RESUMO
Red fruits and their juices are rich sources of polyphenols, especially anthocyanins. Some studies have shown that such polyphenols can inhibit enzymes of the carbohydrate metabolism, such as α-amylase and α-glucosidase, that indirectly regulate blood sugar levels. The presented study examined the in vitro inhibitory activity against α-amylase and α-glucosidase of various phenolic extracts prepared from direct juices, concentrates, and purees of nine different berries which differ in their anthocyanin and copigment profile. Generally, the extracts with the highest phenolic content-aronia (67.7 ± 3.2 g GAE/100 g; cyanidin 3-galactoside; chlorogenic acid), pomegranate (65.7 ± 7.9 g GAE/100 g; cyanidin 3,5-diglucoside; punicalin), and red grape (59.6 ± 2.5 g GAE/100 g; malvidin 3-glucoside; quercetin 3-glucuronide)-showed also one of the highest inhibitory activities against α-amylase (326.9 ± 75.8 µg/mL; 789.7 ± 220.9 µg/mL; 646.1 ± 81.8 µg/mL) and α-glucosidase (115.6 ± 32.5 µg/mL; 127.8 ± 20.1 µg/mL; 160.6 ± 68.4 µg/mL) and, partially, were even more potent inhibitors than acarbose (441 ± 30 µg/mL; 1439 ± 85 µg/mL). Additionally, the investigation of single anthocyanins and glycosylated flavonoids demonstrated a structure- and size-dependent inhibitory activity. In the future in vivo studies are envisaged.
Assuntos
Antocianinas/química , Carboidratos/química , Sucos de Frutas e Vegetais , Hidrolases/antagonistas & inibidores , Fenol/farmacologia , Extratos Vegetais/farmacologia , Ácido Clorogênico/farmacologia , Cromatografia Líquida de Alta Pressão , Flavonoides/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Taninos Hidrolisáveis/farmacologia , Concentração Inibidora 50 , Fenol/química , Pigmentação , Polifenóis/química , Quercetina/análogos & derivados , Quercetina/farmacologia , alfa-Amilases/química , alfa-Glucosidases/químicaRESUMO
M. tuberculosis contains an unusually high number of serine hydrolases by proteome percentage compared to other common bacteria or humans. This letter describes a method to probe the global substrate specificity of mycobacterial serine hydrolases with ester-protected prodrugs of ethambutol, a first-line antibiotic treatment for TB. These compounds were synthesized directly from ethambutol using a selective o-acylation to yield products in high yield and purity with minimal workup. A library of derivatives was screened against M. smegmatis, a non-infectious model for M. tuberculosis, which displayed significantly lowered biological activity compared to ethambutol. Incubation with a general serine hydrolase reactivated each derivative to near-ethambutol levels, demonstrating that esterification of ethambutol should provide a simple screen for mycobacterial hydrolase activity.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Ésteres/farmacologia , Etambutol/farmacologia , Hidrolases/antagonistas & inibidores , Pró-Fármacos/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Ésteres/síntese química , Ésteres/química , Etambutol/síntese química , Etambutol/química , Hidrolases/metabolismo , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium smegmatis/efeitos dos fármacos , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Pró-Fármacos/síntese química , Pró-Fármacos/química , Relação Estrutura-AtividadeRESUMO
Aqueous extracts of young (7-day-old) Malay cherry (Lepisanthes alata) leaves were incorporated into wheat and rice flours to evaluate their inhibitory activities against α-amylase and α-glucosidase. HPLC-ESI/MS² results showed that the active components in young leaves were proanthocyanidins with lower mean degrees of polymerisation (≤10). The IC50 of the aqueous extracts of young leaves were 2.50 ± 0.03 and 12.91 ± 0.29 µg/mL, against α-amylase and α-glucosidase, which make them less active compared to the mature leaves. In contrast, total proanthocyanidins in aqueous extracts decreased as the leaves matured, indicating that the compounds in the mature leaves have much higher activity. However, there was no significant difference in the digestibility of wheat noodles incorporated with the aqueous extracts from either young or mature leaves. Interestingly, with regard to rice noodles, their digestibility was mostly reduced by incorporating aqueous extracts of young leaves compared to using mature leaves.
