A 100K well screen for a muscarinic receptor using the Epic® label-free system – a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors

Seven-transmembrane receptors (7TMRs) are a family of proteins of great interest as therapeutic targets because of their abundance on the cell surface, diverse effects in modulating cell behavior and success as a key class of drugs. We have evaluated the Epic^® label-free system for the purpose of identifying antagonists of the muscarinic M3 receptor. We compared the data generated from the label-free technology with data for the same compounds in a calcium flux assay. We have shown that this technology can be used for high throughput screening (HTS) of 7TMRs and as an orthogonal approach to enable rapid evaluation of HTS outputs. A number of compounds have been identified which were not found in a functional HTS measuring the output from a single pathway, which may offer new approaches to inhibiting responses through this receptor.     

Affinity selection-mass spectrometry screening techniques for small molecule drug discovery

Affinity selection-mass spectrometry (AS-MS) techniques assess the binding of candidate molecules to immobilized or soluble receptors, and these methods are gaining acceptance in high throughput screening laboratories as valuable complements to traditional drug discovery technologies. A diversity of receptor types have been evaluated by AS-MS, including those that are difficult to screen using traditional biochemical approaches. AS-MS techniques that couple liquid chromatography-MS with size-based separation methods, such as ultrafiltration, gel permeation, or size-exclusion chromatography, are particularly amenable to the demands of MS-based screening and have demonstrated the greatest success across a broad range of drug targets. MS measurements of receptor function have many of the same advantages as AS-MS screening and are increasingly used for drug discovery as well.     

Application of photoaffinity crosslinking in determining the interaction between calcitonin and its receptor

Summary Understanding the molecular mechanism underlying how the peptide ligands bind to their receptors with subsequent receptor activation and cellular response is of great long-term value in designing receptor-targeted drugs. This is more difficult for class-II G protein-coupled receptors as only minimal structural data is available and their natural peptide ligands contain a large and diffuse pharmacophore. To address this problem, photoaffinity labeling studies have been developed to identify the spatial proximity between the photophore-modified ligand and its receptor. This minireview looks at the application of this approach in determining the proximal sites between class-II G protein-coupled receptor peptide ligands and their corresponding receptors, including parathyroid hormone, secretin and vasoactive intestinal polypeptide. More specifically, we will highlight interaction sites between positions 19, 16 and 26 of calcitonin with C¹³⁴−K¹⁴¹, and F¹³⁷ and T³⁰ of the receptor, respectively.     

High-content analysis of proapoptotic EphA4 dependence receptor functions using small-molecule libraries

Small-molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of proapoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of an SMC library was performed using stable EphA4-expressing NIH 3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.     

Multiplex GPCR assay in reverse transfection cell microarrays

G protein-coupled receptors (GPCRs) are a superfamily of proteins that include some of the most important drug targets in the pharmaceutical industry. Despite the success of this group of drugs, there remains a need to identify GPCR-targeted drugs with greater selectivity, to develop screening assays for validated targets, and to identify ligands for orphan receptors. To address these challenges, the authors have created a multiplexed GPCR assay that measures greater than 3000 receptor: ligand interactions in a single microplate. The multiplexed assay is generated by combining reverse transfection in a 96-well plate format with a calcium flux readout. This assay quantitatively measures receptor activation and inhibition and permits the determination of compound potency and selectivity for entire families of GPCRs in parallel. To expand the number of GPCR targets that may be screened in this system, receptors are cotransfected with plasmids encoding a promiscuous G protein, permitting the analysis of receptors that do not normally mobilize intracellular calcium upon activation. The authors demonstrate the utility of reverse transfection cell microarrays to GPCR-targeted drug discovery with examples of ligand selectivity screening against a panel of GPCRs as well as dose-dependent titrations of selected agonists and antagonists.     

A study of comparative modelling,simulation and molecular dynamics of CXCR3 receptor with lipid bilayer

The G-coupled receptors seen on the cell surface are composites with a lipid bilayer. The chemokines are kind of G-coupled receptor which majorly involved in the activation and downstream signalling of the cell. In general, many G-coupled receptors lack their 3D structures which become a hurdle in the drug designing process. In this study, comparative modelling of the CXCR3 receptor was carried out, structure evaluation was done using various tools and softwares. Additionally, molecular dynamics and docking were performed to prove the structural quality and architecture. Interestingly, the studies like toggle switch mechanism, lipid dynamics, virtual screening were carried out to find the potent antagonist for the CXCR3 receptor. During virtual screening 14,303 similar molecules were retrieved among them only four compounds have an ability to interact with a crucial amino acid residue of an antagonist. Hence, these screened compounds can serve as a drug candidate for a CXCR3 receptor, but further in vitro and in vivo studies are ought to do to prove its same efficacy.     

