Intrinsic thermodynamics of ethoxzolamide inhibitor binding to human carbonic anhydrase XIII

Abstract

BackgroundHuman carbonic anhydrases (CAs) play crucial role in various physiological processes including carbon dioxide and hydrocarbon transport, acid homeostasis, biosynthetic reactions, and various pathological processes, especially tumor progression. Therefore, CAs are interesting targets for pharmaceutical research. The structure-activity relationships (SAR) of designed inhibitors require detailed thermodynamic and structural characterization of the binding reaction. Unfortunately, most publications list only the observed thermodynamic parameters that are significantly different from the intrinsic parameters. However, only intrinsic parameters could be used in the rational design and SAR of the novel compounds.ResultsIntrinsic binding parameters for several inhibitors, including ethoxzolamide, trifluoromethanesulfonamide, and acetazolamide, binding to recombinant human CA XIII isozyme were determined. The parameters were the intrinsic Gibbs free energy, enthalpy, entropy, and the heat capacity. They were determined by titration calorimetry and thermal shift assay in a wide pH and temperature range to dissect all linked protonation reaction contributions.ConclusionsPrecise determination of the inhibitor binding thermodynamics enabled correct intrinsic affinity and enthalpy ranking of the compounds and provided the means for SAR analysis of other rationally designed CA inhibitors.

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

“…The pK a and enthalpies of active site hydroxide protonation could be compared to other CA isoforms ( Table 2). The CA I had a pKa 8.4, CA II-7.1 [53], CA XIII-8.3 [50], CA XII-7.0 [51], and for CA VB the pK a was 7.2. The increase in pK a for CA I and CA XIII was largely due to the more exothermic enthalpy of protonation (-41 and -44 kJ mol -1 , respectively) as compared to other CAs (* -30 kJ mol -1 ).…”

“…The standard error of the K d measurements is approximately 1.69 of the K d value isoforms [50][51][52][53][54] and are explained by the binding-linked protonation equilibria. First, a sulfonamide compound has to undergo deprotonation in order to bind to the Zn in the active center of the enzyme.…”

Intrinsic binding of 4‐substituted‐2,3,5,6‐tetrafluorobenezenesulfonamides to native and recombinant human carbonic anhydrase VI

“…, the observed enthalpies of binding are essentially useless in the analysis because they are greatly dependent on buffer and pH. Therefore, it is even more important to distinguish intrinsic enthalpies and entropies from the observed values .…”

Peer Review #1 of "Crystal structure correlations with the intrinsic thermodynamics of human carbonic anhydrase inhibitor binding (v0.1)"

“…Manuscript to be reviewed including the Gibbs energies (observem affinities), enthalpies, and entropies of the protein-ligand pairs were determined by the fluorescent thermal shift assay (Pantoliano et al, 2001;Lo et al, 2004;Matulis et al, 2005;Cimmperman & Matulis, 2011) and isothermal titration calorimetry (Wiseman et al, 1989;Harding & Chowdhry, 2001;Broecker, Vargas & Keller, 2011). The measured observem thermodynamic parameters of binding depend on the assay conditions such as pH and buffer because the binding of sulfonamide to CA is linked to several protonation reactions, the protonation of hydroxide bound to the Zn(II) in the active site of CA, the deprotonation of sulfonamide group of the inhibitor, and the (de)protonation of the buffer (Baker & Murphy, 1996;Krishnamurthy et al, 2008b;Baranauskienė & Matulis, 2012;. For the structure-activity relationship and correlation of thermodynamic parameters with the X-ray structures, the intrinsic thermodynamic parameters of sulfonamide anion binding to the Zn-bound water form of CA were calculated.…”