RESUMEN
As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.
Asunto(s)
Bacillus subtilis , Glucosamina , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Ingeniería de Proteínas , Glucosamina/biosíntesis , Glucosamina/metabolismo , Glucosamina/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Bacillus subtilis/genética , Simulación del Acoplamiento Molecular , Fructosa/metabolismo , Fructosa/química , Fructosa/biosíntesis , Simulación de Dinámica Molecular , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Dominio CatalíticoRESUMEN
Candida albicans is one of the most common species of fungus with life-threatening systemic infections and a high mortality rate. The outer cell wall layer of C. albicans is packed with mannoproteins and glycosylated polysaccharide moieties that play an essential role in the interaction with host cells and tissues. The glucosamine-6-phosphate synthase enzyme produces N-acetylglucosamine, which is a crucial chemical component of the cell wall of Candida albicans. Collectively, these components are essential to maintain the cell shape and for infection. So, its disruption can have serious effects on cell growth and morphology, resulting in cell death. Hence, it is considered a good antifungal target. In this study, we have performed an in silico approach to analyze the inhibitory potential of some polyphenols obtained from plants. Those can be considered important in targeting against the enzyme glucosamine-6-phosphate synthase (PDB-2VF5). The results of the study revealed that the binding affinity of complexes theaflavin and 3-o-malonylglucoside have significant docking scores and binding free energy followed by significant ADMET parameters that predict the drug-likeness property and toxicity of polyphenols as potential ligands. A molecular dynamic simulation was used to test the validity of the docking scores, and it showed that the complex remained stable during the period of the simulation, which ranged from 0 to 100 ns. Theaflavins and 3-o-malonylglucoside may be effective against Candida albicans using a computer-aided drug design methodology that will further enable researchers for future in vitro and in vivo studies, according to our in silico study.Communicated by Ramaswamy H. Sarma.
Asunto(s)
Candida albicans , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora) , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Polifenoles/farmacología , Antifúngicos/farmacología , Antifúngicos/química , Simulación de Dinámica Molecular , Simulación del Acoplamiento MolecularRESUMEN
The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Retroalimentación Fisiológica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Hexosaminas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Estrés del Retículo Endoplásmico , Mutación con Ganancia de Función , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Cinética , Fosforilación , Unión Proteica , Dominios Proteicos , Serina/genética , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
BACKGROUND: The frequent use of antibacterial agents leads to antimicrobial resistance, which is one of the biggest threats to global health today. Therefore, the discovery of novel antimicrobial agents is still urgently needed to overcome the severe infections caused by these putative pathogens resistant to currently available drugs. OBJECTIVE: The present work was aimed to synthesize and investigate the preliminary structureactivity relationships (SARs) of isoxazoline and pyrazoline derivatives as antimicrobial agent. METHODS: Target compounds were obtained in a multistep reaction synthesis and the antimicrobial activity was investigated in several species; two-gram negative (Escherichia coli and Pseudomonas aeruginosa), two-gram positive (Staphylococcus aureus and Bacillus subtilis) and one fungi (Candida albicans), using cup-plate agar diffusion method. The most potent compounds were docked into glucosamine-6-phosphate synthase (GlcN-6-P), the molecular target enzyme for antimicrobial agents, using Autodock 4.2 program. RESULTS: Herein, thirteen novel target compounds were synthesized in moderate to good isolated yield. Based on the SARs, two compounds (2c and 5c) were found to be potent antimicrobial agents on all tested targets, recording potency higher than amoxicillin, the standard antimicrobial drug. Compound 2b identified as selective for gram-negative, while compound 7a found to be selective for gram-positive. The hit compounds (2c, 5a, 5c and 5d) were subjected to a docking study on glucosamine-6-phosphate synthase (GlcN-6-P). All hits were found to bind to the orthosteric (active) site of the enzyme, which might represent a competitive mechanism of inhibition. CONCLUSION: The newly synthesized heterocyclic compounds could serve as potent leads for the development of novel antimicrobial agents.
