Stopped-flow assay of the inhibition of CA enzymatic activity (SFA)

Inhibitors of proteins that possess enzymatic activity and thus are enzymes are often discovered by performing biochemical assays that determine the inhibition of enzymatic activity. Such assays must be developed individually for each enzyme and are often quite tedious and material and time-consuming. The CA activity is often determined by the stopped-flow spectrophotometric method by following the decrease in absorbance of a pH indicator (e.g., phenol red) because of the drop in pH due to the protons appearing from the CO₂ hydration reaction catalyzed by the CA, Eq. (1). The assay is well-described elsewhere (Khalifah et al., Reference Khalifah, Strader, Bryant and Gibson1977; Smirnovienė et al., Reference Smirnovienė, Smirnovas and Matulis2017) and its limitations will be discussed below.

Isothermal titration calorimetry (ITC)

ITC is one of the most commonly used techniques to determine protein–ligand interactions (Chaires, Reference Chaires2008; Krimmer and Klebe, Reference Krimmer and Klebe2015; Callies and Daranas, Reference Callies and Daranas2016; Falconer, Reference Falconer2016; Renaud et al., Reference Renaud, Chung, Danielson, Egner, Hennig, Hubbard and Nar2016; Vega et al., Reference Vega, Abian and Velazquez-Campoy2016). However, the technique requires a relatively large amount of pure protein and compound. The technique is isothermal and thus can be fully performed at a physiological temperature, e.g., resembling the conditions in the human body. The reaction is performed by titrating the protein with the compound in the calorimeter cell that measures the heat evolved by the interaction of known quantities of binding partners. The heat is a universal observable in any reaction. Method development is dictated solely by the components of titration and hence straightforward. However, it is not always straightforward to interpret the data obtained. The heat of reaction determined by the calorimeter is equal to the thermodynamic parameter enthalpy, under two conditions that there is no work performed and the pressure is constant. Thus ITC provides a direct route to a determination of the enthalpy of interaction. The experiment is performed in such a way that the ligand is injected in aliquots and thus the evolved heats are proportional to the fraction of ligand bound to the protein. From the direct determination of ligand-bound fraction, one can calculate the equilibrium binding/dissociation constant (and hence affinity) for the interaction. Knowledge of the equilibrium constant permits the calculation of the change in Gibbs energy of binding:

(2)$$\Delta G = -RT\,{\rm ln}(K_b)$$

The standard entropy change upon interaction can also be determined from the same experiment by subtracting the Gibbs energy from enthalpy:

(3)$$\Delta S = \lpar {\Delta H-\Delta G} \rpar /T$$

The binding of 1 (EZA, all compound structures are shown in Supplementary Table 1) to the catalytic domains of catalytically active human recombinant CAs as determined by ITC is shown in Fig. 5. It is clear that, for example, 1 bound CA I significantly weaker than CA II under the assay conditions. Furthermore, the observed enthalpy of interaction was significantly more exothermic (more negative) for CA II than for CA I. For every reaction, the molar ratio necessary to reach the midpoint of titration was approximately 1.0. Thus, essentially all 100% of the protein was able to bind the ligand to achieve saturation. This indicates that both the protein and compound preparations were highly pure.

Fig. 5. Examples of ITC data of 1 binding to catalytically active recombinant human CA isoforms at pH 8, 25 °C as determined using the VP-ITC calorimeter. Insets show the raw curves with unflattened baselines, without user-biased interference. Experiments were performed in 50 mM sodium phosphate buffer containing 100 mM NaCl and 2% DMSO as previously described in: CA I and CA II (Morkūnaitė et al., Reference Morkūnaitė, Gylytė, Zubrienė, Baranauskienė, Kišonaitė, Michailovienė, Juozapaitienė, Todd and Matulis2015), CA IV (Mickevičiūtė et al., Reference Mickevičiūtė, Timm, Gedgaudas, Linkuvienė, Chen, Waheed, Michailovienė, Zubrienė, Smirnov, Čapkauskaitė, Baranauskienė, Jachno, Revuckienė, Manakova, Gražulis, Matulienė, Di Cera, Sly and Matulis2017), CA VB (Kasiliauskaitė et al., Reference Kasiliauskaitė, Časaitė, Juozapaitienė, Zubrienė, Michailovienė, Revuckienė, Baranauskienė, Meškys and Matulis2016), CA VI (Kazokaitė et al., Reference Kazokaitė, Milinavičiūtė, Smirnovienė, Matulienė and Matulis2015), CA VII (Pilipuitytė and Matulis, Reference Pilipuitytė and Matulis2015), CA IX (Linkuvienė et al., Reference Linkuvienė, Matulienė, Juozapaitienė, Michailovienė, Jachno and Matulis2016b), CA XII (Jogaitė et al., Reference Čapkauskaitė, Zubrienė, Smirnov, Torresan, Kišonaitė, Kazokaitė, Gylytė, Michailovienė, Jogaitė, Manakova, Gražulis, Tumkevičius and Matulis2013), and CA XIII (Baranauskienė and Matulis, Reference Baranauskienė and Matulis2012). All panels are drawn at the same scale to help visualize the differences both in enthalpies and affinities of binding. No data have been determined at comparable conditions for CA III, CA VA, and CA XIV.

Fluorescent thermal shift assay

FTSA, also commonly termed DSF, or ThermoFluor (when performed in a high-throughput plate format) is the assay that determines the melting (unfolding or denaturation) temperature T _m of the protein in the absence and the presence of increasing concentrations of a ligand. The T _m may be determined by fluorescence or by differential scanning calorimetry (DSC) (Brandts and Lin, Reference Brandts and Lin1990). The assay may follow the intrinsic protein fluorescence of tryptophan or tyrosine residues as the protein unfolds and the environment of these residues changes. Alternatively, the fluorescence of a solvatochromic probe such as ANS (1,8-anilinonaphthalene sulfonate) (Anderson and Weber, Reference Anderson and Weber1969; Slavik, Reference Slavik1982; Matulis and Lovrien, Reference Matulis and Lovrien1998; Matulis et al., Reference Matulis, Baumann, Bloomfield and Lovrien1999; Cimmperman and Matulis, Reference Cimmperman and Matulis2011) or Sypro Orange (Lo et al., Reference Lo, Aulabaugh, Jin, Cowling, Bard, Malamas and Ellestad2004; Niesen et al., Reference Niesen, Berglund and Vedadi2007) can be followed as the probe binds to the exposed hydrophobic residues upon protein unfolding. FTSA is a less-widely used technique than ITC, but its use has significantly increased when compound libraries are screened to determine the best binders to target proteins (Pantoliano et al., Reference Pantoliano, Petrella, Kwasnoski, Lobanov, Myslik, Graf, Carver, Asel, Springer, Lane and Salemme2001; Yanchunas et al., Reference Yanchunas, Langley, Tao, Rose, Friborg, Colonno and Doyle2005; McDonnell et al., Reference McDonnell, Yanchunas, Newitt, Tao, Kiefer, Ortega, Kut, Burford, Goldfarb, Duke, Shen, Metzler, Doyle, Chen, Tarby, Borzilleri, Vaccaro, Gottardis, Lu, Crews, Kim, Lombardo and Roussel2009). The assay is relatively easy and robust and does not require assay development for a particular protein–ligand system. However, data interpretation sometimes may not be fully straightforward (Cimmperman et al., Reference Cimmperman, Baranauskienė, Jachimovičiūtė, Jachno, Torresan, Michailovienė, Matulienė, Sereikaitė, Bumelis and Matulis2008; Cimmperman and Matulis, Reference Cimmperman and Matulis2011).

In the assay, a series of wells with the constant protein concentration are prepared and various compound concentrations are added. The mixture is heated and the melting curves at all compound concentrations are recorded simultaneously in approximately 1 hour. The melting curves are fit to the 2-state (native-unfolded) model (Matulis et al., Reference Matulis, Kranz, Salemme and Todd2005; Cimmperman et al., Reference Cimmperman, Baranauskienė, Jachimovičiūtė, Jachno, Torresan, Michailovienė, Matulienė, Sereikaitė, Bumelis and Matulis2008) providing the T _ms at various compound concentrations. These T _m dependencies on compound concentration are fit to the model that includes the thermodynamic parameters of protein unfolding and ligand binding (Cimmperman et al., Reference Cimmperman, Baranauskienė, Jachimovičiūtė, Jachno, Torresan, Michailovienė, Matulienė, Sereikaitė, Bumelis and Matulis2008) yielding the affinity of the interaction (the dissociation constant K _d).

The binding affinities of 1 to all 12 catalytically active human CA isoforms determined by FTSA are shown in Fig. 6. The plots show the 1 dosing curves (where the compound concentration is on a logarithmic scale), while the insets show the melting curves with various added inhibitor concentrations. Compound 1 stabilized each CA isoform to a different extent. For example, CA III was stabilized by only several degrees, thus showing that this isoform binds the ligand weakly. However, CA IX was stabilized by more than 10° indicating strong interaction. In addition, the assay yielded stabilities of all CA isoforms in the absence of ligand at these assay conditions. It should be kept in mind that direct comparison between the ΔT _ms is usually valid only when the proteins are of similar molecular mass.

Fig. 6. Example of FTSA data of 1 binding to all 12 catalytically active recombinant human CA isoforms at pH 6. The insets show raw melting curves obtained by following ANS fluorescence. The experiments were performed at pH 6 in universal buffer made of 50 mM sodium phosphate, 50 mM sodium acetate, and 25 mM sodium borate containing 100 mM NaCl and up to 2% DMSO and 50 µM ANS as previously described: CA I and CA II (Morkūnaitė et al., Reference Morkūnaitė, Gylytė, Zubrienė, Baranauskienė, Kišonaitė, Michailovienė, Juozapaitienė, Todd and Matulis2015), CA IV (Mickevičiūtė et al., Reference Mickevičiūtė, Timm, Gedgaudas, Linkuvienė, Chen, Waheed, Michailovienė, Zubrienė, Smirnov, Čapkauskaitė, Baranauskienė, Jachno, Revuckienė, Manakova, Gražulis, Matulienė, Di Cera, Sly and Matulis2017), CA VB (Kasiliauskaitė et al., Reference Kasiliauskaitė, Časaitė, Juozapaitienė, Zubrienė, Michailovienė, Revuckienė, Baranauskienė, Meškys and Matulis2016), CA VI (Kazokaitė et al., Reference Kazokaitė, Milinavičiūtė, Smirnovienė, Matulienė and Matulis2015), CA VII (Pilipuitytė and Matulis, Reference Pilipuitytė and Matulis2015), CA IX (Linkuvienė et al., Reference Linkuvienė, Matulienė, Juozapaitienė, Michailovienė, Jachno and Matulis2016b), CA XII (Jogaitė et al., Reference Jogaitė, Zubrienė, Michailovienė, Gylytė, Morkūnaitė and Matulis2013), CA XIII (Baranauskienė and Matulis, Reference Baranauskienė and Matulis2012), and CA XIV (Juozapaitienė et al., Reference Juozapaitienė, Bartkutė, Michailovienė, Zakšauskas, Baranauskienė, Satkūnė and Matulis2016).

