Scintillation Proximity Assay Research Articles

Receptor activity-modifying protein modulation of parathyroid hormone-1 receptor function and signaling.

Receptor activity-modifying proteins (RAMPs) are known to modulate the pharmacology and function of several G-protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R). However, the precise effects of different RAMPs on PTH1R signalling and trafficking remain poorly understood. This study investigated the impact of RAMP2 and RAMP3 on PTH1R function using a range of PTH and PTH-related protein (PTHrP)-derived ligands. We employed FRET imaging to assess PTH1R interactions with RAMPs. Cell surface expression of PTH1R was evaluated in the presence of RAMPs. PTH1R-mediated cAMP accumulation, β-arrestin recruitment, and calcium signalling were measured in response to various ligands. Antibody-capture scintillation proximity assays were used to examine G-protein activation patterns. PTH1R preferentially interacted with RAMP2 and, to a lesser extent, RAMP3, but not with RAMP1. RAMP3 co-expression reduced cell surface expression of PTH1R. RAMP2 significantly enhanced PTH1R-mediated signalling responses to PTH (1-34), PTHrP (1-34), PTH (1-84), and PTH (1-17) analogue ZP2307, while RAMP3 co-expression attenuated or abolished these responses. Full-length PTHrP analogues exhibited lower potency and efficacy than PTHrP (1-34) in activating PTH1R. RAMP2 increased the potency and/or efficacy of these analogues, whereas RAMP3 reduced these responses. RAMP2 differentially modulated G-protein activation by PTH1R in a ligand-dependent manner, with PTH (1-34) and PTHrP (1-34) inducing distinct patterns of G-protein subtype activation. These findings highlight the complex role of RAMPs in regulating PTH1R signalling and trafficking, revealing differential effects of RAMP2 and RAMP3 on receptor function. The data suggest that targeting the PTH1R/RAMP2 complex may be a promising strategy for developing novel bone anabolic therapies by leveraging biased agonism and functional selectivity. Further research using physiologically relevant models is needed to elucidate the therapeutic potential of this approach.

Development of a scintillation proximity assay for [3H]epibatidine binding sites of Tetronarce californica muscle-type nicotinic acetylcholine receptor

The therapy of intoxication with distinct organophosphorus (OP) compounds is still limited today. Especially chemical warfare agents like tabun and soman as well as novichok intoxications are difficult to address using established oxime therapeutics. These neurotoxins inhibit acetylcholinesterase (AChE), a pivotal enzyme in the synaptic cleft. The following accumulation of acetylcholine in the synaptic cleft leads to a dysfunctional, desensitized state of nicotinic acetylcholine receptors (nAChR). Without adequate treatment, the resulting cholinergic crisis leads to death by respiratory arrest. Consequently, the research approach for new therapeutic options needs to be expanded. A promising option would be substances interacting directly with nAChRs. Therefore, screening methods for new drug candidates are needed, with affinity assays playing an important role. In the present work, a saturation and competition scintillation proximity assay (SPA) for binding studies at [3H]epibatidine binding sites, conventionally classified as orthosteric binding sites of the muscle type nAChR was developed. This method offers several advantages over other assay technologies because no separation as well as washing steps are required to remove unbound ligands. Assay precision and solvent tolerance were validated according to the guidelines for validation of bioanalytical methods of the Food and Drug Administration (FDA) and European Medicines Agency (EMA). The newly developed binding assay was successfully implemented on an automated pipetting platform and is suitable for high-throughput-screening of receptor-ligand interactions at the nAChR. Furthermore, it allows to investigate/quantify competition of highly toxic agents such as nerve agents or structurally similar pesticides at the orthosteric binding site. Related to further pharmacological results, the affinity to [3H]epibatidine binding sites can provide additional information on whether potential drug candidates would be suitable for treatment of nerve agent poisoning.

Ligand bias and inverse agonism on 5-HT2A receptor-mediated modulation of G protein activity in post-mortem human brain.

