Comparative CO2 and SiO2 hydratase activity of an enzyme from the siliceous demosponge Suberitesdomuncula.

Silicase, an enzyme that catalyzes the hydrolysis of silicon-oxygen bonds, is a crucial player in breaking down silicates into silicic acid, particularly in organisms like aquatic sponges with siliceous skeletons. Despite its significance, our understanding of silicase remains limited. This study comprehensively examines silicase from the demosponge Suberites domuncula, focusing on its kinetics toward CO2 as a substrate, as well as its silicase and esterase activity. It investigates inhibition and activation profiles with a range of inhibitors and activators belonging to various classes. By comparing its esterase activity to human carbonic anhydrase II, we gain insights into its enzymatic properties. Moreover, we investigate silicase's inhibition and activation profiles, providing valuable information for potential applications. We explore the evolutionary relationship of silicase with related enzymes, revealing potential functional roles in biological systems. Additionally, we propose a biochemical mechanism through three-dimensional modeling, shedding light on its catalytic mechanisms and structural features for both silicase activity and CO2 hydration. We highlight nature's utilization of enzymatic expertise in silica metabolism. This study enhances our understanding of silicase and contributes to broader insights into ecosystem functioning and Earth's geochemical cycles, emphasizing the intricate interplay between biology and the environment.

A comprehensive investigation of the anion inhibition profile of a ß-carbonic anhydrase from Acinetobacter baumannii for crafting innovative antimicrobial treatments.

This study refers to the intricate world of Acinetobacter baumannii, a resilient pathogenic bacterium notorious for its propensity at antibiotic resistance in nosocomial infections. Expanding upon previous findings that emphasised the bifunctional enzyme PaaY, revealing unexpected γ-carbonic anhydrase (CA) activity, our research focuses on a different class of CA identified within the A. baumannii genome, the ß-CA, designated as 𝛽-AbauCA (also indicated as CanB), which plays a crucial role in the resistance mechanism mediated by AmpC beta-lactamase. Here, we cloned, expressed, and purified the recombinant 𝛽-AbauCA, unveiling its distinctive kinetic properties and inhibition profile with inorganic anions (classical CA inhibitors). The exploration of 𝛽-AbauCA not only enhances our understanding of the CA repertoire of A. baumannii but also establishes a foundation for targeted therapeutic interventions against this resilient pathogen, promising advancements in combating its adaptability and antibiotic resistance.

Cloning, expression, and purification of an α-carbonic anhydrase from Toxoplasma gondii to unveil its kinetic parameters and anion inhibition profile.

Toxoplasmosis, induced by the intracellular parasite Toxoplasma gondii, holds considerable implications for global health. While treatment options primarily focusing on folate pathway enzymes have notable limitations, current research endeavours concentrate on pinpointing specific metabolic pathways vital for parasite survival. Carbonic anhydrases (CAs, EC 4.2.1.1) have emerged as potential drug targets due to their role in fundamental reactions critical for various protozoan metabolic processes. Within T. gondii, the Carbonic Anhydrase-Related Protein (TgCA_RP) plays a pivotal role in rhoptry biogenesis. Notably, α-CA (TcCA) from another protozoan, Trypanosoma cruzi, exhibited considerable susceptibility to classical CA inhibitors (CAIs) such as anions, sulphonamides, thiols, and hydroxamates. Here, the recombinant DNA technology was employed to synthesise and clone the identified gene in the T. gondii genome, which encodes an α-CA protein (Tg_CA), with the purpose of heterologously overexpressing its corresponding protein. Tg_CA kinetic constants were determined, and its inhibition patterns explored with inorganic metal-complexing compounds, which are relevant for rational compound design. The significance of this study lies in the potential development of innovative therapeutic strategies that disrupt the vital metabolic pathways crucial for T. gondii survival and virulence. This research may lead to the development of targeted treatments, offering new approaches to manage toxoplasmosis.

Leveraging SARS-CoV-2 Main Protease (Mpro) for COVID-19 Mitigation with Selenium-Based Inhibitors.

