RESUMEN
Biological carbon dioxide (CO2 ) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.
Asunto(s)
Dióxido de Carbono , Formiato Deshidrogenasas , Aminoácidos/metabolismo , Azidas , Sitios de Unión , Dióxido de Carbono/química , Cianatos , Formiato Deshidrogenasas/química , Formiatos/química , Oxidación-ReducciónRESUMEN
Thiazole derivatives are known inhibitors of alkaline phosphatase, but various side effects have reduced their curative efficacy. Conversely, compounds bearing azomethine linkage display a broad spectrum of biological applications. Therefore, combining the two scaffolds in a single structural unit should result in joint beneficial effects of both. A new series of azomethine-clubbed thiazoles (3a-i) was synthesized and appraised for their inhibitory potential against human tissue non-specific alkaline phosphatase (h-TNAP) and human intestinal alkaline phosphatase (h-IAP). Compounds 3c and 3f were found to be most potent compounds toward h-TNAP with IC50 values of 0.15 ± 0.01 and 0.50 ± 0.01 µM, respectively, whereas 3a and 3f exhibited maximum potency for h-IAP with IC50 value of 2.59 ± 0.04 and 2.56 ± 0.02 µM, respectively. Molecular docking studies were also performed to find the type of binding interaction between potential inhibitor and active sites of enzymes. The enzymes inhibition kinetics studies were carried out to define the mechanism of enzyme inhibition. The current study leads to discovery of some potent inhibitors of alkaline phosphatase that is promising toward identification of compounds with druggable properties.
Asunto(s)
Fosfatasa Alcalina , Inhibidores Enzimáticos , Tiazoles , Humanos , Fosfatasa Alcalina/antagonistas & inhibidores , Fosfatasa Alcalina/química , Inhibidores Enzimáticos/farmacología , Simulación del Acoplamiento Molecular , Relación Estructura-Actividad , Tiazoles/farmacologíaRESUMEN
Deoxyshikonin, a natural naphthoquinone compound extracted from Lithospermum erythrorhizon Sieb. et Zucc (Boraginaceae), has a wide range of pharmacological activities, including anti-tumor, anti-bacterial and wound healing effects. However, the inhibitory effect of deoxyshikonin on cytochrome P450 (CYP) remains unclear. This study investigated the potential inhibitory effects of deoxyshikonin on CYP1A2, 2B1/6, 2C9/11, 2D1/6, 2E1 and 3A2/4 enzymes in human and rat liver microsomes (HLMs and RLMs) by the cocktail approach in vitro. The single-point inactivation experiment showed that deoxyshikonin presented no time-dependent inhibition on CYP activities in HLMs and RLMs. Enzyme inhibition kinetics indicated that in HLMs, deoxyshikonin was not only a competitive inhibitor of CYP1A2 and 2E1, but also a mixed inhibitor of CYP2B6, 2C9, 2D6 and 3A4, with Ki of 2.21, 1.78, 1.68, 0.20, 4.08 and 0.44 µM, respectively. In RLMs, deoxyshikonin not only competitively inhibited CYP2B1 and 2E1, but also exhibited mixed inhibition on CYP1A2, 2C11, 2D1 and 3A2, with Ki values of no more than 18.66 µM. In conclusion, due to the low Ki values of deoxythiokonin on CYP enzymes in HLMs, this may lead to drug-drug interactions (DDI) and potential toxicity.
RESUMEN
Acetylcholinesterase (AChE) inhibitors and calcium channel blockers are considered effective therapies for Alzheimer's disease. AChE plays an essential role in the nervous system by catalyzing the hydrolysis of the neurotransmitter acetylcholine. In this study, the inhibition of the enzyme AChE by Sarcorucinine-D, a pregnane type steroidal alkaloid, was investigated with experimental enzyme kinetics and molecular dynamics (MD) simulation techniques. Kinetics studies showed that Sarcorucinine-D inhibits two cholinesterases-AChE and butyrylcholinesterase (BChE)-noncompetitively, with Ki values of 103.3 and 4.66 µM, respectively. In silico ligand-protein docking and MD simulation studies conducted on AChE predicted that Sarcorucinine-D interacted via hydrophobic interactions and hydrogen bonds with the residues of the active-site gorge of AChE. Sarcorucinine-D was able to relax contractility concentration-dependently in the intestinal smooth muscles of jejunum obtained from rabbits. Not only was the spontaneous spasmogenicity inhibited, but it also suppressed K+-mediated spasmogenicity, indicating an effect via the inhibition of voltage-dependent Ca2+ channels. Sarcorucinine-D could be considered a potential lead molecule based on its properties as a noncompetitive AChE inhibitor and a Ca2+ channel blocker.