Development of a homogeneous binding assay for histamine receptors

Histamine is critically involved in a wide range of physiological and pathological processes through its actions at different receptors. Thus, histamine receptors have been actively pursued as therapeutic targets in the pharmaceutical industry for the treatment of a variety of diseases. There are currently four histamine receptors that have been cloned, all of which are G protein-coupled receptors. Studies from both academia and pharmaceutical companies have identified compounds that modulate the function of specific histamine receptors. These efforts led to the successful introduction of histamine H(1) and H(2) receptor antagonists for the treatment of allergy and excess gastric acid secretion, respectively. Histamine H(3) receptor ligands are currently under investigation for the treatment of obesity and neurological disorders. The recently identified histamine H(4) receptor is preferentially expressed in the immune tissues, suggesting a potential role in normal immune functions and possibly in the pathogenesis of inflammatory diseases. Even with the long history of histamine research and the important applications of histamine receptor ligands, assays to measure the affinity of compounds binding to histamine receptors are still routinely analyzed using a filtration assay, a very low-throughput assay involving washing and filtration steps. This article describes a simple, robust, and homogeneous binding assay based on the scintillation proximity assay (SPA) technology that provides results equivalent to those obtained using the more complex filtration assay. The SPA format is easily adapted to high-throughput screening because it is amenable to automation. In summary, this technique allows high-throughput screening of compounds against multiple histamine receptors and, thus, facilitates drug discovery efforts.     

Anti-cancer activity and mechanistic features of a NK cell activating molecule

Natural cytotoxicity receptors (NCRs) are major activating receptors involved in NK cytotoxicity. NCR expression varies with the activation state of NK cells, and the expression level correlates with NK cells’ natural cytotoxicity. In this study, we found that Gö6983, a PKC inhibitor, induced a remarkable increase of NCR expression on primary NK cells, but other PKC inhibitors and NK cell stimulators such as IL-2 and PMA, did not. Gö6983 increased the expression of NCR in a time- and concentration-dependent manner. Furthermore, Gö6983 strongly upregulated the surface expression of death ligands FasL and TRAIL, but not cytotoxic molecules perforin and granzyme B. Unlike two other NK stimulating molecules, IL-2, and PMA, Gö6983 did not induce NK cell proliferation. Up-regulation of NCRs and death ligands on NK cells by Gö6983 resulted in a significant enhancement of NK cytotoxicity against various cancer cell lines. Most importantly, administration of Gö6983 effectively inhibited pulmonary tumor metastasis in mice in a dose-dependent manner. These results suggest that Gö6983 functions as an NK cell activating molecule (NKAM); this NKAM is a novel anti-cancer and anti-metastasis drug candidate because it enhances NK cytotoxicity against cancer cells in vivo as well as in vitro.     

Netrin-1-independent adenosine A2b receptor activation regulates the response of axons to netrin-1 by controlling cell surface levels of UNC5A receptors

Growth cone response to the bifunctional guidance cue netrin-1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C-alpha (PKCα) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin-1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein-coupled receptors, may regulate responses to netrin-1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCα and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin-1 receptor, the G protein-coupled receptor adenosine A2b receptor (A2bR) results in PKCα-dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin-1 and converts repulsion to attraction. We show these A2bR-mediated alterations in axonal response are not because of netrin-1 because netrin-1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKCα-dependent UNC5A surface loss. Our results demonstrate that netrin-1-independent A2bR signaling governs the responsiveness of a neuron to netrin-1 by regulating the levels of cell surface UNC5A receptor.     

Receptor protein tyrosine phosphatases and cancer: New insights from structural biology

There is general agreement that many cancers are associated with aberrant phosphotyrosine signaling, which can be caused by the inappropriate activities of tyrosine kinases or tyrosine phosphatases. Furthermore, incorrect activation of signaling pathways has been often linked to changes in adhesion events mediated by cell surface receptors. Among these receptors, receptor protein tyrosine phosphatases (RPTPs) both antagonize tyrosine kinases as well as engage extracellular ligands. A recent wealth of data on this intriguing family indicates that its members can fulfill either tumor suppressing or oncogenic roles. The interpretation of these results at a molecular level has been greatly facilitated by the recent availability of structural information on the extra- and intracellular regions of RPTPs. These structures provide a molecular framework to understand how alterations in extracellular interactions can inactivate RPTPs in cancers or why the overexpression of certain RPTPs may also participate in tumor progression.     