Assuntos
Hidrolases/metabolismo , Folhas de Planta/química , Sapindaceae/química , Inibidores de Glicosídeo Hidrolases/química , Inibidores de Glicosídeo Hidrolases/farmacologia , Hidrolases/antagonistas & inibidores , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Amido/metabolismo , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismo , alfa-Glucosidases/metabolismoRESUMO
Peptidyl arginine deiminase 4 (PAD4) is a nuclear enzyme that converts arginine residues to citrulline. Although increasingly implicated in inflammatory disease and cancer, the mechanism of action of PAD4 and its functionally relevant pathways remains unclear. E2F transcription factors are a family of master regulators that coordinate gene expression during cellular proliferation and diverse cell fates. We show that E2F-1 is citrullinated by PAD4 in inflammatory cells. Citrullination of E2F-1 assists its chromatin association, specifically to cytokine genes in granulocyte cells. Mechanistically, citrullination augments binding of the BET (bromodomain and extra-terminal domain) family bromodomain reader BRD4 (bromodomain-containing protein 4) to an acetylated domain in E2F-1, and PAD4 and BRD4 coexist with E2F-1 on cytokine gene promoters. Accordingly, the combined inhibition of PAD4 and BRD4 disrupts the chromatin-bound complex and suppresses cytokine gene expression. In the murine collagen-induced arthritis model, chromatin-bound E2F-1 in inflammatory cells and consequent cytokine expression are diminished upon small-molecule inhibition of PAD4 and BRD4, and the combined treatment is clinically efficacious in preventing disease progression. Our results shed light on a new transcription-based mechanism that mediates the inflammatory effect of PAD4 and establish the interplay between citrullination and acetylation in the control of E2F-1 as a regulatory interface for driving inflammatory gene expression.
Assuntos
Citrulina/metabolismo , Fator de Transcrição E2F1/química , Fator de Transcrição E2F1/metabolismo , Inflamação/metabolismo , Acetilação , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular , Citocinas/genética , Fator de Transcrição E2F1/genética , Regulação da Expressão Gênica , Células HL-60 , Humanos , Hidrolases/antagonistas & inibidores , Hidrolases/genética , Hidrolases/metabolismo , Inflamação/genética , Inflamação/imunologia , Masculino , Camundongos , Camundongos Endogâmicos DBA , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , RNA Interferente Pequeno/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Moringa stenopetala has been used in traditional health systems to treat diabetes mellitus. One of the successful methods to prevent of the onset of diabetes is to control postprandial hyperglycemia by the inhibition of α-glucosidase and pancreatic α-amylase activities, resulting in the aggressive delay of the carbohydrate digestion of absorbable monosaccharides. The aim of the present study is to investigate the effect of the extract of the leaves of Moringa stenopetala on α-glucosidase, pancreatic α-amylase, pancreatic lipase, and pancreatic cholesterol esterase activities, and, therefore find out the relevance of the plant in controlling blood sugar and lipid levels. METHODS: The dried leaves of Moringa stenopetala were extracted with hydroalcoholic solvent and dried using rotary vapor under reduced pressure. The dried extracts were determined for the total phenolic compounds, flavonoid content and condensed tannins content by using Folin-Ciocateu's reagent, AlCl3 and vanillin assay, respectively. The dried extract of plant-based food was further quantified with respect to intestinal α-glucosidase (maltase and sucrase) inhibition and pancreatic α-amylase inhibition by glucose oxidase method and dinitrosalicylic (DNS) reagent, respectively. RESULTS: The phytochemical analysis indicated that flavonoid, total phenolic, and condensed tannin contents in the extract were 71.73 ± 2.48 mg quercetin equivalent/g of crude extract, 79.81 ± 2.85 mg of gallic acid equivalent/g of crude extract, 8.82 ± 0.77 mg catechin equivalent/g of crude extract, respectively. The extract inhibited intestinal sucrase more than intestinal maltase with IC50 value of 1.47 ± 0.19 mg/ml. It also slightly inhibited pancreatic α-amylase, pancreatic lipase and pancreatic cholesterol esterase. CONCLUSION: The result demonstrated the beneficial biochemical effects of Moringa stenopetala by inhibiting intestinal α-glucosidase, pancreatic cholesterol esterase and pancreatic lipase activities. A daily supplement intake of the leaves of Moringa stenopetala may help in reducing hyperglycemia and hyperlipidemia.
