RESUMEN
The application of sound wave technology to different plant species has revealed that variations in the Hz, sound pressure intensity, treatment duration, and type of setup of the sound source significantly impact the plant performance. A study conducted on cotton plants treated with Plant Acoustic Frequency Technology (PAFT) highlighted improvements across various growth metrics. In particular, the treated samples showed increases in the height, size of the fourth expanded leaf from the final one, count of branches carrying bolls, quantity of bolls, and weight of individual bolls. Another study showed how the impact of a 4 kHz sound stimulus positively promoted plant drought tolerance. In other cases, such as in transgenic rice plants, GUS expression was upregulated at 250 Hz but downregulated at 50 Hz. In the same way, sound frequencies have been found to enhance the osmotic potential, with the highest observed in samples treated with frequencies of 0.5 and 0.8 kHz compared to the control. Furthermore, a sound treatment with a frequency of 0.4 kHz and a sound pressure level (SPL) of 106 dB significantly increased the paddy rice germination index, as evidenced by an increase in the stem height and relative fresh weight. This paper presents a complete, rationalized and updated review of the literature on the effects of sound waves on the physiology and growth parameters of sound-treated plants.
RESUMEN
Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation. We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5' end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.
RESUMEN
Peroxidases are widespread key antioxidant enzymes that catalyse the oxidation of electron donor substrates in parallel with the decomposition of H2O2. In this work, a novel tomato peroxidase, named SAAP2, was isolated from MicroTom cell cultures, purified, and characterised. The enzyme was identified with 64% sequence coverage as the leprx21 gene product (suberization-associated anionic peroxidase 2-like) from Solanum lycopersicum, 334 amino acids long. Compared to other plant peroxidases, SAAP2 was more active at elevated temperatures, with the optimal temperature and pH at 90 °C and 5.0, respectively. Furthermore, the enzyme retained more than 80% of its maximal activity over the range of 70-80 °C and the presence of NaCl (1.0-4.5 M). It also exhibited broad pH versatility (65% relative activity over the pH range 2.0-7.0), acid-tolerance (80% residual activity after 22 h at pH 2.0-7.0), high thermostability (50% residual activity after 2 h at 80 °C) and proteolytic resistance. SAAP2 exhibited exceptional resistance under thermo-acidic conditions compared to the horseradish peroxidase benchmark, suggesting that it may find potential applications as a supplement or anti-pollution agent in the food industry.
Asunto(s)
Extremófilos , Peroxidasa , Peróxido de Hidrógeno , Peroxidasas , Peroxidasa de Rábano Silvestre , Técnicas de Cultivo de Célula , ColorantesRESUMEN
In this paper, the efficiency of the carbonic anhydrase (CA) enzyme in accelerating the hydration of CO2 is evaluated using a measurement system which consists of a vessel in which a gaseous flow of mixtures of nitrogen and CO2 is bubbled into water or water solutions containing a known quantity of CA enzyme. The pH value of the solution and the CO2 concentration at the measurement system gas exhaust are continuously monitored. The measured CO2 level allows for assessing the quantity of CO2, which, subtracted from the gaseous phase, is dissolved into the liquid phase and/or hydrated to bicarbonate. The measurement procedure consists of inducing a transient and observing and modelling the different kinetics involved in the steady-state recovery with and without CA. The main contribution of this work is exploiting dynamical system theory and chemical kinetics modelling for interpreting measurement results for characterising the activity of CA enzymes. The data for model fitting are obtained from a standard bioreactor, in principle equal to standard two-phase bioreactors described in the literature, in which two different techniques can be used to move the process itself away from the steady-state, inducing transients.
