Real-Time Binding Kinetics of Small Molecules to CA IX in Live Suspension Cells Using SPR Microscopy.

Membrane-associated carbonic anhydrase (CA IX) is overexpressed in multiple cancers, making it a compelling target for therapeutics, yet measuring small molecule binding is challenging outside its native environment. Surface Plasmon Resonance Microscopy (SPRM) enables label-free kinetic measurements on whole cells, revealing critical insights that are often missed by conventional assays that require receptor purification. Here, we pioneer the use of SPRM to study kinetic interactions of five sulfonamide-based small molecule inhibitors (Acetazolamide, Sulfanilamide Furosemide, Dansylamide, and 4-Carboxybenzenesulfonamide-(4-CBS)) with CA IX on live Ramos B suspension cells. SPRM measurements were in close agreement with the literature and demonstrated a low coefficient of variation (% CV) of 6.8%. Additionally, Sulfanilamide demonstrated a 16-fold stronger affinity in its native membrane-bound state than in its purified state. This pioneering study establishes SPRM for label-free kinetic measurements of small molecule interactions on live suspension cells in vitro.