Surface plasmon resonance

SPR is also a commonly used technique to determine protein–ligand interactions and has been extensively reviewed (Myszka and Rich, Reference Myszka and Rich2000; Patching, Reference Patching2014; Olaru et al., Reference Olaru, Bala, Jaffrezic-Renault and Aboul-Enein2015; Renaud et al., Reference Renaud, Chung, Danielson, Egner, Hennig, Hubbard and Nar2016). The technique determines the protein–ligand association and dissociation rates. Therefore, it is often used to determine the kinetics of interactions. The inverse of the dissociation rate is equal to the residence time, a commonly used characteristic of the drug lead–protein pair. We will go in more detail on the assay in the section on sulfonamide–CA interaction kinetics.

Advantages and limitations of SFA, FTSA, and ITC

Here we emphasize that for the most complete picture of a studied binding reaction a multi-technique-based approach is recommended. We use all of the above-described assays for many CA inhibitors. Each technique has numerous advantages and disadvantages (Renaud et al., Reference Renaud, Chung, Danielson, Egner, Hennig, Hubbard and Nar2016). However, due to limitations in time and material, and the necessity to test as many chemical compounds as possible, some techniques may be more suited than others.

In Fig. 7, we compare the advantages and limitations of SFA, FTSA, and ITC. Panels on the left show simulated data while examples of observed data are shown on the right. The main limitation of SFA is that the IC ₅₀ may only be determined down to about P _t/2 (half of the protein concentration) (Copeland, Reference Copeland2005). Despite being one of the fastest known enzymes, the concentration of any CA isoform cannot be reduced below 10 nM even for the most active CA II and CA IX and, therefore, the IC ₅₀ cannot be determined below approximately 5 nM. The dotted and solid curves in Fig. 7a show the position of the dosing curves if the protein concentration was 10 nM. The lines almost completely coincide with each other and, therefore, the compounds with picomolar affinities cannot be distinguished from nanomolar ones. In order to distinguish the curves, one would need to significantly reduce the protein concentration. This cannot be done in most cases and thus the K _i of CA inhibitors cannot be determined below approximately 2 nM.

Fig. 7. Comparison of SFA, FTSA, and ITC techniques, their advantages and limitations. Panels on the left show simulated curves, while on the right – the examples of measured data. The figure is adapted from Smirnovienė et al. (Reference Smirnovienė, Smirnovas and Matulis2017). (a) The application of Hill equation at protein concentration P _t = 10nM and various values of apparent IC ₅₀. The assay would not distinguish between 1 nM and 1 pM compounds because P _t cannot be reduced to less than 1 nM or 1 pM to obtain distinguishable dosing curves of nanomolar (solid line) or picomolar (dotted line) affinity. (b) Experimental data of an interaction between CA II-307 at several compound concentrations. Data points were fitted according to the Hill equation with varied Hill coefficient. The inset shows the raw absorbance curves at several compound concentrations. (c) The simulated FTSA dosing curves for different binding affinities (K _d from 1 pM to 1 mM) are shown. These curves were generated using the following set of parameters: T = 37 °C, P _t = 10 µM, enthalpy of CA II unfolding is 690 kJ mol⁻¹, heat capacity of unfolding is 17 kJ mol⁻¹ K⁻¹, enthalpy of binding is −42 kJ mol⁻¹, heat capacity of binding is −0.8 kJ mol⁻¹ K⁻¹ , and the melting temperature T _m without added ligand is 60 °C. (d) Experimental FTSA data of CA I-323 (squares), CA II-10 (circles), and CA XIII-310 (triangles). The insets show raw melting curves at 0 and 200 µM inhibitor concentrations. (e) Simulated ITC curves using a single binding site model of different binding affinities at P _t = 10 µM. (f) Experimental ITC curves of the same binding interactions as in panel D. Affinity of CA I-323 is too strong to be determined by ITC but FTSA assay provides accurate K _d.

Furthermore, some isoforms possess significantly lower specific enzymatic activity and thus their concentration in the assay must be increased. For example CA I is approximately 10-times less active than CA II. Therefore, the lowest K _i that can be determined for inhibitors of CA I at the same conditions is approximately 20 nM. This may lead to incorrect assignment of inhibitor selectivity towards CA II over CA I based on SFA alone.

Another significant disadvantage of SFA is that, as shown in Fig. 7b, if the protein concentration is varied, the dosing curves also shift and thus the K _i could appear to vary dependent on the concentration used in the assay. If the experiment is performed at a single CA concentration, the obtained values may be inaccurate. Therefore, SFA should be used with care keeping in mind the applied protein concentration in the assay that limits the K _i reachable in the assay.

In FTSA (Fig. 7c), there are essentially no limitations in affinity determination, thus picomolar and millimolar compounds may be easily distinguished in the same assay. At the simulated conditions, the picomolar compound would shift the temperature by over 20 °C, while the millimolar compound – by only 1° or 2° dependent on the added concentration. In order to detect millimolar compounds, it would be necessary to add a 10 mM compound. Figure 7d shows examples of the observed data. The limitation of FTSA is that the assay cannot be used for highly stable proteins that melt at temperatures over 90 °C. However, CAs denature around 55–65 °C. Thus, FTSA has a significant advantage over SFA because strongly-interacting picomolar compound K _ds may be determined correctly by FTSA and not by SFA.

The main disadvantage of FTSA compared with SFA is that it does not determine whether the compound is actually an inhibitor of the enzyme or just a binder. Ligands potentially may bind to various sites on CA surface, stabilize the protein, and be erroneously assigned to be an inhibitor of CA. Therefore, the SFA is a necessary confirmatory technique to demonstrate that the compound is actually an inhibitor of enzymatic activity. However, the affinity of an inhibitor will still be determined more accurately by FTSA than SFA. Since primary sulfonamides bind to the active site in a well-established way, both techniques yield the same K _d or K _i when the assays are properly performed.

The simulated curves of the third assay, ITC, are shown in Fig. 7e. The assay has optimal accuracy when the curve is of sigmoidal shape, such as the dashed curves (K _d = 100 − 1000 nM). If the compound binds weakly, for example in the micromolar range, the curve becomes too flat and may be indistinguishable from the curve of compound dilution, thus potentially leading to erroneous assignment of the observed data to a binding reaction. In such a situation the protein concentration must be raised, if the solubility of both components permits, expanding the window of affinity determination. On the other hand, if the compound has a K _d of 1 nM or tighter, the curve is too steep and the K _d again cannot be determined. It may be stated that ‘the binding is 10 nM or tighter’, but the actual affinity cannot be determined. In such a situation, the protein solution may be diluted as long as the signal remains sufficiently above the noise level.

The main advantage of ITC is that it is the only assay that may be used to directly determine the enthalpy of protein–ligand binding. It is also the most accurate technique to obtain the entropy and heat capacity of interaction. The change in enthalpy is most accurately determined when the binding is tight, i.e. nanomolar or tighter. It is nearly impossible to determine the change in enthalpy of weakly binding compounds by direct ITC titration (displacement titration may be sometimes used instead). Raising the protein concentration increases the range, but may not be practical in some cases.

Due to the above-described limitations, it is good to combine FTSA-determined K _d and the enthalpy determined by ITC. It is not possible to accurately determine subnanomolar K _d values from ITC without applying displacement ITC techniques that are not discussed here in detail, but have been introduced and reviewed previously (Krainer et al., Reference Krainer, Broecker, Vargas, Fanghänel and Keller2012; Krainer and Keller, Reference Krainer and Keller2015).

Comparison of the observed K _d,obs values obtained by the above-described four techniques is shown in Fig. 8. The FTSA is the most robust technique in our opinion, there is essentially no substrate (CO₂) present (concentration of CO₂ from the air is significantly below the K _M of any CA), and the assay is practically without limits of the range (accessing K _d,obss from at least pM to mM or greater). Therefore all compound interaction with CA isoforms has been determined by FTSA. Only a fraction of the compound affinities has been determined by the other three techniques.

Fig. 8. Comparison of the experimentally determined K _d,obs obtained by four techniques: fluorescent thermal shift assay (FTSA), isothermal titration calorimetry (ITC), stopped-flow assay of the inhibition of CA enzymatic activity, CO₂ hydration (SFA), and the surface plasmon resonance (SPR). All K _ds have been determined by FTSA as the most robust and reliable technique and compared with the results obtained for a portion of compounds by ITC, SFA, and SPR. The FTSA practically does not have limits or range of reliable determinations, the compound K _ds may be determined from pM to mM affinity range in a single experiment, while the other three techniques have limitations of the range where reliable determinations may be made, shown with arrows and shaded in grey.

ITC cannot accurately determine the K _d below 1 nM if we keep the protein concentration around 10 μM because the Wiseman c factor, equal to the product of the K _b and protein molar concentration for 1 : 1 binding reactions, which shows the steepness of the titration curve (see Fig. 7 and should be in the range of approximately 5–500), (Wiseman et al., Reference Wiseman, Williston, Brandts and Lin1989) reaches 1000. Some increase of the accessible range may be achieved by reducing the protein concentration. Here we see (Fig. 8a) that there is a decent correlation between the affinities determined by FTSA and ITC in the 100 nM K _d range, but the ITC-determined values tend to show reduced affinities relative to FTSA in the 1–10 nM range. ITC thus tends to underestimate the single-digit nanomolar affinities. It is also important that the spread of data may reach up to 10-fold in affinity when comparing ITC with FTSA.

There was a quite good correlation between the affinities determined by FTSA and SFA (Fig. 8b). As discussed above, it is important to use SFA to confirm that the compounds are inhibitors of the enzymatic activity. In the case of CA inhibitors, because all compounds, reviewed in this paper, are primary sulfonamides with a well-described mode of binding by a number of techniques, we do not perform the SFA for all compounds due to several reasons. First, the assay cannot determine the IC ₅₀s below the protein concentration, which is approximately 10 nM (Smirnovienė et al., Reference Smirnovienė, Smirnovas and Matulis2017). Second, the assay is significantly more costly both in time and materials to justify its performance for all studied compounds.

The bottom panel (Fig. 8c) compares the affinities obtained by FTSA and SPR. The SPR technique is commonly used to determine both the affinity and the kinetics of compound association and dissociation from CA (Redhead et al., Reference Redhead, Satchell, Morkūnaitė, Swift, Petrauskas, Golding, Onions, Matulis and Unitt2015; Talibov et al., Reference Talibov, Linkuvienė, Matulis and Danielson2016). Similarly to the other three techniques, the spread or uncertainty of the data is quite similar.

Table 1 lists and provides some examples of K _d,obs and K _i values obtained by SFA, FTSA, ITC, and SPR (Smirnovienė et al., Reference Smirnovienė, Smirnovas and Matulis2017). In cases, where the four techniques yielded different values, the bold numbers show the correct determination of the affinities. For some ligands of average affinity, all four techniques yielded the same value. For example, the binding and inhibition constants of 187 interactions with CA I, CA II, CA VII, CA XII, and CA XIII were essentially the same by all techniques. However, for example, the binding affinities of 323 to CA I, and of compounds 369 and 341 to CA IX could be correctly determined only by FTSA. The remaining three assays, SFA, ITC, and SPR, confirmed that the interaction is very strong, but could not reach the actual affinity value due to above-described limitations.