The implementation of innovative approaches is crucial in an ongoing endeavor to mitigate the impact of COVID-19 pandemic. The present study examines the strategic application of the SARS-CoV-2 Main Protease (Mpro) as a prospective instrument in the repertoire to combat the virus. The cloning, expression, and purification of Mpro, which plays a critical role in the viral life cycle, through heterologous expression in Escherichia coli in a completely soluble form produced an active enzyme. The hydrolysis of a specific substrate peptide comprising a six-amino-acid sequence (TSAVLQ) linked to a p-nitroaniline (pNA) fragment together with the use of a fluorogenic substrate allowed us to determine effective inhibitors incorporating selenium moieties, such as benzoselenoates and carbamoselenoates. The new inhibitors revealed their potential to proficiently inhibit Mpro with IC50-s in the low micromolar range. Our study contributes to the development of a new class of protease inhibitors targeting Mpro, ultimately strengthening the antiviral arsenal against COVID-19 and possibly, related coronaviruses.

Novel thiazolone-benzenesulphonamide inhibitors of human and bacterial carbonic anhydrases.

A small library of novel thiazolone-benzenesulphonamides has been prepared and evaluated for their ability to inhibit three human cytosolic carbonic anhydrases (hCA I, hCA II, and hCA VII) and three bacterial carbonic anhydrases (MscCAß, StCA1, and StCA2). All investigated hCAs were inhibited by the prepared compounds 4a-4j in the low nanomolar range. These compounds were effective hCA I inhibitors (KIs of 31.5-637.3 nM) and excellent hCA II (KIs in the range of 1.3-13.7 nM) and hCA VII inhibitors (KIs in the range of 0.9-14.6 nM). The most active analog in the series, 4-((4-oxo-5-propyl-4,5-dihydrothiazol-2-yl)amino)benzenesulphonamide 4d, strongly inhibited bacterial MscCAß, with KI of 73.6 nM, considerably better than AAZ (KI of 625 nM). The tested compounds displayed medium inhibitory potency against StCA1 (KIs of 69.2-163.3 nM) when compared to the standard drug (KI of 59 nM). However, StCA2 was poorly inhibited by the sulphonamides reported here, with KIs in the micromolar range between 275.2 and 4875.0 nM.

Inhibition studies with simple and complex (in)organic anions of the γ-carbonic anhydrase from Mammaliicoccus (Staphylococcus) sciuri, MscCAγ.

The γ-carbonic anhydrase (CA, EC 4.2.1.1) from the pathogenic bacterium, Mammaliicoccus (Staphylococcus) sciuri (MscCAγ) was recently cloned and purified by our groups. Here we investigated inhibition of this enzyme with (in)organic simple and complex anions, in the search of inhibitors with potential applications. The most effective inhibitors (KIs in the micromolar range) were peroxydisulfate and trithiocarbonate, whereas submillimolar inhibition was observed with N,N-diethyldithiocarbamate and phenylboronic acid (KIs of 0.5-0.9 mM). Thiocyanate, hydrogensulfide, bisulphite, stannate, divanadate, tetraborate, perrhenate, perruthenate, hexafluorophosphate, triflate and iminodisulfonate showed KIs of 1.0-13.7 mM. Cyanate, cyanide, azide, carbonate, nitrate, tellurate, selenocyanide, tetrafluoroborate, sulfamide, sulphamic acid and phenylarsonic acid were weaker inhibitors, with KIs in the range of 25.2-95.5 mM, whereas halides, bicarbonate, nitrite, sulphate, perchlorate and fluorosulfonate did not show inhibitory action up until 100 mM concentrations in the assay system. Finding more effective MscCAγ inhibitors may be helpful to fight drug resistance to antibiotics.

The gram-negative bacterium Escherichia coli as a model for testing the effect of carbonic anhydrase inhibition on bacterial growth.

Carbonic anhydrases, catalysing the reversible CO2 hydration reaction, contribute in all living organisms to the maintenance of stable metabolic functions depending on intracellular concentrations of carbon dioxide, bicarbonate, and protons. Recent studies have examined how CAs affect bacterial lifecycle, considering these enzymes druggable targets due to interference with their activities by using inhibitors or activators. Here, we propose Escherichia coli cells as a model for testing the effect of acetazolamide (AZA), a potent CA inhibitor, on bacterial survival by evaluating E. coli growth through its glucose consumption. AZA, at concentrations higher than 31.2 µg/mL, was able to impair E. coli growth and glucose uptake. AZA is a good inhibitor of the two recombinant E. coli CAs, the ß-CA CynT2, and the γ-CA EcoCAγ, with KIs of 227 and 248 nM, respectively. This study provides a proof-of-concept, low-cost method for identifying effective CA inhibitors capable of impairing bacterial metabolism.

