Sulfonamide Inhibition Studies of the ß-Class Carbonic Anhydrase CAS3 from the Filamentous Ascomycete Sordaria macrospora.

A new ß-class carbonic anhydrase was cloned and purified from the filamentous ascomycete Sordaria macrospora, CAS3. This enzyme has a higher catalytic activity compared to the other two such enzymes from this fungus, CAS1 and CAS2, which were reported earlier, with the following kinetic parameters: kcat of (7.9 ± 0.2) × 105 s-1, and kcat/Km of (9.5 ± 0.12) × 107 M-1∙s-1. An inhibition study with a panel of sulfonamides and one sulfamate was also performed. The most effective CAS3 inhibitors were benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfanilamide, methanilamide, and benzene-1,3-disulfonamide, with KIs in the range of 54-95 nM. CAS3 generally shows a higher affinity for this class of inhibitors compared to CAS1 and CAS2. As S. macrospora is a model organism for the study of fruiting body development in fungi, these data may be useful for developing antifungal compounds based on CA inhibition.

A class of carbonic anhydrase IX/XII - selective carboxylate inhibitors.

A small series of 2,4-dioxothiazolidinyl acetic acids was prepared from thiourea, chloroacetic acid, aromatic aldehydes, and ethyl-2-bromoacetate. They were assayed for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms of human (h) origin, the cytosolic hCA I and II, and the transmembrane hCA IX and XII, involved among others in tumorigenesis (hCA IX and XII) and glaucoma (hCA II and XII). The two cytosolic isoforms were not inhibited by these carboxylates, which were also rather ineffective as hCA IX inhibitors. On the other hand, they showed submicromolar hCA XII inhibition, with KIs in the range of 0.30-0.93 µM, making them highly CA XII-selective inhibitors.

First report on two-fold classification of Plasmodium falciparum carbonic anhydrase inhibitors using QSAR modeling approaches.

Quantitative structure-activity relationship (QSAR)-based classification approach is one of the important chemometric tools in drug discovery process for categorizing the target protein inhibitors into more active and less active classes. In this background, we have presented here a novel approach of two-fold QSAR-based classification modeling for the Plasmodium falciparum carbonic anhydrase (PfCA) inhibitors using 2D-QSAR and linear discriminant analysis (LDA) methods. The logic of applying this concept is to ensure more accurate classification of compounds and to draw some concrete conclusion about structure-activity relations for further work, in absence of 3D-protein structure and lack of sufficient experimental data using the PfCA target. The 2D-QSAR modeling analysis suggested the importance of electrotopological, electronic, extended topochemical atom, and spatial (Jurs) indices for modeling the inhibitory activity against PfCA. The LDA model analysis showed that spatial (Jurs), electrotopological and thermodynamic indices were the discriminating features to differentiate the inhibitors into more active and less active groups. The classification ability of both the models for training and test sets was checked by different qualitative validation parameters such as sensitivity, specificity, accuracy, recall, precision, F-measure and G-means. The classification results revealed that the developed models were significant in classifying the more active inhibitors as compared to the less active inhibitors of both training and test sets. The structural features unveiled from these two models could be utilized for the selection of more active compounds against PfCA in the database screening process.

Thermodynamic benchmark study using Biacore technology.

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.

Comparative analysis of 10 small molecules binding to carbonic anhydrase II by different investigators using Biacore technology.

In this benchmark study, 26 investigators were asked to characterize the kinetics and affinities of 10 sulfonamide inhibitors binding to the enzyme carbonic anhydrase II using Biacore optical biosensors. A majority of the participants collected data that could be fit to a 1:1 interaction model, but a subset of the data sets obtained from some instruments were of poor quality. The experimental errors in the k(a), k(d), and K(D) parameters determined for each of the compounds averaged 34, 24, and 37%, respectively. As expected, the greatest variation in the reported constants was observed for compounds with exceptionally weak affinity and/or fast association rates. The binding constants determined using the biosensor correlated well with solution-based titration calorimetry measurements. The results of this study provide insight into the challenges, as well as the level of experimental variation, that one would expect to observe when using Biacore technology for small molecule analyses.

Development of quantitative structure-activity relationship and classification models for a set of carbonic anhydrase inhibitors.