Whereas biased agonism on the 5-HT2A receptor has been ascribed to hallucinogenic properties of psychedelics, no information about biased inverse agonism on this receptor is available. In schizophrenia, increased 5-HT2A receptor constitutive activity has been suggested, highlighting the therapeutic relevance of inverse agonism. This study characterized the modulation of G protein activity promoted by different drugs, commonly considered as 5-HT2A receptor antagonists, in post-mortem human brain cortex. Modulation of [35S]GTPγS binding to different subtypes of Gα proteins exerted by different 5-HT2A receptor drugs was determined by scintillation proximity assays in brain from human, WT and 5-HT2A receptor KO mice. MDL-11,939 was the only drug having no effect on the basal activity of 5-HT2A receptor. Altanserin and pimavanserin decreased basal activation of Gi1, but not Gq/11 proteins. This effect was blocked by MDL-11,939 and absent in 5-HT2A receptor KO mice. Volinanserin showed 5-HT2A receptor-mediated inverse agonism both on Gi1 and Gq/11 proteins. Ketanserin exhibited 5-HT2A receptor partial agonism exclusively on Gq/11 proteins. On the other hand, eplivanserin and nelotanserin displayed inverse agonism on Gq/11 and/or Gi1 proteins, which was insensitive to MDL-11,939 and was present in KO mice suggesting a role for another receptor. The results reveal the existence of constitutively active 5-HT2A receptors in human pre-frontal cortex and demonstrate different pharmacological profiles of various 5-HT2A receptor drugs previously considered antagonists. These findings indicate that altanserin and pimavanserin possess biased inverse agonist profile towards 5-HT2A receptor activation of Gi1 proteins.

Abstract 3233: EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma

Abstract METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant epigenetic modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of numerous hematological and solid tumor cancers. EPICS has discovered and optimized small molecule inhibitors of METTL3 (“M3i”). The aim of the current study is to evaluate the efficacy of M3i in various CDX mouse tumor models. M3i compound potency was evaluated in vitro by measuring enzymatic inhibition of METTL3/14 by scintillation proximity assay (IC50 = 2 nM). The anti-proliferative effects of M3i were measured in cell viability assays following a 72h treatment of A549 (NSCLC, IC50 = 190 nM), SKOV3 (ovarian, IC50 = 9 nM) and FaDu (hypopharyngeal squamous cell carcinoma, IC50 = 24 nM) cells. Complementary data in all three cell lines demonstrates a clear concentration-response for a target engagement biomarker (m6A; IC50 = 2-8 nM, across cell lines) as well as M3i-induced changes in biomarkers of oncogenic drivers. M3i (30 mg/kg, IP, QD) was tested in vivo in mouse models using FaDu (subcutaneous implant) in addition to A549 and SKOV3 (both orthotopic implants); all animal models were performed by LabCorp (MI, USA). Daily M3i treatment significantly prolonged survival in all three disease models consistent with efficacy on tumor growth inhibition as measured by imaging (A549, SKOV3) or direct measurement of tumor volume in the case of subcutaneous implant (FaDu). M3i has previously been shown to be effective in mouse CDX and PDX models of AML. The current results extend the application of METTL3 inhibitors to include the treatment of various types of solid tumors. The efficacy of M3i as a single-agent on tumor growth inhibition and survival advances the validation of METTL3 as a cancer target and provides additional evidence for the applicability of mRNA epigenetic mechanisms to the treatment of solid tumors. Citation Format: Graeme Fraser, Catherine Sorlet, Nicolas Parmentier, Elodie Brunel, Christian Coremans, Sarah Rorive, Anne-France Hartiel, Killian Oukoloff, Guillaume Dutheuil. EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3233.

