelicans contained significant concentrations of microcystins and saxitoxins. Cylindrospermopsin concentrations were detected in all tissues except from the brain. Our results suggest that cyanotoxins are a plausible cause for this bird mass mortality episode in the Karla Reservoir.

Involvement of the sigma1 (sigma1) receptor in the anti-amnesic, but not antidepressant-like, effects of the aminotetrahydrofuran derivative ANAVEX1-41.

BACKGROUND AND PURPOSE: Tetrahydro-N, N-dimethyl-5, 5-diphenyl-3-furanmethanamine hydrochloride (ANAVEX1-41) is a potent muscarinic and sigma(1) (sigma (1)) receptor ligand. The sigma (1) receptor modulates glutamatergic and cholinergic responses in the forebrain and selective agonists are potent anti-amnesic and antidepressant DRUGS. WE HAVE HERE ANALYSED THE SIGMA (1) COMPONENT IN THE BEHAVIOURAL EFFECTS OF ANAVEX1-41. EXPERIMENTAL APPROACH: Binding of ANAVEX1-41 to muscarinic and sigma (1) receptors were measured using cell membranes. Behavioural effects of ANAVEX1-41 were tested in mice using memory (spontaneous alternation, passive avoidance, water-maze) and antidepressant-like activity (forced swimming) procedures. KEY RESULTS: In vitro, ANAVEX1-41 was a potent muscarinic (M(1)>M(3), M(4)>M(2) with K(i) ranging from 18 to 114 nM) and selective sigma (1) ligand (sigma (1), K(i)=44 nM; sigma (2), K(i)=4 microM). In mice, ANAVEX1-41 failed to affect learning when injected alone (0.03-1 mg kg(-1)), but attenuated scopolamine-induced amnesia with a bell-shaped dose response (maximum at 0.1 mg kg(-1)). The sigma (1) antagonist BD1047 blocked the anti-amnesic effect of ANAVEX1-41 on both short- and long-term memories. Pretreatment with a sigma (1) receptor-directed antisense oligodeoxynucleotide prevented effects of ANAVEX1-41 only in the passive avoidance procedure, measuring long-term memory. ANAVEX1-41 reduced behavioural despair at 30 and 60 mg kg(-1), without involving the sigma (1) receptor, as it was not blocked by BD1047 or the antisense oligodeoxynucleotide. CONCLUSIONS AND IMPLICATIONS: ANAVEX1-41 is a potent anti-amnesic drug, acting through muscarinic and sigma (1) receptors. The latter component may be involved in the enhancing effects of the drug on long-term memory processes.

Risk of tuberculosis in dialysis patients: association of tuberculin and 2,4-dinitrochlorobenzene reactivity with risk of tuberculosis.

BACKGROUND: Dialysis patients are at increased risk of tuberculosis (TB) and anergy due to attenuated cellular immunity. AIM: To define specific risks of TB in anergic and non-anergic dialysis patients. METHODS: A total of 272 dialysis patients were enrolled in this prospective study over a 36-month follow-up. Entering the study, participants had Mantoux and 2,4-dinitrochlorobenzene skin tests and their cell-mediated immunity (CMI)-index was estimated. Patients were classified as anergic (CMI-index

Microglial activation and progressive brain changes in schizophrenia.

Schizophrenia is a debilitating disorder that typically begins in adolescence and is characterized by perceptual abnormalities, delusions, cognitive and behavioural disturbances and functional impairments. While current treatments can be effective, they are often insufficient to alleviate the full range of symptoms. Schizophrenia is associated with structural brain abnormalities including grey and white matter volume loss and impaired connectivity. Recent findings suggest these abnormalities follow a neuroprogressive course in the earliest stages of the illness, which may be associated with episodes of acute relapse. Neuroinflammation has been proposed as a potential mechanism underlying these brain changes, with evidence of increased density and activation of microglia, immune cells resident in the brain, at various stages of the illness. We review evidence for microglial dysfunction in schizophrenia from both neuroimaging and neuropathological data, with a specific focus on studies examining microglial activation in relation to the pathology of grey and white matter. The studies available indicate that the link between microglial dysfunction and brain change in schizophrenia remains an intriguing hypothesis worthy of further examination. Future studies in schizophrenia should: (i) use multimodal imaging to clarify this association by mapping brain changes longitudinally across illness stages in relation to microglial activation; (ii) clarify the nature of microglial dysfunction with markers specific to activation states and phenotypes; (iii) examine the role of microglia and neurons with reference to their overlapping roles in neuroinflammatory pathways; and (iv) examine the impact of novel immunomodulatory treatments on brain structure in schizophrenia.

