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l-Carnosine (&beta, Ala-l-His) and several other histidine-containing peptides, including two N-methylated forms on the imidazole ring (l-anserine and l-balenine), two derivatives modified on the carboxyl function (carcinine and l-carnosinamide), two analogues differing in the length of the N-terminal residue (l-homocarnosine and Gly-l-His) and the N-acetyl derivatives, were investigated as activators of four isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The four human isoforms hCA I, II, VA and IX were activated in the low to high micromolar range, with a rather complex structure activity relationship. A performed computational study allowed us to rationalize these results and to propose a binding mode of these activators within the enzyme active site. Similarly to other CA activators, the here studied peptides could find relevant pharmacological applications such as in the management of CA deficiencies, for therapy memory and enhancing cognition or for artificial tissues engineering.

Obesity is a metabolic syndrome leading to several health problems such as hypertension, heart attack, type II diabetes, and even cancer. Carbonic anhydrase VA (CAVA) is a mitochondrial enzyme which is directly associated with the glucose homeostasis and considered as a promising target for obesity and other associated diseases in humans. So far, numerous inhibitors have been designed to inhibit the catalytic activity of CAVA with an assumption for its possible therapeutic uses against type II diabetes and other metabolic diseases. Among these, sulphonamide inhibitors and various non-classical inhibitors are extensively used. The focus of this review is to understand the mechanism and role CAVA in glucose homeostasis to ascertain as a potential drug target of obesity. We have further highlighted different types of inhibitors and their mode of binding and possible consequences with an aim to investigate possible therapeutic used for the treatment of obesity and associated diseases. Along with classical inhibitors, various non-classical inhibitors have proved to be potential inhibitors of CAV which may be employed to combat obesity. Certain phytochemicals are utilized as therapeutic molecules to fight obesity. These phytochemicals have been discussed in detail here.

A 7-day-old female neonate presents to a local hospital with lethargy, grunting, fast breathing, and abnormal movements after a short history of poor feeding overnight. She was born at 39 weeks’ gestation via cesarean section with a birthweight of 3,100 g to a 34-year-old woman. The parents are nonconsaguineous and the pregnancy was a result of in vitro fertilization with their own gametes. Antenatal screening and scans were normal. Cesarean section was performed because the labor did not progress; the neonate did not need any resuscitation. She nursed with the mother and went home breastfeeding on day 3 after birth. There is no family history of early neonatal deaths or neurologic dysfunction. The neonate is brought to our tertiary neonatal center after stabilization with fluid bolus, broad-spectrum antibiotics, levetiracetam for suspected seizures, and mechanical ventilation. Initial differential diagnosis includes late-onset sepsis with meningitis and inborn error of metabolism with encephalopathy. Examination shows no dysmorphic features or neurocutaneous markers, and the head circumference is 32.6 cm. The infant is stuporous with paucity of spontaneous movements, depressed neonatal reflexes, and hypotonia. She is well perfused with normal heart sounds and femoral pulses and has no oxygen requirement. There is no pallor or jaundice and there is no organomegaly. Arterial …

Obesity is quickly becoming an increasing problem in the developed world. One of the major fundamental causes of obesity and diabetes is mitochondria dysfunction due to faulty metabolic pathways which alter the metabolic substrate flux resulting in the development of these diseases. This paper examines the role of mitochondrial carbonic anhydrase (CA) isozymes in the metabolism of pyruvate, acetate, and succinate when specific isozyme inhibitors are present. Using a sensitive electrochemical approach of wired mitochondria to analytically measure metabolic energy conversion, we determine the resulting metabolic difference after addition of an inhibitory compound. We found that certain sulfonamide analogues displayed broad spectrum inhibition of metabolism, where others only had significant effect on some metabolic pathways. Pyruvate metabolism always displayed the most dramatically affected metabolism by the sulfonamides followed by fatty acid metabolism, and then finally succinate metabolism. This allows for the possibility of using designed sulfonamide analogues to target specific mitochondrial CA isozymes in order to subtly shift metabolism and glucogenesis flux to treat obesity and diabetes.