Asunto(s)
Antibacterianos/farmacología , Antifúngicos/farmacología , Isoxazoles/farmacología , Pirazoles/farmacología , Antibacterianos/síntesis química , Antibacterianos/metabolismo , Antifúngicos/síntesis química , Antifúngicos/metabolismo , Bacterias/efectos de los fármacos , Candida albicans/efectos de los fármacos , Dominio Catalítico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Isoxazoles/síntesis química , Isoxazoles/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Pirazoles/síntesis química , Pirazoles/metabolismo , Relación Estructura-ActividadRESUMEN
Glycoconjugates play a central role in several cellular processes, and alteration in their composition is associated with numerous human pathologies. Substrates for cellular glycosylation are synthesized in the hexosamine biosynthetic pathway, which is controlled by the glutamine:fructose-6-phosphate amidotransfera-se (GFAT). Human isoform 2 GFAT (hGFAT2) has been implicated in diabetes and cancer; however, there is no information about structural and enzymatic properties of this enzyme. Here, we report a successful expression and purification of a catalytically active recombinant hGFAT2 (rhGFAT2) in Escherichia coli cells fused or not to a HisTag at the C-terminal end. Our enzyme kinetics data suggest that hGFAT2 does not follow the expected ordered bi-bi mechanism, and performs the glucosamine-6-phosphate synthesis much more slowly than previously reported for other GFATs. In addition, hGFAT2 is able to isomerize fructose-6-phosphate into glucose-6-phosphate even in the presence of equimolar amounts of glutamine, which results in unproductive glutamine hydrolysis. Structural analysis of a three-dimensional model of rhGFAT2, corroborated by circular dichroism data, indicated the presence of a partially structured loop in the glutaminase domain, whose sequence is present in eukaryotic enzymes but absent in the E. coli homolog. Molecular dynamics simulations suggest that this loop is the most flexible portion of the protein and plays a key role on conformational states of hGFAT2. Thus, our study provides the first comprehensive set of data on the structure, kinetics, and mechanics of hGFAT2, which will certainly contribute to further studies on the (patho)physiology of hGFAT2.
Asunto(s)
Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Humanos , Cinética , Simulación de Dinámica Molecular , Conformación Proteica , Dominios Proteicos , Multimerización de ProteínaRESUMEN
G-6-P synthase enzyme has been involved in the synthesis of the microbial cell wall, and its inhibition may lead to the antimicrobial effect. In the present study, we designed a library of amygdalin derivatives, and two most active derivatives selected on the basis of various parameters viz. dock score, binding energy, and ADMET data using molecular docking software (Schrodinger's Maestro). The selected derivatives were synthesized and evaluated for their antioxidant and antimicrobial potential against several Gram (+ ve), Gram (-ve), as well as fungal strains. The results indicated that synthesized compounds exhibited good antioxidant, antimicrobial, and better preservative efficacy in food preparation as compared to the standard compounds. No significant differences were observed in different parameters as confirmed by Kruskal-Wallis test (p < 0.05). Docking results have been found in good correlation with experimental wet-lab data. Moreover, the mechanistic insight into the docking poses has also been explored by binding interactions of amygdalin derivative inside the dynamic site of G-6-P synthase.
Asunto(s)
Amigdalina/farmacología , Conservantes de Alimentos/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Conservadores Farmacéuticos/farmacología , Aloe/química , Amigdalina/síntesis química , Amigdalina/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Conservantes de Alimentos/química , Jugos de Frutas y Vegetales , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Ligandos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Conservadores Farmacéuticos/químicaRESUMEN
OBJECTIVE: To improve enzyme activity of Glucosamine-6-phosphate synthase (Glms) of Bacillus subtilis by site saturation mutagenesis at Leu593, Ala594, Lys595, Ser596 and Val597 based on computer-aided semi-rational design. RESULTS: The results indicated that L593S had the greatest effect on the activity of BsGlms and the enzyme activity increased from 5 to 48 U/mL. The mutation of L593S increased the yield of glucosamine by 1.6 times that of the original strain. The binding energy of the mutant with substrate was reduced from - 743.864 to - 768.246 kcal/mol. Molecular dynamics simulation results showed that Ser593 enhanced the flexibility of the protein, which ultimately led to increased enzyme activity. CONCLUSION: We successfully improved BsGlms activity through computer simulation and site saturation mutagenesis. This combination of methodologies may fit into an efficient workflow for improving Glms and other proteins activity.
