Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX

https://doi.org/10.1016/j.bbagen.2016.01.007Get rights and content

Highlights

  • The pKa of CA IX was determined to be 6.8, the enthalpy of protonation of CA IX was −24 kJ/mol at 25 °C.

  • Intrinsic binding thermodynamics – structure correlations were drawn for 40 novel CA IX inhibitors.

  • Affinities of the CA IX inhibitors achieved the Kd_intr of 0.01 nM and the Kd_obs of 2 nM.

Abstract

Background

Human carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding.

Methods

The observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry.

Results

The pKa of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was − 24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM.

Conclusions

The intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship.

General significance

It is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations.

Introduction

The measurements of the affinity of protein–ligand interaction are an important phase of target-based rational drug design. Compound hits from the high-throughput screening are often ranked according to their affinities towards the disease targets. However, often there are various binding-linked reactions, primarily protonation reactions, that affect the observed affinity. Therefore it is important to distinguish the ‘intrinsic’ affinity from the ‘observed’ affinity. Furthermore, it is important to select the lead binders not only according to their affinities but also according to other thermodynamic parameters of binding, primarily enthalpy and entropy [1]. Here we determine the intrinsic binding enthalpies, entropies and Gibbs energies (affinities) of a series of substituted benzenesulfonamides to carbonic anhydrase IX (CA IX) and characterize this anticancer target protein.

CA IX is a zinc-containing metalloenzyme that reversibly converts carbon dioxide to bicarbonate ion and acid protons. It is one of the 12 catalytically active CA isoforms present in the human body. The protein is composed of several domains: the signaling sequence, the proteoglycan (PG) domain, the catalytic domain, transmembrane helix and an intracellular domain. The PG domain is required for focal adhesion of tumor cells [2]. The catalytic activity of CA IX is similar to or slightly exceeds that of CA II [3], [4], [5], [6], [7], [8]. The expression of CA IX occurs only in few normal human tissues, the gastrointestinal tract epithelium [9], [10], [11], ovarian coelomic epithelium, pancreatic ductal cells, cells of the hair follicles and fetal rete testis [12], [13]. In most cases, CA IX expression is strongly associated with tumors [9], [14]. Increased CA IX expression has been shown to participate in the acidification of the extracellular environment [15] and help tumor cells survive, migrate and invade [2].

The most common CA inhibitors are various sulfonamides, many of them are clinically used to treat diseases related to unbalanced expression of CAs [16]. Novel CA inhibitors were often designed by attaching a tail to the pharmacophoric benzenesulfonamide headgroup [17], [18], [19]. Benzenesulfonamide makes hydrogen bonds with the zinc ion and Thr199 [20] while different substituents interact with other residues and solvent molecules in the CA active site [19], [18], [21].

CA IX has become an attractive therapeutic target since the first report showing that the inhibition of this enzyme reduces the growth of cancer cells [22]. Numerous compounds exhibit high affinity towards CA IX, but also strongly bind to undesired off-target CAs showing side effects. Several partially selective CA IX inhibitors have been synthesized and their binding and inhibition constants to CA IX were measured [23], [24], [25]. However, only the ‘observed’ parameters were determined that depend on experimental conditions such as the buffer and its pH. Here we emphasize the importance of the ‘intrinsic’ binding parameters that were determined by subtracting the linked protonation events from the observed reactions [26].

Thermodynamic parameters provide an insight into the energetic reasons of the binding reaction. Gibbs energy (ΔG) shows the overall affinity of the interaction while the changes in enthalpy (ΔH) and entropy (ΔS) upon binding provide additional molecular information about the process. The ΔH shows the heat evolved during complex formation (at constant temperature and pressure). It is related to the strength and number of formed and broken bonds [1]. The entropy indicates an increase and decrease of the degrees of freedom of the free and bound ligand, protein and water. The decrease of enthalpy and increase of entropy contributes favorably to the binding process. These parameters of binding may also provide insight into the structure of water at the binding interface [27].