Mathematical models are developed to find quantitative structure-activity relationships that correlate chemical structure and inhibition toward three carbonic anhydrase (CA) isozymes: CA I, II, and IV. Numerical descriptors are generated to encode important topological, geometric, and electronic features of molecular structure. After descriptor generation, multiple linear regression, and computational neural network (CNN) analyses are performed on various descriptor subsets to find superior models for prediction. Committees of five CNNs were utilized to average final predicted values for the 142-compound data set. For inhibitors of CA I, an 8-5-1 CNN committee produced a training set rms error of 0.105 log K(i) (r(2) = 0.994) and prediction set rms error of 0.208 log K(i) (r(2) = 0.980). Training and prediction set rms errors of 0.140 log K(i) (r(2) = 0.992) and 0.231 log K(i) (r(2) = 0.971), respectively, were produced by a 9-5-1 CNN committee for inhibitors of CA II. For prediction of CA IV inhibitors, an 8-5-1 CNN committee produced training and prediction set rms errors of 0.147 log K(i) (r(2) = 0.992) and 0.211 log K(i) (r(2) = 0.991), respectively. In addition, classification models were built using k-nearest neighbor (kNN) analysis to solve two- and three-class problems for inhibitors of CA IV. A three-descriptor classification model proved superior in labeling compounds as active or inactive inhibitors for the two-class problem. Training and prediction set percent classification rates of 100% and 87.1%, respectively, were obtained. For the three-class (active/moderate/inactive) problem, a five-descriptor model was deemed optimal producing a training set percent classification rate of 98.8% and prediction set rate of 79.0%.

The inhibitory effect of diuretics on carbonic anhydrases.

A classification of diuretics mainly comprises mercurials; carbonic anhydrase inhibitors, thiazide diuretics, loop diuretics, inhibitors of renal epithelial Na+ channels and antagonists of mineralocorticoid receptors. We studied in this paper the relationship between diuretics and carbonic anhydrase (CA). Our in vitro and in vivo results show that all diuretics inhibit carbonic anhydrase II and renal CA IV. Further, our data show that they also inhibit epithelial cell CA in the renal tubules. The changes in intracellular pH (pHi) induced by these diuretics through CA inhibition would influence: a) the coupling to their receptors affecting information transmission to the epithelial cells of renal tubules as well as diuretic response; b) the decrease of Na+ exchanger (thiazide), of Na+ - K+ - 2Cl- relation (loop diuretics), Na+ channel blocking in distal and collecting tubules (amiloride, triamterene), as well as the antagonism between spironolactone and aldosterone at the mineralocorticoid receptor level, suggest that this competition might also be produced on CA II and on renal CA IV, which, in turn, could be influenced by pH-induced changes, the binding of the diuretic to its membrane receptor as well as the activity of the brush membrane or cytosolic pump. Furosemide and indapamide, diuretics known to have vasodilating effects, induce the fall of blood pressure that parallels the decrease of CA I activity. These results show the involvement of CA in the mechanism of action of the diuretics and in their actions associated with vasodilating effects. pH changes resulting from the action of CA contribute to the action of diuretics. All diuretics inhibit CA isozymes.

A new class of carbonic anhydrase inhibitor.

Aliphatic sulfonamides, as CH3SO2NH2, are very weak inhibitors of the carbonic anhydrases (KI congruent to 10(-4) M) and are extremely weak acids (Ka congruent to 10(-10.5) M). We now find CF3SO2NH2 a very potent inhibitor of carbonic anhydrase II (KI = 2 x 10(-9) M) and a much stronger acid (Ka = 10(-5.8) M). It freely dissolves in water, a 2% solution yielding pH 3.7, the strongest known sulfonamide acid. CHCl3/aqueous partition with this solution is very low, 0.006. The plot of CH3SO2NH2 and eight hydrophilic halo-alkyl congeners gives a linear relation over 5 orders of magnitude, pKI increasing as pKa declines. Transcorneal permeability of CF3SO2NH2 in rabbits is very high; from one drop on the cornea it rapidly gains access to the ciliary process, where it fully inhibits carbonic anhydrase and gives the maximum pressure drop (for any drug) of 6 mm Hg at 30-60 min. Action is short due to rapid disappearance of free drug from the eye at the rate of aqueous humor flow. Analyses are made of binding of CF3SO2NH2 to carbonic anhydrase and melanin in ciliary process. CF3SO2NH2 is not attacked by glutathione, is 75% bound to plasma protein, and is not taken into the renal secretory system. Excretion rate follows from glomerular filtration and tubular reabsorption. Intravenous injection thus leads to prolonged plasma levels (half-life, 24 h), a general diffusion into tissues and alkalinization of the urine, as with "classic" inhibitors. Drug is bound to carbonic anhydrase in red cells and decays with half-life of 4 days. The rapid and effective binding to carbonic anhydrase of this small hydrophilic molecule shows that complex lipophilic structures are not necessary for powerful inhibition of the enzyme. It does not appear essential for a sulfonamide to occupy a "hydrophobic pocket" in the active site cavity to react effectively at the zinc center.