Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze reversible hydration of carbon dioxide to the bicarbonate ion. The enzyme has been studied since 1930s and has become a classical model protein in biochemistry
[1–5]. Recently, the interest in the enzyme has risen again. It is known now that CAs are present both in prokaryotes and eukaryotes, and are encoded by five gene families: the α-CAs (predominantly in vertebrates), the β-CAs (bacteria, algae, other), γ-CAs (archaea), δ-CAs and ζ-CAs (in some marine diatoms)
[6–8]. All human CAs belong to the alpha class. There are 12 catalytically active human CA isoforms: I, II, III, VII, and XIII are cytosolic, IV, IX, XII, and XIV are associated with cell membrane, VA and VB occur in mitochondria, and VI is secreted. There are 3 CA-related proteins VIII, X, and XI that are devoid of catalytic activity due to the absence of zinc. Human CAs were reviewed in detail, including their widely different catalytic and kinetic properties, response to inhibitors, expression patterns, and application for drug design
Human CAs are widely distributed in many tissues and organs. The CAs play a crucial role in CO2 and HCO3
- transport, pH and CO2 homeostasis, electrolyte secretion, biosynthetic reactions, and tumor progression. Therefore the CAs became interesting targets for pharmaceutical research
. However, the main drawback of available compounds is insufficient selectivity towards particular CA isozyme.
Rational design of compounds with desired binding properties requires detailed investigation of the structure-activity relationships (SAR) of the newly designed compounds. Both the thermodynamics of binding and the structure of protein-ligand complex are required for detailed understanding of the reaction and search and rational design of drug-like molecules. It is important to go beyond the determination of the K
and determine the full thermodynamic profile, especially the enthalpy of binding
. Here we describe the intrinsic thermodynamic analysis of well known CA inhibitors, ethoxzolamide (EZA), trifluoromethanesulfonamide (TFMSA), and acetazolamide (AZM) binding to human CA XIII isozyme.
Human CA XIII isozyme has been characterized
. The enzyme expression in human tissues showed that CA XIII is found in several organs including the thymus, kidney, submandibular gland, small intestine, and most notably in reproductive organs suggesting involvement in the fertilization process
[7, 11]. The CO2 hydration activity showed CA XIII to be a catalyst of medium efficiency
. Inhibition profiles demonstrated that CA XIII is similar to CA II
. The crystal structure of hCA XIII and its complex with acetazolamide has been solved providing crucial structural data for inhibitor SAR
Manuscripts reporting novel CA inhibitors usually list only the observed thermodynamic parameters of binding or the observed inhibition constants (K
[8, 14–16]. Such data is useful for the design of novel inhibitors. However, it should not be used for the SAR and correlations with structures. Proper SAR requires correlation of structures only with the intrinsic binding parameters that are dissected from linked reactions occurring simultaneously with the binding reaction. Most often, such linked reactions are protonation-deprotonation reactions occurring upon ligand binding. In the case of CAs, both the inhibitor, and the CA molecule may or may not exhibit a linked protonation reaction upon inhibitor binding. Observed and intrinsic binding parameters coincide only in some rare cases when the inhibitor is deprotonated and the zinc-linked hydroxide anion is protonated. Such situation is possible only with some CA isozymes and only with the inhibitors with the pK
s significantly below 7. Most sulfonamide inhibitors have their pK
s in the range between 8 and 11. Therefore, only small fraction of the compound is in the form that binds the protein. Despite the fact that the linked protonation reactions are largely worked-out, they are rarely used in the SAR of CA inhibitors
By ‘intrinsic’ we mean the parameters that describe actual binding reaction. In the case of sulfonamide inhibitor binding to CAs, the actual binding components are the deprotonated sulfonamide and the CA with protonated Zn-bound water molecule. Only these two forms bind each other. However, in most cases, other forms are the most abundant in solution – sulfonamide is usually protonated and the CA contains unprotonated water molecule. The concentration of the forms that actually bind is small relative to other forms and it takes energy to convert them to active forms. The observed binding energy is therefore diminished and does not resemble true (intrinsic) energetic parameters.
Determination of the intrinsic binding constant (K
, Gibbs free energy Δ
), intrinsic enthalpy (Δ
), intrinsic entropy (Δ
), and the intrinsic heat capacity of binding (Δ
) requires significant effort and a number of measurements at various pHs and temperatures. Here we use isothermal titration calorimetry (ITC) and thermal shift assay (TSA, also called ThermoFluor®, differential scanning fluorimetry) to measure inhibitor binding to CAs. ITC has been routinely used to measure ligand-protein binding thermodynamics
. However, it is not appropriate for determining weak (millimolar) or very tight dissociation constants (subnanomolar K
s require displacement ITC
) and consumes extensive amounts of protein and time. In contrast, TSA is a rapid screening method used in pharmaceutical industry for the identification of binders and requires lower amounts of protein
[22–27]. The method is based on the protein melting temperature (T
) shift that occurs upon ligand binding. The T
is observed by following intrinsic or extrinsic fluorescence changes upon heat-induced protein unfolding. The employment of two techniques to determine binding reactions reduces the error of the measurements. Here we apply both techniques to determine the observed binding parameters and then estimate the intrinsic parameters that could be used for CA inhibitor SAR analysis.