Zinc Enzyme Research Articles

Interference of gadolinium-based contrast agents with the measurement of serum creatinine by the Jaffe reaction

Interference of gadolinium-based contrast agents with the measurement of serum creatinine by the Jaffe reaction

Molecular dynamics simulations on the critical states of the farnesyltransferase enzyme

Protein farnesyltransferase (FTase) is a particularly interesting zinc enzyme that promotes the transfer of a 15-carbons isoprenoid farnesyl group from farnesyl diphosphate (FPP) to a number of peptide substrates with a typical-CAAX motif at the carboxyl-terminus, where C represents the cysteine residue that is farnesylated. This enzyme has been the subject of great attention in anticancer research, as several proteins known to be involved in human cancer development are thought to serve as substrates for FTase and to require farnesylation for proper biological activity. Several FTase inhibitors have advanced into clinical testing. However, despite the progress in the field several functional and mechanistic doubts on the FTase catalytic activity have persisted. This work describes the application of molecular dynamics simulations using specifically designed molecular mechanical parameters to the four key-intermediate states formed during the FTase catalytic mechanism–FTase resting state, binary complex (FTase-FPP), ternary complex (FTase-FPP-Peptide), and product complex (FTase-Product). The study involves a comparative analysis of several important molecular aspects for which are vital not only motion but also the conformational sampling of both enzyme and substrate as well as their interaction, and especially the effect of the solvent. These include the radial distribution function of the water molecules around the catalytically important zinc metal atom, the conformations of the substrate and product molecules and the corresponding RMSF values, critical hydrogen bonds and several catalytically relevant distances. These results are discussed in light of recent experimental and computational evidence, yielding new insights into the elusive catalytic mechanism of this enzyme.

Concomitant upregulation of matrix metalloproteinase-2 in lesions and circulating plasma of oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disorder characterized by a T cell-mediated immune response against epithelial cells. Matrix metalloproteinases (MMPs) are an important group of zinc enzymes and are thought to play an important role in the degradation of components of the extracellular matrix. The aim of this study was to assess the expression of MMP-2 and MMP-9 in both tissue specimens and circulating plasma. Twelve cases of OLP were collected. In addition, six cases with chronic inflammation and six normal subjects were also recruited. Oral tissue specimens were collected for measurement of MMPs by immunohistochemistry, and sera prepared from peripheral blood were used for gelatin zymography to determine MMP levels in plasma. The level of MMP-2 in patients with OLP was significantly higher than that in the other two groups, both in tissues and sera (P < 0.0001). However, there was no difference in the expression of MMP-9 among these groups. MMP-2 overexpression in OLP is consistent with its upregulation in peripheral serum. This result also indicates that MMP-2 might play a role in the pathogenesis of OLP.

Reaction Mechanism of the Dinuclear Zinc Enzyme N-Acyl-l-homoserine Lactone Hydrolase: A Quantum Chemical Study

N-acyl-L-homoserine lactone hydrolase (AHL lactonase) is a dinuclear zinc enzyme responsible for the hydrolytic ring opening of AHLs, disrupting quorum sensing in bacteria. The reaction mechanism is investigated using hybrid density functional theory. A model of the active site is designed on the basis of the X-ray crystal structure, and stationary points along the reaction pathway are optimized and analyzed. Two possible mechanisms based on two different substrate orientations are considered. The calculations give support to a reaction mechanism that involves two major chemical steps: nucleophilic attack on the substrate carbonyl carbon by the bridging hydroxide and ring opening by direct ester C-O bond cleavage. The roles of the two zinc ions are analyzed. Zn1 is demonstrated to stabilize the charge of the tetrahedral intermediate, thereby facilitating the nucleophilic attack, while Zn2 stabilizes the charge of the alkoxide resulting from the ring opening, thereby lowering the barrier for the C-O bond cleavage.

A thiabendazole sulfonamide shows potent inhibitory activity against mammalian and nematode α-carbonic anhydrases

A sulfonamide derivative of the antihelmintic drug thiabendazole was prepared and investigated for inhibition of the zinc enzyme carbonic anhydrase CA (EC 4.2.1.1). Mammalian isoforms CA I–XIV and the nematode enzyme of Caenorhabditis elegans CAH-4b were included in this study. Thiabendazole-5-sulfonamide was a very effective inhibitor of CAH-4b and CA IX ( K Is of 6.4–9.5 nm) and also inhibited effectively isozymes CA I, II, IV–VII, and XII, with K Is in the range of 17.8–73.2 nM. The high resolution X-ray crystal structure of its adduct with isozyme II evidenced the structural elements responsible for this potent inhibitory activity.

