Proposal

Senior Project Proposal

Lauren Varanese

November 22, 2014

1. Title of Project:

The Flu and You: The Beginning of the End

2. Statement of Purpose:

Every fall, a wave of sickness hits, and many people miss school or work and stay home. In 2013, the projected losses from the flu were $16.3 billion (Oursler). On average, between 5% and 20% of people catch the flu each year, and more than 200,000 people are hospitalized from flu-related complications ("Seasonal Influenza Q&A.").  If a cure is found for the flu, then thousands of lives could be saved, along with billions of dollars from missed work days, medications, vaccines, and hospital bills. In my project, I hope to find a form of the drug amanatadine that will block a protein channel in the membrane of the influenza A virus.  Fifty years ago, amanatadine successfully blocked these channels, but nine years ago, the influenza virus mutated, effectively counteracting amanatadine (Kolocouris et al.).  However, different drugs similar to amanatadine could possibly block the current mutated channels.

I hope to answer the following questions: Why did the influenza A virus mutate? Could there be a drug that will block the mutated channels? Furthermore, is there a chance that the virus will mutate again, making the new drug ineffective? To learn more, I will work in a lab, reconstituting viral proteins into liposomes,  tiny vesicles made out of the same material as cell membranes or the viral capsule (“What is a Liposome?”). Channels in the liposomes uptake protons at around 40 protons per second, and I hope to find a drug that will decrease this rate to around 4 protons per second. The drugs that reduce the uptake of protons will hopefully block the virus.

3. Background:

I have loved Biology and Chemistry since I first took it in 7th grade. Taking so many Biology and Chemistry courses have allowed me to narrow down my interests to the study of DNA, the epigenome, and diseases. I like studying the interactions between organisms, and I have always wondered why particular drugs work or how similar DNA can code for completely different organisms. Using my interest in the workings of microscopic molecules, I hope to gain a better understanding of viral interactions with cells while learning more about myself in the process. I’m interested in discovering if a cure for the flu is possible, and when we can expect to receive this treatment.  I’ve researched the drug research and approval process in my Organic Chemistry class, and I’m excited to see the beginnings of this process, starting with the drug discovery and research. Although I’ve never worked in a lab before, I have visited multiple labs, including the one I’ll be working in at BYU.

4. Prior Research:

In the past four decades, the drug amantadine—which consists of an adamantane backbone with an amino group replacing one of the four methynes—counteracted the influenza A virus (“Amantadine”). The amantadine works as an inhibitor, blocking the ion channel formed by the viral M2 protein (Kamps and Hoffmann). Now, however, most influenza A viruses are resistant to the amantadine class of antiviral drugs in the United States and multiple Asian countries (Nelson et al.). For example, in Hong Kong, in 2002, only 8.2% of the influenza A virus was immune to amantadine, but it increased to 73.8% in 2004 (Nelson et al.). Furthermore, there was 100% resistance in the influenza virus from multiple Asian countries, but the sample sizes that were tested were small (Nelson et al.). In America, this resistance has increased from under 2% in 2003 to over 90% in 2006 (Nelson et al.).  In 2009, 100% of all tested seasonal H3N2 flu viruses and pandemic flu viruses were found to have resistance to adamantane derivatives; amantadine is no longer recommended in the United States for treatment of the influenza A virus ("2008-2009 Influenza”).

This resistant strain was detected in Hong Kong in as early as 2003, and reappeared throughout 2005 (Nelson et al.). At first, this mutation was thought to be aided by the temperate regions of Asian countries, where viruses can easily mutate and grow (Nelson et al.).  However, the resistance to amantadine has not increased recently in countries that use amantadine drugs, suggesting that the viruses mutate for other reasons other than drug selection pressure (Nelson et al.)

The influenza virus A has mutated so that the M-segment  associated with a beneficial H-segment mutation, allowing for its proliferance even without drug selection pressure (Nelson et al.). The mutation in the M-segment interferes with amantadine’s ability to block the M2 ion channel activity and viral reproduction (Nelson et al.). Because of this mutation,  researchers are trying to find a new form of amdamantane that will block viral replication.

Researchers are looking for adamantane derivatives  because they are known to interfere with the M2 ion channel in the influenza virus. The M2 protein has been shown to majorly contribute to the budding and replication of the virus, so interfering with this protein could inhibit the reproduction of the influenza A virus (Rossman et al.). Simply removing the M2 protein from the virus has been shown to drastically reduce the virus’s ability to reproduce, reducing the budding and budding efficiency, among other effects (McCown and Pekosz). Furthermore, cholesterol was shown to help the viral replication process (Rossman et al.). Removing cholesterol could impact the influenza virus’s ability to reproduce. Amdamantane derivatives  could interact with the M2 protein and prevent the replication of the influenza A virus, so even if the virus mutates, it will still not be able to replicate, effectively prohibiting the virus from surviving and reproducing.

5. Significance:

Every flu season, thousands of people are sick, and many more people pay to get a flu shot. Worldwide, between 250,00 and 500,000 people die from the flu each year, and between 3 and 5 million people have a serious case of the flu (Duda). About 41.5% of adults, along with 72% of healthcare professionals, spend money on receiving a vaccination (Rienberg). By trying to find a drug that will counteract the influenza A virus, millions of dollars can be saved, along with countless hours of being sick every year.

I hope that I can add to the research already being done, aiding their efforts, hopefully speeding up the process of finding a drug that works against the influenza A virus. Testing different forms of amantadine can do this, since my results could determine whether a drug form should be researched more or ignored. Once I obtain my results, I hope to share  them with other scientists that are currently working on the same problem. By working together, the successful drug can hopefully be found sooner. The sooner that a cure is found, the sooner that thousands of lives and billions of dollars can be saved.

