Thursday, December 8, 2016

A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins.

World wide, over 240 million people are infected with Hepatitis B (HBV), a liver cell infecting virus capable of causing degeneration, such as cirrhosis, and cancers such as hepatocellular carcinoma. Unlike HIV or Hepatitis C infections, which carry the genetic code for about 10 viral proteins, HBV only carries four genes, making it very difficult to target with drug treatment. Two broad categories of antiviral treatment do exist for HBV infections, nucleoside analogs, which block the viruses ability to translate its genes in to proteins and replicate, and interferon’s, which amplify the body’s immune response to the virus presence. Unfortunately, these treatment options both have drawbacks like causing the development of drug resistant viruses and intolerable side effects for the infected patient. Because combining antiviral treatments has been shown to be very effective in managing infections such as HIV, it is important today for researchers to identify safe molecules with novel antiviral inhibition modes. Recently, researchers at the National Institute of Infectious disease in Tokyo, Japan have found that a chemical called proanthocyanidin has shown strong potential in inhibiting Hepatitis B and Hepatitis D infection both alone and in conjunction with a commonly used nucleoside analog. 
            Proanthocyanidin, referred to here as PAC, is a chemical found in grape seed extract that is made up of multiple flavonoid units. Through screening many molecules, researchers were able to identify PAC for its ability to inhibit HBV infection. After identification of its inhibitory quality, researchers pressed on to answer some important questions regarding PAC’s mode of inhibition. First, researchers studied what step of the viral replication cycle PAC disturbed, then what host-virus interaction it affected, whether PAC interacted with liver hepatocytes or HBV virions, which specific region of the host or virus receptor PAC interacted with, and finally how PAC performed as antiviral therapy when combined with a nucleoside analog. The results they found offered a very promising outlook for PAC as a non-toxic antiviral treatment option that works through a novel mechanism.
The LHB membrane protein found on the surface of Hepatitis B. This protein is responsible for binding to the NCTP receptor on hepatocytes and facilitating viral entry. 
            Through numerous chemical tests performed in cell cultures that mimicked the human liver, researchers deduced that PAC and related analogs interact with a specific 7 amino acid sections in the preS1 region of a large protein found on the virus envelop surface. This interaction prevented the viral protein LHB from binding to hepatocyte receptors, thus preventing viral entry necessary for productive infection. Researchers first determined that PAC prevented virus entry by treating cell cultures with PAC before infection and after infection. The results showed that only cells treated before introduction of virus avoided infection. By inferring the method of entry inhibition from previously identified, yet toxic antiviral molecules, the researchers were able to design a chemical test to specifically monitor the interaction of the LHB preS1 domain and its cell receptor counterpart, a sodium dependent bile uptake protein (NTCP) found on the liver cell surface. The introduction of PAC to this system prevented the specific interaction between LHB and NTCP. Researchers questioned which side of the LHB-NTCP interaction PAC was inhibiting. They were able to answer this question by infecting culture cells previously treated with PAC and infecting culture cells with viral particles previously treated with PAC. The results showed that viral particles treated with PAC before being introduced to cells showed limited capacity to infect, whereas cells treated with PAC were still susceptible to infection. Finally, by introducing mutations to the preS1 domain of the viral protein LHB responsible for receptor binding and virus particle entry, researchers were able to locate PAC-preS1 interactions to amino acids 9-16. Taken together this study showed that PAC works as a viral-entry-inhibiting-molecule that prevents the preS1 domain, specifically amino acids 9-16, of envelop protein LHB from binding to the NTCP receptor found on hepatocytes, and thus prevents viral particles from entering cells and infecting them.
            As an antiviral treatment, PAC and its analogs have been shown to safely inhibit viral infection in both HBV and HDV, and to work effectively in conjunction with nucleoside analogs to manage infection. In tests with tenofovir, a nucleoside analog used to treat HBV, PAC was shown to inhibit viral production and spread of infection without any toxic effect for cells. This result has important implications for multidrug treatment, as PAC works to affect viral particles through a novel inhibition pathway. PAC is also a naturally occurring molecule that is already found in supplements of grape seed extract at concentrations high enough to exhibit its antiviral properties. This is a very significant finding for the millions of people currently infected with HBV looking to manage infection and people at risk of contracting an HBV infection as it is readily available, safe to ingest daily, and cost effective as it does not require chemical synthesis. While this work is very promising, PAC has not been confirmed to effectively interact with virus cells in live humans. This means that the most important research that needs to be carried out in the future is studies of the effectiveness of PAC and its derivatives at inhibiting viral infection and spread in live organisms rather than cell culture.

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