RESUMEN
INTRODUCTION: With rapid elevation in population, urbanization and industrialization, the environment is exposed to uncontrolled discharge of effluents filled with broad-spectrum toxicity, persistence and long-distance transmission anthropogenic compounds, among them heavy metals. That put our ecosystem on the verge or at a stake of drastic ecological deterioration, which eventually adversely influence on public health. Therefore, this study employed marine fungal strain Rhodotorula sp. MZ312369 for Zn2+ and Cr6+ remediation using the promising calcium carbonate (CaCO3) bioprecipitation technique, for the first time. RESULTS: Initially, Plackett-Burman design followed by central composite design were applied to optimize carbonic anhydrase enzyme (CA), which succeeded in enhancing its activity to 154 U/mL with 1.8-fold increase comparing to the basal conditions. The potentiality of our biofactory in remediating Zn2+ (50 ppm) and Cr6+ (400 ppm) was monitored through dynamic study of several parameters including microbial count, CA activity, CaCO3 weight, pH fluctuation, changing the soluble concentrations of Ca2+ along with Zn2+ and Cr6+. The results revealed that 9.23 × 107 ± 2.1 × 106 CFU/mL and 10.88 × 107 ± 2.5 × 106 CFU/mL of cells exhibited their maximum CA activity by 124.84 ± 1.24 and 140 ± 2.5 U/mL at 132 h for Zn2+ and Cr6+, respectively. Simultaneously, with pH increase to 9.5 ± 0.2, a complete removal for both metals was observed at 168 h; Ca2+ removal percentages recorded 78.99% and 85.06% for Zn2+ and Cr6+ remediating experiments, respectively. Further, the identity, elemental composition, functional structure and morphology of bioremediated precipitates were also examined via mineralogical analysis. EDX pattern showed the typical signals of C, O and Ca accompanying with Zn2+ and Cr6+ peaks. SEM micrographs depicted spindle, spherical and cubic shape bioliths with size range of 1.3 ± 0.5-23.7 ± 3.1 µm. Meanwhile, XRD difractigrams unveiled the prevalence of vaterite phase in remediated samples. Besides, FTIR profiles emphasized the presence of vaterite spectral peaks along with metals wavenumbers. CONCLUSION: CA enzyme mediated Zn2+ and Cr6+ immobilization and encapsulation inside potent vaterite trap through microbial biomineralization process, which deemed as surrogate ecofriendly solution to mitigate heavy metals toxicity and restrict their mobility in soil and wastewater.
Asunto(s)
Biodegradación Ambiental , Carbonato de Calcio , Anhidrasas Carbónicas , Cromo , Rhodotorula , Zinc , Zinc/metabolismo , Anhidrasas Carbónicas/metabolismo , Cromo/metabolismo , Carbonato de Calcio/metabolismo , Carbonato de Calcio/química , Rhodotorula/enzimología , Concentración de Iones de Hidrógeno , Contaminantes Químicos del Agua/metabolismoRESUMEN
Aim: A series of isocoumarin-chalcone hybrids were prepared and assays for the inhibition of four isoforms of human carbonic anhydrase (hCA; EC 4.2.1.1), hCA I, II, IX and XII. Materials & methods: Isocoumarin-chalcone hybrids were synthesized by condensing acetyl-isocoumarin with aromatic aldehydes. They did not significantly inhibit off-target cytosolic isoforms hCA I and II (KI >100 µM) but acted as low micromolar or submicromolar inhibitors for the tumor-associated isoforms hCA IX and XII. Results & conclusion: Our work provides insights into a new and scarcely investigated chemotype which provides interesting tumor-associated CA inhibitors, considering that some such derivatives like sulfonamide SLC-0111 are in advanced clinical trials for the management of metastatic advanced solid tumors.
