Timothy McClain and Professor David Busath, Department of Physiology and Developmental Biology
Goal/Purpose of the Project
The purpose of this project was to research and test various drug compounds to prevent influenza virus infection. A specific target of my research is M2 protein channels that aid in the uncoating of the viral coat. Recent mutations have rendered formerly effective drugs almost completely useless. With recent outbreaks and mutations of the virus, the demand for antiviral drugs has never been greater. The goal of the project was to test several different compounds that had been selected based on unique characteristics, designed to adjust to the mutation, and prevent further infection.
Procedure
Since the infection of a cell with a virus will lead to “hijacking” the cellular machinery and increasing the level of virus within the host, targeting M2 protein channels is an effective way to block those initial steps. Each drug used was developed with the specific intent to block these channels and adjust for each mutation. Separate preparations of each individual drug had been made and categorized according to concentration and property.
The model used for testing each of these compounds is Xenopus Laevis Oocytes, an excellent cell for viral expression. Weekly preparations of oocytes were made by injecting viral mRNA into the cell and waiting for expression. This is monitored by using electrodes to measure membrane permeability to H+ ions after a given period of time. An influx of these ions can depolarize the cell enough to initiate the “uncoating” process mentioned earlier. If the electrode readings demonstrated a significant change in membrane potential, drug was then administered to see if it could block the depolarization process.
Results
These experiments and the drug compounds hold much promise and potential. Several facets of this experiment contribute to its overall success, and likewise, are also several different points for potential hindrances (much of which I experienced).
Initially, our mRNA stock was considered to be contaminated because we could not culture a significant sample for injection. After some time, older mRNA was found to be a more pure sample and was amplified for experimentation.
The sensitive nature of the oocyte itself proved to be a difficult factor during experimentation. Oocytes are the largest single eukaryotic cell that closely resemble human cells and are therefore the best model for viral expression. Yet, because of its size and sensitivity, many things can cause the oocytes to give poor results.
Contributing to oocyte health is the fact that frogs ovulate in season. Therefore it is important to keep them in constant cycles of light, dark, and temperature so as to ensure regular ovulation for experimentation. As I noticed fluctuations in egg quality, further investigation showed that despite constant light and dark cycles used to represent an optimal environment for ovulation, the frogs are still innately sensitive to seasonal production of eggs, with peak production in spring and summer (1). This was a factor in developing results as my availability for experimentation was somewhat limited to fall and winter semesters. I saw this correlation with the results found in my data.
Lastly, we noticed a discrepancy in the surgery protocol for the frogs and that egg quality was affected by the frequency of surgery. The frogs required roughly three to four months post surgery to recover and allow healthy production of subsequent eggs. Rather, we were performing surgeries every six weeks in a schedule that didn’t allow the frogs enough time to fully recover and generate the quality of eggs necessary for effective experimentation. If oocytes are unhealthy upon injection, they will not have the necessary recovery for full expression of mRNA while maintaining membrane integrity. This was manifested as each impaling would show subpar membrane potentials and/or egg quality. As a result, we have modified our protocols and are expecting a better environment for viral expression. Despite these setbacks, the drug compounds continue to be tested on this and other models; both of which have produced some further preliminary results. Between intermittent setbacks, I have seen occasional blocks in M2 channels that are promising and encourage further experimentation. Included is a figure taken to demonstrate the potential of one such compound (named compound B so as to protect the identity of the drug). (Figure 1) In addition, the information we gathered regarding frequency of frog
surgeries will be incorporated into protocols for the future benefit of fellow researchers. It is with this hope and perseverance that I continue to experiment and improve this procedure so as to find an effective solution to the influenza epidemic. I will research till the end of this semester, continuing to implement the new protocols and gather viable results for publishing.
References
- Green, S. L. “Factors Affecting Oogenesis in the South African Clawed Frog (Xenopus Laevis).” National Center for Biotechnology Information. U.S. National Library of Medicine, n.d. Web. 15 Aug. 2002.