Thermodynamic characterization of human carbonic anhydrase VB stability and intrinsic binding of compounds Aist _ e Kasiliauskait _ e 1 • Vida C ˇ asait _ e 2 • Vaida Juozapaitien _ e 1 • Asta Zubrien _ e 1 • Vilma Michailovien _ e 1 • Jurgita Revuckien _ e 1 • Lina Baranauskien _ e 1 • Rolandas Mesˇkys 2 • Daumantas Matulis 1 Received: 10 April 2015 / Accepted: 21 September 2015 / Published online: 8 October 2015 Ó Akade´miai Kiado´, Budapest, Hungary 2015 Abstract The thermodynamics of low molecular weight synthetic sulfonamide inhibitor binding to carbonic anhy- drase (CA) VB was determined by the isothermal titration calorimetry (ITC) and the fluorescent thermal shift assay (FTSA). ITC provided the enthalpic and entropic contri- butions 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 com- pounds 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 benzenesul- fonamides were investigated as high affinity inhibitors of CA VB. Thermodynamic equilibrium binding measure- ments such as ITC and FTSA provide only the observed parameters. Dissection of binding-linked reactions is nec- essary 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 esti- mated and they reached 1 pM, one of the strongest binding reactions observed between any protein–ligand binding. Keywords ThermoFluor Ò Á Differential scanning fluorimetry Á Isothermal titration calorimetry Á Fluorescent thermal shift assay Á Carbonic anhydrase Á Linked protonation reactions Introduction Protein–ligand interaction thermodynamics is one of the main unsolved scientific tasks. Ability to predict detailed structure–thermodynamics correlations would enable faster discovery of drugs targeting a particular protein. In prac- tice, however, drug design companies still prefer high- throughput screening approaches to discover chemical compounds that would bind a protein target with high affinity. Globular and water-soluble proteins unfold and denature upon heating. Compounds that bind proteins stabilize them against thermal denaturation and thus shift the unfolding transition toward higher temperature. The shift is depen- dent on the affinity and also on the concentration of com- pound. An assay based on this stabilization effect on proteins has been designed and is termed the Fluorescent Thermal Shift Assay (FTSA, also called ThermoFluor Ò [1] and differential scanning fluorimetry [2–5], DSF) [6–8] when protein unfolding is followed by the intrinsic tryp- tophan fluorescence or extrinsic fluorescence of a solva- tochromic probe such as 1,8-anilino-naphthalenesulfonate (ANS) [9, 10] or Sypro Orange [11, 12]. One of the difficulties to experimentally determine the thermodynamics of protein–ligand binding is that the binding reaction is often accompanied with linked reac- tions [13, 14]. Most often observed reactions involve pro- ton binding/release (protonation/deprotonation) upon ligand binding [15]. Any direct measuring of affinity and & Daumantas Matulis matulis@ibt.lt; daumantas.matulis@bti.vu.lt 1 Department of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University, V. A. Graicˇiu¯no 8, 02241 Vilnius, Lithuania 2 Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University, Mokslininku˛ 12, 08662 Vilnius, Lithuania 123 J Therm Anal Calorim (2016) 123:2191–2200 DOI 10.1007/s10973-015-5073-3