The “tail approach” has become a milestone in human carbonic anhydrase inhibitor (hCAI) design for various therapeutics, including antiglaucoma agents. Besides the classical hydrophobic/hydrophilic division of hCAs active site, several subpockets have been identified at the middle/outer active sites rim, which could be targeted to increase the CAI isoform selectivity. This postulate is explored here by three-tailed benzenesulfonamide CAIs (TTI) to fully exploit such amino acid differences among hCAs. In this proof-of-concept study, an extensive structure–activity relationship (SAR) study was carried out with 32 such benzenesulfonamides differing in tails combination that were assayed for hCAs I, II, IV, and XII inhibition. A structural study was undertaken by X-ray crystallography and in silico tools to assess the ligand/target interaction mode. The most active and selective inhibitors against isoforms implicated in glaucoma were assessed in a rabbit model of the disease achieving an intraocular pressure-lowering action comparable to the clinically used dorzolamide.

Nitric oxide (NO) promotes vasodilation through the activation of guanylate cyclase, resulting in the relaxation of the smooth muscle vasculature and a subsequent decrease in blood pressure. Therefore, its regulation is of interest for the treatment and prevention of heart disease. An example is pulmonary hypertension which is treated by targeting this NO/vasodilation pathway. In bacteria, plants and fungi, nitrite (NO₂⁻) is utilized as a source of NO through enzymes known as nitrite reductases. These enzymes reduce NO₂⁻to NO through a catalytic metal ion, often copper. Recently, several studies have shown nitrite reductase activity of mammalian carbonic anhydrase II (CAII), yet the molecular basis for this activity is unknown. Here we report the crystal structure of copper-bound human CAII (Cu–CAII) in complex with NO₂⁻at 1.2 Å resolution. The structure exhibits Type 1 (T-1) and 2 (T-2) copper centers, analogous to bacterial nitrite reductases, both required for catalysis. The copper-substituted CAII active site is penta-coordinated with a `side-on' bound NO₂⁻, resembling a T-2 center. At the N terminus, several residues that are normally disordered form a porphyrin ring-like configuration surrounding a second copper, acting as a T-1 center. A structural comparison with both apo- (without metal) and zinc-bound CAII (Zn–CAII) provides a mechanistic picture of how, in the presence of copper, CAII, with minimal conformational changes, can function as a nitrite reductase.

Carbonic anhydrase II (CAII) is a metalloenzyme that catalyzes the reversible hydration/dehydration of CO₂/HCO₃⁻. In addition, CAII is attributed to other catalytic reactions, including esterase activity. Aspirin (acetyl-salicylic acid), an everyday over-the-counter drug, has both ester and carboxylic acid moieties. Recently, compounds with a carboxylic acid group have been shown to inhibit CAII. Hence, we hypothesized that Aspirin could act as a substrate for esterase activity, and the product salicylic acid (SA), an inhibitor of CAII. Here, we present the crystal structure of CAII in complex with SA, a product of CAII crystals pre-soaked with Aspirin, to 1.35Å resolution. In addition, we provide kinetic data to support the observation that CAII converts Aspirin to its deacetylated form, SA. This data may also explain the short half-life of Aspirin, with CAII so abundant in blood, and that Aspirin could act as a suicide inhibitor of CAII.