MACUB (2021) Conference

Abstract: The prevalence of drug-resistant tuberculosis is becoming a global health concern as tuberculosis is one of the leading causes of death from infectious disease worldwide. Developing new drugs is essential in preventing the spread of drug-resistant tuberculosis strains. The Sacchettini lab recently identified 8918, a molecule that inhibits phosphopantetheinyl transferase (PptT), an enzyme essential to the lipid biosynthesis in Mycobacterium tuberculosis (Mtb). The use of 8918 to inhibit PptT led to Mtb cell death proving that PptT is a strong drug target; however, toxicity associated with 8918 makes it undesirable for further drug development. Recent screening identified a potent enzyme inhibitor of PptT, which unfortunately lacked whole-cell activity. Crystal structure of PptT in complex with this inhibitor indicated the complete displacement of co-purified with PptT substrate CoA out of the active site, so it was chosen as a starting point for a fragment-based approach. We screened a fragment library of 880 compounds through nano-DSF and identified 26 molecules that increased the Tm by 3°C or higher, and 16 hits that decreased Tm by 3°C or lower. The top Tm shifting molecules are going through a co-crystallization with PptT process to obtain binding details information. Fragment binding information will be used to optimize our inhibitor for better cell permeability and to design new PptT inhibitors which may lead to developing a new effective drug treatment for tuberculosis.

Abstract: Non-Small Cell Lung Cancer (NSCLC) is the most common type of Lung cancer found in 80-85% of the cases. The Environmental Risk Factors for NSCLC are: smoking, secondhand smoke, the consumption of occupational carcinogens, e.g., asbestos and radon, and malnutrition, such as the lack of vitamin C. Genetics and Epigenetics alterations also lead to NSCLC through mutations that activate the Oncogene RAS and inactivates the Tumor Suppressor Gene (TSG) P53. These changes affect the cell cycle, causing the cells to proliferate and avoid apoptosis leading them to form malignant tumors. The current treatment is surgical removal, and many tumors resist radiation therapy and chemotherapy treatment. The objective of this study is to investigate the effectiveness of an alternative treatment for Large Cell Carcinoma (LCC) {a subtype of NSCLC} on H460 lung cancer cells using Sulforaphane (SFN) extract as the treatment compound. SFN, found at high level in broccoli seeds and sprouts, has been recently studied as a novel anticancer compound. We hypothesized that SFN will inhibit the proliferation of H460 cells via reducing RAS signaling and increasing P53 activity. We examined the effectiveness of SFN on H460 cells through Cell Proliferation Assays, Trypan Blue staining, Fluorescence Apoptosis Assays, and Western Blot analysis for the determination of expression levels of P53. Our results indicate that SFN induces cell cycle arrest and apoptosis through the re-activation of the P53 gene and the inactivation of the RAS gene.

Abstract:

Pentamidine is a water-soluble diamidine that has been used for over eighty years. It is a potentially toxic antiprotozoal and an antifungal drug known to treat African trypanosomiasis (African Sleeping Sickness). Pentamidine works by inhibiting DNA synthesis with RNA polymerase activity. It does this by entering the protozoal cell and binding to DNA. The goal of this research was to make analogs of pentamidine that would have a greater biological effect, better oral bioavailability, and be less toxic by locking the acylic structure of pentamidine into a macrocycle using the Grubbs Reaction. An advanced intermediate in the synthesis of a 15-membered ring analog has been prepared.

