Development of Novel Quinoline-Based Sulfonamides as Selective Cancer-Associated Carbonic Anhydrase Isoform IX Inhibitors.

A new series of quinoline-based benzenesulfonamides (QBS) were developed as potential carbonic anhydrase inhibitors (CAIs). The target QBS CAIs is based on the 4-anilinoquinoline scaffold where the primary sulphonamide functionality was grafted at C4 of the anilino moiety as a zinc anchoring group (QBS 13a-c); thereafter, the sulphonamide group was switched to ortho- and meta-positions to afford regioisomers 9a-d and 11a-g. Moreover, a linker elongation approach was adopted where the amino linker was replaced by a hydrazide one to afford QBS 16. All the described QBS have been synthesized and investigated for their CA inhibitory action against hCA I, II, IX and XII. In general, para-sulphonamide derivatives 13a-c displayed the best inhibitory activity against both cancer-related isoforms hCA IX (KIs = 25.8, 5.5 and 18.6 nM, respectively) and hCA XII (KIs = 9.8, 13.2 and 8.7 nM, respectively), beside the excellent hCA IX inhibitory activity exerted by meta-sulphonamide derivative 11c (KI = 8.4 nM). The most promising QBS were further evaluated for their anticancer and pro-apoptotic activities on two cancer cell lines (MDA-MB-231 and MCF-7). In addition, molecular docking simulation studies were applied to justify the acquired CA inhibitory action of the target QBS.

Structurally various sorbicillinoids from the deep-sea sediment derived fungus Penicillium sp. SCSIO06871.

Two new hybrid sorbicillinoids (1 and 5), three new bisorbicillinoids (2-4), and three monomeric sorbicillinoids (6-8), along with eighteen known sorbicillinoids (9-26) were isolated from cultures of the deep-sea sediment derived fungus Penicillium sp. SCSIO06871. Their structures and absolute configurations were elucidated based upon the extensive spectroscopic analysis, X-ray crystallography analysis and the comparison of the experimental and calculated ECD data. Bisorbicillpyrone A (4) is the first example of bisorbicillinoid containing an α-pyrone derivative unit. All of the isolated compounds were evaluated for their antibacterial, antifungal and enzyme inhibitory activities against α-glycosidase and acetylcholinesterase (AChE) in vitro. Compound 6 displayed more potent inhibitory activity against α-glycosidase than acarbose with IC50 value of 36.0 µM and compounds 4, 12, 18, 22, 23 exhibited moderate inhibitory activity with IC50 values ranging from 115.8 to 208.5 µM. Compounds 10 and 22 showed weak enzyme inhibitory activities against AChE with 55.1% and 51.1% inhibitions at concentration of 50 µg/mL, respectively. Besides, compounds 11 and 12 exhibited significant antibacterial activities against Staphylococcus aureus with MIC values of 10.0 and 5.0 µg/mL, respectively. The hypothetical biosynthetic pathway of the isolated sorbicillinoids with three different structural types was discussed.

Inhibition of α-, ß- and γ-carbonic anhydrases from the pathogenic bacterium Vibrio cholerae with aromatic sulphonamides and clinically licenced drugs - a joint docking/molecular dynamics study.

The binding mode of aromatic sulphonamides and clinically licenced drugs to the three carbonic anhydrase (CA, EC 4.2.1.1) isoforms from the human pathogen V. cholerae was here thouroghly characterised by a joint docking and molecular dynamics in silico protocol. In fact, VchCA, VchCAß, and VchCAγ are crucial in the pathogen life cycle and growth and represent innovative targets to fight V. cholerae proliferation overcoming the spreading chemoresistance to the available drugs. A set of 40 sulphonamides/sulfamates VchCAs inhibitors was studied using the proteins homology built 3 D models unveiling the key and stable interactions responsible for a potent CA inhibition. This study has the aim to offer insights and guidelines for the future rational design of potent and selective inhibitors targeting CA isoforms from V. cholerae or other human pathogens.

Identification of Novel Carbonic Anhydrase IX Inhibitors Using High-Throughput Screening of Pooled Compound Libraries by DNA-Linked Inhibitor Antibody Assay (DIANA).

