Pharmacological Profiling of Chemokine Receptor–Directed Compounds Using High-Content Screening

Abstract

High-content screening, typically defined as automated fluorescence microscopy combined with image analysis, is now well established as a means to study test compound effects in cellular disease-modeling systems. In this work, the authors establish several high-content screening assays in the 384-well format to measure the activation of the CC-type chemokine receptors 2B and 3 (CCR2B, CCR3). As a cellular model system, the authors use Chinese hamster ovary cells, stably transfected with 1 of the respective receptors. They characterize receptor stimulation by human monocyte chemoattractant protein-1 for CCR2B and by human eotaxin-1 for CCR3: Receptor internalization and receptor-induced phosphorylation of ERK1/2 (pERK) were quantified using fluorescence imaging and image analysis. The 4 assay formats were robust, displayed little day-to-day variability, and delivered good Z′ statistics for both CCRs. For each of the 2 receptors, the authors evaluated the potency of inhibitory compounds in the internalization format and the pERK assay and compared the results with those from other assays (ligand displacement binding, Ca 2+ mobilization, guanosine triphosphate exchange, chemotaxis). Both physiological agonists and test compounds differed significantly with respect to potencies and efficacies in the various profiling assays. The diverse assay formats delivered partially overlapping and partially complementary information, enabling the authors to reduce the probability of test compound-related technology artifacts and to specify the mode of action for individual test compounds. Transfer of the high-content screening format to a fully automated medium-throughput screening platform for CCR3 enabled the profiling of large compound numbers with respect to G protein signaling and possible tolerance-inducing liabilities. (Journal of Biomolecular Screening 2008:40-53)

Eosinophil Chemokines and Clara Cell Protein 16 Production in Nasal Mucosa of Patients with Persistent Allergic Rhinitis

“…Previous investigations have established that eotaxins induce maximal transendothelial eosinophil migration, with respect to the other chemokines in the following order of strength: eotaxin-2>RANTES>MCP-4>IL-5>LTB4 [16][17][18][19]. Many chemokines act by binding to the several receptors, whereas all eotaxins operate by stimulation of the same receptor, CCR3 [20]. Previous studies indicated epithelial cells of the nasal mucosa as the primary source of eotaxin-2 and RANTES [21,22].…”

High-Content Analysis of CCR2 Antagonists on Human Primary Monocytes

“…the Fgrains value was calculated as described previously. 6 IC 50 determination and statistical data analysis. Using the graphpad prism software version 4.03 (graphpad software, la Jolla, ca) or a proprietary high-throughput screening (hts) application, dose-response curves were fitted to the following equation:…”

“…similarly, in the case of ccr2, divergent apparent potencies have previously been observed for two reference ccr2 antagonists with respect to inhibition of mcp-1 binding, gtpγs binding, increase of cytosolic ca 2+ , erk phosphorylation, and chemotaxis. 6 in recent years, high-throughput fluorescence microscopic imaging in combination with automated image analysis, also referred to as high-content analysis (hca), 7 was introduced into pharmaceutical drug discovery programs. in hca assays, the biomolecules of interest are labeled with various fluorophores that can be monitored in parallel at different wavelengths.…”

“…the latter is difficult to accomplish with already existing assay formats, such as those based on fluorescence-activated cell sorting (Facs). 19 the use of primary human monocytes expressing the human ccr2 isoforms at physiologic densities in contrast to nonphysiological recombinant mammalian cell hosts overexpressing single ccr2 variants 6 should further increase the pharmacotherapeutic predictivity of the internalization assay regimen described here.…”

Screening and Mechanism of Trapping Ligand Antagonist Peptide for Chemokine Receptor US28 of Human Cytomegalovirus

“…To date, there have been a variety of chemokine receptor antagonists entering phases of animal experiments or clinical studies [10][11][12] Recently, various small molecules with a similar structure to chemokines have been used as antagonists to inhibit inflammatory responses. These small molecules bind to the chemokine receptor, but do not initiate cytoplasmic signal transduction cascades.…”