“Screening Small Molecule Ligands with the Core Encapsidation Signal of the HIV-1 RNA Genome”
Human Immunodeficiency Virus Type 1 (HIV-1) has become a worldwide epidemic with a high rate of infection. HIV attacks and destroys the infection-fighting CD4 cells of the immune system. Without treatment, HIV can gradually destroy the immune system and advance to AIDS, Acquired Immunodeficiency Syndrome. Current treatments target proteins that are utilized in several stages of the HIV-1 life cycle. But due to the virus’s high mutation rate, these drugs can only work for an extended amount of time.
The purpose of this project is to develop therapeutics that target functional RNA elements within the highly-conserved 5′-untranslated region (UTR). The core encapsidation signal (CES) was identified as a minimal region within the 5’- UTR that is independently capable of directing the packaging of the virus’s unspliced dimeric genome. Our research focuses on understanding how and in which orientation certain classes of ligands bind to the recently determined CES structure.
We use nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to characterize the ligand binding sites and their affinity. SOFAST HMQCs were performed on non-ligand bound and ligand bound CES and the resulting spectra were compared. Ligands that resulted in significant changes in spectrum relative to the non-ligand bound CES were identified as binders. ITC runs were performed with the ligand-RNA complex. One specific class of ligand that showed significant changes to the spectra were aminoglycosides (AMG). Our SOFAST HMQCs correlate hydrogens with isotopically labeled nitrogen on the adenosines of our construct, with our HMQC we are able to see significant disappearance and shifting of peaks that we can attribute to our small molecule ligand binding to our RNA. With NMR assignment, we are able to determine which residue it correlates on our three dimensional CES construct. With ITC runs we can extrapolate ligand binding characteristics. From our results we got a high Ka value which is the association constant. From this we know our ligand is binding tightly to our RNA. We also got a high N of around 8.42, which correlates to the number of binding sites. And finally we get ΔH, which is our change in enthalpy which is overall exothermic. From our data we can conclude that this class of aminoglycosides are binding to our CES structure very tightly and maybe in a specific orientation. While aminoglycosides bind to CES, it is not known that they function as RNA inhibitors for HIV-1 genome packaging. With this data, in vivo studies can be performed to examine any inhibitory effects. Our structural based drug design findings can possibly aid in the development of a new drug that specifically targets the RNA genome, so that HIV-1 infected patients no longer need a drug cocktail to fight against this virus.
What work did you present at the 2016 Annual Biomedical Research Conference for Minority Students (ABRCMS)?I presented a poster in the biochemistry/structural biology category. My project was focused on measuring the binding interaction between small molecule ligands and the Core Encapsidation Signal of HIV-1 RNA as a potential drug target.
What did you gain from participating in the conference?I gained a lot of networking skills. I was able to successfully communicate my research project to not only the judges, but other graduate, post docs, and principal investigators from other school. I gained a lot of insight from students who presented their research in other fields. I also gained insight from keynote speakers who expanded my view on scientific disciplines, and guided me in figuring out what I want to do and how I should go about doing it. I saw first-hand the impact the scientific community has on the world.
How did you find your mentor for this research? How did you know this was the project you wanted to do?With my scholarship program, I was able to do rotations in several labs. I searched through the UMBC of professors in the biology and chemistry department who did research on campus. I had no research prior to being an undergraduate student and did not know what to expect. But I did my best to be fully prepared and to be adaptable to learning new things. I kept my options open and actually found a lab where the social and intellectual environment is fantastic. Now I really enjoy what I do in lab.
Who is your mentor?Dr. Michael Summers and graduate student Ms. Julie Nyman.
How much time do you put into it?I put a lot of time into the research. My lab hours range from 10-13 hours a week. But even outside of lab, I read research papers that are related to my project.
What academic background did you have before you started?I did my rotations during the school year and officially started my freshman summer, having just finished a whole year of general courses in calculus, biology, and chemistry. I had a solid general understanding of basic chemistry, biology, and physics which helped a lot in the methodologies of the lab.
How much does your mentor help you with your research?My mentor helps tremendously with my research. Each of my mentors ask questions about my progress, initiative and understanding of the project almost every day, which is very encouraging and it is a very good way to train undergraduates in the lab.
What has been the hardest part about your research?The hardest part is being able to find another route to solving problems. This is a newer project in the lab, so during my summer it was a lot of troubleshooting problems. At times, I did not get the expected results or the results didn’t make sense. Instead of giving up or trying to do the same thing over again, I had to start thinking more analytically about things and basically tracing back through the steps to see if there is something different to try.
What was the most unexpected thing?I actually ended up liking research. I never thought of doing anything within the structural biology field let alone in biochemistry/chemistry department. But I ended up liking it a whole lot. Every day I go into lab excited to learn new things and run new experiments.
How does your research relate to your work in other classes?Since I had a general background in a couple fields of science, I only understood the basics in lab and never really knew why we did certain things. Now that I am a sophomore, I am taking classes like organic chemistry and genetics which play a big role in the techniques I use in lab. Cloning, transformation, minipreps, growing cell culture, all the way to understanding NMR data. At it is great because once I made a connection between my research and my classes, it made learning a whole lot easier.
What else are you involved in on campus, outside of your research?Outside of my research I am a UMBC gospel choir member and Biology 141 Learning Assistant.
What is your advice to other students about getting involved in research?Two big things. One, be open minded in what you want to do. When you start in the process in getting involved in research, you might have one specific field that you would want to work in and you have your mind set on that. But expand, you never know what you might like and what you might not like. Get experience and gain knowledge in many fields before you start narrowing down. Secondly, get to know your lab environment and truly understand the way you learn. In a research lab, you might have to do things more independently and you have to solve things yourself or you might be in a bigger lab which have more resources for you to go to. It is all in the way you learn and how much of a social environment you want.
What are your career goals?My career goals include obtaining my MD/PhD. I am not too sure what field I would be interested in but structural or developmental biology are some options. I may in the future want to open my own research lab, or have a teaching position at a university or be a clinical researcher. I have a while to think of what I want to do.