A series of isocoumarinchalcone hybrids was prepared and assays for the inhibition of four isoforms of the metalloenzyme carbonic anhydrase (CA; EC 4.2.1.1), i.e., human (h) isoforms hCA I, II, IX and XII. Isocoumarins were less investigated as inhibitors of this enzyme. Here we show that the isocoumarinchalcone hybrids do not significantly inhibit the off-target cytosolic isoforms hCA I and II (KIs >100 µM) but act as low micromolar inhibitors for the tumor-associated isoforms hCA IX and XII. Our work thus provides insights into a new and scarcely investigated chemotype which may provide interesting tumor-associated CA inhibitors, because some such compounds, e.g., the sulfonamide SLC-0111, are presently in advanced clinical trials for the management of metastatic advanced solid tumors.
Asunto(s)
Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Isocumarinas , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/síntesis química , Humanos , Anhidrasas Carbónicas/metabolismo , Isocumarinas/química , Isocumarinas/farmacología , Isocumarinas/síntesis química , Chalcona/química , Chalcona/farmacología , Relación Estructura-Actividad , Isoenzimas/metabolismo , Isoenzimas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Estructura Molecular , Chalconas/química , Chalconas/farmacología , Chalconas/síntesis químicaRESUMEN
A robust and stable carbonic anhydrase (CA) system is indispensable for effectively sequestering carbon dioxide to mitigate climate change. While microbial surface display technology has been employed to construct an economically promising cell-displayed CO2-capturing biocatalyst, the displayed CA enzymes were prone to inactivation due to their low stability in harsh conditions. Herein, drawing inspiration from biomineralized diatom frustules, we artificially introduced biosilica shell materials to the CA macromolecules displayed on Escherichia coli surfaces. Specifically, we displayed a fusion of CA and the diatom-derived silica-forming Sil3K peptide (CA-Sil3K) on the E. coli surface using the membrane anchor protein Lpp-OmpA linker. The displayed CA-Sil3K (dCA-Sil3K) fusion protein underwent a biosilicification reaction under mild conditions, resulting in nanoscale self-encapsulation of the displayed enzyme in biosilica. The biosilicified dCA-Sil3K (BS-dCA-Sil3K) exhibited improved thermal, pH, and protease stability and retained 63 % of its initial activity after ten reuses. Additionally, the BS-dCA-Sil3K biocatalyst significantly accelerated the CaCO3 precipitation rate, reducing the time required for the onset of CaCO3 formation by 92 % compared to an uncatalyzed reaction. Sedimentation of BS-dCA-Sil3K on a membrane filter demonstrated a reliable CO2 hydration application with superior long-term stability under desiccation conditions. This study may open new avenues for the nanoscale-encapsulation of enzymes with biosilica, offering effective strategies to provide efficient, stable, and economic cell-displayed biocatalysts for practical applications.
Asunto(s)
Dióxido de Carbono , Anhidrasas Carbónicas , Escherichia coli , Dióxido de Silicio , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/genética , Dióxido de Carbono/metabolismo , Dióxido de Carbono/química , Escherichia coli/genética , Escherichia coli/metabolismo , Dióxido de Silicio/química , Biocatálisis , Estabilidad de Enzimas , Secuestro de Carbono , Concentración de Iones de Hidrógeno , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Carbonato de Calcio/química , Carbonato de Calcio/metabolismo , Enzimas Inmovilizadas/metabolismo , Enzimas Inmovilizadas/químicaRESUMEN
Microalgae biotechnology holds great potential for mitigating CO2 emissions, yet faces challenges in commercialization due to suboptimal photosynthetic efficiency. This study presents an innovative approach to improve CO2 mass transfer efficiency in microalgae using carbonic anhydrase (CA) in an internal LED flexible air-lift photobioreactor. Optimal conditions initial inoculation with 3.55 × 106 cells/mL and 20 % CO2 concentration, complemented by white LED lighting in Chlorella sp. CA regulated intracellular composition, enhancing chlorophyll, lipid, and protein contents. Metabolomics revealed elevated malic and succinic acids, associated with increased Ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) and Acetoacetyl coenzyme A (Acetyl-CoA) activities, facilitating efficient carbon fixation. CA also mitigated cellular oxidative stress by reducing reactive oxygen species (ROS). Furthermore, CA improved extracellular electron acceptor with currents surpassed CK. This CA-based microalgae biotechnology provides a foundation for future commercial applications, addressing CO2 emissions.