Isothermal titration calorimetry (ITC) provides all these three parameters in a single experiment. ITC directly measures the heat evolved (ΔH) while the Kb can be estimated from the slope of ITC curve and ΔG can be calculated using the equation:ΔG=RTlnKbwhere R is the universal gas constant and T is the absolute temperature. Entropy can be estimated by subtraction:TΔS=ΔHΔG.

ITC experiments determine the observed thermodynamic parameters that may depend on various factors such as pH, buffer, salt, temperature, etc. However, it is important to determine the intrinsic thermodynamic parameters of binding that would be observed in the hypothetical absence of any linked reactions. For example, if a protein should bind a proton in order to be able to bind the ligand, the observed energy would be lower by the amount necessary to protonate the protein. This energy would be pH-dependent and increased 10-fold upon increasing the pH of the medium by one unit (when pH > pKa).

Fluorescent thermal shift assay (FTSA, also called Thermofluor, differential scanning fluorimetry, DSF) can be conveniently used to characterize protein stabilities in various buffers [28], [29] and determine the ligand binding affinities based on a shift in protein melting temperature (Tm) [30], [31], [32], [33]. The Tm is observed when the fluorescence of a solvatochromic dye changes upon heat-induced protein unfolding [34], [35], [31], [36], [37], [38].

During sulfonamide inhibitor binding to CAs, there are two linked protonation reactions, the protonation of the protein and deprotonation of the ligand. Such reactions may reduce the affinity thousands of times. Determination of the intrinsic thermodynamic parameters (Kb_intr, ΔGintr, ΔHintr, ΔSintr) requires multiple measurements in several buffers at various pHs. Upon protonation, the protons are picked up from or released to the buffer and the effect should be subtracted to determine the actual binding reaction [27], [39], [40], [41], [42], [43], [44], [45].

Section snippets

Proteins

The cDNA of human carbonic anhydrase IX (CA IX) was purchased from RZPD Deutsches Ressourcenzentrum für Genomforschung GmbH (Germany). To prevent the dimerization of CA IX monomers, the site-directed mutagenesis of cysteine (41) to serine was performed using the sense (5′-TCGCCGCCTTCAGCCCGGCCCTG-3) and antisense (5′-CAGGGCCGGGCTGAAGGCGGCGA-3′) mutagenic primers according to the procedure described in QuikChange Site-Directed Mutagenesis instruction manual (Stratagene).

Expression of CA IX and CA

Intrinsic thermodynamic parameters of compound binding to CA IX

The intrinsic Gibbs energies (ΔbGintr) of 40 sulfonamide compound binding to the catalytic domain of CA IX were determined. Compounds are benzenesulfonamides with benzimidazole moiety divided into four groups: the para-substituted benzensulfonamides without chlorine in meta-position (1(aj)) and bearing chlorine atom (2(aj)) and the meta-substituted benzenesulfonamides without chlorine atom in para-position (3(aj)) and with the Cl (4(aj)). The ΔbGintr of each compound binding to CA IX are

Discussion

In the studies of protein-ligand binding thermodynamics it is important to determine whether the protein or the ligand undergoes a binding-linked reaction that could influence the measurements. The most common binding-linked reaction is the protonation (or deprotonation) of the ligand or protein. Sulfonamide ligand binding to carbonic anhydrases at nearly all conditions involves binding-linked protonation of the Zn(II)-bound hydroxide and deprotonation of the compound sulfonamide group [54].

Competing interests

The authors declare that they have no competing interests.

Author's contributions

VL made all thermodynamic measurements and analysis, wrote the manuscript. JM, VJ and JJ cloned and expressed the proteins from mammalian cells. VM purified the protein. DM supervised the project and wrote the manuscript.

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Acknowledgments

This research was funded by a grant (no. SEN-04/2015) from the Research Council of Lithuania.

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