Browsing by Author "Das, Joydip"
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Item A Rhodium-Catalyzed [(4+2)+2] Cycloaddition of Allenediene for the Synthesis of Medium-Sized Rings(2018-08) Wang, Dihuang 1991-; Gilbertson, Scott R.; May, Jeremy A.; Do, Loi H.; Das, Joydip; Halasyamani, P. ShivThe synthesis of the compounds with medium-sized rings, especially those with a 5,8-fused ring system embedded into the structure, had long been pursued due to their ubiquitousness in numerous natural products and pharmaceutical molecules. These rings could be efficiently synthesized using metal-catalyzed higher order cycloaddition. In our studies, a previously-reported Rh-catalyzed [4+2+2] cycloaddition was developed and optimized to generate fused systems with a cyclooctadiene core from allenedienes and external alkynes. Upon screening of various metal complexes, ligands and solvents, a rhodium-BozPHOS complex was found to have the best reactivity. This cycloaddition between allenedienes and alkynes showed moderate yield and good substrate tolerance, showing good synthetic potential.Item Binding and Signaling Differences between Prostaglandin E1 and E2 Mediated by Prostaglandin E Subtype Receptors(2010-08) Chillar, Annirudha; Ruan, Ke-He; Chow, Diana Shu-Lian; Gao, Xiaolian; Williams, Louis; Das, JoydipProstaglandin E1 (PGE1) and E2 (PGE2) are ligands for the prostaglandin E2 receptor (EP) family, which consists of four subtype receptors, designated as EP1, EP2, EP3 and EP4. Interestingly, PGE2 mediates inflammation whereas PGE1 acts as an anti-inflammatory factor. However, the molecular basis of their opposite actions on the same set of EP receptors is poorly understood. To study the ligand recognition differences, a potential high throughput mutagenesis and constrained peptide was used. A peptide constrained to a conformation of the second-extracellular loop of human prostaglandin-E2 (PGE2) receptor subtype 3 (hEP3) was synthesized. The contacts between the peptide residues at S211 and R214, and PGE2 were first identified by NMR spectroscopy. The results were used as a guide for site-directed mutagenesis of the hEP3 protein. The S211L and R214L mutants expressed in HEK293 cells lost binding to [3H]PGE2. This study found that the non-conserved S211 and R214 of the hEP3 are involved in PGE2 recognition. The mutant S211L was able to give a calcium signal with PGE1, but not with PGE2. This implied that the corresponding residues in other subtype receptors could be important in distinguishing the different configurations of PGE2 and PGE1 ligand recognition sites. Direct transfection of point mutants in the EP1 receptor extracellular loop (using PCR products) was evaluated in HEK293 cells. Twenty-four EP1 extracellular loop mutants (alanine scan) were generated using phosporylated primers and ligase. The PCR product was directly transfected into HEK293 cells and the [3H]-PGE2 binding and PGE1 and PGE2 calcium signaling assay evaluated. Three mutants, A104G, P105A and P184A, showed reduced [3H]PGE2 binding, but could not differentiate between PGE1 and PGE2 in the calcium signaling assay. However, we propose that this novel high throughput mutagenesis approach using direct PCR product transfection can be integrated into a high throughput screening machine in the future. The PGE1 and PGE2 binding affinity on the four human recombinant EPs expressed in the live HEK293 as stable cell lines was determined by [3H]PGE2 binding. The PGE1 and PGE2 signaling on the four EPs was determined by the calcium (Ca2+) and cyclic AMP signaling. The Kd for [3H]PGE2 was calculated using saturation kinetic experiments. The IC50 of PGE2 and PGE1 were calculated from [3H]PGE2 displacement experiments using cold PGE2 and PGE1, respectively. PGE2 showed higher affinity or preference for EP3 and EP4 as compared to that of EP1 and EP2. PGE1 also showed a higher Ca2+ signal in EP1 as compared to that of PGE2. There was a two-log concentration difference between PGE2 and PGE1 for generation of Ca2+ signal in EP4. There was no difference in cAMP accumulation with PGE1 and PGE2. Leukotriene C4/D4/E2 levels were higher in the EP1 stable cell line