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Dióxido de Carbono , Anhidrasas Carbónicas , Microalgas , Fotobiorreactores , Dióxido de Carbono/metabolismo , Microalgas/metabolismo , Anhidrasas Carbónicas/metabolismo , Ciclo del Carbono , Chlorella , Fotosíntesis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Microbially induced carbonate precipitation (MICP) immobilizes toxic metals and reduces their bioavailability in aqueous systems. However, its application in the treatment of acid mine drainage (AMD) is poorly understood. In this study, the genomes of Sporosarcina sp. UB5 and UB10 were sequenced. Urease, carbonic anhydrases, and metal resistance genes were identified and enzymatic assays were performed for their validation. The geochemical mechanism of precipitation in AMD was elucidated through geo-mineralogical analysis. Sporosarcina sp. UB5 was shown to be a new genomospecies, with an average nucleotide identity < 95 % (ANI) and DNA-DNA hybridization < 70 % (DDH) whereas UB10 is close to S. pasteurii. UB5 contained two urease operons, whereas only one was identified in UB10. The ureolytic activities of UB5 and UB10 were 122.67 ± 15.74 and 131.70 ± 14.35 mM NH4+ min-1, respectively. Both strains feature several carbonic anhydrases of the α, ß, or γ families, which catalyzed the precipitation of CaCO3. Only Sporosarcina sp. UB5 was able to immobilize metals and neutralize AMD. Geo-mineralogical analyses revealed that UB5 directly immobilized Fe (1-23 %), Mn (0.65-1.33 %) and Zn (0.8-3 %) in AMD via MICP and indirectly through adsorption to calcite and binding to bacterial cell walls. The MICP-treated AMD exhibited high removal rates (>67 %) for Ag, Al, As, Ca, Cd, Co, Cu, Fe, Mn, Pb, and Zn, and a removal rate of 15 % for Mg. This study provides new insights into the MICP process and its applications to AMD treatment using autochthonous strains.
Asunto(s)
Minería , Sporosarcina , Ureasa , Sporosarcina/genética , Sporosarcina/metabolismo , Ureasa/metabolismo , Precipitación Química , Carbonatos/química , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/genética , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/químicaRESUMEN
Native mass spectrometry (MS) is a powerful analytical technique to directly probe noncovalent protein-protein and protein-ligand interactions. However, not every MS platform can preserve proteins in their native conformation due to high energy deposition from the utilized ionization source. Most small molecules approved as drugs and in development interact with their targets through noncovalent interactions. Therefore, rapid methods to analyze noncovalent protein-ligand interactions are necessary for the early stages of the drug discovery pipeline. Herein, we describe a method for analyzing noncovalent protein-ligand complexes by IR-MALDESI-MS with analysis times of â¼13 s per sample. Carbonic anhydrase and the kinase domain of Bruton's tyrosine kinase are paired with known noncovalent binders to evaluate the effectiveness of native MS by IR-MALDESI.