Therapeutic applications of glycosidic carbonic anhydrase inhibitors

The zinc enzymes carbonic anhydrases (CAs, EC 4.2.1.1) are very efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate and hence play an important physiological role. In humans, 16 different isozymes have been described, some of them being involved in various pathological disorders. Several of these isozymes are considered as drug targets, and the design of selective inhibitors is a long-standing goal that has captured the attention of researchers for 40 years and has lead to clinical applications against different pathologies such as glaucoma, epilepsy, and cancer. Among the different strategies developed for designing selective CA inhibitors (CAIs), the "sugar approach" has recently emerged as a new attractive and versatile tool. Incorporation of glycosyl moieties in different aromatic/heterocyclic sulfonamide/sulfamides/sulfamates scaffolds has led to the development of numerous and very effective inhibitors of potential clinical value. The clinical use of a highly active carbohydrate-based CA inhibitor, i.e., topiramate, constitutes an interesting demonstration of the validity of this approach. Other carbohydrate-based compounds also demonstrate promising potential for the treatment of ophthalmologic diseases. This review will focus on the development of this emerging sugar-based approach for the development of CAIs.

Dual Inhibitors of Matrix Metalloproteinases and Carbonic Anhydrases: Iminodiacetyl-Based Hydroxamate−Benzenesulfonamide Conjugates

Matrix metalloproteinases (MMPs) and carbonic anhydrases (CAs) are two classes of zinc enzymes with different roles and catalytic targets, such as the degradation of most of the extracellular matrix (ECM) proteins and the regulation of the CO(2)/HCO(3)(-) equilibrium in the cells, respectively. Both families have isoforms which were proved to be involved in several stages of carcinogenic processes, and so the selective inhibition of these enzymes might be of interest in cancer therapy. We report herein the design, synthesis, and in vitro evaluation of a series of compounds possessing the iminodiacetic acid as the main backbone and two functional groups attached, namely, the hydroxamic acid and the arylsulfonamide (ArSO(2)NH(2)) moieties, to enable the inhibition of MMPs and CAs, respectively. These compounds were demonstrated to strongly inhibit both MMPs and CAs, some of them from the nanomolar to subnanomolar range. Furthermore, a docking study for MMPs was reported for the most promising compound in order to investigate its binding interactions with the different MMPs.

Entrapment of Carbon Dioxide in the Active Site of Carbonic Anhydrase II

The visualization at near atomic resolution of transient substrates in the active site of enzymes is fundamental to fully understanding their mechanism of action. Here we show the application of using CO(2)-pressurized, cryo-cooled crystals to capture the first step of CO(2) hydration catalyzed by the zinc-metalloenzyme human carbonic anhydrase II, the binding of substrate CO(2), for both the holo and the apo (without zinc) enzyme to 1.1A resolution. Until now, the feasibility of such a study was thought to be technically too challenging because of the low solubility of CO(2) and the fast turnover to bicarbonate by the enzyme (Liang, J. Y., and Lipscomb, W. N. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3675-3679). These structures provide insight into the long hypothesized binding of CO(2) in a hydrophobic pocket at the active site and demonstrate that the zinc does not play a critical role in the binding or orientation of CO(2). This method may also have a much broader implication for the study of other enzymes for which CO(2) is a substrate or product and for the capturing of transient substrates and revealing hydrophobic pockets in proteins.

