Treatment of NSAPs-rich petrochemical wastewaters using a two-stage combined process of fungi and activated sludge.

A two-stage biological process using fungi and bacteria was set-up and tested for treating a petrochemical wastewater containing naphthalene sulphonic acid polymers. The fungal treatment was carried out through a trickling filter inoculated with Pleurotus ostreatus attached on Luffa cylindrica acting as both physical support and carbon source. The fungal reactor was operated in non-sterile conditions setting two pH values (5 and 6) and two hydraulic retention times (2 d and 3 d). The effluent was then sent to an activated sludge reactor operating the second stage of the treatment. Using an HPLC-based technique, it was observed that the fungal reactor was capable of reducing the polymerization grade of naphthalene sulphonic acid polymers up to 35%, thus increasing significantly the biodegradability of the petrochemical wastewater, from the initial 9% to 46%. The two-stage process allowed to remove about 50% of the total COD much higher than 9% that can be achieved with activated sludge alone. The use of Luffa cylindrica as support for fungi allowed to limit bacterial contamination of the trickling filter and enhanced enzymatic production (on average 20 U/L of Laccase) without any release of non-biodegradable by-products in the effluent. Extraction and PCR-amplification of fungal DNA was carried out along with over 70 d running process in order to monitor the changes of the fungal community inside the reactors. Results showed that Meyerozyma, Fusarium and Thricoderma, spp. developed inside the reactor with Thricoderma, spp. representing the main constituent of fungal biomass at the end of the experiment.

Dioxygen, an unexpected carbonic anhydrase ligand.

Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, grouped into seven different classes, which catalyze the reaction of CO2 hydration to bicarbonate and protons. All of the fifteen human isoforms reported to date belong to the α-class and contain zinc as a cofactor. The structure of human Zn,Cu-CA II has been solved which contains a copper ion bound at its N-terminal, coordinated to His4 and His64. In the active site a dioxygen molecule is coordinated to the zinc ion. Since dioxygen is a rather unexpected CA ligand, molecular dynamics (MD) simulations were performed which suggested a superoxide character of the zinc bound O2.

Discovery of Novel Nonsteroidal Anti-Inflammatory Drugs and Carbonic Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Management of Rheumatoid Arthritis.

Herein we report the design as well as the synthesis of a new series of dual hybrid compounds consisting of the therapeutically used nonsteroidal-anti-inflammatory drugs (NSAIDs; i.e., indometacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase inhibitors) and the carbonic anhydrase inhibitor (CAIs) fragments of the sulfonamide type. Such compounds are proposed as new tools for the management of ache symptoms associated with rheumatoid arthritis (RA) and related inflammation diseases. The majority of the hybrids reported were effective in inhibiting the ubiquitous human (h) CA I and II as well as the RA overexpressed hCAs IX and XII isoforms, with KI values comprised of the low-medium nanomolar ranges. The antihyperalgesic activity of selected compounds was assessed by means of the paw-pressure and incapacitance tests using an in vivo RA model, and among them the hybrids 6B and 8B showed potent antinociceptive effects lasting up to 60 min after administration.

Sulphonamide inhibition studies of the ß-carbonic anhydrase from the bacterial pathogen Clostridium perfringens.

The ß-carbonic anhydrases (CAs, EC 4.2.1.1) from the pathogenic bacterium Clostridium perfringens (CpeCA) was recently characterised kinetically and for its anion inhibition profile. In the search of effective CpeCA inhibitors, possibly useful to inhibit the growth/pathogenicity of this bacterium, we report here an inhibition study of this enzyme with a panel of aromatic, heterocyclic and sugar sulphonamides/sulphamates. Some sulphonamides, such as acetazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, sulthiame and 4-(2-hydroxymethyl-4-nitrophenyl-sulphonamido)ethylbenzenesulphonamide were effective CpeCA inhibitors, with KIs in the range of 37.4-71.6 nM. Zonisamide and saccharin were the least effective such inhibitors, whereas many other aromatic and heterocyclic sulphonamides were moderate - weak inhibitors with KIs ranging between 113 and 8755 nM. Thus, this study provides the basis for developing better clostridial enzyme inhibitors with potential as antiinfectives with a new mechanism of action.

