Human CAII Research Articles

Effects of Phosphorylation on the Activity, Inhibition and Stability of Carbonic Anhydrases.

Carbonic anhydrases (CAs) are a metalloenzyme family that have important roles in cellular processes including pH homeostasis and have been implicated in multiple pathological conditions. Small molecule inhibitors have been developed to target carbonic anhydrases, but the effects of post-translational modifications (PTMs) on the activity and inhibition profiles of these enzymes remain unclear. Here, we investigate the effects of phosphorylation, the most prevalent carbonic anhydrase PTM, on the activities and drug-binding affinities of human CAI and CAII, two heavily modified active isozymes. Using serine to glutamic acid (S > E) mutations to mimic the effect of phosphorylation, we demonstrate that phosphomimics at a single site can significantly increase or decrease the catalytic efficiencies of CAs, depending on both the position of the modification and the CA isoform. We also show that the S > E mutation at Ser50 of hCAII decreases the binding affinities of hCAII with well-characterized sulphonamide inhibitors including by over 800-fold for acetazolamide. Our findings suggest that CA phosphorylation may serve as a regulatory mechanism for enzymatic activity, and affect the binding affinity and specificity of small, drug and drug-like molecules. This work should motivate future studies examining the PTM-modification forms of CAs and their distributions, which should provide insights into CA physiopathological functions and facilitate the development of 'modform-specific' carbonic anhydrase inhibitors.

Efficient Synthesis with Green Chemistry Approach of Novel Pharmacophores of Imidazole-Based Hybrids for Tumor Treatment: Mechanistic Insights from In Situ to In Silico.

Simple SummaryHere, we report the eco-friendly synthesis and antitumor potential of the imidazole hybrids of pyrimidine. The results showed that all the compounds possess excellent inhibition of tumors, promoting enzymes hCA-IX and hCA-II. Furthermore, the selectivity index showed that compounds 7, 10, and 11 are selective inhibitors of hCA-IX, while compound 2 is a selective inhibitor of hCA-IX. More importantly, all the active inhibitors are toxic to the breast cancer cell line and non-cytotoxic for the normal breast cell line. These compounds would be a suitable choice to investigate in the in vivo models to check their efficacy against these particular targets. These newly identified human carbonic anhydrase inhibitors have potential to be considered as therapeutic leads for the treatment of CA-related diseases, especially for breast and lung tumors and glaucoma. Furthermore, lead optimization and preclinical and clinical investigations of these compounds are necessary to develop potential drug entities for the treatment of cancer.Imidazole-based pyrimidine hybrids are considered a remarkable class of compounds in pharmaceutical chemistry. Here, we report the anticancer bioactivities of eleven imidazole-based pyrimidine hybrids (1–11) that specifically target cytosolic carbonic anhydrase (CAs) isoenzymes, including human CA-II and human CA-IX (hCA-II, and hCA-IX). A highly eco-friendly aqueous approach was used for the formation of a carbon–carbon bond by reacting aromatic nitro group substitution of nitroimidazoles with carbon nucleophiles. The in vitro results indicate that this new class of compounds (1–11) includes significant inhibitors of hCA IX with IC50 values in the range of 9.6 ± 0.2–32.2 ± 1.0 µM, while hCA II showed IC50 values in range of 11.6 ± 0.2–31.1 ± 1.3 µM. Compound 2 (IC50 = 12.3 ± 0.1 µM) showed selective inhibition for hCA-II while 7, 8, and 10 (IC50 = 9.6–32.2 µM) were selective for hCA-IX. The mechanism of action was investigated through in vitro kinetics studies that revealed that compounds 7, 3, 11, 10, 4, and 9 for CA-IX and 1, 2, and 11 for CA-II are competitive inhibitors with dissociation constant (Ki) in the range of 7.32–17.02 µM. Furthermore, the in situ cytotoxicity of these compounds was investigated in the human breast cancer cell line MDA-MB-231 and compared with the normal human breast cell line, MCF-10A. Compound 5 showed excellent anticancer/cytotoxic activity in MDA-MB-231 with no toxicity to the normal breast cells. In addition, in silico molecular docking was employed to predict the binding mechanism of active compounds with their targets. This in silico observation aligned with our experimental results. Our findings signify that imidazole-based hybrids could be a useful choice to design anticancer agents for breast and lung tumors, or antiglaucoma compounds, by specific inhibition of carbonic anhydrases.

