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SRI s James Noah on Building Better Assays for Influenza Drug Discovery

James Noah
Research biochemist
Southern Research Institute

At A Glance

Name: James Noah

Position: Research biochemist, department of biochemistry, drug discovery, Southern Research Institute, Birmingham, Ala.

Background: PhD, biochemistry, North Carolina State University, 1999; Postdoc, University of Texas, Austin.

The Southern Research Institute last week announced it had been awarded $1.4 million from the National Institute of Allergy and Infectious Disease to develop high-throughput screening tools to expedite the search for antiviral drugs effective against influenza, including the highly pathogenic H5N1 avian flu strain.

At the center of this initiative is James Noah, a research biochemist in the emerging infectious disease section at SRI. Noah is helping coordinate a consortium of research institutes in an assay development project for influenza therapeutics that will span primary cell-based assay techniques to secondary biochemical analyses that could eventually spawn lead candidates and pre-clinical work. Noah took a few moments this week to discuss the project with CBA News.

Tell me a little about your research group and how it fits into the Southern Research Institute.

At Southern Research Institute, we really have a bunch of facilities available for investigating everything from discovery of a new effective compound to clinical trials. Our particular group is in the drug-discovery division. We consist of people investigating cancer; bacteria such as tuberculosis and black plague, [Yersinia] pestis; and we also have a large group of virologists that are looking into viruses such as influenza, hantavirus, West Nile virus, some of the viruses that cause yellow fever, and SARS. That brings it down to the focus of our particular group. With the advent of influenza and the avian influenza strains that are transmissible to humans, influenza has jumped to the forefront in importance. The government has put out the Bioshield initiative, mandated by the President, to try and discover cures for influenza. This particular initiative wanted to develop high-throughput assays that could be used to screen large compound libraries for efficacy against influenza virus. That is the project that we have applied for and received [money for].

Our group consists of several virologists, and a biochemist — myself. Together we will be working with collaborators at the University of Alabama, Birmingham; Rice University; University of Texas; and Rutgers University to develop key assays — primary assays that are cell-based — for primary screening of large compound libraries, and then secondary mechanistic-based biochemical assays for mechanistic investigations of compounds that have been shown effective in the primary screens. We hope to bring these assays online in an HTS format, and then make them available to the scientific community for large-scale influenza screening. This particular proposal is strictly for the development of these assays, and not the large-scale screening itself.

This is an interesting approach — using cell-based assays for primary screening, and biochemical assays for secondary screening. This is a bit of a change from the traditional pharma-based drug screening procedure.

Most of the pharmaceutical industry has identified important targets in various organisms or viruses that have been key to searching for new drugs. For example, the neuraminidase of influenza is identified as a very important target, and the in silico research that was done to develop the neuraminidase inhibitor has been very fruitful, but a lot of the screening against neuraminidase itself has not been as fruitful. In our case, we want to look at a primary assay that is very broad in its capabilities. By broad, I mean able to screen a broad variety or classes of compounds, and also screen against broad effects of these compounds. We want an assay that will look at something that will inhibit not just neuraminidase, but any viral component — the M2 ion channel that is essential for virus infectivity; the polymerase that is essential for replication; NS1 protein that's essential for modulating the cell mRNA levels. If we can find an assay that can screen any class of compound initially, plus have the advantage of screening out toxicity issues, then I think we will have an advantage. We can take our hits from this assay and then funnel them through the secondary biochemical assay. If you have a biochemical assay, and then you identify classes of compounds that are very effective against the target, but are highly toxic to the whole cell, then you don't have a very effective drug. We're approaching it from the other side and looking at the whole cell as a modulator of both efficacy and toxicity.

How does the primary cell-based assay work?

Basically, we take a cell line that is susceptible to influenza infection — will be lysed by the virus — and put it into conditions where influenza can infect the cells. We then put compounds from many compound libraries, and put those individual compounds into individual wells on a 384-well plate. Then we add the virus, wait three days, and over that time, the virus will undergo multiple cycles of replication. So we add very small amounts of virus — not nearly enough to infect every cell at once. Instead we infect maybe one in 200 cells, initially. The virus will replicate, lyse the cells, go on and infect other cells, so over a period of 72 hours, we see a very strong window between cells that are uninfected, and cells that are infected and killed by the virus. Because we have such a large window, we can determine, relatively accurately, which compounds prevented the virus propagation. We simply do a live-dead cell assay to determine which compounds are effective.