1,3-Oxazole-based selective picomolar inhibitors of cytosolic human carbonic anhydrase II alleviate ocular hypertension in rabbits: Potency is supported by X-ray crystallography of two leads.

Two lead 1,3-oxazole-based carbonic anhydrase inhibitors (CAIs) earlier identified as selective, picomolar inhibitors of hCA II (a cytosolic target for treatment of glaucoma) have been investigated further. Firstly, they were found to be conveniently synthesized on multigram scale, which enables further development. These compounds were found to be comparable in efficacy to dorzolamide eye drops when applied in the eye drop form as well. Finally, the reasons for unusually high potency of these compounds became understood from their high-resolution X-ray crystallography structures. These data significantly expand our understanding of heterocycle-based primary sulfonamides, many of which have recently emerged from our labs - particularly, from the corneal permeability standpoint.

Carbonic anhydrase activation enhances object recognition memory in mice through phosphorylation of the extracellular signal-regulated kinase in the cortex and the hippocampus.

Rats injected with by d-phenylalanine, a carbonic anhydrase (CA) activator, enhanced spatial learning, whereas rats given acetazolamide, a CA inhibitor, exhibited impairments of fear memory consolidation. However, the related mechanisms are unclear. We investigated if CAs are involved in a non-spatial recognition memory task assessed using the object recognition test (ORT). Systemic administration of acetazolamide to male CD1 mice caused amnesia in the ORT and reduced CA activity in brain homogenates, while treatment with d-phenylalanine enhanced memory and increased CA activity. We provided also the first evidence that d-phenylalanine administration rapidly activated extracellular signal-regulated kinase (ERK) pathways, a critical step for memory formation, in the cortex and the hippocampus, two brain areas involved in memory processing. Effects elicited by d-phenylalanine were completely blunted by co-administration of acetazolamide, but not of 1-N-(4-sulfamoylphenyl-ethyl)-2,4,6-trimethylpyridinium perchlorate (C18), a CA inhibitor that, differently from acetazolamide, does not cross the blood brain barrier. Our results strongly suggest that brain but not peripheral CAs activation potentiates memory as a result of ERK pathway enhanced activation.

The crystal structures of native hydroquinone 1,2-dioxygenase from Sphingomonas sp. TTNP3 and of substrate and inhibitor complexes.

The crystal structure of hydroquinone 1,2-dioxygenase, a Fe(II) ring cleaving dioxygenase from Sphingomonas sp. strain TTNP3, which oxidizes a wide range of hydroquinones to the corresponding 4-hydroxymuconic semialdehydes, has been solved by Molecular Replacement, using the coordinates of PnpCD from Pseudomonas sp. strain WBC-3. The enzyme is a heterotetramer, constituted of two subunits α and two ß of 19 and 38kDa, respectively. Both the two subunits fold as a cupin, but that of the small α subunit lacks a competent metal binding pocket. Two tetramers are present in the asymmetric unit. Each of the four ß subunits in the asymmetric unit binds one Fe(II) ion. The iron ion in each ß subunit is coordinated to three protein residues, His258, Glu264, and His305 and a water molecule. The crystal structures of the complexes with the substrate methylhydroquinone, obtained under anaerobic conditions, and with the inhibitors 4-hydroxybenzoate and 4-nitrophenol were also solved. The structures of the native enzyme and of the complexes present significant differences in the active site region compared to PnpCD, the other hydroquinone 1,2-dioxygenase of known structure, and in particular they show a different coordination at the metal center.

Design and Synthesis of Novel Nonsteroidal Anti-Inflammatory Drugs and Carbonic Anhydrase Inhibitors Hybrids (NSAIDs-CAIs) for the Treatment of Rheumatoid Arthritis.

We report the synthesis of a series of hybrid compounds incorporating 6- and 7-substituted coumarins (carbonic anhydrase, CA inhibitors) derivatized with clinically used NSAIDs (indomethacin, sulindac, ketoprofen, ibuprofen, diclofenac, ketorolac, etc., cyclooxygenase inhibitors) as agents for the management of rheumatoid arthritis (RA). Most compounds were effective in inhibiting the RA overexpressed hCA IX and XII, with KI values in the low nanomolar-subnanomolar ranges. The antihyperalgesic activity of such compounds was assessed by means of the paw-pressure and incapacitance tests using an in vivo RA model. Among all tested compounds, the 7-coumarine hybrid with ibuprofen showed potent and persistent antihyperalgesic effect up to 60 min after administration.

