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Tuberculosis (TB) is an infectious disease that primarily affects the lungs of humans and accounts for Mycobacterium tuberculosis (Mtb) bacteria as the etiologic agent. In this study, we introduce a computational framework designed to identify the important chemical features crucial for the effective inhibition of Mtb ß-CAs. Through applying a mechanistic model, we elucidated the essential features pivotal for robust inhibition. Using this model, we engineered molecules that exhibit potent inhibitory activity and introduce relevant novel chemistry. The designed molecules were prioritized for synthesis based on their predicted pKi values via the QSAR (Quantitative Structure-Activity Relationship) model. All the rationally designed and synthesized compounds were evaluated in vitro against different carbonic anhydrase isoforms expressed from the pathogen Mtb; moreover, the off-target and widely human-expressed CA I and II were also evaluated. Among the reported derivatives, 2, 4, and 5 demonstrated the most valuable in vitro activity, resulting in promising candidates for the treatment of TB infection. All the synthesized molecules exhibited favorable pharmacokinetic and toxicological profiles based on in silico predictions. Docking analysis confirmed that the zinc-binding groups bind effectively into the catalytic triad of the Mtb ß-Cas, supporting the in vitro outcomes with these binding interactions. Furthermore, molecules with good prediction accuracies according to previously established mechanistic and QSAR models were utilized to delve deeper into the realm of systems biology to understand their mechanism in combating tuberculotic pathogenesis. The results pointed to the key involvement of the compounds in modulating immune responses via NF-κß1, SRC kinase, and TNF-α to modulate granuloma formation and clearance via T cells. This dual action, in which the pathogen's enzyme is inhibited while modulating the human immune machinery, represents a paradigm shift toward more effective and comprehensive treatment approaches for combating tuberculosis.
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Autotrophic bacteria are able to fix CO2 in a great diversity of habitats, even though this dissolved gas is relatively scarce at neutral pH and above. As many of these bacteria rely on CO2 fixation by ribulose 1,5-bisphospate carboxylase/oxygenase (RubisCO) for biomass generation, they must compensate for the catalytical constraints of this enzyme with CO2-concentrating mechanisms (CCMs). CCMs consist of CO2 and HCO3- transporters and carboxysomes. Carboxysomes encapsulate RubisCO and carbonic anhydrase (CA) within a protein shell and are essential for the operation of a CCM in autotrophic Bacteria that use the Calvin-Benson-Basham cycle. Members of the genus Thiomicrospira lack genes homologous to those encoding previously described CA, and prior to this work, the mechanism of function for their carboxysomes was unclear. In this paper, we provide evidence that a member of the recently discovered iota family of carbonic anhydrase enzymes (ιCA) plays a role in CO2 fixation by carboxysomes from members of Thiomicrospira and potentially other Bacteria. Carboxysome enrichments from Thiomicrospira pelophila and Thiomicrospira aerophila were found to have CA activity and contain ιCA, which is encoded in their carboxysome loci. When the gene encoding ιCA was interrupted in T. pelophila, cells could no longer grow under low-CO2 conditions, and CA activity was no longer detectable in their carboxysomes. When T. pelophila ιCA was expressed in a strain of Escherichia coli lacking native CA activity, this strain recovered an ability to grow under low CO2 conditions, and CA activity was present in crude cell extracts prepared from this strain. IMPORTANCE: Here, we provide evidence that iota carbonic anhydrase (ιCA) plays a role in CO2 fixation by some organisms with CO2-concentrating mechanisms; this is the first time that ιCA has been detected in carboxysomes. While ιCA genes have been previously described in other members of bacteria, this is the first description of a physiological role for this type of carbonic anhydrase in this domain. Given its distribution in alkaliphilic autotrophic bacteria, ιCA may provide an advantage to organisms growing at high pH values and could be helpful for engineering autotrophic organisms to synthesize compounds of industrial interest under alkaline conditions.