Asunto(s)
Bacillus subtilis/enzimología , Glucosamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Bacillus subtilis/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Simulación por Computador , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Homología Estructural de ProteínaRESUMEN
Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies.
Asunto(s)
Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Hexosaminas/metabolismo , Retroalimentación Fisiológica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glicosilación , Hexosaminas/química , Humanos , Ligandos , Conformación Proteica , Proteostasis , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
A library of novel pyrazole-imidazo[1,2-α]pyridine scaffolds was designed and synthesized through a one-pot three-component tandem reaction. The structures of synthesized conjugates were confirmed by spectroscopic techniques (NMR, IR and HRMS). In vitro antibacterial evaluation of the twelve synthesized molecules (7a, 8a-k) against methicillin-resistant Staphylococcus aureus and normal strains of Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia and Pseudomonas aeruginosa established 8b, 8d, 8e, 8h and 8i as potent antibacterial agents with superior minimum bactericidal concentration, compared with standard drug ciprofloxacin. Molecular docking studies of all active compounds into the binding site of glucosamine-6-phosphate synthase were further performed in order to have a comprehensive understanding of putative binding modes within the active sites of the receptor.
Asunto(s)
Antibacterianos/síntesis química , Inhibidores Enzimáticos/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Pirazoles/síntesis química , Piridinas/síntesis química , Bibliotecas de Moléculas Pequeñas/síntesis química , Antibacterianos/farmacología , Sitios de Unión , Ciprofloxacina/farmacología , Ciprofloxacina/normas , Evaluación Preclínica de Medicamentos , Farmacorresistencia Bacteriana Múltiple , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Humanos , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Estructura Molecular , Pirazoles/farmacología , Piridinas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Relación Estructura-ActividadRESUMEN
A series of novel covalent cholesterol-spiro pyrrolidine/pyrrolizidine heterocyclic hybrids possessing biologically active oxindole, indanedione, and acenaphthylene-1-one have been synthesized by the reaction of C3-ß-cholesteroalacrylate with heterocyclic di- and tri-ketones. All the sixteen compounds were obtained as a single isomer in good yield through a stereo- and regio- selective 1,3-dipolar cycloaddition methodology. Stereochemistry of the spiranic cycloadducts has been established by spectroscopic analysis and the regioselectivity outcome of the spiro adducts has been accomplished by DFT calculations at B3LYP/6-31G (d,p) level study. In vitro antibacterial activity of the newly synthesized cycloadducts were evaluated against highly pathogenic Gram-positive and Gram-negative bacteria and the most active compounds 5a, 13, and 14 underwent automated in silico molecular docking analysis in order to validate their effective orientation as a inhibitors bound in the active site of glucosamine-6-phosphate synthase (1XFF) enzyme by employing AutoDock Tools.
Asunto(s)
Antibacterianos/farmacología , Colesterol/análogos & derivados , Colesterol/farmacología , Inhibidores Enzimáticos/farmacología , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Antibacterianos/síntesis química , Bacterias/efectos de los fármacos , Dominio Catalítico , Reacción de Cicloadición , Teoría Funcional de la Densidad , Inhibidores Enzimáticos/síntesis química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Modelos Químicos , Simulación del Acoplamiento Molecular , Unión Proteica , Compuestos de Espiro/síntesis química , EstereoisomerismoRESUMEN
Site-directed mutagenesis of the CaGFA1 gene encoding glucosamine-6-phosphate synthase from Candida albicans was performed. Desensitization of the enzyme to inhibition by UDPGlcNAc was achieved upon T487I and H492F substitutions at the UDP-GlcNAc binding site, exchange of D524, S525 and S527 for Ala at the dimer:dimer interface and construction of the tail-lock array (L434R and L460A) at the C-tail region. The first two sets if mutageneses but not the last one resulted in conversion of the tetrameric enzyme into its dimeric form. Evidence for links and communication between the UDP-GlcNAc binding site and the dimer-dimer contact areas are presented. The CaGfa1-T487IH492F and CaGfa1-KHSH-D524AS525AS527A muteins are the first examples of the successful conversion of eukaryotic GlcN-6-P synthase into its prokaryotic-like version upon rational site-directed mutagenesis.