Asunto(s)
Acetilcolinesterasa , Butirilcolinesterasa , Acetilcolinesterasa/metabolismo , Animales , Butirilcolinesterasa/química , Canales de Calcio , Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Cinética , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , ConejosRESUMEN
For the first time, α-glucosidase, α-amylase, aldose reductase, and glycation at multiple stages inhibitory assays were used to explore the antidiabetic potential of whole unripe jackfruit (peel with pulp, flake, and seed). Two polyphenols (phenolic acids) with strong antihyperglycaemic activity were isolated from the methanol extract of whole jackfruit flour (MJ) using activity-guided repeated fractionation on a silica gel column chromatography. The bioactive compounds isolated were identified as 3-(3,4-Dihydroxyphenyl)-2-propenoic acid (caffeic acid: CA) and 4-Hydroxy-3,5-dimethoxybenzoic acid (syringic acid: SA) after various physicochemical and spectroscopic investigations. CA (IC50: 8.0 and 26.90 µg/mL) and SA (IC50: 7.5 and 25.25 µg/mL) were identified to inhibit α-glucosidase and α-amylase in a competitive manner with low Ki values. In vitro glycation experiments further revealed that MJ and its components inhibited each stage of protein glycation as well as the generation of intermediate chemicals. Furthermore, CA (IC50: 3.10) and SA (IC50: 3.0 µg/mL) inhibited aldose reductase effectively in a non-competitive manner, respectively. The binding affinity of these substances towards the enzymes examined has been proposed by molecular docking and molecular dynamics simulation studies, which may explain their inhibitory activities. The found potential of MJ in antihyperglycaemic activity via inhibition of α-glucosidase and in antidiabetic action via inhibition of the polyol pathway and protein glycation is more likely to be related to the presence of the phenolic compounds, according to our findings.
Asunto(s)
Artocarpus , alfa-Glucosidasas , Aldehído Reductasa , Artocarpus/metabolismo , Inhibidores Enzimáticos/química , Harina , Cinética , Simulación del Acoplamiento Molecular , Polifenoles/farmacología , alfa-Amilasas , alfa-Glucosidasas/metabolismoRESUMEN
In this work, a new strain of Bacillus amyloliquefaciens SY07 isolated from a traditional fermented soybean food was reported to possess remarkable α-glucosidase inhibitor-producing ability. Different culture media were applied for the proliferation of B. amyloliquefaciens SY07, and it was found that fermented okara broth presented the highest α-glucosidase inhibitory activity, while Luria-Bertani medium showed a negative effect. The extract from fermented okara broth acted in a dose-dependent manner to inhibit α-glucosidase activity, with an IC50 value of 0.454 mg/mL, and main inhibitors in the fermentation extract presented a reversible, uncompetitive pattern according to Lineweaver-Burk plots. Moreover, 1-deoxynojirimycin, a recognized α-glucosidase inhibitor, was found in the extract. Results indicated that B. amyloliquefaciens SY07 could utilize okara, a by-product from the soy processing industry, to generate α-glucosidase inhibitors effectively, and be regarded as a novel excellent microbial candidate for safe, economical production of potential functional foods or ingredients with hypoglycemic effect.
Asunto(s)
Bacillus amyloliquefaciens/metabolismo , Fermentación , Glycine max/metabolismo , Inhibidores de Glicósido Hidrolasas/metabolismo , Proteínas de Plantas/metabolismo , Polisacáridos/metabolismo , 1-Desoxinojirimicina/metabolismo , 1-Desoxinojirimicina/farmacología , Reactores Biológicos , Alimentos Funcionales , Inhibidores de Glicósido Hidrolasas/farmacología , Humanos , Alimentos de Soja/microbiología , Glycine max/microbiología , alfa-Glucosidasas/metabolismoRESUMEN
Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of l-proline biosynthesis by catalyzing the reduction of L-Δ1-pyrroline-5-carboxylate (L-P5C) to l-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10. To uncover biochemical and structural insights into human PYCR2, we characterized the steady-state kinetics of the wild-type enzyme along with two protein variants, Arg119Cys and Arg251Cys, that were previously identified in patients with microcephaly and hypomyelination. Kinetic measurements with PYCR2 suggest a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P)+ releasing before L-Pro. Both disease-related variants are catalytically impaired. Depending on whether NADPH or NADH was used, the catalytic efficiency of the R119C protein variant was 40 or 366 times lower than that of the wild-type enzyme, while the catalytic efficiency of the R251C protein variant was 7 or 26 times lower than that of the wild-type enzyme. In addition, thermostability and circular dichroism measurements suggest that the R251C protein variant has a pronounced folding defect. These results are consistent with the involvement of Arg119Cys and Arg251Cys in disease pathology.
