Agonist EC50 Research Articles

Clinical and functional consequences of GRIA variants in patients with neurological diseases.

AMPA receptors are members of the glutamate receptor family and mediate a fast component of excitatory synaptic transmission at virtually all central synapses. Thus, their functional characteristics are a critical determinant of brain function. We evaluate intolerance of each GRIA gene to genetic variation using 3DMTR and report here the functional consequences of 52 missense variants in GRIA1-4 identified in patients with various neurological disorders. These variants produce changes in agonist EC50, response time course, desensitization, and/or receptor surface expression. We predict that these functional and localization changes will have important consequences for circuit function, and therefore likely contribute to the patients' clinical phenotype. We evaluated the sensitivity of variant receptors to AMPAR-selective modulators including FDA-approved drugs to explore potential targeted therapeutic options.

Structure-Activity Studies of 1H-Imidazo[4,5-c]quinolin-4-amine Derivatives as A3 Adenosine Receptor Positive Allosteric Modulators.

We previously reported 1H-imidazo[4,5-c]quinolin-4-amines as A3 adenosine receptor (A3AR) positive allosteric modulators (PAMs). A3AR agonists, but not PAMs, are in clinical trials for inflammatory diseases and liver conditions. We synthesized new analogues to distinguish 2-cyclopropyl antagonist 17 (orthosteric interaction demonstrated by binding and predicted computationally) from PAMs (derivatives with large 2-alkyl/cycloalkyl/bicycloalkyl groups). We predicted PAM binding at a hydrophobic site on the A3AR cytosolic interface. Although having low Caco-2 permeability and high plasma protein binding, hydrophobic 2-cyclohept-4-enyl-N-3,4-dichlorophenyl, MRS7788 18, and 2-heptan-4-yl-N-4-iodophenyl, MRS8054 39, derivatives were orally bioavailable in rat. 2-Heptan-4-yl-N-3,4-dichlorophenyl 14 and 2-cyclononyl-N-3,4-dichlorophenyl 20 derivatives and 39 greatly enhanced Cl-IB-MECA-stimulated [35S]GTPγS binding Emax, with only 12b trending toward decreasing the agonist EC50. A feasible route for radio-iodination at the p-position of a 4-phenylamino substituent suggests a potential radioligand for allosteric site binding. Herein, we advanced an allosteric approach to developing A3AR-activating drugs that are potentially event- and site-specific in action.

Allosteric modulation of the adenosine A2A receptor by cholesterol.

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A2AR while decreasing the agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor's active state population and a G-protein-bound precoupled state. 19F NMR of fluorinated cholesterol analogs reveals transient interactions with A2AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

Evidence for an effect of receptor density on ligand occupancy and agonist EC50

Drug-receptor interaction theory predicts that proportional receptor occupancy is a function of ligand concentration as defined by a ligand-receptor affinity constant, and is independent of receptor density. However, we previously observed that the EC50 of 5-HT reduced as the density of 5-HT3 receptors increased, suggesting an effect of receptor density on occupancy. The current study was designed to maximise variability in experimentally observed currents and confirm this apparent contradiction prospectively. Xenopus oocytes were injected with RNA encoding 5-HT3A receptors under conditions designed to achieve varying receptor expression levels and 5-HT-evoked currents measured using two electrode voltage clamp. Results from 99 oocytes showed that as the maximal peak current increased from 0.05 µA to 12.1 µA there was a 3.7-fold reduction in EC50. Since occupancy and conductance are directly related in this system, this indicates that for a given concentration of 5-HT, proportional occupancy increases with increased receptor density. We conclude that normalising data masks this correlation, and can result in reduced accuracy of pharmacological measurements. We propose a mechanistic explanation for our observations.

