Effects Of Positive Allosteric Modulators Research Articles

The Effects of Positive Allosteric Modulators of α7-nAChR on Social Play Behavior in Adolescent Rats Prenatally Exposed to Valproic Acid.

There is still no effective treatment that addresses the core symptoms of autism spectrum disorders (ASD), including social and communication deficits. A comprehensive body of evidence points to the cholinergic system, including alpha7-nicotinic acetylcholine receptors (α7-nAChRs), as a potential target of pharmacotherapy. A promising approach is based on positive allosteric modulators (PAMs) of these receptors due to their advantages over direct agonists. Nevertheless, α7 n-AChR ligands have not been widely studied in the context of autism. Therefore, using one of the most widely used rodent models of ASD, that is, prenatal exposure to valproic acid (VPA), we examined the impact of α7-nAChR PAMs (PNU-120596 and CCMI) on socio-communicative behavior during social play in adolescent male and female rats. The current study demonstrated that PAM treatment affected certain aspects of socio-communicative behavior in adolescent rats. Accordingly, PNU-120596 ameliorated deficient play abilities in VPA-exposed males, as revealed by increased play time during a social encounter. In addition, this compound enhanced the emission of ultrasonic vocalizations that accompanied playful interactions. Moreover, we observed the overall effect of PNU-120596 on non-playful forms of social behavior (i.e., social exploration) and acoustic parameters (i.e., the duration) of emitted calls. The present results suggest the ability of α7-nAChR PAMs to facilitate socio-communicative behavior in adolescent rats.

Insights into the Structure-Activity Relationship of Glycosides as Positive Allosteric Modulators Acting on P2X7 Receptors.

P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells). The highest positive modulator activity was with ginsenoside–compound K (CK), containing a monosaccharide (glucose) attached at carbon-20. Ginsenoside-20(S)-Rg3, containing a disaccharide group (glucose-glucose) at carbon-3, displayed positive modulator activity with a reduced EC50 for ATP and increased maximal response at human P2X7. The epimer 20(R)-Rg3 was inactive. A similar stereo-specific pattern was observed for 20(S)-Rh2. Ginsenoside-F1, highly similar to ginsenoside-CK but containing a single additional hydroxyl group, was also inactive at P2X7. Computational docking suggests hydrophobic residues in the pocket are involved in steric discrimination between triterpenoids, whereas the position and identity of the carbohydrate group are important for positive modulator activity at human P2X7. Ginsenosides containing monosaccharide attachments perform better than di- or trisaccharide glycosides. Additional modifications to the triterpenoid scaffold at carbon-6 are not tolerated. Gypenosides from plant sources other than Panax ginseng (gypenoside XVII, gypenoside XLIX, stevenleaf) can also act as positive allosteric modulators of P2X7. We also investigated the effect of positive allosteric modulators on endogenous P2X7 in THP-1 monocytes and confirmed our findings in a calcium response assay. A cell viability assay showed potentiation of ATP-induced cell death with ginsenoside-CK in THP-1 and HEK-hP2X7 cells.SIGNIFICANCE STATEMENTGinsenosides are active as positive allosteric modulators at P2X7, and this study determines the chemical features important for mediating this effect. The position and identity of the sugar group is important for activity, as is the position of a number of hydroxyl groups on the triterpenoid scaffold. Diastereomers of ginsenoside-Rg3 and ginsenoside-Rh2 demonstrate the importance of the location of hydroxyl groups relative to the hydrophobic face of the predicted binding pocket.

P.3.e.005 - The involvement of GABAB receptors in antipsychotic-like effects of positive allosteric modulator of muscarinic acetylcholine M4 receptors

P.3.e.005 - The involvement of GABAB receptors in antipsychotic-like effects of positive allosteric modulator of muscarinic acetylcholine M4 receptors

A Scale of Agonism and Allosteric Modulation for Assessment of Selectivity, Bias, and Receptor Mutation.

An index of agonism is described that can be used to quantify agonist receptor selectivity, bias, cell-based agonism, and the effects of receptor mutation on signaling. The parameter is derived from agonist concentration-response curves and comprises the maximal response to the agonist (max) and the EC50 in the form of Δlog(max/EC50). This parameter is derived from equations describing agonists as positive allosteric facilitators of receptor-signaling protein interaction. A similar index is also derived to quantify the potentiating effects of positive allosteric modulators, which can be used to quantify in situ positive allosteric modulator activity in vivo. These indices lend themselves to statistical analysis and are system-independent in that the effects of the system processing of agonist response and differences in assay sensitivity and receptor expression are cancelled. The various applications of the Δlog(max/EC50) scale are described for each pharmacologic application.

