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Hit Identification and Pharmalogical ProfilingThe unique nature of the CellKey™ System information enables novel applications, while the universality and ease of use of the platform provides a powerful tool for generation of pharmacologically relevant data with streamlined assay development. The CellKey™ System is a single platform for conducting pharmacological evaluation (hit confirmation, potency ranking, efficacy analysis, selectivity analysis, Schild analysis) of ligands across a spectrum of receptors and cell types irrespective of the coupling mechanism.
Example of hit confirmation for human dopamine D2L agonists. The activities of 48 representative compounds are shown. Those compounds showing a response 3 standard deviations above the buffer mean (indicated by the dotted line) were defined as hits.
Compounds surviving hit confirmation were examined for D2L selectivity and potency. Compound agonist activities were tested on four different cell lines, each over-expressing a different human dopamine receptor subtype [D1 (Gs), D2L (Gi), D4.4 (Gi), and D5 (Gs)]. Dopamine served as a representative non-selective compound (left), whereas quinpirole was identified as a D2L-selective agonist (right).
Schild analysis of a Gs-protein coupled receptor. Schild analysis using dopamine as agonist and 2-CMP-dibenzafoxepin (2-CMP-D) as antagonist on CHOD1 (Gs) cells. Left: As concentration of antagonist is increased, a rightward shift in the EC50 of the agonist is observed. Right: From the relationship between the shifted EC50 values and the concentration of antagonist, the pA2 value was determined to be -8.25. The advanced ability of the CellKey™ System allows researchers to measure a range of both increases and decreases in impedance as a result of receptor activation. This enables the comprehensive detection of agonists, antagonists, partial and inverse agonists, and allosteric modulators in a native setting in a single assay.
Chemokine-mediated response of primary human cells to IL-8. The EC50 for primary human neutrophils using CDS was 6.2 x 10-10 M. The results of quadruplicate experiments are shown. Data are normalized to the 12.5 nM dose of IL-8. The exquisite sensitivity of the CellKey™ System allows the routine measurement of activation of endogenously expressed receptors, even in primary cells. The results from assaying receptors in primary cells are thought to better match the cellular response in human target tissues. Thus, the CellKey™ System may yield results that are more predictive of the efficacy and safety in vivo. The CellKey™ System has also been shown to yield data comparable to that obtained from labor-intensive and time-consuming chemotaxis assays. These assays are difficult to use in a screening environment. The CellKey™ assay provides data that is similar to a chemotaxis assay but in an extremely robust and reproducible 96-well format.
Partial agonists are detected for the muscarinic m1 receptor. Concentration response curves are shown for a panel of ligands selective for the muscarinic m1 receptor. Arecaidine, arecoline, McN-A343 and pilocarpine are the four partial agonists identified for the transfected m1 receptor in CHO cells. CDS has also been used to identify partial agnoists for the transfected human D2L receptor and the endogenous hamster serotonin 5HT1B receptor. The ability to generate pharmacology on native receptors expressed in their native cells allows the scientist to bring real biology to drug discovery and has the potential to reveal compounds different from those obtained from more artificial systems. For More InformationDocuments marked with an asterisk (*) contain customer approved collaboration data or new research data, thus require a simple, one-time registration. Or, if you have already registered, you must be logged in. Application Bulletin
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