Cellular membrane imaging of the dynamic binding-dissociation of rhodamine-benzenesulfonamide bioconjugate with carbonic anhydrase IX for inhibitor screening

Fluorescent displacement assay has been used as an effective method to screen target protein inhibitors, but there is a lack of reports on the detection of small molecule inhibitor activity by tracking the binding and dissociation of fluorescent substrates to target proteins in living cells. In this paper, we synthesized a cell membrane-impermeable benzenesulfonamide-conjugated rhodamine 6 G derivative, CA532, which dynamically binds to cells expressing carbonic anhydrase IX (CAIX) for fluorescence imaging of cell membranes, while small molecule inhibitors competitively displace CA532 from CAIX, resulting in a decrease in the fluorescence intensity on the cell membrane. The activity of different inhibitors cause concentration-dependent dynamic binding and dissociation of protein-substrate complexes, and the associated diverse replacement processes can be fully revealed by fluorescence imaging of cell membranes, which also enables CA532 to screen CAIX inhibitors in situ in living cells.