Asunto(s)
Enfermedad/genética , Mutación , Pirrolina Carboxilato Reductasas/genética , Estabilidad de Enzimas , Humanos , Cinética , Estructura Secundaria de Proteína , Pirrolina Carboxilato Reductasas/química , Pirrolina Carboxilato Reductasas/metabolismo , TemperaturaRESUMEN
L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M-1 s-1) than PYCR1 (13.7 M-1 s-1). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C.
Asunto(s)
Pirrolina Carboxilato Reductasas/metabolismo , Tiazolidinas/metabolismo , Sitios de Unión/fisiología , Cisteína/metabolismo , Humanos , Cinética , Prolina/metabolismo , Pirroles/metabolismoRESUMEN
Bioassay-guided fractionation led to the isolation of a series of triterpenoids (1-46) including 12 new ones (1-12) from the mushroom Inonotus obliquus. The structures of all the compounds were elucidated by spectroscopic analysis as well as by comparison with literature data. Triterpenoids 1-3, 6, 7, 16, 24, 25, 27, 38, 43, 44 and 46 showed strong α-glucosidase inhibition, with IC50 values from 11.5 to 81.8 µM. Their structure-activity relationships were discussed. Inonotusol F (24) showed the strongest inhibitory activity and it presented noncompetitive inhibition against α-glucosidase. Molecular docking and molecular dynamics stimulation further demonstrated that GLU302 and PHE298 were key amino acids for the inhibition of inonotusol F (24) towards α-glucosidase. This study indicates the vital role of triterpenoids in explaining hypoglycemic effect of Inonotus obliquus and provides important evidence for further development and utilization of this mushroom.
Asunto(s)
Agaricales/química , Inhibidores de Glicósido Hidrolasas/farmacología , Hipoglucemiantes/farmacología , Triterpenos/farmacología , alfa-Glucosidasas/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/aislamiento & purificación , Cinética , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Triterpenos/química , Triterpenos/aislamiento & purificaciónRESUMEN
Type 2 Diabetes mellitus is a chronic disease considered one of the most severe global health emergencies. Chlorogenic acid (1) has been shown to delay intestinal glucose absorption by inhibiting the activity of α-glucosidase (α-Glu) and α-amylase (α-Amy). In the present work, eleven chlorogenic acid amides have been synthesized and evaluated for their antioxidant properties (as DPPH and ORAC) and inhibition activity towards the two enzymes and, with the aim to obtain dual-action antidiabetic agents. The two most promising hypoglycemic compounds, bearing a tertiary amine function on an alkyl chain (8) and a benzothiazole scaffold (11), showed IC50 values lower than that of (1) (45.5 µM α-Glu; 105.2 µM α-Amy). Amides 8 and 11 were by far more potent α-Glu inhibitors than the antidiabetic drug acarbose (IC50 = 268.4 µM) and about twice less active toward α-Amy than acarbose (IC50 = 34.4 µM). Kinetics experiments on amides 8 and 11 indicated these compounds as mixed-type inhibitors of α-Glu with K'i values of 13.3 and 6.3 µM, respectively. The amylase inhibition occurred with a competitive mechanism in the presence of 8 (Ki = 79.7 µM) and with a mixed-type mechanism with 11 (Ki = 19.1 µM; K'i = 93.6 µM). Molecular docking analyses supported these results, highlighting the presence of additional binding sites in both enzymes. Fluorescence experiments confirmed the grater affinity of amides 8 and 11 towards the two enzymes respect to (1). Moreover, a significant enhancement in acarbose efficacy was observed when inhibition assays were performed adding acarbose and amide 11. The above outcomes pinpointed the benzothiazole-based amide 11 as a promising candidate for further studies on type 2 diabetes treatment, both alone or combined with acarbose.