Asunto(s)
Dióxido de Carbono/metabolismo , Anhidrasas Carbónicas/metabolismo , Modelos Químicos , Reactores Biológicos , Dióxido de Carbono/química , Anhidrasas Carbónicas/química , Concentración de Iones de Hidrógeno , CinéticaRESUMEN
A series of urea/thiourea substituted benzoxaboroles was investigated for the inhibition of the three carbonic anhydrases encoded by Vibrio cholerae (VchCAα, VchCAß, and VchCAγ). In particular, benzoxaborole derivatives were here first assayed for the inhibition of a γ-class CA, extending the panel of CA classes that benzoxaboroles efficiently target beyond α and ß. Inhibition profiles demonstrated that VchCAα was significantly more inhibited compared to VchCAγ and, in turn, more efficiently modulated than VchCAß. Among the many selective benzoxaborole ligands detected against VchCAα over the off-target hCA II, compound 18, a p-NO2-phenylthiourea derivative, even exhibited a fully selective inhibition profile against the three VchCAs over hCA II. A comprehensive ligand/target interaction study was performed in silico for all three VchCA isoforms providing the first molecular modeling investigation with inhibitors of a γ-class CA to the best of our knowledge. The present study reinforces the rationale behind the use of benzoxaboroles as innovative antibacterial agents with a new mechanism of action, furnishing suggestions for the rational design of new potent and selective inhibitors targeting V. cholerae CAs over human off-target ones.
RESUMEN
Carbonic anhydrases from Vibrio cholerae (VchCAs) play a significant role in bacterial pathophysiological processes. Therefore, their inhibition leads to a reduction of gene expression virulence and bacterial growth impairment. Herein, we report the first ligand-based pharmacophore model as a computational tool to study selective inhibitors of the ß-class of VchCA. By a virtual screening on a collection of sulfonamides, we retrieved 9 compounds that were synthesized and evaluated for their inhibitory effects against VchCAß as well as α- and γ-classes of VchCAs and selectivity over human ubiquitous isoforms hCA I and II. Notably, all tested compounds were active inhibitors of VchCAs. The N-(4-sulfamoylbenzyl)-[1,1'-biphenyl]-4-carboxamide (20e) stood out as the most exciting inhibitor toward the ß-class (K i = 95.6 nM), also showing a low affinity against the tested human isoforms. By applying docking procedures, we described the binding mode of the inhibitor 20e within the catalytic cavity of the modeled open conformation of VchCAß.
RESUMEN
Carbonic anhydrases (CAs, EC 4.2.1.1) are ubiquitous metalloenzymes involved in biosynthetic processes, transport, supply, and balance of CO2/HCO3 - into the cell. In Bacteria, CAs avoid the depletion of the dissolved CO2/HCO3 - from the cell, providing them to the central metabolism that is compromised without the CA activity. The involvement of CAs in the survival, pathogenicity, and virulence of several bacterial pathogenic species is recent. Here, we report the kinetic properties of the recombinant γ-CA (EcoCAγ) encoded in the genome of Escherichia coli. EcoCAγ is an excellent catalyst for the physiological CO2 hydration reaction to bicarbonate and protons, with a kcat of 5.7 × 105 s-1 and kcat/KM of 6.9 × 106 M-1 s-1. The EcoCAγ inhibition profile with a broad series of known CA inhibitors, the substituted benzene-sulphonamides, and clinically licenced drugs was explored. Benzolamide showed a KI lower than 100 nM. Our study reinforces the hypothesis that the synthesis of new drugs capable of interfering selectively with the bacterial CA activity, avoiding the inhibition of the human α -CAs, is achievable and may lead to novel antibacterials.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Escherichia coli/enzimología , Sulfonamidas/farmacología , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Estructura Molecular , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Coumarins are widely diffused secondary metabolites possessing a plethora of biological activities. It has been established that coumarins represent a peculiar class of human carbonic anhydrase (hCA) inhibitors having a distinct mechanism of action involving a non-classical binding with amino acid residues paving the entrance of hCA catalytic site. Herein, we report the synthesis of a small series of new coumarin derivatives 7-11, 15, 17 prepared via classical Pechmann condensation starting from resorcinol derivatives and suitable ß-ketoesters. The evaluation of inhibitory activity revealed that these compounds possessed nanomolar affinity and high selectivity towards tumour-associated hCA IX and XII over cytosolic hCA I and hCA II isoforms. To investigate the binding mode of these new coumarin-inspired inhibitors, the most active compounds 10 and 17 were docked within hCA XII catalytic cleft.