In vitro Inhibition Profiles and Molecular Docking Analysis of Benzohydrazide Derivatives on Red Blood Cell Carbonic Anhydrases Isozymes.

Carbonic anhydrases (CAs, EC 4.2.1.1) are metalloenzymes that contain zinc ions on the active side and convert carbon dioxide to bicarbonate in metabolism. Human CA-I and CA-II, which are the most abundant CA isozymes in erythrocytes, have been therapeutic targets in the treatment of glaucoma, hypertension, ulcer, osteoporosis, and, neurological disorders. Benzohydrazides are biologically active compounds, and their various pharmacological effects have been reported. In light of this, the objective of this study was to investigate the in vitro effects of benzohydrazide derivatives on the activities of hCA-I and hCA-II, determine the compounds as selective inhibitors for these isoenzymes, and estimate the inhibition mechanism through molecular docking studies. In this work, we synthesized the 10 different derivatives of benzohydrazide containing various functional group of different positions. As a result, all benzohydrazide derivatives inhibited both isozymes in vitro and 2-amino 3- nitro benzohydrazide (10) was found to be the most efficient inhibitor of both hCA isozymes with the IC50 values of 0.030 and 0.047 μM, respectively. In the molecular docking studies, 3-amino 2- methyl benzohydrazide (3) had the lowest estimated free binding energies against hCA isozymes as -6.43 and -6.13 kcal/mol. In this study, hCA-I & II isozymes were isolate from human erythrocytes. CA isozymes are one of these target enzymes. WBC hope that the benzohydrazide derivatives, can guide remedies targeting carbonic anhydrase.

In vitro and in silico interactions of antiulcer, glucocorticoids and urological drugs on human carbonic anhydrase I and II isozymes.

Carbonic anhydrases (CAs, Enzyme Commission 4.2.1.1) convert carbon dioxide to bicarbonate in metabolism and use Zn2+ ions as a cofactor for their catalytic activity. The activators or inhibitors of CA-I and CA-II, which are the most abundant CA isozymes in erythrocytes, have pharmacological applications in medicine. So, investigation of drug-protein interaction of these isozymes is significant. On this basis, the objective of this study was to clarify the primer effects of widely used drugs on the activity of human CA-I and CA-II enzymes and elucidate the inhibition mechanism through molecular docking studies. For this aim isozymes were purified from human erythrocytes by affinity chromatography technique. Then inhibition profiles of antiulcer, glucocorticoids, and urological drugs were investigated. As a result, while budesonide had the highest inhibitory potency on hydratase activity of hCA-I with the IC50 of 0.08mM, levofloxacin showed the highest inhibition effect on hCA-II with the IC50 of 0.886mM. The most effective inhibitor on the esterase activity of isozymes was found as fluticasone propionate with the Ki values of 0.0365±0.016mM and 0.054±0.018mM respectively. However, by molecular docking study, it was estimated that budesonide showed maximum inhibition potency for both isozymes with the free binding energy of -7.58 and -6.97kcal/mol, respectively. Consequently, it was observed that some of the drugs studied did not show any inhibitory effect. Drug-enzyme interactions were also estimated by molecular docking. This study could contribute to the discovery of new drug candidates and as well as target proteins.

Carbonic anhydrase II does not regulate nitrite-dependent nitric oxide formation and vasodilation.