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In this study, some new hydrazone derivatives (2a-g) was designed, synthesized for first time, and evaluated as multitarget inhibitors of AChE, BChE, hCA I and hCA II. The chemical structures of new hybrids were confirmed by elemental analysis and some spectroscopic techniques. All tested compounds showed low nanomolar inhibition with IC50 values of in the range of 30.4 to 264.0 nM against hCA I, 23.2 to 251.6 nM against hCA II, 12.1 to 114.3 nM against AChE, and 76.4 to 134.0 nM against BChE. These compounds inhibited hCA I and AChE more than acetazolamide (AZA) and neostigmine. Among them, compounds 2c and 2e, which have a linear structure, were determined to be the most active inhibitor candidates against these selected enzymes. Molecular docking studies were carried out on the compounds (2a-g), revealing their binding interactions with the active site of AChE, BChE, hCA I and hCA II thus supporting the experimental findings. Additionally, in silico absorption, distribution, metabolism, and excretion (ADME) prediction studies of the obtained compounds (2a-g) with in silico approaches were carried out to determine their solubility, whether they have the potential to cross the blood-brain barrier (BBB), values ââsuch as GI absorption and drug likeness principles.
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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.
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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
Chronic pain is common in our population, and most of these patients are inadequately treated, making the development of safer analgesics a high priority. Knee osteoarthritis (OA) is a primary cause of chronic pain and disability worldwide, and lower extremity OA is a major contributor to loss of quality-adjusted life-years. In this study we tested the hypothesis that a novel JDNI8 replication-defective herpes simplex-1 viral vector (rdHSV) incorporating a modified carbonic anhydrase-8 transgene (CA8*) produces analgesia and treats monoiodoacetate-induced (MIA) chronic knee pain due to OA. We observed transduction of lumbar DRG sensory neurons with these viral constructs (vHCA8*) (~40% of advillin-positive cells and ~ 50% of TrkA-positive cells colocalized with V5-positive cells) using the intra-articular (IA) knee joint (KJ) route of administration. vHCA8* inhibited chronic mechanical OA knee pain induced by MIA was dose- and time-dependent. Mechanical thresholds returned to Baseline by D17 after IA KJ vHCA8* treatment, and exceeded Baseline (analgesia) through D65, whereas negative controls failed to reach Baseline responses. Weight-bearing and automated voluntary wheel running were improved by vHCA8*, but not negative controls. Kv7 voltage-gated potassium channel-specific inhibitor XE-991 reversed vHCA8*-induced analgesia. Using IHC, IA KJ of vHCA8* activated DRG Kv7 channels via dephosphorylation, but negative controls failed to impact Kv7 channels. XE-991 stimulated Kv7.2-7.5 and Kv7.3 phosphorylation using western blotting of differentiated SH-SY5Y cells, which was inhibited by vHCA8* but not by negative controls. The observed prolonged dose-dependent therapeutic effects of IA KJ administration of vHCA8* on MIA-induced chronic KJ pain due to OA is consistent with the specific activation of Kv7 channels in small DRG sensory neurons. Together, these data demonstrate for the first-time local IA KJ administration of vHCA8* produces opioid-independent analgesia in this MIA-induced OA chronic pain model, supporting further therapeutic development.
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Deficiency of endogenous hydrogen peroxide and insufficient intracellular acidity are usually two important factors limiting chemodynamic therapy (CDT). Here we report a glutathione-responsive nanomedicine that can provide a suitable environment for CDT by inhibiting dual-enzymes simultaneously. The nanomedicine is constructed by encapsulation of a novel hydrogen sulfide donor in nanomicelle assembled by glutathione-responsive amphiphilic polymer. In response to intracellular glutathione, the nanomedicine can efficiently release the active ingredients hydrogen sulfide, carbonic anhydrase inhibitor and ferrocene. The hydrogen sulfide can increase the concentrations of hydrogen peroxide and lactic acid by inhibiting catalase and enhancing glycolysis. The carbonic anhydrase inhibitor can further induce intratumoral acidosis by inhibiting the function of carbonic anhydrase IX. Therefore, the nanomedicine can provide more efficient reaction conditions for the ferrocene-mediated Fenton reaction to generate abundant toxic hydroxyl radicals. In vivo results show that the combination of enhanced CDT and acidosis can effectively inhibit tumor growth. This design of nanomedicine provides a promising dual-enzyme inhibiting strategy to enhance antitumor efficacy of CDT.