Events responsible for cerebrovascular disease in diabetes are not fully understood. Pericyte loss is an early event that leads to endothelial cell death, microaneurysms, and cognitive impairment. A biochemical mechanism underlying pericyte loss is rapid respiration (oxidative metabolism of glucose). This escalation in respiration results from free influx of glucose into insulin-insensitive tissues in the face of high glucose levels in the blood. Rapid respiration generates superoxide, the precursor to all reactive oxygen species (ROS), and results in pericyte death. Respiration is regulated by carbonic anhydrases (CAs) VA and VB, the two isozymes expressed in mitochondria, and their pharmacologic inhibition with topiramate reduces respiration, ROS, and pericyte death. Topiramate inhibits both isozymes; therefore, in the earlier studies, their individual roles were not discerned. In a recent genetic study, we showed that mitochondrial CA VA plays a significant role in regulation of reactive oxygen species and pericyte death. The role of CA VB was not addressed. In this report, genetic knockdown and overexpression studies confirm that mitochondrial CA VA regulates respiration in pericytes, whereas mitochondrial CA VB does not contribute significantly. Identification of mitochondrial CA VA as a sole regulator of respiration provides a specific target to develop new drugs with fewer side effects that may be better tolerated and can protect the brain from diabetic injury. Since similar events occur in the capillary beds of other insulin-insensitive tissues such as the eye and kidney, these drugs may also slow the onset and progression of diabetic disease in these tissues.

We sought to validate global microarray results indicating the differential expression of 383 genes in Peripheral Blood Mononuclear Cells (PBMCs) from patients with pancreatic cancer (PC) and to further evaluate their PC diagnostic potential.In total, 177 patients were recruited (47 healthy controls (HC), 35 chronic pancreatitis (CP) patients, and 95 PC patients). PBMC expressions of six genes from our previous study (ANXA3, ARG1, CA5B, F5, SSBP2, and TBC1D8) along with four new genes (MIC1, NGAL, MUC1, and MUC16) were analyzed using multiplex Q-RT PCR.Differential expressions of 5 of the 6 genes previously identified by PBMC microarray were validated in this study. Multivariate models for PBMC gene expression were attempted to determine if any combination was diagnostically superior to CA19-9 alone. We found that addition of PBMC CA5B, F5, SSBP2, and MIC1 expression levels to CA19-9 significantly improved CA19-9's diagnostic abilities when comparing resectable PC to CP patients (p=0.023).Results of our previous study were validated, indicating reproducibility of PC-associated PBMC expression profiling. We identified a score-based model that can differentiate resectable PC from CP better than CA19-9, potentiating that PBMC differential expression analysis may offer a novel tool for early PC diagnosis.

Diabetes mellitus causes cerebral microvasculature deterioration and cognitive decline. The specialized endothelial cells of cerebral microvasculature comprise the blood-brain barrier, and the pericytes (PC) that are in immediate contact with these endothelial cells are vital for blood-brain barrier integrity. In diabetes, increased mitochondrial oxidative stress is implicated as a mechanism for hyperglycemia-induced PC loss as a prerequisite leading to blood-brain barrier disruption. Mitochondrial carbonic anhydrases (CA) regulate the oxidative metabolism of glucose and thus play an important role in the generation of reactive oxygen species and oxidative stress. We hypothesize that the inhibition of mitochondrial CA would reduce mitochondrial oxidative stress, rescue cerebral PC loss caused by diabetes-induced oxidative stress, and preserve blood-brain barrier integrity. We studied the effects of pharmacological inhibition of mitochondrial CA activity on streptozotocin-diabetes-induced oxidative stress and PC loss in the mouse brain. At 3 wk of diabetes, there was significant oxidative stress; the levels of reduced glutathione were lower and those of 3-nitrotyrosine, 4-hydroxy-2-trans-nonenal, and superoxide dismutase were higher. Treatment of diabetic mice with topiramate, a potent mitochondrial CA inhibitor, prevented the oxidative stress caused by 3 wk of diabetes. A significant decline in cerebral PC numbers, at 12 wk of diabetes, was also rescued by topiramate treatment. These results provide the first evidence that inhibition of mitochondrial CA activity reduces diabetes-induced oxidative stress in the mouse brain and rescues cerebral PC dropout. Thus, mitochondrial CA may provide a new therapeutic target for oxidative stress related illnesses of the central nervous system.