Asunto(s)
Espectrometría de Masas , Ligandos , Espectrometría de Masas/métodos , Anhidrasas Carbónicas/química , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/análisis , Unión Proteica , Agammaglobulinemia Tirosina Quinasa/metabolismo , Agammaglobulinemia Tirosina Quinasa/química , Agammaglobulinemia Tirosina Quinasa/antagonistas & inhibidores , Humanos , Proteínas/química , Proteínas/metabolismo , Proteínas/análisisRESUMEN
A wide range of 3-selenylindoles were synthesized via an eco-friendly approach that uses Oxone® as the oxidant in the presence of a catalytic amount of iodine. This mild and economical protocol showed broad functional group tolerance and operational simplicity. A series of novel selenylindoles bearing a benzenesulfonamide moiety were also synthesized and evaluated as carbonic anhydrase inhibitors of the human (h) isoforms hCa I, II, IX, and XII, which are involved in pathologies such as glaucoma and cancer. Several derivatives showed excellent inhibitory activity towards these isoforms in the nanomolar range, lower than that shown by acetazolamide.
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Bencenosulfonamidas , Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Indoles , Yodo , Oxidación-Reducción , Sulfonamidas , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/síntesis química , Sulfonamidas/química , Sulfonamidas/farmacología , Sulfonamidas/síntesis química , Humanos , Anhidrasas Carbónicas/metabolismo , Indoles/química , Indoles/farmacología , Indoles/síntesis química , Yodo/química , Compuestos de Organoselenio/química , Compuestos de Organoselenio/farmacología , Compuestos de Organoselenio/síntesis química , Relación Estructura-Actividad , Estructura MolecularRESUMEN
A new series of piperazine derivatives were synthesized and studied with the aim of obtaining dual inhibitors of P-glycoprotein (P-gp) and carbonic anhydrase XII (hCA XII) to synergistically overcome the P-gp-mediated multidrug resistance (MDR) in cancer cells expressing the two proteins, P-gp and hCA XII. Indeed, these hybrid compounds contain both P-gp and hCA XII binding groups on the two nitrogen atoms of the heterocyclic ring. All compounds showed good inhibitory activity on each protein (P-gp and hCA XII) studied individually, and many of them showed a synergistic effect in the resistant HT29/DOX and A549/DOX cell lines which overexpress both the target proteins. In particular, compound 33 displayed the best activity by enhancing the cytotoxicity and intracellular accumulation of doxorubicin in HT29/DOX and A549/DOX cells, thus resulting as promising P-gp-mediated MDR reverser with a synergistic mechanism. Furthermore, compounds 13, 27 and 32 induced collateral sensitivity (CS) in MDR cells, as they were more cytotoxic in resistant cells than in the sensitive ones; their CS mechanisms were extensively investigated.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Piperazinas , Humanos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Piperazinas/farmacología , Piperazinas/química , Piperazinas/síntesis química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Anhidrasas Carbónicas/metabolismo , Doxorrubicina/farmacología , Doxorrubicina/química , Piperazina/química , Piperazina/farmacología , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Células HT29 , Relación Estructura-Actividad , Línea Celular Tumoral , Estructura Molecular , Células A549RESUMEN
BACKGROUND: Carbonic anhydrase (CA) enzymes facilitate the reversible hydration of CO2 to bicarbonate ions and protons. Identifying efficient and robust CAs and expressing them in model host cells, such as Escherichia coli, enables more efficient engineering of these enzymes for industrial CO2 capture. However, expression of CAs in E. coli is challenging due to the possible formation of insoluble protein aggregates, or inclusion bodies. This makes the production of soluble and active CA protein a prerequisite for downstream applications. RESULTS: In this study, we streamlined the process of CA expression by selecting seven top CA candidates and used two bioinformatic tools to predict their solubility for expression in E. coli. The prediction results place these enzymes in two categories: low and high solubility. Our expression of high solubility score CAs (namely CA5-SspCA, CA6-SazCAtrunc, CA7-PabCA and CA8-PhoCA) led to significantly higher protein yields (5 to 75 mg purified protein per liter) in flask cultures, indicating a strong correlation between the solubility prediction score and protein expression yields. Furthermore, phylogenetic tree analysis demonstrated CA class-specific clustering patterns for protein solubility and production yields. Unexpectedly, we also found that the unique N-terminal, 11-amino acid segment found after the signal sequence (not present in its homologs), was essential for CA6-SazCA activity. CONCLUSIONS: Overall, this work demonstrated that protein solubility prediction, phylogenetic tree analysis, and experimental validation are potent tools for identifying top CA candidates and then producing soluble, active forms of these enzymes in E. coli. The comprehensive approaches we report here should be extendable to the expression of other heterogeneous proteins in E. coli.