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Despite its prominence, the ability to engineer Cupriavidus necator H16 for inorganic carbon uptake and fixation is underexplored. We tested the roles of endogenous and heterologous genes on C. necator inorganic carbon metabolism. Deletion of ß-carbonic anhydrase can had the most deleterious effect on C. necator autotrophic growth. Replacement of this native uptake system with several classes of dissolved inorganic carbon (DIC) transporters from Cyanobacteria and chemolithoautotrophic bacteria recovered autotrophic growth and supported higher cell densities compared to wild-type (WT) C. necator in batch culture. Strains expressing Halothiobacillus neopolitanus DAB2 (hnDAB2) and diverse rubisco homologs grew in CO2 similarly to the wild-type strain. Our experiments suggest that the primary role of carbonic anhydrase during autotrophic growth is to support anaplerotic metabolism, and an array of DIC transporters can complement this function. This work demonstrates flexibility in HCO3- uptake and CO2 fixation in C. necator, providing new pathways for CO2-based biomanufacturing.
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In the search of new cymantrenyl- and ferrocenyl-sulfonamides as potencial inhibitors of human carbonic anhydrases (hCAs), four compounds based on N-ethyl or N-methyl benzenesulfonamide units have been obtained. These cymantrenyl (1a-b) and ferrocenyl (2a-b) derivatives were prepared by the reaction between aminobenzene sulfonamides ([NH2-(CH2)n-(C6H4)-SO2-NH2)], where n = 1, 2) with cymantrenyl sulfonyl chloride (P1) or ferrocenyl sulfonyl chloride (P2), respectively. All compounds were characterized by conventional spectroscopic techniques and cyclic voltammetry. In the solid state, the molecular structures of compounds 1a, 1b, and 2b were determined by single-crystal X-ray diffraction. Biological evaluation as carbonic anhydrases inhibitors were carried out and showed derivatives 1b y 2b present a higher inhibition than the drug control for the Human Carbonic Anhydrase (hCA) II and IX isoforms (KI = 7.3 nM and 5.8 nM, respectively) and behave as selective inhibition for hCA II isoform. Finally, the docking studies confirmed they share the same binding site and interactions as the known inhibitors acetazolamide (AAZ) and agree with biological studies.
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Utilizing carbonic anhydrase (CA) to catalyze CO2 hydration offers a sustainable and potent approach for carbon capture and utilization. To enhance CA's reusability and stability for successful industrial applications, enzyme immobilization is essential. In this study, delignified bamboo cellulose served as a renewable porous scaffold for immobilizing CA through oxidation-induced cellulose aldehydation followed by Schiff base linkage. The catalytic performance of the resulting immobilized CA was evaluated using both p-NPA hydrolysis and CO2 hydration models. Compared to free CA, immobilization onto the bamboo scaffold increased CA's optimal temperature and pH to approximately 45⯰C and 9.0, respectively. Post-immobilization, CA activity demonstrated effective retention (>60â¯%), with larger scaffold sizes (i.e., 8â¯mm diameter and 5â¯mm height) positively impacting this aspect, even surpassing the activity of free CA. Furthermore, immobilized CA exhibited sustained reusability and high stability under thermal treatment and pH fluctuation, retaining >80â¯% activity even after 5 catalytic cycles. When introduced to microalgae culture, the immobilized CA improved biomass production by ~16â¯%, accompanied by enhanced synthesis of essential biomolecules in microalgae. Collectively, the facile and green construction of immobilized CA onto bamboo cellulose block demonstrates great potential for the development of various CA-catalyzed CO2 conversion and utilization technologies.
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Carbonic anhydrase VA (CA-VA) deficiency is a rare cause of hyperammonemia caused by biallelic mutations in CA5A. Most patients present with hyperammonemic encephalopathy in early infancy to early childhood, and patients usually have no further recurrence of hyperammonemia with a favorable outcome. This retrospective cohort study reports 18 patients with CA-VA deficiency caused by homozygosity for a founder mutation, c.59G>A p.(Trp20*) in CA5A. The reported patients show significant intrafamilial and interfamilial variability, and display atypical clinical features. Two adult patients were asymptomatic, 7/18 patients had recurrent hyperammonemia, 7/18 patients developed variable degree of developmental delay, 9/11 patients had hyperCKemia, and 7/18 patients had failure to thrive. Microcephaly was seen in three patients and one patient developed a metabolic stroke. The same variant had been reported already in a single South Asian patient presenting with neonatal hyperammonemic encephalopathy and subsequent development of seizures and developmental delay. This report highlights the limitations of current understanding of the pathomechanisms involved in this disorder, and calls for further evaluation of the possible role of genetic modifiers in this condition.
