Browsing by Subject "antiviral screening"
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(2013)Estimated 180 million people worldwide are infected by hepatitis C virus. It causes liver diseases which are often asymptomatic. Chronic infections can lead to liver cirrhosis, transplantation and hepatocellular carcinoma. Drug development was slow until 1999 when the first cell culture model with autonomously replicating subgenomic HCV replicon was developed. It expresses the viral proteins that are necessary in HCV replication. The current interest in exploring new medicines is concentrated to the essential viral proteins, such as the NS3/4A protease, NS3 helicase, NS5A and NS5B RNA polymerase. HCV belongs to the Hepacivirus genus. Due to its high variability there are at least seven genotypes and several subtypes. Genotype 1 is the most common and the most difficult to treat. The current standard of care continues 24-48 weeks and consists mainly of pegylated interferon alpha and nucleoside analogue ribavirin, both non-specific HCV medicines with severe adverse effects. In 2011 two new direct-acting antivirals, protease inhibitors telaprevir and boceprevir, were approved for the treatment of HCV. A vaccine against HCV has not yet been developed. The aim of this study was to optimize and validate a robust cell-based assay for screening of replication inhibitors against HCV. Genetically modified Huh-7 cells harbor a subgenomic HCV replicon expressing only the viral proteins needed in viral replication. In addition, the replicon encodes a firefly luciferase as a reporter gene. The amount of expressed luciferase is directly correlated with the amount of HCV replication making the replicon system suitable for HTS. The optimized and validated method was used for screening HCV replication inhibitors from a library containing 113 marine-derived substances. Marine environment has been in recent years a very interesting source for finding new drug candidates. This study was part of international MAREX project which aims to discover new active molecules from marine resources. A total of 37 samples (32.7%) exhibited antiviral activity over 50%. A cytotoxicity evaluation in ATP assay was performed with these samples. 10 samples (27.0%) exhibited cytotoxicity below 20%, of which six were synthetic samples and four were extracts. Compounds with high antiviral activity, low cytotoxicity and clear dose-response in further studies should be tested with a cell line expressing the full-length HCV genome. The structural proteins can exhibit some characteristics which inactivate the compound identified as active in the replicon system.
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(2011)Alphaviruses are positive-stranded RNA-viruses and they belong to the family of Togaviridae. Alphaviruses are spread by mosquitoes of family Aedes. Alphaviruses have spread on all continents except Antarctica. So far 29 alphaviral species have been identified and they can be divided in two groups, Old and New world viruses, by their geographical distribution and by diseases they cause. Chikungunya virus (CHIKV) is one of the Old World alphaviruses and it has been found in Africa and Asia. However, due to the global warming, Chikungunya is also spreading to southern Europe. In humans, it causes fever, headache, rash and joint pain, which can last for several years and be very painful. In small children, Chikungunya can cause neurological symptoms such as encephalitis. Genome of alphaviruses encodes for four structural proteins and four non-structural proteins (nsP), of which nsP3 contains a macro domain. Macro domains are conserved in most kingdoms of life but their function has not been elucidated. It has been shown that macro domains bind ADP-ribose and its derivatives and it has been shown that nsP3-protein has an important role in alphavirus replication. The aim of the study was to study the use of compounds which bind to macro domain protein as antiviral agents. 45 compounds were chosen for antiviral studies by molecular modeling. These compounds were expected to bind to macro domain proteins. In a competitive binding assay five compounds inhibited more than 50 % poly-ADP-ribose (PAR) binding to MDO1-macro domain protein, which was the protein on which the molecular modeling was performed. When the competitive binding assay was performed with SFV macro domain (nsP3), only one compound inhibited poly-ADP-ribose binding more than 50 %. In SFV-antiviral assay seven compounds had inhibition percentage higher than 50 %. In a CHIKV replicon assay five compounds had more than 50 % inhibition on replicon expression. We also studied possible inhibition mechanism by studying whether the compounds inhibit the virus to enter the cell. Almost all compounds included in this assay inhibited the virus entry to some extent. In general, the inhibition of PAR binding and antiviral activity did not correlate among the studied compounds. Even though compounds which had antiviral potency did not inhibit PAR binding to macro domains, potential antiviral agents were found which deserve further investigation as virus entry inhibitors.
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