The Role of the M3 Helix in AChR Gating and PAM Action

We are examining the M3 transmembrane helix of mouse muscle AChRs with regard to C↔O gating and the effects of positive allosteric modulators (PAMs). The relative ground and transition state gating energies were estimated from single-channel currents of AChRs having M3 mutations. Diliganded gating rate and equilibrium constants were measured for ∼100 mutations in 5 subunits (α, β, δ, e and γ; 20 mM choline or 500 μM ACh, −100 mV, HEK cells). In general: i) the effects of the mutations were small (≤1 kcal/mol), ii) the effects were similar among subunits, iii) the mutations in different subunits were independent, iv) the mutations have no effect on the energy from the agonist affinity change (≤∼0.3 kcal/mol) and v) most phi values were low (∼0.3). Crystal structures of AChR homologues in detergents suggest that the low phi positions in M3 are interaction sites for PAMs. We investigated the actions of PNU-120596 and IVM (1-1000nM). In WT AChRs (adult and fetal) these ligands increase substantially the frequency and lifetime of constitutive openings in a manner consistent with action at a low-phi position such as in M3. Such potentiation was also apparent at low [agonist] (<1μM ACh or choline), but at higher [agonist] gating kinetics became complex and the potentiation was less clear. The PAMs also increased substantially the frequency and lifetime of constitutive openings in some of the M3 mutants (to W).

Effects of positive allosteric modulators (PAMs) of nicotinic receptors on intracellular Ca2+ concentration [Ca2+]i in mouse embryonic mesencephalic neurons in primary culture

Effects of positive allosteric modulators (PAMs) of nicotinic receptors on intracellular Ca2+ concentration [Ca2+]i in mouse embryonic mesencephalic neurons in primary culture

P.2.024 The effects of positive allosteric modulators of α7 nicotinic receptors on rats' performance in the odour span test

Numerous studies utilizing drug self-administration have shown the importance of conditioned cues in maintaining and reinstating addictive behaviors. However, most used simple cues that fail to replicate the complexity of cues present in human craving and addiction. We have recently shown that music can induce behavioral and neurochemical changes in rats following classical conditioning with psychostimulants. However, such effects have yet to be characterized utilizing operant self-administration procedures, particularly with regard to craving and relapse. The goal of the present study was to validate the effectiveness of music as a contextual conditioned stimulus using cocaine in an operant reinstatement model of relapse. Rats were trained to lever press for cocaine with a musical cue, and were subsequently tested during reinstatement sessions to determine how musical conditioning affected drug seeking behavior. Additionally, in vivo microdialysis was used to determine basolateral amygdala involvement during reinstatement. Lastly, tests were conducted to determine whether the putative anti-addictive agent 18-methoxycoronaridine (18-MC) could attenuate cue-induced drug seeking behavior. Our results show that music-conditioned animals exhibited increased drug seeking behaviors when compared to controls during reinstatement test sessions. Furthermore, music-conditioned subjects exhibited increased extracellular dopamine in the basolateral amygdala during reinstatement sessions. Perhaps most importantly, 18-MC blocked musical cue-induced reinstatement. Thus, music can be a powerful contextual conditioned cue in rats, capable of inducing changes in both brain neurochemistry and drug seeking behavior during abstinence. The fact that 18-MC blocked cue-induced reinstatement suggests that α3β4 nicotinic receptors may be involved in the mechanism of craving, and that 18-MC may help prevent relapse to drug addiction in humans.