Abstract LB-061: CRD5500: A versatile small molecule STING agonist amenable to bioconjugation as an ADC

Abstract BACKGROUND Innate immune signaling mediated through STING (Stimulator of Interferon Genes) generates a Type I interferon (Type 1 IFN) signal that increases T cell infiltration into cold or non-inflamed tumors leading to significant regression (Woo et al., 2015). Comprehensive data from one of our STING agonists was presented at the AACR Tumor Immunology meeting in 2017. Here we describe CRD5500, a versatile next generation compound that can be dosed either by intra-tumoral route, systemically (IV or SC) or as an antibody drug conjugated to Trastuzumab (ADC). The ADC approach allows for tumor targeting through systemic delivery with low systemic exposure of the payload and should result in a higher Therapeutic Index (TI) in the clinic. METHODS We have described the discovery of small molecule activators of human STING that generate potent anti-tumor innate and adaptive responses in recently published patents. CRD 5500 was evaluated in (1) in reporter gene assays on HEK293T cells containing stably transfected hSTING polymorphs, (2) by immunoblots to confirm pSTING, pTBK1, pIRF3, (3) by real-time PCR and ELISA monitoring induction of Type I cytokines in human tumor cell lines, isolated human PBMCs and dendritic cells (hMo-DC). Direct STING binding was demonstrated by (1) Cellular Thermal Shift Assays (CETSA) and (2) in vitro kinase assay. Anti-tumor activity of CRD5500 was demonstrated by multiple routes in syngenic tumors containing human STING. Libraries of linkers were used to permit conjugation of CRD5500 with Trastuzumab. Prior to conjugation, penultimate constructs (Pre-Ab) were screened for their ability to preferentially release CRD5500 in tumor homogenate with reduced plasma exposure. Antibody drug conjugates were synthesized at Abzena, UK and were tested in HER2 expressing cells to assess release of free payload . RESULTS Treatment of an isolated cell free system containing purified recombinant STING and TBK1 leads to STING phosphorylation confirming that CRD-5500 is a direct STING binder. Evidence of direct binding also comes from CETSA. CRD-5500 activates all five common hSTING variants with agonist EC50 comparable to CDNs in the pIRF3 reporter assay. Treatment of hSTING transfected cells, human tumor cell lines or hPBMC with CRD5500 leads to phosphorylation of IRF3, TBK1 and STING. CRD-5500 causes the maturation of hDCs and the release of innate and adaptive inflammatory cytokines such as IFNβ and TNFα from hDCs and hPBMCs. In vivo administration of CRD5500 IT or systemically caused tumor regression in CT26 syngeneic tumors containing human STING. CRD5500 demonstrated the abscopal effect and when combined with check point blockade dramatically reduced tumor mass. Interestingly, when Pre-Ab constructs that were not conjugated to antibody were delivered IV, they combined with an anti-CTLA4 antibody to eliminate tumors in the hSTING.CT26 syngenic tumor model. Conjugation with Trastuzumab was demonstrated and the ADC thus formed was screened in HER2 expressing cell lines. CONCLUSIONS The ability of hSTING agonists to generate Type 1 IFN anti-tumor immune responses makes them a promising therapeutic option either as a single agent or in combination with other therapies. CRD5500 is a small molecule STING agonist that can be delivered in multiple formats making it well suited for further development as an anti-cancer agent. Citation Format: Monali Banerjee, Sourav Basu, Sandip Middya, Ritesh Shrivastava, Rajib Ghosh, David C. Pryde, Dharmendra Yadav, Gopal Bhattacharya, Thanilsana Soram, Kavita Puniya, Ritika Raina, Vijay Kadam, Sujay Garai, Priti Sharma, Anuj Singh, Vaibhav Shinde, Nidhi Rawat, Anindita Middya, Arghyotri Sinha, Sunita Chandel, Ganesh Narisipuram, Abhisek Chatterjee, Nagaswamy Mane, Arjun Surya. CRD5500: A versatile small molecule STING agonist amenable to bioconjugation as an ADC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-061.

VU6007477, a Novel M1 PAM Based on a Pyrrolo[2,3-b]pyridine Carboxamide Core Devoid of Cholinergic Adverse Events.

