research communications
Ibuprofen: a weak inhibitor of carbonic anhydrase II
aDepartment of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA
*Correspondence e-mail: rmckenna@ufl.edu
Carbonic anhydrases (CAs) are drug targets for a variety of diseases. While many clinically relevant CA inhibitors are sulfonamide-based, novel CA inhibitors are being developed that incorporate alternative zinc-binding groups, such as carboxylic acid moieties, to develop CA isoform-specific inhibitors. Here, the X-ray 50 values and were compared with those of other carboxylic acid binders. This study discusses the potential development of CA inhibitors utilizing the carboxylic acid moiety.
of human CA II (hCA II) in complex with the carboxylic acid ibuprofen [2-(4-isobutylphenyl)propanoic acid, a common over-the-counter nonsteroidal anti-inflammatory drug] is reported to 1.54 Å resolution. The binding of ibuprofen is overlaid with the structures of other in complex with hCA II to compare their inhibition mechanisms by direct or indirect (via a water) binding to the active-site zinc. Additionally, enzyme-inhibition assays using ibuprofen, nicotinic acid and ferulic acid were performed with hCA II to determine their ICKeywords: carbonic anhydrases; carboxylic acid-based inhibitors; ibuprofen; X-ray crystallography; kinetics.
PDB reference: carbonic anhydrase II in complex with ibuprofen, 8dj9
1. Introduction
Carbonic anhydrases (CAs) are a family of metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO2) and water to bicarbonate () and a proton via a coordinated metal ion (Steiner et al., 1975). Within this family of metalloenzymes, the α-class zinc-containing human CAs are the most widely studied and understood, and include human carbonic anhydrase II (hCA II), which is widespread in many tissue types and is especially prominent in erythrocytes (Frost & McKenna, 2013). The CA mechanism of catalysis occurs in two distinct steps: hydration/dehydration of CO2 (1) via the zinc-bound solvent (ZBS) and recycling of the ZBS from water to hydroxide (2) via the proton-shuttling residue His64 at the rim of the active site (Fisher et al., 2010):
Many of the human CA isoforms have been shown to be therapeutic targets in diseases such as glaucoma, edema and altitude sickness, and are also being developed for the treatment of certain cancers (Combs et al., 2020; Maren, 1967; Supuran, 2008; Frost & McKenna, 2013). Hence, a focus of CA research is on the development of CA inhibitors (CAIs), which dates back to the 1930s. Initial studies demonstrated sulfanilamide (p-aminobenzenesulfonamide) to be a potent inhibitor of the enzyme, with its mode of inhibition being directly attributed to the sulfonamide moiety (SO2NH2; Keilin & Mann, 1940). In further studies, the development of heterocyclic sulfonamide inhibitors with increased potency resulted in inhibitors with nanomolar binding constants (Miller et al., 1950). The sulfonamide moiety is known to inhibit CAs by directly binding to the active-site zinc, which is coordinated by three histidines (His94, His96 and His119; hCA II numbering) and a ZBS in a tetrahedral geometry, displacing the catalytic ZBS. The sulfonamide-based CAIs are termed classical CA inhibitors as they are the most widely studied pharmacophore in CAIs (Supuran, 2016). However, there has been a change in emphasis in the last few decades to develop CA isoform-specific inhibitors for the clinical treatment of a variety of diseases, including certain cancers (Bonardi et al., 2022; Bozdag et al., 2014; Mboge et al., 2021; Vannozzi et al., 2022). A major hurdle in the development of effective CAIs is the similarity between the human CA isoforms, which leads to off-target binding. Thus, new isoform-specific CAIs that bind differentially depending on the amino acids that line the active sites are currently under development (Bozdag et al., 2022; Lomelino et al., 2016).
