Class Of CA Inhibitors Research Articles

Selenocarbamates As a Prodrug-Based Approach to Carbonic Anhydrase Inhibition.

A study on the activity of selenocarbamates as a novel chemotype acting as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors is reported. Undergoing CA‐mediated hydrolysis, selenocarbamates release selenolates behaving as zinc binding groups and effectively inhibiting CAs. A series of selenocarbamates characterised by high molecular diversity and complexity have been studied against different human CA isoforms such as hCA I, II, IX and XII. Selenocarbamates behave as masked selenols with potential biological applications as prodrugs for CAs inhibition‐based strategies. X‐ray studies provided insights into the binding mode of this novel class of CA inhibitors.

The Effect of Substituted Benzene-Sulfonamides and Clinically Licensed Drugs on the Catalytic Activity of CynT2, a Carbonic Anhydrase Crucial for Escherichia coli Life Cycle.

Proteins are relevant antimicrobial drug targets, and among them, enzymes represent a significant group, since most of them catalyze reactions essential for supporting the central metabolism, or are necessary for the pathogen vitality. Genomic exploration of pathogenic and non-pathogenic microorganisms has revealed genes encoding for a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the physiologically crucial reversible reaction of the carbon dioxide hydration to bicarbonate and protons. Herein, we investigated the sulfonamide inhibition profile of the recombinant β-CA (CynT2) identified in the genome of the Gram-negative bacterium Escherichia coli. This biocatalyst is indispensable for the growth of the microbe at atmospheric pCO2. Surprisingly, this enzyme has not been investigated for its inhibition with any class of CA inhibitors. Here, we show that CynT2 was strongly inhibited by some substituted benzene-sulfonamides and the clinically used inhibitor sulpiride (KIs in the range of 82–97 nM). This study may be relevant for identifying novel CA inhibitors, as well as for another essential part of the drug discovery pipeline, such as the structure–activity relationship for this class of enzyme inhibitors.

Anion Inhibition Profile of the β-Carbonic Anhydrase from the Opportunist Pathogenic Fungus Malassezia Restricta Involved in Dandruff and Seborrheic Dermatitis.

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyze the crucial physiological CO2 hydration/dehydration reaction (CO2 + H2O ⇌ HCO3− + H+) balancing the equilibrium between CO2, H2CO3, HCO3− and CO32−. It has been demonstrated that their selective inhibition alters the equilibrium of the metabolites above affecting the biosynthesis and energy metabolism of the organism. In this context, our interest has been focalized on the fungus Malassezia restricta, which may trigger dandruff and seborrheic dermatitis altering the complex bacterial and fungal equilibrium of the human scalp. We investigated a rather large number of inorganic metal-complexing anions (a well-known class of CA inhibitors) for their interaction with the β-CA (MreCA) encoded by the M. restricta genome. The results were compared with those obtained for the two human α-CA isoforms (hCAI and hCAII) and the β-CA from Malassezia globosa. The most effective MreCA inhibitors were diethyldithiocarbamate, sulfamide, phenyl arsenic acid, stannate, tellurate, tetraborate, selenocyanate, trithiocarbonate, and bicarbonate. The different KI values obtained for the four proteins investigated might be attributed to the architectural features of their catalytic site. The anion inhibition profile is essential for better understanding the inhibition/catalytic mechanisms of these enzymes and for designing novel types of inhibitors, which may have clinical applications for the management of dandruff and seborrheic dermatitis.

Inhibition of Bacterial Carbonic Anhydrases as a Novel Approach to Escape Drug Resistance.