Assuntos
Inibidores Enzimáticos/farmacologia , Inibidores de Glicosídeo Hidrolases , Hidrolases/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Moringa/química , Pâncreas/enzimologia , Extratos Vegetais/farmacologia , Animais , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/enzimologia , Inibidores Enzimáticos/química , Flavonoides/análise , Flavonoides/farmacologia , Humanos , Hidrolases/metabolismo , Hipoglicemiantes/química , Intestinos/efeitos dos fármacos , Intestinos/enzimologia , Lipase/antagonistas & inibidores , Lipase/metabolismo , Pâncreas/efeitos dos fármacos , alfa-Amilases Pancreáticas/antagonistas & inibidores , alfa-Amilases Pancreáticas/metabolismo , Fenóis/análise , Fenóis/farmacologia , Extratos Vegetais/química , Folhas de Planta/química , Ratos , Esterol Esterase/antagonistas & inibidores , Esterol Esterase/metabolismo , Suínos , alfa-Glucosidases/metabolismoRESUMO
In light of the major contribution of the reactive warhead to the binding energy trend in reversible covalent transition-state analog inhibitors of serine and cysteine hydrolases, would it be possible to rationally design and quickly filter such warheads, especially for large-scale screening? The previously defined W1 and W2 covalent descriptors quantitatively account for the energetic effect of the covalent bonds reorganization, accompanying enzyme-inhibitor covalent binding. The quantum mechanically calculated W1 and W2 reflect the warhead binding energy by modeling of the enzyme-inhibitor reaction core. Here, we demonstrate the use of these descriptors for warhead filtering, and examine its scope and limitations. The W1 and W2 descriptors provide a tool for rational design of various warheads as universal building blocks of real inhibitors without the requirement of 3D structural information about the target enzyme or QSAR studies. These warheads could then be used as hit structural templates in the subsequent optimization of inhibitors recognition sites.
Assuntos
Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Hidrolases/antagonistas & inibidores , Inibidores de Proteases/química , Inibidores de Proteases/farmacologia , Sítios de Ligação , Hidrolases/metabolismo , Estrutura Molecular , Inibidores de Proteases/isolamento & purificação , Relação Quantitativa Estrutura-AtividadeRESUMO
The protein arginine deiminases (PADs) catalyze the post-translational hydrolysis of peptidyl-arginine to form peptidyl-citrulline in a process termed deimination or citrullination. PADs likely play a role in the progression of a range of disease states because dysregulated PAD activity is observed in a host of inflammatory diseases and cancer. For example, recent studies have shown that PAD2 activates ERα target gene expression in breast cancer cells by citrullinating histone H3 at ER target promoters. To date, all known PAD inhibitors bind directly to the enzyme active site. PADs, however, also require calcium ions to drive a conformational change between the inactive apo-state and the fully active calcium bound holoenzyme, suggesting that it would be possible to identify inhibitors that bind the apoenzyme and prevent this conformational change. As such, we set out to develop a screen that can identify PAD2 inhibitors that bind to either the apo or calcium bound form of PAD2. Herein, we provide definitive proof of concept for this approach and report the first PAD inhibitor, ruthenium red (Ki of 17 µM), to preferentially bind the apoenzyme.