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Anhidrasas Carbónicas , Biología Computacional , Escherichia coli , Solubilidad , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/enzimología , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/genética , Biología Computacional/métodos , Filogenia , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Dióxido de Carbono/metabolismoRESUMEN
We study the carbonic anhydrase (CA) pathway using autochthonous CA-producing bacteria as a means of inducing calcite precipitation, which acts as a biocement to improve the engineering soil properties. Forty different microbial strains producing CA were isolated from the foundation soil of a railway embankment in Prickwillow, UK. Three of the best CA-producing strains were selected and identified by DNA sequencing as Bacillus licheniformis, Bacillus toyonensis and Bacillus pumilus with CA activity values respectively of 1.79 U/ml, 1.42 U/ml and 1.55 U/ml. To optimise the treatments, we investigated the effect of pH, temperature, zinc co-factor and cementation solution molarity on the growth and CA activity and bioprecipitates, with CO2 added in the form of bicarbonate. Scanning electron microscope (SEM) analysis of the bioprecipitates showed that these had characteristic morphologies of calcite and vaterite crystals. The formation of calcite was further corroborated by FT-IR and Raman analysis of bioprecipitates. The precultured bacteria were injected into the fine-grained soil together with cementation solution. Unconfined compressive strength in treated soil increased up to 1 MPa, and its calcium carbonate content increased by 2.78%. This, as well as the stability of the treated soil upon water immersion, proved the biocementation of the fine-grained soil. These findings suggest the potential of employing the CA biocementation route for soil stabilisation pending further development of the technique.
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Carbonato de Calcio , Anhidrasas Carbónicas , Suelo , Carbonato de Calcio/química , Anhidrasas Carbónicas/metabolismo , Suelo/química , Microbiología del Suelo , Bacterias/enzimologíaRESUMEN
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 ð½-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 ð½-AbauCA, unveiling its distinctive kinetic properties and inhibition profile with inorganic anions (classical CA inhibitors). The exploration of ð½-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.
Asunto(s)
Acinetobacter baumannii , Aniones , Antibacterianos , Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Pruebas de Sensibilidad Microbiana , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/efectos de los fármacos , Anhidrasas Carbónicas/metabolismo , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Aniones/farmacología , Aniones/química , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Estructura MolecularRESUMEN
INTRODUCTION: Cholera is a bacterial diarrheal disease caused by pathogen bacteria Vibrio cholerae, which produces the cholera toxin (CT). In addition to improving water sanitation, oral cholera vaccines have been developed to control infection. Besides, rehydration and antibiotic therapy are complementary treatment strategies for cholera. ToxT regulatory protein activates transcription of CT gene, which is enhanced by bicarbonate (HCO3-). AREAS COVERED: This review delves into the genomic blueprint of V. cholerae, which encodes for α-, ß-, and γ- carbonic anhydrases (CAs). We explore how the CAs contribute to the pathogenicity of V. cholerae and discuss the potential of CA inhibitors in mitigating the disease's impact. EXPERT OPINION: CA inhibitors can reduce the virulence of bacteria and control cholera. Here, we reviewed all reported CA inhibitors, noting that α-CA from V. cholerae (VchCAα) was the most effective inhibited enzyme compared to the ß- and γ-CA families (VchCAß and VchCAγ). Among the CA inhibitors, acyl selenobenzenesulfonamidenamides and simple/heteroaromatic sulfonamides were the best VchCA inhibitors in the nM range. It was noted that some antibacterial compounds show good inhibitory effects on all three bacterial CAs. CA inhibitors belonging to other classes may be synthesized and tested on VchCAs to harness cholera.