Molecular characterization of the AMPA-receptor potentiator S70340 in rat primary cortical culture: Whole-genome expression profiling

To improve our understanding of the molecular events underlying the effects of positive allosteric modulators of the alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid ( S)-AMPA-type glutamate receptors, gene expression profiles of primary cortical culture were measured by Agilent-Microarray technique under ( S)-AMPA (1 μM) stimulation for 0.5, 6, 24 and 48 h in the presence or absence of S70340 (30 μM), an allosteric potentiator of AMPA receptors. ( S)-AMPA and S70340 treatment alone have little effect on gene expression whereas as early as 6 h, their combination induced a large number of genes known to decrease apoptosis and mediate cell survival. Pathway analyses of ( S)-AMPA + S70340 treatment-mediated gene expression from 6 to 48 h further suggested the activation of cellular functions including neuron differentiation and neurite outgrowth. A proportion of genes implicated in these functions encode proteins involved in environmental cues and are expressed in growth cones, such as extracellular matrix component proteins and filopodia microfilament-associated proteins. Time course analysis of mRNA expression combined with in silico promoter analysis revealed an enrichment in the cAMP response element (CRE) among co-regulated genes. This study demonstrated that S70340-mediated AMPA potentialisation activated genes and functional processes involved in neuroprotective and cognitive effects and describes putative new functional biomarkers.

Prevention of ketamine-induced working memory impairments by AMPA potentiators in a nonhuman primate model of cognitive dysfunction

Working memory impairments are a core aspect of schizophrenia, yet current medicines do not address such cognitive dysfunction. We have developed a model of these working memory deficits by acutely disrupting glutamatergic synaptic transmission by administration of the N-methyl- d-aspartate (NMDA) antagonist ketamine in the nonhuman primate. The current studies evaluated the effect of positive allosteric modulators (“potentiators”) of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors on the working memory and behavioral effects of ketamine. AMPA receptors mediate fast excitatory synaptic transmission throughout the brain and play a critical role in the activity-dependent regulation of NMDA receptors. We find that positive modulation of AMPA receptors with LY451646 (0.1–1.0 mg/kg, SC) and structurally distinct PF-4778574 (0.01 mg/kg, SC) robustly ameliorates ketamine-induced working memory impairments without altering behavioral effects of acute ketamine we consider related to positive- and negative-like symptoms. These results support AMPA receptor potentiators as a potential adjunctive treatment for cognitive impairment associated with schizophrenia (CIAS).

Novel positive allosteric modulators of GABAA receptors: Do subtle differences in activity at α1 plus α5 versus α2 plus α3 subunits account for dissimilarities in behavioral effects in rats?

Over the last years, genetic studies have greatly improved our knowledge on the receptor subtypes mediating various pharmacological effects of positive allosteric modulators at GABA A receptors. This stimulated the development of new benzodiazepine (BZ)-like ligands, especially those inactive/low-active at GABA A receptors containing the α 1 subunit, with the aim of generating more selective drugs. Hereby, the affinity and efficacy of four recently synthesized BZ site ligands: SH-053-2′N, SH-053-S-CH3-2′F, SH-053-R-CH3-2′F and JY-XHe-053 were assessed. They were also studied in behavioral tests of spontaneous locomotor activity, elevated plus maze, and water maze in rats, which are considered predictive of, respectively, the sedative, anxiolytic, and amnesic influence of BZs. The novel ligands had moderately low to low affinity and mild to partial agonistic efficacy at GABA A receptors containing the α 1 subunit, with variable, but more pronounced efficacy at other BZ-sensitive binding sites. While presumably α 1 receptor-mediated sedative effects of GABA A modulation were not fully eliminated with any of the ligands tested, only SH-053-2′N and SH-053-S-CH3-2′F, both dosed at 30 mg/kg, exerted anxiolytic effects. The lack of clear anxiolytic-like activity of JY-XHe-053, despite its efficacy at α 2- and α 3-GABA A receptors, may have been partly connected with its preferential affinity at α 5-GABA A receptors coupled with weak agonist activity at α 1-containing subtypes. The memory impairment in water-maze experiments, generally reported with BZ site agonists, was completely circumvented with all four ligands. The results suggest that a substantial amount of activity at α 1 GABA A receptors is needed for affecting spatial learning and memory impairments, while much weaker activity at α 1- and α 5-GABA A receptors is sufficient for eliciting sedation.