Asunto(s)
Candida albicans/enzimología , Proteínas Fúngicas/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Mutagénesis Sitio-Dirigida , Sitios de Unión , Candida albicans/genética , ADN de Hongos/genética , Escherichia coli/genética , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Estructura Cuaternaria de ProteínaRESUMEN
Nodulation outer protein M (NopM) is an IpaH family type three (T3) effector secreted by the nitrogen-fixing nodule bacterium Sinorhizobium sp. strain NGR234. Previous work indicated that NopM is an E3 ubiquitin ligase required for an optimal symbiosis between NGR234 and the host legume Lablab purpureus Here, we continued to analyze the function of NopM. Recombinant NopM was biochemically characterized using an in vitro ubiquitination system with Arabidopsis thaliana proteins. In this assay, NopM forms unanchored polyubiquitin chains and possesses auto-ubiquitination activity. In a NopM variant lacking any lysine residues, auto-ubiquitination was not completely abolished, indicating noncanonical auto-ubiquitination of the protein. In addition, we could show intermolecular ubiquitin transfer from NopM to C338A (enzymatically inactive NopM form) in vitro Bimolecular fluorescence complementation analysis provided clues about NopM-NopM interactions at plasma membranes in planta NopM, but not C338A, expressed in tobacco cells induced cell death, suggesting that E3 ubiquitin ligase activity of NopM induced effector-triggered immunity responses. Likewise, expression of NopM in Lotus japonicus caused reduced nodule formation, whereas expression of C338A showed no obvious effects on symbiosis. Further experiments indicated that serine residue 26 of NopM is phosphorylated in planta and that NopM can be phosphorylated in vitro by salicylic acid-induced protein kinase (NtSIPK), a mitogen-activated protein kinase (MAPK) of tobacco. Hence, NopM is a phosphorylated T3 effector that can interact with itself, with ubiquitin, and with MAPKs.
Asunto(s)
Proteínas Bacterianas/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Proteínas Recombinantes/genética , Simbiosis/genética , Ubiquitina-Proteína Ligasas/química , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas Bacterianas/química , Regulación Bacteriana de la Expresión Génica , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Lotus/genética , Lotus/microbiología , MAP Quinasa Quinasa 1/química , MAP Quinasa Quinasa 1/genética , Fijación del Nitrógeno/genética , Fosforilación , Poliubiquitina/química , Poliubiquitina/genética , Proteínas Recombinantes/química , Sinorhizobium/enzimología , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación/genéticaRESUMEN
Glucosamine-6-phosphate synthase (EC 2.6.1.16) is responsible for catalysis of the first and practically irreversible step in hexosamine metabolism. The final product of this pathway, uridine 5' diphospho N-acetyl-d-glucosamine (UDP-GlcNAc), is an essential substrate for assembly of bacterial and fungal cell walls. Moreover, the enzyme is involved in phenomenon of hexosamine induced insulin resistance in type II diabetes, which makes of it a potential target for anti-fungal, anti-bacterial and anti-diabetic therapy. The crystal structure of isomerase domain from human pathogenic fungus Candida albicans has been solved recently but it doesn't reveal the molecular mechanism details of inhibition taking place under UDP-GlcNAc influence, the unique feature of eukaryotic enzyme. The following study is a continuation of the previous research based on comparative molecular dynamics simulations of the structures with and without the enzyme's physiological inhibitor (UDP-GlcNAc) bound. The models used for this study included fructose-6-phosphate, one of the enzyme's substrates in its binding pocket. The simulation results studies demonstrated differences in mobility of the compared structures. Some amino acid residues were determined, for which flexibility is evidently different between the models. Importantly, it has been confirmed that the most fixed residues are related to the inhibitor binding process and to the catalysis reaction. The obtained results constitute an important step towards understanding of the inhibition that GlcN-6-P synthase is subjected by UDP-GlcNAc molecule.
