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

Area of Science:

• Biochemistry
• Chemical Biology
• Biophysics

Background:

• Membrane-associated carbonic anhydrase (CA IX) is a cancer target, but studying its interactions with small molecules is difficult.
• Conventional assays often require receptor purification, potentially altering binding characteristics.

Purpose of the Study:

• To pioneer the use of Surface Plasmon Resonance Microscopy (SPRM) for label-free kinetic measurements of small molecule inhibitors binding to CA IX on live suspension cells.
• To compare the binding affinity of sulfonamide inhibitors to membrane-bound CA IX versus purified CA IX.

Main Methods:

• Utilized Surface Plasmon Resonance Microscopy (SPRM) for label-free kinetic measurements.
• Studied five sulfonamide-based small molecule inhibitors (Acetazolamide, Sulfanilamide, Furosemide, Dansylamide, and 4-Carboxybenzenesulfonamide) against CA IX on live Ramos B suspension cells.
• Ensured SPRM measurements showed low coefficient of variation (6.8%) and agreed with literature values.

Main Results:

• SPRM successfully measured kinetic interactions of small molecule inhibitors with membrane-bound CA IX on live cells.
• Sulfanilamide exhibited a 16-fold higher affinity for CA IX in its native membrane-bound state compared to its purified state.
• SPRM measurements demonstrated high reproducibility and agreement with existing data.

Conclusions:

• SPRM is established as a viable technique for label-free kinetic analysis of small molecule interactions with membrane proteins on live suspension cells.
• Studying inhibitors in their native membrane environment provides more accurate insights into their binding affinities.
• This approach offers a valuable tool for developing targeted cancer therapeutics.