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Antibacterianos , Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Cólera , Vibrio cholerae , Vibrio cholerae/enzimología , Inhibidores de Anhidrasa Carbónica/farmacología , Cólera/tratamiento farmacológico , Cólera/microbiología , Humanos , Antibacterianos/farmacología , Anhidrasas Carbónicas/metabolismo , Animales , Virulencia , Toxina del Cólera/farmacología , Toxina del Cólera/antagonistas & inhibidores , Vacunas contra el Cólera/farmacología , Desarrollo de MedicamentosRESUMEN
Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells1,2. Here we describe that Drosophila crystal cells-myeloid-like immune cells called haemocytes-control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.
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Catecol Oxidasa , Proteínas de Drosophila , Drosophila melanogaster , Precursores Enzimáticos , Hemocitos , Oxígeno , Transición de Fase , Respiración , Animales , Femenino , Masculino , Transporte Biológico , Anhidrasas Carbónicas/metabolismo , Catecol Oxidasa/metabolismo , Cobre/metabolismo , Cristalización , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Drosophila melanogaster/inmunología , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Precursores Enzimáticos/metabolismo , Hemocianinas/metabolismo , Hemocitos/inmunología , Hemocitos/metabolismo , Homeostasis , Concentración de Iones de Hidrógeno , Hiperoxia/metabolismo , Hipoxia/metabolismo , Larva/anatomía & histología , Larva/citología , Larva/inmunología , Larva/metabolismo , Oxígeno/metabolismoRESUMEN
Background: Carbonic anhydrase VI (CA VI) is crucial in regulating oral pH and predicting susceptibility to dental caries. The hypothesis posits that caries activity may alter the CA VI function, diminishing its capacity to regulate pH effectively and potentially exacerbating cariogenic challenges. This 1-year cohort study sought to investigate the enzymatic activity of salivary CA VI and buffering capacity following a 20% sucrose rinse in 4 to 6.5-year-old children. Method: This research involved 46 volunteers categorized into three groups based on their caries status after follow-up: caries-free (CFee), arrested caries (CArrested), and caries active (CActive). Children underwent visible biofilm examination and saliva collection for salivary flow rate, buffering capacity, and CA VI analyses before and after a 20% sucrose rinse. Results: A reduction in the buffering capacity was observed after sucrose rinse in all groups. The CA VI activity decreased significantly in CFee and CArrested groups after sucrose rinse, although it did not change in the CActive group. An improvement in the buffering capacity and salivary flow rate was found at follow-up when compared with the baseline. After 1-year follow-up, buffering capacity and salivary flow rate increased in all groups, whilst the CA VI activity reduced only in CFree and CArrested children. Conclusion: Sucrose rinse universally reduces the salivary buffering capacity, while caries activity may disrupt CA VI activity response during a cariogenic challenge. After a year, increased salivary flow enhances buffering capacity but not CA VI activity in caries-active children.
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Anhidrasas Carbónicas , Caries Dental , Saliva , Sacarosa , Humanos , Saliva/enzimología , Saliva/química , Sacarosa/metabolismo , Niño , Anhidrasas Carbónicas/metabolismo , Masculino , Femenino , Estudios Longitudinales , Preescolar , Tampones (Química) , Concentración de Iones de Hidrógeno , Antisépticos BucalesRESUMEN
This study investigates the co-precipitation of calcium and barium ions in hypersaline wastewater under the action of Bacillus licheniformis using microbially induced carbonate precipitation (MICP) technology, as well as the bactericidal properties of the biomineralized product vaterite. The changes in carbonic anhydrase activity, pH, carbonate and bicarbonate concentrations in different biomineralization systems were negatively correlated with variations in metal ion concentrations, while the changes in polysaccharides and protein contents in bacterial extracellular polymers were positively correlated with variations in barium concentrations. In the mixed calcium and barium systems, the harvested minerals were vaterite containing barium. The increasing concentrations of calcium promoted the incorporation and adsorption of barium onto vaterite. The presence of barium significantly increased the contents of O-CO, N-CO, and Ba-O in vaterite. Calcium promoted barium precipitation, but barium inhibited calcium precipitation. After being treated by immobilized bacteria, the concentrations of calcium and barium ions decreased from 400 and 274 to 1.72 and 0 mg/L (GB/T15454-2009 and GB8978-1996). Intracellular minerals were also vaterite containing barium. Extracellular vaterite exhibited bactericidal properties. This research presents a promising technique for simultaneously removing and recycling hazardous heavy metals and calcium in hypersaline wastewater.
