Spectroscopic characterization of furosemide binding to human carbonic anhydrase II

This study reports the interaction between furosemide and human carbonic anhydrase II (hCA II) using fluorescence, UV-vis and circular dichroism (CD) spectroscopy. Fluorescence data indicated that furosemide quenches the intrinsic fluorescence of the enzyme via a static mechanism and hydrogen bonding and van der Walls interactions play the major role in the drug binding. The binding average distance between furosemide and hCA II was estimated on the basis of the theory of F?rster energy transfer. Decrease of protein surface hydrophobicity was also documented upon furosemide binding. Chemical modification of hCA II using N-bromosuccinimide indicated decrease of the number of accessible tryptophans in the presence of furosemide. CD results suggested the occurance of some alterations in α-helical content as well as tertiary structure of hCA II upon drug binding.     

Intrinsic thermodynamics of inhibitor binding to human carbonic anhydrase IX

BackgroundHuman carbonic anhydrase 9th isoform (CA IX) is an important marker of numerous cancers and is increasingly interesting as a potential anticancer drug target. Various synthetic aromatic sulfonamide-bearing compounds are being designed as potent inhibitors of CA IX. However, sulfonamide compound binding to CA IX is linked to several reactions, the deprotonation of the sulfonamide amino group and the protonation of the CA active site Zn(II)-bound hydroxide. These linked reactions significantly affect the affinities and other thermodynamic parameters such as enthalpies and entropies of binding.MethodsThe observed and intrinsic affinities of compound binding to CA IX were determined by the fluorescent thermal shift assay. The enthalpies and entropies of binding were determined by the isothermal titration calorimetry.ResultsThe pK_a of CA IX was determined to be 6.8 and the enthalpy of CA IX-Zn(II)-bound hydroxide protonation was − 24 kJ/mol. These values enabled the analysis of intrinsic thermodynamics of a library of compounds binding to CA IX. The most strongly binding compounds exhibited the intrinsic affinity of 0.01 nM and the observed affinity of 2 nM.ConclusionsThe intrinsic thermodynamic parameters of compound binding to CA IX helped to draw the compound structure to thermodynamics relationship.General significanceIt is important to distinguish the intrinsic from observed parameters of any disease target protein interaction with its inhibitors as drug candidates when drawing detailed compound structure to thermodynamics correlations.     

A nuclear-magnetic-resonance study of the binding of novel N-hydroxybenzenesulphonamide carbonic anhydrase inhibitors to native and cadmium-111-substituted carbonic anhydrase

Various ring- and nitrogen-substituted benzenesulphonamides have been prepared and tested as potential inhibitors of carbonic anhydrase. N-Methoxysulphonamides showed no inhibitory activity, as predicted by the classic work of Krebs on N-substituted inhibitors. By contrast, N-hydroxysulphonamides proved to be very effective inhibitors of carbonic anhydrase. Using 111Cd-NMR it has been possible to analyse the molecular interaction of 4-fluoro-N-hydroxybenzenesulphon[15N]amide, with 111Cd-substituted bovine carbonic anhydrase. A large cadmium-111:nitrogen-15 spin-coupling shows that this inhibitor is directly bound to the metal via its nitrogen rather than through an oxygen atom. The mode of this binding is similar to that for the unsubstituted sulphonamide inhibitor, 4-fluorobenzenesulphon[15N]amide. The 111Cd-chemical shift of the signal for the inhibited enzyme shows that the N-hydroxysulphonamide is bound as its anion. From the relative intensities of free and complexed enzyme signals it can be deduced that the cadmium enzyme complex with the N-hydroxysulphonamide has a longer life-time than that formed with the unsubstituted sulphonamide. By contrast, native zinc-containing bovine carbonic anhydrase shows similar I50 values with both of these sulphonamides. Attempts to monitor the binding using 15N-NMR were unsuccessful, possibly due to a very long relaxation time for the nitrogen nucleus in the N-hydroxysulphonamide when bound to the enzyme leading to loss of the 15N signal.     