PRELIMINARY DATA ON THE ENDOGENOUS OUABAIN IN IDIOPATHIC PULMONARY ARTERIAL HYPERTENSION PATIENTS

Objective: The aim is to detect the endogenous ouabain (EO) in idiopathic pulmonary arterial hypertension (IPAH) patients. Design and method: EO is a steroid hormone secreted by the adrenal glands associated with adverse cardiovascular outcomes. However, EO plays other roles as brain protection against traumatic injury and seems involved in the adaptive response to hypoxia. Recently, we detected, for the first time, EO in a healthy human group of acute hypoxia (elite apnea divers) and in diving animals (common bottlenose dolphins, phocids and otariids, and loggerhead sea turtles). This study complements the above as we considered a human model of chronic hypoxia. We collected blood samples from five Caucasian males and measured EO with a Scintillation Proximity Assay. Results: We found that IPAH patients had higher plasma concentrations levels of EO than control subjects. In addition, EO plasma concentrations were negatively correlated with the mean pulmonary arterial pressure and total pulmonary vascular resistance (p-values <0.05). A positive correlation trend was observed with the cardiac index, a marker of healthy cardiac output. The results could suggest that high concentrations of EO are predictive of a better adaptation of the right ventricular afterload. Conclusions: Although these are preliminary results, they match data coming from other studies on clinical conditions that frequently occur in comorbidity with IPAH. It can represent a helpful hint for future investigations involving other pathological conditions for possible therapeutic and diagnostic approaches.

Characterization of substrates and inhibitors of the human heterodimeric transporter 4F2hc-LAT1 using purified protein and the scintillation proximity radioligand binding assay.

Amino acids have diverse and essential roles in many cellular functions such as in protein synthesis, metabolism and as precursors of different hormones. Translocation of amino acids and derivatives thereof across biological membranes is mediated by amino acid transporters. 4F2hc-LAT1 is a heterodimeric amino acid transporter that is composed of two subunits belonging to the SLC3 (4F2hc) and SLC7 (LAT1) solute carrier families. The ancillary protein 4F2hc is responsible for the correct trafficking and regulation of the transporter LAT1. Preclinical studies have identified 4F2hc-LAT1 as a valid anticancer target due to its importance in tumor progression. The scintillation proximity assay (SPA) is a valuable radioligand binding assay that allows the identification and characterization of ligands of membrane proteins. Here, we present a SPA ligand binding study using purified recombinant human 4F2hc-LAT1 protein and the radioligand [3H]L-leucine as tracer. Binding affinities of different 4F2hc-LAT1 substrates and inhibitors determined by SPA are comparable with previously reported K m and IC 50 values from 4F2hc-LAT1 cell-based uptake assays. In summary, the SPA is a valuable method for the identification and characterization of ligands of membrane transporters including inhibitors. In contrast to cell-based assays, where the potential interference with other proteins such as endogenous transporters persists, the SPA uses purified protein making target engagement and characterization of ligands highly reliable.

35S]GTPγS (Guanosine-5'-O-(γ-thio)triphosphate-[35S]) Binding Scintillation Proximity Assay Experiments in Postmortem Brain Tissue.

Heterotrimeric guanine nucleotide-binding proteins (G proteins) are the very first effector in signal transduction events triggered by G-protein-coupled receptors (GPCRs). One of the most widely used approaches for determining GPCR activity in native tissue is based on the binding of [35S]GTPγS. Classically, an heterogeneous procedure including a filtration step has been used, but a modification of the protocol including an immunoprecipitation step has allowed the specific discrimination of the contribution of the different Gα subunit subtypes to the effect of each ligand. Nowadays, that the concept of functional selectivity has been demonstrated for several ligands and GPCRs, information obtained from this methodological approach will be very useful for broadening the knowledge ofGPCRs signaling profiles and describing the effect of different ligands over them. In this chapter we will describe the detailed protocol of antibody-capture [35S]GTPγS scintillation proximity assay (SPA) in order to provide the reader with comprehensive guidelines to study receptor-mediated functional activation of different Gα-protein subtypes in native mammalian brain membranes. In addition, advantages and limitations of this method will be described, as well as future direction in the application of this approach indicated.

