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Viruses are very small infectious agents consisting of nucleic acids (RNA or DNA) wrapped with the shield made of proteins or capsid. The virus lives solely as a parasite, it has no its own metabolism but it uses metabolism of the host for the synthesis and assembly of their active particles – virions.

The Life Cycle of The Virus

The life cycle of the virus is very simple. The virus enters the body and finds the suitable cells. Then it enters the cells by penetrating the cell membrane and releases its genetic material, DNA or RNA. The genetic material of the virus enters the nucleus of the host cell and uses host enzymes for the synthesis of cellular mRNA which controls the synthesis of new elements for the construction of new viruses through the ribosome. These protein components are expressed on the cell surface. Replicated viral genetic material gets out of the host cell nucleus and travels to the surface of the cell, where using already created protein elements thousands of new viruses are assembled.


The main difficulty in designing of antiviral drugs is the nature of virus itself, as an obligate intracellular parasite, which has the reproductive cycle completely related to infected cell functioning. One of the most important tasks is to get the selectivity in antiviral drugs action.

Antiviral Drugs

Antiviral drugs act mainly by blocking the synthesis of viral DNA or RNA by inhibition of certain enzymes, while others block some other important steps in the life cycle of the virus. It is important to stress that there are antivirotics only for a few viruses, for other viruses there are vaccines and immunoglobulins, while for some viruses there is simply no effective treatment.
According to research conducted at The University of Iowa, antiviral drugs prevent the replication and spread of influenza virus type A by binding to the proton channel through which the virus penetrates the healthy cells. These findings are published 4th February 2012 in the journal Nature.

The New Research on Antivirotics Mechanism of Action

Mei Hong and John D. Corbett, a professor of chemistry at the University of Iowa and the Ames Laboratory associate at the Department of Energy in the United States, said that these results clarify results of previous, often contradictory studies and should direct the development of new antiviral drugs, primarily for various types of influenza, including pandemic H1N1.

Two papers released in 2008 in the journal Nature offer two different conclusions about where the antiviral drug Amantadine binds to a flu virus and how to prevent infection of the healthy cells. The work based on the x-ray shows that the drug binds to the lumen of the proton channel, travels inside the channel and blocks it, thus inactivating the virus. Another work is based on the technology of nuclear magnetic resonance (NMR) and it concluded that the drug binds to the viral surface protein, near the proton channel, and the virus is inactivated indirectly by changing the structure of the proton channel.

Hong’s research indicates that Amantadine, when in pharmacologically relevant amount of one molecule per channel, connects to the inner surface of the proton channel. However, research has shown that the drug, at a high concentration in the membrane, binds also to a secondary active site located on the surface of the viral proteins near the channel. "Our research is based on nuclear magnetic resonance in solid and irrefutably shows that the drug binds to the lumen of the channel, while the secondary active site binds only when it is in excess," says Hong. "The previous research, which relied on NMR in liquid, was using as much as 200 times the amount of medication needed, which would explain their results on how the drug binds to the active site of surfactants. The solution to this confusion means that chemists can now focus on developing drugs that will target real active site of the channel. "

Here's how the influenza virus uses proton channel and how Amantadine blocks the channel: viral infection begins with viruses binding to healthy cells. Healthy cell surrounds the flu virus and draw it into the cell using the process called endocytosis. Once it is inside the cell, the virus uses the protein called M2 to open a channel to a healthy cell. Through this channel protons from the healthy cells enter the virus and increase its acidity. This in turn drives the release of viral genetic material into the healthy cell. The virus then takes over cell resources to reproduce and spread viral particles. When Amantadine binds to the cell and blocks the M2 proton channel, the process stops, and the virus is no longer able to infect the cell.


Hong and his team have developed powerful techniques to study the proton channel using NMR spectroscopy, the technology of medical imaging using magnetic resonance. These technique allows them a detailed insight into the position of antiviral drug within the proton channel, and the scientists have been using it successfully to analyze the structure of the protein active site and to determine very accurately the distance between the drug and the protein. Scientists have also discovered that Amantadine rotates while binding to the active site within the proton channel. That means it do not fill the channel. Hong says this opens up a lot of room for development of new drugs that could block the channel better, thus stopping the flu, and also avoiding the development of resistance to the drug.

Klaus Schmidt-Rohr, a chemistry professor at the University of Iowa and the Ames Laboratory chemist superior, Sarah Cady, PhD student and external associate of the chemistry department of the University of Iowa, William DeGrado, George W. Raiziss professor of biochemistry and biophysics and a professor of chemistry at the University of Pennsylvania; Cinque S. Soto, a doctoral student in the department of biochemistry and biophysics at the University Pennsylvania and Jun Wang, a graduate student in the Department of Chemistry at the University of Pennsylvania, are other contributors to this study. The study was financed through donations. National Science Foundation has donated 687 411 dollars, 616 $ 295 donated by the National Institutes of Health.

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Mutation of influenza virus M2 proton channel and drug resistance

The previous article focuses on how the anti-viral drug amantadine targets the M2 proton channel of the H1N1 influenza A virus. However, one of the major issues with designing effective anti-viral drugs is the constant mutation of viruses which can render once effective drugs relatively powerless. This has been the fate of amantadine and another adamantane-based drug, rimantadine. Both of these drugs formerly had a 90% bioactivity against H1N1 influenza A virus, however this has been lost due to viral mutation rates, mainly at sites S31N, V27A and L26F in the channel pore. Thus there was a necessity to develop new anti-viral drugs against the M2 channel. Availability of the NMR structure of the M2 channel, as referred to in the previous article, provides the structural basis for design of such drugs. This NMR structure provided information on the M2 channel gating mechanism and the inhibiting mechanism.

New adamantine-based inhibitors were designed which hold two adjacent helices of the H1N1-M2 tetramer via two inhibitor pharmacophore groups outside the channel. Such newly designed inhibitors are hoped to address the problem of H1N1 influenza A virus drug resistance. Work is ongoing in designing new adamantane-based drugs, for example polycyclic amines initially designed as ring-rearranged analogues of amantadine. Some of these secondary amines had inhibitory activity against influenza A M2 function. It was observed that some H1N1 viruses such as the A/PR/8/34 strain are particularly sensitive to the kind of subtle increase in endosomal pH caused by the amines.

There are issues associated with advancing drugs directed against the mutated M2 channels to clinical trial. These issues include lack of a robust assay for discovery of new candidates. Recently, a yeast growth restoration assay has been proposed as a potentially robust and sensitive high-throughput screen for M2 channel inhibitors. Results were promising as in a screen of over 250,000 pure chemicals and semi-purified fractions, 21 active compounds were identified which included comprising amantadine, rimantadine and 13 related adamantanes. In addition some non-adamantanes were identified including hexamethylene amiloride and a triazine derivative. The search continues for effective drugs against mutated H1N1 influenza A virus M2 proton channels, but there are promising signs.

Sources

BALGI, A.D. et al., 2013. Inhibitors of the influenza A virus M2 proton channel discovered using a high-throughput yeast growth restoration assay. Plos One, 8(2), pp. e55271-e55271

DU, Q. et al., 2010. Designing inhibitors of M2 proton channel against H1N1 swine influenza virus. Plos One, 5(2), pp. e9388-e9388

TORRES, E. et al., 2013. Role of the viral hemagglutinin in the anti-influenza virus activity of newly synthesized polycyclic amine compounds. Antiviral Research