Assessing the distribution of parameters in models of ligand-receptor interaction: to log or not to log.

It is quite common to see experimental data analysed according to a variety of models of ligand-receptor interaction. Often, parameters derived from such models are compared statistically. The most commonly employed statistical analyses contain explicit assumptions about the underlying distributions of the model parameters being compared, yet the validity of these assumptions is not often ascertained. In this article, Arthur Christopoulos describes a general approach to Monte Carlo simulation of data, and outlines how the analysis of such simulated data may be used to address the question of the distribution of model parameters. The results of such an exercise can guide the researcher to the appropriate choice of statistical test or data transform.

Dynamic mechanisms of non-classical antagonism by competitive AT(1) receptor antagonists.

Selective competitive angiotensin AT(1) receptor antagonists exhibit diverse patterns of antagonism of angiotensin-II-mediated responses in functional assays. These range from the classical parallel rightward shift of agonist concentration-response curves with no depression of the maximum response to an apparently straightforward insurmountable antagonism with complete depression of the maximum response and no rightward shift. This article reviews some earlier equilibrium-based models that have been used to explain the insurmountable antagonism, and suggests that a kinetic model might provide a more satisfactory account of the observations. Such a model might provide deeper insights into the pharmacology of G-protein-coupled receptors than the more popular equilibrium models.

Novel GPCR paradigms at the µ-opioid receptor.

UNLABELLED: Opioids, such as morphine, are the most clinically useful class of analgesic drugs for the treatment of acute and chronic pain. However, the use of opioids is greatly limited by the development of severe adverse side effects. Consequently, drug discovery efforts have been directed towards improving the therapeutic profile of opioid-based treatments. Opioid receptors are members of the family of GPCRs. As such, the recent GPCR paradigms of biased agonism and allosterism may provide novel avenues for more effective analgesics. Biased agonism (or functional selectivity) has been described for all the opioid receptor family members. Furthermore, the first allosteric modulators of opioid receptors have very recently been described. However, identification and quantification of biased agonism in a manner that is informative to medicinal chemists and drug discovery programmes still remains a challenge. In this review, we examine the progress, to date, towards identification and quantification of biased agonism and allosterism at the µ-opioid receptor in the context of its implications for the discovery of better and safer analgesics. LINKED ARTICLES: This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.

Biased allosteric modulation at the CaS receptor engendered by structurally diverse calcimimetics.

BACKGROUND AND PURPOSE: Clinical use of cinacalcet in hyperparathyroidism is complicated by its tendency to induce hypocalcaemia, arising partly from activation of calcium-sensing receptors (CaS receptors) in the thyroid and stimulation of calcitonin release. CaS receptor allosteric modulators that selectively bias signalling towards pathways that mediate desired effects [e.g. parathyroid hormone (PTH) suppression] rather than those mediating undesirable effects (e.g. elevated serum calcitonin), may offer better therapies. EXPERIMENTAL APPROACH: We characterized the ligand-biased profile of novel calcimimetics in HEK293 cells stably expressing human CaS receptors, by monitoring intracellular calcium (Ca(2+) i ) mobilization, inositol phosphate (IP)1 accumulation, ERK1/2 phosphorylation (pERK1/2) and receptor expression. KEY RESULTS: Phenylalkylamine calcimimetics were biased towards allosteric modulation of Ca(2+) i mobilization and IP1 accumulation. S,R-calcimimetic B was biased only towards IP1 accumulation. R,R-calcimimetic B and AC-265347 were biased towards IP1 accumulation and pERK1/2. Nor-calcimimetic B was unbiased. In contrast to phenylalkylamines and calcimimetic B analogues, AC-265347 did not promote trafficking of a loss-of-expression, naturally occurring, CaS receptor mutation (G(670) E). CONCLUSIONS AND IMPLICATIONS: The ability of R,R-calcimimetic B and AC-265347 to bias signalling towards pERK1/2 and IP1 accumulation may explain their suppression of PTH levels in vivo at concentrations that have no effect on serum calcitonin levels. The demonstration that AC-265347 promotes CaS receptor receptor signalling, but not trafficking reveals a novel profile of ligand-biased modulation at CaS receptors The identification of allosteric modulators that bias CaS receptor signalling towards distinct intracellular pathways provides an opportunity to develop desirable biased signalling profiles in vivo for mediating selective physiological responses.