The DNA-linked inhibitor antibody assay (DIANA) has been recently validated for ultrasensitive enzyme detection and for quantitative evaluation of enzyme inhibitor potency. Here we present its adaptation for high-throughput screening of human carbonic anhydrase IX (CAIX), a promising drug and diagnostic target. We tested DIANA's performance by screening a unique compound collection of 2816 compounds consisting of lead-like small molecules synthesized at the Institute of Organic Chemistry and Biochemistry (IOCB) Prague ("IOCB library"). Additionally, to test the robustness of the assay and its potential for upscaling, we screened a pooled version of the IOCB library. The results from the pooled screening were in agreement with the initial nonpooled screen with no lost hits and no false positives, which shows DIANA's potential to screen more than 100,000 compounds per day.All DIANA screens showed a high signal-to-noise ratio with a Z' factor of >0.89. The DIANA screen identified 13 compounds with Ki values equal to or better than 10 µM. All retested hits were active also in an orthogonal enzymatic assay showing zero false positives. However, further biophysical validation of identified hits revealed that the inhibition activity of several hits was caused by a single highly potent CAIX inhibitor, being present as a minor impurity. This finding eventually led us to the identification of three novel CAIX inhibitors from the screen. We confirmed the validity of these compounds by elucidating their mode of binding into the CAIX active site by x-ray crystallography.

Coumarins from Magydaris pastinacea as inhibitors of the tumour-associated carbonic anhydrases IX and XII: isolation, biological studies and in silico evaluation.

In an in vitro screening for human carbonic anhydrase (hCA) inhibiting agents from higher plants, the petroleum ether and ethyl acetate extracts of Magydaris pastinacea seeds selectively inhibited hCA IX and hCA XII isoforms. The phytochemical investigation of the extracts led to the isolation of ten linear furocoumarins (1-10), four simple coumarins (12-15) and a new angular dihydrofurocoumarin (11). The structures of the isolated compounds were elucidated based on 1 D and 2 D NMR, MS, and ECD data analysis. All isolated compounds were inactive towards the ubiquitous cytosolic isoform hCA I and II (Ki > 10,000 nM) while they were significantly active against the tumour-associated isoforms hCA IX and XII. Umbelliprenin was the most potent coumarin inhibiting hCA XII isoform with a Ki of 5.7 nM. The cytotoxicity of the most interesting compounds on HeLa cancer cells was also investigated.

Inhibition of α-, ß-, γ-, and δ-carbonic anhydrases from bacteria and diatoms with N'-aryl-N-hydroxy-ureas.

The inhibition of α-, ß-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N'-aryl-N-hydroxy-ureas is reported. The α-/ß-CAs from V. cholerae (VchCAα and VchCAß) were effectively inhibited by some of these derivatives, with KIs in the range of 97.5 nM - 7.26 µM and 52.5 nM - 1.81 µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (KIs of 4.75 - 8.87 µM). The ß-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAß) was not inhibited by these compounds (KIs > 10 µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (KIs of 59.8 nM - 6.42 µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (KIs of 33.3 nM - 8.74 µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (KIs > 10 µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.

Inhibition of the ß-carbonic anhydrase from the dandruff-producing fungus Malassezia globosa with monothiocarbamates.

A series of monothiocarbamates (MTCs) was investigated for the inhibition of the ß-class carbonic anhydrase (CAs, EC 4.2.1.1) from the fungal parasite Malassezia globosa, MgCA. These MTCs incorporate various scaffolds, among which aliphatic amine with 1-4 carbons atom in their molecule, morpholine, piperazine, as well as phenethylamine and benzylamine derivatives. All the reported MTCs displayed a better efficacy in inhibiting MgCA compared to the clinically used sulphonamide drug acetazolamide (KI of 74 µM), with KIs spanning between 1.85 and 18.9 µM. The homology model of the enzyme previously reported by us was used to rationalize the results by docking some of these MTCs within the fungal CA active site. This study might be useful to enrich the knowledge of the MgCA inhibition profile, eliciting novel ideas pertaining the design of modulators with potential efficacy in combatting dandruff or other fungal infections.

Bioactive Natural Product and Superacid Chemistry for Lead Compound Identification: A Case Study of Selective hCA III and L-Type Ca2+ Current Inhibitors for Hypotensive Agent Discovery.

Dodoneine (Ddn) is one of the active compounds identified from Agelanthusdodoneifolius, which is a medicinal plant used in African pharmacopeia and traditional medicine for the treatment of hypertension. In the context of a scientific program aiming at discovering new hypotensive agents through the original combination of natural product discovery and superacid chemistry diversification, and after evidencing dodoneine's vasorelaxant effect on rat aorta, superacid modifications allowed us to generate original analogues which showed selective human carbonic anhydrase III (hCA III) and L-type Ca2+ current inhibition. These derivatives can now be considered as new lead compounds for vasorelaxant therapeutics targeting these two proteins.

