Traditionally known for its toxicity, hydrogen sulfide (H₂S) also possesses physiological roles as an endogenous gaseous signaling molecule in multiple biological processes. Previous research has demonstrated changes in levels of H₂S-producing enzymes during oxidative stress, hypoxia, and inflammation in various tissues including the liver and heart. H₂S protects cells from cytotoxicity in part by promoting the synthesis of glutathione, neutralizing reactive oxygen species, and inhibiting apoptosis signaling pathways. Thiol methyltransferases TMT1A and TMT1B can methylate endogenous H₂S to methanethiol. TMT1B has been shown to have a potential role in mediating the toxic effects of methanethiol. Gene silencing of TMT1B was found to significantly alleviate the observed cytotoxicity induced by methanethiol in human bronchial epithelial cells (16HBE). Methanethiol may induce harm to human respiratory tract cells, and understanding the mechanisms involved, including the role of TMT1B, could potentially lead to insights for mitigating these harmful effects. Doxorubicin (Dox) is a widely used chemotherapy drug for the treatment of various cancers but can induce oxidative stress in cells. In my preliminary experiments, I assessed the cell viability of HepG2 liver cells that were supplemented with various concentrations of NaSH (H₂S donor) and sodium methanethiolate (NaSMe, MeSH donor), followed by Dox treatment. Supplementing the cells with H₂S significantly increased cell viability in the presence of doxorubicin, while the methanethiol had no effect. The goal of my project is to identify H₂S-dependent protective pathways during cellular stress in HepG2 versus cardiomyocytes. My preliminary data indicates that both H₂S and its metabolite, methanethiol, may alter cellular responses following treatment of exogenous compounds that induce cellular stress such as CoCl₂, hydrogen peroxide and Dox. My goal is to pinpoint genes altered during the stress response. This understanding of H₂S-dependent pathways may pave the way for designing novel therapeutics that maintain or enhance H₂S levels.

Human Immunodeficiency Virus (HIV) is a rapidly evolving pathogen with no effective vaccine for eliciting broad protection against HIV infection. The HIV Envelope protein (Env) is a trimeric glycoprotein that is responsible for host-cell membrane fusion and infection initiation. As the only protein on the HIV virion surface, Env is the sole target for neutralizing antibodies. Characterizing the local structural dynamics of Env provides valuable insight into HIV host-virus interaction mechanisms. HDX-MS is an excellent tool for determining structural dynamics by measuring local backbone amide solvent accessibility. Generally, less structured protein regions uptake deuterium more rapidly compared to buried regions or those that are stabilized by secondary structure. We can use mass spectrometry to measure the kinetics of deuterium uptake for peptides throughout the Env protein. HDX-MS provides a detailed portrait of local structural dynamics and order, effectively identifying switching between completely closed prefusion and more open conformational states. A particular HIV Env isolate, A4, is of interest due to its unusually dynamic nature compared to other well-studied Env isolates, such as BG505. Dynamic Env exhibit more conformational flexibility, allowing them to sample various intermediary conformations between open and closed. We hypothesize that this attribute could increase HIV resistance to broadly neutralizing antibodies (bnAbs) that selectively target the closed Env conformation to prevent virus entry in immune cells. We may be able to correlate antibody binding to local dynamics measured in A4 versus BG505 Env trimers to verify this hypothesis. Biolayer interferometry will be applied to quantify antibody association and dissociation rates, as well as binding affinities. These studies will advance existing knowledge in Env-based vaccine therapeutics to improve immune responses to HIV.