Assuntos
Cálcio/química , Sistemas de Liberação de Medicamentos , Hidrolases/metabolismo , Rutênio Vermelho/química , Rutênio Vermelho/farmacologia , Sítios de Ligação , Bioensaio , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Desiminases de Arginina em ProteínasRESUMO
RATIONALE: Neutrophil extracellular trap (NET) formation promotes vascular damage, thrombosis, and activation of interferon-α-producing plasmacytoid dendritic cells in diseased arteries. Peptidylarginine deiminase inhibition is a strategy that can decrease in vivo NET formation. OBJECTIVE: To test whether peptidylarginine deiminase inhibition, a novel approach to targeting arterial disease, can reduce vascular damage and inhibit innate immune responses in murine models of atherosclerosis. METHODS AND RESULTS: Apolipoprotein-E (Apoe)(-/-) mice demonstrated enhanced NET formation, developed autoantibodies to NETs, and expressed high levels of interferon-α in diseased arteries. Apoe(-/-) mice were treated for 11 weeks with daily injections of Cl-amidine, a peptidylarginine deiminase inhibitor. Peptidylarginine deiminase inhibition blocked NET formation, reduced atherosclerotic lesion area, and delayed time to carotid artery thrombosis in a photochemical injury model. Decreases in atherosclerosis burden were accompanied by reduced recruitment of netting neutrophils and macrophages to arteries, as well as by reduced arterial interferon-α expression. CONCLUSIONS: Pharmacological interventions that block NET formation can reduce atherosclerosis burden and arterial thrombosis in murine systems. These results support a role for aberrant NET formation in the pathogenesis of atherosclerosis through modulation of innate immune responses.
Assuntos
Aterosclerose/prevenção & controle , Inibidores Enzimáticos/uso terapêutico , Hidrolases/antagonistas & inibidores , Imunidade Inata/efeitos dos fármacos , Ornitina/análogos & derivados , Animais , Doenças da Aorta/tratamento farmacológico , Doenças da Aorta/etiologia , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/tratamento farmacológico , Aterosclerose/enzimologia , Aterosclerose/etiologia , Aterosclerose/imunologia , Aterosclerose/patologia , Autoanticorpos/biossíntese , Autoanticorpos/imunologia , Citrulina/análise , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/farmacologia , Espaço Extracelular , Histonas/metabolismo , Hidrolases/fisiologia , Interferon-alfa/biossíntese , Interferon-alfa/genética , Selectina L/análise , Lipídeos/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutropenia/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Ornitina/farmacologia , Ornitina/uso terapêutico , Processos Fotoquímicos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína-Arginina Desiminase do Tipo 4 , Receptor de Interferon alfa e beta/deficiência , Seio Aórtico/patologia , Túnica Íntima/patologiaRESUMO
Characterization of polyphenolic compounds in the stems of P. multiflorum was conducted using HPLC, high resolution LC-MS and LC-MSn. Proanthocyanidins in particular were isolated in 4.8% yield using solvent extraction followed by Sephadex LH-20 fractionation. HPLC analysis using a diol column revealed oligomers (from dimer to nonamer) as minor components, with (epi)catechin monomeric units predominating, and oligomers with higher degree of polymerization being dominant. Thiolysis treatment of the proanthocyanidins using mercaptoacetic acid produced thioether derivatives of (epi)catechin as the major product and a mean value of the degree of polymerization of 32.6 was estimated from the ratio of terminal and extension units of the (epi)catechin. The isolated proanthocyanidins were shown to strongly inhibit α-amylase with an acarbose equivalence (AE) value of 1,954.7 µmol AE/g and inhibit α-glucosidase with an AE value of 211.1 µmol AE/g.
