RESUMEN
Diabetes mellitus is a metabolic disease characterized by hyperglycemia, which can be counteracted by the inhibition of α-glucosidase (α-Glu) and α-amylase (α-Amy), enzymes responsible for the hydrolysis of carbohydrates. In recent decades, many natural compounds and their bioinspired analogues have been studied as α-Glu and α-Amy inhibitors. However, no studies have been devoted to the evaluation of α-Glu and α-Amy inhibition by the neolignan obovatol (1). In this work, we report the synthesis of 1 and a library of new analogues. The synthesis of these compounds was achieved by implementing methodologies based on: phenol allylation, Claisen/Cope rearrangements, methylation, Ullmann coupling, demethylation, phenol oxidation and Michael-type addition. Obovatol (1) and ten analogues were evaluated for their in vitro inhibitory activity towards α-Glu and α-Amy. Our investigation highlighted that the naturally occurring 1 and four neolignan analogues (11, 22, 26 and 27) were more effective inhibitors than the hypoglycemic drug acarbose (α-Amy: 34.6 µM; α-Glu: 248.3 µM) with IC5O value of 6.2-23.6 µM toward α-Amy and 39.8-124.6 µM toward α-Glu. Docking investigations validated the inhibition outcomes, highlighting optimal compatibility between synthesized neolignans and both the enzymes. Concurrently circular dichroism spectroscopy detected the conformational changes in α-Glu induced by its interaction with the studied neolignans. Detailed studies through fluorescence measurements and kinetics of α-Glu and α-Amy inhibition also indicated that 1, 11, 22, 26 and 27 have the greatest affinity for α-Glu and 1, 11 and 27 for α-Amy. Surface plasmon resonance imaging (SPRI) measurements confirmed that among the compounds studied, the neolignan 27 has the greater affinity for both enzymes, thus corroborating the results obtained by kinetics and fluorescence quenching. Finally, in vitro cytotoxicity of the investigated compounds was tested on human colon cancer cell line (HCT-116). All these results demonstrate that these obovatol-based neolignan analogues constitute promising candidates in the pursuit of developing novel hypoglycemic drugs.
Asunto(s)
Inhibidores de Glicósido Hidrolasas , Lignanos , alfa-Amilasas , alfa-Glucosidasas , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/metabolismo , alfa-Glucosidasas/metabolismo , Inhibidores de Glicósido Hidrolasas/síntesis química , Inhibidores de Glicósido Hidrolasas/farmacología , Inhibidores de Glicósido Hidrolasas/química , Lignanos/farmacología , Lignanos/química , Lignanos/síntesis química , Relación Estructura-Actividad , Humanos , Estructura Molecular , Relación Dosis-Respuesta a Droga , Simulación del Acoplamiento Molecular , Hipoglucemiantes/farmacología , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/químicaRESUMEN
Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.
Asunto(s)
Colágeno Tipo I/química , Hidroxiprolina/química , Prolina/química , Serina/química , Animales , Dicroismo Circular , Peces , Desnaturalización Proteica , TemperaturaRESUMEN
Three new arylalkenyl α,ß-unsaturated δ-lactones, cryptobrachytones A-C (1-3), together with one known analogue kurzilactone (4), were isolated from the leaves and twigs of Cryptocarya brachythyrsa. Their structures were elucidated based on extensive spectroscopic data and electronic circular dichroism (ECD) analysis. All the isolates were evaluated in vitro for anti-proliferative activity against a panel of five human cancer cell lines and one human normal cell, respectively, and the results showed 1, 2 and 4 possessing significant selective cytotoxicity toward the human cancer cell lines with IC50 values from 5.41 to 15.43⯵M. This is the first study for C. brachythyrsa.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Cryptocarya/química , Lactonas/farmacología , Hojas de la Planta/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Línea Celular Tumoral , China , Humanos , Lactonas/aislamiento & purificación , Estructura Molecular , Fitoquímicos/aislamiento & purificación , Fitoquímicos/farmacologíaRESUMEN
This study reports crude glycerol fermentation by G. oxydans for dihydroxyacetone (DHA) production, and intensification of fermentation with sonication. Fermentation was carried out using both free and immobilized cells (on polyurethane foam support) for initial glycerol concentrations of 20, 30 and 50â¯g/L. Sonication at 20% duty cycle enhanced glycerol consumption by 60-84% with no significant change in cell morphology. Lesser DHA yield in crude glycerol fermentation was attributed to possible formation of inhibitory products. Slight reduction in DHA yield for initial glycerol concentration of 50â¯g/L was attributed to substrate inhibition. Higher DHA productivity was obtained for immobilized cells. Circular dichroism analysis of intracellular proteins obtained from ultrasound-treated G. oxydans revealed significant reduction in α-helix and ß-sheet content. These conformational changes in protein structure could augment activity of intracellular glycerol dehydrogenase, which is manifested in terms of enhanced metabolism of glycerol by G. oxydans.