As such, alternative zinc-binding moieties other than et al., 2016). Interestingly, not all carboxylic acid-based inhibitors show the same binding mode within the CA active site (Figs. 1a and 1d; Lomelino & McKenna, 2019). The carboxylic acid inhibitors either bind directly (Figs. 1b and 1e) or indirectly (Figs. 1c and 1f) to the zinc. In the case of direct binding, an O atom of the carboxylic acid displaces the catalytic ZBS (Langella et al., 2016) and therefore prevents the first step in the enzymatic mechanism (1), nucleophilic attack of the hydroxide on the CO2 substrate (Figs. 1b and 1e; Fisher et al., 2010). In the indirect binding mode, in contrast, the carboxylic acid binds to the zinc via a bridging hydrogen bond to the ZBS (Figs. 1c and 1f; Lomelino & McKenna, 2019). In this mode, it is thought that the second step in the enzymatic mechanism (2) is blocked, with the inhibitor stabilizing the ZBS, which blocks access to the zinc within the active site.
are being explored to target the CA active site. Some examples include polyamines, sulfocoumarins and (LomelinoIbuprofen, nicotinic acid and ferulic acid are all medically administered enzyme inhibitors that contain a carboxylic acid moiety. Ibuprofen has analgesic, antipyretic and anti-inflammatory properties, which is why it is typically utilized by patients with inflammation and arthritis (Dornan & Reynolds, 1974). Nicotinic acid is typically used to treat dyslipidemia states by increasing the concentration of plasma HDL cholesterol (Bodor & Offermanns, 2008). Ferulic acid has been linked to a wide variety of functions such as anticancer, antimicrobial, antidiabetic, antioxidant and anti-inflammatory properties (Zduńska et al., 2018). Ferulic acid has also been linked to wound healing and enhanced angiogenesis, and is used in cosmetics, food and pharmaceutical applications (Zduńska et al., 2018).
This paper presents the structure of hCA II in complex with the carboxylic acid-based inhibitor ibuprofen determined by X-ray crystallography. In addition, inhibition studies of hCA II with ibuprofen as well as with the carboxylic acid-based compounds nicotinic acid and ferulic acid were performed. Based on these structural and activity observations, combined with other literature reports, the implications of the direct and indirect binding modes between carboxylic acid-based inhibitors and hCA II are discussed.
2. Materials and methods
2.1. Macromolecule production
The enzyme hCA II was expressed in Escherichia coli BL21 pLysS (DE3) cells from New England Biolabs and was purified as described previously (Pinard et al., 2013; Tanhauser et al., 1992). The cells were grown in LB medium (Fisher) and protein expression was induced using 1 mM isopropyl β-D-1-thiogalactopyranoside (Fisher) and 1 mM zinc sulfate (Fisher) for 3 h (Fisher et al., 2009). The cell pellets were lysed with a microfluidizer (Microfluidics LM10) after harvesting via centrifugation (Beckman J-10). The protein was loaded onto a p-aminomethylbenzenesulfonamide–agarose (Sigma) column and hCA II was eluted with 0.4 M sodium azide (Fisher). hCA II was buffer-exchanged into 50 mM Tris pH 8.0 (Fisher) and concentrated to 10 mg ml−1. The protein purity was checked with by SDS–PAGE (Fisher).
2.2. Crystallization
The crystallization conditions for hCA II were set up as described previously (Lomelino & McKenna, 2019). In brief, a 1:1 ratio of protein (10 mg ml−1) and precipitant solution (1.6 M sodium citrate, 50 mM Tris pH 7.8) in a drop of 5 µl in volume was suspended over 500 µl precipitant solution (Fisher) by the hanging-drop vapor-diffusion method using a 24-well VDXm Plate with sealant (Hampton Research). The plate was incubated at room temperature and crystal growth was noted within a week. The hCA II crystals were soaked with 1 µl 100 mM ibuprofen (Sigma; a final concentration of 17 mM) for 1 h prior to crystal mounting. A 20% glycerol (Fisher) precipitant solution was used as a cryoprotectant to transfer the hCA II crystals before flash-cooling in liquid nitrogen.