Asunto(s)
Acarbosa/farmacología , Amidas/farmacología , Antioxidantes/farmacología , Ácido Clorogénico/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Inhibidores de Glicósido Hidrolasas/farmacología , Hipoglucemiantes/farmacología , Acarbosa/química , Amidas/síntesis química , Amidas/química , Animales , Antioxidantes/síntesis química , Antioxidantes/química , Compuestos de Bifenilo/antagonistas & inhibidores , Ácido Clorogénico/síntesis química , Ácido Clorogénico/química , Diabetes Mellitus Tipo 2/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores de Glicósido Hidrolasas/síntesis química , Inhibidores de Glicósido Hidrolasas/química , Hipoglucemiantes/síntesis química , Hipoglucemiantes/química , Simulación del Acoplamiento Molecular , Estructura Molecular , Páncreas/enzimología , Picratos/antagonistas & inhibidores , Saccharomyces cerevisiae/enzimología , Relación Estructura-Actividad , Porcinos , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/metabolismo , alfa-Glucosidasas/metabolismoRESUMEN
Enzyme activity modulation by synthetic compounds provide strategies combining the inhibitory and therapeutic mode of action of the confirmed inhibitors. However, natural modulators could offer a valuable alternative for synthetic ones for the treatment of different chronic diseases (diabetes, hypertension, cancer); due to the numerous side effects of the latter. In vitro screening assays were conducted for Psidium guajava leaf methanolic extract against three metabolism-related enzymes; α-amylase, tyrosinase, and hyaluronidase. The obtained results showed that the examined extract retained weak and moderate multitarget inhibition against α-amylase, tyrosinase, and hyaluronidase, respectively; however, the leaf fractions exhibited stronger inhibitions for the three investigated enzymes. Fractionation of P. guajava leaf extract revealed that anthraquinones and ellagic acid are of the major active compounds with inhibitory activities for α-amylase, tyrosinase, and hyaluronidase. Kinetic studies showed that quinalizarin inhibition is competitive for both α-amylase and hyaluronidase, and ellagic acid inhibition for tyrosinase and hyaluronidase is competitive and un-competitive, respectively. The molecular docking studies of quinalizarin and ellagic acid with α-amylase, tyrosinase, and hyaluronidase showed high binding energies with different bonds stabilizing the ligand-protein complex. Compiling all obtained results led to conclude that both P. guajava leaf fractions, quinalizarin and ellagic acid, have multitarget activities with potential therapeutic applications in many metabolic disorders.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Fenoles/farmacología , Extractos Vegetales/farmacología , Hojas de la Planta/química , Psidium/química , Agaricales/enzimología , Animales , Aspergillus oryzae/enzimología , Bovinos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Hialuronoglucosaminidasa/antagonistas & inhibidores , Hialuronoglucosaminidasa/metabolismo , Simulación del Acoplamiento Molecular , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/metabolismo , Fenoles/química , Fenoles/aislamiento & purificación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/metabolismoRESUMEN
Angiotensin-converting enzyme (ACE) is best known for its formation of the vasopressor angiotensin II that controls blood pressure but is also involved in other physiological functions through the hydrolysis of a variety of peptide substrates. The enzyme contains two catalytic domains (nACE and cACE) that have different affinities for ACE substrates and inhibitors. We investigated whether nACE inhibitor backbones contain a unique property which allows them to take advantage of the hinging of nACE. Kinetic analysis showed that mutation of unique nACE residues, in both the S2 pocket and around the prime subsites (S') to their C-domain counterparts, each resulted in a decrease in the affinity of nACE specific inhibitors (SG6, 33RE and ketoACE-13) but it required the combined S2_S' mutant to abrogate nACE-selectivity. However, this was not observed with the non-domain-selective inhibitors enalaprilat and omapatrilat. High-resolution structures were determined for the minimally glycosylated nACE with the combined S2_S' mutations in complex with the ACE inhibitors 33RE (1.8â Å), omapatrilat (1.8â Å) and SG6 (1.7â Å). These confirmed that the affinities of the nACE-selective SG6, 33RE and ketoACE-13 are not only affected by direct interactions with the immediate environment of the binding site, but also by more distal residues. This study provides evidence for a more general mechanism of ACE inhibition involving synergistic effects of not only the S2, S1' and S2' subsites, but also residues involved in the sub-domain interface that effect the unique ways in which the two domains stabilize active site loops to favour inhibitor binding.
