“EVALUATING THE EFFECT OF NOVEL DRUGS ON LPS-INDUCED INFLAMMATION USING ENZYME-LINKED IMMUNOSORBENT ASSAY”

Neuroinflammation plays a key role in many neurodegenerative diseases and is characterized by the over-activation of immune cells within the central nervous system. Activated microglia and astrocytes release pro-inflammatory cytokines such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF- α), as well as reactive oxygen species that contribute to neuronal damage. These inflammatory responses are mediated through signaling pathways including the NF-κB pathway and the NLRP3 inflammasome.
Alzheimer’s disease (AD) is the most common neurodegenerative disease and the leading cause of dementia worldwide. In AD, accumulation of β-amyloid plaques (Aβ) stimulates chronic neuroinflammatory responses and contributes to neuronal dysfunction and disease progression. Elevated levels of inflammatory cytokines, particularly IL-1β, have been strongly associated with Aβ plaque deposition and the onset of AD.
Based on this relationship, targeting neuroinflammatory signaling pathways might be a promising therapeutic strategy for AD. PD2244, a novel compound synthesized by P2D Biosciences, is hypothesized to reduce neuroinflammation by suppressing the production of pro-inflammatory cytokines involved in AD pathology. Specifically, PD2244 is expected to decrease IL-1β production by inhibiting either the NF-κB pathway or the NLRP3 inflammasome.
To test this hypothesis, PD2244 was tested on several cells related to AD pathology, including microglial, neuronal, and monocytic. These cells were pretreated with increasing concentrations of PD2244 prior to inflammatory stimulation. IL-1β production was quantified using Enzyme Linked Immunosorbent Assay (ELISA), a highly sensitive technique commonly used to detect pro-inflammatory cytokine production.

As antibiotic resistance continues to rise, the development of new antibiotics is more important than ever. However, converting an active antibiotic compound into a clinically viable pharmaceutical requires optimization of the drug’s pharmacokinetic profile, including improving its stability, permeability, and targeted delivery to pathogens. One strategy to overcome these delivery challenges is the use of prodrugs—inactive or chemically modified derivatives of a parent compound that are converted to their active form in vivo, most often through enzymatic cleavage. Effective prodrug modifications can improve drug delivery to pathogens or enhance permeability through the hydrophobic bacterial cell membrane while maintaining or improving the activity of the parent compound. Our goal is to categorize novel prodrug structures by assessing efficacy of penicillin prodrug derivatives. This project analyzes the activity of penicillin prodrugs synthesized in the Montchamp Lab against the gram-positive bacterial species Bacillus anthracis and vancomycin-resistant Enterococcus faecalis using MIC (Minimum Inhibitory Concentration) assays. Penicillin G is a well-known antibiotic with a single acidic site, providing a convenient parent compound for synthesis and well-established MIC values. We determined that, consistent with hypothesis, prodrug structures with increasing numbers of enzymatic activation “triggers” exhibited increased antibiotic activity. For example, structures containing two benzene groups showed greater activity than those containing one, and both showed greater activity than nonderivatized Penicillin G. Knowledge of these promising prodrug structures will guide the future synthesis of antibiotics with more challenging pharmacokinetics to improve drug delivery and efficacy.

Partner and Localizer of BRCA2 (PALB2) is a necessary linker protein between BRCA1 and BRCA2 that directs the cells towards homologous recombination in the presence of double-strand breaks (DSB). When this linkage is disrupted, the cell routes the repair towards non-homologous end joining or single-stranded annealing, which are not as efficient or accurate in their repair of DSB. With inefficient DNA repair, mutations accumulate that increase the risk of the development of cancer. It has been documented that mutation L35P in PALB2 is pathogenic and leads to a decrease in HR in cells. However, it is unknown if the loss of leucine at this position is causing a decrease in BRCA1 binding or if it is the introduction of a proline into the coiled coil region that is disrupting the secondary structure, thereby inhibiting binding. We are studying 5 variants of unknown significance (VUS) from PALB2 that are within the coiled coil and are also proline substitutions. One of these mutations is within the binding interface and the other four are on the backside of the coil opposite the predicted binding interface. We aim to answer if it is the introduction of a proline that is destroying the secondary structure and preventing binding. Isothermal titration calorimetry data suggests that all proline variants thus far, regardless of proximity to the interface, inhibit binding with BRCA1. We will pair binding data with the secondary structure analysis and thermal stability of these variants (using circular dichroism) to better connect variant structure with PALB2 dysfunction. 