Effects of Positive Allosteric Modulators on Single-Cell Oscillatory Ca2+Signaling Initiated by the Type 5 Metabotropic Glutamate Receptor

Agonist stimulation of the type 5 metabotropic glutamate (mGlu5) receptor initiates robust oscillatory changes in cytosolic Ca2+ concentration ([Ca2+]i) in single cells by rapid, repeated cycles of phosphorylation/dephosphorylation of the mGlu5 receptor, involving protein kinase C and as-yet-unspecified protein phosphatase activities. An emergent property of this type of Ca2+ oscillation-generating mechanism (termed "dynamic uncoupling") is that once a threshold concentration has been reached to initiate the Ca2+ oscillation, its frequency is largely insensitive to further increases in orthosteric agonist concentration. Here, we report the effects of positive allosteric modulators (PAMs) on the patterns of single-cell Ca2+ signaling in recombinant and native mGlu5 receptor-expressing systems. In a Chinese hamster ovary cell-line (CHO-lac-mGlu5a), none of the mGlu5 receptor PAMs studied [3,3'-difluorobenzaldazine (DFB), N-{4-chloro-2-[(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) methyl]phenyl}-2-hydroxy-benzamide (CPPHA), 3-cyano-N-(1, 3-diphenyl-1H-prazol-5-yl)benzamide (CDPPB), S-(4-fluoro-phenyl)-{3-[3-(4-fluoro-phenyl)-[1,2,4]oxadiazol-5-yl]-piperidinl-1-yl}-methanone (ADX47273)], stimulated a Ca2+ response when applied alone, but each PAM concentration-dependently increased the frequency, without affecting the amplitude, of Ca2+ oscillations induced by glutamate or quisqualate. Therefore, PAMs can cause graded increases (and negative allosteric modulator-graded decreases) in the Ca2+ oscillation frequency stimulated by orthosteric agonist. Initial data in rat cerebrocortical astrocytes demonstrated that similar effects of PAMs could be observed in a native cell background, although at high orthosteric agonist concentrations, PAM addition could much more often be seen to drive rapid Ca2+ oscillations into peak-plateau responses. These data demonstrate that allosteric modulators can "tune" the Ca2+ oscillation frequency initiated by mGlu5 receptor activation, and this might allow pharmacological modification of the downstream processes (e.g., transcriptional regulation) that is unachievable through orthosteric ligand interactions.

Benzodiazepine receptor ligands modulate ethanol drinking in alcohol-preferring rats

The effects of benzodiazepine receptor ligands with different intrinsic activity profiles were studied on voluntary ethanol consumption in the selectively bred alcohol-preferring AA (Alko, Alcohol) rat line, and compared to those of an opiate antagonist, naloxone, and a serotonin uptake inhibitor, citalopram. The rats were first allowed to develop a strong preference for 10% (v/v) ethanol solution in tap water over plain water until their ethanol consumption stabilized. Thereafter, the period when ethanol solution was available for the rats was gradually reduced to 4 h, 3 times a week, every second working day. The acute effects of positive allosteric modulators (agonists) of the γ-aminobutyric acid type A (GABA A)/benzodiazepine receptor [midazolam, abecarnil, ethyl 5-benzyloxy-4-methoxymethyl-β-carboline-3-carboxylate (ZK 91296), bretazenil, and 2 2,5-dihydro-2-(4-methylphenyl)-3 H-pyrazolo[4,3-C]quinolin-3(5 H)-one (CGS 9895)] and of negative allosteric modulators [inverse agonists, ethyl 8-azido-5,6-dihydro-5-methyl-6-oxo-4 H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate (Ro 15-4513) and t-butyl 5,6-dihydro-5-methyl-6-oxo-4 H-imidazo[1,5-a]thieno[2,3-f][1,4]diazepine-3-carboxylate (Ro 19-4603)] were tested after i.p. injections of three different drug doses using saline injections as a control treatment. The benzodiazepine agonists had rather modest effects on ethanol intake, measured 1 and 4 h after the injections, whereas the inverse agonists and naloxone strongly decreased ethanol consumption. Acute citalopram had no clear effect on ethanol drinking, but it slightly decreased the consumption of novel food during the 4-h session, as did all other benzodiazepine agonists except bretazenil. Neither the inverse agonists nor naloxone had any significant effect on food intake. Three additional groups of rats were repeatedly administered fixed doses of either abecarnil, naloxone (both at 5 mg/kg, i.p.), or saline (1 ml/kg) before five consecutive sessions. Naloxone significantly decreased ethanol consumption at 1-h and 4-h measuring points, whereas abecarnil tended to increase the drinking at 4 h. Food consumption at 4 h was slightly decreased by both compounds. The results suggest that the GABA A receptor may be involved in the regulation of voluntary ethanol drinking in alcohol-preferring AA rats, but do not indicate that these rats would consume ethanol because of its anxiolytic effects in a way that benzodiazepine agonists (full or partial) could substitute for it.