Asunto(s)
Anhidrasa Carbónica IX/antagonistas & inhibidores , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Simulación del Acoplamiento Molecular , Neoplasias/enzimología , Umbeliferonas/farmacología , Antígenos de Neoplasias/metabolismo , Anhidrasa Carbónica IX/metabolismo , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Relación Estructura-Actividad , Umbeliferonas/síntesis química , Umbeliferonas/químicaRESUMEN
A ß-carbonic anhydrase (CA, EC 4.2.1.1) from the widespread bacterium Escherichia coli (EcoCAß), encoded by the CynT2 gene, has been investigated for its catalytic properties and enzymatic activation by a panel of amino acids and amines. EcoCAß showed a significant catalytic activity for the hydration of CO2 to bicarbonate and a proton, with a kinetic constant kcat of 5.3 × 105 s- and a Michaelis-Menten constant KM of 12.9 mM. The most effective EcoCAß activators were L- and D-DOPA, L-Tyr, 4-amino-Phe, serotonin and L-adrenaline, with KAs from 2.76 to 10.7 µM. L-His, 2-pyridyl-methylamine, L-Asn and L-Gln were relatively weak activators (KAs from 36.0 to 49.5 µM). D-His, L- and D-Phe, L- and D-Trp, D-Tyr, histamine, dopamine, 2-(aminoethyl)pyridine/piperazine/morpholine, L-Asp, L- and D-Glu have KAs from 11.3 to 23.7 µM. Endogenous CA activators may play a role in bacterial virulence and colonisation of the host.
Asunto(s)
Aminas/metabolismo , Aminoácidos/metabolismo , Anhidrasas Carbónicas/metabolismo , Escherichia coli/enzimología , Catálisis , Activación Enzimática , Cinética , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Antibacterianos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Proteínas de Escherichia coli/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacología , Proteínas de Transporte de Anión/antagonistas & inhibidores , Proteínas de Transporte de Anión/genética , Antibacterianos/química , Benceno/química , Dióxido de Carbono/química , Dióxido de Carbono/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/genética , Evaluación Preclínica de Medicamentos/métodos , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/genética , Humanos , Relación Estructura-ActividadRESUMEN
The interconversion of CO2 and HCO3- is catalyzed by a superfamily of metalloenzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1), which maintain the equilibrium between dissolved inorganic CO2 and HCO3-. In the genome of Escherichia coli, a Gram-negative bacterium typically colonizing the lower intestine of warm-blooded organisms, the cyn operon gene includes the CynT gene, encoding for a ß-CA, and CynS gene, encoding for the cyanase. CynT (ß-CA) prevents the depletion of the cellular bicarbonate, which is further used in the reaction catalyzed by cyanase. A second ß-CA (CynT2 or Can or yadF), as well as a γ and ι-CAs were also identified in the E. coli genome. CynT2 is essential for bacterial growth at atmospheric CO2 concentration. Here, we characterized the kinetic properties and the anion inhibition profiles of recombinant CynT2. The enzyme showed a good activity for the physiological CO2 hydratase reaction with the following parameters: kcat = 5.3 × 105 s-1 and kcat/KM = of 4.1 × 107 M-1 s-1. Sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethyldithiocarbamate were the most effective CynT2 inhibitors (KI = 2.5 to 84 µM). The anions allowed for a detailed understanding of the interaction of inhibitors with the amino acid residues surrounding the catalytic pocket of the enzyme and may be used as leads for the design of more efficient and specific inhibitors.