Coumarins inhibit η-class carbonic anhydrase from Plasmodium falciparum.

Coumarins were discovered to act as inhibitors of α-carbonic anhydrases (CAs, EC 4.2.1.1) after undergoing hydrolysis mediated by the esterase activity of the enzyme to the corresponding 2-hydroxycinnamic acids. Other classes of CAs among the eight currently known do not possess esterase activity or this activity was poorly investigated. Hence, we decided to look at the potential of coumarins as inhibitors of the η-CA from the malaria-producing protozoan Plasmodium falciparum, PfaCA. A panel of simple coumarins incorporating hydroxyl, amino, ketone or carboxylic acid ester moieties in various positions of the ring system acted as low to medium micromolar PfaCA inhibitors, whereas their affinities for the cytosolic off-target human isoforms hCA I and II were in a much higher range. Thus, we confirm that η-CAs possess esterase activity and that coumarins effectively inhibit this enzyme. Elaboration of the simple coumarin scaffolds investigated here may probably lead to more effective PfaCA inhibitors.

Heterologous expression and biochemical characterisation of the recombinant ß-carbonic anhydrase (MpaCA) from the warm-blooded vertebrate pathogen malassezia pachydermatis.

Warm-blooded animals may have Malassezia pachydermatis on healthy skin, but changes in the skin microenvironment or host defences induce this opportunistic commensal to become pathogenic. Malassezia infections in humans and animals are commonly treated with azole antifungals. Fungistatic treatments, together with their long-term use, contribute to the selection and the establishment of drug-resistant fungi. To counteract this rising problem, researchers must find new antifungal drugs and enhance drug resistance management strategies. Cyclic adenosine monophosphate, adenylyl cyclase, and bicarbonate have been found to promote fungal virulence, adhesion, hydrolase synthesis, and host cell death. The CO2/HCO3-/pH-sensing in fungi is triggered by HCO3- produced by metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1). It has been demonstrated that the growth of M. globosa can be inhibited in vivo by primary sulphonamides, which are the typical CA inhibitors. Here, we report the cloning, purification, and characterisation of the ß-CA (MpaCA) from the pathogenic fungus M. pachydermatis, which is homologous to the enzyme encoded in the genome of M. globosa and M. restricta, that are responsible for dandruff and seborrhoeic dermatitis. Fungal CAs could be thus considered a new pharmacological target for combating fungal infections and drug resistance developed by most fungi to the already used drugs.

A comparative study of carbonic anhydrase activity in lymphocytes from colorectal cancer tissues and adjacent healthy counterparts.

Several carbonic anhydrase (CA, EC 4.2.1.1) isoforms play an essential role in processes connected to tumorigenesis, as they efficiently accelerate the hydration of carbon dioxide to bicarbonate and proton. In this context, examples are CA IX and CA XII, which were proved to be upregulated in many solid malignancies. On the other hand, cancer and the immune system are inextricably linked, and targeting the immune checkpoints recently was shown to efficiently improve the treatment of malignancies. In this study, we have investigated the expression of CA isoforms in tumour-infiltrating lymphocytes (TILs) that, according to the immunosurveillance theory, were suggested to have a crucial role in the development of colorectal cancer (CRC). T lymphocytes isolated from healthy surrounding mucosa showed a higher CA activity compared to those present in tumour and peripheral blood in the same patients. CA I and II were confirmed as enzyme isoforms involved in the process, as determined by proteomic analysis of corresponding TIL samples. These preliminary findings suggest a dysregulation of the local immune response in the CRC tissues and a loss of effective anticancer mechanisms mediated by CAs therein.

Synthesis and biological evaluation of sulfonamide-based compounds as inhibitors of carbonic anhydrase from Vibrio cholerae.