2.3. Data collection and processing
X-ray crystallographic diffraction data were collected on the BL9-2 beamline at Stanford Synchrotron Radiation Lightsource (SSRL) using a PILATUS 6M detector. An exposure time of 0.5 s, an oscillation angle of 1° and a crystal-to-detector distance of 270 mm were used to collect a 180-image data set. XDS was used to index and integrate the diffraction data (Kabsch, 2010) and AIMLESS from the CCP4 program package was used to reduce and scale the data in P21 (Evans & Murshudov, 2013; Winn et al., 2011). was performed using the wild-type hCA II structure with PDB code 3ks3 as a search model to determine the initial phases (Avvaru et al., 2010). The interactive graphical software Coot (Emsley et al., 2010) was used to modify the model and inspect the electron-density maps, while Phenix (Leibschner et al., 2019) was used to refine the model and generate restraint files for ibuprofen. Interactions were determined via LigPlot (Laskowski & Swindells, 2011) and figures were generated in PyMOL (version 0.9.4; Schrödinger).
2.4. Kinetics
A colorimetric substrate, 4-nitrophenyl acetate (pNPA), was used to perform esterase assays that measured the inhibition constants of ibuprofen, nicotinic acid (Acros Organics) and ferulic acid (Sigma) in the presence of hCA II. An ester bond within pNPA (Sigma) is cleaved by hCA II, forming 4-nitrophenol that absorbs strongly at 348 nm. This allows the reaction to be monitored spectroscopically (Tashian et al., 1964). Inhibition experiments varied the concentrations of ibuprofen, nicotinic acid and ferulic acid between 2 and 50 mM. These inhibitors were incubated with 50 µl 0.1 mg ml−1 hCA II at room temperature for 30 min. 200 µl of 3 mM pNPA in 3% acetone (Sigma) was added to the sample and immediately scanned in a Synergy HTX BioTek plate reader at 348 nm absorbance for 10 min. Acetazolamide (Sigma) was used as a positive control for inhibition. The data were analyzed using Prism (version 9.2.0; GraphPad). A line of best fit was fitted to the data.
3. Results
3.1. X-ray crystallography
The 8dj9 (Table 1, Fig. 2). While ibuprofen exists as two isomers (R and S), only the S isomer binds to hCA II. This is important as this is the pharmacologically active isomer (Geisslinger et al., 1989).
of hCA II in complex with ibuprofen was determined to a resolution of 1.54 Å and deposited in the PDB as entry
‡Rmeas = . §Rp.i.m. = . ¶Rwork = . ††Rfree = (using data omitted from (5%). |
For the inhibition of hCA II by ibuprofen, the carboxylic acid directly binds to the zinc and displaces the ZBS (Fig. 2b). This binding mode is similar to that in previously described direct complexes of carboxylic acid-based inhibitors with hCA II (Boone et al., 2014). The ibuprofen binds between the hydrophobic and hydrophilic sides of the active site. The hydrophilic residues Thr199 and Thr200 form hydrogen bonds to a water within the active site that also forms a hydrogen bond to the carboxylic acid of ibuprofen (Fig. 2c). The hydrophobic residues Val121, Phe131, Val143, Leu198 and Trp209 and the hydrophilic residue Gln92 all make significant intermolecular interactions with the ring and tail portion of ibuprofen (Fig. 2c). Hence, ibuprofen predominately interacts within the hydrophobic pocket of hCA II, with an occluded interface binding surface area of 240 Å2 (Table 2).
|
3.2. Inhibition
In addition to the interconversion of CO2 and , hCA II also has an esterase activity that can be used as a CA functional assay (Tashian et al., 1964). A colorimetric probe such as 4-nitrophenyl acetate (pNPA) is used as a substrate to monitor the reaction when the enzyme cleaves the ester bond to form 4-nitrophenyl, which is spectroscopically absorbent at 348 nm (Bua et al., 2020; Uda et al., 2015). This assay was used to measure the catalytic rate of the enzyme in the presence of ibuprofen, nicotinic acid and ferulic acid. The three inhibitors were initially tested at concentrations of 50, 30, 20, 10, 5 and 2 mM. Additional experiments were performed between concentrations of 2 and 20 mM to obtain further data points near the 50% inhibition concentration. The data were plotted to a nonlinear regression using Prism to determine IC50 values (Fig. 3). The average standard deviations at each concentration for ibuprofen, nicotinic acid and ferulic acid are 3.5, 3.4 and 2.8% of the respectively. The calculated IC50 values of ibuprofen, nicotinic acid and ferulic acid are 12.8 ± 1.1, 9.6 ± 1.0 and 10.6 ± 1.0 mM, respectively, with a 95% confidence interval. The lines of best fit for all three inhibitors have an R2 value above 0.94.