Kinetic properties and affinities for sulfonamide inhibitors of an α-carbonic anhydrase (CruCA4) involved in coral biomineralization in the Mediterranean red coral Corallium rubrum.

We report the kinetic properties and sulfonamide inhibition profile of an α-carbonic anhydrase (CA, EC 4.2.1.1), named CruCA4, identified in the red coral Corallium rubrum. This isoform is involved in the biomineralization process leading to the formation of a calcium carbonate skeleton. Experiments performed on the recombinant protein show that the enzyme has a "moderate activity" level. Our results are discussed compared to values obtained for other CA isoforms involved in biomineralization. This is the first study describing the biochemical characterization of an octocoral CA.

Microwave-assisted extraction, HPLC analysis, and inhibitory effects on carbonic anhydrase I, II, VA, and VII isoforms of 14 blueberry Italian cultivars.

The multi-component fingerprint and the biological evaluation of plant-derived material are indispensable for the pharmaceutical field, in food quality control procedures, and in all plant-based products. We investigated the quantitative content of biologically active compounds (anthocyanins and chlorogenic acid) of microwave-assisted blueberry extracts from 14 different Italian cultivars, using validated high-performance liquid chromatography-photodiode array detector (HPLC-PDA) method and routinely instrument configuration. The carbonic anhydrase (CA, EC 4.2.1.1) inhibition profiles against several pharmacologically relevant CA isoforms of blueberry extracts and some bioactive compounds were also investigated. The various cultivars showed a highly variable content in anthocyanins and chlorogenic acid, and their CA inhibitory effects were also highly variable. Overall these data prove that antioxidant natural products found in blueberries may be useful for designing pharmacological agents in which various CAs are involved, e.g., antiobesity, antitumor, or anticonvulsants agents.

Discovering novel carbonic anhydrase type IX (CA IX) inhibitors from seven million compounds using virtual screening and in vitro analysis.

Carbonic anhydrase type IX (CA IX) enzyme is mostly over expressed in different cancer cell lines and tumor tissues. Potent CA IX inhibitors can be effective for adjusting the pH imbalance in tumor cells. In the present work, we represented the successful application of high throughput virtual screening (HTVS) of large dataset from ZINC database included of ∼7 million compounds to discover novel inhibitors of CA IX. HTVS and molecular docking were performed using consequence Glide/standard precision (SP), extra precision (XP) and induced fit docking (IFD) molecular docking protocols. For each compound, docking code calculates a set of low-energy poses and then exhaustively scans the binding pocket of the target with small compounds. Novel CA IX inhibitor candidates were suggested based on molecular modeling studies and a few of them were tested using in vitro analysis. These compounds were determined as good inhibitors against human CA IX target with Ki in the range of 0.85-1.58 µM. In order to predict the pharmaceutical properties of the selected compounds, ADME (absorption, distribution, metabolism and excretion) analysis was also carried out.

Cloning, characterization and anion inhibition studies of a γ-carbonic anhydrase from the Antarctic cyanobacterium Nostoc commune.

We report the cloning and catalytic activity of a γ-carbonic anhydrase (CA, EC 4.2.1.1) isolated from the Antarctic cyanobacterium Nostoc commune, NcoCA. The enzyme has a significant catalytic activity for the physiologic reaction, CO2 hydration to bicarbonate and protons, with a k(cat) of 9.5×10(5) s(-1) and a k(cat)/K(m) of 8.3×10(7) M(-1) × s(-1), being the most catalytically efficient γ-CA investigated so far. An anion inhibition study of NcoCA with inorganic/organic anions is also reported here. Fluoride, sulfate, perchlorate and tetrafluoroborate did not inhibit appreciably NcoCA, whereas the other halides, pseudohalides, bicarbonate, nitrate, nitrite and many complex inorganic anions showed inhibition in the millimolar range. The best NcoCA inhibitors detected so far were diethyldithiocarbamate (K(I) of 0.80 mM) as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (K(I)s in the range of 70-90 µM). Since γ-CAs are present in carboxysomes, being involved in photosynthesis, this study may be relevant for a better understanding of such processes in some Antarctic organisms.