Benzalkonium Chlorides (BACs) are widely used as an antimicrobial disinfectant in a variety of food and consumer goods processing. Exposure to BACs has increased significantly due to the COVID-19 pandemic. BACs have been reported in common foods like fruits, milk, and other dairy products, raising concerns about the impact of BACs on human health via oral exposure. Recent work in our lab has reported that BACs are metabolized by cytochrome P450 (CYPs) 4Fs and 2D6 in the liver. However, there is a gap in knowledge regarding how BACs and BAC metabolites are distributed throughout the body, post-oral exposure. We hypothesize that insight into BAC disposition and distribution following an oral exposure route could lead to valuable knowledge of BAC accumulation and subsequent toxicity. In this study, we exposed male and female C57BL/6 mice to deuterated C12- and C16-BACs at 120 μg/g/day for one week via a gel food diet. We harvested liver, lung, heart, spleen, and intestinal section tissues at the end of the study, as well as fecal samples at two time points, and a singular urine time point. Through a targeted BAC and BAC metabolite quantitation analysis using liquid chromatography-mass spectrometry, we found omega-oxidation of the alkyl chain to carboxylic acid followed by beta-oxidation to be a major route of metabolism. Additionally, we found that the liver and big intestine had a higher metabolizing capacity than other tissues and the C16 BACs were preferentially metabolized compared to the C12 BACs. This work provided a deeper look into the disposition and metabolism of BACs and revealed organs that are susceptible to BAC exposure for future studies

There are three enzymes that S-methylate thiols: thiopurine methyltransferase (TPMT) and two enzymes that our lab has recently identified, alkyl thiol methyltransferase 1A and 1B (TMT1A and TMT1B). These enzymes have been studied predominantly because of their role in drug metabolism. Thiopurines, potent chemotherapeutics, are inactivated through thiol methylation by TPMT. While there are not many drugs that contain alkyl thiols, nearly every alkyl thiol-containing drug is metabolized via S-methylation, presumably mediated by TMT1A or TMT1B. With any drug used there is a possibility of drug-drug interactions (DDI) that can lead to a drug concentration in the cell that is above the therapeutic index, leading to toxicity. Thus, it is important to have a method that allows for quick determination of possible DDIs in the body. In this work, using ligation cloning and nickel affinity chromatography, I recombinantly expressed and purified TPMT from bacteria. I then developed an absorbance-based high throughput assay to compare the substrate specificity of TPMT with that of recombinant TMT1A and TMT1B. I determined that TMT1A and TMT1B preferentially methylate alkyl thiols, while TPMT exclusively methylates thiols involved in a conjugated electron system. Although these enzymes serve a crucial role as drug metabolizing enzymes, it is not known if these enzymes have a function beyond drug metabolism. To study the endogenous role of these enzymes beyond drug metabolism, I am utilizing my developed assay to screen for compounds that can specifically inhibit TMT1A, TMT1B, and TPMT. In addition, the assay is optimized in order to screen for potential drug-drug interactions that might result due to interactions with TPMT. Based on literature, I expect most benzoic acid derivatives and similar structured compounds to result in DDIs.

Perineuronal nets are extracellular matrix structures comprised of chondroitin and dermatan sulfate-glycosaminoglycans (CS/DS-GAGs). Histological imaging of brain PNNs is achieved using Wisteria floribunda agglutinin (WFA) labeling of PNN CS/DS-GAGs, while composition can be determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Although these methods are used to determine PNN CS/DS-GAG abundance and composition, it’s unknown whether brain fixation or processing influence these outcomes. We first explored whether tissue processing, using cryosectioning (CRYO) or paraffin embedding (PE), influence PNN analyses. Ten mice were perfused with PBS and post-fixed in 4% paraformaldehyde (PFA). Brains were cut sagittal, and one hemisphere was prepared as floating tissues (CRYO) and the second hemisphere was processed as direct mounted tissues (PE). Histochemical analyses show a 78.9% reduction in hippocampal WFA+ PNNs in the PE processed hemisphere compared to CRYO processed side. LC-MS/MS analysis of hippocampal CS/DS isomers also showed differences between each method. In a second cohort of mice, we determined that fixative (4% PFA vs 10% formalin) did not influence hippocampal WFA or CS/DS isomers between groups, suggesting tissue processing (not fixative) influences PNN analyses. We then explored whether we could correct for these CS/DS baseline differences. By comparing CS/DS isomers isolated from CRYO vs PE processed tissues within each mouse, we discovered reproducible correction factors for each isomer. Adjusting the CRYO group using these factors normalizes baseline compositional differences between CRYO and PF groups. To determine translational relevance, we compared hippocampal CS/DS isomers between three CRYO vs PE prepared non-human primate (M. nemestrina) tissues and observe similar baseline CS/DS differences. Adjusting the CRYO prepared group using corrections factors normalizes the baseline composition. These results provide strong, translational evidence that tissue processing greatly influences both PNN glycan histology and composition analyses, and that corrections must be made to account for baseline differences before comparing groups.