Clinically used antibiotics act through one of these four mechanisms: cell wall biosynthesis inhibition, inhibition of protein biosynthesis, interference with DNA and RNA synthesis and the folate pathway. The metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) widespread in microorganisms and present as three genetically distinct families may be considered for the design of antiinfective agents with a different mechanism of action compared to the clinically used antibiotics. CAs are crucial for the life cycle of the pathogen, interfering with pH regulation and biosynthetic processes in which CO2 or bicarbonate are substrates. CA inhibition was shown to lead to debilitation or growth defects of several pathogenic bacteria. CAs catalyzes the interconversion between carbon dioxide to bicarbonate, leading to the formation of protons, and thus affecting pH homeostasis. Several classes of CA inhibitors (CAIs) are known to date, among which the metal complexing anions, the unsubstituted sulfonamides, the dithiocarbamates, etc., which bind to the Zn(II) ion of the enzyme either by substituting the non-protein zinc ligand or add to the metal coordination sphere. Effective inhibitors for many bacterial CAs belonging to the α-, β-, and γ-CA classes were detected, some of which inhibited bacterial growth in vivo. Few of the inhibitors investigated so far were also selective for the bacterial over the human CA isoforms, which may pose problems for their wide clinical applications. Structure-based drug design campaigns might lead to the achievement of the desired selectivity/ potency for preferentially inhibiting bacterial but not the host CAs.

An Overview of the Selectivity and Efficiency of the Bacterial Carbonic Anhydrase Inhibitors.

The possibility to develop new antibacterial agents raised much interest recently. The main classes of antibiotics clinically used nowadays act towards the inhibition of four classical targets: a) cell wall biosynthesis; b) protein biosynthesis; c) DNA and RNA biosynthesis; d) folate biosynthesis. Recently, carbonic anhydrases (CAs, EC 4.2.1.1) started to be investigated in detail in pathogenic bacteria, in the search for antibiotics with a novel mechanism of action, since it has been demonstrated that in many bacteria, CAs are essential for the life cycle of the organism and that their inhibition leads to growth impairment or growth defects of the pathogen. CAs catalyze a simple but physiologically relevant reaction in all life kingdoms, carbon dioxide hydration to bicarbonate and protons. Several classes of CA inhibitors (CAIs) are known to date: the metal complexing anions and the unsubstituted sulfonamides, which bind to the Zn(II) ion of the enzyme either by substituting the non-protein zinc ligand or add to the metal coordination sphere, generating trigonal- bipyramidal species are the classical, most frequently investigated ones. In many cases effective inhibitors were detected, some of which also inhibited the bacterial growth in vivo. However, very few of the detected inhibitors were also selective for the bacterial over the human, off target isoforms such as hCA II. Using structure-based drug design processes, we estimate that it will be possible to achieve the desired selectivity for inhibiting preferentially the bacterial but not the host CA isoforms.

Anion inhibition study of the β-class carbonic anhydrase (PgiCAb) from the oral pathogen Porphyromonas gingivalis

The oral pathogenic bacterium Porphyromonas gingivalis, encodes for two carbonic anhydrase (CA, EC 4.2.1.1) one belonging to the β-class (PgiCAb) and another one to the γ-class (PgiCA). This last enzyme has been characterized earlier for its inhibition profile with various classes of CA inhibitors, such as the sulfonamides and anions, whereas for PgiCAb such data were not yet reported. Here we show that PgiCAb has a good catalytic activity for the CO2 hydration reaction, with kcat 2.8×105s−1 and kcat/Km of 1.5×107M−1×s−1, being inhibited by cyanate and diethyldithiocarbamate in the submillimolar range (KIs of 0.23–0.76mM) and more efficiently by sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KIs of 60–78μM). The anion inhibition profile of the two P. gingivalis enzymes is very different. Identification of selective inhibitors of PgiCAb/PgiCA may lead to pharmacological tools useful for understanding the physiological role(s) of these enzymes, since this bacterium is the main causative agent of periodontitis and few treatment options are presently available.

Chemometric modeling of breast cancer associated carbonic anhydrase IX inhibitors belonging to the ureido-substituted benzene sulfonamide class

Ureido-substituted benzene sulfonamides are the most important class of CA inhibitors which significantly inhibited the formation of primary tumors and metastases. Here, we present quantitative structure activity relationships (QSAR) study on a pool of 27 such inhibitors. A heuristic algorithm selected the best multiple linear regression (MLR) equation, showing the correlation between the observed values and the calculated values of activity. The calculated values of activity were in good agreement with the experimental values. The novelty of this work consists in not only exploring the structural attributes of bioactive molecules but also in predicting in silico the structures of new compounds which may show antimetastatic activity. The not yet synthesized such molecules (i.e. the prediction set) included many compounds showing a higher computed activity compared to the reported such derivatives, but they have been however not yet assayed.