upon stimulation with PGE2, but not PGE1. An anti-inflammatory molecule, 20 hydroxy lipoxin B4, peak was observed using mass spectroscopy with PGE1 and not PGE2. We also used a newly engineered hybrid enzyme (COX-2-10aa-mPGES-1) linking COX-2 and mPGES-1 together thus adopting the full biological activity of COX-2 and mPGES-1 in directly converting AA to PGE2. This enzyme was genetically introduced into HEK293 cells. These cells expressing the COX-2-10aa-mPGES-1 were producing higher level of PGE2 using endogenous AA as confirmed by LC/MS analysis. A new mouse model of cancer was developed by subcutaneous injection of these cells into Balb/c/nu/nu mice. A 100% (8 out 8) occurrence rate of cancer mass was detected in these cells. In contrast, 30% occurrence of cancer mass were determined for the groups of the cells co-expressing the individual COX-2 and mPGES-1. The presence of EP1 and EP2 stable cell line growth around these tumor masses confirmed their involvement in cancer. In conclusion, the Ca2+ signal indicated that the EP1 is likely the dominant and ligand-differentiating receptor in terms of signaling in tissues that co-express the EPs (cancer cells). PGE2 is likely to cause inflammation through leukotrienes and PGE1 is likely to be anti-inflammatory due to its ability to produce Lipoxin B4. High throughput mutagenesis for producing multiple-point mutations using direct PCR product transfection is a promising new method for the future. The experiments on nude mice indicated that the sole coupling of COX-2 to mPGES-1 is a powerful cancer-advancing factor, which implies that the coupling of COX-2 to mPGES-1 is a promising target for anti-cancer drug development. EP1 and EP2 receptors were identified as the likely receptors, to induce cancer. This study provides a molecular basis to understand the biological functions of PGE1 and PGE2 through their binding and signaling properties.Item Development of Allosteric Non-covalent KRAS Inhibitors that Disrupt Effector Binding and Development of Small Molecule Inhibitors to Modulate Liver X Receptor Activity(2022-04) Thawalama Gamage, Kasuni Dilsha; Gilbertson, Scott R.; May, Jeremy A.; Harth, Eva M.; Xu, Shoujun; Das, JoydipSynthetic chemistry plays an important role in searching for new medications which are therapeutically useful in treating disease. The development of a new small molecule drug for a specific biological target mainly derives from two sources: natural products or synthetic collections of molecules. A traditional drug-like molecule can be an effective enzyme inhibitor and allosteric modifier or can target extracellular proteins or intracellular receptors. This dissertation presents our effort and accomplishments in the synthesis of small-molecule inhibitors that target mutant KRAS function and molecules that modulate liver x receptor (LXR) activity. Activating somatic KRAS mutations are associated with more than >30% of all human cancers. However, KRAS has long been considered as an undruggable target due to the competitive inhibition of its catalytic site. After decades of failures, researchers are seeing promising approaches to develop drugs that target KRAS. Our collaborator has identified a novel pyrazolopyrimidine-based KRAS inhibitor that binds to activated KRAS. Depending on its structural features a library of small molecule inhibitors was synthesized by attaching various functional groups in five main regions of the structure. Additionally, the chemistry to synthesize PROTAC molecules with high-affinity for the target that allow for the attachment of E3 ligase ligands by click reaction will be discussed. Pancreatic ductal adenocarcinoma (PDAC) is the predominant form of pancreatic cancer, the third leading cause of cancer deaths in the US. Due to ineffective therapies, a new strategy is clearly needed to treat PDAC patients. It has been found LXRs are highly expressed in PDAC cell lines. Therefore, modulation of LXR activity via small molecule ligands could constitute a new therapeutic strategy to target PDAC tumors. In a screen of a focused library, two novel LXR ligands (3A4 and 1E5) were identified with potent antitumor activity. Herein, we