Asunto(s)
Inhibidores de la Enzima Convertidora de Angiotensina/química , Inhibidores de la Enzima Convertidora de Angiotensina/metabolismo , Dominio Catalítico , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Peptidil-Dipeptidasa A/química , Peptidil-Dipeptidasa A/metabolismo , Presión Sanguínea/fisiología , Cristalografía por Rayos X , Glicosilación , Humanos , Cinética , Ligandos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Peptidil-Dipeptidasa A/genética , Unión Proteica , Conformación Proteica en Lámina beta/genética , Sistema Renina-Angiotensina/fisiologíaRESUMEN
OBJECTIVE: Laccase is one of the best known biocatalysts which degrade wide varieties of complex molecules that are both non-cyclic and cyclic in structure. The study focused on enzyme kinetics of a purified laccase from Trametes hirsuta L. fungus and its application on biotransformation of a carcinogenic molecule 1,4-dioxane. RESULTS: Laccase was purified from white-rot fungus T. hirsuta L. which showed specific activity of 978.34 U/mg after the purification fold of 54.08. The stable laccase activity (up to 16 h) is shown at 4-6 pH and 20-40 °C temperature range. The purified enzyme exhibited significant stability for 10 metal ions up to 10 mM concentration, except for Fe2+ and Hg2+. The Cu2+ ion induced laccase activity up to 142% higher than the control at 10 mM concentration. The laccase enzyme kinetic parameters Km was 20 ± 5 µM and 400 ± 60 µM, whereas Kcat was 198.29 ± 0.18/s and 80.20 ± 1.59/s for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and guaiacol respectively. The cyclic ether 1,4-dioxane (100 ppm) was completely degraded in presence of purified laccase within 2 h of incubation and it was confirmed by HPLC and GC analysis. The oxidation reaction was accelerated by 25, 22, 6 and 19% in presence of 1 mM syringaldehyde, vanillin, ABTS and guaiacol mediators respectively. CONCLUSIONS: In this study, fungal laccase (a natural biocatalyst) based degradation of synthetic chemical 1,4-dioxane was reported for the first time. This method has added advantages over the multiple methods reported earlier being a natural remedy.
Asunto(s)
Dioxanos/metabolismo , Proteínas Fúngicas , Lacasa , Trametes/enzimología , Biodegradación Ambiental , Biotransformación , Dioxanos/análisis , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Cinética , Lacasa/química , Lacasa/metabolismoRESUMEN
The inhibition of α-glucosidase activity is a prospective approach to attenuate postprandial hyperglycemia in the treatment of type 2 diabetes mellitus (T2DM). Herein, the inhibition of α-glucosidase by three compounds T1 -T3 of Akebia trifoliata stem, namely hederagenin (T1 ), 3-epiakebonoic acid (T2 ), and arjunolic acid (T3 ) were investigated using enzyme kinetics and molecular docking analysis. The three triterpenoids exhibited excellent inhibitory activities against α-glucosidase. T1 -T3 showed the strongest inhibition with IC50 values of 42.1±5.4, 19.6±3.2, and 11.2±2.3â µM, respectively, compared to the acarbose positive control (IC50 =106.3±8.2). Enzyme inhibition kinetics showed that triterpenoids T1 -T3 demonstrated competitive, mixed, and noncompetitive-type inhibition against α-glucosidase, respectively. The inhibition constant (Ki ) values were 21.21, 7.70, and 3.18â µM, respectively. Docking analysis determined that the interaction of ligands T1 -T3 and α-glucosidase was mainly forced by hydrogen bonds and hydrophobic interactions, which could result in improved binding to the active site of the target enzyme. The insulin resistant (IR)-HepG2â cell model used in this study (HepG2 cells exposed to 10-7 â M insulin for 24 h) and glucose uptake assays showed that compounds T1 -T3 had no cytotoxicity with concentrations ranging from 6.25 to 25â µM and displayed significant stimulation of glucose uptake in IR-HepG2 cells. Thus, triterpenoids T1 -T3 showed dual therapeutic effects of α-glucosidase inhibition and glucose uptake stimulation and could be used as potential medicinal resources to investigate new antidiabetic agents for the prevention or treatment of diabetes.