Table 1. Comparison of binding and inhibition constants of CA I, CA II, CA VI, CA VII, CA IX, CA XII, and CA XIII obtained by SFA (pH 7.0–7.5, 25 °C), FTSA, ITC (pH 7.0, 37 °C), and SPR (pH 7.0–7.4, 25 °C). SFA. The used K _M values were, for CA I – 1.4 mM, CA II – 4.7 mM, CA VI – 6.9 mM, CA VII – 11.4 mM, CA IX – 6.9 mM, CA XII – 12 mM, and CA XIII – 13.8 mM, taken from Supuran (Reference Supuran2008). The IC ₅₀ is the 50% inhibition concentration of enzymatic activity obtained by Hill fit of the data. K _i is the inhibition constant obtained after application of the Cheng–Prusoff equation to the IC ₅₀. The K _d,obs is the dissociation constant obtained via Morrison equation. FTSA. The K _d,obs is the observed dissociation constant obtained by FTSA at the CA concentration 5 to 10 µM. ITC. The K _d,obs is the observed dissociation constant obtained by ITC at the CA concentration of 4 to 10 µM. SPR. The K _d,obs is the observed dissociation constant obtained by SPR at the CA concentration for immobilization of 75 µg ml⁻¹ for CA I, 100 µg ml⁻¹ for CA II, 25 µg ml⁻¹ for CA VII, 100 µg ml⁻¹ for CA IX, 25 µg ml⁻¹ for CA XII, 75μg ml⁻¹ for CA XIII as described by Talibov et al. (Reference Talibov, Linkuvienė, Matulis and Danielson2016). Values are shown in the regular font when the results match among three methods within an approximate error of ±2 fold. Values are shown in bold to point the most reliable value when results are significantly different among the methods. Values are in italic to emphasize unreliable results when the methods should not be used due to their limitations (for SFA – concentration of the enzyme above the concentration of inhibitor, for ITC – Wiseman c-factor outside the required range). SFA, FTSA, and ITC data are taken from the references indicated next to the compound number. The standard error of K _d measurements is ±2 times (Petrauskas et al., Reference Petrauskas, Baranauskienė, Zubrienė and Matulis2016; Linkuvienė et al., Reference Linkuvienė, Krainer, Chen and Matulis2016a)

Volume change upon protein–ligand binding

The above-described assays provide means to determine inhibition and inhibitor binding thermodynamic and kinetic parameters. However, the picture of protein–ligand interaction can be significantly enhanced by the determination of volumetric properties of the interactions. Unfortunately, the majority of studies are limited to the techniques, which exploit only the temperature as a thermodynamic variable and avoid studying pressure effects because such techniques are much more laborious.

There are several techniques that are capable of determining the volume of protein–ligand binding: the fluorescent pressure shift assay (PressureFluor, FPSA) (Toleikis et al., Reference Toleikis, Cimmperman, Petrauskas and Matulis2011, Reference Toleikis, Sirotkin, Skvarnavičius, Smirnovienė, Roumestand, Matulis and Petrauskas2016; Skvarnavičius et al., Reference Skvarnavičius, Toleikis, Grigaliūnas, Smirnovienė, Norvaišas, Cimmperman, Matulis and Petrauskas2017), vibrating tube densitometry(Barbosa et al., Reference Barbosa, Taboada and Mosquera2003; Son et al., Reference Son, Shek, Dubins and Chalikian2012; Toleikis et al., Reference Toleikis, Sirotkin, Skvarnavičius, Smirnovienė, Roumestand, Matulis and Petrauskas2016), and the high-pressure NMR (Wilton et al., Reference Wilton, Kitahara, Akasaka, Pandya and Williamson2009; Roche et al., Reference Roche, Caro, Norberto, Barthe, Roumestand, Schlessman, Garcia, García-Moreno and Royer2012; Toleikis et al., Reference Toleikis, Sirotkin, Skvarnavičius, Smirnovienė, Roumestand, Matulis and Petrauskas2016). They can determine the change of system volume and compressibility caused by the bound ligand. The volumetric assays have been described previously by Carey et al. (Reference Carey, Knowles and Gibson1977); Barbosa et al. (Reference Barbosa, Taboada and Mosquera2003); Chalikian and Filfil (Reference Chalikian and Filfil2003); Wilton et al. (Reference Wilton, Kitahara, Akasaka, Pandya and Williamson2009); Toleikis et al. (Reference Toleikis, Cimmperman, Petrauskas and Matulis2011, Reference Toleikis, Cimmperman, Petrauskas and Matulis2012, Reference Toleikis, Sirotkin, Skvarnavičius, Smirnovienė, Roumestand, Matulis and Petrauskas2016); Roche et al. (Reference Roche, Caro, Norberto, Barthe, Roumestand, Schlessman, Garcia, García-Moreno and Royer2012); Petrauskas et al. (Reference Petrauskas, Gylytė, Toleikis, Cimmperman and Matulis2013); Voloshin et al. (Reference Voloshin, Medvedev, Smolin, Geiger and Winter2015); Skvarnavičius et al. (Reference Skvarnavičius, Toleikis, Grigaliūnas, Smirnovienė, Norvaišas, Cimmperman, Matulis and Petrauskas2017). However, there is relatively little data available in the literature on any protein–ligand system, not mentioning the CA, and it is difficult to judge yet if these approaches will be useful in the design of ligands with desired binding properties.

Intrinsic affinity of binding

Sulfonamides are by far the largest class of compounds that are inhibitors of CAs. However, despite the long history and importance of sulfonamides as CA inhibitors, there still remains uncertainty in their binding mechanism. As first identified by Taylor et al. (Reference Taylor, King and Burgen1970), the binding kinetics of sulfonamide inhibitor to CA II is dependent on pH and the dependence has a U-shape, interaction is fastest near neutral pH and slows down towards acidic and alkaline regions. Application of FTSA, ITC, and SPR techniques also showed that the binding affinity is strongest near neutral pH and decreases both in the acidic and alkaline regions. The affinity decreases exactly tenfold with one pH unit both in the acidic and alkaline regions. This indicates that the CA-sulfonamide binding reaction is coupled to at least two protonation–deprotonation reactions. As pointed by Taylor et al. (Reference Taylor, King and Burgen1970), there are four possible ways for the sulfonamide compound to bind the CA, shown in Eqs. (4)–(7).

(4)$$\eqalign{{{\rm CA} &- {\rm Zn}^{{\rm II}}{\rm - OH}^-+ {\rm RSO}_2{\rm NH}_2}\cr & {\rightleftharpoons {\rm CA} - {\rm Zn}^{{\rm II}} - {\rm NH}_2{\rm SO}_2{\rm R} + {\rm OH}^-$$}}

(5)$$\eqalign{{{\rm CA} &- {\rm Zn}^{{\rm II}}{\rm - OH}^- + {\rm RSO}_2{\rm NH}^-}\cr & {\rightleftharpoons {\rm CA} - {\rm Zn}^{{\rm II}} - {\rm NH} - {\rm SO}_2{\rm R + O}{\rm H}^- }}$$

(6)$$\eqalign{{{\rm CA} &- {\rm Zn}^{{\rm II}} - {\rm H}_2{\rm O + RS}{\rm O}_2{\rm NH}_2}\cr & {&\rightleftharpoons {\rm CA} - {\rm Zn}^{{\rm II}} - {\rm NH}_2{\rm SO}_2{\rm R} + {\rm H}_2{\rm O}}} $$

(7)$$\eqalign{{\rm CA - Z}n^{{\rm II}}{\rm -} {\rm H}_2{\rm O\ +\ RS}{\rm O}_2{\rm N}{\rm H}^-}\cr &\quad\rightleftharpoons {\rm CA - Z}{\rm n}^{{\rm II}}{\rm - NH - S}{\rm O}_2{\rm R\ +} {\rm H}_2{\rm O\;} $$

The first possibility is that the electrostatically neutral (protonated) sulfonamide replaces the OH⁻ bound to the Zn^II in the active site of CA (Eq. (4)). Second, that the negatively charged sulfonamide replaces the OH⁻ (Eq. (5)), third, that neutral sulfonamide displaces neutral water molecule (Eq. (6)), and the fourth, that the deprotonated sulfonamide (negatively charged SO₂NH⁻) replaces the H₂O molecule coordinated by the Zn^II in the active site of CA (Eq. (7)). The second and third reactions have been already shown by the authors to be inconsistent with the U-shape dependence on pH. However, both the first and the fourth reactions seemed plausible and were difficult to distinguish.

We are now convinced that the fourth reaction (Eq. (7)) is the only or at least a dominant reaction that takes place when sulfonamides bind to a CA, meaning that the intrinsic binding reaction is between the negatively charged sulfonamide to such form of CA where the fourth ligand bound to the Zn^II in the active site is electrostatically neutral water molecule. The first indication that the fourth reaction occurs was the spectral evidence on Co-containing CA enzyme (Engberg and Lindskog, Reference Engberg and Lindskog1984; Tu and Silverman, Reference Tu and Silverman1986). However, a more recent neutron-diffraction crystal structure of acetazolamide bound to CA II from McKenna laboratory (Fisher et al., Reference Fisher, Boone, Biswas, Venkatakrishnan, Aggarwal, Tu, Agbandje-McKenna, Silverman and McKenna2012b) directly showed that the sulfonamide amino group possesses only one proton and thus the group is deprotonated and negatively charged when bound to the CA II. Our data are consistent with this conclusion. However, it is difficult to distinguish the fourth possibility from the first using thermodynamic methods alone.

At neutral pH 7, most sulfonamides are almost fully protonated since their pK _as are usually around 9 or 10, in the form that is unable to bind. However, a small fraction is deprotonated and binds to CA. When the deprotonated form is depleted, additional sulfonamide deprotonates to keep the fraction unchanged. This additional sulfonamide also binds to CA and so on until the enzyme is fully saturated with the inhibitor.

