Abstract
A homogeneous phase protein-based assay for the high throughput screening of drugs was developed using enhanced green fluorescent protein (EGFP) as the reporter. For that, a fusion protein between calmodulin (CaM) and EGFP was constructed in order to monitor the conformational changes induced in CaM upon binding to tricyclic anti-depressant drugs. In the presence of Ca
2+, CaM undergoes a conformational change exposing a hydrophobic pocket that interacts with CaM-binding proteins, peptides, and drugs. Further, the conformational changes induced in CaM upon binding to Ca
2+ and the target analyte drug, leads to a change in the microenvironment of EGFP concomitant with 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. Further, the response of CaM–EGFP fusion protein in the presence of Ca
2+ to increasing concentrations of phenothiazines and structurally related tricyclic anti-depressants was investigated. Dose-response curves for various tricyclic anti-depressants were prepared. Moreover, this assay can serve as a model system for other homogeneous binding assays for pharmaceuticals employing genetically fused binding proteins with reporter proteins and may find applications in the high throughput screening of tricyclic anti-depressants.
