Inhibition of Carbonic Anhydrase II by Thioxolone: A Mechanistic and Structural Study

Abstract

This paper examines the functional mechanism of thioxolone, a compound recently identified as a weak inhibitor of human carbonic anhydrase II by Iyer et al. (2006) J. Biomol. Screening 11, 782-791. Thioxolone lacks sulfonamide, sulfamate, or hydroxamate functional groups that are typically found in therapeutic carbonic anhydrase (CA) inhibitors, such as acetazolamide. Analytical chemistry and biochemical methods were used to investigate the fate of thioxolone upon binding to CA II, including Michaelis-Menten kinetics of 4-nitrophenyl acetate esterase cleavage, liquid chromatography-mass spectrometry (LC-MS), oxygen-18 isotope exchange studies, and X-ray crystallography. Thioxolone is proposed to be a prodrug inhibitor that is cleaved via a CA II zinc-hydroxide mechanism known to catalyze the hydrolysis of esters. When thioxolone binds in the active site of CA II, it is cleaved and forms 4-mercaptobenzene-1,3-diol via the intermediate S- (2,4-thiophenyl)hydrogen thiocarbonate. The esterase cleavage product binds to the zinc active site via the thiol group and is therefore the active CA inhibitor, while the intermediate is located at the rim of the active-site cavity. The time-dependence of this inhibition reaction was investigated in detail. Because this type of prodrug inhibitor mechanism depends on cleavage of ester bonds, this class of inhibitors may have advantages over sulfonamides in determining isozyme specificity. A preliminary structure-activity relationship study with a series of structural analogues of thioxolone yielded similar estimates of inhibition constants for most compounds, although two compounds with bromine groups at the C1 carbon of thioxolone were not inhibitory, suggesting a possible steric effect.

Bidentate Zinc Chelators for α‐Carbonic Anhydrases that Produce a Trigonal Bipyramidal Coordination Geometry

“…A similar CA II complex has been determined crystallographically and was recently published by Barrese et al[20] Investigating the inhibition profile and binding mode of thioxolone 6 in CA II, they observed that the inhibitor, most likely a prodrug, is hydrolyzed upon binding to form the fragment 4-thiobenzene-1,3-diol 7 ( K i =148 µm)[20] (Figure 4). However, superposition of 7 with 4 shows significant differences in the binding mode, with 7 binding in a monodentate fashion (Figure 3b, c).…”

Exploring the Influence of the Protein Environment on Metal-Binding Pharmacophores

“…38 To determine whether the interactions perturbing 1,2-HOPTO binding apply to other MBPs containing similar donor sets, methyl derivatives of TP and 2MP analogous to 4-CH 3 -1,2-HOPTO were obtained and their binding to hCAII investigated. While 3-CH 3 -TP has similar inhibitory activity to unsubstituted TP ( K i = 2.1 μM vs 3.5 μM), the 4-methyl analogue of 2MP is significantly more potent (0.52 vs 3.1 μM).…”

Multiple Binding Modes of Inhibitors to Carbonic Anhydrases: How to Design Specific Drugs Targeting 15 Different Isoforms?

“…100 The inhibition mechanism of this molecule was not immediately clear analyzing its chemical structure but crystallographic studies helped to clarify it. 100 In particular, in hCA II crystals soaked with 1, the latter was not found in the active site, which instead contained a 4-mercaptobenzene-1,3-diol molecule (2) bound to the catalytic zinc ion (Figure 9). This observation led to hypothesize that when thioxolone binds to the hCA II active site, it is cleaved forming 4-mercaptobenzene-1,3-diol via the intermediate S-(2,4-thiophenyl)hydrogen thiocarbonate (see Scheme 2) utilizing, as hypothesized for other esters, 101 a hCA II zinc-hydroxide mechanism.…”