Similarly, only a part of the Zn^II-coordinated molecules are in H₂O form because the pK _as of most human CAs is approximately 7. Thus, approximately half of CA fraction is deprotonated and must undergo protonation in order to bind the sulfonamide anion. When these linked reactions (Eq. (8)) occur, the proton must be uptaken from the buffer or released to the buffer (Fig. 9).

$$\eqalign{&{\rm Intrinsic:\;\ CA - Z}{\rm n}^{{\rm II}}{\rm -} {\rm H}_2{\rm O} + {\rm RS}{\rm O}_2{\rm N}{\rm H}^-\cr &\quad\quad\quad\quad\quad\rightleftharpoons {\rm CA - Z}{\rm n}^{{\rm II}}{\rm - NH - S}{\rm O}_2{\rm R} + {\rm H}_2{\rm O}$$

$${\rm Ligand}:{\rm RS}{\rm O}_2{\rm N}{\rm H}^- + {\rm \;} {\rm H}^ + {\rm \;} \rightleftharpoons {\rm \;\ RS}{\rm O}_2{\rm N}{\rm H}_2$$

(8)$${\rm Protein}:{\rm CA - Z}{\rm n}^{{\rm II}}{\rm - O}{\rm H}^- + {\rm H}^ + \rightleftharpoons {\rm \;\ CA - Z}{\rm n}^{{\rm II}}{\rm -} {\rm H}_2{\rm O}$$

$${\rm Buffer}:{\rm Buf} + {\rm H}^ + \rightleftharpoons {\rm Buf}{\rm H}^ + {\rm \;} $$

$${\rm Observed:}\,{\rm Intrinsic\ +\ Ligand\ +\ Protein\ +\ Buffer}$$

Fig. 9. The model of the linked reactions (also shown as Eq. (8)), occurring upon sulfonamide ligand binding to CA. A sum of several binding-linked reactions occur that we observe by any experimental technique, but the Quantitative Structure–Activity Relationship (QSAR)-type analysis requires that we analyze only the intrinsic reaction and correlate the intrinsic affinity and other intrinsic parameters with the structure of the ligand and the ligand–protein complex. The linked contributing reactions must be subtracted from the observed ones to obtain the intrinsic reaction.

The fraction of deprotonated sulfonamide is dependent on the sulfonamide amino group pK _a,SA and can be calculated using Eq. (9):

(9)$$f_{{\rm RS}{\rm O}_2{\rm N}{\rm H}^-} = \displaystyle{{{10}^{{\rm pH}-{\rm p}K_{a,{\rm SA}}}} \over {1 + {10}^{{\rm pH}-{\rm p}K_{a{\rm, SA}}}}}$$

Similarly, the fraction of binding-ready CA depends on pH and the pK _a,CA of the water molecule in the active site and can be calculated using Eq. (10):

(10)$$f_{{\rm CAZn}{\rm H}_2{\rm O}} = \displaystyle{{{10}^{\,pK_{a,{\rm CA}}-{\rm pH}}} \over {1 + {10}^{\,pK_{a,{\rm CA}}-{\rm pH}}}}$$

The affinity of interaction will be reduced by the amount how much the fractions are reduced. In other words, the observed binding constant K _b,obs indicates us a diminished K _b,int due to reduced fractions. The intrinsic K _b,int can be calculated using Eq. (11):

(11)$$K_{b,int} = \displaystyle{{K_{b,obs}} \over {\,f_{{\rm CAZn}{\rm H}_2{\rm O}} \times f_{{\rm RS}{\rm O}_2{\rm N}{\rm H}^-}}}$$

Figure 10 shows the relationship between the intrinsic and observed affinities and the dependence of the latter on pH. The intrinsic affinity is independent of pH, but because there are a reduced fraction of both interacting components at both acidic and alkaline pHs, there is energy needed to overcome this linked reaction and therefore the observed affinity is reduced

Fig. 10. Relationship between the intrinsic (independent of pH, shown as horizontal line) and observed affinities (filled circles fit by the solid line of overturned U-shape) of 1 binding to CA IX. The observed affinity is reduced both at acidic and alkaline pHs due to reduced fractions of available binding-ready species (negatively charged sulfonamide, shown as solid red line, and CA, bearing the neutral water molecule, shown as a dashed red line).

In turn, the intrinsic Gibbs energy of binding can be calculated using Eq. (12):

(12)$$\Delta G_{int} = -RT\,{\rm ln}(K_{b,int})$$

The change in the standard Gibbs energy of ligand binding dependence on pH has a U-shape, similar to the K _d, but upside down as K _b. Figure 11 shows that the U-shapes are obtained for all catalytically active human CAs. All experimental techniques discussed in this paper (FTSA, SFA, ITC, and SPR), yield the U-shape inhibitor binding Gibbs energy dependence on pH. In all cases, the affinity is strongest in near-neutral pH range and decreases in both directions. The observed affinity comes closest to the intrinsic value at near-neutral pH, but usually does not reach it and thus is almost never observed experimentally.

Fig. 11. The dependence of ΔG _obs on pH determined by FTSA (filled circles) and ITC (open circles) methods at 25 °C. Circles show the binding of 1 and squares of 303 (to CA III and CA VA). Solid curve is a fit of the model, solid line is intrinsic Gibbs energy of binding, dashed line is a fraction of ligand, dotted line is a fraction of protein. Experiments were performed in universal buffer made of 50 mM sodium phosphate, 50 mM sodium acetate, and 25 mM sodium borate containing 100 mM NaCl and up to 2% DMSO as previously described in: CA I and CA II (Morkūnaitė et al., Reference Morkūnaitė, Gylytė, Zubrienė, Baranauskienė, Kišonaitė, Michailovienė, Juozapaitienė, Todd and Matulis2015), CA IV (Mickevičiūtė et al., Reference Mickevičiūtė, Timm, Gedgaudas, Linkuvienė, Chen, Waheed, Michailovienė, Zubrienė, Smirnov, Čapkauskaitė, Baranauskienė, Jachno, Revuckienė, Manakova, Gražulis, Matulienė, Di Cera, Sly and Matulis2017), CA VB (Kasiliauskaitė et al., Reference Kasiliauskaitė, Časaitė, Juozapaitienė, Zubrienė, Michailovienė, Revuckienė, Baranauskienė, Meškys and Matulis2016), CA VI (Kazokaitė et al., Reference Kazokaitė, Milinavičiūtė, Smirnovienė, Matulienė and Matulis2015), CA VII (Pilipuitytė and Matulis, Reference Pilipuitytė and Matulis2015), CA IX (Linkuvienė et al., Reference Linkuvienė, Matulienė, Juozapaitienė, Michailovienė, Jachno and Matulis2016b), CA XII (Jogaitė et al., Reference Čapkauskaitė, Zubrienė, Smirnov, Torresan, Kišonaitė, Kazokaitė, Gylytė, Michailovienė, Jogaitė, Manakova, Gražulis, Tumkevičius and Matulis2013), CA XIII (Baranauskienė and Matulis, Reference Baranauskienė and Matulis2012), and CA XIV (Juozapaitienė et al., Reference Juozapaitienė, Bartkutė, Michailovienė, Zakšauskas, Baranauskienė, Satkūnė and Matulis2016). All panels are drawn at the same scale to help visualize the differences in affinities of binding.

Intrinsic enthalpy of binding

The change in the observed enthalpy on the binding, similarly to ΔG, also has contributions from all linked processes, including inhibitor deprotonation, protonation of the CA-bound hydroxide, and the compensating buffer effects. Therefore, to get the change in the intrinsic enthalpy on binding it is necessary to subtract all linked processes as shown in Eq. (13):

(13)$$\Delta H_{int} = \Delta H_{obs}-n_{{\rm SA}}\Delta H_{\,pr,{\rm SA}}-n_{{\rm CA}}\Delta H_{\,pr,{\rm CA}} + n_{buf}\Delta H_{\,pr,buf}$$

where ΔH _obs – the observed enthalpy of binding obtained by direct ITC titration, ΔH _pr,SA – the enthalpy of protonation of deprotonated inhibitor, ΔH _pr,CA – the enthalpy of protonation of the CA-bound OH⁻, and ΔH _pr,buf – the enthalpy of buffer protonation. The ns denote the numbers of protons uptaken or released:

(14)$$n_{{\rm SA}} = f_{{\rm RS}{\rm O}_2{\rm N}{\rm H}^-}-1$$

is the number of protons released from the inhibitor to buffer (n _inh = −1 to 0),

(15)$$n_{{\rm CA}} = 1-f_{{\rm CAZn}{\rm H}_2{\rm O}}$$

is the number of protons uptaken by the CA-bound OH⁻ (n _CA = 0 to 1),

(16)$$n_{buf} = n_{{\rm SA}} + n_{{\rm CA}}$$

is the net sum of protons uptaken by the buffer both from the ligand and protein or released by the buffer to the ligand or protein (n _buf = −1 to 1).

The enthalpy of protonation of TRIS base is −47.45 kJ mol⁻¹ and of ${\rm HPO}_4^{2-} $ is −5.1 kJ mol⁻¹ at 25 °C (Goldberg et al., Reference Goldberg, Kishore and Lennen2002).

If we perform sulfonamide–CA ITC titrations in various buffers at various pHs as shown in Fig. 12, an X-shape dependence is observed as shown in Fig. 13. At low pH, the observed enthalpies (ΔH _obs) in phosphate buffer are low in magnitude, while in TRIS – significantly more exothermic. The difference between the observed enthalpies is equal to the difference between the enthalpies of protonation of the two buffers. In alkaline region, the situation is reversed – the enthalpies in phosphate became much more exothermic, while in TRIS – less exothermic. The red solid line shows the position of the intrinsic enthalpy (ΔH _int) of 1 binding to CA I. The intrinsic enthalpy is independent of pH. The dashed line shows a hypothetical situation for the observed enthalpy dependence on pH if the buffer had exactly zero enthalpy of protonation (ΔH _pr,buf = 0). The difference between the dashed line and the intrinsic enthalpy in the acidic region is equal to the enthalpy of inhibitor protonation (ΔH _pr,SA), while in the alkaline region – to the enthalpy of protonation of the CA-bound OH⁻ (ΔH _pr,CA).

Fig. 12. ITC data of 1 binding to CA IX in two buffers at several pHs at 25 °C. The observed enthalpies depended both on the buffer and on the pH used in the experiment. In such cases, it is necessary to obtain the intrinsic enthalpy to perform any meaningful Structure–Activity Relationship (SAR)-type study. The inset shows a typical ITC raw data curve.

Fig. 13. The observed enthalpies obtained by ITC of 1 binding to CA I in sodium phosphate or TRIS chloride buffer as a function of pH at 25 °C. Blue datapoints show the ΔH _obs in phosphate buffer, while black – in TRIS. Solid black and blue lines are global fits to the above-described model (fit in the way where the pK _as are consistent with the FTSA data for each isoform) yielding the intrinsic enthalpy of binding (ΔH _int, red line) that is independent of pH. Dashed line shows a hypothetical situation how the observed enthalpy dependence should look like if the enthalpy of buffer protonation was equal to zero.

The same procedure has been performed with every purified CA isoform and the data is shown in Fig. 14. All of the nine isoforms where the data has been obtained exhibited similar X-shaped curves, but since the protein pK _as were slightly different and the intrinsic enthalpies of binding were also different among isoforms, they yielded slightly different curves. It should be noted that the precision of the enthalpies is really high, but there still were some discrepancies between the observed data and the model curves, especially at the acidic and alkaline pH. We assume that these discrepancies are due to protein destabilization. However, an attempt was made to determine many more data points for one isoform in one buffer. The data for CA XIII in phosphate buffer are shown in Fig. 15.