Kinetic and in silico analysis of thiazolidin-based inhibitors of α-carbonic anhydrase isoenzymes

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, salicylic acid derivatives (acting as drug or prodrugs). A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I and II (cytosolic) in a different manner, is reported here. Kinetic measurements allowed us to identify thiazolidin-based compounds as submicromolar-low micromolar inhibitors of these two CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II active site allowed us to understand the inhibition mechanism. This new class of inhibitors bind differently compared to other classes of inhibitors known to date: they were found between the phenol-binding site, filling thus the middle of the enzyme cavity.

Kinetic and docking studies of phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX and XII evidence a new binding mode within the enzyme active site

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, coumarins (acting as prodrugs) and polyamines. A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) in a different manner, is reported here. Kinetic measurements allowed us to identify hydroxy-/methoxy-substituted benzoic acids as well as di-/tri-methoxy benzenes as submicromolar–low micromolar inhibitors of the four CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II allowed us to understand the inhibition mechanism. This new class of inhibitors binds differently compared to all other classes of inhibitors known to date: they were found between the phenol-binding site and the coumarin-binding site, filling thus the middle of the enzyme cavity. They exploit different interactions with amino acid residues and water molecules from the CA active site compared to other classes of inhibitors, offering the possibility to design CAIs with an interesting inhibition profile compared to the clinically used sulfonamides/sulfamates.

Polyamines Inhibit Carbonic Anhydrases by Anchoring to the Zinc-Coordinated Water Molecule

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, phenols, and coumarins. Polyamines such as spermine, spermidine, and many synthetic congeners are described to constitute a novel class of CA inhibitors (CAIs), interacting with the different CA isozymes with efficiency from the low nanomolar to millimolar range. The main structure-activity relationship for these CAIs have been delineated: the length of the molecule, number of amine moieties, and their functionalization are the main parameters controlling activity. The X-ray crystal structure of the CA II-spermine adduct allowed understanding of the inhibition mechanism. Spermine anchors to the nonprotein zinc ligand through a network of hydrogen bonds. Its distal amine moiety makes hydrogen bonds with residues Thr200 and Pro201, which further stabilize the adduct. Spermine binds differently compared to sulfonamides, phenols, or coumarins, rendering possible to develop CAIs with a diverse inhibition mechanism, profile, and selectivity for various isoforms.

Carbonic anhydrase inhibitors. Inhibition of cytosolic isoforms I, II, III, VII and XIII with less investigated inorganic anions

An inhibition study of the cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isoforms I, II, III, VII and XIII with anions such as stannate(IV), selenate(VI), tellurate(VI), perosmate(VIII), persulfate, pyrophosphate(V), pyrovanadate(V), tetraborate, persulfate, perrhenate(VII), perrutenate(VII), selenocyanate, iminodisulfonate, fluorosulfate and trithiocarbonate is reported. Trithiocarbonate was the best inhibitor detected, showing affinities of 8.7–9.9 μM for CA I–III, of 36.15 mM for CA VII and of 0.43 mM for CA XIII. Considering trithiocarbonate as lead, we show that compounds incorporating the new zinc-binding group CS 2 - , such as among other the dithiocarbamates, are even more active inhibitors, with submicromolar inhibitory activity. New classes of CA inhibitors are being detected based on the CS 2 - zinc-binding group.