Allosteric modulation of G protein-coupled receptors.

G protein-coupled receptors (GPCRs) constitute the largest receptor superfamily in the human genome and represent the most common targets of drug action. Classic agonist and antagonist ligands that act at GPCRs tend to bind to the receptor's orthosteric site, that is, the site recognized by the endogenous agonist for that receptor. However, it is now evident that GPCRs possess additional, extracellular, allosteric binding sites that can be recognized by a variety of small molecule modulator ligands. Allosteric modulators offer many advantages over classic orthosteric ligands as therapeutic agents, including the potential for greater GPCR-subtype selectivity and safety. However, the manifestations of allosterism at GPCRs are many and varied and, in the past, traditional screening methods have generally failed to detect many allosteric modulators. More recently, there have been a number of major advances in high throughput screening, including the advent of cell-based functional assays, which have led to the discovery of more allosteric modulator ligands than previously appreciated. In addition, a number of powerful analytical techniques have also been developed exclusively for detecting and quantifying allosteric effects, based on an increased awareness of various mechanisms underlying allosteric modulator actions at GPCRs. Together, these advances promise to change the current paucity of GPCR allosteric modulators in the clinical setting and yield novel therapeutic entities for the treatment of numerous disorders.

Pharmacological analysis of cannabinoid receptor activity in the rat vas deferens.

1. The interaction between the cannabinoid agonists, WIN 55,212-2 or CP 55,940 with the CB(1) receptor-selective antagonists, SR141716A or LY320135 was investigated using the rat electrically-stimulated vas deferens bioassay. 2. Tissues were stimulated by single-field pulses (150 V, 0.5 ms) delivered every 30 mins. In the presence of nifedipine (3 microM), agonists elicited a concentration-dependent inhibition of the contractile response, with pEC(50) values of 7.93 and 6.84 for WIN 55,212-2 and CP 55,940, respectively. 3. SR141716A and LY320135 caused parallel dextral displacements of the agonist concentration-response curves. However, the shift of the agonist curves by either antagonist was accompanied by a concentration-dependent enhancement of basal (agonist-independent) tissue contraction. 4. Addition of the amidase inhibitor, phenylmethylsulphonylfluoride (200 microM), resulted in a significant reduction of the basal twitch response, an effect consistent with the presence of tonic receptor activation mediated by the endogenous cannabinoid, anandamide. 5. In light of these findings, we propose a theoretical model of competitive agonist-antagonist interaction in the presence of endogenous agonist tone that was used to derive an optimized analytical approach for the determination of antagonist potency estimates under conditions of tonic receptor activation. 6. This approach yielded pK(B) estimates for SR141716A and LY320135 that were in good agreement with their activity at cannabinoid CB(1) receptors. 7. It is concluded that the rat vas deferens contains prejunctional cannabinoid CB(1) receptors that are under tonic activation from endogenous substances; under these conditions our analytical approach is preferable to the standard methods for the determination of antagonist potency.

Qualitative and quantitative assessment of relative agonist efficacy.