Although it has been reported that bovine carbonic anhydrase CAII is capable of generating NO from nitrite, the function and mechanism of CAII in nitrite-dependent NO formation and vascular responses remain controversial. We tested the hypothesis that CAII catalyses NO formation from nitrite and contributes to nitrite-dependent inhibition of platelet activation and vasodilation. The role of CAII in enzymatic NO generation was investigated by measuring NO formation from the reaction of isolated human and bovine CAII with nitrite using NO photolysis-chemiluminescence. A CAII-deficient mouse model was used to determine the role of CAII in red blood cell mediated nitrite reduction and vasodilation. We found that the commercially available purified bovine CAII exhibited limited and non-enzymatic NO-generating reactivity in the presence of nitrite with or without addition of the CA inhibitor dorzolamide; the NO formation was eliminated with purification of the enzyme. There was no significant detectable NO production from the reaction of nitrite with recombinant human CAII. Using a CAII-deficient mouse model, there were no measurable changes in nitrite-dependent vasodilation in isolated aorta rings and in vivo in CAII-/- , CAII+/- , and wild-type mice. Moreover, deletion of the CAII gene in mice did not block nitrite reduction by red blood cells and the nitrite-NO-dependent inhibition of platelet activation. These studies suggest that human, bovine and mouse CAII are not responsible for nitrite-dependent NO formation in red blood cells, aorta, or the systemic circulation.

Activation and inhibition effects of some natural products on human cytosolic CAI and CAII

Carbonic anhydrases (CAs) play a significant function in diverse pathological and physiological processes. Their inhibitors and activators are suitable molecules to use as a drug in the treatment of different disease. In the present study, seven natural compounds, namely didymin, retusin isoquercitrin, silymarin, verbascoside, teucroside, and 3′-O-methylhypolaetin 7-O-[6′′′-O-acetyl-β-D-allopyranosyl-(1→2)]-6′′-O-acetyl-β-D-glucopyranoside were isolated from Mentha spicata, Sideritis libanotica linearis, Platanus orientalis, Teucrium chamaedrys subsp. chamaedrys, and Silybum marianum. The influences of compounds on the carbonic anhydrase I(hCAI) and II(hCAII) purified from human erythrocytes were tested. Five phenolic compounds acted as an inhibitor on the activity of hCAI, and IC50 values were computed between 18.16 and 172.5 μM. Isozyme hCAII is only inhibited by silymarin with an IC50 value of 43.12 μM. This isoenzyme was effectively activated by five natural compounds with AC50 values in the range of 2.98–18.53 μM. To understand the binding patterns of molecules that show activation effect against hCAII, molecular docking was done using Leadit 2.3.2 software, and calculated between −19.05 and −14.42 (kJ/mol) binding energies. Both in vitro and in silico results demonstrated that the best activators against hCAII were teucroside and isoquercitrin, with AC50 values of 2.98 and 3.17 μM, and binding energies −19.05 and −18.01 (kJ/mol), respectively. According to the ADME results, retusin demonstrated physicochemical and pharmacokinetic properties specific to the drug candidates.

Evaluation of Inhibition Effects of Some Cardiovascular Therapeutics on Human Erythrocyte Carbonic Anhydrase Isoenzymes

Abstract Carbonic anhydrase enzyme plays a vital role in metabolic events such as acid-base regulation and respiration. In our research, it is tried to determine the inhibitory influences of the cardiovascular therapeutics esmolol hydrochloride, amiodarone hydrochloride and lidocaine hydrochloride on human erythrocytes carbonic anhydrases (hCAI and II). In accordance with this purpose, carbonic anhydrase isoenzymes were purified from human erythrocytes by using affinity chromatography method. Enzyme purity was checked by SDS-PAGE electrophoresis method. After the aldose reductase enzymes were purified, the inhibitory affects of cardiovascular therapeutics on these enzymes, using esterase activity, which is the method of measuring in vitro activity, were examined. The three cardiovascular therapeutics dose-dependently decreased activity of hCAs. IC 50 values of amiodarone hydrochloride, esmolol hydrochloride and lidocaine hydrochloride were found to be, respectively, 0.91 mM, 5 mM, 5.8 mM for hCA-I and 0.41 mM, 3.5 mM, and 6.36 mM for hCA-II. Our results proved that, under in vitro conditions, cardiovascular therapeutics significantly inhibit human CA-I and II activities. So, irregular use of these medicines may cause serious adverse effects in terms of human health.