Herein, we report the chemical optimization of a new series of M1 positive allosteric modulators (PAMs) based on a novel pyrrolo[2,3-b]pyridine core, developed via scaffold hopping and iterative parallel synthesis. The vast majority of analogs in this series proved to display robust cholinergic seizure activity. However, by removal of the secondary hydroxyl group, VU6007477 resulted with good rat M1 PAM potency (EC50 = 230 nM, 93% ACh max), minimal M1 agonist activity (agonist EC50 > 10 μM), good CNS penetration (rat brain/plasma K p = 0.28, K p,uu = 0.32; mouse K p = 0.16, K p,uu = 0.18), and no cholinergic adverse events (AEs, e.g., seizures). This work demonstrates that within a chemical series prone to robust M1 ago-PAM activity, SAR can result, which affords pure M1 PAMs, devoid of cholinergic toxicity/seizure liability.

Abstract B43: Novel small-molecule human STING agonists generate robust Type I interferon responses in tumors

Abstract Background: A significant proportion of human cancers grow unchecked by the immune system. These cold or non-inflamed tumors do not have a T cell infiltrate and it has been shown that activation of innate immune signaling through STING (Stimulator of Interferon Genes) generates a Type I interferon (Type 1 IFN) signal that increases T cell infiltration into these tumors leading to significant regression (Woo et al., Ann Rev Immunol 2015). Activation of STING using small molecules is a novel therapeutic approach gaining significant traction in the translational immuno-oncology research community. Methods: We used a pharmacophore based approach to identify small molecule human STING agonists. Our compounds were assessed (1) in reporter gene assays on HEK293T cells containing stably transfected hSTING polymorphs, (2) by immunoblots to confirm pSTING, pTBK1, pIRF3, (3) by real-time PCR monitoring induction of Type I cytokines in human tumor cell lines, isolated human PBMCs and dendritic cells (hDC). Promising lead compounds were further evaluated for anti-tumor activity in c57/BL6 syngeneic and xenografted SCID mice humanized with hPBMC and dendritic cells from human donors. Results: We have designed and synthesized potent non-macrocyclic, non-nucleoside human STING agonists. Our lead compound CRD-100 described here has good oral bioavailability and drug like properties (Patents filed). Treatment of an isolated cell free system containing purified recombinant STING and TBK1 leads to STING phosphorylation confirming that CRD-100 is a direct STING binder. Evidence of direct binding also comes from low temperature isothermal calorimetry. CRD-100 activates all five common hSTING variants with agonist EC50 comparable to CDNs in the pIRF3 reporter assay. Treatment of hSTING transfected cells, human tumors cell lines or hPBMC with CRD-100 leads to phosphorylation of IRF3, TBK1 and STING. CRD-100 also causes the maturation of hDCs and the release of innate and adaptive inflammatory cytokines such as IFNβ and TNFα from hPBMCs. Once fortnightly doses of CRD-100 delivered intra-tumorally caused tumor regression in syngeneic and xenografted tumors. Conclusions: The ability of hSTING agonists to generate anti-tumor immune responses in cold tumors in murine models makes them a promising therapeutic option either as a single agent or in combination with existing or developing therapies. CRD-100 has conventional small molecule drug like properties making it an attractive drug candidate for advanced development as an immune mediated anti-cancer agent. Disclosure of Chemical Structures: We will not be disclosing chemical structures at AACR. Citation Format: Monali Banerjee, Sandip Middya, Sourav Basu, Ritesh Shrivastava, Rajib Ghosh, Dharmendra Yadav, Thanilsana Soram, Ritika Raina, David C. Pryde, Kavita Puniya, Navin Pandit, Rubeenaparveen R. Mansuri, Vijay Kadam, Sabyasachi Debnath, Sunny Goon, Anuj Singh, Anindita Middya, Nidhi Rawat, Arjun Surya. Novel small-molecule human STING agonists generate robust Type I interferon responses in tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B43.