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Productos Biológicos/farmacología , Inhibidores de Glicósido Hidrolasas/farmacología , Hipoglucemiantes/farmacología , Extractos Vegetales/farmacología , Ranunculales/química , Triterpenos/farmacología , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Productos Biológicos/química , Productos Biológicos/aislamiento & purificación , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Ensayos de Selección de Medicamentos Antitumorales , Glucosa/metabolismo , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Células Hep G2 , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/aislamiento & purificación , Resistencia a la Insulina , Conformación Molecular , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Triterpenos/química , Triterpenos/aislamiento & purificación , alfa-Glucosidasas/metabolismoRESUMEN
Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of Kii 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteínas Fúngicas/antagonistas & inhibidores , Luteolina/farmacología , Monofenol Monooxigenasa/antagonistas & inhibidores , Moringa oleifera/química , Agaricales/química , Agaricales/enzimología , Alpinia/química , Ácido Ascórbico/química , Ácido Ascórbico/aislamiento & purificación , Ácido Ascórbico/farmacología , Pruebas de Enzimas , Inhibidores Enzimáticos/química , Proteínas Fúngicas/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Cinética , Luteolina/química , Luteolina/aislamiento & purificación , Monofenol Monooxigenasa/aislamiento & purificación , Panax/química , Extractos Vegetales/química , Extractos Vegetales/farmacología , Hojas de la Planta/química , Pironas/química , Pironas/aislamiento & purificación , Pironas/farmacología , Rizoma/química , Semillas/química , Vitis/químicaRESUMEN
Chemical probes that covalently modify cysteine residues in a protein-specific manner are valuable tools for biological investigations. Covalent fragments are increasingly implemented as probe starting points, but the complex relationship between fragment structure and binding kinetics makes covalent fragment optimization uniquely challenging. We describe a new technique in covalent probe discovery that enables data-driven optimization of covalent fragment potency and selectivity. This platform extends beyond the existing repertoire of methods for identifying covalent fragment hits by facilitating rapid multiparameter kinetic analysis of covalent structure-activity relationships through the simultaneous determination of Ki , kinact and intrinsic reactivity. By applying this approach to develop novel probes against electrophile-sensitive kinases, we showcase the utility of the platform in hit identification and highlight how multiparameter kinetic analysis enabled a successful fragment-merging strategy.
Asunto(s)
Acrilamida/farmacología , Cisteína/farmacología , Colorantes Fluorescentes/farmacología , Fosfotransferasas/antagonistas & inhibidores , Acrilamida/química , Cristalografía por Rayos X , Cisteína/química , Colorantes Fluorescentes/química , Humanos , Cinética , Modelos Moleculares , Estructura Molecular , Fosfotransferasas/metabolismo , Relación Estructura-Actividad , TermodinámicaRESUMEN
Phenylglyoxal (PGO), known to cause post-translational modifications of Arg residues, was used to highlight the role of arginine residues of the F1FO-ATPase, which may be crucial to yield the mitochondrial permeability transition pore (mPTP). In swine heart mitochondria PGO inhibits ATP hydrolysis by the F1FO-ATPase either sustained by the natural cofactor Mg2+ or by Ca2+ by a similar uncompetitive inhibition mechanism, namely the tertiary complex (ESI) only forms when the ATP substrate is already bound to the enzyme, and with similar strength, as shown by the similar K'i values (0.82 ± 0.07 mM in presence of Mg2+ and 0.64 ± 0.05 mM in the presence of Ca2+). Multiple inhibitor analysis indicates that features of the F1 catalytic sites and/or the FO proton binding sites are apparently unaffected by PGO. However, PGO and F1 or FO inhibitors can bind the enzyme combine simultaneously. However they mutually hinder to bind the Mg2+-activated F1FO-ATPase, whereas they do not mutually exclude to bind the Ca2+-activated F1FO-ATPase. The putative formation of PGO-arginine adducts, and the consequent spatial rearrangement in the enzyme structure, inhibits the F1FO-ATPase activity but, as shown by the calcium retention capacity evaluation in intact mitochondria, apparently favours the mPTP formation.