Assuntos
Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Caules de Planta/química , Polygonum/química , Proantocianidinas/análise , Amido/metabolismo , Antraquinonas/análise , Cromatografia Líquida de Alta Pressão , Flavonoides/análise , Hidrolases/metabolismo , Cinética , Espectrometria de Massas , Fenóis/análise , Fenóis/química , Extratos Vegetais/química , Polifenóis/isolamento & purificação , Polifenóis/farmacologia , Propanóis/análise , Espectrometria de Massas por Ionização por Electrospray , Estilbenos/análise , Compostos de Sulfidrila/química , alfa-Amilases/antagonistas & inibidores , alfa-Amilases/metabolismoRESUMO
Brassinin hydrolase (BHAb), an inducible enzyme produced by the plant pathogen Alternaria brassicicola under stress conditions, catalyzes the hydrolysis of the methyl dithiocarbamate group of the phytoalexin brassinin, to indolyl-3-methanamine, methane thiol and carbonyl sulfide. Thirty four substrate inspired compounds, bioisosteres of brassinin and a range of related compounds, were evaluated as potential substrates and inhibitors of BHAb for the first time. While six compounds containing thiocarbamate, carbamate and carbonate groups displayed inhibitory activity against BHAb, only two were found to be substrates (thionecarbamate and dithiocarbamate). Methyl naphthalen-1-yl-methyl carbamate, the most potent inhibitor of the six, and methyl N'-(1-methyl-3-indolylmethyl)carbamate inhibited BHAb through a reversible noncompetitive mechanism (K(i)=89±9 and 695±60µM, respectively). Importantly, these carbamate inhibitors were resistant to degradation by A. brassicicola. Carbonates were also inhibitory of BHAb, but a quick degradation by A. brassicicola makes their potential use as crop protectants less likely. Overall, these results indicate that indolyl and naphthalenyl carbamates are excellent lead structures to design new paldoxins that could inhibit the detoxification of brassinin by A. brassicicola.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Tiocarbamatos/química , Alternaria/enzimologia , Antifúngicos/síntese química , Antifúngicos/química , Antifúngicos/farmacologia , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Hidrolases/metabolismo , Indóis/química , Cinética , Especificidade por Substrato , Tiocarbamatos/síntese química , Tiocarbamatos/farmacologiaRESUMO
Rheumatoid arthritis is associated with the development of autoantibodies to citrullinated self-proteins. Citrullinated synovial proteins, which are generated via the actions of the protein arginine deiminases (PADs), are known to develop in the murine collagen-induced arthritis (CIA) model of inflammatory arthritis. Given these findings, we evaluated whether N-α-benzoyl-N5-(2-chloro-1-iminoethyl)-L-ornithine amide (Cl-amidine), a recently described pan-PAD inhibitor, could affect the development of arthritis and autoimmunity by treating mice in the CIA model with Cl-amidine on days 0-35. Cl-amidine treatment reduced total synovial and serum citrullination, decreased clinical disease activity by â¼50%, and significantly decreased IgG2a anti-mouse type II collagen Abs. Additionally, histopathology scores and total complement C3 deposition were significantly lower in Cl-amidine-treated mice compared with vehicle controls. Synovial microarray analyses demonstrated decreased IgG reactivity to several native and citrullinated epitopes compared with vehicle controls. Cl-amidine treatment had no ameliorative effect on collagen Ab-induced arthritis, suggesting its primary protective mechanism was not mediated through effector pathways. Reduced levels of citrullinated synovial proteins observed in mice treated with Cl-amidine are consistent with the notion that Cl-amidine derives its efficacy from its ability to inhibit the deiminating activity of PADs. In total, these results suggested that PADs are necessary participants in the autoimmune and subsequent inflammatory processes in CIA. Cl-amidine may represent a novel class of disease-modifying agents that modulate aberrant citrullination, and perhaps other immune processes, necessary for the development of inflammatory arthritis.
