A new study published in the August 2 issue of PLoS Pathogens could potentially lead to the development of new antiviral drugs that also avoid the problem of drug resistance.

Researchers from St. Jude Children’s Research Hospital discovered that compounds could block an enzyme that is universal to all influenza viruses.

The team reports that certain drugs can precisely target and block an enzyme that is crucial for replication of the influenza virus, and because all strains of influenza viruses need the same functioning enzyme, they believe that their findings will assist in developing drugs that can effectively treat new influenza virus strains that may prove resistant to current antiviral drugs.

Developing a new vaccine for new strains of influenza can take many months, and experts are concerned that large numbers of people could be hospitalized and that the effects could be catastrophic if highly virulent strains emerge, which are or become resistant to current drugs.

The team investigated drugs that are aimed at inhibiting polymerase, a dual-purpose enzyme complex produced by the influenza virus. During replication, this polymerase produces copies of the viral genome and also assembles mRNA (messenger RNA) molecules that are coded for viral proteins that the virus requires in order to take over the cell’s function and produce more of the virus.

The researchers targeted an RNA-snipping enzyme called endonuclease, a key sub-unit of the polymerase complex, which enables the virus to disguise its messenger RNA. This enables the mRNA to become incorporated into the cell’s protein-making machinery by cutting cellular nRNA apart and retaining the ‘cap’ segment, the ‘identification tag’ for the cell’s machinery, and subsequently, the polymerase attaches this cap to its own mRNA.

Senior author Stephen White, DPhil., chair of the St. Jude Structural Biology department said:

“Inhibitors of the polymerase complex would make excellent drug candidates. It is a good target because these polymerases are essentially the same across many strains, and also because the virus absolutely needs the polymerase to make copies of itself. The polymerase doesn’t have very many similarities to other polymerases in cells, so it should be fairly specific for the flu polymerase.”

He continues explaining that viruses have readily developed resistance to already existing antiviral drugs, since these drugs target viral proteins that a virus can readily change without having to compromise its viability.

The team based their study on findings of earlier research, which had mapped the molecular structure of the endonuclease, showing its active site, in which the chemical reaction of cutting the mRNA molecules apart occurs. They engineered a version of the endonuclease so that they were able to readily determine whether compounds would block the active site, testing it in six compounds they knew or predicted to inhibit the active site. Three of these compounds were developed earlier by Merck as viral inhibitors but subsequently ruled out as drug candidates because of their properties, even though they showed some effectiveness, whilst the three other compounds were developed in the laboratory of one of the authors, Thomas Webb, Ph.D., a member of the Chemical Biology and Therapeutics department at St. Jude.

Webb constructed a molecule called the ‘warhead’ as the basis for the compounds, which was designed to fit precisely into a central pocket of the active site. This warhead provides the basis for producing antiviral drug candidates, whilst other molecular segments were designed to fit into neighboring pockets.

Once it was evident that the six compounds displayed activity against the endonuclease, the team conducted a detailed structural analysis of how the compounds fit the active site by using X-ray crystallography.

X-ray crystallography is a commonly used analytical technique, whereby X-rays are directed through a crystallized protein, and the pattern of diffracted X-rays is subsequently analyzed to draw the structure.

The research provided evidence of the warhead binding to the active site and also revealed new information on other surrounding pockets into which the compounds fit and which pockets are conserved by different strains.

First author Rebecca DuBois, Ph.D., a postdoctoral research fellow at St. Jude’s Structural Biology department, declared:

“By analyzing the structure of the active site with the drugs bound to it, we have identified a number of pockets inside the active site of the protein. We can use these structures to develop drugs that will specifically target certain pockets. Now that we know which pockets are really conserved, we can predict the best way to avoid the development of resistance by viral strains.”

When a viral strain mutates to change the pocket’s structure, resistance arises and eliminates the drug’s ability to bind and inhibit the active site.

White states that his team will build on their findings to develop improved compounds, which could be drug candidates for pre-clinical and clinical trials. In a collaboration headed by Webb, they will be working with a pharmaceutical company to further develop and test drugs with the support of the National Institutes of Health.

Once these drugs have been approved for clinical use, they would prove very valuable and could be widely applied as a frontline treatment for the virus. DuBois commented: “You could use these drugs in any situation where a person is hospitalized for influenza. Whether used alone or in combination with existing influenza medications, we would expect them to be highly effective.”

He continued saying that other viruses have polymerases that work just like that of the influenza virus, such as Hantavirus and the lymphocytic choriomeningitis virus and that the drug molecules designed for influenza could therefore also potentially be used to treat viruses like these.

Written by Grace Rattue