Fluorescence-Based Assay for Carbonic Anhydrase Inhibitors

Summary Carbonic anhydrases are implicated in a number of diseases, including glaucoma, cancer, epilepsy, and others. Here, we present a fluorescence-based assay that utilizes the indicator-displacement assay concept and can be used to prescreen inhibitor candidates to treat such conditions. We designed and synthesized fluorescent indicators that bind carbonic anhydrase and display fluorescence quenching in the presence of the enzyme. Their fluorescence is, however, recovered when a competitive inhibitor is added. To investigate the utility of this dye-displacement system and its potential for use in high-throughput screening, we designed a fluorescence microarray. This confirmed the potential of the approach for both qualitative and quantitative analysis. Using AutoDock Vina and AutoDock4 Zn , we compared the results of the assay with ligand docking calculations. We show that the dye-displacement arrays are more accurate and sensitive than docking calculations and that these two tools can be used together to efficiently screen libraries of potential carbonic anhydrase inhibitors in a high-throughput manner.