Pharmacological Characterization of Purified Full-Length Dopamine Transporter from Drosophila melanogaster

The dopamine transporter (DAT) is a member of the neurotransmitter:sodium symporter (NSS) family, mediating the sodium-driven reuptake of dopamine from the extracellular space thereby terminating dopaminergic neurotransmission. Our current structural understanding of DAT is derived from the resolutions of DAT from Drosophila melanogaster (dDAT). Despite extensive structural studies of purified dDAT in complex with a variety of antidepressants, psychostimulants and its endogenous substrate, dopamine, the molecular pharmacology of purified, full length dDAT is yet to be elucidated. In this study, we functionally characterized purified, full length dDAT in detergent micelles using radioligand binding with the scintillation proximity assay. We elucidate the consequences of Na+ and Cl- binding on [3H]nisoxetine affinity and use this to evaluate the binding profiles of substrates and inhibitors to the transporter. Additionally, the technique allowed us to directly determine a equilibrium binding affinity (Kd) for [3H]dopamine to dDAT. To compare with a more native system, the affinities of specified monoamines and inhibitors was determined on dDAT, human DAT and human norepinephrine transporter expressed in COS-7 cells. With our gathered data, we established a pharmacological profile for purified, full length dDAT that will be useful for subsequent biophysical studies using dDAT as model protein for the mammalian NSS family of proteins.

Pancreatic Cancer Cell-Conditioned, Human-Derived Primary Myotubes Display Increased Leucine Turnover, Increased Lipid Accumulation, and Reduced Glucose Uptake.

Metabolic alterations occurring in cancer cells have been seen to also occur in other tissues than cancerous tissue. For instance, cachexia, peripheral insulin resistance, or both are commonly seen in patients with cancer. We explored differences in substrate use in myotubes conditioned with the medium from a pancreatic cancer cell line, PANC-1, or primary human pancreatic cells, hPECs. Protein turnover was assessed using scintillation proximity assay, glucose and oleic acid handling were analyzed by substrate oxidation assay. We performed qPCR to study gene expression and immunoblotting and proteomic analyses to study protein expression. PANC-1-conditioned myotubes had an imbalance in protein turnover with decreased accumulation, increased decay, and decreased MYH2 gene expression. Glucose uptake decreased despite increased insulin-stimulated Akt phosphorylation. Fatty acid uptake increased, whereas fatty acid oxidation was unchanged, leading to accumulation of intracellular lipids (TAG) in PANC-1-conditioned myotubes. Secretome analyses revealed increased release of growth factors and growth factor receptor from PANC-1 cells, potentially affecting muscle cell metabolism. Myotubes exposed to pancreatic cancer cell medium displayed altered energy metabolism with increased protein/leucine turnover and lipid accumulation, while glucose uptake and oxidation reduced. This indicates production and release of substances from pancreatic cancer cells affecting skeletal muscle.

Synthesis of biotinylated-LPG as a chemical biology tool enabling discovery of ALCAT1 modulators

As a mitochondrial signature phospholipid, cardiolipin (CL) is required for membrane structure, respiration, dynamics, fragmentation, and mitophagy. Alteration of CL by reactive oxygen species (ROS) can cause mitochondrial dysfunction, which is implicated in the pathogenesis of many diseases. The enzyme ALCAT1 (acyl-CoA: lysocardiolipin acyltransferase-1) facilitates the conversion of CL by incorporating polyunsaturated fatty acids into lysocardiolipin. Accumulating evidence suggests that overexpression of ALCAT1 is involved in pathological cardiolipin remodeling and mitochondrial bioenergetics. Few ALCAT1 modulators are reported in the literature, and the enzymatic activity was tested via a low-throughput TLC (thin layer chromatography) assay. To identify small molecule ALCAT1 inhibitors, a robust assay was needed to enable a full deck high throughput screen. Scintillation proximity assay (SPA) was the method of choice because it permits the rapid and sensitive measurement of a broad range of biological processes in a homogeneous system. A biotinylated ALCAT1 substrate was required as a chemical biology tool in developing SPA. Among a panel of phospholipids, lysophosphatidyl glycerol (LPG) was identified as the best substrate for ALCAT1. Herein we report the synthesis of biotinylated-LPG analogs with varied linker lengths and their activity towards ALCAT1.