Asunto(s)
Acetilglucosamina/química , Candida albicans/enzimología , Fructosafosfatos/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Aminoácidos/química , Candida albicans/química , Catálisis , Glucosamina/análogos & derivados , Glucosamina/química , Glucosa-6-Fosfato/análogos & derivados , Glucosa-6-Fosfato/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Humanos , Cinética , Simulación de Dinámica Molecular , Unión Proteica , Conformación ProteicaRESUMEN
The biological activity of heterocyclic compounds depends on their structure, the type of hetero atoms in the ring and on the type of substituents present. In this paper, some heterocyclic compounds with coumarin moieties S1-S5 and novobiocin known as coumarin antibiotic were subjected to the molecular docking studies as important tools for drug discovery. Glucosamine-6-phosphate synthase is selected as a suitable target in this study. In silico studies reveal that all synthesized compounds S1-S5 are good inhibitors of GlcN-6 and the docking results are in agreement with in vitro antibacterial evaluation of compounds S1-S5.
Asunto(s)
Cumarinas/química , Inhibidores Enzimáticos/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Simulación del Acoplamiento Molecular , Animales , Sitios de Unión , Cumarinas/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Ligandos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
6-Sulfo-6-deoxy-D-glucosamine (GlcN6S), 6-sulfo-6-deoxy-D-glucosaminitol (ADGS) and their N-acetyl and methyl ester derivatives have been synthesized and tested as inhibitors of enzymes catalyzing reactions of the UDP-GlcNAc pathway in bacteria and yeasts. GlcN6S and ADGS at micromolar concentrations inhibited glucosamine-6-phosphate (GlcN6P) synthase of microbial origin. The former was also inhibitory towards fungal GlcN6P N-acetyl transferase, but at millimolar concentrations. Both compounds and their N-acetyl derivatives exhibited antimicrobial in vitro activity, with MICs in the 0.125-2.0 mg mL-1 range. Antibacterial but not antifungal activity of GlcN6S was potentiated by D-glucosamine and a synergistic antibacterial effect was observed for combination of ADGP and a dipeptide Nva-FMDP.
Asunto(s)
Antiinfecciosos/síntesis química , Antiinfecciosos/farmacología , Glucosamina/síntesis química , Glucosamina/farmacología , Tioazúcares/farmacología , Antiinfecciosos/química , Antiinfecciosos/metabolismo , Técnicas de Química Sintética , Glucosamina/química , Glucosamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Espacio Intracelular/metabolismo , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Conformación Proteica , Tioazúcares/síntesis química , Tioazúcares/química , Tioazúcares/metabolismoRESUMEN
BACKGROUND: Synthesis, characterization and investigation of antibacterial activity of ten novel Schiff base derivatives of 4-formylbenzoic acid is presented. Their structures were determined using 1H and 13CNMR, EI(+)-MS and elemental analyses. Additionally, DFT calculations of interaction energies in complexes of the novel drugs and DNA bases are carried out. OBJECTIVE: Design and synthesis of thiazole derivatives with benzoic acid scaffold to obtain compounds with an improved antibacterial activity. METHOD: The examined compounds were screened in vitro for antibacterial activity using the broth microdilution method. Geometrical parameters of the investigated complexes were optimized within the Density Functional Theory (DFT) approximation using the B3LYP functional and the 6-311G** basis set. The docking simulations were performed using the FlexX docking module. RESULTS: Among the derivatives, compound 4b showed very strong bacterial activity against staphylococci, MIC 1.95-3.91 µg/ml, micrococci, MIC 0.98 µg/ml, and Bacillus spp., MIC 7.81-15.62 µg/ml. The compounds 4c, 4d, 4e and 4j also showed high bioactivity against staphylococci, MIC 3.91-31.25 µg/ml, and micrococci, MIC 0.98-15.62 µg/ml. Interaction energy values for investigated guanine complexes are about 2 kcal/mol lower than for the corresponding cytosine complexes. Molecular docking studies of all compounds on the active sites of bacterial enzymes indicated gyrase B as possible target. CONCLUSION: To conclude, an efficient and economic method for the synthesis of thiazoles containing benzoic acid moiety has been developed. The results of antibacterial screenings reveal that some obtained compounds show high to very strong antibacterial activity. The DFT calculations showed that interaction of the obtained drugs with guanine is stronger than with cytosine. Molecular docking studies of all compounds on the active sites of bacterial enzymes indicated gyrase B as possible target.