Eriocitrin and Apigenin as New Carbonic Anhydrase VA Inhibitors from a Virtual Screening of Calabrian Natural Products.

In this work, we performed a structure-based virtual screening against five carbonic anhydrase isoforms using, as a ligand library, natural components of Citrus bergamia (Bergamot) and Allium cepa var. Tropea (red onion) sources, which are some typical Calabrian products. The most relevant Bergamot and red onion components, identified as potentially new hits by means of the computational work, were submitted to in vitro tests in order to confirm the ability to exert the predicted biological activity. Apigenin and eriocitrin were identified as new potent inhibitors of human carbonic anhydrase VA isozyme.

Monoclonal antibodies raised against 167-180 aa sequence of human carbonic anhydrase XII inhibit its enzymatic activity.

Abstract Human carbonic anhydrase XII (CA XII) is a single-pass transmembrane protein with an extracellular catalytic domain. This enzyme is being recognized as a potential biomarker for different tumours. The current study was aimed to generate monoclonal antibodies (MAbs) neutralizing the enzymatic activity of CA XII. Bioinformatics analysis of CA XII structure revealed surface-exposed sequences located in a proximity of its catalytic centre. Two MAbs against the selected antigenic peptide spanning 167-180 aa sequence of CA XII were generated. The MAbs were reactive with recombinant catalytic domain of CA XII expressed either in E. coli or mammalian cells. Inhibitory activity of the MAbs was demonstrated by a stopped flow CO2 hydration assay. The study provides new data on the surface-exposed linear CA XII epitope that may serve as a target for inhibitory antibodies with a potential immunotherapeutic application.

Inhibition of carbonic anhydrase isozymes I and II with natural products extracted from plants, mushrooms and honey.

Different natural products and secondary metabolites from mushrooms, teas, honeys, mosses, plants and seaweeds were investigated for their in vitro inhibitory effects on human carbonic anhydrase (hCA, E.C.4.2.1.1) isoforms I and II. Inhibition data were correlated with the total phenol content in the extract and investigated with the pure compounds believed to be responsible for this activity. Methanolic extracts were prepared for 17 such pure chemicals present in the natural products and for 41 diverse natural products. The IC(50) values were in the range of 0.11-66.50 µg/mL against hCA I and of 0.09-54.54 µg/mL against hCA II, respectively. The total phenol content was in the range of 0.02-1318.96 (as milligrams of gallic acid equivalents) per gram of sample. These data offer new insights on possible novel classes of CA inhibitors based on natural products, possessing a range of chemical structures not present in the classical inhibitors with pharmacological applications, such as the sulfonamides and sulfamates.

Honey, pollen, and propolis extracts show potent inhibitory activity against the zinc metalloenzyme carbonic anhydrase.

Three different honey extracts from the endemic plant in the Black Sea region Rhododendron ponticum, were investigated for their inhibitory effects against the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1), more precisely the human (h) isoforms hCA I and hCA II. Hexane, methanol, ethanol, and water solid-phase extractions (SPEs) showed inhibitory activity towards the two CA isozymes which were related to the total phenolic content. The highest inhibitory effects (0.036-0.039 mg/mL) were those of propolis methanolic extract. Among the three different samples investigated here, the aqueous extracts showed lower inhibitory effects compared to the organic solvent SPE extracts (in the range of 1.150- 5.144 mg/mL). The studied honey extracts constitute an interesting source of phenolic derivatives that might serve to identify lead compounds, targeting the physiologically relevant enzymes CA I and CA II.

A new norlignan from Taxodium ascendens.

A new norlignan, (2R,3R,4S,5S)-2,4-bis(4-hydroxyphenyl)-3,5-dihydroxy-tetrahydropyran (1), together with 9 known compounds were isolated from the branches and leaves of Taxodium ascendens. Their structures were mainly determined on the basis of MS, IR, 1D and 2D NMR spectral evidences. Methanol extract showed inhibitory activity on carbonic anhydrase II with an IC(50) value of 4.27 microg/ml, acetone extract and methanol extract inhibited activity of cathepsin B with IC(50) values of 2.12 and 3.71 microg/ml, respectively.

Carbonic anhydrase inhibitors. Inhibition of the zinc and cobalt gamma-class enzyme from the archaeon Methanosarcina thermophila with anions.

