Background: Carbonic anhydrases (CAs) regulate pH homeostasis via the reversible
hydration of CO2, thereby emerging as essential enzymes for many vital functions.
Among 12 catalytically active CA isoforms in humans, CA IX has become a relevant
therapeutic target because of its role in cancer progression. Only two CA IX inhibitors
have entered clinical trials, mostly due to low affinity and selectivity properties.
Objective: The current review presents the design, development, and identification of the
selective nano- to picomolar CA IX inhibitors VD11-4-2, VR16-09, and VD12-09.
Methods and Results: Compounds were selected from our database, composed of over
400 benzensulfonamides, synthesized at our laboratory, and tested for their binding to 12
human CAs. Here we discuss the CA CO2 hydratase activity/inhibition assay and several
biophysical techniques, such as fluorescent thermal shift assay and isothermal titration
calorimetry, highlighting their contribution to the analysis of compound affinity and structure-
activity relationships. To obtain sufficient amounts of recombinant CAs for inhibitor
screening, several gene cloning and protein purification strategies are presented, including
site-directed CA mutants, heterologous CAs from Xenopus oocytes, and native endogenous
CAs. The cancer cell-based methods, such as clonogenicity, extracellular acidification,
and mass spectrometric gas-analysis are reviewed, confirming nanomolar activities
of lead inhibitors in intact cancer cells.
Conclusions: Novel CA IX inhibitors are promising derivatives for in vivo explorations.
Furthermore, the simultaneous targeting of several proteins involved in proton flux upon
tumor acidosis and the disruption of transport metabolons might improve cancer management.