report the synthesis and structure-activity relationship of the series of 3A4 and 1E5 derivatives. The chemistry and bioassay results that indicate general trends of the functional groups on the activity of the ligands will be discussed.Item Dual Impacts of Newly Engineered COX-PGIS Hybrid Enzyme on Cardiovascular and Neuronal Protection(2018-05) Ling, Qinglan; Ruan, Ke-He; Aronowski, Jaroslaw A.; Das, Joydip; Eikenburg, Douglas C.; Gao, XiaolianStatement of the problem: Non-steroidal anti-inflammation drugs (NSAIDs) are widely used for varieties of inflammatory conditions by inhibiting COX pathway and decreasing the production of downstream prostaglandins, such as PGE2 and PGI2. As neuroinflammation may lead to Alzheimer’s disease (AD), NSAIDs have also been suggested as a prevention or treatment for AD. However, clinical studies did not show effectiveness. To further understand the mechanisms and eventually achieve the goal, it is important to understand the crosstalk of prostanoids in the hippocampus, a major brain area impacted in AD. So far, a major obstacle in that was to mimic the biosynthesis of each prostanoid. Thus, this study intended to accomplish three aims. Specific aim 1: To study the crosstalk of prostanoids in a neuronal cell line by using hybrid enzymes to redirect the synthesis of prostanoids to PGE2 and PGI2, respectively. Specific aim 2: To investigate the crosstalk of prostanoids in primary hippocampal neurons. Specific aim 3: To examine the dual impacts of newly engineered COX-PGIS hybrid enzyme on cardiovascular and neuronal protection in vivo. Procedure or methods: Here, we created Single-Chain Hybrid Enzyme Complexes (SCHECs), COX-2-10aa-mPGES-1 and COX-1-10aa-PGIS, which could control cellular AA metabolites to the desired PGE2 or PGI2, respectively. By using the two hybrid enzymes, as well as synthetic PGE2 and PGI2 analogues, we investigated the crosstalk of PGE2 and PGI2 in hippocampal neuronal cell line, primary hippocampal neurons, and in vivo Alzheimer’s disease model. Results: The hippocampal neurons expressing SCHECs redirected AA metabolism to a PGI2, or PGE2. Overproduced PGI2 exerted survival protection and resistance to Aβ-induced neurotoxicity. The protection mediated by PGI2 might be through IP receptor. Our PGI2-producing transgenic mice exhibited resistance to AA-induced thrombotic stroke and angiotensin-II-induced vascular constriction. The hybrid of PGI2-producing transgenic mice and AD mice showed restored long-term memory in two behavioral tasks. Conclusions: These findings suggested that the vascular mediators, PGI2 and PGE2, exerted significant regulatory influences on neuronal protection (by PGI2), or damage (by PGE2) in the hippocampus, and raised a concern that the widely uses of aspirin in cardiovascular diseases may exert negative impacts on neurodegenerative protection.Item Identification of the Activator Binding Residues in the Second Cysteine-Rich Regulatory Domain of Protein Kinase C Theta (PKCθ)(2012-08) Rahman, Ghazi 1973-; Das, Joydip; Prasad, B. V. Venkatar; Ruan, Ke-He; Williams, Louis; Briggs, James M.Protein kinase C theta (PKCθ) is a serine threonine kinase, which is predominantly expressed in the T-cells and is selectively translocated in the immunological synapse upon activation. Active PKCθ initiates the downstream activation of the immunological responses against the intruders. Selective PKCθ inhibition may manage autoimmune disorders. PKCθ belongs to novel class of PKCs. Diacylglycerol (DAG) and phorbol esters are the common activators, which bind to the C1 domains of the novel and conventional PKCs. Design of PKCθ selective inhibitors targeting its activator binding C1 domain, requires the knowledge of C1 domain structure and the activator binding residues. PKCθ C1 domain consists of twin cysteine-rich subdomains, C1A and C1B, of which C1B plays a major role in the activation followed by the membrane anchoring PKC. To this end we determined the crystal structure of PKCθC1B subdomain at 1.63 Å, which showed a similar overall structure to that of PKCC1B, except that the orientation of the Trp-253 residue is towards the membrane and the