ABSTRACT
Hepatitis C virus is one of the causative agents of non-A non-B hepatitis. Since one of viral proteins, NS3, has serine protease activity indispensable for virus maturation. NS3 serine protease is considered to be a suitable target for anti-HCV reagents. We report an assay of HCV NS3 protease in living cells. We designed peptide substrates bearing one of the sequences of HCV NS3 protease cleavage sites sandwiched with fluorescent proteins CFP and YFP. Substrates were expressed and cleaved efficiently in HeLa cells by cotransfection with HCV NS3 protease. The relationship between the progress of cleavage reaction and the change in fluorescence of the substrate emitted from living cells was confirmed. As a group of candidates for inhibitor of HCV NS3 protease, we chose RNA aptamers, nucleic acid ligands selected from a completely random RNA pool by in vitro selection. We found that 3 classes of aptamers, G9-I, II and III, bound NS3 protease specifically and inhibited cleavage in vitro. We studied the effect of RNA aptamers introduced into HeLa cells. The addition of G9-II RNA in the medium at a concentration of 2.5 micro g/ml reduced cleavage by one-third that of control.
Subject(s)
Fluorescent Dyes/chemistry , Hepatitis C/enzymology , RNA/pharmacology , Serine Proteinase Inhibitors/pharmacology , Blotting, Western , Drug Evaluation, Preclinical , Escherichia coli/metabolism , Fluorescent Antibody Technique , HeLa Cells , Humans , Plasmids/genetics , RNA/chemistryABSTRACT
One hundred fifty-two methanol and water extracts of different parts of 71 plants commonly used in Sudanese traditional medicine were screened for their inhibitory effects on hepatitis C virus (HCV) protease (PR) using in vitro assay methods. Thirty-four extracts showed significant inhibitory activity (>/=60% inhibition at 100 microg/mL). Of these, eight extracts, methanol extracts of Acacia nilotica, Boswellia carterii, Embelia schimperi, Quercus infectoria, Trachyspermum ammi and water extracts of Piper cubeba, Q. infectoria and Syzygium aromaticum, were the most active (>/=90% inhibition at 100 microg/mL). From the E. schimperi extract, two benzoquinones, embelin (I) and 5-O-methylembelin (II), were isolated and found as potent HCV-PR inhibitors with IC(50) values of 21 and 46 microM, respectively. Inhibitory activities of derivatives of I against HCV-PR as well as their effects on other serine proteases were also investigated.
Subject(s)
Endopeptidases/drug effects , Hepacivirus/drug effects , Hepacivirus/enzymology , Plant Extracts/pharmacology , Plants, Medicinal , Protease Inhibitors/pharmacology , Chromatography, High Pressure Liquid , Hepatitis C/prevention & control , Humans , Medicine, Traditional , Plant Extracts/chemistry , Protease Inhibitors/chemistry , SudanABSTRACT
Forty-eight methanol and aqueous extracts from Sudanese plants were screened for their inhibitory activity on viral replication. Nineteen extracts showed inhibitory effects on HIV-induced cytopathic effects (CPE) on MT-4 cells. The extracts were further screened against HIV-1 protease (PR) using an HPLC assay method. Of the tested extracts, the methanol extracts of Acacia nilotica (bark and pods), Euphorbia granulata (leaves), Maytenus senegalensis (stem-bark) and aqueous extracts of A. nilotica (pods) and M. senegalensis (stem-bark) showed considerable inhibitory effects against HIV-1 PR. Inhibitory principles were isolated from M. senegalensis and their activities were also discussed.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Protease Inhibitors/pharmacology , HIV Protease/metabolism , HIV-1/drug effects , Plant Extracts/pharmacology , Plants, Medicinal/chemistry , Virus Replication/drug effects , Chromatography, High Pressure Liquid , Humans , Phenols/analysis , Phenols/pharmacology , Plant Epidermis/chemistry , Spectrophotometry, Ultraviolet , Sudan , Tannins/analysis , Tannins/pharmacologyABSTRACT
A new highly oxygenated triterpene named ganoderic acid alpha has been isolated from a methanol extract of the fruiting bodies of Ganoderma lucidum together with twelve known compounds. The structures of the isolated compounds were determined by spectroscopic means including 2D-NMR. Ganoderiol F and ganodermanontriol were found to be active as anti-HIV-1 agents with an inhibitory concentration of 7.8 micrograms ml-1 for both, and ganoderic acid B, ganoderiol B, ganoderic acid C1, 3 beta-5 alpha-dihydroxy-6 beta-methoxyergosta-7,22-diene, ganoderic acid alpha, ganoderic acid H and ganoderiol A were moderately active inhibitors against HIV-1 PR with a 50% inhibitory concentration of 0.17-0.23 mM.