Results, meta-analysis and a first evaluation of UNOxR, the urinary nitrate-to-nitrite molar ratio, as a measure of nitrite reabsorption in experimental and clinical settings.

We recently found that renal carbonic anhydrase (CA) is involved in the reabsorption of inorganic nitrite (NO2-), an abundant reservoir of nitric oxide (NO) in tissues and cells. Impaired NO synthesis in the endothelium and decreased NO bioavailability in the circulation are considered major contributors to the development and progression of renal and cardiovascular diseases in different conditions including diabetes. Isolated human and bovine erythrocytic CAII and CAIV can convert nitrite to nitrous acid (HONO) and its anhydride N2O3 which, in the presence of thiols (RSH), are further converted to S-nitrosothiols (RSNO) and NO. Thus, CA may be responsible both for the homeostasis of nitrite and for its bioactivation to RSNO/NO. We hypothesized that enhanced excretion of nitrite in the urine may contribute to NO-related dysfunctions in the renal and cardiovascular systems, and proposed the urinary nitrate-to-nitrite molar ratio, i.e., UNOxR, as a measure of renal CA-dependent excretion of nitrite. Based on results from clinical and experimental animal studies, here, we report on a first evaluation of UNOxR. We determined UNOxR values in preterm neonates, healthy children, and adults, in children suffering from type 1 diabetes mellitus (T1DM) or Duchenne muscular dystrophy (DMD), in elderly subjects suffering from chronic rheumatic diseases, type 2 diabetes mellitus (T2DM), coronary artery disease (CAD), or peripheral arterial occlusive disease (PAOD). We also determined UNOxR values in healthy young men who ingested isosorbide dinitrate (ISDN), pentaerythrityl tetranitrate (PETN), or inorganic nitrate. In addition, we tested the utility of UNOxR in two animal models, i.e., the LEW.1AR1-iddm rat, an animal model of human T1DM, and the APOE*3-Leiden.CETP mice, a model of human dyslipidemia. Mean UNOxR values were lower in adult patients with rheumatic diseases (187) and in T2DM patients of the DALI study (74) as compared to healthy elderly adults (660) and healthy young men (1500). The intra- and inter-variabilities of UNOxR were of the order of 50% in young and elderly healthy subjects. UNOxR values were lower in black compared to white boys (314 vs. 483, P = 0.007), which is in line with reported lower NO bioavailability in black ethnicity. Mean UNOxR values were lower in DMD (424) compared to healthy (730) children, but they were higher in T1DM children (1192). ISDN (3 × 30mg) decreased stronger UNOxR compared to PETN (3 × 80mg) after 1day (P = 0.046) and after 5days (P = 0.0016) of oral administration of therapeutically equivalent doses. In healthy young men who ingested NaNO3 (0.1mmol/kg/d), UNOxR was higher than in those who ingested the same dose of NaCl (1709 vs. 369). In LEW.1AR1-iddm rats, mean UNOxR values were lower than in healthy rats (198 vs. 308) and comparable to those in APOE*3-Leiden.CETP mice (151).