Assuntos
Amidinas/uso terapêutico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/imunologia , Inibidores Enzimáticos/uso terapêutico , Hidrolases/antagonistas & inibidores , Imunossupressores/uso terapêutico , Ornitina/análogos & derivados , Animais , Artrite Experimental/patologia , Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Autoanticorpos/biossíntese , Autoanticorpos/toxicidade , Citrulina/metabolismo , Colágeno Tipo II/antagonistas & inibidores , Colágeno Tipo II/imunologia , Hidrolases/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos DBA , Ornitina/uso terapêutico , Peptídeos Cíclicos/imunologia , Peptídeos Cíclicos/metabolismo , Desiminases de Arginina em Proteínas , Índice de Gravidade de Doença , Membrana Sinovial/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/patologiaRESUMO
Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110+ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (> 80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activity-based assay in a library-versus-library format to screen 70+ SHs against 140+ structurally diverse carbamates. Lead inhibitors were discovered for â¼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.
Assuntos
Carbamatos/farmacologia , Descoberta de Drogas/métodos , Inibidores Enzimáticos/química , Hidrolases/antagonistas & inibidores , Serina , Bibliotecas de Moléculas Pequenas/farmacologia , Carbamatos/uso terapêutico , Domínio Catalítico , Avaliação Pré-Clínica de Medicamentos , Fluoretos , Humanos , Sondas Moleculares , Fosfatos , Ligação Proteica , Proteoma , Especificidade por SubstratoRESUMO
Protein Arginine Deiminase (PAD) activity is dysregulated in numerous diseases, e.g., Rheumatoid Arthritis. Herein we describe the development of a fluorescence polarization-Activity Based Protein Profiling (fluopol-ABPP) based high throughput screening assay that can be used to identify PAD-selective inhibitors. Using this assay, streptonigrin was identified as a potent, selective, and irreversible PAD4 inactivator.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Hidrolases/antagonistas & inibidores , Hidrolases/metabolismo , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Humanos , Concentração Inibidora 50 , Desiminases de Arginina em Proteínas , Estreptonigrina/farmacologiaAssuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/isolamento & purificação , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Proteômica/métodos , Animais , Células COS , Chlorocebus aethiops , Inibidores Enzimáticos/química , Hidrolases/metabolismo , Estrutura MolecularRESUMO
A new approach was developed for the synthesis of 5'-(Z)-substituted 4',5'-unsaturated adenosines, potential inhibitors against S-adenosyl-L-homocysteine hydrolase (SAHase). A highly stereoselective radical-mediated sulfur-extrusive stannylation was employed as a key step in the present approach. Inhibitory activity of the compounds against SAHase is also presented.
Assuntos
Adenosina/síntese química , Adenosina/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Hidrolases/antagonistas & inibidores , Adenosina/análogos & derivados , Adenosil-Homocisteinase , Avaliação Pré-Clínica de MedicamentosRESUMO
Moffatt oxidation of 2',3'-O-isopropylideneuridine (1a) and treatment of the crude 5'-aldehyde with formylmethylene-stabilized Wittig reagent gave the vinylogously extended 7'-aldehyde2a. Condensation of 2a with ethoxycarbonyl- or dibromomethylene phosphorane reagents gave the conjugated dienes 6a and 4a, respectively. Deacetonization gave diene ester 7a [5'(E),7'(E); with s-trans conformation] and dibromodiene 5a [5'(E)], respectively. Analogously, 2',3'-O-isopropylideneadenosine (1b) was Wittig-extended into the conjugated dibromodiene 5b [5'(E)] and dienoic ester 7b [5'(E),7'(E)]. Furthermore, palladium-catalyzed coupling of the vinyl 6'(E)-stannanes 14 with (E) and (Z) ethyl 3-iodoacrylate gave stereodefined access to dienoic esters 7 (E,E) and 16 (E,Z). Incubation of AdoHcy hydrolase with 100 microM of 5b resulted in partial inhibition of the enzyme without any apparent change in the enzyme's nicotinamide adenine dinucleoside (NAD(+)) content. In contrast, 7b and 16b produced time- and concentration-dependent inactivation of S-adenosyl-L-homocysteine (AdoHcy) hydrolase producing significant decreases in the enzyme's NAD(+) content. However, 7b and 16b upon incubation with AdoHcy hydrolase were not metabolized suggesting that these compounds are type I mechanism-based inhibitors. No specific antiviral activity was noted for 5a,b, 7a,b, and 16a,b against any of the viruses tested; dibromodiene 5b proved cytotoxic at a concentration > or =6.7 microM and cytostatic at > or =11 microM, while dienoic esters 16a,b showed activity against both varicella-zoster virus (at 10 microM, 16a) and cytomegalovirus (at 10 microM, 16a; 18 microM, 16b).
