A biomarker of hypoxic large solid tumors, carbonic anhydrase IX (CAIX)—a dimeric membrane protein, expressed in many cancers, localized on the surface of cancer cells—promotes extracellular acidification and tumor progression by catalyzing the reversible hydration of carbon dioxide to bicarbonate and acid proton, playing essential roles in physiological processes such as acid-base homeostasis, calcification, and carbon metabolism. Due to its critical role in maintaining an acidic tumor microenvironment and its association with cancer progression and invasion, CAIX represents a promising therapeutic target. Inhibitors of CAIX must be highly selective to avoid interactions with other CA isoforms and minimize side effects to become drug candidates. In this study, we applied a competitive binding model to directly quantify the affinity of CAIX inhibitors in live cancer cells. Our designed high-affinity compound, labeled with fluorescein, was used with various ratios of tested unlabeled compounds, yielding a dose-response curve and the dissociation constant (K d ) for CAIX expressed in cancer cells. The identified high-affinity ligands exhibited picomolar K d , which matched the K d obtained from affinity-purified recombinant protein assays. The compounds bound in a wide range of affinities from micromolar to picomolar, with the same K d for purified protein and live cell-expressed protein. This competitive binding approach provided a framework for determining the K d of CAIX inhibitors, linking in vitro biochemical assays and cell biology. The efficacy of a newly developed CAIX-selective inhibitor was evaluated using both 2D and 3D cancer cell models, demonstrating the target-engaging effect at nanomolar concentrations.
Kvietkauskaitė et al. (2026) studied this question.