Abstract: This project is to develop therapies to bypass challenges to effective and continuous drug delivery to the brain, for the treatment of glioblastoma multiforme (GBM). Currently, individuals diagnosed with GBM have a short life expectancy of 12-14 months. Our approach has the potential to deliver one single dose of gene therapy directly to the GBM tumor environment and block the production of cancer-driving genes. Epidermal growth factor receptor (EGFR) is dysregulated in 57% of all GBM. Our approach uses an adeno-associated virus gene transfer vector encoding RNA therapeutics targeting critical elements of the EGFR pre-mRNA transcript. We have examined the ‘pre-mRNA structurome’ of EGFR to evaluate the accessibility of targetable regions. To advance our therapeutic strategy, we have analyzed the secondary structure of the EGFR transcript using selective 2’ hydroxyl acylation and primer extension followed by mutational profiling (SHAPE-MaP). SHAPE-MaP reactivity profiles were generated revealing the structure of splicing and cryptic polyadenylation signal (PAS) elements within the targeted region. We identified enhancer binding motifs surrounding the 5’ splice site and hidden elements of a cryptic polyadenylation signal. Based on these structural profiles, we generated RNA therapies that interact with structural elements to unravel the hidden polyadenylation signal with the potential to activate expression of the short therapeutic isoform. In this project, we cloned these therapies into our therapeutic delivery platform and tested their efficacy to alter EGFR gene expression in tissue culture cells. Currently, we are evaluating in vitro, the therapeutic RNA interaction with the target sequence of the EGFR pre-mRNA transcript.

Abstract: Human Never in Mitosis Kinase 2 (Nek2) is a serine/threonine-protein kinase and a core component of the human centrosome. Nek2 is known for its critical role in regulating centrosome disjunction through phosphorylation of C-NAP-1 and beta-catenin. It plays an integral role in promoting spindle checkpoint assembly and mediating mitotic events. Aberrant activity of Nek2 kinase has been associated with the highly invasive behavior of metastatic tumors and drug resistance. Novel inhibitory agents of Nek2 kinase are thus urgently needed for the development of targeted anti-cancer therapeutics. Currently, no clinical agent targeting Nek2 kinase has been developed yet. Using a fly model in Drosophila melanogaster, we have shown that the overexpression of Nek2 kinase can promote activation of the Akt/PI3K pathway and cancer metastasis. We also identified a novel non-toxic quinoline-based pharmacophore from the inhibitory EGFR candidate library that inhibited Nek2 function in-vivo and reduced metastasis. The present work describes the development of a dual-action inhibitor library using the quinoline-based pharmacophore that inhibits Nek2 kinase as well as EGFR kinase potently. We anticipated that that developed inhibitory molecules will possess desirable anti-cancer activities in several triple-negative breast cancer cell lines where the expressions of both Nek2 and EGFR kinase are abnormally high.

Abstract:

Biological science’s quest has always been to understand the existence of life and to gain a deep understanding of the relationship between living organisms and how they interact both within and outside of themselves. GFP protein expression is an experiment that can help to better understand how character traits are developed from DNA encoded genes. In this experiment, the GFP gene or DNA particle in the form of plasmid is taken from Jellyfish (Aequorea victoria). This plasmid containing GFP gene and other characteristics are then inserted into bacteria cells. The cells are allowed to grow and then observed for the specific traits of interest, in this case; resistance to antibiotic ampicillin and the ability to glow under UV light. Plasmids are extrachromosomal circular DNA molecules. They have their own origin of replication and carry genes independent of the chromosomal DNA. The pGLO plasmid is designed to carry the gene for GFP protein and the BLA gene. The plasmid consists of Ori where replication begins and ensures that the plasmid is replicated in each daughter cell. It also has a BLA gene which is responsible for the antibiotic resistance by producing beta-lactamase that neutralizes a group of antibiotics to which ampicillin belong. There is the AraC gene which controls or regulates the arabinose operon. The arabinose operon in not active unless there is a presence of arabinose sugar therefore arabinose serves as the inducer for the arabinose operon. It is modified to express the GFP protein in the presence pf arabinose. The GFP gene is downstream of the AraC gene. It codes for the green fluorescent protein. Its expression depends on the activation of the AraC gene.

The experiment is used to illustrate the central dogma of biology from DNA to protein expression in observable traits. There are many important uses for this experiment. It is useful in understanding gene regulation mechanisms. It can also be useful in medicine to understand how bacteria resistance can occur, gene therapy for treating diseased genes. In agriculture, useful and desirable traits can be introduced into plants and animals.