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by memory loss, cognitive decline, and chronic neuroinflammation. Inflammatory signaling pathways such as nuclear factor-κB (NF-κB) play a critical role in the progression of neurodegeneration by regulating the expression of pro-inflammatory cytokines such as TNF-α and IL-1β. Targeting NF-κB signaling therefore represents a promising therapeutic strategy for reducing inflammation associated with AD. This study evaluated the effects of several novel anti-inflammatory compounds provided by P2D Biosciences and Dr. Geen’s research lab on TNF-α–induced NF-κB activation. HEK293 cells were transfected with an NF-κB responsive PRDII luciferase reporter and a CMV luciferase control, followed by treatment with novel compounds and stimulation with TNF-α. Luciferase activity was measured to quantify the effect of our molecules on TNF-a-induced NF-κB transcriptional activation. Results demonstrated dose-dependent reductions in NF-κB activation for several compounds, suggesting potential anti-inflammatory activity. These findings contribute to ongoing efforts to identify novel small molecules capable of modulating NF-κB signaling and may support future therapeutic development targeting neuroinflammation in Alzheimer’s disease.

BRCA1 is widely recognized for its role in maintaining genomic stability, particularly through its involvement in several DNA repair pathways and chromatin regulation. While mutations in BRCA1 are strongly associated with increased cancer risk in humans, the broader cellular consequences of BRCA1 mutations under environmental stress remain unclear. The goal of the project was to investigate how loss or alteration of BRC-1, the Caenorhabditis elegans (C. elegans) homolog of BRCA1, affects stress responses at the organismal level, with focus on oxidative stress and reactive oxygen species (ROS) accumulation.

Using C. elegans as a model organism allowed me to study the stress responses within a system where development, reproduction, and genome stability are tightly connected. A key advantage of using C. elegans is that the organism is transparent. This allows for visualization and quantification of fluorescence to measure ROS within the worms, and to see how these values vary depending on BRC-1 status. Three strains were compared: 1) wild-type (N2), 2) a BRC-1 mutant (syb5376) predicted to disrupt nucleosome interaction and H2A monoubiquitylation while retaining other enzymatic functions, and 3) double knockout strain (xoe4) lacking functional BRC-1 entirely. The comparison of these worms with various levels of BRC-1 activity allowed for the investigation of how each of these different strains responded to various oxidative stressors. The broader aim of the project was specifically to look at how altered BRC-1 nucleosome ubiquitylation affects the cell's ability to deal with ROS, and compare this with the wild-type and complete knockout.

Several key questions were used to guide the research: 1) Whether stress responses differed between wild-type, mutant, and knockout strains, 2) How oxidative stressors altered ROS levels in each of the strains, and 3) Whether the syb5376 and xoe4 strains behaved similarly or exhibited distinct patterns compared to wild-type worms. Across multiple experimental conditions, the double knockout strain consistently showed the most elevated fluorescence, indicating increased ROS accumulation and reduced ability to manage oxidative stress due to the lack of BRC-1. The syb5376 mutant displayed an intermediate effect, suggesting partial impairment of regulation when lacking nucleosome interaction. These findings support the idea that BRC-1 plays a protective role under stress conditions and that disruption of nucleosome ubiquitylation may compromise the cellular response to oxidative damage and lead to a higher accumulation of ROS within cells.

Looking at the bigger picture, these results align with the broader understanding that BRCA1 loss does not immediately lead to cancer, but rather increases vulnerability when cells are challenged by environmental or metabolic stressors. Increased ROS levels can lead to DNA damage, and without proper chromatin remodeling and repair coordination, cells may struggle to restore their genomic integrity. The differences observed between the mutant and knockout strains further suggest that mutation type matters in determining the severity of stress sensitivity and the overall impact that this will have on the cell and organism as a whole.