Systematic N-methylation of oxytocin: Impact on pharmacology and intramolecular hydrogen bonding network

Oxytocin (OT) is a peptide hormone agonist of the OT receptor (OTR) that plays an important role in social behaviors such as pair bonding, maternal bonding and trust. The pharmaceutical development of OT as an oral peptide therapeutic has been hindered historically by its unfavorable physicochemical properties, including molecular weight, polarity and number of hydrogen bond donors, which determines poor cell permeability. Here we describe the first systematic study of single and multiple N-methylations of OT and their effect on physicochemical properties as well as potency at the OT receptor. The agonist EC50 and percent effect for OTR are reported and show that most N-methylations are tolerated but with some loss in potency compared to OT. The effect of N-methylation on exposed polarity is assessed through the EPSA chromatographic method and the results validated against NMR temperature coefficient experiments and the determination of NMR solution structures. We found that backbone methylation of residues not involved in IMHB and removal of the N-terminal amine can significantly reduce the exposed polarity of peptides, and yet retain a significant OTR agonist activity. The results of this study also expose the potential challenge of using the N-methylation strategy for the OT system; while exposed polarity is reduced, in some cases backbone methylation produces a significant conformational change that compromises agonist activity. The data presented provides useful insights on the SAR of OT and suggests future design strategies that can be used to develop more permeable OTR agonists based on the OT framework.

Analysis of functional selectivity through G protein-dependent and -independent signaling pathways at the adrenergic α(2C) receptor.

Although G protein-coupled receptors (GPCRs) are traditionally categorized as Gs-, Gq-, or Gi/o-coupled, their signaling is regulated by multiple mechanisms. GPCRs can couple to several effector pathways, having the capacity to interact not only with more than one G protein subtype but also with alternative signaling or effector proteins such as arrestins. Moreover, GPCR ligands can have different efficacies for activating these signaling pathways, a characteristic referred to as biased agonism or functional selectivity. In this work our aim was to detect differences in the ability of various agonists acting at the α2C type of adrenergic receptors (α2C-ARs) to modulate cAMP accumulation, cytoplasmic Ca(2+) release, β-arrestin recruitment and receptor internalization. A detailed comparative pharmacological characterization of G protein-dependent and -independent signaling pathways was carried out using adrenergic agonists (norepinephrine, phenylephrine, brimonidine, BHT-920, oxymetazoline, clonidine, moxonidine, guanabenz) and antagonists (MK912, yohimbine). As initial analysis of agonist Emax and EC50 values suggested possible functional selectivity, ligand bias was quantified by applying the relative activity scale and was compared to that of the endogenous agonist norepinephrine. Values significantly different from 0 between pathways indicated an agonist that promoted different level of activation of diverse effector pathways most likely due to the stabilization of a subtly different receptor conformation from that induced by norepinephrine. Our results showed that a series of agonists acting at the α2C-AR displayed different degree of functional selectivity (bias factors ranging from 1.6 to 36.7) through four signaling pathways. As signaling via these pathways seems to have distinct functional and physiological outcomes, studying all these stages of receptor activation could have further implications for the development of more selective therapeutics with improved efficacy and/or fewer side effects.

Crystal Structure and Pharmacological Characterization of a Novel N-Methyl-d-aspartate (NMDA) Receptor Antagonist at the GluN1 Glycine Binding Site

NMDA receptors are ligand-gated ion channels that mediate excitatory neurotransmission in the brain. They are tetrameric complexes composed of glycine-binding GluN1 and GluN3 subunits together with glutamate-binding GluN2 subunits. Subunit-selective antagonists that discriminate between the glycine sites of GluN1 and GluN3 subunits would be valuable pharmacological tools for studies on the function and physiological roles of NMDA receptor subtypes. In a virtual screening for antagonists that exploit differences in the orthosteric binding site of GluN1 and GluN3 subunits, we identified a novel glycine site antagonist, 1-thioxo-1,2-dihydro-[1,2,4]triazolo[4,3-a]quinoxalin-4(5H)-one (TK40). Here, we show by Schild analysis that TK40 is a potent competitive antagonist with Kb values of 21-63 nM at the GluN1 glycine-binding site of the four recombinant GluN1/N2A-D receptors. In addition, TK40 displayed >100-fold selectivity for GluN1/N2 NMDA receptors over GluN3A- and GluN3B-containing NMDA receptors and no appreciable effects at AMPA receptors. Binding experiments on rat brain membranes and the purified GluN1 ligand-binding domain using glycine site GluN1 radioligands further confirmed the competitive interaction and high potency. To delineate the binding mechanism, we have solved the crystal structure of the GluN1 ligand-binding domain in complex with TK40 and show that TK40 binds to the orthosteric binding site of the GluN1 subunit with a binding mode that was also predicted by virtual screening. Furthermore, the structure reveals that the imino acetamido group of TK40 acts as an α-amino acid bioisostere, which could be of importance in bioisosteric replacement strategies for future ligand design.