Abstract: Although chemotherapy, radiation, immunotherapy, and surgery can be effective cancer treatments, cancer research is always on a mission to find a more efficient, less expensive way to treat cancer. The use of peptides in cancer-related treatments is not as common as the treatments mentioned above, but is a very promising cancer research field. Cell-penetrating peptides have the ability to deliver anti-cancer therapeutics to cancer cells. Inducible cAMP Early Repressor (ICER) is a transcription factor that is found in all eukaryotes. Deregulation of ICER protein is a common phenomena in many cancers, including skin cancer melanomas. ICER possesses tumor suppressing abilities. Overexpression of ICER blocks cells in mitosis, eliciting cell death and therefore, halting the tumorigenicity of cancer cells. Our data shows that ICER possesses cell penetrating peptide properties and is able to penetrate melanoma cells in culture. This cell-penetrating characteristic of ICER protein could potentially be used to eliminate tumor cells by apoptosis, setting the stage for the development of novel treatments for cancer.

Abstract:

The Von-Hippel Lindau (VHL) tumor suppressor gene produces a protein known as pVHL, which regulates HIF-a by targeting it for ubiquitin-mediated degradation1. pVHL also regulates integrins, such as alpha-5 and beta-1, which are proteins essential for cell adhesion and migration. Mutations in VHL result in the upregulation of integrins, thereby promoting metastasis. Integrins are taken into the cell via endocytosis and recycled in order to travel and fuse with a new part of the cellular membrane, enabling cancer cells to move forward and invade body tissues.

Past experiments performed in the lab demonstrated that bafilomycin, a lysosomal inhibitor, blocks the regulation of integrins by pVHL. This indicates that pVHL requires lysosomes to regulate integrins and may be involved in the mechanism that targets integrins to the lysosome for degradation. A recently performed experiment illustrated that lysosomes are necessary for tight junctions, which link cells together, suggesting that the degradation of integrins by lysosomes is important for cell linkage. This semester, RNA interference will be used to specifically lower the levels of alpha-5 integrin. Once confirmed by Western blotting, the goal is to see whether some of pVHL’s tumor suppressor properties, such as tight junction formation and reduced cell migration, are due to alpha-5 regulation.

The overall purpose of this research project is to understand the mechanism by which pVHL regulates integrins. Since pVHL ubiquitinates target proteins like HIF-a, other pVHL substrates that could be involved in the lysosomal targeting of alpha-5 integrin are being considered. Sequestosome-1 (SQSTM1) and sorting nexin-17 (SNX17) are characterized as potential target proteins involved in the degradation of integrins. SQSTM1 is a possible target because it interacts with atypical protein kinase C, which is identified as a target of pVHL2. The interest in SNX17 is due to its possible role in protecting integrins from degradation by separating lysosomal and recycling pathways3. It is hypothesized that when these target proteins are present, integrins are recycled. However, when these target proteins are present in low levels or even absent, integrins are sent to the lysosome. SQSTM1 and SNX17 will be tested as possible target proteins of pVHL by first performing a Western blot to observe whether there are high levels in VHL- cells and low or absent levels in VHL+ cells. If these differences are observed, a proteosomal inhibitor will be utilized to see if the decrease is due to regulation by pVHL. Another method of investigation is to examine whether pVHL ubiquitinates integrins. Some integrins have ubiquitination patterns that may allow pVHL to target them to the lysosome. This will be tested by transfecting a tagged ubiquitin molecule and observing whether alpha-5 integrin is ubiquitinated. Alpha-5 integrin will be extracted from the cells through immunoprecipitation and a Western blot will be performed to observe the levels of ubiquitin. If pVHL ubiquitinates integrins, levels of ubiquitinated alpha-5 integrin will be higher in VHL+ cells in comparison to the VHL- cells.