Reaction mechanism of the binuclear zinc enzyme glyoxalase II – A theoretical study

The glyoxalase system catalyzes the conversion of toxic methylglyoxal to nontoxic d-lactic acid using glutathione (GSH) as a coenzyme. Glyoxalase II (GlxII) is a binuclear Zn enzyme that catalyzes the second step of this conversion, namely the hydrolysis of S-d-lactoylglutathione, which is the product of the Glyoxalase I (GlxI) reaction. In this paper we use density functional theory method to investigate the reaction mechanism of GlxII. A model of the active site is constructed on the basis of the X-ray crystal structure of the native enzyme. Stationary points along the reaction pathway are optimized and the potential energy surface for the reaction is calculated. The calculations give strong support to the previously proposed mechanism. It is found that the bridging hydroxide is capable of performing nucleophilic attack at the substrate carbonyl to form a tetrahedral intermediate. This step is followed by a proton transfer from the bridging oxygen to Asp58 and finally C-S bond cleavage. The roles of the two zinc ions in the reaction mechanism are analyzed. Zn2 is found to stabilize the charge of tetrahedral intermediate thereby lowering the barrier for the nucleophilic attack, while Zn1 stabilizes the charge of the thiolate product, thereby facilitating the C-S bond cleavage. Finally, the energies involved in the product release and active-site regeneration are estimated and a new possible mechanism is suggested.

Carbonic anhydrase inhibitors. Biphenylsulfonamides with inhibitory action towards the transmembrane, tumor-associated isozymes IX possess cytotoxic activity against human colon, lung and breast cancer cell lines

Reaction of 4,4-biphenyl-disulfonyl chloride with aromatic/heterocyclic sulfonamides also incorporating a free amino group, such as 4-aminobenzenesulfonamide, 4-aminoethyl-benzenesulfonamide, 6-chloro-4-aminobenzene-1,3-disulfonamide or 5-amino-1,3,4-thiadiazole-2-sulfonamide afforded bis-sulfonamides which have been tested as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4..2.1.1). The compounds were rather modest inhibitors of isozymes CA I and XII, but were more efficient as inhibitors of the cytosolic CA II and transmembrane, tumor-associated CA IX (inhibition constants in the range of 21–129 nM gainst hCA II, and 23–79 nM against hCA IX, respectively). The new bis-sulfonamides also showed inhibition of growth of several tumor cell lines (ex vivo), with GI50 values in the range of 0.74–10.0 μg/mL against the human colon cancer cell line HCT116, the human lung cancer cell line H460 and the human breast cancer cell line MCF-7.

A promising structural zinc enzyme model for CO 2 fixation and calcification

A new ligand, namely, N-{tris([2-[(1-methylbenzimidazol-2-yl)ethyl]methyl]amino)-2-oxoethyl}iminodiacetic acid is synthesized and characterized and used to prepare a zinc complex as a promising model for the active site of the nacreous protein in mollusc shells. Preliminary investigation of CO 2 fixation and calcification is studied with regard to the influence of the p K a value of the coordinated water molecule and the carboxylate groups.

Structure-Based Dissection of the Active Site Chemistry of Leukotriene A4 Hydrolase: Implications for M1 Aminopeptidases and Inhibitor Design

M1 aminopeptidases comprise a large family of biologically important zinc enzymes. We show that peptide turnover by the M1 prototype, leukotriene A4 hydrolase/aminopeptidase, involves a shift in substrate position associated with exchange of zinc coordinating groups, while maintaining the overall coordination geometry. The transition state is stabilized by residues conserved among M1 members and in the final reaction step, Glu-296 of the canonical zinc binding HEXXH motif shuffles a proton from the hydrolytic water to the leaving group. Tripeptide substrates bind along the conserved GXMEN motif, precisely occupying the distance between Glu-271 and Arg-563, whereas the Arg specificity is governed by a narrow S1 pocket capped with Asp-375. Our data provide detailed insights to the active site chemistry of M1 aminopeptidases and will aid in the development of novel enzyme inhibitors.

A macrophage cell model for selective metalloproteinase inhibitor design.

The desire to inhibit zinc-dependent matrix metalloproteinases (MMPs) has, over the course of the last 30 years, led to the development of a plethora of MMP inhibitors that bind directly to the active-site metal. With one exception, all of these drugs have failed in clinical trials, due to many factors, including an apparent lack of specificity for MMPs. To address the question of whether these inhibitors are selective for MMPs in a biological setting, a cell-based screening method is presented to compare the relative activities of zinc, heme iron, and non-heme iron enzymes in the presence of these compounds using the RAW264.7 macrophage cell line. We screened nine different zinc-binding groups (ZBGs), four established MMP inhibitors (MMPis), and two novel MMP inhibitors developed in our laboratory to determine their selectivities against five different metalloenzymes. Using this model, we identified two nitrogen donor compounds--2,2'-dipyridylamine (DPA) and triazacyclononane (TACN)--as the most selective ZBGs for zinc metalloenzyme inhibitor development. We also demonstrated that the model could predict known nonspecific interactions of some of the most commonly used MMPis, and could also give cross-reactivity information for newly developed MMPis. This work demonstrates the utility of cell-based assays in both the design and the screening of novel metalloenzyme inhibitors.