Asunto(s)
Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/química , Escherichia coli/enzimología , Proteínas de Transporte de Anión/metabolismo , Aniones , Arsenicales , Ácidos Borónicos/química , Dióxido de Carbono/química , Anhidrasa Carbónica I/química , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Catálisis , Ditiocarba/química , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano , Humanos , Concentración de Iones de Hidrógeno , Cinética , Isoformas de Proteínas , Estructura Secundaria de Proteína , Protones , Proteínas Recombinantes/química , Ácidos Sulfónicos/química , Vibrio cholerae/metabolismoRESUMEN
Carbonic anhydrase (CA) is a zinc enzyme that catalyzes the reversible conversion of carbon dioxide to bicarbonate and proton. Currently, CA inhibitors are widely used as antiglaucoma, anticancer, and anti-obesity drugs and for the treatment of neurological disorders. Recently, the potential use of CA inhibitors to fight infections caused by protozoa, fungi, and bacteria has emerged as a new research line. In this article, the X-ray crystal structure of ß-CA from Burkholderia pseudomallei was reported. The X-ray crystal structure of this new enzyme was solved at 2.7 Å resolution, revealing a tetrameric type II ß-CA with a "closed" active site in which the zinc is tetrahedrally coordinated to Cys46, Asp48, His102, and Cys105. B. pseudomallei is known to encode at least two CAs, a ß-CA, and a γ-CA. These proteins, playing a pivotal role in its life cycle and pathogenicity, offer a novel therapeutic opportunity to obtain antibiotics with a different mechanism of action. Furthermore, the new structure can provide a clear view of the ß-CA mechanism of action and the possibility to find selective inhibitors for this class of CAs.
Asunto(s)
Proteínas Bacterianas , Burkholderia pseudomallei/enzimología , Anhidrasa Carbónica II , Inhibidores de Anhidrasa Carbónica/química , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica II/química , Dominio Catalítico , Cristalografía por Rayos X , Estructura Cuaternaria de ProteínaRESUMEN
The carbonic anhydrases (CAs, EC 4.2.1.1) catalyse a simple but physiologically crucial reversible reaction, the carbon dioxide hydration with the production of bicarbonate and protons. In the last years, and especially, to the rapid emergence of the bacterial antibiotic resistance that is occurring worldwide, the understanding of the function of bacterial CAs has increased significantly. Recently, a new CA-class (ι-CA) was discovered in the marine diatom T. pseudonana. It has been reported that bacterial genomes may contain genes with relevant homology to the diatom ι-class CA. Still, the catalytic activity of the enzyme encoded by the gene was not investigated. Thus, herein, for the first time, we cloned, expressed, and purified the recombinant bacterial ι-CA (acronym BteCAι) identified in the genome of Burkholderia territorii. The recombinant BteCAι resulted in a good catalyst for the hydration of CO2 to bicarbonate and protons, with a kcat of 3.0 × 105 s -1 and kcat/KM of 3.9 × 107 M -1 s -1, and is also sensitive to inhibition by the sulphonamide acetazolamide. Furthermore, with the aid of the protonography, it has been demonstrated that BteCAι can be present as a dimer. This result is corroborated by the construction of a molecular model of BteCAι, which showed that the enzyme is formed by two equivalent monomers having a structure similar to a butterfly.
Asunto(s)
Acetazolamida/farmacología , Burkholderia/enzimología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Sulfonamidas/farmacología , Acetazolamida/química , Secuencia de Aminoácidos , Inhibidores de Anhidrasa Carbónica/química , Anhidrasas Carbónicas/genética , Anhidrasas Carbónicas/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Estructura Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
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 â 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.