Inhibition Profiling of Human Carbonic Anhydrase II by High-Throughput Screening of Structurally Diverse, Biologically Active Compounds

Human carbonic anhydrase II (CA II), a zinc metalloenzyme, was screened against 960 structurally diverse, biologically active small molecules. The assay monitored CA II esterase activity against the substrate 4-nitrophenyl acetate in a format allowing high-throughput screening. The assay proved to be robust and reproducible with a hit rate of approximately 2%. Potential hits were further characterized by determining their IC(50) and K(d) values and tested for nonspecific, promiscuous inhibition. Three known sulfonamide CA inhibitors were identified: acetazolamide, methazolamide, and celecoxib. Other hits were also found, including diuretics and antibiotics not previously identified as CA inhibitors, for example, furosemide and halazone. These results confirm that many sulfonamide drugs have CA inhibitory properties but also that not all sulfonamides are CA inhibitors. Thus many, but not all, sulfonamide drugs appear to interact with CA II and may target other CA isozymes. The screen also yielded several novel classes of nonsulfonamide inhibitors, including merbromin, thioxolone, and tannic acid. Although these compounds may function by some nonspecific mechanism (merbromin and tannic acid), at least 1 (thioxolone) appears to represent a genuine CA inhibitor. Thus, this study yielded a number of potentially new classes of CA inhibitors and preliminary experiments to characterize their mechanism of action.

Carbonic anhydrase inhibitors: Novel sulfonamides incorporating 1,3,5-triazine moieties as inhibitors of the cytosolic and tumour-associated carbonic anhydrase isozymes I, II and IX

A new series of aromatic benzenesulfonamides incorporating 1,3,5-triazine moieties in their molecules is reported. This series was obtained by reaction of cyanuric chloride with sulfanilamide, homosulfanilamide or 4-aminoethylbenzenesulfonamide. The prepared dichlorotriazinyl-benzenesulfonamides were subsequently derivatized by reacting them with various nucleophiles, such as ammonia, hydrazine, primary and secondary amines, amino acid derivatives or phenol. The library of sulfonamides incorporating triazinyl moieties was tested for the inhibition of three physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isozymes, the cytosolic hCA I and II, and the transmembrane, tumour-associated hCA IX. The new compounds inhibited hCA I with inhibition constants in the range of 31–8500 nM, hCA II with inhibition constants in the range of 14–765 nM and hCA IX with inhibition constants in the range of 1.0–640 nM. Structure–activity relationship was straightforward and rather simple in this class of CA inhibitors, with the compounds incorporating compact moieties at the triazine ring (such as amino, hydrazino, ethylamino, dimethylamino or amino acyl) being the most active ones, and the derivatives incorporating such bulky moieties ( n-propyl, n-butyl, diethylaminoethyl, piperazinylethyl, pyridoxal amine or phenoxy) being less effective hCA I, II and IX inhibitors. Some of the new derivatives also showed selectivity for inhibition of hCA IX over hCA II (selectivity ratios of 23.33–32.00), thus constituting excellent leads for the development of novel approaches for the management of hypoxic tumours.

Carbonic anhydrase inhibitors: Sulfonamides as antitumor agents?

Novel sulfonamide inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1) were prepared by reaction of aromatic or heterocyclic sulfonamides containing amino, imino, or hydrazino moieties with N, N-dialkyldithiocarbamates in the presence of oxidizing agents (sodium hypochlorite or iodine). The N, N-dialkylthiocarbamylsulfenamido-sulfonamides synthesized in this way behaved as strong inhibitors of human CA I and CA II (hCA I and hCA II) and bovine CA IV (bCA IV). For the most active compounds, inhibition constants ranged from 10 −8 to 10 −9 M (for isozymes II and IV). Three of the derivatives belonging to this new class of CA inhibitors were also tested as inhibitors of tumor cell growth in vitro. These sulfonamides showed potent inhibition of growth against several leukemia, non-small cell lung, ovarian, melanoma, colon, CNS, renal, prostate and breast cancer cell lines. With several cell lines, GI 50 values of 10–75 nM were observed. The mechanism of antitumor action with the new sulfonamides reported here remains obscure, but may involve inhibition of CA isozymes which predominate in tumor cell membranes (CA IX and CA XII), perhaps causing acidification of the intercellular milieu, or inhibition of intracellular isozymes which provide bicarbonate for the synthesis of nucleotides and other essential cell components (CA II and CA V). Optimization of these derivatives from the SAR point of view, might lead to the development of effective novel types of anticancer agents.