4. Discussion
In this study, the structure of hCA II in complex with ibuprofen has been determined to 1.54 Å resolution using X-ray crystallography (Fig. 2). Esterase inhibition activity studies were used to obtain the IC50 values for ibuprofen, nicotinic acid and ferulic acid, which were determined to be 15.3, 10.4 and 8.6 mM, respectively (Fig. 3). Unlike nicotinic acid and ferulic acid, ibuprofen has a chiral branch between the carboxylic acid and benzene ring moieties which creates some slight steric binding issues within the active site of hCA II. Additionally, ibuprofen binds directly to the zinc, displacing the ZBS. On the other hand, both nicotinic acid and ferulic acid do not have a chiral branch linker and bind through a hydrogen bond to the ZBS, rather than directly to the zinc (Lomelino & McKenna, 2019). These differences in both the conformational nature and the binding mode contribute to the weaker binding of ibuprofen. These observations lead to the hypothesis that one or either of these features may account for the differences in CA inhibition between the three carboxylic acid-based compounds studied here and previously.
While hCA II is not the target of ibuprofen in a treatment regimen, it does bind at concentrations that are physiologically relevant. With a standard dose of 200 mg and with the average human having 5 l of blood (Cooper et al., 1977; Dean, 2005), the effective physiological concentration of ibuprofen in the blood would be ∼0.2 mM. Considering the calculated IC50 of 13 mM, it is unlikely that much ibuprofen would bind to hCA II in vivo. As such, no side effects would be expected due to interactions with hCA II.
To further investigate the carboxylic acid binding modes, the structures of an additional 15 carboxylic acid moiety-based hCA II inhibitors were examined. Analysis of the PDB (Berman et al., 2003) revealed that the carboxylic acid compounds followed the direct and indirect binding modes described here. Ibuprofen (this study; green; PDB entry 8dj9), a saccharin derivative (teal; PDB entry 5clu), an ethanoic acid derivative (chocolate; PDB entry 5fnj), another ethanoic acid derivative (wheat; PDB entry 5flq), propenoic acid (pink; PDB entry 5ehv) and cholate (olive; PDB entry 4n16) bound via direct binding to the zinc (Boone et al., 2014; Langella et al., 2016; Woods et al., 2016; Fig. 4a). In contrast, a heteroaryl-pyrazole carboxylic acid derivative (orange; PDB entry 6b4d), an enoic acid derivative (deep teal; PDB entry 5eh8), another enoic acid derivative (warm pink; PDB entry 5fls), 3-phenoxybenzoic acid (pale yellow; PDB entry 5flt), 2,6-dihydroxybenzoic acid (violet purple; PDB entry 4e3f), 2,5-dihydroxybenzoic acid (lemon; PDB entry 4e3d), p-hydroxybenzoic acid (lime green; PDB entry 4e3g), 2-sulfanylbenzoic acid (light pink; PDB entry 4e4a), ferulic acid (marine; PDB entry 6mby), nicotinic acid (deep blue; PDB entry 6mbv), salicylic acid (dark red; PDB entry 6ux1) and 2-hydroxybenzoic acid (brown; PDB entry 5m78) were observed to bind indirectly to the ZBS (Andring et al., 2020; Cadoni et al., 2017; Lomelino & McKenna, 2019; Martin & Cohen, 2012; Woods et al., 2016; Fig. 4b). Ibuprofen (this study; green) almost superimposed onto the previously determined structure of cholic acid (olive) bound to hCA II (Boone et al., 2014; Fig. 4a).