Ensemble Simulations and Experimental Free Energy Distributions: Evaluation and Characterization of Isoxazole Amides as SMYD3 Inhibitors.

Optimization of binding affinities for ligands to their target protein is a primary objective in rational drug discovery. Herein, we report on a collaborative study that evaluates various compounds designed to bind to the SET and MYND domain-containing protein 3 (SMYD3). SMYD3 is a histone methyltransferase and plays an important role in transcriptional regulation in cell proliferation, cell cycle, and human carcinogenesis. Experimental measurements using the scintillation proximity assay show that the distributions of binding free energies from a large number of independent measurements exhibit non-normal properties. We use ESMACS (enhanced sampling of molecular dynamics with approximation of continuum solvent) and TIES (thermodynamic integration with enhanced sampling) protocols to predict the binding free energies and to provide a detailed chemical insight into the nature of ligand–protein binding. Our results show that the 1-trajectory ESMACS protocol works well for the set of ligands studied here. Although one unexplained outlier exists, we obtain excellent statistical ranking across the set of compounds from the ESMACS protocol and good agreement between calculations and experiments for the relative binding free energies from the TIES protocol. ESMACS and TIES are again found to be powerful protocols for the accurate comparison of the binding free energies.

A Scintillation Proximity Assay for Real-Time Kinetic Analysis of Chemokine-Chemokine Receptor Interactions.

Chemokine receptors are extensively involved in a broad range of physiological and pathological processes, making them attractive drug targets. However, despite considerable efforts, there are very few approved drugs targeting this class of seven transmembrane domain receptors to date. In recent years, the importance of including binding kinetics in drug discovery campaigns was emphasized. Therefore, kinetic insight into chemokine–chemokine receptor interactions could help to address this issue. Moreover, it could additionally deepen our understanding of the selectivity and promiscuity of the chemokine–chemokine receptor network. Here, we describe the application, optimization and validation of a homogenous Scintillation Proximity Assay (SPA) for real-time kinetic profiling of chemokine–chemokine receptor interactions on the example of ACKR3 and CXCL12. The principle of the SPA is the detection of radioligand binding to receptors reconstituted into nanodiscs by scintillation light. No receptor modifications are required. The nanodiscs provide a native-like environment for receptors and allow for full control over bilayer composition and size. The continuous assay format enables the monitoring of binding reactions in real-time, and directly accounts for non-specific binding and potential artefacts. Minor adaptations additionally facilitate the determination of equilibrium binding metrics, making the assay a versatile tool for the study of receptor–ligand interactions.

Endogenous Ouabain in Human and Animal Models of Hypoxia

Endogenous ouabain (EO) is a steroid hormone secreted by the adrenal glands, usually associated with adverse cardiovascular effects. However, recent studies have highlighted its possible role in blood pressure control and in cardio-renal damage, and it seems to be involved in the adaptive response to hypoxia. The aim of this study is to detect the EO in human and animal models of hypoxia. We collected blood samples from seven competitive elite apnea divers, 11 noncompetitive elite apnea divers, and 26 healthy control subjects. Animal blood samples were collected from 16 common bottlenose dolphins (Tursiops truncatus), two phocids and two otariids kept under human care, and 11 wild loggerhead sea turtles (Caretta caretta) hosted in two rescue centers. We measured EO plasma concentrations with a Scintillation Proximity Assay. In elite apnea divers and healthy control subjects, EO plasma concentrations were positively correlated to weight (p < 0.05). Elite apnea divers showed statistically significant (p < 10-6) higher EO plasma concentrations compared to healthy subjects without any diving experience or experience in other sports activities involving breath-holding. In dolphins, EO plasma concentrations were positively correlated to age, total length, and weight (p < 0.05). In loggerhead sea turtles, EO plasma concentrations were negatively correlated to total length and weight (p < 0.05). In pinnipeds, correlation analysis was not performed due to the small number of animals. Herein, we demonstrate, for the first time, that different taxa, phylogenetically distant from each other and which perform apneas without reporting neurological damages, express EO. Our findings, although preliminary, are in line with the recently emerging hypothesis on a possible role of EO in the adaptive response to hypoxia and represent a helpful hint for future investigations aimed to identify novel molecules useful to treat very disabling pathological conditions such as idiopathic pulmonary arterial hypertension or obstructive sleep apnea.