Fig. 14. ITC data of 1 binding to CA isoforms at 25 °C. Filled circles – ΔH _obs determined in sodium phosphate buffer, open circles – ΔH _obs determined in TRIS buffer, dotted line shows value of ΔH _int of 1 binding, solid curves are fits of experimental data, while the dashed curve is a fit of a hypothetical situation if there were no buffer or the enthalpy of buffer protonation (ΔH _pr,buf) was equal to zero. The fits were global with the FTSA U-shapes keeping the sulfonamide and CA pK _as the same. Experiments were performed in either 50 mM sodium phosphate or 50 mM TRIS chloride buffer containing 100 mM NaCl and up to 2% DMSO as previously described in: CA I and CA II (Morkūnaitė et al., Reference Morkūnaitė, Gylytė, Zubrienė, Baranauskienė, Kišonaitė, Michailovienė, Juozapaitienė, Todd and Matulis2015), CA IV (Mickevičiūtė et al., Reference Mickevičiūtė, Timm, Gedgaudas, Linkuvienė, Chen, Waheed, Michailovienė, Zubrienė, Smirnov, Čapkauskaitė, Baranauskienė, Jachno, Revuckienė, Manakova, Gražulis, Matulienė, Di Cera, Sly and Matulis2017), CA VB (Kasiliauskaitė et al., Reference Kasiliauskaitė, Časaitė, Juozapaitienė, Zubrienė, Michailovienė, Revuckienė, Baranauskienė, Meškys and Matulis2016), CA VI (Kazokaitė et al., Reference Kazokaitė, Milinavičiūtė, Smirnovienė, Matulienė and Matulis2015), CA VII (Pilipuitytė and Matulis, Reference Pilipuitytė and Matulis2015), CA IX (Linkuvienė et al., Reference Linkuvienė, Matulienė, Juozapaitienė, Michailovienė, Jachno and Matulis2016b), CA XII (Jogaitė et al., Reference Čapkauskaitė, Zubrienė, Smirnov, Torresan, Kišonaitė, Kazokaitė, Gylytė, Michailovienė, Jogaitė, Manakova, Gražulis, Tumkevičius and Matulis2013), CA XIII (Baranauskienė and Matulis, Reference Baranauskienė and Matulis2012). All panels are drawn at the same scale to help visualize the differences in enthalpies of binding. No data has been determined at comparable conditions for CA III, CA VA, and CA XIV. The overall pattern of the X-shapes is similar, but details are different due to different pK _a,CA and ΔH _int for each isoform.

Fig. 15. The enthalpy changes upon compound 303 binding to CA XIII at various pHs. The curve was fit using the following parameters: pK _a,SA = 6.25, pK _a,CA = 8.3, ΔH _int = −37 kJ mol⁻¹ , ΔH _pr,SA = −28 kJ mol⁻¹ , ΔH _pr,CA = −40 kJ mol⁻¹. The open and closed symbols show the data obtained by two independent researchers. The solid curve matched the data resembling all bends in the curve except the discrepancy in the alkaline region. Possibly the protein is slightly destabilized in this region. The horizontal straight line shows the change in the intrinsic enthalpy upon binding if we assume that the reaction occurs according to Eq. (7). However, the same indistinguishable curve would be obtained for Eq. (4) and Eq. (7). The intrinsic enthalpy would then be different. The thermodynamic data alone cannot distinguish the models and we base our decision that Eq. (7) is correct based on crystallographic data.

Most researchers have based the choosing of the lead compound for development on the observed parameters, observed affinity or observed enthalpy. From the first glance, it may seem that it is sufficient to experimentally determine the physiologically relevant compound affinities. Thus, one may be interested in the affinities observed at the pH 7.4. It seems that it is most relevant to correlate the compound chemical structures with the binding data obtained at such pH. However, this is only partially true and may lead to erroneous conclusions. For example, if the binding affinities of two compounds are compared where the compounds have highly different pK _as (e.g., 8 and 11), then if the compound with the pK _a 8 is 1000-fold stronger binder than the compound with pK _a 11, one may assume that the first compound is better attached to the CA. This conclusion is incorrect because the higher affinity is entirely explained by the difference in the pK _a and both compounds would recognize the protein surface with identical affinity.

As shown in this section, the observed enthalpies are not really informative, instead, it is the intrinsic enthalpy change that describes the interaction. The buffer, ligand, and protein contributions may be so large that they may overwhelm the enthalpy data and the compounds would thus be ranked in a completely wrong order. Therefore, it is essential to determine the mechanism and calculate intrinsic enthalpies to be able to rank compounds in the order of increasingly exothermic enthalpy.

In this work, we attempted to determine intrinsic thermodynamic parameters of a series of sulfonamide compounds to the 12 catalytically active human CA isoforms. Intrinsic affinities and intrinsic enthalpies were obtained for the structure–thermodynamics correlations.

Protonation p K _a and enthalpy of CA–Zn^II-bound water molecule of all 12 human CA isoforms

It is not straightforward to determine the pK _a and the enthalpy of protonation of the Zn^II-bound hydroxide/water molecule. Differently, from the compounds, it is not possible to pH-titrate the protein and record with spectrophotometer or titration calorimeter. Instead, in order to determine the pK _a researchers have determined the pH dependence of inhibition of enzymatic activity or replaced Zn with Co that exhibited visible spectral changes upon the change in pH. However, it is disputable to which extent such non-natural CA represents the native CA.

In addition, we have determined the pK _as of most CA isoforms by fitting the inhibitor affinities at various pHs as explained earlier in the discussion of U-shaped dependencies. The enthalpies of protonation were determined via the global fit of the X-shaped curves obtained by ITC titration of an inhibitor in two buffers. Both the pK _a and the enthalpy of protonation had to be obtained only once with one inhibitor and then can be applied for other inhibitors to obtain the intrinsic energetics of binding to a CA.

These data are summarized in Table 2 which shows the thermodynamic parameters of protonation of the hydroxide anion bound to catalytically active CA isoforms. The pK _as of human CAs have been determined previously by using numerous techniques, see the references in Table 2. Their values span between approximately 6 and 8.5. The values determined by FTSA and ITC were close to the values previously determined by other techniques. However, there were several outliers of up to 1 pK _a unit. It seems that CA I and CA XIII have rather high pK _as, above 8.0. Remaining ones are essentially the same as determined previously. The standard deviation of combined FTSA and ITC determinations in the pH dependencies described above do not exceed 0.2 pH or pK _a units. This accuracy is highly dependent on the correct calibration of the pH-meters and standard buffer preparations. The Gibbs energy changes upon protonation were recalculated according to Eq. (12). The enthalpies were the result of the global fit of ITC titrations of each CA isoform with 1 at a series of pH as explained above. The entropies were obtained by subtracting the Gibbs energy changes from enthalpies (Eq. (3)). The entropy values carry a largest standard deviation, a sum of deviations of the enthalpy and Gibbs energy.

Table 2. Thermodynamic parameters of protonation of CA-Zn^II-bound hydroxide anion (Eq. (8), third reaction) for all 12 catalytically active CA isoforms. The pK _a values determined by various methods and taken from the earlier literature are listed in the second column together with the references. Other values were determined by FTSA and ITC with the references listed in the first column next to the CA isoforms. The uncertainty of the pK _a values determined by FTSA and ITC is approximately 0.2 pH units, while for the change in Gibbs energies and enthalpies it is approximately 2 kJ mol⁻¹

Determination of the pK _a and the change of enthalpy of protonation of the sulfonamide inhibitor amino group

Sulfonamide inhibitor amino group deprotonates with a pK _a between approximately 6 and 11. The actual value depends on the chemical structure of the compound, the presence of electron-withdrawing and donating groups, the size of the aromatic conjugated system and other factors. In order to calculate the intrinsic parameters of these sulfonamide inhibitor binding to CAs, it was necessary to determine the pK _as and enthalpies of their protonation.

The sulfonamide amino group pK _a may be roughly estimated from the proton NMR spectrum. The spectrum is determined for every newly synthesized compound thus the information is readily available. The chemical shift of the amino group protons correlates approximately linearly with the spectrophotometrically measured pK _a (Fig. 16).

Fig. 16. Correlation between the pK _as of sulfonamide amino group (determined spectrophotometrically) and the chemical shift of the sulfonamide amino group protons as determined by NMR in deuterated DMSO solvent. Three series of compounds are distinguished in three colors and symbol shapes, VD series are mostly fluorinated benzenesulfonamides, EA and E series are mostly chlorinated compounds. The correlation is approximately linear. The plot may be used to obtain an estimate of the amino group pK _a from proton NMR data when characterizing newly synthesized primary sulfonamides.

To determine the pK _a of the sulfonamide, the compound solution may be pH-titrated and the midpoint pH of the titration would correspond to the pK _a, as shown in Fig. 17 (Ladbury and Doyle, Reference Ladbury and Doyle2004; Morkūnaitė et al., Reference Morkūnaitė, Gylytė, Zubrienė, Baranauskienė, Kišonaitė, Michailovienė, Juozapaitienė, Todd and Matulis2015). However, as shown by the Whitesides group (Snyder et al., Reference Snyder, Mecinović, Moustakas, Thomas, Harder, Mack, Lockett, Héroux, Sherman and Whitesides2011), it is easier to determine the pK _a by preparing the solution of the compound in a series of buffers, made at 0.5 pH unit, and recording the UV spectrum of the solution at each pH. The spectrum changes around the pK _a and it is easy to find the wavelengths of the largest change. The absorbance values at those pH are then used and plotted as a function of pH and normalized to the unit as shown in Fig. 17a and c.

Fig. 17. Determination of the pK _a and enthalpy of protonation (ΔH _pr,SA) of the sulfonamide amino group. Left panels (a), (c), and (e) show the spectrophotometric, while right panels (b), (d), and (f) show the ITC data. (a) UV-Vis spectra of the compound solution in buffers of various pH. (c) The normalized absorbance change at a chosen wavelength or ratio of two wavelengths. (e) Determination of the ΔH _pr,SA via van't Hoff relationship, the dependence of the pK _a on temperature. (b) ITC raw data of titrating a base-neutralized (with 1.5 equiv.) sulfonamide with HNO₃. (d) Integrated ITC curves of the sulfonamide titration with acid. (f) Determination of the heat capacity of protonation by performing the ITC titrations at various temperatures. The ΔC _p values for 310 and 369 were equal to 107 J mol⁻¹ K⁻¹ and 88 J mol⁻¹ K⁻¹, respectively.

The enthalpies of sulfonamide amino group protonation may be determined by titrating the compound solution with an acid in an ITC calorimeter at a constant temperature. The compound in a solid pure form exists as a protonated electrostatically neutral sulfonamide. Therefore, it must be deprotonated by applying a sufficient amount of base to achieve the pH sufficiently above the pK _a for accurate determination. We usually apply 50% surplus of the base and therefore the resultant ITC curves appear as two-step ITC reactions. Titration of such solution with a strong acid provides a direct measurement of the enthalpy of protonation as shown in Fig. 17d.

Alternatively, the enthalpy of protonation may be determined by applying the van't Hoff relationship and performing the pK _a determination at several temperatures as shown in Fig. 17e. However, such determination is not as accurate as ITC because it is assumed here that the enthalpy of protonation is independent of temperature in the studied temperature range. To some extent this assumption is correct, but the enthalpy is temperature-dependent as seen from the ITC determinations at various temperatures (Fig. 17f). There is a non-zero heat capacity associated with the protonation process. The heat capacity change upon protonation is positive (Matulis and Todd, Reference Matulis, Todd, Ladbury and Doyle2004; Morkūnaitė et al., Reference Morkūnaitė, Gylytė, Zubrienė, Baranauskienė, Kišonaitė, Michailovienė, Juozapaitienė, Todd and Matulis2015) as usually is the case of ionic binding reactions.