Is this a commercially available assay?


How long has it been available?

Our assay has just been available since January. Some of the components that we use in the assay have been available longer, but this assay was just recently developed.

So is the grant money for developing that assay further, or for some of the follow-on secondary assays?

The grant money we just won was for developing this assay; however, we were so interested in getting this assay online that we spent internal money in order to develop it, and then we'll recover that from the grant.

After these primary screens, how do some of the biochemical assays fit in?

There are several influenza components that are critical for virus infection and propagation of new virus. One of those components is called the M2 ion channel, which is a proton channel that acidifies the interior of the virus core. When the virus attaches to the cell, the cell membrane engulfs the virus in an endosome, the interior of which is acidic. That M2 channel will pump hydrogen ions into the virus interior from the endosome, allowing the virus to uncoat and make its way to the nucleus. That M2 channel function is critical for the initial stages of infection.

Traditional inhibitors — one of which is amantadine — were discovered back in the 1940s. It has been used very frequently, [but] there are some neurological side effects, and frequent use has caused the production of resistant strains of the virus. Our particular assay uses an amantadine-resistant strain in order to screen out amantadine-like inhibitors. We are searching for new inhibitors for this M2 channel to stop this virus infection at an early stage.

Another piece of the virus that is very important is neuraminidase. This surface protein functions at the very end of the cycle, and is responsible for release of the virus from the cell membrane. The virus is attached to the cell membrane by sialic acid residues, and the neuraminidase will cleave these. However, if you have an inhibitor to neuraminidase, the virus cannot detach from the cell, so infection is stopped at a late stage. Tamiflu and Relenza are two of the neuraminidase inhibitors on the market, and are very effective; however, we are seeing an increase in resistant strains, and there have been some reports about bird flu — the highly pathogenic strain of avian influenza — that has also been resistant. So in this case, new inhibitors of neuraminidase are needed, so we're going to develop a neuraminidase assay.

Two related assays we're developing are looking at the function of the influenza virus polymerase. It's a hollow enzyme, three separate proteins that come together to have three separate functions in the virus replication. The influenza virus cannot make complete mRNAs inside the cells on its own — it has to steal premier components from host messenger RNAs. In this case, it's the capped 5' end of host mRNAs that are stolen by the polymerase, and this particular function is a combination of three different activities: recognition of the cap, cutting of the host mRNA, and polymerization of the new mRNA that contains the viral sequences. We are targeting both the recognition factor and the cleavage factor, and trying to develop assays to screen against those particular functions. Polymerase function is ubiquitous in many of the enzymes in our cells, and they function by the same mechanism. So although one could find a polymerase inhibitor, it might also inhibit important functions inside human cells. However, the cap recognition and endonuclease function have been shown to be very specific to the influenza virus polymerase.

The final assay we're looking at is against the NS1 protein, which is produced inside the cell upon infection, and will shut down cellular mRNA processing by preventing 3' end processing of host mRNAs. So not only does the virus steal the 5' end, many of the mRNAs that make it to the nucleus aren't processed efficiently because of this protein. In collaboration with researchers from University of Texas and Rutgers University, we'll be looking at the function of this protein, and whether we can find small-molecule inhibitors of this.

Have you uncovered promising hits with the primary screens yet?

Yes — we've done a library of about 10,000 compounds, and found about 35 hits out of that. We're beginning characterization of those hits right now. The assay itself functions well, and is clearly able to differentiate effective compounds.

SRI was named one of the National Institutes of Health's Molecular Libraries Screening Center Network centers. Does this work overlap with that at all?

We do have a collaboration with them, and that project is led by Gary Piazza at SRI. We were fortunate to be chosen for that. The project involved the development of a wide selection of assays to different targets, and then submission of those assays to the MLSCN database. Those assays are then disseminated across all the screening centers, and screened against somewhere in the vicinity of 500,000 compounds. We do take part in that, but this particular [influenza] project is not part of that one. However, it is in the same spirit — the assays for the MLSCN project have to be in HTS format, and readily usable. The assays of the type we're doing for BioShield would certainly qualify for that project.

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