A whole-cell assay for the high throughput screening of calmodulin antagonists |
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Authors: | Emre Dikici Sapna K Deo Sylvia Daunert |
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Institution: | (1) Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA;(2) Present address: Department of Chemistry and Chemical Biology, Indiana University-Purdue University, Indianapolis, IN 46202-3274, USA |
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Abstract: | Cell-based screening systems for pharmaceuticals are desired over molecular biosensing systems because of the information
they provide on toxicity and bioavailability. However, the majority of sensing systems developed are molecular biosensing
type screening systems and cannot be easily adapted to cell-based screening. In this study, we demonstrate that protein-based
molecular sensing systems that employ a fluorescent protein as a signal transducer are amenable to cell-based sensing by expressing
the protein molecular sensing system in the cell and employing these cells for screening of desired molecules. To achieve
this, we expressed a molecular sensing system based on the fusion protein of calmodulin (CaM) and enhanced green fluorescent
protein (EGFP) in bacterial cells, and utilized these cells for the screening of CaM antagonists. In the presence of Ca2+, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides,
and drugs. This conformational change induced in CaM leads to a change in the microenvironment of EGFP, resulting in a change
in its fluorescence intensity. The observed change in fluorescence intensity of EGFP can be correlated to the concentration
of the analyte present in the sample. Dose-response curves for various tricyclic antidepressants were generated using cells
containing CaM-EGFP fusion protein. Additionally, we demonstrate the versatility of our system for studying protein-protein
interactions by using cells to study the binding of a peptide to CaM. The study showed that the CaM-EGFP fusion protein within
the intact cells responds similarly to that of the isolated fusion protein, hence eliminating the need for any isolation and
purification steps. We have demonstrated that this system can be used for the rapid screening of various CaM antagonists that
are potential antipsychotic drugs. |
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Keywords: | Calmodulin Calmodulin antagonists Enhanced green fluorescent protein Whole-cell assay Protein-protein interactions Tricyclic antidepressants High throughput screening |
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