Mitochondrial carbonic anhydrase VA (CAVA) catalyzes the hydration of carbon dioxide to produce proton and bicarbonate which is primarily expressed in the mitochondrial matrix of liver, and involved in numerous physiological processes including lipogenesis, insulin secretion from pancreatic cells, ureagenesis, gluconeogenesis, and neuronal transmission. To understand the effect of pH on the structure, function, and stability of CAVA, we employed spectroscopic techniques such as circular dichroism, fluorescence, and absorbance measurements in wide range of pH (from pH 2.0 to pH 11.5). CAVA showed an aggregation at acidic pH range from pH 2.0 to pH 5.0. However, it remains stable and maintains its secondary structure in the pH range, pH 7.0-pH 11.5. Furthermore, this enzyme has an appreciable activity at more than pH 7.0 (7.0

An inhibition study of the human cytosolic isozymes I, and II, the mitochondrial isoform VA, and the tumor-associated, transmembrane isozyme IX of carbonic anhydrase (CA, EC 4.2.1.1) with a library of aromatic/heteroaromatic/polycyclic difluoromethanesulfonamides is reported. Most of the inhibitors were derivatives of benzenedifluoromethanesulfonamide incorporating substituted-phenyl moieties, or were methylsulfonamide and difluoromethyl-sulfonamide derivatives of the sulfamates COUMATE and EMATE, respectively. Except for the methylsulfonamide-COUMATE derivative which behaved as a potent CA II inhibitor (KI of 32 nM), these sulfonamides were moderate inhibitors of all isozymes, with inhibition constants in the range of 96–5200 nM against hCA I, of 80–670 nM against hCA II, and of 195–9280 nM against hCA IX, respectively. Remarkably, some derivatives, such as 3-bromophenyl-difluoromethanesulfonamide, showed a trend to selectively inhibit the mitochondrial isoform CA VA, showing selectivity ratios for inhibiting CA VA over CA II of 3.53; over CA I of 6.84 and over CA IX of 9.34, respectively, although it is a moderate inhibitor (KI of 160 nM). Some of these derivatives may be considered as leads for the design of isozyme selective CA inhibitors targeting the mitochondrial isozyme CA VA, with potential use as anti-obesity agents.

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes; the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V, and the tumor-associated, transmembrane hCA IX, with anions isosteric and isoelectronic with sulfate, nitrate, and carbonate; such as chlorate, perchlorate, bromate, iodate, periodate, silicate, bismuthate, vanadate, molybdate, and wolframate is reported. Apparently, the geometry of the inhibitor (tetrahedral or trigonal) does not influence its binding to the Zn(II) ion of the enzyme active site, but the nature of the central element is the most important factor influencing potency. Isozymes hCA I and II are best inhibited by chlorate, perchlorate, and silicate, together with the anions structurally related to sulfate, sulfamate, and sulfamidate, but sulfate itself is a weak inhibitor (inhibition constant of 74 mM against hCA I and 183 mM against hCA II). Molybdate is a very weak hCA I inhibitor (K(I) of 914 mM) but it interacts with hCA II (K(I) of 27.5mM). Isozyme IV is well inhibited by sulfate (K(I) of 9 mM), sulfamate, and sulfamidate (in the low micromolar range), but not by perchlorate (K(I) of 767 mM). The mitochondrial isozyme V has the lowest affinity for sulfate (K(I) of 680 mM) and carbonate (K(I) of 95 mM) among all the investigated isozymes, suggesting on one hand its possible participation in metabolon(s) with sulfate anion exchanger(s), and on the other hand an evolutionary adaptation to working at higher pH values (around 8.5 in mitochondria) where rather high amounts of carbonate in equilibrium with bicarbonate may be present. Metasilicate, isosteric to carbonate, is also about a 10 times weaker inhibitor of this isozyme as compared to other CAs investigated here (K(I) of 28.2 mM). Surprisingly, the tumor-associated isozyme IX is resistant to sulfate inhibition (K(I) of 154 mM) but has affinity in the low micromolar range for carbonate, sulfamate, and sulfamidate (K(I) in the range of 8.6-9.6 microM). This constitutes another proof that this isozyme best works at acidic pH values present in tumors, being inhibited substantially at higher pH values when more carbonate may be present. Bromate and chlorate are quite weak CA IX inhibitors (K(I) s of 147-274 mM).