GC-MS determination of nitrous anhydrase activity of bovine and human carbonic anhydrase II and IV

The most widely recognized activity of the large family of the metalloenzyme carbonic anhydrases (CAs) is the diffusion-controlled hydration of CO2 to HCO3− and one proton, and the less rapid dehydration of HCO3− to CO2: CO2 + H2O ⇆ HCO3− + H+. CAs also catalyze the reaction of water with other electrophiles such as aromatic esters, sulfates and phosphates, thus contributing to lending to CAs esterase, sulfatase and phosphatase activity, respectively. Renal CAII and CAIV are involved in the reabsorption of nitrite, the autoxidation product of the signalling molecule nitric oxide (NO): 4 NO + O2 + 2 H2O → 4 ONO− + 4 H+. Bovine and human CAII and CAIV have been reported to exert nitrite reductase and nitrous anhydride activity: 2 NO2− + 2 H+ ⇆ [2 HONO] ⇆ N2O3 + H2O. In the presence of L-cysteine, NO may be formed. In the literature, these issues are controversial, mainly due to analytical shortcomings, i.e., the inability to detect authentic HONO and N2O3. Here, we present a gas chromatography-mass spectrometry (GC-MS) assay to unambiguously detect and quantify the nitrous anhydrase activity of CAs. The assay is based on the hydrolysis of N2O3 in H218O to form ON18O− and 18ON18O−. After pentafluorobenzyl bromide derivatization and electron capture negative-ion chemical ionization of the pentafluorobenzyl nitro derivatives, quantification is performed by selected-ion monitoring of the anions with mass-to-charge (m/z) ratios of 46 (ONO−), m/z 48 (ON18O− and 18ONO−), m/z 50 (18ON18O−) and m/z 47 (O15NO−, internal standard).

Design, synthesis, in silico and biological evaluation of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazine carboxamides.

A series of novel 2-(4-(4-substituted piperazin-1-yl)benzylidene)hydrazinecarboxamide derivatives has been successfully designed and synthesized to evaluate their potential as carbonic anhydrase (CA) inhibitors. The inhibitory potential of synthesized compounds against human CAI and CAII was evaluated. Compounds 3a-n exhibited [Formula: see text] values between [Formula: see text] against CAI and [Formula: see text] against CAII. Compound 3g was the most active inhibitor, with an [Formula: see text] value of [Formula: see text] against CAII. Molecular docking studies of compound 3g with CAII showed this compound fits nicely in the active site of CAII and it interacts with the zinc ion ([Formula: see text]) along with three histidine residues in the active site. Molecular dynamics simulation studies of compound 3g complexed with CAII also showed essential interactions which were maintained up to 40ns of simulation. In vivo sub-acute toxicity study using 3g (300mg/kg) was found non-toxic in adult Wistar rats.

Some Anti-Inflammatory Agents Inhibit Esterase Activities of Human Carbonic Anhydrase Isoforms I and II: An In Vitro Study.

Carbonic anhydrases (CAs) are known as a drug-target enzymes. The inhibitors of the enzyme are important compounds for discovering new therapeutic agents and understanding in detail protein-drug interactions at the molecular level. For this purpose, the invitro effects of some anti-inflammatory agents such as tenoxicam, fluorometholone acetate, and dexamethasone were investigated on esterase activity of human erythrocyte CA-I and CA-II in this study. hCA-I and hCA-II were purified by affinity chromatography with a yield of 47.25% and 87%, and a specific activity of 642.8EU/mg proteins and 5576.9EU/mg proteins, respectively. SDS-PAGE was performed to determine the purity of the enzymes. Inhibitory effects of the drugs on hCA-I and hCA-II were determined by spectrophotometric method. IC50 values for hCA-I and hCA-II were 0.198, 2.18, 11.7, 0.11, 17.5 and 14μm using tenoxicam, fluorometholone acetate, and dexamethasone, respectively. For fluorometholone acetate and dexamethasone, Ki values from Lineweaver-Burk plots were obtained as 1.044 and 21.2μm (noncompetitive) for hCA-I and 9.98 and 8.66μm (non-competitive) for hCA-II. In conclusion, tenoxicam, fluorometholone acetate, and dexamethasone showed potent inhibitory effects on esterase activity of hCA-I and hCA-II isozymes under invitro conditions.