Asunto(s)
Bacterias/enzimología , Anhidrasas Carbónicas/metabolismo , Chlorella/metabolismo , Bacterias/citología , Bacterias/crecimiento & desarrollo , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , HumanosRESUMEN
The critical CO2 hydration reaction to bicarbonate and protons is catalyzed by carbonic anhydrases (CAs, EC 4.2.1.1). Their physiological role is to assist the transport of the CO2 and HCO3- at the cellular level, which will not be ensured by the low velocity of the uncatalyzed reaction. CA inhibition may impair the growth of microorganisms. In the yeasts, Candida albicans and Malassezia globosa, the activity of the unique ß-CA identified in their genomes was demonstrated to be essential for growth of the pathogen. Here, we decided to investigate the sulfonamide inhibition profile of the homologous ß-CA (MreCA) identified in the genome of Malassezia restricta, an opportunistic pathogen triggering dandruff and seborrheic dermatitis. Among 40 investigated derivatives, the best MreCA sulfonamide inhibitors were dorzolamide, brinzolamide, indisulam, valdecoxib, sulthiam, and acetazolamide (KI < 1.0 µM). The MreCA inhibition profile was different from those of the homologous enzyme from Malassezia globosa (MgCA) and the human isoenzymes (hCA I and hCA II). These results might be useful to for designing CA inhibitor scaffolds that may selectively inhibit the dandruff-producing fungi.
RESUMEN
A series of 2-thio- and 2-seleno-acetamides bearing the benzenesulfonamide moiety were evaluated as Carbonic Anhydrase (CA, EC 4.2.1.1) inhibitors against different pathogenic bacteria such as the Vibrio cholerae (VchCA-α and VchCA-ß), Burkholderia pseudomallei (BpsCA-ß and BpsCA-γ), Mycobacterium tuberculosis (Rv3723-ß) and the Salmonella enterica serovar Typhimurium (StCA2-ß). The molecules represent interesting leads worth developing as innovative antibacterial agents since they possess new mechanism of action and isoform selectivity preferentially against the bacterial expressed CAs. The identification of potent and selective inhibitors of bacterial CAs may lead to tools also useful for deciphering the physiological role(s) of such proteins.
Asunto(s)
Acetamidas/química , Bacterias/efectos de los fármacos , Bacterias/patogenicidad , Infecciones Bacterianas/tratamiento farmacológico , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/química , Sulfonamidas/química , Bacterias/enzimología , Infecciones Bacterianas/microbiología , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Compuestos de Organoselenio/química , Relación Estructura-Actividad , Compuestos de Sulfhidrilo/química , Sulfonamidas/farmacología , BencenosulfonamidasRESUMEN
Acidity, hypoxia and increased release of exosomes are severe phenotypes of tumours. The regulation of pH in tumours involves the interaction of several proteins, including the carbonic anhydrases which catalyze the formation of bicarbonate and protons from carbon dioxide and water. Among CA isoforms, CA IX is over-expressed in a large number of solid tumours, conferring to cancer cells a survival advantage in hypoxic and acidic microenvironment, but there isn't evidence that CA IX expression could have a real clinical impact. Therefore, in this study for the first time the expression and activity of CA IX have been investigated in the plasmatic exosomes obtained from patients with prostate carcinoma (PCa). For this purpose, the study was performed through different methodological approaches, such as NTA, western blot analysis, enzyme activity assay, Nanoscale flow cytometry, ELISA, confocal microscopy. The results showed that PCa exosomes significantly overexpressed CA IX levels and related activity as compared to healthy donors. Furthermore, CA IX expression and activity were correlated to the exosome intraluminal pH, demonstrating for the first time that PCa exosomes are acidic. Our data suggest the possible use of the exosomal CA IX expression and activity as a biomarker of cancer progression in PCa.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Anhidrasa Carbónica IX/biosíntesis , Exosomas/metabolismo , Neoplasias de la Próstata/sangre , Anciano , Antígenos de Neoplasias/sangre , Anhidrasa Carbónica IX/sangre , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , Masculino , Microscopía Confocal , Persona de Mediana EdadRESUMEN
The inhibition of δ- and η-class carbonic anhydrases (CAs; EC 4.2.1.1) was poorly investigated so far. Only one δ-CA, TweCA from the diatom Thalassiosira weissflogii, and one η-CA, PfCA, from Plasmodium falciparum, have been cloned and characterised to date. To enrich δ- and η-CAs inhibition profiles, a panel of 22 phenols was investigated for TweCA and PfCA inhibition. Some derivatives showed effective, sub-micromolar inhibition of TweCA (KIs 0.81-65.4 µM) and PfCA (KIs 0.62-78.7 µM). A subset of compounds demonstrated a significant selectivity for the target CAs over the human physiologically relevant ones. This study promotes the identification of new potent and selective inhibitors of TweCA and PfCA, which could be considered as leads for finding molecular probes in the study of carbon fixation processes (in which TweCA and orthologue enzymes are involved) or drug candidates in the treatment of malaria.