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Cancers share common cellular and physiological features. Little is known about whether distinctive gene expression patterns can be displayed at the single-cell level by gene families in cancer cells. The expression of gene homologs within a family can exhibit concurrence and exclusivity. Concurrence can promote all-or-none expression patterns of related genes and underlie alternative physiological states. Conversely, exclusive gene families express the same or similar number of homologs in each cell, allowing a broad repertoire of cell identities to be generated. We show that gene families involved in the cell-cycle and antigen presentation are expressed concurrently. Concurrence in the DNA replication complex MCM reflects the replicative status of cells, including cell lines and cancer-derived organoids. Exclusive expression requires precise regulatory mechanism, but cancer cells retain this form of control for ion homeostasis and extend it to gene families involved in cell migration. Thus, the cell adhesion-based identity of healthy cells is transformed to an identity based on migration in the population of cancer cells, reminiscent of epithelial-mesenchymal transition.
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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.
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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
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
The present research focused on the tail-approach synthesis of novel extended thiazolotriazoles (8a-8j) and triazolothiadiazines (11a-11j) including aminotriazole intermediate 10. After successful synthesis, all the compounds were evaluated for their inhibition potential against cytosolic isoforms of human carbonic anhydrase (hCA I, II), tumor-linked transmembrane isoforms (hCA IX, XII), and cathepsin B. As per the inhibition data, the newly synthesized compounds showed poor inhibition against hCA I. Many of the compounds showed effective inhibition toward hCA IX and/or XII in low nanomolar concentration. Despite the strong to moderate inhibition of hCA II by these compounds, more than half of them demonstrated better inhibition against hCA IX and/or XII, comparatively. Further, insights of CA inhibition data of these extended analogs and their comparison with earlier reported thiazolotriazole and triazolothiadiazine derivatives might help in the rational design of novel potent and selective hCA IX and XII inhibitors. The novel compounds were also found to possess anti-cathepsin B potential at a low concentration of 10-7 M. Broadly, compounds of series 11a-11j presented more effective inhibition against cathepsin B than their counterparts in series 8a-8j. Moreover, these in vitro results with respect to cathepsin B inhibition were also supported by the in silico insights obtained via molecular modeling studies.
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A small library of 79 substituted phenylsulfonamidoalkyl sulfamates, 1b-79b, was synthesized starting from arylsulfonyl chlorides and amino alcohols with different numbers of methylene groups between the hydroxyl and amino moieties yielding intermediates 1a-79a, followed by the reaction of the latter with sulfamoyl chloride. All compounds were screened for their inhibitory activity on bovine carbonic anhydrase II. Compounds 1a-79a showed no inhibition of the enzyme, in contrast to sulfamates 1b-79b. Thus, the inhibitory potential of compounds 1b-79b towards this enzyme depends on the substituent and the substitution pattern of the phenyl group as well as the length of the spacer. Bulkier substituents in the para position proved to be better for inhibiting CAII than compounds with the same substituent in the meta or ortho position. For many substitution patterns, compounds with shorter spacer lengths were superior to those with long chain spacers. Compounds with shorter spacer lengths performed better than those with longer chain spacers for a variety of substitution patterns. The most active compound held inhibition constant as low as Ki = 0.67 µM (for 49b) and a tert-butyl substituent in para position and acted as a competitive inhibitor of the enzyme.