The effects of some tumor markers on human erythrocyte (HCA-I and HCA-II), bovine erythrocyte (BCA) and bovine lung (CA-IV) carbonic anhydrase enzyme activities in vitro.

The influence of prostatic acid phosphatase (PAP) and human chorionic gonadotropin (HCG), tumor markers have been investigated on human erythrocyte carbonic anhydrase (HCA-I and HCA-II) and bovine erythrocyte (BCA) and bovine lung carbonic anhydrase (CA-IV) in vitro. Tumor markers are substances that can often be detected in higher-than-normal amounts in the blood, urine, or body tissues of some patients with certain types of cancer. Tumor markers are produced either by the tumor itself or by the body in response to the presence of cancer or certain benign (noncancerous) conditions. In addition to their role in cancer diagnosis, some tumor marker levels are measured before treatment to help doctors plan appropriate therapy. All of the tumor markers were determined to have inhibition effect, on human CA-I, CA-II, bovine erythrocyte CA (BCA) and bovine lung CA-IV isoenzymes. The effect of each tumor marker on CA was investigated by Wilbur-Andersen method modified by Rickly et al Inhibition effects of two different tumor markers on human CA-I, CA-II, bovine erythrocyte CA (BCA) and bovine lung CA-IV isoenzymes were determined by using the CO2-Hydratase method by plotting activity % vs (tumor markers). I50 values of tumor markers exhibiting inhibition effects were found by means of these graphs (Tab.1, Fig. 2, Ref. 20).

Lack of Pathogenic Mutation in the Human CAII Gene in an Individual Suffering Renal Tubular Acidosis

Renal tubular acidosis (RTA) is a rare genetic disorder. It has four clinical types, and type 3 demonstrates a mixed pattern of tubular dysfunction. The causative gene for type 3 RTA (CAII) is located on the 8q22 locus and encodes a protein called carbonic anhydrase II. In this study, we analyzed the entire exons and flanking regions of the CAII gene in a child suffering renal tubular acidosis with an autosomal recessive pattern that was diagnosed with type3 RTA. DNA was extracted from the blood sample of the patient and his parents by the salting out extraction method. The exons and flanking regions of the CAII gene were amplified using polymerase chain reaction (PCR). We performed exon direct sequencing by forward and reverse primers, which were designed by primer3 program. No mutation was found following the screening of the entire coding sequence of the CAII gene. It is likely that another gene might be involved in this case. In other words, other types of RTA have to be considered. Keywords : Renal Tubular Acidosis, distal, type 3; Gene; Polymerase Chain Reaction; Sequence Analysis, DNA.

Carbonic anhydrase inhibition. Insight into the characteristics of zonisamide, topiramate, and the sulfamide cognate of topiramate

Some useful therapeutic agents inhibit certain carbonic anhydrase (CA) isozymes to varying degrees. We have conducted enzyme kinetics studies in a 4-nitrophenyl acetate (4-NPA) hydrolysis assay with the marketed antiepileptic drugs topiramate (1) and zonisamide (2) to determine if their full inhibition of human CA-II and CA-I requires extended preincubation conditions. We found that neither 1 nor 2 requires appreciable preincubation with either enzyme to manifest full inhibitory activity. We also examined the sulfamide cognate of topiramate (3) to characterize its CA inhibitory activity, and confirmed that it is a very weak inhibitor, unlike 1 or 2. In a CO2 hydration assay, 3 behaved as a very weak, partial inhibitor of CA-II and CA-I. We conclude that topiramate (1), zonisamide (2), and sulfamide 3 do not require extended exposure to human CA-I or CA-II to manifest full inhibitory activity (4-NPA assay).