Historically, the ability of a ligand to bind to its receptor and the ability to subsequently activate that receptor have been described as the properties of affinity and intrinsic efficacy, respectively. These properties were originally believed to be independent of one another; both are possessed by ligands classed as "agonists," and they have served as the quantitative foundation of the drug and receptor classification process. Although affinity has been interpreted readily in physicochemical terms, equivalent molecular models for efficacy remain elusive. In recent times, there has been a significant paradigm shift in our understanding of the interrelationship between affinity and intrinsic efficacy, particularly on theoretical grounds, yet the actual methods available to measure these parameters remain largely operational. Nevertheless, a number of approaches, based on both functional measurements and radioligand binding studies, are available to quantify agonist efficacy on a relative scale and, to date, these remain the most practical. This commentary discusses the most common of these methods, their advantages and limitations, the dependence of the expression of agonism on the chosen assay system, and the impact of recent biochemical and molecular biological advances on the study of efficacy. Additionally, some of the more contemporary theories regarding the molecular nature of efficacy are briefly discussed, as well as the caveats that always must be borne in mind when any determinations of relative agonist efficacy are made.

Kinetic studies of co-operativity at atrial muscarinic M2 receptors with an "infinite dilution" procedure.

The effects of two competitive antagonists and two allosteric ligands on the rate of dissociation of [3H]N-methylscopolamine ([3H]NMS) were studied at atrial muscarinic acetylcholine M2 receptors by the technique of "infinite dilution." The dissociation rate for [3H]NMS, initiated by diluting the incubation mixture in a 100-fold volume of buffer, was 0.61 +/- 0.10 min-1. Addition of the competitive antagonists, atropine or NMS, to the dilution medium did not alter the observed [3H]NMS dissociation rate. In contrast, gallamine and the bisquaternary, heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl-ammonium bromide) (C7/3'-phth), produced a concentration-dependent slowing of the dissociation rate of [3H]NMS, with IC50 values of 7.5 microM and 196 nM, respectively. Gallamine exhibited an increased modulatory potency when equilibration with the tissue was allowed prior to dilution. The findings showed that the influence of low concentrations of allosteric modulators on the [3H]NMS dissociation rate may be demonstrated separately from any effects on association rate, and that the contact time with the allosteric ligand may influence the extent of these effects.

Characterization of the subtype selectivity of the allosteric modulator heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl) ammonium bromide (C7/3-phth) at cloned muscarinic acetylcholine receptors.

The present study investigated the interaction between the muscarinic acetylcholine receptor (mAChR) allosteric modulator heptane-1,7-bis-(dimethyl-3'-phthalimidopropyl) ammonium bromide (C(7)/3-phth) and the orthosteric antagonist [3H]N-methylscopolamine ([3H]NMS) at the five cloned human mAChRs expressed in Chinese hamster ovary cells. Equilibrium binding studies, using two different concentrations of radioligand, showed the interaction between C(7)/3-phth and [3H]NMS to be characterized by different degrees of negative cooperativity, depending on the receptor subtype. The modulator exhibited the highest affinity (85 nM) for the unoccupied M2 receptor and the lowest affinity for the unoccupied M5 receptor, the latter being approximately 100-fold lower. In contrast, the highest degree of negative cooperativity was observed at the M5 receptor, whereas lowest negative cooperativity was found at the M1 and M4 receptors. Non-equilibrium dissociation kinetic studies also confirmed the allosteric properties of C(7)/3-phth at all five mAChRs and yielded independent estimates of the modulator affinity for the occupied receptor. The latter estimates showed good agreement with those calculated using parameter values determined from the equilibrium experiments. The present results extend previous findings that C(7)/3-phth is a potent allosteric modulator at mAChRs, particularly the M2 subtype, and also highlight the effects of cooperativity on apparent drug-receptor subtype selectivity.

Interaction of anandamide with the M(1) and M(4) muscarinic acetylcholine receptors.

