A fluorescence polarization-based assay for peptidyl prolyl cis/trans isomerase cyclophilin A

Abstract Peptidyl prolyl cis / trans isomerase cyclophilin A (CypA) serves as a cellular receptor for the important immunosuppressant drug, cyclosporin A. In addition, CypA and its enzyme family have been found to play critical roles in a variety of biological processes, including protein trafficking, HIV and HCV infection/replication, and Ca 2+ -mediated intracellular signaling. For these reasons, cyclophilins have emerged as potential drug targets for several diseases. Therefore, it is extremely important to screen for novel small molecule cyclophilin inhibitors. Unfortunately, the biochemical assays reported so far are not adaptable to a high-throughput screening format. Here, we report a fluorescence polarization-based assay for human CypA that can be adapted to high-throughput screening for drug discovery. The technique is based on competition and uses a fluorescein-labeled cyclosporin A analog and purified human CypA to quantitatively measure the binding capacity of unlabeled inhibitors. Detection by fluorescence polarization allows real-time measurement of binding ratios without separation steps. The results obtained demonstrated significant correlation among assay procedures, suggesting that the application of fluorescence polarization in combination with CypA is highly advantageous for the accurate assessment of inhibitor binding.