Reconsidering anion inhibitors in the general context of drug design studies of modulators of activity of the classical enzyme carbonic anhydrase

Abstract

Inorganic anions inhibit the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1) generally by coordinating to the active site metal ion. Cyanate was reported as a non-coordinating CA inhibitor but those erroneous results were subsequently corrected by another group. We review the anion CA inhibitors (CAIs) in the more general context of drug design studies and the discovery of a large number of inhibitor classes and inhibition mechanisms, including zinc binders (sulphonamides and isosteres, dithiocabamates and isosteres, thiols, selenols, benzoxaboroles, ninhydrins, etc.); inhibitors anchoring to the zinc-coordinated water molecule (phenols, polyamines, sulfocoumarins, thioxocoumarins, catechols); CAIs occluding the entrance to the active site (coumarins and derivatives, lacosamide), as well as compounds that bind outside the active site. All these new chemotypes integrated with a general procedure for obtaining isoform-selective compounds (the tail approach) has resulted, through the guidance of rigorous X-ray crystallography experiments, in the development of highly selective CAIs for all human CA isoforms with many pharmacological applications.

Keywords: carbonic anhydrase; cyanate; inhibition mechanisms; inhibitor; sulphonamide.

Figure 1.

Active site view of the superimposed hCA II adducts with its main substrates: CO₂ (green, pdb 2VVA) and bicarbonate (pink, pdb 2VVB) . The zinc ion is shown as a grey sphere with its three coordinated protein ligands (green/red), His94, 96 and 119. Amino acid residues involved in the binding of the substrates/inhibitors, at the bottom of the active site, are also shown.

Figure 2.

Representation of the superimposed X-ray crystallographic data for hCA II adducts with cyanate (cyan, pdb 4E5Q³¹) bromide (magenta, pdb 1RAZ³²) azide (blue, pdb 1RAY³²) O₂ (presumably as superoxide anion or radical anion; red, pdb 5EOI³³) with a tetrahedrally coordinated active site zinc ion (grey sphere). The key active site zinc ligands (His94, 96 and 119) and “gate-keeping” residues (Thr199, Glu106) are also shown.

Figure 3.

Representation of the superimposed hCA II adducted to cyanate (cyan, pdb 4E5Q³¹) bicarbonate (pink, pdb 2VVB²⁴) urea (as anion) (white, pdb 1BV3 [39]), and trithiocarbonate (light blue, pdb 3K7K⁴⁰) The zinc ion (grey sphere), its protein ligands (His94, 96 and 119) and gate-keeping residues (Thr199, Glu106) are also shown.

Figure 4.

Representation of the binding of A) O₂⁻ to a Zn²⁺/Cu²⁺ hCA II (pdb 5EOI³³) and B) nitrite to Cu²⁺ substituted Cu₂-hCA II (pdb 6PDV⁴³) Distances of the closest atom of the anion to the metal ion are: Zn-O (in the O₂ adduct) of 1.88 Å; Cu-O (nitrite adduct) of 2.14 Å.

Figure 5.

Active site view of A) hCA IX catalytic domain adducted to SLC-0111 (magenta) and B) hCA II adducted to the three tailed inhibitor 4 (in green). H-bonds are represented as black dashed lines. The Zn(II) ion (grey sphere) and some important amino acid residues involved in the binding of inhibitors are shown.

Figure 6.

Historical overview on the discovery of the main chemotypes with CA inhibitory activity.

Figure 7.

Active site view of hCA II complexed to A) selenol 11 (pdb 6hX5) , B) ninhydrin 10 (predicted in silico) and C) catechol derivative 12 (pdb 6YRI) . H-bonds are represented as black dashed lines. The Zn(II) ion (grey sphere) and residues involved in its coordination and some active site residues near the binding of inhibitors are shown in CPK colours. Water molecules are shown as red spheres.

Figure 8.

hCAs as drug targets^,,. From edoema and glaucoma, to obesity, neuropathic pain, hypoxic cancers, cerebrovascular diseases and oxidative stress, many isoforms are involved in diverse pathologies for which isoform-selective inhibitors showed a relevant potential to be translated to clinical entities.

Chart 1.

Structure of the compounds 1–14 discussed in this review.