Assuntos
Adenosina/análogos & derivados , Adenosina/síntese química , Alcadienos/síntese química , Antineoplásicos/síntese química , Antivirais/síntese química , Carboidratos/química , Hidrolases/antagonistas & inibidores , Uridina/análogos & derivados , Uridina/síntese química , Adenosina/química , Adenosina/farmacologia , Adenosil-Homocisteinase , Alcadienos/química , Alcadienos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antivirais/química , Antivirais/farmacologia , Linhagem Celular , Citomegalovirus/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Herpesvirus Humano 3/efeitos dos fármacos , Humanos , Uridina/química , Uridina/farmacologiaRESUMO
BACKGROUND: To establish a SAH hydrolase antiviral screening in vitro model for screening of broad spectrum antiviral agents. METHODS: SAH hydrolase was purified from rat livers by (NH4) 2SO4 fractionation, DEAE52,hydroxyapatite and Sephadex G-100 chromatography successively. The activity of SAH hydrolase was estimated by radio labeled substrate in synthesis direction by TLC. RESULTS: Purified SAH hydrolase showed a single band in SDS-PAGE electrophoresis with silver nitrate staining, the apparent molecular weight is 45 000. The Km for adenosine is (6.32 +- 0.17) micromol/L. The IC50 of S-DNPA, a known inhibitor of SAH hydrolase, was 7.6 micromol/L estimated in our system. The structure and activity relationships shown by racemic and regiosomer analogs of S-DHPA indicated that the structural specificity of SAH hydrolase was high. 42 compounds had been screened in the system and no compound showed more inhibitory activity against SAH hydrolase than S-DNPA. CONCLUSIONS: An in vitro antiviral screening model has been established using SAH hydrolase. It can also be used to study kinetics of enzyme inhibition.
Assuntos
Antivirais , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos , Hidrolases/antagonistas & inibidores , Adenina/análogos & derivados , Adenina/química , Adenina/farmacologia , Adenosil-Homocisteinase , Animais , Antivirais/química , Feminino , Hidrolases/isolamento & purificação , Hidrolases/metabolismo , Técnicas In Vitro , Fígado/enzimologia , Ratos , Ratos WistarRESUMO
Treatment of the 6-aldehyde derived by Moffatt oxidation of 3-O-benzoyl-1,2-O-isopropylidene-alpha-D-ribo-hexofuranose (2c) with the dibromo- or bromofluoromethylene Wittig reagents generated in situ with tetrabromomethane or tribromofluoromethane, triphenylphosphine, and zinc gave the dihalomethyleneheptofuranose analogues 3b and 3d, respectively. Acetolysis, coupling with adenine, and deprotection gave 9-(7,7-dibromo-5,6, 7-trideoxy-beta-D-ribo-hept-6-enofuranosyl)adenine (5a) or its bromofluoro analogue 5b. Treatment of 5a with excess butyllithium provided the acetylenic derivative 9-(5,6, 7-trideoxy-beta-D-ribo-hept-6-ynofuranosyl)adenine (6). The doubly homologated vinyl halides 5a and 5b and acetylenic 6 adenine nucleosides were designed as putative substrates of the "hydrolytic activity" of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. Incubation of AdoHcy hydrolase with 5a, 5b, and 6 resulted in time- and concentration-dependent inactivation of the enzyme (K(i): 8.5 +/- 0.5, 17 +/- 2, and 8.6 +/- 0.5 microM, respectively), as well as partial reduction of enzyme-bound NAD(+) to E-NADH. However, no products of the "hydrolytic activity" were observed indicating these compounds are type I mechanism-based inhibitors. The compounds displayed minimal antiviral and cytostatic activity, except for 6, against vaccinia virus and vesicular stomatitis virus (IC(50): 15 and 7 microM, respectively). These viruses typically fall within the activity spectrum of AdoHcy hydrolase inhibitors.