The crystal structure of F65A/Y131C murine alpha-carbonic anhydrase V (CAV), covalently modified at cysteine residues with 4-chloromethylimidazole, is reported at 1.88 A resolution. This modification introduces a methylimidazole (MI) group at residue C131 in the active site with important consequences. F65A/Y131C-MI CAV exhibits an up to 3-fold enhancement of catalytic activity over that of wild-type CAV [Earnhardt, J. N., Wright, S. K., Qian, M., Tu, C., Laipis, P. J., Viola, R. E., and Silverman, D. N. (1999) Arch. Biochem. Biophys. 361, 264-270]. In this modified CAV variant, C131-MI acts as a proton shuttle, facilitating the deprotonation of a zinc-bound water molecule to regenerate the nucleophilic zinc-bound hydroxide ion. A network of three hydrogen-bonded water molecules, across which proton transfer likely proceeds, bridges the zinc-bound water molecule and the C131-MI imidazole group. The structure of F65A/Y131C-MI CAV is compared to structures of Y64H/F65A murine CAV, wild-type human alpha-carbonic anhydrase II, and the gamma-carbonic anhydrase from Methanosarcina thermophilain an effort to outline common features of catalytic proton shuttles.

The dehydration of bicarbonate catalyzed by carbonic anhydrase is accompanied by the transfer of a proton from solution to the zinc-bound hydroxide. We have investigated the properties of proton transfer from donors in solution, mostly derivatives of imidazole and pyridine, to a truncated mutant of carbonic anhydrase V with replacements that render the active site cavity less sterically constrained, Tyr 64 →> Ala and Phe 65 →> Ala. Catalysis was measured by determining the rate of exchange of 18O between the CO2-HCO3- system and water, and rate constants for proton transfer were estimated as the rate-limiting step in the release of H218O from the enzyme to solution. Each proton donor enhanced catalytic activity in a saturable manner. The resulting rate constants for proton transfer when compared with the values of pKa of the donor and acceptor gave a Brønsted plot of high curvature. These data could also be described by Marcus theory which showed an intrinsic barrier for intermolecular proton transfer near 0.8 kcal/mol and a work term or thermodynamic contribution to the free energy of reaction near 10 kcal/mol. This low intrinsic kinetic barrier for proton transfer is very similar to nonenzymic bimolecular proton transfer between nitrogen and oxygen acids and bases in solution. However, the significant thermodynamic contribution suggests appreciable involvement of solvent and active-site organization prior to proton transfer. These Marcus parameters are very similar to those describing intramolecular proton transfer from His 64 in carbonic anhydrase, suggesting similarities in the intra- and intermolecular proton transfer processes.Key words: carbonic anhydrase, proton transfer, Marcus theory, carbon dioxide.

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.

The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5) mutation, collapse of fins (cof), which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders.

This paper presents result of quantitative structure-activity relationships (QSAR) study realized with the PRECLAV, omega, brood and MOPAC software. The dependent property is the inhibitory activity against human carbonic anhydrase mitochondrial isoforms VA and VB. The calibration set includes 17 aromatic/heterocyclic sulphonamides incorporating phenacetyl, pyridylacetyl and thienylacetyl tails with three clinically used CA inhibitors namely AZA, TPM and ZNS molecules. The prediction set contains 24 others not yet synthesized substituted aromatic/heterocyclic sulphonamides having unknown observed values of activity. In the presence of prediction set, the predictive quality of QSAR of hCA VA (r(2) = 0.9789, F = 418.115, r(2)(CV) = 0.9689) and hCA VB (r(2) = 0.9768; F = 379.717; r(2)(CV) = 0.9637) is large. The obtained models suggest a slightly different inhibition mechanism for the two isoforms. Large percentage, in weight, of CONH molecular fragments seems to be favourable to inhibitory activity of both VA and VB.

A library of 10 novel benzenesulfonamides containing triazole-tethered phenyl 'tail' moieties were synthesized by a Cu(I) catalyzed 1,3-dipolar cycloaddition reaction (DCR) (i.e., click chemistry) between 4-azido benzenesulfonamide and a panel of variously substituted phenyl acetylenes. These compounds were very effective inhibitors (low nanomolar) of the human mitochondrial carbonic anhydrase isozymes VA and VB. Mitochondrial carbonic anhydrases are potential targets for anti-obesity therapies, acting to reduce lipogenesis through a novel mechanism of action. The inhibitors reported here should prove valuable as lead compounds to further investigate the potential of CA inhibition for this novel therapeutic application.

