RESUMEN
Endocrine-disrupting chemicals are a major public health problem throughout the world. In the human body, these compounds functionalize the same as sexual hormones, inducing precocious puberty, gynecomastia, etc. To help prevent this occurrence, a simple detection system is needed. In this study, a nonylphenol ethoxylate (NPE)-specific aptamer was selected by reduced graphene oxide-systematic evolution of ligands by exponential enrichment. A random ssDNA library was incubated with rGO for adsorption, followed by elution with the target molecule. As a result of screening, a DNA aptamer was found that specifically bounds to the target with high binding affinity (Kd = 100.9 ± 13.2 nM) and had a low limit of detection (LOD = 696 pM). Furthermore, this NPE-binding aptamer bounds selectively to the target. Characterization of the aptamer was confirmed by measuring the fluorescence signal recovery from rGO. In addition, detection of NPE was performed with several water samples, and the detection accuracy was 100 ± 10%. From these results, we expect that this aptamer could be applied to an on-site detection system for NPE in industrial sites or domestic fields.
Asunto(s)
ADN de Cadena Simple/metabolismo , Detergentes/análisis , Glicoles de Etileno/análisis , Grafito/química , Adsorción , Aptámeros de Nucleótidos/química , ADN de Cadena Simple/química , Biblioteca de Genes , Humanos , Límite de Detección , Modelos Moleculares , Conformación de Ácido Nucleico , Técnica SELEX de Producción de AptámerosRESUMEN
Anthrax is caused by Bacillus anthracis, a bacterium that is able to secrete the toxins protective antigen, edema factor and lethal factor. Due to the high level of secretion from the bacteria and its severe virulence, lethal factor (LF) has been sought as a biomarker for detecting bacterial infection and as an effective target to neutralize toxicity. In this study, we found three aptamers, and binding affinity was determined by fluorescently labeled aptamers. One of the aptamers exhibited high affinity, with a Kd value of 11.0⯱â¯2.7â¯nM, along with low cross reactivity relative to bovine serum albumin and protective antigen. The therapeutic functionality of the aptamer was examined by assessing the inhibition of LF protease activity against a mitogen-activated protein kinase kinase. The aptamer appears to be an effective inhibitor of LF with an IC50 value of 15⯱â¯1.5⯵M and approximately 85% cell viability, suggesting that this aptamer provides a potential clue for not only development of a sensitive diagnostic device of B. anthracis infection but also the design of novel inhibitors of LF.
Asunto(s)
Aptámeros de Nucleótidos/metabolismo , Toxinas Bacterianas/antagonistas & inhibidores , ADN de Cadena Simple/metabolismo , Animales , Antígenos Bacterianos/metabolismo , Aptámeros de Nucleótidos/toxicidad , Bacillus anthracis/química , Toxinas Bacterianas/metabolismo , ADN de Cadena Simple/toxicidad , Ensayo de Inmunoadsorción Enzimática , MAP Quinasa Quinasa 1/química , MAP Quinasa Quinasa 1/metabolismo , Ratones , Unión Proteica , Proteolisis , Células RAW 264.7 , Técnica SELEX de Producción de AptámerosRESUMEN
Antibiotics are useful for improving the living conditions of livestock. However, residual antibiotics induce several human diseases such as food-borne illness and infection of carbapenem-resistant Enterobacteriaceae (CRE). In this study, the identification of a benzylpenicillin-specific aptamer was selected by rGO-SELEX (reduced Graphene Oxide-Systematic Evolution of Ligands by EXponential enrichment). A random ssDNA library was incubated with rGO for adsorption and eluted with benzylpenicillin. As a result of the selection process, a DNA aptamer was found that specifically bound to benzylpenicillin with high binding affinity, Kd = 383.4 nM, and had a low limit of detection (LOD) of 9.2 nM. The characterization of the aptamer was performed through the fluorescence recovery signal from rGO surface. In addition, detection of benzylpenicillin was performed in pretreated milk samples, and its detection accuracy was shown to be 100± 10%. This represented that BBA1 was used for fluorescence aptasensor system in real sample. Furthermore, this benzylpenicillin binding aptamer showed high specificity against other antibiotics except for ampicillin. With these advantageous characteristics, we expect that this aptamer could be applied to an on-site detection system for residual benzylpenicillin.