This study reports our continued efforts to identify inhibitors capable of targeting carbonic anhydrases (CAs) expressed in bacteria. Based on previously identified chemotypes, we designed and synthesized new analogs that were screened toward the α, ß, and γ classes encoded in Vibrio cholerae (Vch). The Ki values measured in the stopped-flow hydrase assay revealed that very simple structural modifications might induce a relevant impact on the inhibitory effects as well as the selectivity profile over ubiquitous human isozymes (hCA I/II). Unfortunately, the best active VchCA inhibitors demonstrated a dramatic loss of hCA II selectivity when compared to previously reported compounds. Among the new series of sulfonamides, several molecules proved to be about sevenfold more potent against VchCAγ than the reference compound acetazolamide, thus furnishing new insights for further development of inhibitors targeting CAs expressed in bacteria.

May Sulfonamide Inhibitors of Carbonic Anhydrases from Mammaliicoccus sciuri Prevent Antimicrobial Resistance Due to Gene Transfer to Other Harmful Staphylococci?

Mammaliicoccus sciuri, previously known as Staphylococcus sciuri, is a Gram-positive bacterium involved in gene transfer phenomena that confer resistance to multiple antibiotics. These plasmid-encoded genes can be easily transferred to other pathogenic staphylococci. Because antibiotic resistance is rising, inhibiting M. sciuri proliferation may be a credible strategy for restricting antimicrobial resistance gene transfer to other pathogenic bacteria. Recently, it has been shown that blocking bacterial carbonic anhydrases (CAs, EC 4.2.1.1), metalloenzymes sustaining bacterial metabolic activities, can reduce pathogen survival and fitness. Here, the recombinant M. sciuri γ-CA (MscCAγ) has been cloned and purified, utilizing the DNA recombinant technology. Its kinetic properties for the CO2 hydration reaction, as well as the sulfonamide inhibition profile, were investigated and compared with those reported earlier for MscCAß (previously described as SauBCA) and the two off-target human CA isoforms (hCA I and hCA II). The recombinant MscCAγ showed significant hydratase activity. Moreover, the MscCAγ sulfonamide inhibitory profile was different from that of MscCAß, implying that a varied amino acid set typifies the catalytic pocket of the two enzymes. These differences provide additional evidence for the possibility of developing novel CA class-specific inhibitors.

Benzenesulfonamides Incorporating Hydantoin Moieties Effectively Inhibit Eukaryoticand Human Carbonic Anhydrases.

A series of novel 1-(4-benzenesulfonamide)-3-alkyl/benzyl-hydantoin derivatives were synthesized and evaluated for the inhibition of eukaryotic and human carbonic anhydrases (CAs, EC 4.2.1.1). The prepared compounds were screened for their hCA inhibitory activities against three cytosolic isoforms as well as two ß-CAs from fungal pathogens. The best inhibition was observed against hCA II and VII as well as Candida glabrata enzyme CgNce103. hCA I and Malassezia globosa MgCA enzymes were, on the other hand, less effectively inhibited by these compounds. The inhibitory potency of these compounds against CAs was found to be dependent on the electronic and steric effects of substituent groups on the N3-position of the hydantoin ring, which included alkyl, alkenyl and substituted benzyl moieties. The interesting results against CgNce103 make the compounds of interest for investigations in vivo as potential antifungals.

Anion inhibition studies of the Zn(II)-bound ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii.

Burkholderia territorii, a Gram-negative bacterium, encodes for the ι-class carbonic anhydrase (CA, EC 4.2.1.1) BteCAι, which was recently characterised. It acts as a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat value of 3.0 × 105 s-1 and kcat/KM value of 3.9 × 107 M-1 s-1. No inhibition data on this new class of enzymes are available to date. We report here an anion and small molecules inhibition study of BteCAι, which we prove to be a zinc(II)- and not manganese(II)-containing enzyme, as reported for diatom ι-CAs. The best inhibitors were sulphamic acid, stannate, phenylarsonic acid, phenylboronic acid and sulfamide (KI values of 6.2-94 µM), whereas diethyldithiocarbamate, tellurate, selenate, bicarbonate and cyanate were submillimolar inhibitors (KI values of 0.71-0.94 mM). The halides (except iodide), thiocyanate, nitrite, nitrate, carbonate, bisulphite, sulphate, hydrogensulfide, peroxydisulfate, selenocyanate, fluorosulfonate and trithiocarbonate showed KI values in the range of 3.1-9.3 mM.

