Fluorescence-based thermal shift assay (FTSA) was used to measure compound–protein binding affinity. This assay quantifies protein melting temperatures (Tm) at various concentrations of the added compound. Protein unfolding is monitored by detecting the fluorescence of the solvatochromic dye 8-anilino-1-naphtalensulfonate (ANS), whose fluorescence increases upon protein unfolding [1517]. Dependence of the protein Tm on ligand concentration was used to determine the dissociation constant Kd. In this study, FTSA samples contained 5–20 μM protein, 50 μM ANS, 0–300 μM compound in 50 mM sodium phosphate or 25 mM Hepes buffer containing 50 mM NaCl, pH 7.5, and 2% (v/v) DMSO. Experiments were performed in Corbett Rotor-Gene 6000 instrument. Blue channel was used for excitation at 365 ± 20 nm and detection at 460 ± 15 nm of the ANS fluorescence. The heating rate of 1°/min was applied.

Affinities between 20 compounds and the 12 catalytically active isoforms of CA were determined by FTSA. Each Kd was estimated from a binding experiment consisting of 12 samples with different compound concentrations prepared by a 1.5x serial dilution (the last one consisted of a protein sample without the compound). The experiments were repeated at least twice, and averages are listed. The standard deviation analysis has been performed as previously described [18]. The data analysis was performed as previously described [19], and the equations used to fit the FTSA data and determine the Kd of compound interaction with each CA isoform are provided in the S1 File.