The M(1) and M(4) muscarinic acetylcholine receptors are the most abundant muscarinic receptor subtypes in the brain, and are involved in learning and memory. Because cannabinoid receptors are also abundantly expressed in similar brain regions and mediate opposite effects to acetylcholine on cognition, the present study investigated whether the endocannabinoid agonist, anandamide, and its metabolically stable derivative, methanandamide, directly modified the binding properties of the human M(1) and M(4) receptors individually expressed in CHO cell membranes. Experiments utilized the antagonists, [(3)H]N-methylscopolamine and [(3)H]quinuclidinyl benzilate. When acetylcholine was used as the inhibiting ligand, shallow, biphasic isotherms were observed at both receptors, characterised by similar apparent dissociation constants for high and low affinity binding at each receptor but with a greater proportion of high affinity sites at the M(4) (40-45%) than at the M(1) receptor (17-20%). In contrast, anandamide and methanandamide inhibited the binding of both radioligands over a narrow (low micromolar) concentration range, with monophasic isotherms characterized by Hill coefficients significantly greater than 1 at both receptors. These effects were not due to the vehicle used. Further saturation binding analyses found anandamide able to significantly reduce the apparent affinity and maximal density of binding sites labeled by [(3)H]quinuclidinyl benzilate. Interestingly, no significant inhibition of radioligand binding was noted using the synthetic cannabinoid agonist, WIN55212-2, or the cannabinoid CB(1) receptor antagonist, SR141716A. These data thus provide evidence for a direct role of anandamides in modulating muscarinic receptor binding properties through a non-competitive mechanism that is unrelated to their actions on cannabinoid receptors.

Regulation of acetylcholine binding by ATP at the muscarinic M(1) receptor in intact CHO cells.

ATP may have a modulatory effect on cholinergic transmission, as it is known that ATP is released as a co-transmitter with acetylcholine from nerve terminals. The ability of ATP to influence the binding of acetylcholine to the M(1) muscarinic acetylcholine receptor expressed in intact CHO cells was investigated. In competition binding experiments, acetylcholine completely inhibited the binding of [3H]N-methylscopolamine, but yielded a shallow competition isotherm that was best described in terms of two affinity states. When these experiments were repeated in the presence of 1 mM ATP, the acetylcholine competition curve was better described in terms of a single, low-affinity state with a Hill slope not significantly different from unity. This modulatory effect of ATP was completely reversed by the addition of the P(2) purinoceptor antagonist, suramin, to the assay medium. When the competition between the muscarinic receptor antagonist, atropine, and [3H]N-methylscopolamine was investigated, however, ATP was unable to modulate the binding of atropine, which was consistent with a one-site binding model in each instance. In contrast to the intact cell studies, ATP did not affect either affinity state of acetylcholine binding when studied in homogenate preparations. The results of the present study indicate that ATP, acting via endogenously expressed purinoceptors, is able to influence agonist binding to the M(1) muscarinic acetylcholine receptor via a cross-talk that requires the functional integrity of intact CHO cells.

[3H]N-methylscopolamine dissociation from muscarine receptors affected by low concentrations of allosteric modulators.

The ability of allosteric ligands to modulate the dissociation rate of [3H]N-methylscopolamine from atrial muscarinic receptors in the presence of varying concentrations of unlabelled N-methylscopolamine or atropine was evaluated. Gallamine, at a concentration approximating its KD value, slowed the dissociation of [3H]N-methylscopolamine in the presence of ca. 30 x KD of both unlabelled NMS or atropine. This was less evident when concentrations of ca. 1000 x KD of the unlabelled antagonists were employed. Similar findings were made with another allosteric modulator. These results indicate that gallamine can act allosterically at low concentrations.

Pharmacological analysis of the mode of interaction of McN-A-343 at atrial muscarinic M2 receptors.

The mode of interaction of the muscarinic M2 receptor partial agonist, McN-A-343 (4-(m-chlorophenylcarbamoyloxy)-2-butynyltrimethylammonium), was investigated in the electrically-driven guinea-pig left atrium. Comparison of the negative inotropic responses to this agent with those of the full agonist, carbachol, followed by operational model-fitting, yielded estimates of the dissociation constant (KA) and efficacy (tau) of McN-A-343. Following partial receptor inactivation with propylbenzylcholine mustard, the properties of McN-A-343 as an inhibitor of the carbachol-mediated responses were assessed. Analysis of the data, according to both competitive and allosteric models of interaction, favored the former model. The log KB of McN-A-343 as an antagonist was - 4.65+/-0.06, and was not significantly different from the log KA of this agent as an agonist.