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Bario , Calcio , Precipitación Química , Aguas Residuales , Aguas Residuales/química , Bario/química , Calcio/química , Calcio/metabolismo , Bacillus/metabolismo , Carbonato de Calcio/química , Carbonato de Calcio/metabolismo , Contaminantes Químicos del Agua/metabolismo , Contaminantes Químicos del Agua/química , Reciclaje , Carbonatos/química , Anhidrasas Carbónicas/metabolismo , Purificación del Agua/métodosRESUMEN
A novel class of compounds designed to hit two anti-tumour targets, G-quadruplex structures and human carbonic anhydrases (hCAs) IX and XII is proposed. The induction/stabilisation of G-quadruplex structures by small molecules has emerged as an anticancer strategy, disrupting telomere maintenance and reducing oncogene expression. hCAs IX and XII are well-established anti-tumour targets, upregulated in many hypoxic tumours and contributing to metastasis. The ligands reported feature a berberine G-quadruplex stabiliser scaffold connected to a moiety inhibiting hCAs IX and XII. In vitro experiments showed that our compounds selectively stabilise G-quadruplex structures and inhibit hCAs IX and XII. The crystal structure of a telomeric G-quadruplex in complex with one of these ligands was obtained, shedding light on the ligand/target interaction mode. The most promising ligands showed significant cytotoxicity against CA IX-positive HeLa cancer cells in hypoxia, and the ability to stabilise G-quadruplexes within tumour cells.
Asunto(s)
Antineoplásicos , Anhidrasa Carbónica IX , Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , G-Cuádruplex , Humanos , G-Cuádruplex/efectos de los fármacos , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis química , Relación Estructura-Actividad , Estructura Molecular , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasa Carbónica IX/metabolismo , Anhidrasas Carbónicas/metabolismo , Proliferación Celular/efectos de los fármacos , Ligandos , Células HeLa , Antígenos de Neoplasias/metabolismo , Modelos MolecularesRESUMEN
New sulfonamide-triazole-glycoside hybrids derivatives were designed, synthesised, and investigated for anticancer efficacy. The target glycosides' cytotoxic activity was studied with a panel of human cancer cell lines. Sulfonamide-based derivatives, 4, 7 and 9 exhibited promising activity against HepG-2 and MCF-7 (IC50 = 8.39-16.90 µM against HepG-2 and 19.57-21.15 µM against MCF-7) comparing with doxorubicin (IC50 = 13.76 ± 0.45, 17.44 ± 0.46 µM against HepG-2 and MCF-7, rescpectively). To detect the probable action mechanism, the inhibitory activity of these targets was studied against VEGFR-2, carbonic anhydrase isoforms hCA IX and hCA XII. Compoumds 7 and 9 gave favorable potency (IC50 = 1.33, 0.38 µM against VEGFR-2, 66, 40 nM against hCA IX and 7.6, 3.2 nM against hCA XII, respectively), comparing with sorafenib and SLC-0111 (IC50 = 0.43 µM, 53 and 4.8 nM, respectively). Moreover, the docking simulation was assessed to supply better rationalization and gain insight into the binding affinity between the promising derivatives and their targeted enzymes that was used for further modification in the anticancer field.