Why intrinsic data is necessary for making compound structure–thermodynamics correlations

It is often thought that it is sufficient to make a SAR-type analysis based on the observed data. This may be true to some extent when the compounds are very similar and there is no need to make explanations why one or another compound binds stronger or weaker to a particular protein. However, if we would like to understand the physico-chemical reasons behind the observed thermodynamic parameters, e.g., determine the contributions of particular atoms to the binding energetics, it is necessary to operate with the intrinsic parameters.

Figure 18 shows the observed and intrinsic K _ds for a series of compound binding to human CA VI. The compounds were arranged in the order of decreasing intrinsic affinity. It is clear that the intrinsic and observed affinities do not correlate for these compounds. The observed K _d for the first and last compound in the figure are almost the same, but the intrinsic affinities differ by more than 300 fold. Not knowing the intrinsic values would yield completely wrong conclusions about the structural determinants of the compounds that cause the affinity to be greater or smaller.

Fig. 18. The observed and intrinsic K _d of a series of sulfonamide compound interaction with CA VI (Kazokaitė et al., Reference Kazokaitė, Milinavičiūtė, Smirnovienė, Matulienė and Matulis2015). There is clearly no correlation between the observed and intrinsic binding affinities.

Another example (Fig. 19) shows a different situation when the observed binding constants are quite different, but the intrinsic ones are nearly the same. If only the observed data were available, one could make an erroneous conclusion that fluorines substantially increase the affinity of the compound towards CA I due to the contacts between fluorines and the protein. However, in actuality, fluorines increase the affinity by only 1.5-fold (equal to the ratio of the intrinsic binding constants) due to direct contacts and the remaining 100-fold increase in the observed affinity arises simply due to the reduction of the pK _a of the sulfonamide amino group by electron-withdrawing capabilities of the fluorine atoms.

Fig. 19. The observed and intrinsic K _d of two sulfonamide compound interaction with CA I. The compounds are para-substituted benzenesulfonamides, one of them with four fluorines on the benzene ring. The observed affinity showed that the F-bearing compound bound CA I significantly stronger than the non-fluorinated compound. The intrinsic K _ds were practically equal for both compounds, but the observed ones differed by 150-fold mostly due to the reduction of the pK _a of the fluorinated compound and not direct recognition between the F atoms and the protein surface.

The quality of prepared CA proteins

All 12 catalytically active human CA isoforms have been prepared in our laboratory. There was always of a concern how pure those preparations are and whether the preparations exhibit a specific activity that is comparable with the proteins described in the literature.

The proteins have been confirmed by such standard techniques and SDS-PAGE and high-resolution mass spectrometry. They were of correct molecular weight on resolving gels and exhibited a mass that was within a single Da from the mass predicted from the amino acid sequence. However, we think that in addition, it is of most importance to demonstrate that the entire protein preparation is catalytically active by titrating it with an inhibitor and thus demonstrate that the entire preparation of the protein is catalytically active and it is not contaminated by non-active fraction of the same protein that may be indistinguishable by other techniques.

Figure 25 shows the activity titration of CA XIV. First, it is necessary to titrate the active fraction of the protein. The known large concentration of CA XIV, 500 nM, was added to the solution and various concentrations of a strongly-binding inhibitor 1 were added. Two straight lines should intersect at exactly 500 nM as was experimentally observed (Fig. 25) if the inhibitor is of sufficient binding affinity.

Fig. 25. Determination of the catalytically active fraction of the preparation of CA XIV as previously described by Juozapaitienė et al. (Reference Juozapaitienė, Bartkutė, Michailovienė, Zakšauskas, Baranauskienė, Satkūnė and Matulis2016), performed according to Copeland (Reference Copeland2005). Two straight lines intersected at exactly 500 nM concentration equal to the added concentration of CA XIV thus confirming that the entire preparation of CA XIV was enzymatically active. The inset shows the decrease in absorbance lines at various inhibitor concentrations (steep blue lines – no inhibitor, shallow red lines – largest inhibitor concentration added). The red CO₂ curve shows spontaneous hydration of CO₂ in the absence of enzyme.

Protein purity can be further confirmed by demonstrating inhibition by several known inhibitors. Then, it is useful to demonstrate using a strong inhibitor that the compound binding stoichiometry by ITC is near 1. Determination of the protein stability in various buffers and various compounds that are known binders is also useful. Finally, X-ray crystallographic structural characterization of the protein is one of the most widely accepted techniques to show that the protein is of high purity. All these techniques are orthogonal and complementary and add to the overall picture that we need in order to understand the structure–thermodynamics of protein–ligand binding.

The recombinant CAs may not be fully representative of their behavior in live human cells. First, here we produced only the catalytic domains of isoforms that contain transmembrane domains (CA IX, CA XII, and CA XIV). The dimerization of these and other isoforms may also be affected thus changing the compound affinities. Furthermore, different isoforms may be posttranslationally modified in human and not represented in bacterial preparations. Therefore, despite the recombinant proteins being the best models representing proteins in the human body, it should be used with some caution.

However, for several monomeric isoforms that were produced in bacteria in full length and where no glycosylation or other posttranslational modifications are known, the bacterial recombinant isoforms are an excellent model to represent their binding of a potential drug in the human body.

The most interesting and compelling case is of CA VI which can be affinity-purified from human volunteer saliva. We have demonstrated that the CA VI preparations from human saliva and the protein recombinantly prepared from mammalian cells, as previously described by Kazokaitė et al. (Reference Kazokaitė, Milinavičiūtė, Smirnovienė, Matulienė and Matulis2015), exhibited essentially the same affinity towards a large series of sulfonamide compounds (Fig. 26).

Fig. 26. Correlation between observed inhibitor binding affinities to CA VI produced by three different paths (Kazokaitė et al., Reference Kazokaitė, Milinavičiūtė, Smirnovienė, Matulienė and Matulis2015). Human recombinant CA VI was prepared in bacterial and mammalian cell cultures. The native human CA VI was also affinity-purified from human volunteer saliva. Comparing the affinities of the compounds to the three kinds of CA VI we see that affinities are highly similar and identical within an error margin with the exception of bacterial preparation that bound the compounds with systematically higher affinity by approximately 2–3 fold. This difference could be caused by the different dimerization pattern of various CA VI preparations.

Thermal stability of CA isoforms

Figure 27 shows the thermal stabilities of all 12 catalytically active CA isoforms in various buffers as a function of pH. The stability profiles are quite similar among the isoforms, with stability maximal and minimal pH dependence between pH 6 and 9. Isoform CA VI is somewhat exceptional with the pH stability maximum between pH 5 and 6. The most stable isoform is CA IX, the next is CA IV, while CA VII and CA XII are the least stable isoforms. It should be kept in mind that these are stabilities of catalytic domains and may not fully represent native CAs that contain transmembrane parts.

Fig. 27. Characterization of CA isoform stability in various buffers at various pHs. The isoform stability profiles look similar with the greatest stability near pH 7, but there are several notable exceptions, especially CA VI that exhibits stability maximum around slightly acidic pH 5. Note that citrate buffer is destabilizing all CAs because it is complexating Zn^II and thus destabilizing the native conformation of CA. Experiments were performed in 100 mM buffer containing 2 mM NaCl and 0.5% DMSO as previously described in: CA I and CA II (Morkūnaitė et al., Reference Morkūnaitė, Gylytė, Zubrienė, Baranauskienė, Kišonaitė, Michailovienė, Juozapaitienė, Todd and Matulis2015), CA IV (Mickevičiūtė et al., Reference Mickevičiūtė, Timm, Gedgaudas, Linkuvienė, Chen, Waheed, Michailovienė, Zubrienė, Smirnov, Čapkauskaitė, Baranauskienė, Jachno, Revuckienė, Manakova, Gražulis, Matulienė, Di Cera, Sly and Matulis2017), CA VB (Kasiliauskaitė et al., Reference Kasiliauskaitė, Časaitė, Juozapaitienė, Zubrienė, Michailovienė, Revuckienė, Baranauskienė, Meškys and Matulis2016), CA VI (Kazokaitė et al., Reference Kazokaitė, Milinavičiūtė, Smirnovienė, Matulienė and Matulis2015), CA VII (Pilipuitytė and Matulis, Reference Pilipuitytė and Matulis2015), CA IX (Linkuvienė et al., Reference Linkuvienė, Matulienė, Juozapaitienė, Michailovienė, Jachno and Matulis2016b), CA XII (Jogaitė et al., Reference Čapkauskaitė, Zubrienė, Smirnov, Torresan, Kišonaitė, Kazokaitė, Gylytė, Michailovienė, Jogaitė, Manakova, Gražulis, Tumkevičius and Matulis2013), CA XIII (Baranauskienė and Matulis, Reference Baranauskienė and Matulis2012), and CA XIV (Juozapaitienė et al., Reference Juozapaitienė, Bartkutė, Michailovienė, Zakšauskas, Baranauskienė, Satkūnė and Matulis2016). All panels are drawn at the same scale to help visualize the differences in stabilities among CA isoforms.

Interestingly, the citrate buffer exhibits a strong destabilizing effect on most CA isoforms, most likely because citrate binds Zn^II and thus destabilizes CAs by pulling the metal cation from the active site.

CA I

The very first crystal structure of CA reported as the human erythrocyte CA form B, was the structure of CA I (Kannan et al., Reference Kannan, Notstrand, Fridborg, Lovgren, Ohlsson and Petef1975). The crystallization report appeared earlier (Kannan et al., Reference Kannan, Fridborg, Bergsten, Liljas, Lovgren, Petef, Strandberg, Waara, Adler, Falkbring, Göthe and Nyman1972a) and the structure was revisited and refined to the resolution of 2 Å (Kannan et al., Reference Kannan, Ramanadham and Jones1984). The bicarbonate in CA I was found as a monodentate ligand in the tetrahedral coordination sphere of Zn ligands (Kumar and Kannan, Reference Kumar and Kannan1994). This position differs from that in the crystal structure of CA II-Co-${\rm HCO}_3^- $ complex, where cobalt substitutes for the zinc (Haakansson and Wehnert, Reference Haakansson and Wehnert1992). In this structure, the bidentate binding of ${\rm HCO}_3^- $ was found. Further crystal structures of CA I with anionic inhibitors (Kumar et al., Reference Kumar, Kannan and Sathyamurthi1994) revealed differences in the binding of two anions: iodide replaced the Zn-bound water, whereas gold cyanide did not bind to the zinc directly. One of CN⁻ atoms made H-bond with the Zn-bound water. Thus, Au(CN)²⁻ can inhibit CA I because it occupies a substrate binding site, whereas I⁻ directly binds to Zn.

An interesting naturally occurring genetic variant of CA I, Michigan 1(Ferraroni et al., Reference Ferraroni, Tilli, Briganti, Chegwidden, Supuran, Wiebauer, Tashian and Scozzafava2002), where His67 residue is replaced with Arg, could bind an additional zinc ion. An additional Zn-binding site was formed by His64, His200 and Arg67 side chains. Coordination of Zn^II by arginine side chain is rare.