Influenza is a deadly virus that remains a worldwide problem despite vaccinations, modern medicine, and public awareness. After infection, antiviral drugs are the primary treatment option. One common antiviral strategy is to prevent viral entry by targeting the hemagglutinin protein (HA) which sits on the surface of the virus. There are currently 18 known subtypes of HA, resulting in the efficacy of many antiviral drugs being limited. The goal of this project is to computationally design disulfide-closed cyclic peptides that target conserved binding regions on HA and test for broad spectrum activity against multiple HA subtypes. To start, I used RFdiffusion to generate peptides that are docked against the H1 subtype of HA. Then I used ProteinMPNN to optimize the amino acid sequence of the peptides. Afterwards, I used a custom written program named Protein Transform to take the peptides and dock them on the different HA subtypes. I then ran a custom program that uses RoseTTAFold2 (RF2) to predict the root mean square error (rmsd) between the target binding spot and the spot RF2 believes the peptide will stay and score the interface predicted align error (ipae), which is a confidence metric for the binding interaction. Next, I ran a Rosetta program to calculate the interaction energy (ddG) between the protein-peptide complex. The designs were then filtered based on ipae, rmsd, and ddG scores using our lab’s standard filtering thresholds. Our lab then used yeast surface display screening to test the peptides and measure the binding affinity (KD) against the H1 subtype. Then we verified the hits using surface plasmon resonance. We anticipate to see KD values in the range of 10 to 1000 nM. Identifying a lead compound with broad spectrum HA binding is an important step to creating a drug to treat any strain of Influenza.

B-cell lymphoma-extra large (Bcl-xl) is a mitochondrial transmembrane protein that acts as an anti-apoptotic protein by sequestering the apoptosis-inducing proteins Bim, Bak, and Bad. This prevents the release of cytochrome c from the mitochondria, preventing activation of apoptosis pathways. Higher levels of Bcl-xl expression are commonly found in cancer cells. This contributes to the prevention of apoptosis in cancer cells, allowing them to proliferate uncontrollably. Bcl-xl is an incredibly important target for cancer therapeutics. A Bcl-xl binder would inhibit the interaction between Bcl-xl and apoptosis inducing proteins, allowing cancer cells to undergo apoptosis. In my research, I am using deep learning methods to design cyclic peptides that bind to Bcl-xl. To design the binders, I used RFDiffusion - a generative diffusion model - to produce thousands of cyclic peptide binder scaffolds bound to Bcl-xl. I then used a sequence-based deep learning tool to generate multiple sequences for each backbone design. The resulting binders were computationally validated with the highly accurate, machine-learning-based structure prediction tools AlphaFold and RoseTTAFold. Of the 40000 generated cyclic peptides, 2052 were predicted to bind to Bcl-xl based on standard metrics. Along with their excellent metrics, these designs show a high structural similarity and binding location to the known Bcl-xl binders Bim, Bak, and Bad. The designs were clustered by backbone into 350 unique clusters. We synthesized the top designs and identified which peptides display binding to Bcl-xl through a Homogeneous Time-Resolved Fluorescence (HTRF) assay. A successful Bcl-xl binder has the potential to serve as the basis for an effective and affordable cancer therapy.