Structure–Activity Relationships of Benzenesulfonamide‐Based Inhibitors towards Carbonic Anhydrase Isoform Specificity

Carbonic anhydrases (CAs) are implicated in a wide range of diseases, including the upregulation of isoforms CA IX and XII in many aggressive cancers. However, effective inhibition of disease‐implicated CAs should minimally affect the ubiquitously expressed isoforms, including CA I and II, to improve directed distribution of the inhibitors to the cancer‐associated isoforms and reduce side effects. Four benzenesulfonamide‐based inhibitors were synthesized by using the tail approach and displayed nanomolar affinities for several CA isoforms. The crystal structures of the inhibitors bound to a CA IX mimic and CA II are presented. Further in silico modeling was performed with the inhibitors docked into CA I and XII to identify residues that contributed to or hindered their binding interactions. These structural studies demonstrated that active‐site residues lining the hydrophobic pocket, especially positions 92 and 131, dictate the positional binding and affinity of inhibitors, whereas the tail groups modulate CA isoform specificity. Geometry optimizations were performed on each ligand in the crystal structures and showed that the energetic penalties of the inhibitor conformations were negligible compared to the gains from active‐site interactions. These studies further our understanding of obtaining isoform specificity when designing small molecule CA inhibitors.     

Functionalization of Fluorinated Benzenesulfonamides and Their Inhibitory Properties toward Carbonic Anhydrases

Substituted tri‐ and tetrafluorobenzenesulfonamides were designed, synthesized, and evaluated as high‐affinity and isoform‐selective carbonic anhydrase (CA) inhibitors. Their binding affinities for recombinant human CA I, II, VA, VI, VII, XII, and XIII catalytic domains were determined by fluorescent thermal shift assay, isothermal titration calorimetry, and a stopped‐flow CO₂ hydration assay. Variation of the substituents at the 2‐, 3‐, and 4‐positions yielded compounds with a broad range of binding affinities and isoform selectivities. Several 2,4‐substituted‐3,5,6‐trifluorobenzenesulfonamides were effective CA XIII inhibitors with high selectivity over off‐target CA I and CA II. 3,4‐Disubstituted‐2,5,6‐trifluorobenzenesulfonamides bound CAs with higher affinity than 2,4‐disubstituted‐3,5,6‐trifluorobenzenesulfonamides. Many such fluorinated benzenesulfonamides were found to be nanomolar inhibitors of CA II, CA VII, tumor‐associated CA IX and CA XII, and CA XIII. X‐ray crystal structures of inhibitors bound in the active sites of several CA isoforms provide structure–activity relationship information for inhibitor binding affinities and selectivity.     

Synthesis,Molecular Docking Analysis,and Biological Evaluations of Saccharide-Modified Sulfonamides as Carbonic Anhydrase IX Inhibitors

Based on the strategy of the “tail approach”, 15 novel saccharide-modified sulfonamides were designed and synthesised. The novel compounds were evaluated as inhibitors of three human carbonic anhydrase (CA) isoforms, namely cytoplasmic CA II, transmembrane CA IX, and XII. Most of these compounds showed good activity against CAs and high topological polar surface area (TPSA) values, which had a positive effect on the selective inhibition of transmembrane isoforms CA IX and XII. In the in vitro activity studies, compounds 16a, 16b, and 16e reduced the viability of HT-29 and MDA-MB-231 cells with a high expression of CA IX under hypoxia. The inhibitory activity of compound 16e on the human osteosarcoma cell line MG-63 with a high expression of CA IX and XII was better than that of AZM. Moreover, high concentrations of compounds 16a and 16b reversed the acidification of the tumour microenvironment. In addition, compound 16a had a certain inhibitory effect on the migration of MDA-MB-231 cells. All the above results indicate that the saccharide-modified sulfonamide has further research value for the development of CA IX inhibitors.     

Chromene-Containing Aromatic Sulfonamides with Carbonic Anhydrase Inhibitory Properties

Carbonic anhydrases (CAs, EC 4.2.1.1) catalyze the essential reaction of CO₂ hydration in all living organisms, being actively involved in the regulation of a plethora of patho/physiological conditions. A series of chromene-based sulfonamides were synthesized and tested as possible CA inhibitors. Their inhibitory activity was assessed against the cytosolic human isoforms hCA I, hCA II and the transmembrane hCA IX and XII. Several of the investigated derivatives showed interesting inhibition activity towards the tumor associate isoforms hCA IX and hCA XII. Furthermore, computational procedures were used to investigate the binding mode of this class of compounds, within the active site of hCA IX.     