Effect of amino acids and amines on the activity of the recombinant ι-carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii.

We here report a study on the activation of the ι-class bacterial CA from Burkholderia territorii (BteCAι). This protein was recently characterised as a zinc-dependent enzyme that shows a significant catalytic activity (kcat 3.0 × 105 s-1) for the physiological reaction of CO2 hydration to bicarbonate and protons. Some amino acids and amines, among which some proteinogenic derivatives as well as histamine, dopamine and serotonin, showed efficient activating properties towards BteCAι, with activation constants in the range 3.9-13.3 µM. L-Phe, L-Asn, L-Glu, and some pyridyl-alkylamines, showed a weaker activating effect towards BteCAι, with KA values ranging between 18.4 µM and 45.6 µM. Nowadays, no information is available on active site architecture, metal ion coordination and catalytic mechanism of members of the ι-group of CAs, and this study represents another contribution towards a better understanding of this still uncharacterised class of enzymes.

Effect of Sulfonamides and Their Structurally Related Derivatives on the Activity of ι-Carbonic Anhydrase from Burkholderia territorii

Carbonic anhydrases (CAs) are essential metalloenzymes in nature, catalyzing the carbon dioxide reversible hydration into bicarbonate and proton. In humans, breathing and many other critical physiological processes depend on this enzymatic activity. The CA superfamily function and inhibition in pathogenic bacteria has recently been the object of significant advances, being demonstrated to affect microbial survival/virulence. Targeting bacterial CAs may thus be a valid alternative to expand the pharmacological arsenal against the emergence of widespread antibiotic resistance. Here, we report an extensive study on the inhibition profile of the recently discovered ι-CA class present in some bacteria, including Burkholderia territorii, namely BteCAι, using substituted benzene-sulfonamides and clinically licensed sulfonamide-, sulfamate- and sulfamide-type drugs. The BteCAι inhibition profile showed: (i) several benzene-sulfonamides with an inhibition constant lower than 100 nM; (ii) a different behavior with respect to other α, ß and γ-CAs; (iii) clinically used drugs having a micromolar affinity. This prototype study contributes to the initial recognition of compounds which efficiently and selectively inhibit a bacterial member of the ι-CA class, for which such a selective inhibition with respect to other protein isoforms present in the host is highly desired and may contribute to the development of novel antimicrobials.

Inhibitory Effects of Sulfonamide Derivatives on the ß-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 ß-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.

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.

Use of an immobilised thermostable α-CA (SspCA) for enhancing the metabolic efficiency of the freshwater green microalga Chlorella sorokiniana.

There is significant interest in increasing the microalgal efficiency for producing high-quality products that are commonly used as food additives in nutraceuticals. Some natural substances that can be extracted from algae include lipids, carbohydrates, proteins, carotenoids, long-chain polyunsaturated fatty acids, and vitamins. Generally, microalgal photoautotrophic growth can be maximised by optimising CO2 biofixation, and by adding sodium bicarbonate and specific bacteria to the microalgal culture. Recently, to enhance CO2 biofixation, a thermostable carbonic anhydrase (SspCA) encoded by the genome of the bacterium Sulfurihydrogenibium yellowstonense has been heterologously expressed and immobilised on the surfaces of bacteria. Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes, which catalyse the physiologically reversible reaction of carbon dioxide hydration to bicarbonate and protons: CO2 + H2O ⇄ HCO3- + H+. Herein, we demonstrate for the first time that the fragments of bacterial membranes containing immobilised SspCA (M-SspCA) on their surfaces can be doped into the microalgal culture of the green unicellular alga, Chlorella sorokiniana, to significantly enhance the biomass, photosynthetic activity, carotenoids production, and CA activity by this alga. These results are of biotechnological interest because C. sorokiniana is widely used in many different areas, including photosynthesis research, human pharmaceutical production, aquaculture-based food production, and wastewater treatment.

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 ß-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.