Receptor protein tyrosine phosphatases and cancer

There is general agreement that many cancers are associated with aberrant phosphotyrosine signaling, which can be caused by the inappropriate activities of tyrosine kinases or tyrosine phosphatases. Furthermore, incorrect activation of signaling pathways has been often linked to changes in adhesion events mediated by cell surface receptors. Among these receptors, receptor protein tyrosine phosphatases (RPTPs) both antagonize tyrosine kinases as well as engage extracellular ligands. A recent wealth of data on this intriguing family indicates that its members can fulfill either tumor suppressing or oncogenic roles. The interpretation of these results at a molecular level has been greatly facilitated by the recent availability of structural information on the extra- and intracellular regions of RPTPs. These structures provide a molecular framework to understand how alterations in extracellular interactions can inactivate RPTPs in cancers or why the overexpression of certain RPTPs may also participate in tumor progression.     

Live-cell microscopy or fluorescence anisotropy with budded baculoviruses—which way to go with measuring ligand binding to M4 muscarinic receptors?

M₄ muscarinic acetylcholine receptor is a G protein-coupled receptor (GPCR) that has been associated with alcohol and cocaine abuse, Alzheimer''s disease, and schizophrenia which makes it an interesting drug target. For many GPCRs, the high-affinity fluorescence ligands have expanded the options for high-throughput screening of drug candidates and serve as useful tools in fundamental receptor research. Here, we explored two TAMRA-labelled fluorescence ligands, UR-MK342 and UR-CG072, for development of assays for studying ligand-binding properties to M₄ receptor. Using budded baculovirus particles as M₄ receptor preparation and fluorescence anisotropy method, we measured the affinities and binding kinetics of both fluorescence ligands. Using the fluorescence ligands as reporter probes, the binding affinities of unlabelled ligands could be determined. Based on these results, we took a step towards a more natural system and developed a method using live CHO-K1-hM₄R cells and automated fluorescence microscopy suitable for the routine determination of unlabelled ligand affinities. For quantitative image analysis, we developed random forest and deep learning-based pipelines for cell segmentation. The pipelines were integrated into the user-friendly open-source Aparecium software. Both image analysis methods were suitable for measuring fluorescence ligand saturation binding and kinetics as well as for screening binding affinities of unlabelled ligands.     

Advances in membrane receptor screening and analysis

During the last decade there has been significant progress in the development of analytical techniques for the screening of ligand binding to membranes and membrane receptors. This review focuses on developments using label-free assays that facilitate ligand-membrane-receptor screening without the need for chemical-, biological- or radiological-labelled reagents. These assays include acoustic, optical surface plasmon resonance biosensing, sedimentation (analytical ultracentrifugation), chromatographic assays, isothermal titration calorimetry and differential scanning calorimetry. The merits and applications of cell-based screening systems and of different model membrane systems, including planar supported lipid layers, bead-supported membranes and lipid micro-arrays, are discussed. Recent advances involving more established techniques including intrinsic fluorescence, FRET spectroscopy, scintillation proximity assays and automated patch clamping are presented along with applications to peripheral membrane proteins, ion channels and G protein-coupled receptors. Novel high-throughput assays for determination of drug- and protein-partitioning in membranes are also highlighted. To aid the experimenter, a brief synopsis of the techniques commonly employed to purify and reconstitute membranes and membrane receptors is included.     

Minireview: Challenges of High Throughput Screening Against Cell Surface Receptors

Abstract

Excessive or inappropriate activation of cell surface receptors can mediate the development of disease. Receptors, therefore, are a focus for drug discovery activities. Empirical screening is important in the search for novel compounds acting at receptors. Technical developments and the application of molecular biology have facilitated access to receptors of interest and have provided efficient screening methods capable of very high throughput. Reliability in high throughput screening requires the use of appropriate methodology, good screen design and effective validation and quality control processes. Validation should aim to establish that the basic experimental design is sound. In developing software to handle high throughput screening data, a fundamental requirement is to provide performance monitoring and error trapping facilities. Additional requirements are automatic data capture from instruments, on-line data reduction and analysis and transfer of results to central databases. As data volumes increase through effective high throughput screening, conventional interrogation methods become less appropriate and are being augmented by newer computing techniques referred to as knowledge mapping or database mining. Targeting cell surface receptors has been very successful as an approach to drug discovery. If the challenges of high throughput empirical screening are addressed effectively, cell surface receptors will provide new opportunities for improved therapy in the coming years.     

Amplification of agonist stimulation of human G-protein-coupled receptor signaling in yeast

G-protein-coupled receptors (GPCRs) are considered as important targets for drug discovery. The yeast Saccharomyces cerevisiae is an attractive host for high-throughput screening of agonistic ligands for human GPCRs because it can simplify the complicated signaling pathways that are present in mammalian cell lines. Unfortunately, many human GPCRs induce only partial signal activation in yeast cells depending on their coupling efficiency with yeast G-proteins. This problem often results in unsatisfactory detection sensitivity, thereby resulting in a limitation to yeast-based detection systems. Here we introduce a new highly sensitive detection method that provides robust agonist detection of human GPCRs. Our strategy is designed to invoke feedback activation of signals within yeast G-protein signaling pathways. Briefly, agonist stimulation of human GPCRs triggers expression of an artificial signal activator that amplifies signaling. We chose human somatostatin receptor subtype 5 (hSSTR5) as a model of a human GPCR. Investigation of the response of hSSTR5-expressing yeast to various concentrations of somatostatin demonstrated that feedback activation of the signal can successfully improve the detection limit and the maximum level of signaling. This novel approach will enhance the usefulness of yeast-based screening of agonistic ligands for a variety of human GPCRs.     