Subject(s)
Anti-HIV Agents/isolation & purification , Drugs, Chinese Herbal/pharmacology , HIV Protease Inhibitors/isolation & purification , Plants, Medicinal/chemistry , Anti-HIV Agents/chemistry , Anti-HIV Agents/pharmacology , Cell Line , Cytopathogenic Effect, Viral/drug effects , HIV Protease Inhibitors/chemistry , HIV Protease Inhibitors/pharmacology , HIV-1/drug effects , HIV-1/pathogenicity , Humans , Magnetic Resonance Spectroscopy , Mass Spectrometry , Medicine, Chinese Traditional , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/isolation & purification , Reverse Transcriptase Inhibitors/pharmacologyABSTRACT
Effects of methanolic extract of root bark of Beli (Aegle marmelos Corr.), an Ayurvedic crude drug used for heart diseases, and constituents isolated from the extract on spontaneous beating of cultured mouse myocardial cells were examined. The extract at a concentration of 100 micrograms/ml inhibited the beating rate by approximately 50%. Among the isolated constituents, aurapten was the most potent inhibitor; the IC50 of aurapten is 0.6 microgram/ml, which is comparable with that of verapamil, a calcium antagonist. Addition of aurapten at concentrations higher than 1 microgram/ml significantly reduced the ratio of morphologically changed myocardial cells which originated from calcium overload caused by successive treatment with calcium-free and calcium-containing solutions.
Subject(s)
Calcium/metabolism , Heart/drug effects , Myocardium/metabolism , Plants, Medicinal , Animals , Cells, Cultured , Heart Rate/drug effects , Medicine, Ayurvedic , Mice , Mice, Inbred ICRABSTRACT
Rhizomes of five identified plants of the Paris species, Liliaceae, and Rhizoma Paridis which are sold as a crude drug named "Zao Xiu," "Qiyeyizhihua" or other names in nine different markets in China were tested for their effects on cultured cardiomyocytes. In the standard medium, eight methanol extracts out of sixteen at a concentration of 0.2 mg/ml stopped the spontaneous beating of myocardial cell sheets, but these extracts significantly increased the beating rate when the concentration was reduced to one half. In the culture medium with a low calcium concentration, 0.5 mM, the beating rate of the cells decreased to about 60% of that of the control in the standard medium. The addition of five of the extracts to the low calcium medium at a concentration of 0.1 mg/ml caused a stop of cell beating, but the other extracts increased beating rate at least by 10%. These steroidal glycosides isolated from the rhyzomes of P. vietnamensis (Takht.) H. Li also stimulated cell beating. Among them, diosgenin-3)-alpha-L-rhamno-pyranosyl-(1---2)-(alpha-L-arabinofura nosyl-(1---4))-D-glucopranoside (compound 1) was the most effective stimulant for cell beating as well as calcium uptake by the myocardial cells.
Subject(s)
Cardiac Glycosides , Drugs, Chinese Herbal/pharmacology , Animals , Calcium/metabolism , Cardiac Glycosides/isolation & purification , Cells, Cultured , Molecular Structure , Sodium-Potassium-Exchanging ATPase/metabolismABSTRACT
Rhizomes of five identified plants of the PARIS species, Liliaceae, and Rhizoma Paridis which are sold as a crude drug named "Zao Xiu", "Qiyeyizhihua" or other names in nine different markets in China were tested for their effects on cultured cardiomyocytes. In the standard medium, eight methanol extracts out of sixteen at a concentration of 0.2 mg/ml stopped the spontaneous beating of myocardial cell sheets, but these extracts significantly increased the beating rate when the concentration was reduced to one half. In the culture medium with a low calcium concentration, 0.5 mM, the beating rate of the cells decreased to about 60% of that of the control in the standard medium. The addition of five of the extracts to the low calcium medium at a concentration of 0.1 mg/ml caused a stop of cell beating, but the other extracts increased beating rate at least by 10%. Three steroidal glycosides isolated from the rhizomes of P. VIETNAMENSIS (Takht.) H. Li also stimulated cell beating. Among them, diosgenin-3- O-alpha- L-rhamnopyranosyl-(1-->2) - (alpha- L-arabinofuranosyl-(1-->4))- D-glucopranoside (compound 1) was the most effective stimulant for cell beating as well as calcium uptake by the myocardial cells.