Asunto(s)
Antibacterianos/farmacología , Benzoatos/farmacología , Tiazoles/farmacología , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/antagonistas & inhibidores , 3-Oxoacil-(Proteína Transportadora de Acil) Sintasa/química , Adenina/química , Antibacterianos/síntesis química , Antibacterianos/química , Benzoatos/síntesis química , Benzoatos/química , Citosina/química , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Guanina/química , Simulación del Acoplamiento Molecular , Péptido Sintasas/antagonistas & inhibidores , Péptido Sintasas/química , Teoría Cuántica , Estereoisomerismo , Relación Estructura-Actividad , Termodinámica , Tiazoles/síntesis química , Tiazoles/química , Timina/química , Inhibidores de Topoisomerasa II/síntesis química , Inhibidores de Topoisomerasa II/química , Inhibidores de Topoisomerasa II/farmacologíaRESUMEN
Mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) cause the neuromuscular disorder limb-girdle congenital myasthenic syndrome (LG-CMS). One recurrent GFPT1 mutation detected in LG-CMS patients is a c.*22C>A transversion in the 3'-untranslated region (UTR). Because this variant does not alter the GFPT1 open reading frame, its pathogenic relevance has not yet been established. We found that GFPT1 protein levels were reduced in myoblast cells of the patients carrying this variant. In silico algorithms predicted that the mutation creates a microRNA target site for miR-206*. Investigation of the expression of this so far unrecognized microRNA confirmed that miR-206* (like its counterpart miR-206) is abundant in skeletal muscle. MiR-206* efficiently reduced the expression of reporter constructs containing the mutated 3'-UTR while no such effect was observed with reporter constructs containing the wild-type 3'-UTR or when a specific anti-miR-206* inhibitor was added. Moreover, anti-miR-206* inhibitor treatment substantially rescued GFPT1 expression levels in patient-derived myoblasts. Our data demonstrate that the c.*22C>A mutation in the GFPT1 gene leads to illegitimate binding of microRNA resulting in reduced protein expression. We confirm that c.*22C>A is a causative mutation and suggest that formation of microRNA target sites might be a relevant pathomechanism in Mendelian disorders. Variants in the 3'-UTRs should be considered in genetic diagnostic procedures.