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.     

Aromatic Sulfonamides including a Sulfonic Acid Tail: New Membrane Impermeant Carbonic Anhydrase Inhibitors for Targeting Selectively the Cancer-Associated Isoforms

We report here a new drug design strategy for producing membrane-impermeant carbonic anhydrase (CA; EC 4.2.1.1) inhibitors selectively targeting the tumor-associated, membrane-bound human CAs IX and XII over off-target cytosolic isoforms. To date, this approach has only been pursued by including permanent positively charged pyridinium type or highly hydrophilic glycosidic moieties into the structure of aromatic sulfonamide CA inhibitors (CAIs). Aliphatic (propyl and butyl) sulfonic acid tails, deprotonated at physiological pH, were thus incorporated onto a benzenesulfonamide scaffold by a common 1,2,3-triazole linker and different types of spacers. Twenty such derivatives were synthesized and tested for their inhibition of target (hCAs IV, IX, and XII) and off-target CAs (hCAs I and II). Most sulfonate CAIs induced a potent inhibition of hCAs II, IX, and XII up to a low nanomolar K_I range (0.9–459.4 nM) with a limited target/off-target CA selectivity of action. According to the drug design schedule, a subset of representative derivatives was assessed for their cell membrane permeability using Caco-2 cells and a developed FIA-MS/MS method. The complete membrane impermeability of the sulfonate tailed CAIs (≥98%) validated these negatively charged moieties as being suitable for achieving, in vivo, the selective targeting of the tumor-associated CAs over off-target ones.     

Development of intravascular contrast agents for MRI using gadolinium chelates

Two MRI contrast agents (CAs) composed of Gd‐DO3A conjugated to amino acid building blocks derived from glutamic acid ( CA1 ) and lysine ( CA2 ) have been synthesized by using novel alkyne and propionate linkers, and subsequently characterized. In vitro cell viability assays showed insignificant cytotoxicity of both CAs at low concentrations up to 0.2 mM . The longitudinal relaxivities (r₁) of CA1 and CA2 measured at 9.4 T are 6.4 and 5.4 mM ^?1 s^?1 in H₂O at 25 °C, respectively. Both r₁ values are higher than those of CAs in clinical use: Gd‐DTPA (Magnevist, Bayer Schering, Germany) and Gd‐DOTA (Dotarem, Guerbet, France). In vivo imaging in Wistar rats demonstrated considerable signal enhancement (～50 %) in the brain artery by CA2 , but lower signal enhancement (～30 %) by CA1 . In contrast to Dotarem, which showed a similar signal enhancement as CA2 , the enhancement by CA2 remained high (～30 %), even at 52 min post‐injection. This demonstrates that CA2 has a much longer blood half‐life (68.1 min), which could be advantageous for angiography and tissue targeting.     

Exploration of the binding mode of indanesulfonamides as selective inhibitors of human carbonic anhydrase type VII by targeting Lys 91

Convulsions are common neurological disorders in clinical medicine and are triggered by several mechanisms. The enhancement of neuronal excitability can be related, among other factors, to GABAergic depolarization. Carbonic anhydrase (CA) VII contributes to this electrophysiological behavior by providing bicarbonate anion, which can mediate current through channels coupled to GABA(A) receptors. Among the cytosolic CAs, the mechanism of action and inhibition of CA VII is less understood. We present herein the pharmacological evaluation of both enantiomers of an indanesulfonamide compound substituted by a pentafluorophenyl moiety against CA VII and five other human CA isoforms to evaluate their selectivity. The investigated compounds are powerful inhibitors of hCA VII, with K(i) values in the range of 1.7-3.3 nM, but their selectivity needs to be improved. A molecular modeling study was conducted to rationalize the structure-activity relationships and provide useful insight into the future design of selective hCA VII inhibitors.     