ATP Induces NO Production in Hippocampal Neurons by P2X7 Receptor Activation Independent of Glutamate Signaling

To assess the putative role of adenosine triphosphate (ATP) upon nitric oxide (NO) production in the hippocampus, we used as a model both rat hippocampal slices and isolated hippocampal neurons in culture, lacking glial cells. In hippocampal slices, additions of exogenous ATP or 2′(3′)-O-(4-Benzoylbenzoyl) ATP (Bz-ATP) elicited concentration-dependent NO production, which increased linearly within the first 15 min and plateaued thereafter; agonist EC50 values were 50 and 15 µM, respectively. The NO increase evoked by ATP was antagonized in a concentration-dependent manner by Coomassie brilliant blue G (BBG) or by Nω-propyl-L-arginine, suggesting the involvement of P2X7Rs and neuronal NOS, respectively. The ATP induced NO production was independent of N-methyl-D-aspartic acid (NMDA) receptor activity as effects were not alleviated by DL-2-Amino-5-phosphonopentanoic acid (APV), but antagonized by BBG. In sum, exogenous ATP elicited NO production in hippocampal neurons independently of NMDA receptor activity.

Ion Channel Pharmacology Under Flow: Automation Via Well-Plate Microfluidics

Automated patch clamping addresses the need for high-throughput screening of chemical entities that alter ion channel function. As a result, there is considerable utility in the pharmaceutical screening arena for novel platforms that can produce relevant data both rapidly and consistently. Here we present results that were obtained with an innovative microfluidic automated patch clamp system utilizing a well-plate that eliminates the necessity of internal robotic liquid handling. Continuous recording from cell ensembles, rapid solution switching, and a bench-top footprint enable a number of assay formats previously inaccessible to automated systems. An electro-pneumatic interface was employed to drive the laminar flow of solutions in a microfluidic network that delivered cells in suspension to ensemble recording sites. Whole-cell voltage clamp was applied to linear arrays of 20 cells in parallel utilizing a 64-channel voltage clamp amplifier. A number of unique assays requiring sequential compound applications separated by a second or less, such as rapid determination of the agonist EC(50) for a ligand-gated ion channel or the kinetics of desensitization recovery, are enabled by the system. In addition, the system was validated via electrophysiological characterizations of both voltage-gated and ligand-gated ion channel targets: hK(V)2.1 and human Ether-à-go-go-related gene potassium channels, hNa(V)1.7 and 1.8 sodium channels, and (α1) hGABA(A) and (α1) human nicotinic acetylcholine receptor receptors. Our results show that the voltage dependence, kinetics, and interactions of these channels with pharmacological agents were matched to reference data. The results from these IonFlux™ experiments demonstrate that the system provides high-throughput automated electrophysiology with enhanced reliability and consistency, in a user-friendly format.

Disease-causing Mutation in GPR54 Reveals the Importance of the Second Intracellular Loop for Class A G-protein-coupled Receptor Function

The G-protein-coupled receptor (GPCR) GPR54 is essential for the development and maintenance of reproductive function in mammals. A point mutation (L148S) in the second intracellular loop (IL2) of GPR54 causes idiopathic hypogonadotropic hypogonadism, a disorder characterized by delayed puberty and infertility. Here, we characterize the molecular mechanism by which the L148S mutation causes disease and address the role of IL2 in Class A GPCR function. Biochemical, immunocytochemical, and pharmacological analysis demonstrates that the mutation does not affect the expression, ligand binding properties, or protein interaction network of GPR54. In contrast, diverse GPR54 functional responses are markedly inhibited by the L148S mutation. Importantly, the leucine residue at this position is highly conserved among class A GPCRs. Indeed, mutating the corresponding leucine of the alpha(1A)-AR recapitulates the effects observed with L148S GPR54, suggesting the critical importance of this hydrophobic IL2 residue for Class A GPCR functional coupling. Interestingly, co-immunoprecipitation studies indicate that L148S does not hinder the association of Galpha subunits with GPR54. However, fluorescence resonance energy transfer analysis strongly suggests that L148S impairs the ligand-induced catalytic activation of Galpha. Combining our data with a predictive Class A GPCR/Galpha model suggests that IL2 domains contain a conserved hydrophobic motif that, upon agonist stimulation, might stabilize the switch II region of Galpha. Such an interaction could promote opening of switch II of Galpha to facilitate GDP-GTP exchange and coupling to downstream signaling responses. Importantly, mutations that disrupt this key hydrophobic interface can manifest as human disease.