Asunto(s)
Dihidroxiacetona , Gluconobacter oxydans , Sonicación , Fermentación , GlicerolRESUMEN
G13 is a 19-residue cationic antimicrobial peptide derived from granulysin. In order to achieve high-level expression of G13 in Escherichia coli cells, and to reduce downstream processing costs, we introduced an Asp-Pro acid labile bond between the His-Patch thioredoxin and G13 and constructed the recombinant plasmid pThiohisA-DP-G13. The plasmid was transformed into E. coli BL21 (DE3). After induction with isopropyl-ß-d-thiogalactopyranoside for 5 h, the fusion protein accumulated up to 200 mg/L in soluble form. The fusion protein was released by a high pressure homogenizer, cleaved using 13% acetic acid at 50 °C hydrolysis for 72 h. The recombinant G13 (r-G13) was then successively purified by fractional precipitation with ammonium sulfate and trichloroacetic acid, followed by one-step cation exchange chromatography. The purified r-G13 displayed a single band (about 2.2 kDa) as analyzed by Tris-Tricine buffered SDS-PAGE, and its precise molecular weight was confirmed using tandem mass spectrometry. Analysis of r-G13 by circular dichroism (CD) indicated that r-G13 contained predominantly ß-sheet and random coil. Agar plate diffusion assay revealed that the r-G13 exhibited antibacterial activity against both Bacillus subtilis and E. coli.
Asunto(s)
Antibacterianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/biosíntesis , Fragmentos de Péptidos/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Antígenos de Diferenciación de Linfocitos T/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Bacillus subtilis/efectos de los fármacos , Secuencia de Bases , Precipitación Química , Cromatografía por Intercambio Iónico , Escherichia coli/efectos de los fármacos , Expresión Génica , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , SolubilidadRESUMEN
Threonine aldolase (TA) catalyzes a reversible reaction, in which threonine is decomposed into glycine and acetaldehyde. The same enzyme can be used to catalyze aldol reaction between glycine and a variety of aromatic and aliphatic aldehydes, thus creating various alpha-amino-alcohols. Therefore, TA is a very promising enzyme that could be used to prepare biologically active compounds or building blocks for pharmaceutical industry. Rational design was applied to thermophilic TA from Thermotoga maritima to improve thermal stability by the incorporation of salt and disulfide bridges between subunits in the functional tetramer. An activity assay together with CD analysis and Western-blot detection was used to evaluate mutants. Except one, each of the designed mutants preserved activity toward the natural substrate. One of the 10 proposed single point mutants, P56C, displayed significantly enhanced stability compared to the wild type (WT). Its initial activity was not affected and persisted longer than WT, proportionally to increased stability. Additionally one of the mutants, W86E, displayed enhanced activity, with stability similar to WT. Higher activity may be explained by a subtle change in active site availability. Salt bridge formation between glutamic acid at position 86 and arginine at position 120 in the neighboring chain may be responsible for the slight shift of the chain fragment, thus creating wider access to the active site both for the substrate and PLP.