Structural Features that Govern Enzymatic Activity in Carbonic Anhydrase from a Low-Temperature Adapted Fish, Chionodraco hamatus

The carbonic anhydrase (CA) family of zinc metalloenzymes includes many known isozymes that have different subcellular distributions. The study described here focuses on identification of the structural features that define low-temperature adaptation in a Chionodraco hamatus protein, both for the reaction center, at an atomic level, and for the tertiary structure of the protein. To this aim, an x-ray absorption near-edge spectroscopy/Minuit x-ray absorption near-edge spectroscopy analysis of the reaction center was undertaken for both a structurally characterized human CAII and CA of C. hamatus. Higher structural levels were analyzed by sequence comparison and homology modeling. To establish whether the structural insights acquired in fish CAs are general, theoretical models were generated by homology modeling for three temperate-climate-adapted fish CAs. The measured structural differences between the two proteins are discussed in terms of the differences in the electrostatic potential between human CAII and CA of C. hamatus. We conclude that modulation of the interaction between the catalytic water molecule and the zinc ion could depend on the effect of the electrostatic potential distribution.

Development of Quantitative Structure-Activity Relationship for a Set of Carbonic Anhydrase Inhibitors: Use of Quantum and Chemical Descriptors

A set of 24 descriptors consisting of quantum and chemical descriptors have been used to model binding constant (logK) of the benzene sulfonamides to human CAII. Simple as well as multiple regression have indicated that MNC (most negative charge) is the most dominating parameter to be used in modeling log K. Excellent results are obtained in multi-parametric regression. The results are critically discussed using a variety of statistics, which indicated that the hydrophobic term (log P) is not essential to yield excellent models. Keywords: QSAR, Quantum parameter, Hydrophobicity, Regression analysis, Chemical parameter

A distinct carbonic anhydrase in the mucus of the colon of humans and other mammals

We have collected gastrointestinal, mainly colonic, mucus from humans, guinea pigs, rats, and normal and carbonic anhydrase II (CAII)-deficient mice. In the mucus of all species, substantial CA activity was present. Using antibodies against human CA isoforms we found that the human mucus CA differs from cytosolic CAI and CAII, membrane-bound CAIV, and the secreted CAVI of saliva. The high sensitivity of mucus CA to acetazolamide rules out its identity with cytosolic CAIII. Partial sequences obtained from the purified human mucus CA show similarity, but not identity, with human CAI, and clear differences from the other known CAs. Additional evidence concerning the CA isoform present in mucus was obtained for the mucus CA of other species and was derived from: (1) the mucus of CAII-deficient mice, whose high CA activity confirms that it is not CAII that is responsible; (2) the inhibitory effect of iodide, which shows that mucus CA from mice, guinea pig and humans does not have the high anion sensitivity of CAI; (3) the inactivating effect of 0.2% SDS on guinea pig, mouse and human mucus CA, ruling out the SDS-resistant CAIV; and (4) the partitioning of guinea-pig mucus CA into the water phase in Triton X114 phase separation experiments, which also argues against its identity with membrane-bound CAs, such as CAIV. A comparison of colonic mucus CA activity in normal and germ-free rats indicates that the mucus CA is not of bacterial origin but is produced by the gastrointestinal tissues. We conclude (from its immunoreactivity, from iodide inhibition and from partial amino acid sequences) that mucus CA of human origin probably represents an isozyme, which is specific for mucus and is not identical with the known CA isozymes. The results obtained for mucus CA of other species collectively point in the same direction.