Distinct Interactions of Human β1- and β2-Adrenoceptors with Isoproterenol, Epinephrine, Norepinephrine, and Dopamine

Fluorescence studies with purified human beta(2)-adrenoceptor (beta(2)AR) revealed that the endogenous catecholamines, (-)-epinephrine (EPI), (-)-norepinephrine (NE), and dopamine (DOP), stabilize distinct active receptor conformations. However, the functional relevance of these ligand-specific conformations is as yet poorly understood. We addressed this question by studying fusion proteins of the beta(1)-adrenoceptor (beta(1)AR) and beta(2)AR with the short and long splice variants of G(s)alpha (G(s)alpha(S) and G(s)alpha(L)), respectively. Fusion proteins ensure efficient receptor/G-protein coupling and defined stoichiometry of the coupling partners. EPI, NE, DOP, and the prototypical synthetic betaAR agonist, (-)-isoproterenol (ISO), showed marked differences in their efficacies at stabilizing the high-affinity ternary complex at beta(1)AR-G(s)alpha and beta(2)AR-G(s)alpha fusion proteins. Ternary complex formation was more sensitive to disruption by GTP with the beta(2)AR than with the beta(1)AR. Generally, in steady-state GTPase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. Exceptionally, at beta(1)AR-G(s)alpha(L), NE was only a partial agonist. Generally, in adenylyl cyclase assays, ISO, EPI, and NE were full agonists, and DOP was a partial agonist. At beta(2)AR-G(s)alpha(L), NE was only a partial agonist. There was no correlation between efficacy at stabilizing the ternary complex and activating GTPase, and there were also dissociations between K(i) values for high-affinity agonist binding and EC(50) values for GTPase activation. In contrast to synthetic partial agonists, DOP did not exhibit increased efficacy at betaAR-G(s)alpha(L) versus betaAR-G(s)alpha(S) fusion proteins. In conclusion, our data with betaAR-G(s)alpha fusion proteins show that endogenous catecholamines and ISO stabilize distinct conformations in the beta(1)AR and beta(2)AR.

Development of a robust GABAB calcium signaling cell line using β‐lactamase technology and sorting

The GABA(B) receptor is a member of the "family 3" G protein coupled receptors. The GABA(B) receptors modulate activity inwardly rectifying potassium channels and high voltage activated calcium channels. The GABA(B) receptors require heterodimerization between two subunits, GABA(B1) and GABA(B2), for functional expression. A robust functional calcium cell line was developed that contained both the human truncated GABA(B(1b)) and human truncated GABA(B(2)) receptors. The cell line was analyzed and sorted using beta-lactamase as a reporter. Single cell clones were sorted and isolated using flow cytometry based on high beta-lactamase expression. The single cell clones were further tested in a 384-well calcium mobilization assay using the Fluo-4 AM calcium indicator on the fluorescent imaging plate reader system (FLIPR). Twenty-seven clones were grown up from single cell collections and 10 clones demonstrated a high response to GABA stimulation. The 10 clones were re-evaluated based on agonist dose response and EC(50). Clone-16 was identified and utilized in high throughput screening (HTS) assay development. Using sorting and beta-lactamase as a reporter, we were able to develop a robust, functional cell-based, GABA(B), calcium mobilization assay. The cell line described here can be used for high throughput FLIPR screening and also to compare and rank the potency and selectivity of agonists, antagonists and potentiators of the GABA(B) receptor.

