Label-free flow cytometry-based enzyme inhibitor identification
Anna Nickelsen1, Joachim Jose1
1Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, D-48149 Münster, Germany.
Analytica Chimica Acta
|September 18, 2021
Summary
This study introduces a novel, label-free assay for enzyme inhibitor screening. It utilizes intrinsic ligand fluorescence and autodisplay technology for efficient identification of high-affinity inhibitors.
Area of Science:
- Biochemistry
- Enzyme kinetics
- Molecular biology
Background:
- Fluorescence assays for enzyme inhibitors typically require labeling of proteins or ligands.
- Existing methods can be complex and time-consuming due to labeling procedures.
Purpose of the Study:
- To develop a label-free displacement assay for enzyme inhibitor screening.
- To leverage intrinsic fluorescence of ligands and cell-surface enzyme display.
Main Methods:
- Utilized autodisplay technology for cell-surface expression of human protein kinase CK2 in E. coli.
- Employed flow cytometry to quantify the binding of an intrinsically fluorescent CK2 inhibitor (compound 5).
- Assessed competitive binding using a non-fluorescent inhibitor (TBB) to validate the assay.
Main Results:
- Demonstrated successful quantification of intrinsically fluorescent ligand binding to cell-surface CK2.
- Showcased a dose-dependent increase in fluorescence with increasing compound 5 concentrations.
- Confirmed assay validity through competitive inhibition studies with TBB.
Conclusions:
- Presented a simple, label-free binding assay for screening enzyme inhibitors.
- Overcame limitations associated with fluorescent labeling in inhibitor discovery.
- Established a valuable tool for identifying high-affinity enzyme inhibitors efficiently.