CA II

Crystallographic studies of CA II play a very important role in the field of human CAs. First CA II structure was described in 1972 (Kannan et al., Reference Kannan, Liljas, Waara, Bergsten, Lovgren, Strandberg, Bengtsson, Carlbom, Fridborg, Jarup and Petef1972b; Liljas et al., Reference Liljas, Kannan, Bergsten, Waara, Fridborg, Strandberg, Carlbom, Jarup, Lovgren and Petef1972) as the human erythrocyte CA form C. First CA II crystal structures of limited resolution 2 Å were obtained by Liljas et al. (Reference Liljas, Kannan, Bergsten, Waara, Fridborg, Strandberg, Carlbom, Jarup, Lovgren and Petef1972) as early as in 1972 (Liljas et al., Reference Liljas, Kannan, Bergsten, Waara, Fridborg, Strandberg, Carlbom, Jarup, Lovgren and Petef1972), then revisited and refined in 1988 (Eriksson et al., Reference Eriksson, Jones and Liljas1988a). Additional restrained refinement allowed to identify water molecules in the active site, one of them bound to the Zn^II. The structure helped to elucidate the reaction mechanism. High-resolution crystal structures of CA II appeared later and helped to understand the water network in the active site (Fisher et al., Reference Fisher, Maupin, Budayova-Spano, Govindasamy, Tu, Agbandje-McKenna, Silverman, Voth and McKenna2007a; Avvaru et al., Reference Avvaru, Kim, Sippel, Gruner, Agbandje-McKenna, Silverman and McKenna2010b). There was also some variability in crystal forms of CA II, first mentioned by Robbins et al. (Reference Robbins, Domsic, Agbandje-McKenna and McKenna2010).

CA III

The structural studies of CA III isoform were begun, most probably, by the crystallization of the reduced bovine CA III from red muscle (Eriksson and Liljas, Reference Eriksson and Liljas1993). The S-glutathionated form of the rat liver enzyme was also described (Mallis et al., Reference Mallis, Poland, Chatterjee, Fisher, Darmawan, Honzatko and Thomas2000). Two surface cysteines, Cys183 and Cys188, were modified and the significance of this modification in the oxidative stress was discussed.

The mechanism of proton transfer by human CA III was elucidated in mutational and structural studies (Duda et al., Reference Duda, Yoshioka, Govindasamy, An, Tu, Silverman and McKenna2002, Reference Duda, Tu, Fisher, An, Yoshioka, Govindasamy, Laipis, Agbandje-McKenna, Silverman and McKenna2005). The replacement of Phe198 side chain by leucine has increased the catalytic activity of CA III. An interesting feature of the CA III crystal structures is the ordered C-terminal His-tag that is bound in the active site cavity of the crystallographic symmetry-related subunit. In the WT enzyme, there is a stacking between one of the Zn-coordinating histidines and the phenyl group of Phe198. The possible mechanism of proton donor activation is discussed (Duda et al., Reference Duda, Tu, Fisher, An, Yoshioka, Govindasamy, Laipis, Agbandje-McKenna, Silverman and McKenna2005). The role of Lys64 and Arg67 side chains, which are the structural analogs of CA II His64 and Asn67, respectively, in the proton transfer was attempted to clarify (Elder et al., Reference Elder, Fisher, Laipis, Tu, McKenna and Silverman2007).

CA IV

The first crystal structure of the membrane-associated CA IV was reported by Stams et al. (Reference Stams, Nair, Okuyama, Waheed, Sly and Christianson1996). The soluble secreted mutant of CA IV with the C-terminal deletion was crystallized with a limited resolution. The conformation of the N-terminal domain was stabilized by two disulfide bonds: Cys6–Cys11 and Cys23–Cys203. The crystal structures of murine CA IV (also a truncated form) were solved by the same group later and compared with CA II (Stams et al., Reference Stams, Chen, Boriack-Sjodin, Hurt, Liao, May, Dean, Laipis, Silverman and Christianson1998). The protein contains intramolecular disulfide bond typical for trans-membrane and secreted CAs (Cys23–Cys203 in CA I) (Stams et al., Reference Stams, Nair, Okuyama, Waheed, Sly and Christianson1996). The N-terminal insertion of 5 amino acids in the murine CA IV was compared with other CAs (Stams et al., Reference Stams, Nair, Okuyama, Waheed, Sly and Christianson1996, Reference Stams, Chen, Boriack-Sjodin, Hurt, Liao, May, Dean, Laipis, Silverman and Christianson1998).

CA VI

In the only published crystal structure of the catalytic domain of CA VI (Pilka et al., Reference Pilka, Kochan, Oppermann and Yue2012) the intramolecular disulfide bond (Cys42–Cys224) was also found. Such disulfide bonds are typical in membrane-bound CA IV, CA XII, and CA XIV. It is interesting that the octahedrally coordinated Mg^II was found in the active site instead of Zn^II. The NCS dimer was found in the crystal structure and both active sites face each other, differently from CA IX and CA XII. His85 in this structure is a good candidate for the proton shuttle.

CA VII

There are two crystal structures of CA VII found in the PDB database: the unpublished complex with ethoxzolamide 1 (PDB ID: 3MDZ) and the complex with acetazolamide 2 (Fiore et al., Reference Fiore, Truppo, Supuran, Alterio, Dathan, Bootorabi, Parkkila, Monti and De Simone2010a). The mutant with two cysteine residues replaced with serine side chains was used in the crystallization. Intramolecular disulfide bond was found in the crystal (Cys54–Cys178), but authors argued that it is an artifact, because no conserved homologous disulfide was found and such bonds are rare in the cytosolic proteins. 2 makes H-bonds with the protein main chain atoms, water molecules, Gln92 side chain, and van der Waals contacts with the Phe131 side chain.

CA VIII (Carbonic Anhydrase Related Protein, CARP)

The crystal structure of the only structurally characterized CARP, CA VIII, was solved in 2009 (Picaud et al., Reference Picaud, Muniz, Kramm, Pilka, Kochan, Oppermann and Yue2009). The CARP proteins lost their catalytic activity because Zn-coordinating residue His94 (CA II numbering) is replaced by Arg116. The N-terminal Glutamine-rich loop (residues 24–36), a peculiar feature of CA VIII, is partially resolved in the structure. This loop forms contacts with another unique feature of CA VIII, an α3 − β15 loop. Both structural elements contribute to the relatively negatively charged surface of the protein. Two bulky residues restrain a cavity near the active site (Arg116 and Ile224) (homologous to Thr200 of CA II). The residue Glu114 replaces in CA VIII the CA II residue Gln92. Substitutions of the active site residues by more bulky side chains probably preclude the binding of CO₂. Chloride anion is modeled in the crystal structure instead of zinc.

CA IX

The structure of Fab fragment of anti-CA IX mAb M75 with the peptide representing epitope, the proteoglycan-like (PG) segment of CA IX was described (Kral et al., Reference Kral, Mader, Collard, Fabry, Horejsi, Rezacova, Kozisek, Zavada, Sedlacek, Rulisek and Brynda2008) and the problems of epitope recognition by the antibody are discussed. The first crystal structure of the catalytic domain of CA IX with bound AZM drug appeared in Alterio et al. (Reference Alterio, Hilvo, Di Fiore, Supuran, Pan, Parkkila, Scaloni, Pastorek, Pastorekova, Pedone, Scozzafava, Monti and De Simone2009). The protein was produced using the Baculovirus expression system. The residue Cys41, responsible for intermolecular disulfide bond, that stabilizes a dimer, was replaced by serine. The dimerization interface differs from that of CA XII. Active sites of monomers are located on the same side of the dimer. AZM makes several H-bonds with side chains Thr199-200 and Gln92.

Crystallization of CA IX even with a published structure in view, was not an easy task, but finally, it was solved (Leitans et al., Reference Leitans, Kazaks, Balode, Ivanova, Zalubovskis, Supuran and Tars2015). The protein was expressed in yeast and the binding of a series of 2-thiophene-sulfonamide compounds to CA IX was characterized structurally. Binding of one ligand by CA IX was compared with CA II (Leitans et al., Reference Leitans, Sprudza, Tanc, Vozny, Zalubovskis, Tars and Supuran2013). The tail part of thiophenes interacts with the side chain of Phe131 in CA II, that corresponds to the Val130 in CA IX. The presence of phenylalanine in CA II is the main reason for the differences in the binding mode.

CA XII

The first crystal structures of CA XII and CA XII-AZM complex were reported in 2001 (Whittington et al., Reference Whittington, Waheed, Ulmasov, Shah, Grubb, Sly and Christianson2001). In the first structure, acetate ion is present as the fifth ligand of zinc. Its position mimics the binding of bicarbonate. AZM is bound in the other structure of CA XII. As in CA II, AZM interacts with a Phe131 side chain, that is absent in CA XII, the position of ligand differs between two isoforms. CA XII was used for the comparison of binding of various compounds with different human CA isoforms in the search of isoform-specific inhibitors (Čapkauskaitė et al., Reference Čapkauskaitė, Zubrienė, Smirnov, Torresan, Kišonaitė, Kazokaitė, Gylytė, Michailovienė, Jogaitė, Manakova, Gražulis, Tumkevičius and Matulis2013; Dudutienė et al., Reference Dudutienė, Zubrienė, Smirnov, Gylytė, Timm, Manakova, Gražulis and Matulis2013, Reference Dudutienė, Matulienė, Smirnov, Timm, Zubrienė, Baranauskienė, Morkūnaite, Smirnovienė, Michailovienė, Juozapaitienė, Mickevičiūtė, Kazokaitė, Bakšytė, Kasiliauskaitė and Jachno2014, Reference Dudutienė, Zubrienė, Smirnov, Timm, Smirnovienė, Kazokaitė, Michailovienė, Zakšauskas, Manakova, Gražulis and Matulis2015; Zubrienė et al., Reference Zubrienė, Smirnovienė, Smirnov, Morkūnaitė, Michailovienė, Jachno, Juozapaitienė, Norvaišas, Manakova, Gražulis and Matulis2015).

CA XIII

The first published structures of CA XIII contained acetate and AZM bound in the active site (Fiore et al., Reference Fiore, Monti, Hilvo, Parkkila, Romano, Scaloni, Pedone, Scozzafava, Supuran and Simone2009). In the structure with acetate, the zinc is penta-coordinated (with acetate and water molecule). The other structure contains 2. No H-bonds with 2 were evident in the structure. Recently several series of benzenesulfonamide-based inhibitors were analyzed bound in CA XIII in the search for isoform-specific ligands (Čapkauskaitė et al., Reference Čapkauskaitė, Zubrienė, Smirnov, Torresan, Kišonaitė, Kazokaitė, Gylytė, Michailovienė, Jogaitė, Manakova, Gražulis, Tumkevičius and Matulis2013; Dudutienė et al., Reference Dudutienė, Zubrienė, Smirnov, Gylytė, Timm, Manakova, Gražulis and Matulis2013, Reference Dudutienė, Zubrienė, Smirnov, Timm, Smirnovienė, Kazokaitė, Michailovienė, Zakšauskas, Manakova, Gražulis and Matulis2015; Kišonaitė et al., Reference Kišonaitė, Zubrienė, Čapkauskaitė, Smirnov, Smirnovienė, Kairys, Michailovienė, Manakova, Gražulis and Matulis2014).