Engineered mononuclear variants in Bacillus cereus metallo-beta-lactamase BcII are inactive.

Metallo-beta-lactamases (MbetaLs) are zinc enzymes able to hydrolyze almost all beta-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MbetaLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-beta-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms. Despite extensive studies, there is still controversy about the nature of the active BcII species. Here we have designed two mutant enzymes in which each one of the metal binding sites was selectively removed. Both mutants were almost inactive, despite preserving most of the structural features of each metal site. These results reveal that neither site isolated in the MbetaL scaffold is sufficient to render a fully active enzyme. This suggests that only the dinuclear species is active or that the mononuclear variants can be active only if aided by other residues that would be metal ligands in the dinuclear species.

Farnesyltransferase Inhibitors: A Detailed Chemical View on an Elusive Biological Problem

Farnesyltransferase (FTase) is a zinc enzyme that has been the subject of particular attention in anti-cancer research. This enzyme promotes the addition of a farnesyl group from farnesyl diphosphate (FPP) to a cysteine residue of a protein substrate containing a typical -CAAX motif at the carboxyl terminus. Initial interest in FTase inhibition was prompted by the finding that farnesylation was absolutely required for the oncogenic forms of ras proteins to transform cells, as ras proteins have been implicated in around 30% of all human cancers. This discovery led to frenetic search for FTase inhibitors (FTIs), with more than 400 patents registered in less than a decade. However, despite the very promising initial results, the outcome of Phase II and Phase III clinical trials was, is general, rather disappointing, with the most advanced FTIs failing to demonstrate anti-tumor activity in ras dependent cancers, presumably because K-ras, the most frequently mutated form of ras in human cancers, is able to bypass FTI blockade through cross-prenylation by the related enzyme geranylgeranyltransferase I (GGTase I). Surprisingly, several of these compounds were later shown to have anti-tumor activity against non-ras dependent cancers, launching the grounds for a new and exciting era in FTIs research and development, although the precise target for the FTIs activity of these compounds still remains unknown. This review reports the recent progress in the field, presenting a comprehensive summary of the most promising FTIs, in terms of their chemical structure and properties, taking into account the topology of the enzyme's active-site, and the most recent mechanistic results on the catalytic activity of FTase, both at the theoretical and mechanistic level. These features are presented in close linking with the available results on the biological activity of these inhibitors, and with the outcome of the most recent clinical trials.

Carbonic Anhydrase Inhibitors: Bioreductive Nitro-Containing Sulfonamides with Selectivity for Targeting the Tumor Associated Isoforms IX and XII

2-Substituted-5-nitro-benzenesulfonamides incorporating a large variety of secondary/tertiary amines were explored as inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), with the aim of designing bioreductive inhibitors targeting the hypoxia overexpressed, tumor-associated isozymes. The compounds were ineffective inhibitors of the cytosolic isoform I, showed a better inhibition of the physiologically relevant CA II (KIs of 8.8-4975 nM), and strongly inhibited the tumor-associated CA IX and XII (KIs of 5.4-653 nM). Some of these compounds showed excellent selectivity ratios for the inhibition of the tumor-associated isozymes over the cytosolic ones (in the range of 10-1395). The X-ray crystal structure of the adduct of hCA II with the lead molecule 2-chloro-5-nitro-benzenesulfonamide as well as molecular modeling studies for interaction with hCA IX afforded a better understanding of factors governing the discrimination of the two isoforms for this type of bioreductive compound targeting specifically hypoxic tumors.

