Intrinsic Thermodynamics and Structures of 2,4- and 3,4- Substituted Fluorinated Benzenesulfonamides Binding to Carbonic Anhydrases Asta Zubriene ˙, [a] Alexey Smirnov, [a] Virginija Dudutiene ˙, [a] David D. Timm, [a] Jurgita Matuliene ˙, [a] Vilma Michailoviene ˙, [a] Audrius Zaks ˇauskas, [a] Elena Manakova, [b] Saulius Graz ˇulis, [b] and Daumantas Matulis* [a] Introduction Rational drug design, the design of low-molecular-weight com- pounds that would specifically and with high affinity bind a target protein, is not yet possible because of the poorly un- derstood correlation between compound structures and bind- ing thermodynamics. Thermodynamic data can be used to select and optimize lead compounds and to elucidate the driv- ing forces of binding by exploring the effects of different chemical functionalities on binding. [1–4] Thermodynamic studies determine not only the binding affinity of compounds to the target protein but also dissect the enthalpic and entropic con- tributions. The label-free technique, isothermal titration calo- rimetry (ITC), is able to determine the Gibbs energy, enthalpy, and entropy in a single experiment. Interpretation of the thermodynamic data is difficult because the binding is influenced by numerous factors, including hy- drogen bonding, hydrophobic interactions, solvation effects, dynamic structural changes of the protein and/or ligand, and local water structure. Water molecules in the binding interface often strongly alter the binding process by changing the en- thalpy and entropy while having minor effect on the binding affinity. [5–7] Structural and thermodynamic studies have shown that water molecules might make enthalpically favorable/en- tropically unfavorable or, exactly the opposite, enthalpically un- favorable/entropically favorable contributions to binding. [4, 8] Without high-resolution crystal structures of protein–ligand complexes, it would hardly be possible to understand all of the contributions linked to binding. X-ray crystallography data, together with the binding thermodynamics, helps with under- standing of the interactions in protein–ligand complexes. The ITC-observed thermodynamic parameters may depend on many system-specific properties, such as protonation of the ligand, protein, and buffer, and each may contribute to the ob- served enthalpy of binding. [9] ITC experiments that are per- formed without examination of such effects on the binding process can lead to improper interpretation of results. [10] Only the intrinsic thermodynamic parameters that are independent of the experimental conditions (pH value and buffer) can be correlated with the protein–ligand crystal structures and pro- vide insight into molecular recognition. Human carbonic anhydrases (CAs) belong to the alpha CA family and comprise 12 catalytically active isoforms that partici- pate in many physiological and pathological processes. [11–13] The goal of rational drug design is to understand structure– thermodynamics correlations in order to predict the chemical structure of a drug that would exhibit excellent affinity and se- lectivity for a target protein. In this study we explored the con- tribution of added functionalities of benzenesulfonamide in- hibitors to the intrinsic binding affinity, enthalpy, and entropy for recombinant human carbonic anhydrases (CA) CA I, CA II, CA VII, CA IX, CA XII, and CA XIII. The binding enthalpies of compounds possessing similar chemical structures and affini- ties were found to be very different, spanning a range from 90 to + 10 kJ mol 1 , and are compensated by a similar oppos- ing entropy contribution. The intrinsic parameters of binding were determined by subtracting the linked protonation reac- tions. The sulfonamide group pK a values of the compounds were measured spectrophotometrically, and the protonation enthalpies were measured by isothermal titration calorimetry (ITC). Herein we describe the development of meta- or ortho- substituted fluorinated benzenesulfonamides toward the highly potent compound 10 h, which exhibits an observed dis- sociation constant value of 43 pm and an intrinsic dissociation constant value of 1.1 pm toward CA IX, an anticancer target that is highly overexpressed in various tumors. Fluorescence thermal shift assays, ITC, and X-ray crystallography were all ap- plied in this work. [a] Dr. A. Zubriene ˙, A. Smirnov, Dr. V. Dudutiene ˙, D. D. Timm, Dr. J. Matuliene ˙, V. Michailoviene ˙, A. Zaksˇauskas, Prof. D. Matulis Department of Biothermodynamics and Drug Design, Institute of Biotech- nology, Life Sciences Center, Vilnius University, Saule ˙tekio 7, Vilnius 10257 (Lithuania) E-mail : matulis@ibt.lt daumantas.matulis@bti.vu.lt [b] Dr. E. Manakova, Dr. S. Graz ˇulis Department of Protein–DNA Interactions, Institute of Biotechnology, Life Sci- ences Center, Vilnius University, Saule ˙tekio 7, Vilnius 10257 (Lithuania) Supporting information and the ORCID identification number(s) for the author(s) of this article can be found under http://dx.doi.org/10.1002/ cmdc.201600509. ChemMedChem 2016, 11, 1 – 17 # 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 & These are not the final page numbers! ÞÞ These are not the final page numbers! ÞÞ Full Papers DOI: 10.1002/cmdc.201600509