RESUMO
Low-density lipoprotein receptor (LDLR) plays a pivotal role in the control of plasma LDL-cholesterol level. This occurs predominantly at the transcriptional level through two gene regulation elements, named SRE: sterol-responsive element and SIRE: sterol-independent responsive element. We have developed a high-throughput screening using LDLR promoter activation-based assay to search for cholesterol-lowering compounds from a Chinese herb-based natural compound library. With this approach, we identified two compounds, named Daphnetoxin and Gniditrin, from Chinese herb Daphne giraldii Nitsche, which could activate LDLR promoter. Characterization of these compounds showed that they increased the level of LDLR mRNA and consequently up-regulate LDLR expression. The structures of these compounds are different from well-known LDLR promoter activating compounds such as GW707. The results suggested that these herbal compounds could represent good candidates for development of new classes of cholesterol-lowering drugs.
Assuntos
Anticolesterolemiantes/farmacologia , Diterpenos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Avaliação Pré-Clínica de Medicamentos , Regiões Promotoras Genéticas/efeitos dos fármacos , RNA Mensageiro/análise , Receptores de LDL/genética , Regulação para Cima/genéticaRESUMO
AIM: To find human dopamine receptors, especially D1-like receptor specific agonists from Chinese herbs as potential antihypertension drug leads. METHODS: Two D1-like receptor cell lines carrying a beta-lactamase reporter gene, and a D2 receptor cell line coexpressing a promiscuous G protein G15 were constructed using HEK293 cells. A natural compound library made from fractionated samples of herbal extracts was used for high-throughput screening (HTS) against one of the cell lines, HEK/D5R/CRE-blax. The interested hits were evaluated for their activities against various dopamine receptors. RESULTS: Fourteen hits were identified from primary screening, of which 2 of the better hit samples, HD0522 and HD0059, were selected for further material and activity analysis, and to obtain 2 compounds that appeared as 2 single peaks in HPLC, HD0522H01 and HD0059H01. HD0059H01 could activate D1, D2, and D5 receptors, with EC(50 ) values of 2.28 microg/mL, 0.85 microg/mL, and 1.41 microg/mL, respectively. HD0522H01 could only activate D1R and D5R with EC(50 ) values of 2.95 microg/mL and 8.38 microg/mL. CONCLUSION: We established cellbased assays for 3 different human dopamine receptors and identified specific agonists HD0522H01 and HD0059H01 through HTS. The specific agonist to D1-like receptors, HD0522H01, may become a new natural product-based drug lead for antihypertension treatment.
Assuntos
Medicamentos de Ervas Chinesas/farmacologia , Plantas Medicinais/química , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistas , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Medicamentos de Ervas Chinesas/isolamento & purificação , Genes Supressores de Tumor , Humanos , Plasmídeos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D5/agonistas , Receptores de Dopamina D5/genética , Receptores de Dopamina D5/metabolismo , Transfecção , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
More than 200 Chinese medicinal herb extracts were screened for antiviral activities against Severe Acute Respiratory Syndrome-associated coronavirus (SARS-CoV) using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay for virus-induced cytopathic effect (CPE). Four of these extracts showed moderate to potent antiviral activities against SARS-CoV with 50% effective concentration (EC50) ranging from 2.4 +/- 0.2 to 88.2 +/- 7.7 microg/ml. Out of the four, Lycoris radiata was most potent. To identify the active component, L. radiata extract was subjected to further fractionation, purification, and CPE/MTS assays. This process led to the identification of a single substance lycorine as an anti-SARS-CoV component with an EC50 value of 15.7 +/- 1.2 nM. This compound has a CC50 value of 14980.0 +/- 912.0 nM in cytotoxicity assay and a selective index (SI) greater than 900. The results suggested that four herbal extracts and the compound lycorine are candidates for the development of new anti-SARS-CoV drugs in the treatment of SARS.