Bioinorganic chemistry: a short course

Chapter 1: Inorganic Chemistry Essentials. Introduction . Essential Chemical Elements. Metals in Biological Systems: A Survey. Inorganic Chemistry Basics. Biological Metal Ion Complexation. Thermodynamics. Kinetics. Electronic and Geometric Structures of Metals in Biological Systems. Bioorganometallic Chemistry. Electron Transfer. Introduction to Biological Metal Homeostasis, Transport, and Storage. Chapter 2: Biochemistry Fundamentals. Introduction. Proteins. Amino Acid Building Blocks. Protein Structure. Protein Sequencing and Proteomics. Protein Function, Enzymes and Enzyme Kinetics. Nucleic Acids. DNA and RNA Building Blocks. DNA and RNA Molecular Structures. Transmission of Genetic Information. Genetic Mutations and Site-Directed Mutagenesis. Genes and Cloning. Genomics and the Human Genome. The ATP-binding cassette (ABC) Transporter Superfamily. Chapter 3: Instrumental Methods. Introduction. Analytical Instrument-Based Methods. Spectroscopy. X-ray Absorption Spectroscopy (XAS) and Extended X-ray Absorption Fine Structure (EXAFS). Theoretical Aspects and Hardware. Descriptive Examples. X-ray Crystallography. Introduction. Crystallization and Crystal Habits. Theory and Hardware. Descriptive Examples. Electron Paramagnetic Resonance. Theory and Determination of g-values. Hyperfine and Superhyperfine Interactions. Descriptive Examples. Nuclear Magnetic Resonance. Theoretical Aspects. Nuclear Screening and the Chemical Shift. Spin-Spin coupling. Techniques of Spectral Integration and Spin-Spin Decoupling. Nuclear Magnetic Relaxation. The nuclear Overhauser Effect (NOE). Obtaining the NMR spectrum. Two-dimensional NMR Spectroscopy. Correlation Spectroscopy (COSY). Nuclear Overhauser Effect Spectroscopy (NOESY). Descriptive Examples. Mossbauer Spectroscopy. Theoretical Aspects. Quadrupole Splitting and the Isomer Shift. Magnetic Hyperfine Interactions. Descriptive Examples. Atomic Force Microscopy. Fast Methods. Stopped-Flow Kinetic Methods. Flash Photolysis. Time-Resolved Crystallography. Chapter 4: Introduction to Computer-Based Methods. Computer Hardware . Molecular Modeling and Molecular Mechanics. Introduction to MM. Molecular Modeling, Molecular Mechanics and Molecular Dynamics. Biomolecule Modeling. Molecular Modeling Descriptive Examples. Quantum Mechanics-Based Computational Methods. Introduction. Ab-initio Methods. Density Functional Theory. Semi-empirical Methods. Computer Software for Chemistry. Mathematical Software. World Wide Web Online Resources. Nomenclature and Visualization Resources. Online Societies, Literature, Materials, Equipment Webservers. Chapter 5: Group I and II metals in biological systems. Transport and storage of metal ions. Homeostasis involving Na+, K+, Ca2+, Mg2+, Zn2+ (as well as P, Cl, and H). Movement of molecules and ions across membranes. Proton pumps. Ion pumps. Active versus passive transport . Na+/ K+ ATPases. Magnesium dependent biomolecules. Kinases. ATP-pumps. Catalytic RNA and Magnesium. Magnesium in chlorophyll. Calcium dependent biomolecules. Calmodulins and triggering. Biochemistry of nitrogen oxides, nitroxyl, and peroxynitrite ion. Chapter 6: Iron Containing Proteins and Enzymes. Iron-containing proteins with porphyrin ligand systems. Hemoglobin and Myoglobin (first edition material somewhat condensed). Cytochrome P-450: a monooxygenase. Iron-containing cytochromes in photosynthesis. Green plant cytochromes. Cytochrome bc559. Cytochrome bf. Bacterial cytochromes. Cytochrome c2. Cytochrome bc1. Iron-containing cytochromes in the respiratory pathway. Cytochrome c. Cytochrome c oxidase. Non-Heme Iron-containing proteins. MMO-Methane Monooxygenase. Catechol dioxygenases. Iron-oxo clusters. Transferrin for metal transport . Ferritin for metal storage. Hemerythrin. Purple acid phosphatase. Iron-sulfur cluster proteins. Rubredoxin. Ferrodoxins. Aconitase. Chapter 7: Enzymes containing zinc, nickel, and enzymes with multiple metal cofactors. Zinc homeostasis, transport, and storage. Zinc Enzymes. Alkaline phosphatase. Alcohol dehydrogenase. Aspartate transcarbamoylase. Carbonic anhydrase. Carboxypeptidase A and B. Zinc Fingers. Nickel Enzymes. Urease. F430. Enzymes with multiple metal cofactors. Hydrogenases. Carbon monoxide dehydrogenases. Chapter 8 Disease, Disease Diagnosis and Treatment Utilizing Inorganic Species. Metal Transport and Metallochaperones. Disease States. Metal deficiency. Metal toxicity. Copper toxicity. FALS. Wilson and Menkes diseases. Disease Diagnosis. Technetium Imaging Agents. Gadolinium MRI Imaging Agents. Quantum Dots for In vivo Imaging. Metals in Medicine. Manganese superoxide mimetics. Vanadium-based Diabetes Drugs. Platinum-containing Anticancer Agents. Nanomedicine.

