Daniel Bassoni

Screening β-Arrestin Recruitment for the Identification of Natural Ligands for Orphan G-Protein–Coupled Receptors:

A variety of G-protein–coupled receptor (GPCR) screening technologies have successfully partnered a number of GPCRs with their cognate ligands. GPCR-mediated β-arrestin recruitment is now recognized as a distinct intracellular signaling pathway, and ligand-receptor interactions may show a bias toward β-arrestin over classical GPCR signaling pathways. We hypothesized that the failure to identify native ligands for the remaining orphan GPCRs may be a consequence of biased β-arrestin signaling. To investigate this, we assembled 10 500 candidate ligands and screened 82 GPCRs using PathHunter β-arrestin recruitment technology. High-quality screening assays were validated by the inclusion of liganded receptors and the detection and confirmation of these established ligand-receptor pairings. We describe a candidate endogenous orphan GPCR ligand and a number of novel surrogate ligands. However, for the majority of orphan receptors studied, measurement of β-arrestin recruitment did not lead to the identification of cognate ligands from our screening sets. β-Arrestin recruitment represents a robust GPCR screening technology, and ligand-biased signaling is emerging as a therapeutically exploitable feature of GPCR biology. The identification of cognate ligands for the orphan GPCRs and the extent to which receptors may exist to preferentially signal through β-arrestin in response to their native ligand remain to be determined.

Biased Agonism as a Mechanism for Differential Signaling by Chemokine Receptors

Background: Chemokines have been thought to act in a redundant fashion through their shared receptors. Results: Chemokines can display different efficacies for G proteins and β-arrestins, resulting in different chemotactic profiles. Conclusion: Chemokines can behave as biased agonists at their receptors, leading to functionally distinct, not redundant, responses. Significance: Biased agonism plays an important role in biological signaling. Chemokines display considerable promiscuity with multiple ligands and receptors shared in common, a phenomenon that is thought to underlie their biochemical “redundancy.” Their receptors are part of a larger seven-transmembrane receptor superfamily, commonly referred to as G protein-coupled receptors, which have been demonstrated to be able to signal with different efficacies to their multiple downstream signaling pathways, a phenomenon referred to as biased agonism. Biased agonism has been primarily reported as a phenomenon of synthetic ligands, and the biologic prevalence and importance of such signaling are unclear. Here, to assess the presence of biased agonism that may underlie differential signaling by chemokines targeting the same receptor, we performed a detailed pharmacologic analysis of a set of chemokine receptors with multiple endogenous ligands using assays for G protein signaling, β-arrestin recruitment, and receptor internalization. We found that chemokines targeting the same receptor can display marked differences in their efficacies for G protein- or β-arrestin-mediated signaling or receptor internalization. This ligand bias correlates with changes in leukocyte migration, consistent with different mechanisms underlying the signaling downstream of these receptors induced by their ligands. These findings demonstrate that biased agonism is a common and likely evolutionarily conserved biological mechanism for generating qualitatively distinct patterns of signaling via the same receptor in response to different endogenous ligands.

A Novel Encoded Particle Technology that Enables Simultaneous Interrogation of Multiple Cell Types

The authors have developed a cellular analysis platform, based on encoded microcarriers, that enables the multiplexed analysis of a diverse range of cellular assays. At the core of this technology are classes of microcarriers that have unique, identifiable codes that are deciphered using CCD-based imaging and subsequent image analysis. The platform is compatible with a wide variety of cellular imaging-based assays, including calcium flux, reporter gene activation, cytotoxicity, and proliferation. In addition, the platform is compatible with both colorimetric and fluorescent readouts. Notably, this technology has the unique ability to multiplex different cell lines in a single microplate well, enabling scientists to perform assays and data analysis in novel ways.

Measurements of β-Arrestin Recruitment to Activated Seven Transmembrane Receptors Using Enzyme Complementation

The recruitment of arrestins to activated 7TMRs results in the activation of alternative signaling pathways, quenching of G-protein activation, and coupling to clathrin-mediated endocytosis. The nearly ubiquitous involvement of arrestin in 7TMR signaling has spurred the development of several methods for monitoring this interaction in mammalian cells. Nonetheless, few maintain the reproducibility and precision necessary for drug discovery applications. Enzyme fragment complementation technology (EFC) is an emerging protein-protein interaction technology based on the forced complementation of a split enzyme that has proven to be highly effective in monitoring the formation of GPCR-arrestin complexes. In these systems, the target proteins are fused to two fragments of an enzyme that show little or no spontaneous complementation. Interaction of the two proteins forces the complementation of the enzyme, resulting in an enzymatic measure of the protein interaction. This chapter discusses the utility and methods involved in using the PathHunter β-galactosidase complementation system to monitor arrestin recruitment and the advantages of exploiting this pathway in the characterization of 7TMR function.