In vitro screening strategies for nicotinic receptor ligands

A common historical strategy to the discovery of nicotinic receptor ligands has involved the use of radioligand-binding assays for ligand identification in combination with two-electrode voltage clamp in Xenopus oocytes for electrophysiological characterization. More recently, higher-throughput methodologies have replaced these approaches to accommodate screening of large compound libraries and to provide increased capacity for electrophysiological profiling in mammalian cell lines. We, and others, have implemented cell-based screening assays using the fluorometric imaging plate reader (FLIPR™) for primary and lead optimization screening of nicotinic receptor agonists and positive allosteric modulators (PAMs). Using GH4C1 cells expressing the rat α7 nicotinic receptor, both acetylcholine and nicotine produced concentration-dependent elevations of intracellular calcium with EC 50 values of 5.5 and 1.6 μM, respectively. PAM activity was robustly detected using the FLIPR assay; for example, the known α7 receptor PAM 5-hydroxyindole failed to directly activate the receptor but produced a leftward shift of the nicotine concentration–response curve in combination with a potentiation of the maximum evoked response to nicotine. Electrophysiological confirmation of agonist activity was achieved using the Dynaflow rapid perfusion system and patch clamp in the same GH4C1 cell expression system. Estimated EC 50 values for acetylcholine-evoked currents in GH4C1/α7 cells were 55 and 576 μM for area-under-the-curve (AUC) and maximum peak height calculations, respectively. Similarly, PAM activity was confirmed using electrophysiological recordings while also allowing for the mechanistic discrimination of compounds, not possible using the FLIPR assay. Specifically, PAMs capable of slowing the rapid desensitization of α7 receptors to different extents were discernable in these studies. Further improvements in the capacity to screen compounds using electrophysiology has been achieved by implementation of high-throughput gigaohm quality recording systems such as the QPatch and PatchXpress where agonist EC 50 values are highly comparable to those obtained using conventional manual patch clamp.

Targeting AMPA Receptor Gating Processes with Allosteric Modulators and Mutations

Allosteric modulators and mutations that slow AMPAR desensitization have additional effects on deactivation and agonist potency. We investigated whether these are independent actions or the natural consequence of slowing desensitization. Effects of cyclothiazide (CTZ), trichlormethiazide (TCM), and CX614 were compared at wild-type GluR1 and “nondesensitizing” GluR1-L497Y mutant receptors by patch-clamp recording with ultrafast perfusion. CTZ, TCM, or L/Y mutation all essentially blocked GluR1 desensitization; however, the effects of L/Y mutation on deactivation and glutamate EC50 were three to five times greater than for modulators. CTZ and TCM further slowed desensitization of L/Y mutant receptors but paradoxically accelerated deactivation and increased agonist EC50. Results indicate that CTZ and TCM target deactivation and agonist potency independently of desensitization, most likely by modifying agonist dissociation ( k off). Conversely, CX614 slowed desensitization and deactivation without affecting EC50 in both wild-type and L/Y receptors. The S750Q or combined L497Y-S750Q mutations abolished all CTZ and TCM actions without disrupting CX614 activity. Notably, the S/Q mutation also restored L/Y deactivation and EC50 to wild-type levels without restoring desensitization, further demonstrating that desensitization can be modulated independently of deactivation and EC50 by mutagenesis and possibly by allosteric modulators.

The Proto-oncogene SET Interacts with Muscarinic Receptors and Attenuates Receptor Signaling