Asunto(s)
Antiprotozoarios/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Diatomeas/enzimología , Fenoles/farmacología , Plasmodium falciparum/efectos de los fármacos , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , Pruebas de Sensibilidad Parasitaria , Fenoles/síntesis química , Fenoles/química , Plasmodium falciparum/enzimología , Relación Estructura-ActividadRESUMEN
A primary strategy to combat antimicrobial resistance is the identification of novel therapeutic targets and anti-infectives with alternative mechanisms of action. The inhibition of the metalloenzymes carbonic anhydrases (CAs, EC 4.2.1.1) from pathogens (bacteria, fungi, and protozoa) was shown to produce an impairment of the microorganism growth and virulence. As phosphonamidates have been recently validated as human α-CA inhibitors (CAIs) and no phosphorus-based zinc-binding group have been assessed to date against ß-class CAs, herein we report an inhibition study with this class of compounds against ß-CAs from pathogenic bacteria, fungi, and protozoa. Our data suggest that phosphonamidates are among the CAIs with the best selectivity for ß-class over human isozymes, making them interesting leads for the development of new anti-infectives.
Asunto(s)
Amidas/farmacología , Antiinfecciosos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Compuestos Organometálicos/farmacología , Ácidos Fosfóricos/farmacología , Amidas/química , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Bacterias/efectos de los fármacos , Bacterias/enzimología , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/química , Relación Dosis-Respuesta a Droga , Hongos/efectos de los fármacos , Hongos/enzimología , Humanos , Leishmania donovani/efectos de los fármacos , Leishmania donovani/enzimología , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Ácidos Fosfóricos/química , Fósforo/química , Fósforo/farmacología , Relación Estructura-Actividad , Zinc/química , Zinc/farmacologíaRESUMEN
γ-Class carbonic anhydrases (CAs; EC 4.2.1.1) lack of extended inhibition characterization in comparison to α- and ß-class isozymes. For this reason, a panel of 22 phenols was investigated here for the inhibition of the γ-CAs from the pathogenic bacteria Burkholderia pseudomallei (BpsCAγ), Porphyromonas gingivalis (PgiCA), Vibrio cholerae (VchCAγ) and from the antarctic bacteria Pseudoalteromonas haloplanktis (PhaCAγ) and Colwellia psychrerythraea (CpsCAγ). The exploration of the chemical space around the main phenolic group led to the discovery of a number of such derivatives showing effective, sometimes sub-micromolar inhibition against BpsCAγ (KIs 0.45-8.6⯵M), PgiCA (KIs 0.36-9.8⯵M) and VchCAγ (KIs 0.47-9.6⯵M). A subset of compounds even demonstrated a significant selectivity for the target γ-CAs over the human physiologically most relevant isoform CA II. This study enriches the inhibitory profiles database for γ-class CAs and promotes the identification of new potent and selective inhibitors against bacterial isoforms over human off-target ones. These agents are of remarkable interest and importance in the search of novel, worldwide required, antibiotic agents possessing alternative mechanisms of action as a strategy to overcome the spread to antimicrobic resistance.