CA XIV

The structural characterization of mammalian CA XIV was started by the crystal structures of the extracellular catalytic domain of murine CA XIV (Whittington et al., Reference Whittington, Grubb, Waheed, Shah, Sly and Christianson2004). The S–S bond characteristic for other membrane-bound CA (Cys23–Cys203) is found in the murine enzyme. The N-glycosylation is detected at the residue Asn195. Murine CA XIV in the crystal structure is monomeric. In the structure of the CA XIV complex with 2, H-bonds are detected between 2 and Thr199-200. The human CA XIV was first characterized by the crystal structure of its extracellular domain in complex with 2 (Alterio et al., Reference Alterio, Pan, Parkkila, Buonanno, Supuran, Monti and De Simone2013). The H-bonds of 2 with water are found besides H-bonds to Thr199–200.

Summary of the X-ray crystallographic structures of unliganded human CA isoforms

The X-ray crystallographic structures of the 10 out of 12 catalytically active human CA isoforms that are available in the PDB are shown in the Supplementary Fig. 1. There are no structures of CA VA and CA VB available in the PDB. Seven of the 10 structures are monomers, while CA VI, CA IX, and CA XII are dimers.

Studies of CA stability and mimicking the active site of another isoform

Sulfonamides

The relatively small compound trifluoromethane sulfonamide (TFS) binds to CAs very effectively due to its low pK _a (5.9–6.2) and its complex with CA II was crystallized (Haakansson and Liljas, Reference Haakansson and Liljas1994). The position of the ligand was compared with the crystal structure of CA II-2 complex (Vidgren et al., Reference Vidgren, Liljas and Walker1990). It is interesting that the oxygen atoms of sulfonamide in CF₃SO₂NH₂ were directed opposite to oxygen atoms in 2. CF₃ fits into the hydrophobic cleft, whereas the aromatic inhibitor must be oriented outwards. Another small molecule, N-hydroxysulfonamide, is bound in the crystal structure of CA II (Temperini et al., Reference Temperini, Winum, Montero, Scozzafava and Supuran2007c).

Heterocycles

Sulfamides

One of the simplest inhibitors structurally characterized was N-hydroxysulfamide (Temperini et al., Reference Temperini, Winum, Montero, Scozzafava and Supuran2007c), that is the more effective inhibitor of CA I and CA II than sulfamide. The compound is bound to zinc like all other sulfonamides by the nitrogen atom, but not by the hydroxyl group.

Sulfamides comprise yet another variant of the Zn-binding group and are widely represented in CA crystallography. The positioning of these ligands has an additional degree of flexibility with respect to benzenesulfonamides (or other heterocyclesulfonamides, because of the additional rotation around the nitrogen atom).

The borolane-containing benzenesulfamide was co-crystallized with CA II due to its possible use in boron neutron capture in the anticancer therapy (Fiore et al., Reference Fiore, Monti, Innocenti, Winum, Simone and Supuran2010b). Carboranes bound through a flexible linker to the sulfamide (Brynda et al., Reference Brynda, Mader, Sicha, Fabry, Poncova, Bakardiev, Gruner, Cigler and Rezacova2013) also can be effectively bound by CAs and therefore, considered to have pharmaceutical perspectives. The length of linkers was chosen in order to fill the active site by the icosahedral carborane in an optimal way. Crystal structures confirmed the predicted binding position of ligands by van der Waals interactions with the protein side chains. This study was continued further (Mader et al., Reference Mader, Pecina, Cígler, Lepšík, Šícha, Hobza, Grüner, Fanfrlík, Brynda and Řezáčová2014). Here, high-resolution structure of carboranesulfamide bound in CA II was described and used for the modeling of the interaction with CA IX.

A series of nitroimidazole derivatives with sulfamide, sulfonamide, and sulfamate groups for anchoring to CA were discussed in numerous studies. Crystal structure with one of nitroimidazolesulfamides confirmed the inhibition studies (Rami et al., Reference Rami, Dubois, Parvathaneni, Alterio, van Kuijk, Monti, Lambin, De Simone, Supuran and Winum2013). These compounds were chosen as possible chemo- or radiosensitizing agents in an anticancer therapy. The electron density was good and H-bonds of the nitro group with Thr200 and His64 were found.

The other research (De Simone et al., Reference De Simone, Pizika, Monti, Di Fiore, Ivanova, Vozny, Trapencieris, Zalubovskis, Supuran and Alterio2014) described inhibitory properties of other series of compounds: phenylmethylsulfamide with substituents optimized to make interactions in the active site. But-2-yn-1-yloxy tail was found to provide strong interactions with CA II active site.

Sulfamates

Sulfamates like sulfamides are derivatives of the sulfamic acid where the substituent is bound via one of three oxygen atoms at sulfur atom. Most of the sulfamate inhibitors studied are perspective anticancer steroid or non-steroid drugs or their homologs.

Many non-steroid drugs initially designed for other targets possess sulfamate Zn-binding group. One of the examples is the anti-convulsant drug RWJ- 37 497, an analog of topiramate crystallized bound to CA II (Recacha et al., Reference Recacha, Costanzo, Maryanoff and Chattopadhyay2002). The interaction of topiramate that belongs to this group of compounds with CA II is also described (Lopez et al., Reference Lopez, Paul, Hofmann, Morizzi, Wu, Charman, Innocenti, Vullo, Supuran and Poulsen2009) as well as its sulfamide analog (Winum et al., Reference Winum, Temperini, Cheikh, Innocenti, Vullo, Ciattini, Montero, Scozzafava and Supuran2006). The crystal structure of 667-coumate, a potent non-steroidal inhibitor of steroid sulfatase (anticancer drug) based on a coumarine ring bound to CA II was reported and the mechanism of the drug stability in vivo due to its sequestration by CA II in blood was proposed. The tricyclic coumarine was proposed to mimic steroid structure. The electron density of the 7-membered ring was not very good due to flexibility (Lloyd et al., Reference Lloyd, Pederick, Natesh, Woo, Purohit, Reed, Acharya and Potter2005a). In the structures of CA II with aromatase-steroid sulfatase inhibitor analogs possessing sulfamate Zn-binding group (Lloyd et al., Reference Lloyd, Thiyagarajan, Ho, Woo, Sutcliffe, Purohit, Reed, Acharya and Potter2005b), the sulfamate-containing rings in both cases nearly coincided, but the para-substituents differ in binding to the protein. Steroid sulfatase inhibitors were sequestered in blood by binding to CA II via sulfamate moiety. This interaction is favorable from the pharmacokinetics point of view and several structures of CA II complexes with similar ligands were reported (Leese et al., Reference Leese, Leblond, Smith, Newman, Di Fiore, De Simone, Supuran, Purohit, Reed and Potter2006; Woo et al., Reference Woo, Fischer, Sharland, Trusselle, Foster, Chander, Fiore, Supuran, Simone, Purohit, Reed and Potter2008, Reference Woo, Jackson, Putey, Cozier, Leonard, Acharya, Chander, Purohit, Reed and Potter2010; Cozier et al., Reference Cozier, Leese, Lloyd, Baker, Thiyagarajan, Acharya and Potter2010).

Microtubule disruptor crystal structure in complex with CA II (Leese et al., Reference Leese, Jourdan, Gaukroger, Mahon, Newman, Foster, Stengel, Regis-Lydi, Ferrandis, Fiore, Simone, Supuran, Purohit, Reed and Potter2008, Reference Leese, Jourdan, Kimberley, Cozier, Thiyagarajan, Stengel, Regis-Lydi, Foster, Newman, Acharya, Ferrandis, Purohit, Reed and Potter2010) was yet another example of the anticancer drug which bioavailability was increased by interaction with CA. Antiproliferative bis-sulfamates and steroid sulfatase inhibitor-CA II structure (Leese et al., Reference Leese, Leblond, Smith, Newman, Di Fiore, De Simone, Supuran, Purohit, Reed and Potter2006) revealed the binding of two molecules in the active site. One molecule was coordinated at zinc by sulfamate in an unusual way, the other filled the rest of the cavity. The binding of estradiol-sulfamate compounds in CA II and its double mutant, mimic of CA IX, were compared (Sippel et al., Reference Sippel, Stander, Tu, Venkatakrishnan, Robbins, Agbandje-McKenna, Fourie, Joubert and McKenna2011). These compounds could have an antiproliferative effect, and the aim of the study was to find a CA IX-specific inhibitor that binds stronger to CA IX, but still could be sequestered by CA II.

Rather an unusual set of aliphatic mono- and bis-sulfamates were studied in the search of isoform-specific inhibitors (Vitale et al., Reference Vitale, Alterio, Innocenti, Winum, Monti, De Simone and Supuran2009). Four compounds were bound in a similar way in CA II. The second sulfamate was exposed out of the cavity and was mobile as could be judged from the increased B-factors.

Monosaccharides with one of the hydrophilic groups derivatized to sulfamate described as inhibitors of human CAs (Lopez et al., Reference Lopez, Vu, Wang, Wolf, Groenhof, Innocenti, Supuran and Poulsen2011) were also subject to CA II esterase activity. The loss of acyl groups was observed as well as an inversion of stereochemistry. Carbohydrates carrying sulfamate as a Zn-binding group containing various linkers were studied with the goal of designing CA IX-specific compounds (Moeker et al., Reference Moeker, Mahon, Bornaghi, Vullo, Supuran, McKenna and Poulsen2014a). Binding of two compounds with CA II and CA IX mimic (Pinard et al., Reference Pinard, Boone, Rife, Supuran and McKenna2015) was studied crystallographically. Carbohydrate moiety as a tail group was chosen due to its lower membrane permeability. The topic hydrocarbonate-based sulfamates as isoform-selective human CA inhibitors were developed further (Mahon et al., Reference Mahon, Lomelino, Ladwig, Rankin, Driscoll, Salguero, Pinard, Vullo, Supuran, Poulsen and McKenna2015b, Reference Mahon, Lomelino, Salguero, Driscoll, Pinard and McKenna2015c). Together with the analysis of the ligand binding in CA II and CA IX mimic, the artifact of acetylation of the surface lysine and its effect on the protein stability was discussed (Mahon et al., Reference Mahon, Lomelino, Salguero, Driscoll, Pinard and McKenna2015c).

Abbate et al. (Reference Abbate, Winum, Potter, Casini, Montero, Scozzafava and Supuran2004c) reported the crystal structure of antiendocrine drug showing both CA and estrone sulfatase inhibition activity: EMATE-sulfamate complex with CA II.

Natural products: alternative metal-binding groups and products of hydrolysis

Sulfonamides as drugs

Activators

Activator molecules bind mostly at the entrance of the active site and participate in the proton transfer pathway. Activator compounds include amino acids, peptides, and amines that may be easily protonated.