Assuntos
Alcinos/síntese química , Antineoplásicos/síntese química , Antivirais/síntese química , Didesoxiadenosina/síntese química , Inibidores Enzimáticos/síntese química , Hidrolases/antagonistas & inibidores , Adenosil-Homocisteinase , Alcinos/química , Alcinos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Antivirais/química , Antivirais/farmacologia , Didesoxiadenosina/química , Didesoxiadenosina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Placenta/química , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
Ebola (subtype Zaire) viral replication was inhibited in vitro by a series of nine nucleoside analogue inhibitors of S-adenosylhomocysteine hydrolase, an important target for antiviral drug development. Adult BALB/c mice lethally infected with mouse-adapted Ebola virus die 5-7 days after infection. Treatment initiated on day 0 or 1 resulted in dose-dependent protection, with mortality completely prevented at doses > or =0.7 mg/kg every 8 h. There was significant protection (90%) when treatment was begun on day 2, at which time, the liver had an average titer of 3 x 10(5) pfu/g virus and the spleen had 2 x 10(6) pfu/g. Treatment with 2.2 mg/kg initiated on day 3, when the liver had an average titer of 2 x 10(7) pfu/g virus and the spleen had 2 x 10(8) pfu/g, resulted in 40% survival. As reported here, Carbocyclic 3-deazaadenosine is the first compound demonstrated to cure animals from this otherwise lethal Ebola virus infection.
Assuntos
Antivirais/uso terapêutico , Ebolavirus/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Hidrolases/antagonistas & inibidores , Adenosil-Homocisteinase , Animais , Antivirais/química , Chlorocebus aethiops , Modelos Animais de Doenças , Avaliação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Ebolavirus/patogenicidade , Inibidores Enzimáticos/química , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Técnicas In Vitro , Fígado/virologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/virologia , Fatores de Tempo , Tubercidina/análogos & derivados , Tubercidina/uso terapêutico , Células VeroRESUMO
The response of murine T cells to MHC class II determinants on allogeneic cells induces helper T cell activation and the development of cytotoxic T cells. We have recently established that an S-adenosyl-L-homocysteine hydrolase inhibitor, (Z)-5'-fluoro-4',5'-didehydro-5'-deoxyadenosine (MDL 28,842), is a potent immunosuppressive agent which selectively inhibits T cell activation. In this report we characterize the effect of MDL 28,842 on in vitro and in vivo models of transplant rejection. In vitro, MDL 28,842 inhibited the generation of cytotoxic T cells in the murine mixed lymphocyte reaction with an IC50 of less than 0.1 microM. MDL 28,842 (1.0 microM) totally inhibited the generation of cytotoxic T cells when added up to 3 days after the initiation of culture with no apparent cell toxicity. In vivo, MDL 28,842 given by gavage at 5.0, 2.5, or 1.0 mg/kg/day inhibited the increase in popliteal lymph node weight induced by injection of allogeneic spleen cells into the footpad. MDL 28,842 was also evaluated in a model of graft rejection. Skin allografts on animals given MDL 28,842 at 5 mg/kg/day (ip) for the first 6 days following transplantation survived for 12.2 days, compared to 8.7 days for control animals. Cyclosporin A (CSA) given at 5.0 mg/kg/day did not prolong graft survival. The combination of MDL 28,842 and CSA was not any more effective than MDL 28,842 alone. Based on these findings, we suggest that MDL 28,842 is useful in the prevention of allograft rejection.