Inhibitory Effects of Sulfonamide Derivatives on the β-Carbonic Anhydrase (MpaCA) from Malassezia pachydermatis,a Commensal,Pathogenic Fungus Present in Domestic Animals

Fungi are exposed to various environmental variables during their life cycle, including changes in CO₂ concentration. CO₂ has the potential to act as an activator of several cell signaling pathways. In fungi, the sensing of CO₂ triggers cell differentiation and the biosynthesis of proteins involved in the metabolism and pathogenicity of these microorganisms. The molecular machineries involved in CO₂ sensing constitute a promising target for the development of antifungals. Carbonic anhydrases (CAs, EC 4.2.1.1) are crucial enzymes in the CO₂ sensing systems of fungi, because they catalyze the reversible hydration of CO₂ to proton and HCO₃^-. Bicarbonate in turn boots a cascade of reactions triggering fungal pathogenicity and metabolism. Accordingly, CAs affect microorganism proliferation and may represent a potential therapeutic target against fungal infection. Here, the inhibition of the unique β-CA (MpaCA) encoded in the genome of Malassezia pachydermatis, a fungus with substantial relevance in veterinary and medical sciences, was investigated using a series of conventional CA inhibitors (CAIs), namely aromatic and heterocyclic sulfonamides. This study aimed to describe novel candidates that can kill this harmful fungus by inhibiting their CA, and thus lead to effective anti-dandruff and anti-seborrheic dermatitis agents. In this context, current antifungal compounds, such as the azoles and their derivatives, have been demonstrated to induce the selection of resistant fungal strains and lose therapeutic efficacy, which might be restored by the concomitant use of alternative compounds, such as the fungal CA inhibitors.     

Discovery of New Potential Anti‐Infective Compounds Based on Carbonic Anhydrase Inhibitors by Rational Target‐Focused Repurposing Approaches

In academia, compound recycling represents an alternative drug discovery strategy to identify new pharmaceutical targets from a library of chemical compounds available in house. Herein we report the application of a rational target‐based drug‐repurposing approach to find diverse applications for our in‐house collection of compounds. The carbonic anhydrase (CA, EC 4.2.1.1) metalloenzyme superfamily was identified as a potential target of our compounds. The combination of a thoroughly validated docking screening protocol, together with in vitro assays against various CA families and isoforms, allowed us to identify two unprecedented chemotypes as CA inhibitors. The identified compounds have the capacity to preferentially bind pathogenic (bacterial/protozoan) CAs over human isoforms and represent excellent hits for further optimization in hit‐to‐lead campaigns.     

Catalytic action of metallic salts in autoxidation and polymerization. VI. The inhibitive effects of transition-metal N,N-diethyldiselenocarbamates on the thermal oxidative degradation of isotactic polypropylene

Transition-metal (Zn, Ni, Pd, Cu, Cr, Co) N,N-diethyldiselenocarbamates were prepared, and the inhibitive effects of these compounds on the thermal oxidative degradation of isotactic polypropylene, both in the presence and absence of copper stearate as oxidation accelerator, were examined by conventional oxygen uptake. In both cases, all metal selenocarbamates showed a pronounced inhibitive effect on the degradation of the polymer, especially Zn and Ni selenocarbamates. The inhibitive action of these metal selenocarbamates was discussed from the viewpoint of viscosity change and carbonyl and hydroperoxide contents.     

Pull-Down of Metalloproteins in Their Native States by Using Desthiobiotin-Based Probes

One-third of all proteins are estimated to require metals for structural stability and/or catalytic activity. Desthiobiotin probes containing metal binding groups can be used to capture metalloproteins with exposed active-site metals under mild conditions so as to prevent changes in metallation state. The proof-of-concept was demonstrated with carbonic anhydrase (CA), an open active site, Zn²⁺-containing protein. CA was targeted by using sulfonamide derivatives. Linkers of various lengths and structures were screened to determine the optimal structure for capture of the native protein. The optimized probes could selectively pull down CA from red blood cell lysate and other protein mixtures. Pull-down of differently metallated CAs was also investigated.     

