Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro
- a UFU – Federal University of Uberlândia, Institute of Biomedical Science – ICBIM, Uberlândia, MG, Brazil
- b UNESP – São Paulo State University, Institute of Bioscience, Language and Exact Science – IBILCE, Department of Biology, São José do Rio Preto, SP, Brazil
- c School of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
- d UNESP – São Paulo State University, Institute of Chemistry, Department of Organic Chemistry, Araraquara, SP, Brazil
- e Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan
- Received 23 October 2013, Revised 27 November 2014, Accepted 23 December 2014, Available online 31 December 2014
Highlights
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Antiviral effects of 20 Brazilian natural compounds were investigated.
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Four compounds with potent inhibitory activity on HCV replication were identified.
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Antiviral activity was independent of HCV genotype.
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Antiviral effect was not affected by variants described to confer resistance.
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One of the four compounds inhibited HCV IRES-mediated translation.
Abstract
Compounds extracted from plants can provide an alternative approach to new therapies. They present characteristics such as high chemical diversity, lower cost of production and milder or inexistent side effects compared with conventional treatment. The Brazilian flora represents a vast, largely untapped, resource of potential antiviral compounds. In this study, we investigate the antiviral effects of a panel of natural compounds isolated from Brazilian plants species on hepatitis C virus (HCV) genome replication. To do this we used firefly luciferase-based HCV sub-genomic replicons of genotypes 2a (JFH-1), 1b and 3a and the compounds were assessed for their effects on both HCV replication and cellular toxicity. Initial screening of compounds was performed using the maximum non-toxic concentration and 4 compounds that exhibited a useful therapeutic index (favourable ratio of cytotoxicity to antiviral potency) were selected for extra analysis. The compounds APS (EC50 = 2.3 μM), a natural alkaloid isolated from Maytrenus ilicifolia, and the lignans 3∗43 (EC50 = 4.0 μM), 3∗20 (EC50 = 8.2 μM) and 5∗362 (EC50 = 38.9 μM) from Peperomia blanda dramatically inhibited HCV replication as judged by reductions in luciferase activity and HCV protein expression in both the subgenomic and infectious systems. We further show that these compounds are active against a daclatasvir resistance mutant subgenomic replicon. Consistent with inhibition of genome replication, production of infectious JFH-1 virus was significantly reduced by all 4 compounds. These data are the first description of Brazilian natural compounds possessing anti-HCV activity and further analyses are being performed in order to investigate the mode of action of those compounds.
Keywords
- Brazilian plants;
- Natural compounds;
- Antiviral;
- Hepatitis C virus;
- Replication
1. Introduction
Hepatitis C virus (HCV) infection is a worldwide public health problem and it is estimated that the virus infects around 3% of the world population (Shepard et al., 2005). Chronic infection can progress to liver cirrhosis with risk of the development of hepatocellular carcinoma, and causes around 500,000 deaths per year (Alter, 2007, Chevaliez and Pawlotsky, 2007 and Saito et al., 1990). There is no effective vaccine for prevention of HCV infection; however a number of drugs are available for the treatment of infection. Until recently, the standard therapy was based on pegylated interferon (IFN) plus ribavirin (RBV), resulting in a sustained virological response in approximately 50% of patients infected with HCV genotypes 1a/1b and 80% of those infected with genotypes 2 or 3 (Fried et al., 2002, Hadziyannis et al., 2004 and Manns et al., 2001). The availability of new, direct-acting antivirals targeting the NS3 protease, NS5B polymerase and NS5A protein have dramatically improved therapeutic options (Pawlotsky, 2014). However, the high costs and potential for development of resistance presented by existing treatment demonstrate the need for the development of more efficient new antivirals, or combination of therapies for HCV treatment.
Traditional medicines have a long history and there is now a great interest in discovering new molecules from natural sources for the treatment of many human diseases. An extensive variety of natural compounds has demonstrated antiviral action worldwide, including anti-HCV activity (Calland et al., 2012). In this context, compounds extracted from plants can provide an alternative approach to new therapies. Natural compounds present characteristics such as high chemical diversity, lower cost of production and milder or non-existent side effects than conventional treatment (Kitazato et al., 2007). Additionally, most of the drugs used today in the clinic were first discovered from plants and microorganisms (Mann, 2002). Therefore, they present a great opportunity to find novel compounds that can act as antiviral drugs.
The Brazilian flora represents a vast, largely untapped, resource of potential therapeutic compounds. The wide distribution of natural resources in Brazil and the natural diversity of chemical components provide the country with potential bioactive materials (Duarte et al., 2005). Here we investigate the antiviral effects of a panel of Brazilian natural compounds consisting of extracts, fractions and isolated compounds on HCV replication. These data are the first description of Brazilian natural compounds possessing anti-HCV activity.
2. Materials and methods
2.1. Natural compounds
Compounds were extracted from Maytrenus ilicifolia (APS, C, P and M), Peperomia blanda (5-362, 3-20, 3-43, 48-3, F3 and F6) and Piper fuligineum (F8–40). The root bark of M. ilicifolia was collected in the city of Ribeirão Preto (São Paulo State, Brazil, at 21°11′56.1″S; 47°46′42.2″W) in March 2006. The plant was identified by Rita Maria de Carvalho. A voucher specimen (HPM-BR 0059) has been deposited in the Herbarium of the University of Campinas, São Paulo, Brazil ( Santos et al., 2012). The aerial parts of P. blanda were collected at the Reserva da Ripasa, Ibaté – SP, Brazil in January of 2005 and identified by Dr. Elsie Franklin Guimarães. A voucher specimen (Kato-547) has been deposited at the Herbarium of the Institute of Bioscience, São Paulo University, São Paulo – SP, Brazil ( Felippe et al., 2008). The Piper fuligineum species was identified by Dr. Agnes Lamb of the Institute of Botany (IBt of São Paulo, SP, Brazil) and their voucher specimens are deposited in the Herbarium of the Institute of Botany (USP – SP) under the voucher Kato-0720.