Functional approaches to the study of G-protein-coupled receptors in postmortem brain tissue: [35S]GTPγS binding assays combined with immunoprecipitation.

G-protein-coupled receptors (GPCRs) have an enormous biochemical importance as they bind to diverse extracellular ligands and regulate a variety of physiological and pathological responses. G-protein activation measures the functional consequence of receptor occupancy at one of the earliest receptor-mediated events. Receptor coupling to G-proteins promotes the GDP/GTP exchange on Gα subunits. Thus, modulation of the binding of the poorly hydrolysable GTP analog [35S]GTPγS to the Gα-protein subunit can be used as a functional approach to quantify GPCR interaction with agonist, antagonist or inverse agonist drugs. In order to determine receptor-mediated selective activation of the different Gα-proteins, [35S]GTPγS binding assays combined with immunodetection by specific antibodies have been developed and applied to physiological and pathological brain conditions. Currently, immunoprecipitation with magnetic beads and scintillation proximity assays are the most habitual techniques for this purpose. The present review summarizes the different procedures, advantages and limitations of the [35S]GTPγS binding assays combined with selective Gα-protein sequestration methods. Experience of functional coupling of several GPCRs to different Gα-proteins and recommendations for optimal performance in brain membranes are described. One of the biggest opportunities opened by these techniques is that they enable evaluation of biased agonism in the native tissue, which results in high interest in drug discovery. The available results derived from application of these functional methodologies to study GPCR dysfunctions in neuro-psychiatric disorders are also described. In conclusion, [35S]GTPγS binding combined with antibody-mediated immunodetection represents an useful method to separately evaluate the functional activity of drugs acting on GPCRs over each Gα-protein subtype.

Insight into the direct interaction of Na+ with NhaA and mechanistic implications

Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life that are essential in cellular ion homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystals provided insight in the structure of this molecular machine. However, structural data revealing the composition of the binding site for Na+ (or its surrogate Li+) is missing, representing a bottleneck in our understanding of the correlation between the structure and function of NhaA. Here, by adapting the scintillation proximity assay (SPA) for direct determination of Na+ binding to NhaA, we revealed that (i) NhaA is well adapted as the main antiporter for Na+ homeostasis in Escherichia coli and possibly in other bacteria as the cytoplasmic Na+ concentration is similar to the Na+ binding affinity of NhaA, (ii) experimental conditions affect NhaA-mediated cation binding, (iii) in addition to Na+ and Li+, the halide Tl+ interacts with NhaA, (iv) whereas acidic pH inhibits maximum binding of Na+ to NhaA, partial Na+ binding by NhaA is independent of the pH, an important novel insight into the effect of pH on NhaA cation binding.

Predicting pleural metastasis by detecting CD55 using an ultrasound-enhanced scintillation proximity assay

We used an ultrasound-enhanced scintillation proximity assay to accurately determine the levels of complement decay-accelerating factor (CD55) for the early detection and assessment of the progression of pleural metastatic lung cancer. We found that the expression of CD55 in metastatic tumor tissues is 2.8 times higher than that in the serum at the early stages. We also found that the concentration of CD55 in serum decreases with the progression of metastasis. These results suggest that CD55 is a potential biomarker for the prediction of pleural metastatic lung cancer.

Acoustic Mist Ionization-Mass Spectrometry: A Comparison to Conventional High-Throughput Screening and Compound Profiling Platforms.