Benzylaminoethylureido-Tailed Benzenesulfonamides Show Potent Inhibitory Activity against Bacterial Carbonic Anhydrases

A series of benzylaminoethylureido-tailed benzenesulfonamides was analyzed for their inhibition potential against bacterial carbonic anhydrases (CAs) such as VhCA α, β, and γ from Vibrio cholerae, and BpsCA β and γ-CAs from Burkholderia pseudomallei. Growing drug resistance against antibiotics demands alternative targets and mechanisms of action. As CA is essential for the survival of bacteria, such enzymes have the potential for developing new antibiotics. Most of the compounds presented excellent inhibition potential against VhCA γ compared to α and β, with K_i values in the range of 82.5–191.4 nM. Several sulfonamides exhibited excellent inhibition against BpsCA β with K_i values in the range of 394–742.8 nM. Recently it has been demonstrated that sufonamide CA inhibitors are effective against vancomycin-resistant enterococci. These data show that CA inhibition of pathogenic bacteria may lead to a new class of antibiotics.     

Breath figure templated semifluorinated block copolymers with tunable surface properties and binding capabilities

Patterning of functionalized polymeric surfaces enables the adjustment of their characteristics and use in novel applications. We prepared breath figure (BF) films from three semifluorinated diblock copolymers, which all are composed of a polystyrene block and a semifluorinated one to compare their surface properties. “Click” chemistry was employed to one of the polymers, containing a poly(pentafluorostyrene) block to incorporate hydrophilic sugar or carboxylic acid moieties. The structure of the polymer alters the obtained porous morphology of the films. Contact angle (CA) analyses of the BF films reveals that the surface porosity increases water CAs compared with solvent cast films, and, in the case of hydrophobic polymers, leads to significant increase in the CAs of dodecane. The hydrophobicity of the BF films is further amplified by the removal of the topmost layer which leads in some cases to superhydrophobic surfaces. BF films containing glucose units are hydrophilic exhibiting water CAs below 90°. These glycosylated porous surfaces are shown to bind lectin Con A‐FITC or can be labelled with isothiocyanate marker. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015 , 132, 41225.     

Characterisation of Photoaffinity‐Based Chemical Probes by Fluorescence Imaging and Native‐State Mass Spectrometry

Chemical probes are small‐molecule reagents used by researchers for labelling and detection of biomolecules. We present the design, synthesis, and characterisation of a panel of 11 structurally diverse photoaffinity labelling (PAL) probes as research tools for labelling the model enzyme carbonic anhydrase (CA) in challenging environments, including in protein mixtures and cell lysates. We targeted the ubiquitous CA II as well as the two cancer‐associated CAs (CA IX and CA XII) that are of high priority as potential biomarkers of aggressive and/or multidrug‐resistant cancer. We utilise an atypical biophysical approach, native state mass spectrometry, to monitor the initial protein–probe binding and subsequent UV crosslinking efficiency of the protein:probe complex. This mass spectrometry methodology represents a new approach for chemical probe optimisation and development that might have broader applications to chemical probe characterisation beyond this study. This also represents one of the first studies, to the best of our knowledge, in which a comprehensive set of PAL probes has been used to establish the relationship between probe structure, noncovalent protein–probe binding, and covalent protein–probe crosslinking efficiency. Our results demonstrate the benefits of a comprehensive analysis of chemical probe structure–activity relationships to support the development of optimum chemical probes.     

A new formaldehyde-free wood adhesive from renewable materials

A formaldehyde-free adhesive that consists of soy flour (SF) and a new curing agent (CA) was developed and evaluated for making interior plywood. Three types of plywood panels (seven-ply maple/white fir/pine/white fir/pine/white fir/maple, five-ply yellow poplar, and five-ply aspen) were prepared with the SF–CA adhesives and evaluated for their water resistance. The CA was derived from the reaction of epichlorohydrin (ECH) and ammonium hydroxide in water. Effects of the reaction time, reaction temperature, NaOH usage, heat treatment of CA, addition order of reactants in the preparation of the CA, and storage time of the CA on the water resistance of plywood panels bonded with SF–CA adhesives were investigated. The reaction time required for the completion of the reaction significantly decreased as reaction temperatures increased. The addition of NaOH to the SF–CA adhesive improved the water resistance and dry shear strength of the five-ply aspen panels. All plywood panels met the requirements for interior plywood when the CAs were prepared at 45–60 °C no matter whether the CA was heat-treated or not. Mixing ECH and ammonium hydroxide all at once resulted in better water resistance of the resulting plywood panels than adding either of ECH or ammonium hydroxide to the other dropwise. The viscosity of heat-treated CA was comparable to that of untreated CA when the CA was prepared at 50 °C. Both heat-treated and untreated CAs could be stored at room temperature for at least two months without compromising the water resistance of the resulting plywood panels.     