Discovery, synthesis, and molecular pharmacology of selective positive allosteric modulators of the δ-opioid receptor

Allosteric modulators of G protein-coupled receptors (GPCRs) have a number of potential advantages compared to agonists or antagonists that bind to the orthosteric site of the receptor. These include the potential for receptor selectivity, maintenance of the temporal and spatial fidelity of signaling in vivo, the ceiling effect of the allosteric cooperativity which may prevent overdose issues, and engendering bias by differentially modulating distinct signaling pathways. Here we describe the discovery, synthesis, and molecular pharmacology of δ-opioid receptor-selective positive allosteric modulators (δ PAMs). These δ PAMs increase the affinity and/or efficacy of the orthosteric agonists leu-enkephalin, SNC80 and TAN67, as measured by receptor binding, G protein activation, β-arrestin recruitment, adenylyl cyclase inhibition, and extracellular signal-regulated kinases (ERK) activation. As such, these compounds are useful pharmacological tools to probe the molecular pharmacology of the δ receptor and to explore the therapeutic potential of δ PAMs in diseases such as chronic pain and depression.

Identification of Selective Agonists and Positive Allosteric Modulators for µ- and δ-Opioid Receptors from a Single High-Throughput Screen

Hetero-oligomeric complexes of G protein–coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a β-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature.

Characterization of G-Protein Coupled Receptor Modulators Using Homogeneous cAMP Assays

More than two-thirds of all known G-protein coupled receptors are known to modulate the function of adenylate cyclase resulting in altered levels of cAMP. In turn, cAMP fluctuations transform agonist binding events into physiological changes in cell behavior. The advent of nonradioactive, homogeneous methods of measuring intracellular cAMP has enabled the rapid growth of drug discovery and research applications for these GPCR targets. In this chapter, we describe a nonradioactive, chemiluminescent cAMP detection method using enzyme fragment complementation technology to detect a wide range of GPCR modulators which is also suitable for high-throughput screening.

Use of the CellCard™ System for Analyzing Multiple Cell Types in Parallel

The CellCard system enables the analysis of multiple cell types within a single microtiter well. In doing so, the CellCard system not only determines the effect of an experimental condition on a cell type of interest, but also the relative selectivity of that response across nine other cell types. In addition, this approach of cellular multiplexing is a means of miniaturization without the necessity of microfluidic devices. The standard 96-well plate generates ten 96-well plates of data (or, the equivalent of a 960-well plate). Taken together, the CellCard technology enables multiple cell types to be assayed within a single microtiter well allowing for the simultaneous determination of cellular activity and compound selectivity. This chapter will describe a method by which multiple cell types can be simultaneously assayed for biological parameters of interest.

A Pharmacochaperone-Based High-Throughput Screening Assay for the Discovery of Chemical Probes of Orphan Receptors

G-protein-coupled receptors (GPCRs) have varying and diverse physiological roles, transmitting signals from a range of stimuli, including light, chemicals, peptides, and mechanical forces. More than 130 GPCRs are orphan receptors (i.e., their endogenous ligands are unknown), representing a large untapped reservoir of potential therapeutic targets for pharmaceutical intervention in a variety of diseases. Current deorphanization approaches are slow, laborious, and usually require some in-depth knowledge about the receptor pharmacology. In this study we describe a cell-based assay to identify small molecule probes of orphan receptors that requires no a priori knowledge of receptor pharmacology. Built upon the concept of pharmacochaperones, where cell-permeable small molecules facilitate the trafficking of mutant receptors to the plasma membrane, the simple and robust technology is readily accessible by most laboratories and is amenable to high-throughput screening. The assay consists of a target harboring a synthetic point mutation that causes retention of the target in the endoplasmic reticulum. Coupled with a beta-galactosidase enzyme-fragment complementation reporter system, the assay identifies compounds that act as pharmacochaperones causing forward trafficking of the mutant GPCR. The assay can identify compounds with varying mechanisms of action including agonists and antagonists. A universal positive control compound circumvents the need for a target-specific ligand. The veracity of the approach is demonstrated using the beta-2-adrenergic receptor. Together with other existing assay technologies to validate the signaling pathways and the specificity of ligands identified, this pharmacochaperone-based approach can accelerate the identification of ligands for these potentially therapeutically useful receptors.

Abstract 4538: A novel assay platform for the detection of kinase-inhibitor binding in intact mammalian cells.

Kinase binding assays have become an integral part of inhibitor characterization. These tools have been applied in KINOME-wide screening systems as well as the detailed characterization of inhibitor residence time for lead optimization and MOA studies. However in cells, assays are limited to monitoring kinase activity and downstream substrate phosphorylation events. Although these types of cellular assays are critical to move programs forward, their ability to characterize inhibitor function is limited, requires targeted antibody reagents, and knowledge of specific substrates which are often not available. To circumvent these issues and apply the power of binding assays to a cellular format we have devised a method for monitoring compound binding to kinase targets in intact mammalian cells termed InCELL Hunter. The assay is shown to retain the benefits of cellular assays such as assessing compound permeability, toxicity, and target engagement; while exhibiting the generic applicability of a kinase binding assay. The InCELL hunter assay correctly describes type I, type II, and Type III inhibitors with the expected rank order cellular potencies. The assay is activity and substrate independent making it amenable to previously intractable targets and provides novel cellular information that complements both biochemical and existing cellular formats. Citation Format: Abhi Saharia, William Raab, Daniel Bassoni, Jeremy Hunt, Daniel K. Treiber, Tom Wehrmann. A novel assay platform for the detection of kinase-inhibitor binding in intact mammalian cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4538. doi:10.1158/1538-7445.AM2013-4538