width of the activator pocket opening is narrower. The homologous Trp-252 in PKCC1B is oriented away from the membrane with wider activator pocket opening. Using this structure, five possible activator binding residues were identified though the overlaying of the crystal structure, and alignment of the sequences of PKCθC1B and PKCC1B, followed by molecular docking of a library of DAG and phorbol ester analogs into PKCθC1B as receptor. To determine the role of these residues, Y239A, T243A, W253G, L255G and Q258G mutants in isolated PKCθ C1B domain were designed, expressed and purified from E. coli and their binding affinity (Kd or Ki) for phorbol 12, 13-dibutyrate (PDBu) and Sn-1,2-dioctanoylglycerol (DOG) were measured by radioactive PDBu binding assay. All the mutants showed significantly reduced binding affinity for both PDBu and DOG. Among all the mutants, Q258G showed highest reductions in activator binding affinity than the wild type. The extent of reductions in the binding affinity for θC1B mutants of Y239A, W253A and L255G were different however was much lesser than the homologous mutations in C1B. All the five mutants of full length PKCθ were expressed in HEK293 cells and showed reduced phorbol 12-myristate 13-acetate (TPA) and DOG induced membrane translocation compared to wild type. These results provide insights into the PKC C1B activator binding domain, which will aid in future design of PKCθ selective inhibitors.Item Investigating the Role of Serotonin and Ghrelin Receptors in Neurobiology(2019-08) Yang, Yaxing 1990-; Gilbertson, Scott R.; May, Jeremy A.; Teets, Thomas S.; Coltart, Don M.; Das, JoydipDrug addiction remains a significant problem worldwide. A new treatment strategy is needed based on the lack of approved pharmacological treatment. Serotonin (5-hydroxytryptamine, 5-HT) receptors have been implicated in a wide variety of physiological functions both in the central nerve system (CNS) and in the periphery. Our research program has been focused on the control and balance of 5-HT2AR and 5-HT2CR systems. It has been shown that these two receptors are in oppositional control with 5-HT2AR antagonists and 5-HT2CR agonists exerting similar effects on behaviors such as impulse control and reactivity to cues. The existence of the homo and heterodimers of 5-HT2AR with 5-HT2CR may play a role in differentiating signaling in this GPCR system. Accumulating data also indicate that function cross-talk between 5-HT2CR and GHS-R1a (growth hormone secretagogue receptor), and the interaction between 5-HT2CR and GHS-R1a would be a new research field to investigate. Chemistry to access bivalent ligands for the system 5-HT2AR and 5-HT2CR or 5-HT2CR and GHS-R1a, as well as ligands attached to cholesterol and serotonin will be discussed.Item Modeling and Validation of Gene Networks in Breast and Pancreatic Cancer(2022-04-30) Ho, Charles; Lin, Chin-Yo; Das, Joydip; Umetani, Michihisa; Chung, Sang-Hyuk; Feng, QinOmics data have been growing at an extraordinary pace and are on track to hit exabytes of data within the decade. With the ability to quickly sequence whole genomes within a day, there are many opportunities to use this data in the study of disease pathophysiology and in therapy development. With 1.9 million new cases of cancer and an estimated 610,000 deaths each year in the United States, it would be beneficial to have an integrated bioinformatic pipeline to quickly and efficiently integrate this growing amount of publicly available data in cancer related omics research. In our studies, we utilized a multi-omics approach to develop an integrated bioinformatic pipeline which combines machine learning methods, the latest bioinformatic tools, and various large omics data sets for uncovering disease mechanisms. We then took advantage of large patient cohort databases to establish a methodology for testing and validating the clinical relevance of these novel mechanisms. Using this approach, we conducted three studies in breast and pancreatic cancer. First, we focused on the oncogenic mechanisms of alcohol in the development and progression of breast cancer. We conducted a secondary analysis of our previous