Thermodynamic characterization of human carbonic anhydrase VB stability and intrinsic binding of compounds

Abstract

The thermodynamics of low molecular weight synthetic sulfonamide inhibitor binding to carbonic anhydrase (CA) VB was determined by the isothermal titration calorimetry (ITC) and the fluorescent thermal shift assay (FTSA). ITC provided the enthalpic and entropic contributions to the binding affinity of ethoxzolamide to CA VB. FTSA is a high-throughput assay that measures protein thermal stabilization by added ligands. FTSA enabled determination of extremely high affinity of several compounds binding to CA VB. CA VB is one of two isoforms that are expressed in mitochondria, participate in carbon metabolism and pH homeostasis and are implicated in diseases such as obesity. Therefore CA VB is a drug target. Here a series of para-substituted tetrafluoro benzenesulfonamides were investigated as high affinity inhibitors of CA VB. Thermodynamic equilibrium binding measurements such as ITC and FTSA provide only the observed parameters. Dissection of binding-linked reactions is necessary to obtain the intrinsic parameters that in turn could be correlated with the chemical structure of the inhibitors. Intrinsic dissociation constants of the inhibitors were estimated and they reached 1 pM, one of the strongest binding reactions observed between any protein–ligand binding.