title: 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.
authors.affiliation: Department of Integrated Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co KG, Biberach, Germany.
authors.forename: Dorothea
authors.initials: D
authors.lastname: Haasen
authors.forename: Susanne
authors.initials: S
authors.lastname: Merk
authors.forename: Peter
authors.initials: P
authors.lastname: Seither
authors.forename: Domnic
authors.initials: D
authors.lastname: Martyres
authors.forename: Silke
authors.initials: S
authors.lastname: Hobbie
authors.forename: Ralf
authors.initials: R
authors.lastname: Heilker
mesh_terms: D000818:Animals; D016466:CHO Cells; D054413:Chemokine CCL11 / Q000494:pharmacology; D018932:Chemokine CCL2 / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D020935:MAP Kinase Signaling System / Q000187:drug effects; D054390:Receptors, CCR2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D054397:Receptors, CCR3 / Q000819:agonists / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D019707:Receptors, Chemokine / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: C515456:CCL11 protein, human; C111835:CCL2 protein, human; C515334:CCR2 protein, human; C515350:CCR3 protein, human; D054413:Chemokine CCL11; D018932:Chemokine CCL2; D054390:Receptors, CCR2; D054397:Receptors, CCR3; D019707:Receptors, Chemokine; D011994:Recombinant Proteins