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

## TL;DR

This study uses SPRM to measure how small molecules bind to CA IX on live cancer cells, revealing more accurate and detailed results than traditional methods.

## Contribution

The paper pioneers the use of SPRM for label-free kinetic analysis of small molecule binding to CA IX on live suspension cells.

## Key findings

- SPRM measurements showed a low coefficient of variation (% CV) of 6.8%, indicating high reproducibility.
- Sulfanilamide exhibited a 16-fold stronger affinity in its membrane-bound state compared to its purified form.
- SPRM results aligned closely with existing literature, validating its accuracy for in vitro studies.

## Abstract

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.