Theoretical Investigation of the Reaction Mechanism of the Dinuclear Zinc Enzyme Dihydroorotase

The reaction mechanism of the dinuclear zinc enzyme dihydroorotase was investigated by using hybrid density functional theory. This enzyme catalyzes the reversible interconversion of dihydroorotate and carbamoyl aspartate. Two reaction mechanisms in which the important active site residue Asp250 was either protonated or unprotonated were considered. The calculations establish that Asp250 must be unprotonated for the reaction to take place. The bridging hydroxide is shown to be capable of performing nucleophilic attack on the substrate from its bridging position and the role of Zn(beta) is argued to be the stabilization of the tetrahedral intermediate and the transition state leading to it, thereby lowering the barrier for the nucleophilic attack. It is furthermore concluded that the rate-limiting step is the protonation of the amide nitrogen by Asp250 coupled with C-N bond cleavage, which is consistent with previous experimental findings from isotope labeling studies.

The relationship between serum level of thyroid hormones, trace elements and antioxidant enzymes in dromedary camel (Camelus dromedarius)

Thyroid hormones might be able to regulate the activities of superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX). The role of thyroid hormones in metabolic pathways and antioxidant enzyme activities are well known in many species. Nevertheless, there is no report describing probable relationship between thyroid hormones status, erythrocyte antioxidant enzymes and serum profiles of trace elements. This study was undertaken to investigate the relationship between these parameters in Iranian dromedary camels. Blood samples were taken from the jugular vein of 30 clinically healthy Iranian dromedary camels under aseptic conditions during 6 consecutive days of summer. The serum was analyzed for serum profile of thyroid hormones, trace elements, SOD and GPX activity. There were no significant differences in serum thyroid hormones, serum level of zinc (Zn), copper (Cu), manganese (Mn), iron (Fe), selenium and antioxidant enzymes in different days (P > 0.05). There was a significant negative correlation between SOD and Fe (P < 0.01, r = -493). There was no significant correlation between other parameters. In case of copper deficiency likewise the present study, the copper was used to produce more SOD, so there was a decrease in Fe transportation, and it might be a cause of decrease in Fe amount. On the other hand, a relatively small quantity of Fe is present in tissue myoglobin, catalase, peroxidases, and cytochromes. So it might be another cause of decrease in Fe amount. The explanation for these finding is not possible at this moment and further investigations are needed to interpret these changes.

Carbonic Anhydrase Inhibitors: Binding of Indanesulfonamides to the Human Isoform II

Indanesulfonamides are interesting lead compounds for designing selective inhibitors of the different isoforms of the zinc enzyme Carbonic Anhydrase (CA). Herein, we report for the first time the X-ray crystal structure of two such derivatives, namely indane-5-sulfonamide and indane-2-valproylamido-5-sulfonamide, in complex with the physiologically dominant human isoform II. The structural analysis reveals that, although these two inhibitors have quite similar chemical structures, the arrangement of their indane ring within the enzyme active site is significantly diverse. Thus, our findings suggest that the introduction of bulky substituents on the indane-sulfonamide ring may alter the binding mode of this potent class of CA inhibitors, although retaining good inhibitory properties. Accordingly, the introduction of bulky tail moieties on the indane-sulfonamide scaffold may represent a powerful strategy to induce a desired physicochemical property to an aromatic sulfonamide or to obtain inhibitors with diverse inhibition profiles and selectivity for various mammalian CAs.