Asunto(s)
Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/síntesis química , Penicilina G/análisis , Técnica SELEX de Producción de Aptámeros/métodos , Fluorescencia , Grafito , HumanosRESUMEN
Ecumicin is a well-known and potent inhibitor of Mycobacterium tuberculosis. Although the target of ecumicin is caseinolytic protease C1 (ClpC1), the exact mechanism by which ecumicin inhibits ClpC1 has not been identified. To analyze ecumicin's action on ClpC1, site-directed mutagenesis was performed on its binding site. The estimated binding residues within ClpC1 to ecumicin were selected via in silico analysis using molecular docking. The selected residues were mutated by site-directed mutagenesis and the effects on ecumicin binding were analyzed. Mutation at the R83 residue, especially the R83A mutation, in ClpC1 resulted in strong resistance to ATPase activation and inhibition of proteolytic activity. In addition, binding of ecumicin to the R83A ClpC1 N-terminal domain (residues 1-145) was not observed in native gel analysis. These results reveal that the R83 residue plays an important role in the binding of ecumicin. This result provides a basis for the development of an anti-tuberculosis agent based on ecumicin derivatives.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Mutación , Mycobacterium tuberculosis/enzimología , Péptidos Cíclicos/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Activación Enzimática/efectos de los fármacos , Proteínas de Choque Térmico/química , Cinética , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/efectos de los fármacos , Péptidos Cíclicos/farmacología , Unión Proteica , ProteolisisRESUMEN
Overuse of antibiotics has caused serious problems, such as appearance of super bacteria, whose accumulation in the human body through the food chain is a concern. Kanamycin is a common antibiotic used to treat diverse infections; however, residual kanamycin can cause many side effects in humans. Thus, development of an ultra-sensitive, precise, and simple detection system for residual kanamycin in food products is urgently needed for food safety. In this study, we identified kanamycin-binding aptamers via a new screening method, and truncated variants were analyzed for optimization of the minimal sequence required for target binding. We found various aptamers with high binding affinity from 34.7 to 669 nanomolar Kdapp values with good specificity against kanamycin. Furthermore, we developed a reduced graphene oxide (RGO)-based fluorescent aptasensor for kanamycin detection. In this system, kanamycin was detected at a concentration as low as 1 pM (582.6 fg/mL). In addition, this method could detect kanamycin accurately in kanamycin-spiked blood serum and milk samples. Consequently, this simple, rapid, and sensitive kanamycin detection system with newly structural and functional analysis aptamer exhibits outstanding detection compared to previous methods and provides a new possibility for point of care testing and food safety.
RESUMEN
Mycobacterium tuberculosis acetohydroxyacid synthase (MTB-AHAS) has been suggested as a crucial target for antibacterial agents. High-throughput screening of a chemical library was performed to identify potent new inhibitors of MTB-AHAS. Among the 6800 tested compounds, 15 were identified as potent inhibitors, exhibiting >80-90% inhibition of in vitro MTB-AHAS activity at a fixed concentration of 20 µM. Five compounds belonging to the triazolopyrimidine structural class showed greater inhibition potency, with a half-maximum inhibition concentration (IC50 value) in the low micromolar range (0.4-1.24 µM). Furthermore, potent inhibitors demonstrated non-competitive, uncompetitive or mixed-competitive inhibition. Molecular docking experiments with these potent chemicals using a homology model of MTB-AHAS indicated hydrophobic and hydrogen bond interactions with some key herbicide binding site residues with binding energies (ΔG) of -8.04 to -10.68 Kcal/mol, respectively. The binding modes were consistent with inhibition mechanisms, as the chemicals were oriented outside the active site. Importantly, these potent inhibitors demonstrated significant growth inhibition of various clinically isolated multidrug-resistant and extensively drug-resistant M. tuberculosis strains, with 50% minimum inhibitory concentrations (MIC50 values) ranging from 0.2 µg/mL to 0.8 µg/mL, which resemble the MICs of conventional drugs for tuberculosis (isoniazid, 0.1 µg/mL; rifampicin, 0.4 µg/mL). Thus, the identified potent inhibitors show potential as scaffolds for further in vivo studies and might provide an impetus for the development of strong antituberculosis agents targeting MTB-AHAS.