Label-free optical biosensor for ligand-directed functional selectivity acting on beta(2) adrenoceptor in living cells

Recent realization of ligand-directed functional selectivity demands high-resolution tools for studying receptor biology and ligand pharmacology. Here we use label-free optical biosensor to examine the dynamic mass redistribution of human epidermoid A431 cells in response to diverse beta(2)-adrenoceptor ligands. Multi-parameter analysis reveals distinct patterns in activation and signaling of the receptor induced by different agonists. Sequential and co-stimulation assays categorize various ligands for their ability to modulate signaling induced by catechol, a structural component of catecholamines. This study documents multiple ligand-specific states of the beta(2)-adrenoceptor and highlights the power of the biosensor assays for screening pathway-biased ligands.     

Expression - Level Dependent Activation of Recombinant Human Parathyroid Hormone/Parathyroid Hormone-Related Peptide Receptor: Effect of Human Parathyroid Hormone (1-34), (1-31), and (28-48)

Abstract

A stable recombinant Chinese hamster ovary (CHO) cell model system expressing the human type-1 receptor for parathyroid hormone and parathyroid hormone-related peptide (hPTH-R) was established for the analysis of human PTH (hPTH) variants. The cell lines showed receptor expression in the range from 10⁵ to 1.9xl0⁶ receptors per cell. The affinity of the receptors for hPTH-(l-34) was independent of the receptor number per cell (K<j = 8 nmol/1). The induction of cAMP by hPTH-(l-34) is maximal in clones expressing >2xl0⁵ receptors per cell and Ca⁺⁺ signals were maximal in cell lines expressing >1.4xl0⁶ receptors per cell. Second messenger specific inhibitors demonstrated that PTH-induced increases in intracellular cAMP and Ca⁺⁺ are independent and Ca⁺⁺ ions are derived from intracellular stores. The cAMP-specific receptor activator hPTH-(l-31) showed also an increase in intracellular Ca⁺⁺. Even in cell lines expressing more than 10 receptors per cell the Ca⁺⁺/PKC specific activator hPTH-(28-48) did not activate hPTH-Rs. Based on these results, synthesis of further derivatives of PTH is required to identify pathway-specific ligands for the type-1 hPTH-R.     

Functional selectivity of adenosine receptor ligands

Adenosine receptors are plasma membrane proteins that transduce an extracellular signal into the interior of the cell. Basically every mammalian cell expresses at least one of the four adenosine receptor subtypes. Recent insight in signal transduction cascades teaches us that the current classification of receptor ligands into agonists, antagonists, and inverse agonists relies very much on the experimental setup that was used. Upon activation of the receptors by the ubiquitous endogenous ligand adenosine they engage classical G protein-mediated pathways, resulting in production of second messengers and activation of kinases. Besides this well-described G protein-mediated signaling pathway, adenosine receptors activate scaffold proteins such as β-arrestins. Using innovative and sensitive experimental tools, it has been possible to detect ligands that preferentially stimulate the β-arrestin pathway over the G protein-mediated signal transduction route, or vice versa. This phenomenon is referred to as functional selectivity or biased signaling and implies that an antagonist for one pathway may be a full agonist for the other signaling route. Functional selectivity makes it necessary to redefine the functional properties of currently used adenosine receptor ligands and opens possibilities for new and more selective ligands. This review focuses on the current knowledge of functionally selective adenosine receptor ligands and on G protein-independent signaling of adenosine receptors through scaffold proteins.     

Photoaffinity scanning in the mapping of the peptide receptor interface of class II G protein-coupled receptors.

The family of G protein-coupled receptors constitutes about 50% of the therapeutic drug targets used in clinical medicine today, although the mechanisms of ligand binding, activation and signal transduction for G protein-coupled receptors are not yet well defined. This review discusses ongoing research using the photoaffinity scanning method to map the bimolecular interface between class II G protein-coupled receptors and their ligands. Furthermore the available computer model of class II peptide ligand docking into the receptor, based on the positional constraints imposed by the photoaffinity scanning analyses, will be discussed briefly. The ultimate goal of these efforts is to understand the molecular basis of receptor binding and therefore to generate a template for rational drug design.