Drug discovery usually begins with a high-throughput screen (HTS) of thousands to millions of molecules to identify starting points for medicinal chemistry. Conventional HTS platforms require expensive reagents and typically have complex assay formats. HTS platforms based on radioactivity are expensive, both in terms of reagent cost and disposal. Furthermore, nonspecific interferences common to these technologies result in an extensive attrition of hits during validation experiments. Mass spectrometry (MS) is a highly selective, label-free technology that can quantify multiple analytes in a single experiment. However, most commercial MS platforms typically involve a separation or cleanup prior to analysis and are too slow for large-scale screening campaigns. Recently, an MS platform (AMI-MS) was introduced that uses acoustically generated droplets to deliver analyte molecules directly from microtiter plates into the mass spectrometer at subsecond per well sampling rates. Here, we demonstrate the application of AMI-MS by developing an HTS-compatible assay that measures the inhibition of histone acetyltransferase activity. Real-time kinetic measurements from a single well were used to determine enzyme Km and Vmax values. We compare the AMI-MS readout with conventional platforms in single-shot screening and multipoint profiling modes. The AMI-MS assay identified 86% of hits previously identified, with a pIC50 ≥ 5.0, in a scintillation proximity assay (SPA) HTS at a lower hit rate and with a significantly reduced cost per well compared to the SPA-based readout. Furthermore, pIC50s, as measured by AMI-MS, showed a good correlation with values generated by RapidFire-MS. AMI-MS has the potential to provide significant improvements to high-throughput bioassays.

Two Forms of Tyrosyl-tRNA Synthetase from Pseudomonas aeruginosa: Characterization and Discovery of Inhibitory Compounds.

Pseudomonas aeruginosa is a multidrug-resistant (MDR) pathogen and a causative agent of both nosocomial and community-acquired infections. The genes (tyrS and tyrZ) encoding both forms of P. aeruginosa tyrosyl-tRNA synthetase (TyrRS-S and TyrRS-Z) were cloned and the resulting proteins purified. TyrRS-S and TyrRS-Z were kinetically evaluated and the Km values for interaction with Tyr, ATP, and tRNATyr were 172, 204, and 1.5 μM and 29, 496, and 1.9 μM, respectively. The kcatobs values for interaction with Tyr, ATP, and tRNATyr were calculated to be 3.8, 1.0, and 0.2 s-1 and 3.1, 3.8, and 1.9 s-1, respectively. Using scintillation proximity assay (SPA) technology, a druglike 2000-compound library was screened to identify inhibitors of the enzymes. Four compounds (BCD37H06, BCD38C11, BCD49D09, and BCD54B04) were identified with inhibitory activity against TyrRS-S. BCD38C11 also inhibited TyrRS-Z. The IC50 values for BCD37H06, BCD38C11, BCD49D09, and BCD54B04 against TyrRS-S were 24, 71, 65, and 50 μM, respectively, while the IC50 value for BCD38C11 against TyrRS-Z was 241 μM. Minimum inhibitory concentrations (MICs) were determined against a panel of clinically important pathogens. All four compounds were observed to inhibit the growth of cultures of both Gram-positive and Gram-negative bacteria organisms with a bacteriostatic mode of action. When tested against human cell cultures, none of the compounds were toxic at concentrations up to 400 μg/mL. In mechanism of inhibition studies, BCD38C11 and BCD49D09 selectively inhibited TyrRS activity by competing with ATP for binding. BCD37H06 and BCD54B04 inhibited TyrRS activity by a mechanism other than substrate competition.

Pimavanserin exhibits serotonin 5-HT2A receptor inverse agonism for Gαi1- and neutral antagonism for Gαq/11-proteins in human brain cortex