6. Description:

To test different drugs similar to amanatadine, I plan to reconstitute mutant influenza virus proteins into liposomes, which are like viruses but lack viral DNA. After creating the liposomes, I will test a few different versions of amanatdine by measuring the pH of the extracellular solution.  On their own, the liposomes should take up protons, raising the pH of the outside solution. Drugs that block the uptake of the protons will hopefully block the virus. Under normal conditions, the uptake of the channel is 40 protons per second.  I hope to find a drug that will knock down that uptake to fewer than 4 protons per second.

While I search for these drugs, I plan to familiarize myself with current research on the influenza virus, so I can keep up to date with my studies. I will look at current articles about similar research, and I will discuss my findings with my project mentor.  This should help me better understand the virus and hopefully help me with my research. At the end of my research, I should have specific data on the rate of uptake of protons by different forms of amantadine and the pH of the extracellular matrix. I will then compile my data into Microsoft and I’ll present it to my peers.

7. Methodology:

Starting in early February, I will conduct my research as follows:

First, I will learn how to create liposomes using DNA from the influenza A virus. This could take a few weeks.

Next, I will carry out experiments twice a week and analyze the results on off days, so that I will spend at least 15 hours per week in the lab. These experiments will consist of preparing proteoliposomes from thin films, injecting the liposome suspension into an external buffer, and measuring the pH change upon the addition of the activation drug, valinomycin, to polarize the liposomes. The time-course of the pH change is measured using a set of four pH electrodes, each of which is tracked by digital-analog converters and locally composed software on a laboratory computer. Each experiment is performed with and without channels and with and without drugs for each drug being tested.

Then, I will analyze my data; this requires the transfer of the time series electrode signals into a pre-programmed excel spreadsheet, the identification of the initial slope in pH produced by activation, and the calibration by evaluation of the black titrations with known quantities of acid. The rate of proton flux into the liposomes is divided by the nominal number of ion channels in the liposomes.

Each week, I will report my results at a lab meeting to seven other researchers. We will discuss my results, along with other results and new articles on this research. Currently, there are a set of over 50 compounds, all variants of amantadine, that I will draw from and try to test. If everything goes smoothly, I may be able to test up to as many as 30 compounds, one per experiment, at a high dosage in order to identify the promising leads. More realistically, given the usual startup time and repeat of failed experiments, I will probably be able to test around 10 to 15 compounds.

Finally, I will record my results into a spreadsheet of my own, and I will compile all my data. At the end, I will report my results to my mentor and my peers; at this time, I will give a speech discussing my results and the possible implications.

8. Problems:

I expect to have difficulties creating the liposomes since I have to first learn how to do this without any prior knowledge or experience. However, with practice and patience, I hope to be able to effectively create them. As is always the case, there will be some lab errors, but I hope to complete enough trials of each drug so that the errors will be insignificant.  Even though I’ll have access to primary resources for my research, I expect to not fully understand them right away. To solve this problem, I will continue my research online and I will ask my mentor many questions. I will not be able to work in the lab for the full extent of my project time, so to compensate for this, I will work more than 15 hours in the weeks that I have access to the lab. However, due to my time constraints, I might have issues with performing enough trials so that my results are statistically significant.

9. Bibliography:

"2008-2009 Influenza Season Week 35 Ending September 5, 2009." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 27 Aug. 2009. Web. 22 Nov. 2014.

"Amantadine." PubChem. National Center for Biotechnology Information, n.d. Web. 22 Nov. 2014.

Duda, Kristina. "How Many People Die From the Flu Each Year?" About Health. About.com, 24 Oct. 2014. Web. 22 Nov. 2014.

Kamps, Bernd S., and Christian Hoffmann. "Amantadine." Influenza Textbook. Influenza Report, n.d. Web. 22 Nov. 2014.

Kolocouris A, Tzitzoglaki C, Johnson FB, Zell R, Wright AL, Cross TA, Tietjen I, Fedida D, and Bustath DD. Aminoadamantanes with persistent in vitro efficacy against H1N1 (2009) influenza A. J Med Chem 2014, 57(11):4629-4639.

McCown, Matthew, and Andrew Pekosz. "Distinct Domains of the Influenza A Virus M2 Protein Cytoplasmic Tail Mediate Binding to the M1 Protein and Facilitate Infectious Virus Production." Journal of Virology. American Society for Microbiology, Aug. 2006. Web. 22 Nov. 2014.

Nelson MI, Simonsen L, Viboud C, Miller MA, Holmes EC. The origin and global emergence of adamantane resistant A/H3N2 influenza viruses. Virology 2009, 388(2):270-278.

Oursler, Alyssa. "Flu Math: How Much the Sickness Costs Us." InvestorPlace RSS. InvestorPlace Media, LLC, 11 Jan. 2013. Web. 22 Nov. 2014.

Reinberg, Steven. "CDC Reports More Americans Getting Flu Shots." Consumer HealthDay. HealthDay, 26 Sept. 2013. Web. 22 Nov. 2014.

Rossman, Jeremy, Xianghong Jing, George Leser, Victoria Balannik, Lawrence Pinto, and Robert Lamb. "Influenza Virus M2 Ion Channel Protein Is Necessary for Filamentous Virion Formation." Journal of Virology. American Society for Microbiology, n.d. Web. 22 Nov. 2014.

"Seasonal Influenza Q&A." Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 14 Aug. 2014. Web. 22 Nov. 2014.

"What Is a Liposome?" News-Medical.net. AZoM.com, 1 Feb. 2011. Web. 22 Nov. 2014.