Plasma and whole blood pharmacokinetics of topiramate: the role of carbonic anhydrase

Topiramate (TPM) is a broad-spectrum antiepileptic drug with various mechanisms of action including an inhibitory effect on some isozymes of carbonic anhydrase (CA). Binding to CA-I and CA-II, which are highly concentrated in erythrocytes, may affect drug pharmacokinetics. Consequently, the objectives of this study were: (a) to comparatively assess TPM pharmacokinetics in healthy subjects, based on plasma and whole blood data, by simultaneously measuring TPM concentrations in plasma and whole blood following different therapeutic doses; (b) to rigorously establish the affinity of TPM for CA-I and CA-II in order to gain insight into how binding to these isozymes in erythrocytes influences TPM pharmacokinetics. TPM (100, 200 and 400 mg, single dose) was given in a randomized three-way crossover design to 27 healthy subjects and the drug concentrations in plasma and whole blood were simultaneously measured for 168 h after dosing. The pharmacokinetics of TPM in plasma was linear, but TPM clearance from whole blood increased with increasing dose. At low therapeutic concentrations, the blood-to-plasma ratio for TPM decreased from 8 to 2 as its concentration increased, indicating a substantial and saturable binding of TPM to erythrocytes. The kinetics (dissociation binding constant – K d and maximum binding rate – B max) of the binding of TPM to erythrocytes was determined from the measured concentrations of TPM in whole blood and plasma. This analysis indicated the existence of two binding sites with K d values of 0.54 and 140 μM, and B max values of 22 and 124 μmol/L of erythrocyte volume, respectively. These B max values are similar to literature values for the molar concentration of human CA-II (14–25 μmol/L) and CA-I (115–125 μmol/L). TPM inhibition constant ( K i) values for the inhibition of purified human CA obtained using assays based on CO 2 hydration or 4-nitrophenylacetate hydrolysis were 0.62 and 0.49 μM for CA-II, and 91 and 93 μM for CA-I. The results of these studies indicate that virtually all of the binding of TPM to erythrocytes is attributable to CA-I and CA-II. Because CA-I and CA-II are highly concentrated in erythrocytes, a large portion of TPM in whole blood is bound and serves as a depot. This contributes to the lower oral clearance (CL/ F), apparent volume of distribution (Vss/ F) and longer half-life ( t 1/2) that TPM has in blood compared to the CL/ F, Vss/ F and t 1/2, estimated from plasma data. The difference between TPM blood and plasma pharmacokinetics was more profound at low doses (≤100 mg/day).

QSAR studies on benzene sulfonamide carbonic anhydrase inhibitors: need of hydrophobic parameter for topological modeling of binding constants of sulfonamides to human CA-II

The binding constants (log K) of benzene sulfonamides to human CA-II have been modeled using a large series of distance-based topological indices. The need or otherwise of the hydrophobic parameter (log P) for such topological modeling of log K has been examined critically. In both the cases excellent results have been obtained. In multiparametric models involving indicator parameters we observed that introduction of hydrophobic parameter (log P) yields much improved statistics. The results are discussed on the basis of statistical parameters and also by using cross-validation method.

Complementation of the yeast deletion mutant DeltaNCE103 by members of the beta class of carbonic anhydrases is dependent on carbonic anhydrase activity rather than on antioxidant activity.

In recent years, members of the beta class of CAs (carbonic anhydrases) have been shown to complement Delta NCE103, a yeast strain unable to grow under aerobic conditions. The activity required for complementation of Delta NCE103 by tobacco chloroplast CA was studied by site-directed mutagenesis. E196A (Glu196-->Ala), a mutated tobacco CA with low levels of CA activity, complemented Delta NCE103. To determine whether restoration of Delta NCE103 was due to residual levels of CA activity or whether it was related to previously proposed antioxidant activity of CAs [Götz, Gnann and Zimmermann (1999) Yeast 15, 855-864], additional complementation analysis was performed using human CAII, an alpha CA structurally unrelated to the beta class of CAs to which the tobacco protein belongs. Human CAII complemented Delta NCE103, strongly arguing that CA activity is responsible for the complementation of Delta NCE103. Consistent with this conclusion, recombinant NCE103 synthesized in Escherichia coli shows CA activity, and Delta NCE103 expressing the tobacco chloroplast CA exhibits the same sensitivity to H2O2 as the wild-type strain.