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Regiones no Traducidas 3' , Sitios de Unión , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , MicroARNs/genética , Mutación , Síndromes Miasténicos Congénitos/genética , ARN Mensajero/genética , Animales , Secuencia de Bases , Línea Celular , Expresión Génica , Perfilación de la Expresión Génica , Genes Reporteros , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Humanos , MicroARNs/química , Células Musculares/metabolismo , Síndromes Miasténicos Congénitos/metabolismo , Interferencia de ARN , ARN Mensajero/químicaRESUMEN
BACKGROUND: Structural modifications of thiazolidinediones at 3rd and 5th position have exhibited significant biological activities. In view of the facts, and based on in silico studies carried out on thiazolidine-2,4-diones as HIV-1- RT inhibitors, a novel series of 2,4-thiazolidinedione analogs have been designed and synthesized. METHODS: Title compounds were prepared by the reported method. Conformations of the structures were assigned on the basis of results of different spectral data. The assay of HIV-1 RT was done as reported by Silprasit et al. Antimicrobial activity was determined by two fold serial dilution method. Docking study was performed for the highest active compounds by using Glide 5.0. RESULTS: The newly synthesized compounds were evaluated for their HIV-1 RT inhibitory activity. Among the synthesized compounds, compound 24 showed significant HIV-1 RT inhibitory activity with 73% of inhibition with an IC50 value of 1.31 µM. Compound 10 showed highest activity against all the bacterial strains.A molecular modeling study was carried out in order to investigate the possible interactions of the highest active compounds 24, 10 and 4 with the non nucleoside inhibitory binding pocket(NNIBP) of RT, active site of GlcN-6-P synthase and cytochrome P450 14-α-sterol demethylase from Candida albicans (Candida P450DM) as the target receptors respectively using the Extra Precision (XP) mode of Glide software. CONCLUSION: A series of novel substituted 2-(5-benzylidene-2,4-dioxothiazolidin-3-yl)-N-(phenyl)propanamides (4-31) have been synthesized and evaluated for their HIV-1 RT inhibitory activity, antibacterial and antifungal activities. Some of the compounds have shown significant activity. Molecular docking studies showed very good interaction.
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Antibacterianos/farmacología , Fármacos Anti-VIH/farmacología , Antifúngicos/farmacocinética , Diseño de Fármacos , Modelos Moleculares , Inhibidores de la Transcriptasa Inversa/farmacología , Tiazolidinedionas/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/metabolismo , Fármacos Anti-VIH/síntesis química , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Antifúngicos/síntesis química , Antifúngicos/química , Antifúngicos/metabolismo , Sitios de Unión , Candida albicans/efectos de los fármacos , Candida albicans/enzimología , Candida albicans/crecimiento & desarrollo , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Transcriptasa Inversa del VIH/antagonistas & inhibidores , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , Cinética , Ligandos , Pruebas de Sensibilidad Microbiana , Conformación Molecular , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de la Transcriptasa Inversa/síntesis química , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/metabolismo , Relación Estructura-Actividad , Tiazolidinedionas/síntesis química , Tiazolidinedionas/química , Tiazolidinedionas/metabolismoRESUMEN
Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is an essential enzyme involved in cell wall biogenesis that has been proposed as a strategic target for antifungal chemotherapy. Here we describe the cloning and functional characterization of Sporothrix schenckii GFA1 gene which was isolated from a genomic library of the fungus. The gene encodes a predicted protein of 708 amino acids that is homologous to GlcN-6-P synthases from other sources. The recombinant enzyme restored glucosamine prototrophy of the Saccharomyces cerevisiae gfa1 null mutant. Purification and biochemical analysis of the recombinant enzyme revealed some differences from the wild type enzyme, such as improved stability and less sensitivity to UDP-GlcNAc. The sensitivity of the recombinant enzyme to the selective inhibitor FMDP [N(3)-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid] and other properties were similar to those previously reported for the wild type enzyme.
Asunto(s)
Proteínas Fúngicas/aislamiento & purificación , Glucosamina/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/aislamiento & purificación , Plásmidos/metabolismo , Saccharomyces cerevisiae/genética , Sporothrix/química , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Escherichia coli/metabolismo , Fumaratos/química , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expresión Génica , Prueba de Complementación Genética , Biblioteca Genómica , Glucosamina/análogos & derivados , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/antagonistas & inhibidores , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/química , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Cinética , Sistemas de Lectura Abierta , Plásmidos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Sporothrix/enzimología , beta-Alanina/análogos & derivados , beta-Alanina/químicaRESUMEN
Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml(-1) for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein (-1). The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The Km and Vmax of the GlcN-6-P synthase towards D-fructose 6-phosphate were 2.8 mM and 6.9 µmol min(-1) mg(-1), respectively.