Asunto(s)
Antineoplásicos , Inhibidores de Anhidrasa Carbónica , Glicósidos , Simulación del Acoplamiento Molecular , Sulfonamidas , Triazoles , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Humanos , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Sulfonamidas/química , Sulfonamidas/farmacología , Glicósidos/química , Glicósidos/farmacología , Triazoles/química , Triazoles/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Antineoplásicos/farmacología , Antineoplásicos/química , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/antagonistas & inhibidores , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/química , Células MCF-7 , Células Hep G2 , Línea Celular Tumoral , Antígenos de Neoplasias/metabolismo , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Inhibidores de Anhidrasa Carbónica , Anhidrasas Carbónicas , Clonación Molecular , Toxoplasma , Toxoplasma/enzimología , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/genética , Cinética , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Relación Estructura-Actividad , Relación Dosis-Respuesta a Droga , Estructura Molecular , Aniones/química , Aniones/farmacología , Aniones/metabolismoRESUMEN
Carbonic anhydrase (CA) was among the first proteins whose X-ray crystal structure was solved to atomic resolution. CA proteins have essentially the same fold and similar active centers that differ in only several amino acids. Primary sulfonamides are well defined, strong and specific binders of CA. However, minor variations in chemical structure can significantly alter their binding properties. Over 1000 sulfonamides have been designed, synthesized and evaluated to understand the correlations between the structure and thermodynamics of their binding to the human CA isozyme family. Compound binding was determined by several binding assays: fluorescence-based thermal shift assay, stopped-flow enzyme activity inhibition assay, isothermal titration calorimetry and competition assay for enzyme expressed on cancer cell surfaces. All assays have advantages and limitations but are necessary for deeper characterization of these protein-ligand interactions. Here, the concept and importance of intrinsic binding thermodynamics is emphasized and the role of structure-thermodynamics correlations for the novel inhibitors of CA IX is discussed - an isozyme that is overexpressed in solid hypoxic tumors, and thus these inhibitors may serve as anticancer drugs. The abundant structural and thermodynamic data are assembled into the Protein-Ligand Binding Database to understand general protein-ligand recognition principles that could be used in drug discovery.
Asunto(s)
Anhidrasas Carbónicas , Isoenzimas , Unión Proteica , Sulfonamidas , Termodinámica , Humanos , Cristalografía por Rayos X , Anhidrasas Carbónicas/metabolismo , Anhidrasas Carbónicas/química , Isoenzimas/metabolismo , Isoenzimas/química , Ligandos , Sulfonamidas/química , Sulfonamidas/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasa Carbónica IX/metabolismo , Anhidrasa Carbónica IX/química , Modelos MolecularesRESUMEN
The development of carbon capture and storage technologies has resulted in a rising interest in the use of carbonic anhydrases (CAs) for CO2 fixation at elevated temperatures. In this study, we chose to rationally engineer the α-CA (NtCA) from the thermophilic bacterium Nitratiruptor tergarcus, which has been previously suggested to be thermostable by in silico studies. Using a combination of analyses with the DEEPDDG software and available structural knowledge, we selected residues in three regions, namely, the catalytic pocket, the dimeric interface and the surface, in order to increase thermostability and CO2 hydration activity. A total of 13 specific mutations, affecting seven amino acids, were assessed. Single, double and quadruple mutants were produced in Escherichia coli and analyzed. The best-performing mutations that led to improvements in both activity and stability were D168K, a surface mutation, and R210L, a mutation in the dimeric interface. Apart from these, most mutants showed improved thermostability, with mutants R210K and N88K_R210L showing substantial improvements in activity, up to 11-fold. Molecular dynamics simulations, focusing particularly on residue fluctuations, conformational changes and hydrogen bond analysis, elucidated the structural changes imposed by the mutations. Successful engineering of NtCA provided valuable lessons for further engineering of α-CAs.