Effect of pH on structure,function, and stability of mitochondrial carbonic anhydrase VA

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 < pH ≤ 11.5) with maximum activity at pH 9.0. The maximal values of k_cat and k_cat/K_m at pH 9.0 are 3.7?×?10⁶ s^?1 and 5.5?×?10⁷ M^?1 s^?1, respectively. However, this enzyme loses its activity in the acidic pH range. We further performed 20-ns molecular dynamics simulation of CAVA to see the dynamics at different pH values. An excellent agreement was observed between in silico and in vitro studies. This study provides an insight into the activity of CAVA in the pH range of subcellular environment.     

Effect of sulfonamides as carbonic anhydrase VA and VB inhibitors on mitochondrial metabolic energy conversion

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.     

Structural analysis of inhibitor binding to human carbonic anhydrase II.

X-ray crystal structures of carbonic anhydrase II (CAII) complexed with sulfonamide inhibitors illuminate the structural determinants of high affinity binding in the nanomolar regime. The primary binding interaction is the coordination of a primary sulfonamide group to the active site zinc ion. Secondary interactions fine-tune tight binding in regions of the active site cavity >5 A away from zinc, and this work highlights three such features: (1) advantageous conformational restraints of a bicyclic thienothiazene-6-sulfonamide-1,1-dioxide inhibitor skeleton in comparison with a monocyclic 2,5-thiophenedisulfonamide skeleton; (2) optimal substituents attached to a secondary sulfonamide group targeted to interact with hydrophobic patches defined by Phe131, Leu198, and Pro202; and (3) optimal stereochemistry and configuration at the C-4 position of bicyclic thienothiazene-6-sulfonamides; the C-4 substituent can interact with His64, the catalytic proton shuttle. Structure-activity relationships rationalize affinity trends observed during the development of brinzolamide (Azopt), the newest carbonic anhydrase inhibitor approved for the treatment of glaucoma.     

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).     

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 CO(2) 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).     

Characterization of human carbonic anhydrase XII stability and inhibitor binding

Human carbonic anhydrase isozyme XII is a transmembrane protein that is overexpressed in many human cancers. Therefore CA XII is an anticancer drug target. However, there are few compounds that specifically target CA XII. The design of specific inhibitors against CA XII relies on the detailed understanding of the thermodynamics of inhibitor binding and the structural features of the protein–inhibitor complex. To characterize the thermodynamic parameters of the binding of known sulfonamides, namely ethoxzolamide, acetazolamide and trifluoromethanesulfonamide, we used isothermal titration calorimetry and fluorescent thermal shift assay. The binding of these sulfonamides to CA XII was buffer and pH-dependent. Dissection of protonation–deprotonation reactions of both the water molecule bound to the CA XII active site and the sulfonamide group of the inhibitor yielded the intrinsic thermodynamic parameters of binding, such as binding enthalpy, entropy and Gibbs free energy. Thermal shift assay was also used to determine CA XII stabilities at various pH and in the presence of buffers and salts.     

Molecular basis for the origin of differential spectral and binding profiles of dansylamide with human carbonic anhydrase I and II

Sulfonamide derivatives serve as potent inhibitors of carbonic anhydrases (CAs), and a few such inhibitors have been currently used as drugs for the treatment of different pathogenic conditions in humans. In pursuit of designing the isozyme-specific inhibitors of human CAs, we observed that the fluorescence spectral properties and binding profiles of a fluorogenic sulfonamide derivative, 5-(dimethylamino)-1-naphthalenesulfonamide (dansylamide, DNSA), were markedly different between the recombinant forms of human carbonic anhydrase I (hCA I) and II (hCA II). The kinetic evaluation of the overall microscopic pathways for the binding of DNSA to hCA I versus hCA II revealed that the protein isomerization step served as a major determinant of the above discrepancy. Arguments are presented that the detailed structural-functional investigations of enzyme-ligand interactions may provide insights into designing the isozyme-specific inhibitors of CAs.     

An inhibitor-like binding mode of a carbonic anhydrase activator within the active site of isoform II

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor.     

