Inhibition of the ß-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with sulphonamides.

Sulphonamides and their isosteres are classical inhibitors of the carbonic anhydrase (CAs, EC 4.2.1.1) metalloenzymes. The protozoan pathogen Trichomonas vaginalis encodes two such enzymes belonging to the ß-class, TvaCA1 and TvaCA2. Here we report the first sulphonamide inhibition study of TvaCA1, with a series of simple aromatic/heterocyclic primary sulphonamides as well as with clinically approved/investigational drugs for a range of pathologies (diuretics, antiglaucoma, antiepileptic, antiobesity, and antitumor drugs). TvaCA1 was effectively inhibited by acetazolamide and ethoxzolamide, with KIs of 391 and 283 nM, respectively, whereas many other simple or clinically used sulphonamides were micromolar inhibitors or did not efficiently inhibit the enzyme. Finding more effective TvaCA1 inhibitors may constitute an innovative approach for fighting trichomoniasis, a sexually transmitted infection, caused by T. vaginalis.

Fluorinated benzenesulfonamide anticancer inhibitors of carbonic anhydrase IX exhibit lower toxic effects on zebrafish embryonic development than ethoxzolamide.

The toxic effects of two recently discovered inhibitors (VD12-09 and VD11-4-2) that selectively and with extraordinary strong, picomolar binding affinity to human carbonic anhydrase (CA) isoform IX were investigated on zebrafish embryonic development. CA IX has been recently introduced as an anticancer target since it is highly overexpressed in numerous human cancers but nearly absent in normal tissues. Morphological changes in zebrafish treated by the compounds were studied by light-field microscopy and histological analysis. Homology models of zebrafish CA II and CA IX were built to identify the conserved amino acid residues in the active site of zebrafish CAs. The toxicity studies here showed that the LC50 values at 120 hours post-fertilization (hpf) were 13 µM for VD12-09, 120 µM for VD11-4-2, and 9 µM for ethoxzolamide (EZA), a non-selective CA inhibitor commonly used as a drug in clinics. Thus, EZA was the most toxic of the three compounds. The zebrafish embryos exposed to LC50 doses of VD12-09 and VD11-4-2 showed fewer phenotypic abnormalities compared with the embryos exposed to the corresponding dose of EZA. Histochemical studies did not show any gross morphological changes in the embryos treated with VD12-09 and VD11-4-2 unlike EZA. The results of our study indicate that the compounds exhibited 10-fold lower toxicity and induced fewer side effects in zebrafish than EZA. Therefore, the exposure to VD11-4-2 and VD12-09 at concentrations below LC50 did not lead to deleterious effects on the zebrafish embryonic development and thus both inhibitors may be further developed as drugs.

Intrinsic thermodynamics of trifluoromethanesulfonamide and ethoxzolamide binding to human carbonic anhydrase VII.

Human carbonic anhydrase (CA) isozyme VII is a cytosolic protein that is highly expressed in the cortex, hippocampus, and thalamus regions within mammalian brain, and expression disorders can cause epilepsy and several cases of malignant brain tumors. Therefore, CA VII is a potential antiepileptic and anticancer drug target. There are numerous sulfonamides that target CAs nonspecifically. It is important to understand the thermodynamics of inhibitor binding and the structural features of the protein-inhibitor complex in order to design specific inhibitors against CA VII. Isothermal titration calorimetry and fluorescent thermal shift assay were used to characterize the intrinsic thermodynamic parameters of trifluoromethanesulfonamide and ethoxzolamide binding to CA VII. Binding experiments were carried out at various pH in different buffers in order to dissect linked protonation of the water molecule bound to the CA VII active site, deprotonation of the sulfonamide group of the inhibitor, and protonation-deprotonation of buffer. Dissection of all those contributions yielded the intrinsic thermodynamic parameters of binding, such as Gibbs free energy, binding enthalpy, entropy, and protein pKa value. Thermal shift assay was also used to determine CA VII stability at various pH.