Pimavanserin is claimed as the first antipsychotic drug that shows selectivity for serotonin 5-HT2 receptors (5-HT2Rs) and lacks of affinity for dopamine D2 receptors (D2Rs). Cell-based functional assays suggest that pimavanserin and antipsychotics with D2R/5-HT2R affinity could act as inverse agonists of 5-HT2ARs. However, there is not evidence of such pharmacological profile in native brain tissue. 5-HT2ARs are able to engage both canonical Gαq/11- and non-canonical Gαi1-proteins. In the present study, the response to pimavanserin of the 5-HT2AR coupling to Gαq/11- and Gαi1-proteins was measured in membranes of postmortem human prefrontal cortex by antibody-capture [35S]GTPγS binding scintillation proximity assays. Pimavanserin promoted a concentration-dependant inhibition of the 5-HT2AR coupling to Gαi1-proteins whereas the response of Gαq/11-proteins was unaltered, suggesting inverse agonism and neutral antagonism properties, respectively. The inhibition was abolished in the presence of the selective 5-HT2AR antagonist MDL-11,939 and was absent in brain cortex of 5-HT2AR knock-out mice when compared to respective 5-HT2AR wild-type animals. In conclusion, the results demonstrate the existence of constitutive 5-HT2AR activity in human brain for the signalling pathway mediated by Gαi1-proteins. Pimavanserin demonstrates 5-HT2AR functional selectivity and exhibits inverse agonist profile towards Gαi1-proteins, which is considered the effector pathway promoting hallucinogenic responses. In contrast, pimavanserin behaves as neutral antagonist on the 5-HT2AR coupling to the canonical Gαq/11-protein pathway. The results strengthen the relevance of inverse agonism as potential mechanism of antipsychotic activity. Moreover, the existence of functional selectivity of 5-HT2ARs for different Gα-proteins could contribute to better design of 5-HT2AR-related antipsychotic drugs.

The CB1 cannabinoid receptor‐mediated inhibition of cAMP accumulation in neuroblastoma cells: role of different Gi/o protein subtypes

The CB1 cannabinoid receptor (CB1R) is a G protein coupled receptor with robust expression in the nervous system. CB1R signaling is mediated by pertussis toxin (PTX)‐sensitive Gi /o proteins, and leads to inhibition of adenylyl cyclase. Because of the importance of the cyclic AMP pathway in differentiated neuronal functions, the investigation of G protein subtypes involved in the inhibition of adenylyl cyclase activity is of particular interest. The aim of the present study is to investigate the effect of individual Gi/o proteins on CB1R‐promoted inhibition of forskolin‐ stimulated cAMP accumulation in N18TG2 neuroblastoma cells. We utilized [3H]cAMP binding competition assay and cell membranes in ligand‐modulated [35S]GTPγS binding and scintillation proximity assay (SPA) as previously described ( Blume et al 2015 and Eldeeb et al 2017).The CB1R agonist, CP55940 (100 nM) inhibited forskolin‐stimulated cAMP accumulation to 34±8% of forskolin alone. Pretreatment with 100 ng/ml of PTX for 20 hrs attenuated CP55940 induced inhibition to 85±24% of forskolin control. In N18TG2 cells that were stably expressing either PTX‐resistant Gαo, Gαi1, Gαi2 or Gαi3 proteins, treatment of PTX‐pretreated with CP55940 (100 nM) inhibited cAMP accumulation to 38±3% Gαo, 53±4 Gαi1, 76±24 Gαi2 and 28±15% Gαi3 of forsklin alone. Using SPA, CP55940 (100 nM) stimulated [35S]GTPγS binding mediated by Gi/o protein subtypes, Gαo, Gαi1, Gαi2 and Gαi3 in N18TG2 Gαi3 PTX‐resistant cell membranes. In contrast, in PTX‐pretreated membranes CP55940 was able to stimulate [35S] GTPγS binding mediated by Gαi3 only. In conclusion, these data show that Gi/o proteins contribute differently to CB1R‐promoted inhibition of cAMP in N18TG2 cells, with greater contribution by Gαi3. This is consistent with our earlier findings of the high level of Gαi3 expression and its contribution toward total G protein activation in N18TG2 membranes ( Eldeeb et al 2017). The contribution of different G protein subtypes to CB1R signaling is necessary to consider as we evaluate CB1R functions in the nervous system.Support or Funding InformationNIH grants R01‐DA042157, P50‐DA006634