published transcriptome data and used our integrated pipeline to discover alcohol-regulated metabolic genes associated with oncogenic calcium signaling. The role calcium signaling in cancer promoting actions of alcohol was validated experimentally and the clinical relevance of these genes was established by their expression profiles in publicly available patient data. Our second study used a similar pipeline to find gene networks which play an important role in mediating the cytotoxic effects of liver x receptor (LXR) β activation in pancreatic cancers. This study revealed that LXRβ activity up-regulated the genes involved in pro-apoptotic fatty acid production and endoplasmic reticulum stress genes and down-regulated cell cycle genes. Many of these genes showed clinically relevant expression profiles in patient samples. Finally, our third study integrated machine learning with our established pipeline to predict targetable mechanisms in novel predicted pancreatic cancer molecular subtypes. We uncovered three novel subtypes with targetable immune associated mechanisms and subtype-specific gene expression profiles with therapeutic and prognostic implications.Item Pyrido-pyrimidinone Derivatives as RIPK2 Kinase and NOD Signaling Inhibitors(2018-08) Nikhar, Sameershivnath; Cuny, Gregory D.; Das, Joydip; Ruan, Ke-He; Degterev, Alexei; May, Jeremy A.Receptor interacting protein kinase-2 (RIPK2) is an enzyme that transduces pro-inflammatory signaling from the nucleotide binding oligomerization containing proteins (NOD1/2) during bacterial invasion in immune cells. However, NOD 1/2 mutations cause uncontrolled activation of RIPK2, resulted in excessive signaling and cytokine production. Such dysregulated signaling has been associated with inflammatory bowel (Crohn’s disease) and neuro-inflammatory diseases (Multiple Sclerosis), making RIPK2 an important target. Although, several RIPK2 kinase inhibitors have been reported in the literature, they possess limitations including off-target effects, reduced cellular potency, compromised in vivo efficacy and hERG/CYP inhibition. Thus, novel types of RIPK2 inhibitors are needed to address such problems. To this end we have identified a pyrido-pyrimidine based inhibitor (UH15) that has shown potent RIPK2 inhibition in enzymes and cells, but also showed non-selective inhibition of structurally similar activin like kinase-2 (ALK2). Nevertheless, molecular docking studies of UH15 in RIPK2 and ALK2 identified key differences that have been used to optimize the compound towards potent and selective RIPK2 inhibitors. The docking model-based optimization of UH15 identified two sets of analogs that differentiate RIPK2 inhibition in cells based upon size and position of substituents at a specific site on the inhibitor. Compounds that occupy the shallow hydrophobic pocket between the β3/β5 strands and the α-C helix in the kinase domain, showed effective RIPK2 cellular inhibition. The behavior of this subset of UH15 inhibitors correlates to the recently identified CSLP compounds that have shown RIPK2 cellular potency by interfering with RIPK2-XIAP interaction after occupying the hydrophobic pocket. The most potent and selective RIPK2 inhibitor (UH15-15) has shown optimal absorption, distribution, metabolism and excretion (ADME) and pharmacokinetic (PK) properties, making it a suitable probe for delineating the role of RIPK2 in disease models. Furthermore, modifications of the central pyrido-pyrimidine scaffold of UH15 series resulted in several other distinct structural classes of RIPK2 inhibitors. Overall, the identified RIPK2 inhibitors show mechanism based cellular potency, selectivity verses the structurally similar ALK2 kinase and promising pharmacokinetic properties. These compounds will allow investigation of RIPK2 function in disease models and will provide the basis for further optimization and advancement of RIPK2 inhibitors.Item Role of Munc13-1 and Munc13-2 Proteins in Alcoholism, and its Potential Clinical Applications(2017) Badal, Sean; Ghosh, Anamitra; Wooden, Jessica; Leasure, J. Leigh; Das, JoydipMunc13-1 and Munc13-2 are presynaptic proteins that are involved in the vesicular