Hemagglutinin (HA) is a glycoprotein found on the surface of the influenza virus. HA is responsible for binding to sialic acid receptors on host cells and mediating membrane fusion. Because it is involved in these essential functions, HA is targeted by neutralizing antibodies (nAb) against the influenza virus. When the antigen-binding fragment (Fab) domain of a nAb binds to HA, it can neutralize infectivity either by blocking receptor binding of HA, by inhibiting conformational changes required for membrane fusion, or by disrupting HA structure. All of these mechanisms can prohibit the virus from entering the cell. In a hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiment, we attempted to map the epitope-- the site on the antigen where the antibody binds-- of the 5E10 neutralizing antibody on the HA from the 2011 Victoria H3 influenza strain. A deuterium exchange time course was performed and HDX-MS was used to determine the rate at which deuterium is exchanged for hydrogen on specific peptide segments of the HA protein backbone. We hypothesized that the epitope would be an area that becomes more protected, meaning that it would take up less deuterium, due to local structural ordering from the bound Fab fragment. Through our research, we have identified two potential epitope sites. Identifying the 5E10 epitope on H3 HA will tell us about sites of vulnerability that can be targeted by the immune system. Furthermore, it will help us understand the way the antibody can neutralize the virus as well as predict if the antibody would be able to neutralize other strains of flu based on comparisons of amino acid conservation at the epitope. This information can be used by other scientists to design vaccines that direct an immune response to a specific epitope.

Lipid peroxidation has been found to be associated tightly with ferroptosis, a type of programmed cell death. Our lab recently reported that lipids with unconjugated and conjugated double bonds undergo lipid peroxidation via different mechanisms. Importantly, conjugated polyunsaturated fatty acids (PUFAs) were found to be more reactive to lipid peroxidation than their non-conjugated isomers. Since ferroptosis is caused by lipid peroxidation, we hypothesize that the addition of different exogenous lipids will affect the induction of ferroptosis in cancer cells differently. We treated multiple cancer cell lines with a variety of saturated, monounsaturated, polyunsaturated FAs, and other biologically important lipids at varying concentrations to obtain the EC50 values, the concentrations of various lipids where 50% of cells are viable. We then treated the cell lines with ferroptosis inducers in the presence and absence of various lipids at their EC50 concentrations to observe changes in cell viability. We found that while some biologically important lipids protect the cells from ferroptosis, PUFAs enhance ferroptosis induction. Among PUFAs, the conjugated ones show higher potency compared to their nonconjugated counterparts. We then performed flow cytometry to compare lipid peroxidation accumulation between lipid treatments and found that conjugated PUFAs lead to higher lipid peroxidation levels. Additionally, according to the proposed lipid peroxidation mechanism of conjugated PUFAs, conjugated aldehydes could potentially form as secondary oxidation products. Aldehydes are highly electrophilic and react readily with nucleophiles in cells, including DNA and proteins. We performed cell viability assays with unsaturated, nonconjugated, and conjugated aldehydes and found that the conjugated ones are the most toxic to the cancer cells, suggesting that they contribute to the high potency of conjugated PUFAs in inducing ferroptosis. Identifying the effects of biologically important lipids and their oxidation products on the induction of ferroptosis in cancer cells can lead to the development of therapeutic candidates.

While vaccines help prevent infection from SARS-CoV-2, therapeutic drugs remain necessary to treat people who are already infected. In my research, I am developing peptide-based therapeutics. These therapeutics are safe and readily bioavailable due to their low immunogenic response and low production cost. I design peptide inhibitors against the Mpro enzyme, the main protease present in SARS-CoV-2. Mpro normally cleaves the virus’ pp1a and pp1ab viral polyproteins, which are then activated and assist with viral replication and transcription. By creating a competitive inhibitor that binds to Mpro, I can prevent it from activating the pp1a and pp1ab proteins and thereby prevent viral proliferation. The project began with computational design of peptide inhibitors on the Rosetta Macromolecular Modeling suite, a computational platform that provides accurate structure prediction and design of peptides. An effective drug not only finds the active site of Mpro, but is able to bind more strongly to it than the natural substrate. Mpro’s active site is hydrophobic, so my mentor designed the stub, the part of the peptide that fits within the active site, to include three hydrophobic amino acids: alanine, valine, and leucine. From there, she followed Rosetta's Generalized Kinematic Closure Algorithm to develop various cyclic peptides. After filtering these peptides on Rosetta based on shape and chemical complementarity, she proceeded with 50 peptides. Afterwards, I chemically synthesized and purified some of these peptides with High-Performance Liquid Chromatography. I tested their effectiveness in mass spectrometry-based inhibition assays and determined that the gzm_1,2 peptide has an IC50 value (the concentration of peptide required to inhibit 50 percent of Mpro) of 12.68 µM. Through refining the structure of gzm_1,2, I can improve its inhibitory effectiveness and membrane permeability, enabling it to serve as the basis of an effective and affordable medication for people infected with SARS-CoV-2.