Anions represent the second class of inhibitors of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1), in addition to sulfonamides, which possess clinical applications. The first inhibition study of the zinc and cobalt gamma-class enzyme from the archaeon Methanosarcina thermophila (Cam) with anions is reported here. Inhibition data of the alpha-class human isozymes hCA I and hCA II (cytosolic) as well as the membrane-bound isozyme hCA IV with a large number of anionic species such as halides, pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrosulfide, bisulfite, and sulfate, etc., are also provided for comparison. The best Zn-Cam anion inhibitors were hydrogen sulfide and cyanate, with inhibition constants in the range of 50-90 microM, whereas thiocyanate, azide, carbonate, nitrite, and bisulfite were weaker inhibitors (K(I)s in the range of 5.8-11.7 mM). Fluoride, chloride, and sulfate do not inhibit this enzyme appreciably up to concentrations of 200 mM, whereas the substrate bicarbonate behaves as a weak inhibitor (K(I)s of 42 mM). The best Co-Cam inhibitor was carbonate, with an inhibition constant of 9 microM, followed by nitrate and bicarbonate (K(I)s in the range of 90-100 microM). The metal poisons were much more ineffective inhibitors of this enzyme, with cyanide possessing an inhibition constant of 51.5mM, whereas cyanate, thiocyanate, azide, iodide, and hydrogen sulfide showed K(I)s in the range of 2.0-6.1mM. As for Zn-Cam, fluoride, chloride, and sulfate are not inhibitors of Co-Cam. These major differences between the two gamma-CAs investigated here can be explained only in part by the different geometries of the metal ions present within their active sites.

Properties of a carbonic anhydrase inhibitor protein in flounder serum.

The blood serum of the European flounder Platichthys flesus strongly inhibits soluble erythrocytic carbonic anhydrase from the same species. The inhibition is of the uncompetitive type. Hence, the mechanism of the carbonic anhydrase inhibition is different from that of all other known carbonic anhydrase inhibitors. The serum showed no inhibitory effect on carbonic anhydrase from human and bovine red blood cells. By applying the (18)O exchange reaction, it could be demonstrated that the presence of the carbonic anhydrase inhibitor in the extracellular fluid has no effect on carbonic anhydrase in intact red blood cells. Thus, this carbonic anhydrase inhibitor seems to act only within the plasma space of the circulatory system. However, the carbonic anhydrase inhibitor does appear to reduce the bicarbonate permeability of flounder red cells to approximately one-quarter of normal levels as measured by the (18)O exchange reaction. The 28 kDa carbonic anhydrase inhibitor was isolated from the serum by gel filtration. The isolated inhibitor was detected in acrylamide gels as a single band representing a 7 kDa protein. The denaturing conditions used in electrophoresis presumably led to a dissociation of the native protein into subunits.

Analytical chiral separation of the stereoisomers of a novel carbonic anhydrase inhibitor and its deethylated metabolite, and the assignment of absolute configuration of the human metabolite and chiral degradation products.

Several approaches to the separation of four stereoisomers, 1-4, of a novel, topically active, carbonic anhydrase inhibitor, 1, with two chiral centers in the molecule and four isomers, 5-8, of its chiral metabolite, 5, were evaluated. These methods include nonchiral derivatization followed by separation on chiral stationary phases (CSPs) and chiral derivatization and separation on nonchiral columns and on CSPs. Baseline separation of stereoisomers 1-4 was achieved in less than 15 min after chiral derivatization with (S)-(+)-1-(1-naphthyl)ethyl isocyanate (NEIC) and chiral chromatography on a (R)-N-(3,5-dinitrobenzoyl)phenyl glycine (DNBPG) column under normal phase (NP) conditions. Similarly, isomers 5-8 were baseline separated in less than 20 min after derivatization with NEIC and chromatography on nonchiral (nitrophenyl) and chiral [(S)-(3,5-dinitrobenzoyl)leucine; DNBL] columns in series under the same NP chromatographic conditions. Only partial separation of the diastereomeric derivatives was observed on a variety of nonchiral columns. In addition, all other direct and indirect chiral separation approaches gave only partial separation of at least two stereoisomers within the group of 1-4 or 5-8. The details of chiral separations using various methods and separation (alpha) and capacity factors (k') of the derivatized isomers 1-8 on a series of chiral and nonchiral columns are presented. Using these methods, the absolute configuration of the human metabolite of 1 was established as S1S2 (5), and the heat (HD) and light (LD) degradation products of 1 as R1S2 (3) and S1S2 (5), respectively.