Polyamines and α-Carbonic Anhydrases.

Natural products represent a straightforward source for molecular structures bearing a vast array of chemical features and potentially useful for biomedical purposes. Recent examples of this type include the discovery of the coumarins and the polyamine natural products as atypical chemotypes for the inhibition of the metalloenzymes carbonic anhydrases (CAs; EC 4.2.2.1). CA enzymes are established pharmacological targets for important pathologies, which, among others, include glaucoma, hypoxic tumors, and central nervous system (CNS)-affecting diseases. Moreover, they are expressed in many bacteria, fungi and helminths which are the etiological agents of the majority of infectious diseases. In this context, natural products represent the ideal source of new and selective druggable CA modulators for biomedical purposes. Herein we report the state of the art on polyamines of natural origin as well as of synthetic derivatives as inhibitors of human CAs.

Biodegradation of 2-naphthalensulfonic acid polymers by white-rot fungi: Scale-up into non-sterile packed bed bioreactors.

This paper presents a first scale up under non-sterile conditions of the biodegradation process of 2-naphthalensulfonic acid polymers (NSAP) contained in a petrochemical wastewater by two white-rot fungi (Bjerkandera adusta and Pleurotus ostreatus). The biodegradation experiment was conducted first in flasks and then in packed-bed bioreactors filled with inert and biodegradable carriers (straw), the latter acting as both physical support and carbon source. Reactor inoculated with P. ostreatus attached on straw worked under non-sterile conditions for three months showing 30 ± 5% NSAP degradation. Respirometric tests showed that the fungal treatment was also able to significantly increase the biodegradable fraction of the wastewater COD, which rose from 9% to 40%. It was observed that the fungal degradation of the straw in the bed releases non-biodegradable by-products. Taking into account this contribution to nbCOD, the combined treatment of fungi and activated sludge could theoretically be able to reduce the original COD by up to 73%.

Kinetic and X-ray crystallographic investigations of substituted 2-thio-6-oxo-1,6-dihydropyrimidine-benzenesulfonamides acting as carbonic anhydrase inhibitors.

Herein we report an in vitro kinetic evaluation against the most relevant human carbonic anhydrase (hCA, EC 4.2.1.1) isoforms (I, II, IX and XII) of a small series of lactate dehydrogenase (LDH, EC 1.1.1.27) inhibitors. All compounds contain a primary sulfonamide zinc-binding group (ZBG) substituted with the 2-thio-6-oxo-1,6-dihydropyrimidine scaffold. By means of X-ray crystallographic experiments we explored the ligand-enzyme binding modes, thus highlighting the contribution of the 2-thio-6-oxo-1,6-dihydropyrimidine moiety to the stabilization of the complex.

Monothiocarbamates Strongly Inhibit Carbonic Anhydrases in Vitro and Possess Intraocular Pressure Lowering Activity in an Animal Model of Glaucoma.

A series of monothiocarbamates (MTCs) were prepared from primary/secondary amines and COS as potential carbonic anhydrase (CA, EC 4.2.1.1) inhibitors, using the dithiocarbamates, the xanthates, and the trithiocarbonates as lead compounds. The MTCs effectively inhibited the pharmacologically relevant human (h) hCAs isoforms I, II, IX, and XII in vitro and showed KIs spanning between the low and medium nanomolar range. By means of a computational study, the MTC moiety binding mode on the CAs was explained. Furthermore, a selection of MTCs were evaluated in a normotensive glaucoma rabbit model for their intraocular pressure (IOP) lowering effects and showed interesting activity.

Sulfonamide inhibition studies of the γ-carbonic anhydrase from the Antarctic bacterium Colwellia psychrerythraea.

The Antarctic bacterium Colwellia psychrerythraea encodes for a γ-class carbonic anhydrase (CA, EC 4.2.1.1), which was cloned, purified and characterized. The enzyme (CpsCAγ) has a moderate catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a k(cat) 6.0×10(5) s(-1) and a k(cat)/K(m) of 4.7×10(6) M(-1) s(-1). A series of sulfonamides and a sulfamate were investigated as inhibitors of the new enzyme. The best inhibitor was metanilamide (K(I) of 83.5 nM) followed by indisulam, valdecoxib, celecoxib, sulthiame and hydrochlorothiazide (K(I)s ranging between 343 and 491 nM). Acetazolamide, methazolamide as well as other aromatic/heterocyclic derivatives showed inhibition constants between 502 and 7660 nM. The present study may shed some more light regarding the role that γ-CAs play in the life cycle of psychrophilic bacteria as the Antarctic one investigated here, by allowing the identification of inhibitors which may be useful as pharmacologic tools.