Upon closer inspection, the hCA II direct zinc binders revealed two subgroups with distinctive orientations, with propenoic acid (pink), one ethanoic acid derivative (chocolate) and cholate (olive) binding in one orientation (class I), while ibuprofen (green), the other ethanoic acid derivative (wheat) and the saccharin derivative (teal) bind in a second orientation (class II) in the active site (Fig. 4c). In contrast, the indirect binders vary in location and orientation depending on their tail group; the carboxylic acid moieties all bind in the same orientation, with a movement of up to 1.7 Å between the ZBS of different indirect binders (Fig. 4d). Overall, the carboxylic acid-based compounds tend to bind predominately on the hydrophobic side as opposed to the hydrophilic side of the active-site cavity (Figs. 4b and 4d).
When studying enzyme inhibition, the technique and the reported kinetic value can differ depending on the equipment and the assay used by the researchers. There are a variety of reportable constants related to inhibitor binding such as Ki (the inhibition constant), IC50 (the half-maximal inhibitory concentration) and Kd (the dissociation constant) (Table 2). These values can be compared using the equation Ki = IC50/{1 + ([C]/Kd)}. Taking the inhibition data for carboxylic acid hCA II binders from the literature, the direct binders have an average kinetic inhibition (Ki) of 3.4 ± 5.1 mM, while the indirect binders have an average kinetic inhibition value of 5.5 ± 3.8 mM (excluding the lowest value of 0.8 µM, which is 1000-fold lower than the second lowest kinetic value and is associated with a heteroaryl-pyrazole carboxylic acid derivative; PDB entry 6b4d). Therefore, on average the direct binders have a slightly higher affinity for CA compared with the indirect binders, although this difference is not statistically significant. This difference might be due to the carboxylic acid having a higher affinity for zinc than for the ZBS. Furthermore, the carboxylic acid inhibitors are identifed as weak binders; the commonly used sulfonamide hCA II inhibitor acetazolamide has a Ki value of 10 nM (Supuran et al., 2003).
In broad terms, the higher the indirect carboxylic acid affinity, the larger the covered interface area between the compound and the active-site residues (Table 2). However, no trend is observed for the direct binders, which might be because of the smaller sample size of the structures. The average interface between hCA II and direct and indirect binders is 260 ± 40 and 200 ± 30 Å2, respectively. This demonstrates that the direct binders interact more strongly with residues within the active site than indirect binders. This is attributed to the direct binders being buried further within the active-site pocket compared with the indirect binders.
For the indirect carboxylic acid binders, the distance between the zinc and ZBS is shorter for the tighter binders and larger for the weaker binders (Fig. 5, Table 2). However, this trend is less convincing for the direct binders. For the indirect binders the Ki for hCA II increases as the compounds move slightly farther away from the zinc.
The average distance between the O atom of the carboxylic acid group of indirect binders and the ZBS is 2.7 Å, while the distance between the zinc and the O atom of the carboxylic acid group of indirect binders is 3.7 Å. In contrast, the average distance between the O atom of the carboxylic acid group of direct binders and the zinc is 2.0 Å. The distance between the ZBS and the zinc for indirect binders is slightly greater at 2.06 Å (Table 2). This small increase in distance between ZBS and zinc seen in indirect binders could be the rationale for their weaker affinities compared with direct binders.
These data provide information for the design of alternative zinc-binding groups: carboxylic acid moieties for CA inhibitors rather than the classical sulfonamide drugs currently in clinical use. While the sulfonamide-based compounds are several logs better at inhibiting than
the carboxylic acid-based inhibitors rely less on their affinity to bind zinc and more on their interactions with the amino acids that line the active site, giving them greater design potential to better target selection between the different CA isoforms.Acknowledgements
Experiments were performed at SSRL and the authors would like to acknowledge the guidance and expertise of the SSRL staff.
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