Investigation of the supercritical deposition of platinum nanoparticles into carbon aerogels

The preparation of platinum/carbon aerogel (CA) nanocomposites by the supercritical deposition method was investigated. CAs were impregnated with dimethyl(cyclooctadiene)platinum, CODPtMe₂, from supercritical carbon dioxide (scCO₂) solutions and the resulting CODPtMe₂/CA composites were converted to Pt/CA composites. The adsorption isotherms of CODPtMe₂ on CAs were measured and could be represented by the Langmuir model. The results indicated a strong interaction between CODPtMe₂ molecules and the CA surface and that a substantial fraction of the surface of the CAs was covered with CODPtMe₂ molecules at relatively low concentrations. Four different reduction methods were used to convert the CODPtMe₂ impregnated CAs which were: (1) thermal reduction at atmospheric pressure in an inert atmosphere; (2) thermal reduction in scCO₂; (3) chemical reduction in scCO₂ with hydrogen; and (4) chemical reduction at atmospheric pressure with hydrogen. Method 1 gave highly dispersed Pt nanoparticles (1–3 nm) at loadings ranging from 10 to 40 wt.%. The use of hydrogen in Method 4 increased the average particle size by a factor of 2 over Method 1 at the same Pt loading, but the particles still had a narrow unimodal size distribution. When the thermal reduction was carried out in scCO₂, loadings as high as 73% could be obtained. Method 3 generated a composite having a disordered columnar Pt coating and equiaxed particles ≈1 μm in diameter on the external surface of the monolith and dispersed Pt nanoparticles in the interior. The analysis of the reaction products in scCO₂ indicated an autocatalytic reaction. Increasing the Pt loading was found to decrease the surface area of the CA, primarily through blockage of the micropores.     

Synthesis and characterization of a novel polyurethane curing agent modified by a diazafluorene derivative

In this study, we synthesized a novel polyurethane (PU) curing agent (CA) modified by the diazafluorene derivative, 9,9′‐di(4‐hydroxyphenyl)‐9‐1H‐4,5‐diazafluorene, at different hydroxyl ratios. The results of ¹H‐NMR and Fourier transform infrared spectroscopy proved the structure of the fluorine derivative and the modification of CAs. Differential scanning calorimetry and dynamic mechanical analysis showed that the glass‐transition temperature of the PU increased with the addition of the diazafluorene derivative, and the PU cured in the novel CA had more stable and better damping properties under moderate‐temperature conditions (30–80 °C) than the PU cured in the N75 CA. Tan δ was maintained in a relatively lower range shift in the moderate‐temperature range. The average adhesion values of the PU coating cured in the novel CA increased. The scanning electron microscopy morphologies of the PU film and the average adhesion of the coatings showed that the PU cured in the novel CA had good mechanical properties. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46591.     

Selective Inhibition of Helicobacter pylori Carbonic Anhydrases by Carvacrol and Thymol Could Impair Biofilm Production and the Release of Outer Membrane Vesicles

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.     

A Highlight on the Inhibition of Fungal Carbonic Anhydrases as Drug Targets for the Antifungal Armamentarium

Carbon dioxide (CO₂), a vital molecule of the carbon cycle, is a critical component in living organisms’ metabolism, performing functions that lead to the building of compounds fundamental for the life cycle. In all living organisms, the CO₂/bicarbonate (HCO₃⁻) balancing is governed by a superfamily of enzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the pivotal physiological reaction, consisting of the reversible hydration of the CO₂ to HCO₃⁻ and protons. Opportunistic and pathogenic fungi can sense the environmental CO₂ levels, which influence their virulence or environmental subsistence traits. The fungal CO₂-sensing is directly stimulated by HCO₃⁻ produced in a CA-dependent manner, which directly activates adenylyl cyclase (AC) involved in the fungal spore formation. The interference with CA activity may impair fungal growth and virulence, making this approach interesting for designing antifungal drugs with a novel mechanism of action: the inhibition of CAs linked to the CO₂/HCO₃⁻/pH chemosensing and signaling. This review reports that sulfonamides and their bioisosteres as well as inorganic anions can inhibit in vitro the β- and α-CAs from the fungi, suggesting how CAs may be considered as a novel “pathogen protein” target of many opportunistic, pathogenic fungi.