Rosmarinic acid inhibits some metabolic enzymes including glutathione S-transferase, lactoperoxidase, acetylcholinesterase, butyrylcholinesterase and carbonic anhydrase isoenzymes.

Rosmarinic acid (RA) is a natural polyphenol contained in many aromatic plants with promising biological activities. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates. Acetylcholinesterase (AChE, E.C. 3.1.1.7) is intimately associated with the normal neurotransmission by catalysing the hydrolysis of acetylcholine to acetate and choline and acts in combination with butyrylcholinesterase (BChE) to remove acetylcholine from the synaptic cleft. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms, whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and in eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effects of rosmarinic acid on tumour-associated carbonic anhydrase IX and XII isoenzymes, AChE, BChE, LPO and GST enzymes were evaluated. Rosmarinic acid inhibited these enzymes with Kis in the range between micromolar to picomolar. The best inhibitory effect of rosmarinic acid was observed against both AChE and BChE.

The effect of caffeic acid phenethyl ester (CAPE) on metabolic enzymes including acetylcholinesterase, butyrylcholinesterase, glutathione S-transferase, lactoperoxidase, and carbonic anhydrase isoenzymes I, II, IX, and XII.

Caffeic acid phenethyl ester (CAPE) is an active component of honeybee propolis extracts. Carbonic anhydrases (CAs, EC 4.2.1.1) are widespread and intensively studied metalloenzymes present in higher vertebrates including humans as many diverse isoforms. Acetylcholinesterase (AChE) is responsible for acetyl choline (ACh) hydrolysis and plays a fundamental role in nerve impulse transmission by terminating the action of the ACh neurotransmitter at cholinergic synapses and neuromuscular junctions. Butyrylcholinesterase (BChE) is another enzyme abundantly present in the liver and released into blood in a soluble form. Lactoperoxidase (LPO) is an enzyme involved in fighting pathogenic microorganisms whereas glutathione S-transferases (GSTs) are dimeric proteins present both in prokaryotic and eukaryotic organisms and involved in cellular detoxification mechanisms. In the present study, the inhibition effect of CAPE on human carbonic anhydrase (hCA) isoforms I, II, IX, and XII, AChE, BChE, LPO, and GST was evaluated. CAPE inhibited these enzymes with Kis in the range between micromolar to picomolar. The best inhibitory effect was observed against AChE and BChE.

Fluorescent sulfonamide carbonic anhydrase inhibitors incorporating 1,2,3-triazole moieties: Kinetic and X-ray crystallographic studies.

Fluorescent sulfonamide carbonic anhydrase (CA, EC 4.2.1.1) inhibitors (CAIs) were essential for demonstrating the role played by the tumor-associated isoform CA IX in acidification of tumors, cancer progression towards metastasis and for the development of imaging and therapeutic strategies for the management of hypoxic tumors which overexpress CA IX. However, the presently available such compounds are poorly water soluble which limits their use. Here we report new fluorescent sulfonamides 7, 8 and 10 with increased water solubility. The new derivatives showed poor hCA I inhibitory properties, but were effective inhibitors against the hCA II (KIs of 366-127 nM), CA IX (KIs of 8.1-36.9 nM), CA XII (KIs of 4.1-20.5 nM) and CA XIV (KIs of 12.8-53.6 nM). A high resolution X-ray crystal structure of one of these compounds bound to hCA II revealed the factors associated with the good inhibitory properties. Furthermore, this compound showed a three-fold increase of water solubility compared to a similar derivative devoid of the triazole moiety, making it an interesting candidate for ex vivo/in vivo studies.

Thioxocoumarins Show an Alternative Carbonic Anhydrase Inhibition Mechanism Compared to Coumarins.