Binding of human carbonic anhydrase to human hemoglobin

The ability of human carbonic anhydrases to interact with human CO-hemoglobin have been studied with the counter-current distribution technique in aqueous/aqueous biphasic systems. The experimental results show that human carbonic anhydrase II interacts with human CO-hemoglobin whereas human carbonic anhydrase I does not. THe interaction between CO-hemoglobin and carbonic anhydrase II was quantified using the theoretical model developed previously for one-to-one interacting systems. [Backman, L. and Shanbhag, V.P. (1979) J. Chromatogr. 171, 1-13]. The apparent association constant was estimated to be 4.1 x 10(5) l mol-1 at pH 8.0 and 21 degrees C for the association of carbonic anhydrase II and CO-hemoglobin.     

Molecular evolution of the carbonic anhydrase genes: Calculation of divergence time for mouse carbonic anhydrase I and II

Summary A cDNA clone in pBR322 that cross-hybridizes with a mouse carbonic anhydrase form II (CAII) probe has been sequenced and identified as mouse carbonic anhydrase form I (CAI). The 1224-base-pair clone encodes the entire 260-amino-acid protein and appears to contain an Alu-like element in the 3 untranslated region. The deduced amino acid sequence exhibits 77% homology to human CAI and contains 17 of the 20 residues that are considered unique to and invariant for all mammalian CAI isozymes. The results of a detailed comparison of the nucleic acid sequences spanning the coding regions of mouse CAI and rabbit CAI have been used to calibrate an evolutionary clock for the carbonic anhydrases (CAs). These data have been applied to a comparison of the mouse CAI and CAII nucleic acid sequences to calculate the divergence time between the two genes. The divergence-time calculation provides the first estimation of the evolutionary relationship between CAs based entirely on nucleotide sequence comparison.     

Kinetics and mechanism of anionic ligand binding to carbonic anhydrase

The kinetics of complex formation between Co(II)-carbonic anhydrase B and the anions cyanate, thiocyanate and cyanide has been studied at different pH values employing temperature-jump relaxation spectrometry. Formation of the 1:1 complex occurs via binding of the deprotonated state of the anion to an acidic state of the enzyme. The determined formation rate constants range from 10(8) to 3 X 10(9) M-1 s-1 and are two to three orders of magnitude higher than the value estimated for a ligand coordination to the central Co2+, based on a solvate substitution mechanism. These kinetic results strongly indicate that the deprotonated anion binds to an unoccupied coordination position of the protein-bound heavy metal ion in the form of an addition reaction. Upon binding of the anion, the coordination number of the Co2+ in the acidic state of the enzyme is increased from four to five. In the case of cyanide, a 2:1 anion complex is also formed. The formation rate constant is 5 X 10(5) M-1 s-1 which provides good evidence that this binding process is controlled by a solvate substitution mechanism.     

Cell-specific expression of mitochondrial carbonic anhydrase in the human and rat gastrointestinal tract.

Mitochondrial carbonic anhydrase V (CA V) in liver provides HCO3- to pyruvate carboxylase for the first step in gluconeogenesis and HCO3- to carbamyl phosphate synthetase I for the first step in ureagenesis. Because carbamyl phosphate synthetase I and ornithine transcarbamylase are also expressed in enterocytes, we tested the hypothesis that CA V is expressed in the gastrointestinal tract in addition to liver. Polyclonal rabbit antisera were raised against a polypeptide of 17 C-terminal amino acids of human CA V and against purified recombinant mouse isozyme and were used in Western blotting and immunoperoxidase staining of human and rat tissues. Immunohistochemistry showed that CA V is expressed cell-specifically in the alimentary canal mucosa from stomach to rectum. Immunoreactions for CA V were detected in the parietal cells and gastrin-producing G-cells of the stomach and in intestinal enterocytes. Western blotting of human and rat gastrointestinal tissues with isozyme-specific antibodies showed positive signals for CA V with the expected molecular mass. The findings in human tissues paralleled those in rat. The cell-specific pattern of CA V expression suggests a role for CA V in alimentary canal physiology. We propose that mitochondrial CA V participates in the detoxification of ammonia produced in the gastrointestinal tract by providing bicarbonate to carbamyl phosphate synthetase I. (J Histochem Cytochem 47:517-524, 1999)