A series of 2-substituted-1,3,4-thiadiazole-5-sulfamides was prepared and assayed as inhibitors of several carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic CA I and II, the membrane-associated CA IV and the mitochondrial CA VA and VB. The new compounds showed weak inhibitory activity against hCA I (K(I)s of 102 nM-7.42 microM), hCA II (K(I)s of 0.54-7.42 microM) and hCA IV (K(I)s of 4.32-10.05 microM) but were low nanomolar inhibitors of hCA VA and hCA VB, with inhibition constants in the range of 4.2-32 nM and 1.3-74 nM, respectively. Furthermore, the selectivity ratios for inhibiting the mitochondrial enzymes over CA II were in the range of 67.5-415, making these sulfamides the first selective CA VA/VB inhibitors.

The antiepileptic drug zonisamide was considered to act as a weak inhibitor of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) (with a K(I) of 4.3 microM against the cytosolic isozyme II). Here we prove that this is not true. Indeed, testing zonisamide in the classical assay conditions of the CO2 hydrase activity of hCA II, with incubation times of enzyme and inhibitor solution of 15 min, a K(I) of 10.3 microM has been obtained. However, when the incubation between enzyme and inhibitor was prolonged to 1 h, the obtained K(I) was of 35.2 nM, of the same order of magnitude as that of the clinically used sulfonamides/sulfamates acetazolamide, methazolamide, ethoxzolamide and topiramate (K(I)s in the range of 5.4-15.4 nM). The inhibition of the human mitochondrial isozyme hCA V with these compounds has been also tested by means of a dansylamide competition binding assay, which showed zonisamide and topiramate to be effective inhibitors, with K(I)s in the range of 20.6-25.4 nM. The X-ray crystal structure of the adduct of hCA II with zonisamide has also been solved at a resolution of 1.70 A, showing that the sulfonamide moiety participates in the classical interactions with the Zn(II) ion and the residues Thr199 and Glu106, whereas the benzisoxazole ring is oriented toward the hydrophobic half of the active site, establishing a large number of strong van der Waals interactions (4.5 A) with residues Gln92, Val121, Phe131, Leu198, Thr200, Pro202.

The inhibition of five human carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, the membrane-bound hCA IV, the mitochondrial hCA V and the tumour associated, transmembrane hCA IX, with complex anions incorporating fluoride, chloride and cyanide, as well as B(III), Si(IV), P(V), As(V), Al(III), Fe(II), Fe(III), Pd(II), Pt(II), Pt(IV), Cu(I), Ag(I), Au(I) and Nb(V) species has been investigated. Apparently, the most important factors influencing activity of these complexes are the nature of the central metal ion/element, and its charge. Geometry of these compounds appears to be less important, since both linear, tetrahedral, octahedral as well as pentagonal bipyramidal derivatives led to effective inhibitors. However, the five isozymes showed very different affinities for these anion inhibitors. The best hCA I inhibitors were cyanide, dicyanocuprate and dicyanoaurate (K(I)s in the range of 0.5-7.7 microM), whereas the least effective were fluoride and hexafluoroarsenate. The best hCA II inhibitors were cyanide, hexafluoroferrate and tetrachloroplatinate (K(I)s in the range of 0.02-0.51 mM), whereas the most ineffective ones were fluoride, hexafluoroaluminate and chloride. The best hCA IV inhibitors were dicyanocuprate (K(I) of 9.8 microM) and hexacyanoferrate(II) (K(I) of 10.0 microM), whereas the worst ones were tetrafluoroborate and hexafluoroaluminate (K(I)s in the range of 124-126 mM). The most effective hCA V inhibitors were cyanide, heptafluoroniobate and dicyanocuprate (K(I)s in the range of 0.015-0.79 mM), whereas the most ineffective ones were fluoride, chloride and tetrafluoroborate (K(I)s in the range of 143-241 mM). The best hCA IX inhibitors were on the other hand cyanide, heptafluoroniobate and dicyanoargentate (K(I)s in the range of 4 microM-0.33 mM), whereas the worst ones were hexacyanoferrate(III) and hexacyanoferrate(II).