Asunto(s)
Acetolactato Sintasa/antagonistas & inhibidores , Antituberculosos/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/enzimología , Acetolactato Sintasa/química , Antituberculosos/química , Antituberculosos/aislamiento & purificación , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Unión Proteica , Tuberculosis Resistente a Múltiples Medicamentos/microbiologíaRESUMEN
Mycobacterium tuberculosis acetohydroxyacid synthase (M. tuberculosis AHAS) has been proposed to bean essential target for novel herbicide- and chemical-based antibacterial agents. Therefore, here we investigated the roles of multiple conserved herbicide-binding site residues (R318, A146, Q148, M512, and V513) in M. tuberculosis AHAS through site-directed mutagenesis by characterizing the kinetic parameters and herbicide sensitivities of various point mutants. Interestingly, all mutant enzymes showed significantly altered kinetic parameters, specifically reduced affinity towards both the substrate and cofactor. Importantly, mutation of R318 led to a complete loss of AHAS activity, indicating a key role for this residue in substrate binding. Furthermore, all mutants demonstrated significant herbicide resistance against chlorimuron ethyl (CE), with several-fold higher IC50 than that of wild type AHAS. Docking analysis also indicated that binding of CE was slightly affected upon mutation of these residues. Taken together, these data suggest that the residues examined here mediate CE binding and may also be important for the catalytic activity of AHAS. This study will pave the way for future structure-function studies of CE and will also aid the development of novel anti-tuberculosis agents based on this chemical scaffold.
Asunto(s)
Acetolactato Sintasa/genética , Acetolactato Sintasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Herbicidas/metabolismo , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/genética , Acetolactato Sintasa/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Antituberculosos/metabolismo , Antituberculosos/farmacología , Proteínas Bacterianas/química , Dominio Catalítico/genética , Herbicidas/farmacología , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mycobacterium tuberculosis/efectos de los fármacos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Homología Estructural de ProteínaRESUMEN
Acetohydroxyacid synthase (AHAS) catalyzes the first essential biosynthetic step of branched-chain amino acids and is a biologically safe target against Mycobacterium tuberculosis (MTB). In our previous research, we used virtual screening to identify some novel AHAS inhibitors as potent antituberculosis agents. In this study, we synthesized twenty-four additional quinazolinone benzoates and explored their antitubercular activity. Five of these compounds displayed significant MTB-AHAS inhibition and their IC50 values were determined to be in the range of 6.50 µM-12.08 µM. Importantly, these compounds also exhibited strong in vitro activity (MICs in the range of 2.5-10 mg/L) and intracellular activity against clinically isolated extensively drug-resistant strains of M. tuberculosis. Taken together, these results indicated that the quinazolinone benzoate compounds should be regarded as promising lead compounds for the development of potent antituberculosis drugs with a novel mode of action.
Asunto(s)
Acetolactato Sintasa/antagonistas & inhibidores , Antituberculosos/farmacología , Benzoatos/farmacología , Inhibidores Enzimáticos/farmacología , Mycobacterium tuberculosis/efectos de los fármacos , Quinazolinonas/farmacología , Acetolactato Sintasa/metabolismo , Antituberculosos/síntesis química , Antituberculosos/química , Benzoatos/síntesis química , Benzoatos/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Quinazolinonas/síntesis química , Quinazolinonas/química , Relación Estructura-ActividadRESUMEN
Catabolic acetolactate synthase (cALS) from Enterococcus faecalis is a FAD-independent enzyme, which catalyzes the condensation of two molecules of pyruvate to produce acetolactate. Mutational and kinetic analyses of variants suggested the importance of H111, Q112, and Q411 residues for catalysis in cALS. The wild-type and variants were expressed as equally soluble proteins and co-migrated to a size of 60 kDa on SDS-PAGE. Importantly, H111 in cALS, which is widely present as phenylalanine in many other ThDP-dependent enzymes, plays a crucial role in substrate binding. Interestingly, the H111 variants, H111R and H111F, demonstrated altered specific activity of H111 variants with 17- and 26-fold increases in Km, respectively, compared to wild-type cALS. Furthermore, Q112 variants, Q112E, Q112N, and Q112V, exhibited significantly lower specific activity with 70-, 15-, and 10-fold higher Ks for ThDP, respectively. In the case of Q411, the variant Q411E showed a 10-fold rise in Km and a 20-fold increase in Ks for ThDP. Further, the molecular docking results indicated that the binding mode of ThDP was slightly affected in the variants of cALS. Based on these results, we suggest that H111 plays a role in substrate binding, and further suggest that Q112 and Q411 might be involved in ThDP binding of cALS.