A whole-cell assay for the high throughput screening of calmodulin antagonists.
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 Ca(2+), 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.
Dikici
Emre
E
Department of Chemistry, University of Kentucky, Lexington, KY 40506-0055, USA.
Deo
Sapna K
SK
Daunert
Sylvia
S
D000929:Antidepressive Agents, Tricyclic / Q000032:analysis* / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000378:metabolism; D001681:Biological Assay / Q000379:methods*; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence / Q000379:methods; D015336:Molecular Probe Techniques / Q000295:instrumentation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D013997:Time Factors
D000929:Antidepressive Agents, Tricyclic; D002147:Calmodulin; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins