Ibuprofen: a weak inhibitor of carbonic anhydrase II

Abstract

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 crystal structure 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 carboxylic acids 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 IC₅₀ 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.

Keywords: X-ray crystallography; carbonic anhydrases; carboxylic acid-based inhibitors; ibuprofen; kinetics.

Figure 1

Carboxylic acid inhibitor-binding modes of CA. (a), (b), (c) Observed crystallographic data from PDB entries 3ks3, 8dj9 and 5eh8, respectively; (d), (e), (f) schematic models. (a, d) hCA II active site with ZBS, (b, e) direct inhibition in which the carboxylic acid displaces the ZBS and (c, f) indirect inhibition with the carboxylic acid hydrogen-bonded to the ZBS. (a), (b), (c) Zinc is depicted as a magenta sphere, water as a red sphere, the O atoms of the carboxylic acid moiety of the inhibitor are in red and the active-site histidine residues are labeled. (d), (e), (f) Zinc direct metal coordination is depicted as dashed lines, with hydrogen bonds as solid lines; the respective distances are labeled.

Figure 2

Structure of hCA II in complex with ibuprofen. (a) Surface view of the complex, with the hydrophobic face of the hCA II active site in orange, the hydrophilic face in purple and ibuprofen in green. (b) Close-up of the ibuprofen binding site with a 2F _o − F _c electron-density mesh (blue, contoured at 1.0σ). (c) Interactions of ibuprofen with relevant hCA II residues as labeled. Hydrogen bonds are shown as dashed lines, waters as red spheres, zinc ions as magenta spheres and bound ibuprofen as green sticks.

Figure 3

Inhibition of hCA II with ibuprofen (blue circles), nicotinic acid (red squares) and ferulic acid (green triangles). Each compound concentration was run in triplicate; error bars indicate standard deviations. Where error bars are not present the standard deviation is too small for the software to depict. The chemical structures of each compound are shown in the inset.

Figure 4

Carboxylic acid inhibitor–hCA II complex structures. (a) Superposition of direct binding to the zinc: ibuprofen (green; PDB entry 8dj9), 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). (b) Superposition of indirect binding via the ZBS: 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). (c) and (d) are active-site close-up views of (a) and (b), respectively. Zinc is shown as a magenta sphere, the hydrophobic pocket is in orange and the hydrophilic pocket is in purple with PDB codes included for reference.

Figure 5

Inhibition of hCA II by carboxylic acid compounds. Inhibition constants previously reported as IC₅₀, K _i or K _d versus active-site distance. Direct binders are shown as blue circles and indirect binders as red squares. For direct binders the distance from the zinc to the closest O atom in the carboxyl group is measured, while for indirect binders the distance from the ZBS to the zinc is measured.