A series of coumarins and the corresponding 2-thioxocoumarines were prepared and tested for their inhibition profiles against four physiologically relevant human carbonic anhydrases (hCAs, EC 4.2.1.1), isoforms hCA I, II, IX, and XII. The X-ray crystal structure of 6-hydroxy-2-thioxocoumarin bound to hCA II revealed an unprecedented and unexpected inhibition mechanism for this new class of inhibitors when compared to isostructural coumarins. Unlike coumarins which are hydrolyzed by the esterase CA activity to the corresponding 2-hydroxy-cinnamic acid derivatives, the 2-thioxocoumarin was observed intact when bound to hCA II, with its exo-sulfur atom anchored to the zinc-coordinated water molecule, whereas the scaffold establishing favorable contacts with amino acid residues from the active site. This inhibition mechanism is very different from the one observed for hydrolyzed coumarins, which occlude the entrance of the active site cavity. This versatility in the binding mode of coumarins/thioxocoumarins has important consequences for the design of isoform-selective CA inhibitors, some of which are in clinical use or clinical development for various pathologies, among which glaucoma, edema, epilepsy, neuropathic pain, and hypoxic tumors.

Dithiocarbamates with potent inhibitory activity against the Saccharomyces cerevisiae ß-carbonic anhydrase.

Dithiocarbamates (DTCs) prepared from primary or secondary amines, which incorporated amino/hydroxyl-alkyl, mono-/bicyclic aliphatic/heterocyclic rings based on the quinuclidine, piperidine, hydroxy-/carboxy-/amino-substituted piperidine, morpholine and piperazine scaffolds, were investigated for the inhibition of α- and ß-carbonic anhydrases (CAs, EC 4.2.1.1) of pharmacologic relevance, such as the human (h) isoform hCA I and II, as well as the Saccharomyces cerevisiae ß-CA, scCA. The yeast and its ß-CA were shown earlier to be useful models of pathogenic fungal infections. The DTCs investigated here were medium potency hCA I inhibitors (K(I)s of 66.5-910 nM), were more effective as hCA II inhibitors (K(I)s of 8.9-107 nM) and some of them showed excellent, low nanomolar activity against the yeast enzyme, with inhibition constants ranging between 6.4 and 259 nM. The detailed structure activity relationship for inhibition of the yeast and human enzymes is discussed. Several of the investigated DTCs showed excellent selectivity ratios for inhibiting the yeast over the human cytosolic CA isoforms.

Synthesis and inhibition potency of novel ureido benzenesulfonamides incorporating GABA as tumor-associated carbonic anhydrase IX and XII inhibitors.

New ureido benzenesulfonamides incorporating a GABA moiety as a linker between the ureido and the sulfonamide functionalities were synthesized and their inhibition potency determined against both the predominant cytosolic (hCA I and II) and the transmembrane tumor-associated (hCA IX and XII) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1). The majority of these compounds were medium potency inhibitors of the cytosolic isoform hCA I and effective hCA II inhibitors, whereas they showed strong inhibition of the two transmembrane tumor-associated isoforms hCA IX and XII, with KIs in nanomolar range. Only one derivative had a good selectivity for inhibition of the tumor-associated hCA IX target isoform over the cytosolic and physiologically dominant off-target hCA I and II, being thus a potential tool to develop new anticancer agents.

Benzenesulfonamides incorporating bulky aromatic/heterocyclic tails with potent carbonic anhydrase inhibitory activity.

Three series of sulfonamides incorporating long, bulky tails were obtained by applying synthetic strategies in which substituted anthranilic acids, quinazolines and aromatic sulfonamides have been used as starting materials. They incorporate long, bulky diamide-, 4-oxoquinazoline-3-yl- or quinazoline-4-yl moieties in their molecules, and were investigated for the inhibition of four physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms, the cytosolic human (h) hCA I and II, as well as the transmembrane hCA IX and XII. Most of the new sulfonamides showed excellent inhibitory effects against the four isoforms, with KIs of 7.6-322nM against hCA I, of 0.06-85.4nM against hCA II; of 6.7-152nM against hCA IX and of 0.49-237nM against hCA XII; respectively. However no relevant isoform-selective behavior has been observed for any of them, although hCA II and XII, isoforms involved in glaucoma-genesis were the most inhibited ones. The structure-activity relationship for inhibiting the four CAs with these derivatives is discussed in detail.