G protein-coupled receptors mediate cell responses to extracellular stimuli and likely function in the context of a larger signal transduction complex. Utilizing the third intracellular loop of a G protein-coupled receptor in glutathione S-transferase pulldown assays from rat brain lysates coupled with high sensitivity detection methods and subsequent functional studies, we report the identification of SET as a regulator of muscarinic receptor signaling. SET is a putative oncogene reported to inhibit protein phosphatase 2A and regulate gene transcription. SET binds the carboxyl region of the M3-muscarinic receptor i3 loop, and endogenous SET co-immunoprecipitates with intact M3 muscarinic receptor expressed in cells. Small interfering RNA knockdown of endogenous SET in Chinese hamster ovary cells stably expressing the M3 muscarinic receptor augmented receptor-mediated mobilization of intracellular calcium by approximately 35% with no change in agonist EC(50), indicating that interaction of SET with the M3 muscarinic receptor reduces its signaling capacity. SET knockdown had no effect on the mobilization of intracellular calcium by the P2-purinergic receptor, ionomycin, or a direct activator of phospholipase C, indicating a specific regulation of M3 muscarinic receptor signaling. These data provide expanded functionality for SET and a previously unrecognized mechanism for regulation of GPCR signaling capacity.

Amino Acid Residues in the P2X7 Receptor that Mediate Differential Sensitivity to ATP and BzATP

Agonist properties of the P2X7 receptor (P2X7R) differ strikingly from other P2X receptors in two main ways: high concentrations of ATP (> 100 microM) are required to activate the receptor, and the ATP analog 2',3'-O-(4-benzoyl-benzoyl)ATP (BzATP) is both more potent than ATP and evokes a higher maximum current. However, there are striking species differences in these properties. We sought to exploit the large differences in ATP and BzATP responses between rat and mouse P2X7R to delineate regions or specific residues that may be responsible for the unique actions of these agonists at the P2X7R. We measured membrane currents in response to ATP and BzATP at wild-type rat and mouse P2X7R, at chimeric P2X7Rs, and at mouse P2X7Rs bearing point mutations. Wild-type rat P2X7R was 10 times more sensitive to ATP and 100 times more sensitive to BzATP than wild-type mouse P2X7R. We found that agonist EC50 values were determined solely by the ectodomain of the P2X7R. Two segments (residues 115-136 and 282-288), when transposed together, converted mouse sensitivities to those of rat. Point mutations through these regions revealed a single residue, asparagine284, in the rat P2X7R that fully accounted for the 10-fold difference in ATP sensitivity, whereas the 100-fold difference in BzATP sensitivity required the transfer of both Lys127 and Asn284 from rat to mouse. Thus, single amino acid differences between species can account for large changes in agonist effectiveness and differentiate between the two widely used agonists at P2X7 receptors.

Identification of Thr283 as a key determinant of P2X7 receptor function

The ATP-gated P2X(7) receptor is an unusual ion channel that couples to multiple downstream signalling cascades. We noted differences in mouse cDNA sequences that may indicate polymorphisms; the aim of this study was to compare function and expression of these mouse P2X(7) receptor mutations. There are three differences in the sequences of P2X(7) cDNA cloned from mouse NTW8 microglial cells or C57 BL/6 mice: [Phe(11),Ala(221),Met(283)]P2X(7) in the former and [Leu(11),Thr(221),Thr(283)]P2X(7) in the latter. We expressed these receptors and measured membrane currents, ethidium uptake, calcium influx and surface membrane expression. We also carried out these assays on the previously described polymorphism observed between C57 BL/6 and Balb/c mice ([Leu(451)]P2X(7) vs [Pro(451)]P2X(7)). Maximum current densities at [Phe(11),Ala(221),Met(283)]P2X(7) were <12% of those at [Leu(11),Thr(221),Thr(283)]P2X(7) without change in the agonist concentration-response. Replacing methionine with threonine at residue 283 yielded a receptor whose properties were the same as [Leu(11),Thr(221),Thr(283)]P2X(7). Replacing T283 in the rat P2X(7) receptor with methionine yielded currents that were <10% of wildtype and no ethidium uptake was associated with its activation. Maximum current densities and agonist EC(50) values were the same at mouse [Thr(283),Leu(451)]P2X(7) and [Thr(283),Pro(451)]P2X(7) but ethidium uptake and Fluo4 fluorescence were significantly reduced at the [Thr(283),Leu(451)]P2X(7) receptor. There was equivalent surface membrane expression of all P2X(7) receptors. This study has revealed a residue (Thr(283)) in the ectodomain that is critical for P2X(7) receptor function and suggests that the intracellular residue 451 alters downstream signalling independently of ion channel activity.