Asunto(s)
Glioxilatos/metabolismo , Ácidos Mandélicos/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ATPasas de Translocación de Protón/metabolismo , Animales , Calcio/metabolismo , Magnesio/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , PorcinosRESUMEN
The inhibition of soluble epoxide hydrolase (sEH) is a promising therapeutic approach to treat inflammation and other disorders. In our present investigation on searching for sEH inhibitors from traditional Chinese medicines, we found that Alisma orientale displayed inhibition of sEH. We constructed a small library of protostane-type triterpenoids (1-25) isolated from A. orientale, and screened their inhibitory activities. Alismanin B (1), 11-deoxy-25-anhydro alisol E (4), 11-deoxy alisol B (5), and 25-O-ethyl alisol A (15) displayed concentration-dependently inhibitory activities against sEH with IC50 values from 3.40 ± 0.57 µM to 9.57 ± 0.88 µM. 11-Deoxy-25-anhydro alisol E (4) and 11-deoxy alisol B (5) were defined as mixed-type competitive inhibitors with Ki values of 12.6 and 3.48 µM, respectively, based on the result of inhibition kinetics. The potential interaction mechanism of 11-deoxy alisol B (5) with sEH was analyzed by molecular docking and molecular dynamics, revealing that amino acid residues Trp336 and Tyr466 were vital for its inhibitory activity.
Asunto(s)
Alisma/química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Epóxido Hidrolasas/antagonistas & inhibidores , Triterpenos/química , Triterpenos/farmacología , Inhibidores Enzimáticos/aislamiento & purificación , Epóxido Hidrolasas/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Triterpenos/aislamiento & purificaciónRESUMEN
Enzyme activity modulation by synthetic compounds provide strategies combining the inhibitory and therapeutic mode of action of the confirmed inhibitors. However, natural modulators could offer a valuable alternative for synthetic ones for the treatment of different chronic diseases (diabetes, hypertension, cancer) due to the numerous side effects of the latter. In vitro screening assays were conducted for Punica granatum rind methanolic extract against three metabolism-related enzymes: α-amylase, tyrosinase, and hyaluronidase. The obtained results showed that the examined extract retained high multitarget inhibition with inhibition percentages 31.5 ± 1.3%, 75.9 ± 4.7%, and 68.5 ± 5.3% against α-amylase, tyrosinase, and hyaluronidase, respectively. Bioguided fractionation of P. granatum rind extract revealed that quercetin is the major active compound with inhibitory activities: 54.3 ± 2.7%, 94.2 ± 3.5%, and 90.9 ± 2.7% against α-amylase, tyrosinase, and hyaluronidase, respectively. Kinetic studies of enzymes showed that quercetin inhibition was noncompetitive, uncompetitive, and competitive for α-amylase, tyrosinase, and hyaluronidase, respectively. The molecular docking of quercetin with α-amylase and hyaluronidase showed high binding energy with different bonds stabilizing the ligand-protein complex. Compiling all obtained results led to conclude that both P. granatum rind extract and quercetin have multitarget activities with potential therapeutic applications in many metabolic disorders.
Asunto(s)
Aspergillus oryzae/enzimología , Proteínas Fúngicas , Hialuronoglucosaminidasa , Monofenol Monooxigenasa , Fenoles/química , Extractos Vegetales/química , Granada (Fruta)/química , alfa-Amilasas , Animales , Bovinos , Proteínas Fúngicas/antagonistas & inhibidores , Proteínas Fúngicas/química , Hialuronoglucosaminidasa/antagonistas & inhibidores , Hialuronoglucosaminidasa/química , Simulación del Acoplamiento Molecular , Monofenol Monooxigenasa/antagonistas & inhibidores , Monofenol Monooxigenasa/química , alfa-Amilasas/antagonistas & inhibidores , alfa-Amilasas/químicaRESUMEN
Many phenolic compounds, derived from lignin during the pretreatment of lignocellulosic biomass, could obviously inhibit the activity of cellulolytic and hemicellulolytic enzymes. Acetosyringone (AS) is one of the phenolic compounds produced from lignin degradation. In this study, we investigated the inhibitory effects of AS on xylanase activity through kinetic experiments. The results showed that AS could obviously inhibit the activity of xylanase in a reversible and noncompetitive binding manner (up to 50% activity loss). Inhibitory kinetics and constants of xylanase on AS were conducted by the HCH-1 model (ß = 0.0090 ± 0.0009 mM-1). Furthermore, intrinsic and 8-anilino-1-naphthalenesulfonic (ANS)-binding fluorescence results showed that the tertiary structure of AS-mediated xylanase was altered. These findings provide new insights into the role of AS in xylanase activity. Our results also suggest that AS was an inhibitor of xylanase and targeting AS was a potential strategy to increase xylose production.