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Anhidrasa Carbónica II , Inhibidores de Anhidrasa Carbónica , Ácidos Sulfónicos , Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica II/metabolismo , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Inhibidores de Anhidrasa Carbónica/farmacología , Ácidos Sulfónicos/química , Animales , Bovinos , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacología , Sulfonamidas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/farmacología , Estructura MolecularRESUMEN
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
In contemporary medicinal chemistry, employing a singular small molecule to concurrently multi-target disparate molecular entities is emerging as a potent strategy in the ongoing battle against metabolic disease. In this study, we present the meticulous design, synthesis, and comprehensive biological evaluation of a novel series of 1,2,3-triazolylmethylthio-1,3,4-oxadiazolylbenzenesulfonamide derivatives (8a-m) as potential multi-target inhibitors against human carbonic anhydrase (EC.4.2.1.1, hCA I/II), α-glycosidase (EC.3.2.1.20, α-GLY), and α-amylase (EC.3.2.1.1, α-AMY). Each synthesized sulfonamide underwent rigorous assessment for inhibitory effects against four distinct enzymes, revealing varying degrees of hCA I/II, a-GLY, and a-AMY inhibition across the tested compounds. hCA I was notably susceptible to inhibition by all compounds, demonstrating remarkably low inhibition constants (KI) ranging from 42.20 ± 3.90 nM to 217.90 ± 11.81 nM compared to the reference standard AAZ (KI of 439.17 ± 9.30 nM). The evaluation against hCA II showed that most of the synthesized compounds exhibited potent inhibition effects with KI values spanning the nanomolar range 16.44 ± 1.53-70.82 ± 4.51 nM, while three specific compounds, namely 8a-b and 8d, showcased lower inhibitory potency than other derivatives that did not exceed that of the reference drug AAZ (with a KI of 98.28 ± 1.69 nM). Moreover, across the spectrum of synthesized compounds, potent inhibition profiles were observed against diabetes mellitus-associated α-GLY (KI values spanning from 0.54 ± 0.06 µM to 5.48 ± 0.50 µM), while significant inhibition effects were noted against α-AMY, with IC50 values ranging between 0.16 ± 0.04 µM and 7.81 ± 0.51 µM) compared to reference standard ACR (KI of 23.53 ± 2.72 µM and IC50 of 48.17 ± 2.34 µM, respectively). Subsequently, these inhibitors were evaluated for their DPPH· and ABTS+· radical scavenging activity. Moreover, molecular docking investigations were meticulously conducted within the active sites of hCA I/II, α-GLY, and α-AMY to provide comprehensive elucidation and rationale for the observed inhibitory outcomes.
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Bencenosulfonamidas , Inhibidores de Anhidrasa Carbónica , Sulfonamidas , Sulfonamidas/química , Sulfonamidas/farmacología , Humanos , Inhibidores de Anhidrasa Carbónica/farmacología , Inhibidores de Anhidrasa Carbónica/química , Inhibidores de Anhidrasa Carbónica/síntesis química , Simulación del Acoplamiento Molecular , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/química , alfa-Amilasas/metabolismo , Anhidrasa Carbónica I/antagonistas & inhibidores , Anhidrasa Carbónica I/metabolismo , Anhidrasa Carbónica I/química , Anhidrasa Carbónica II/antagonistas & inhibidores , Anhidrasa Carbónica II/metabolismo , Anhidrasa Carbónica II/química , Relación Estructura-ActividadRESUMEN
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
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.
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Lipid metabolism dysregulation can lead to dyslipidemia and obesity, which are major causes of cardiovascular disease and associated mortality worldwide. The purpose of the study was to obtain and characterize six plant extracts (ACE-Allii cepae extractum; RSE-Rosmarini extractum; CHE-Cichorii extractum; CE-Cynarae extractum; AGE-Apii graveolentis extractum; CGE-Crataegi extractum) as promising adjuvant therapies for the prevention and treatment of dyslipidemia and its related metabolic diseases. Phytochemical screening revealed that RSE was the richest extract in total polyphenols (39.62 ± 13.16 g tannic acid/100 g dry extract) and phenolcarboxylic acids (22.05 ± 1.31 g chlorogenic acid/100 g dry extract). Moreover, the spectrophotometric chemical profile highlighted a significant concentration of flavones for CGE (5.32 ± 0.26 g rutoside/100 g dry extract), in contrast to the other extracts. UHPLC-MS quantification detected considerable amounts of phenolic constituents, especially chlorogenic acid in CGE (187.435 ± 1.96 mg/g extract) and rosmarinic acid in RSE (317.100 ± 2.70 mg/g extract). Rosemary and hawthorn extracts showed significantly stronger free radical scavenging activity compared to the other plant extracts (p < 0.05). Pearson correlation analysis and the heatmap correlation matrix indicated significant correlations between phytochemical contents and in vitro antioxidant activities. Computational studies were performed to investigate the potential anti-obesity mechanism of the studied extracts using target prediction, homology modeling, molecular docking, and molecular dynamics approaches. Our study revealed that rosmarinic acid (RA) and chlorogenic acid (CGA) can form stable complexes with the active site of carbonic anhydrase 5A by either interacting with the zinc-bound catalytic water molecule or by directly binding Zn2+. Further studies are warranted to experimentally validate the predicted CA5A inhibitory activities of RA and CGA and to investigate the hypolipidemic and antioxidant activities of the proposed plant extracts in animal models of dyslipidemia and obesity.