A detailed inhibition study of five carbonic anhydrase (CA, EC 4.2.1.1) isozymes with inorganic phosphates, carbamoyl phosphate, the antiviral phosphonate foscarnet as well as formate is reported. The cytosolic isozyme hCA I was weakly inhibited by neutral phosphate, strongly inhibited by carbamoyl phosphate (K(I) of 9.4 microM), and activated by hydrogen- and dihydrogenphosphate, foscarnet and formate (best activator foscarnet, K(A)=12 microM). The cytosolic isozyme hCA II was weakly inhibited by all the investigated anions, with carbamoyl phosphate showing a K(I) of 0.31 mM. The membrane-associated isozyme hCA IV was the most sensitive to inhibition by phosphates/phosphonates, showing a K(I) of 84 nM for PO(4)(3-), of 9.8 microM for HPO(4)(2-), and of 9.9 microM for carbamoyl phosphate. Foscarnet was the best inhibitor of this isozyme (K(I) of 0.82 mM) highly abundant in the kidneys, which may explain some of the renal side effects of the drug. The mitochondrial isozyme hCA V was weakly inhibited by all phosphates/phosphonates, except carbamoyl phosphate, which showed a K(I) of 8.5 microM. Thus, CA V cannot be the isozyme involved in the carbamoyl phosphate synthetase I biosynthetic reaction, as hypothesized earlier. Furthermore, the relative resistance of CA V to inhibition by inorganic phosphates suggests an evolutionary adaptation of this mitochondrial isozyme to the presence of high concentrations of such anions in these energy-converting organelles, where high amounts of ATP are produced by ATP synthetase, from ADP and inorganic phosphates. The transmembrane, tumor-associated isozyme hCA IX was on the other hand slightly inhibited by all these anions.

Among the 14 human isozymes of carbonic anhydrase (CA, EC 4.2.1.1) presently known, the cytosolic hCA II is the most active and plays a host of physiological functions, whereas the mitochondrial hCA V is unique due to its role in several biosynthetic reactions. An inhibition study of these isozymes with a series of sulfonamides is reported here, with the scope to detect lead molecules for the design of isozyme-specific CA inhibitors (CAIs) targeting the mitochondrial isoform. Indeed, recently it has been shown that CA V is a novel target for the drug design of anti-obesity agents among others. Compounds included in this study were mainly ortho-, meta-, and para-substituted-benzenesulfonamides, together with several halogeno-substituted sulfanilamides and disubstituted-benzene-1,3-disulfonamide derivatives. Isozyme V showed an inhibition profile with these sulfonamides different of that of hCA II. Thus, IC(50) values in the range of 80 nM to 74 microM against hCA II, and 0.78-63.7 microM against hCA V with these derivatives have been obtained. Only one compound, 2-carboxymethyl-benzenesulfonamide, was more active against hCA V over hCA II (selectivity ratio of 1.39), whereas all other derivatives investigated here were much better hCA II inhibitors (selectivity ratios CA II/CA V in the range of 0.0008-0.73) than hCA V inhibitors.

The first inhibition study of the mitochondrial isozyme carbonic anhydrase (CA) V (of murine origin) with a series of aromatic and heterocyclic sulfonamides is reported. Inhibition data of the cytosolic isozymes CA I and CA II and the membrane-bound isozyme CA IV with these inhibitors are also provided for comparison. Several low nanomolar CA V inhibitors were detected (KI values in the range of 4-15 nM), most of them belonging to the acylated sulfanilamide, ureido-benzenesulfonamide, 1,3,4-thiadiazole-2-sulfonamide, and aminobenzolamide type of compounds. The clinically used inhibitors acetazolamide, methazolamide, ethoxzolamide, dorzolamide, brinzolamide, and topiramate on the other hand were less effective CA V inhibitors, showing inhibition constants in the range of 47-63 nM. Some of the investigated sulfonamides, such as the ureido-benzenesulfonamides and the acylated sulfanilamides showed higher affinity for CA V than for the other isozymes, CA II included, which is a remarkable result, since most compounds investigated up to now inhibited the cytosolic isozyme CA II better. These results prompt us to hypothesize that the selective inhibition of CA V, or the dual inhibition of CA II and CA V, may lead to the development of novel pharmacological applications for such sulfonamides, for example in the treatment or prevention of obesity, by inhibiting CA-mediated lipogenetic processes.

In addition to sulfonamides, metal complexing anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). The first inhibition study of the mitochondrial isozyme CA V (of murine and human origin) with anions is reported here. Inhibition data of the cytosolic isozymes CA I and CA II as well as the membrane-bound isozyme CA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, nitrate, hydrosulfide, arsenate, sulfamate, and sulfamidate and so on, are also provided for comparison. Isozyme V has an inhibition profile by anions completely different to those of CA I and IV, but similar to that of hCA II, which may have interesting physiological consequences. Similarly to hCA II, the mitochondrial isozymes show micro-nanomolar affinity for sulfonamides such as sulfanilamide and acetazolamide.