priming and subsequent release of glutamate neurotransmitter. Previous research has demonstrated some of the effects of ethanol in specific areas of the brain related to addiction, but the effects of ethanol on the expression of Munc13-1 and Munc13-2 and their glutamatergic effects in different brain regions is unknown. First, we performed immunocytochemistry (ICC) of Munc13-1 and Munc13-2 on ethanol-treated differentiated HT22 cells in-vitro and found that there was a significant change in the overall expression of Munc13-1 in a dose and time-dependent manner. Next, we harvested primary hippocampal neurons, treated them with ethanol in a dose and time-dependent manner, and then measured both Munc13-1 and Munc13-2 protein expression using Western-blot analysis. Here, our results showed that ethanol significantly upregulates the expression of both Munc13-1 and Munc13-2. We then tested the effects of ethanol on Munc13 proteins in-vivo on both wild-type (Wt) and heterozygous Munc13-1 knockout mice, using the drinking in the dark (DID) paradigm. In comparison to our previous ex-vivo data, these results showed that with alcohol exposure there was a significant increase in the expression of Munc13-1 in the hippocampus and cerebellum of Wt mice, but a decrease in the expression of Munc13-2 in the hippocampus, cerebellum and cortex. In the heterozygous Munc13-1 knockout mice, ethanol caused significant compensation for the loss of Munc13-1 in the hippocampus and cerebellum, and also an increase in Munc13-2 expression in the cerebellum and cortex. Since both Munc13-1 and Munc13-2 predominantly control glutamatergic synapses, their modulation by ethanol exposure could potentiate an increase in glutamate release from the pre-synaptic terminals of these regions and stimulate excitation of the Central Nervous System (CNS). This is analogous to the CNS excitation we see with chronic AUD patients experiencing alcohol withdrawal symptoms. By identifying these proteins as drug targets, we may be able to unfold a different mechanism of alleviating alcohol withdrawal symptoms in AUD patients, without the GABAergic adverse effects that usually present with the current mainstay treatment options.Item Structure-Based Design of Selective RIPK1 and RIPK2 Inhibitors(2017-12) Suebsuwong, Chalada 1988-; Gilbertson, Scott R.; Cuny, Gregory D.; Das, Joydip; Coltart, Don M.; Yang, Ding-ShyueKinases regulate various biological functions by post-translational phosphorylation of proteins. Kinase dysfunction is associated with many pathological conditions. Therefore, kinase inhibitors have become an important class of drugs and chemical biology probes for mechanistic studies of diseases. This dissertation focuses on a structural-based design of selective RIPK1 and RIPK2 inhibitors using various approaches. Chapter 1 presents an introduction to protein kinases and a review of kinase inhibitor types for understanding the rationale and design of selective RIPK1 and RIPK2 inhibitors. Chapter 2 describes two design strategies to potent and selective RIPK1 inhibition. The first strategy involves modifications of ponatinib, a type II Abl kinase inhibitor, exploiting differences in the steric and hydrophilic characteristics of Abl and RIPK1 gatekeeper residues. An introduction of tert-butyl on the central phenyl (CS5) caused unfavorable interactions with Abl’s gatekeeper and resulted in significantly improved selectivity for RIPK1 versus Abl/RIPK2/RIPK3. To improve cellular activity, a hybridization strategy linking ponatinib and Nec-1, a type III RIPK1 inhibitor, was pursued. PN10 displayed selectivity for RIPK1 inhibition and better RIPK1 cellular activity than either Nec-1 or ponatinib. In Chapter 3, strategies to develop selective type II RIPK2 inhibitors based on regorafenib, a VEGFR inhibitor, are presented. The first strategy is based on structural differences between RIPK2 and VEGFR around allosteric hydrophobic pocket and gatekeeper residues. The second strategy targeted the activation loop, a region of kinases with diverse amino acid sequences. CSR35 was identified and shown to form an interaction with the activation loop. A RIPK2CSR35 co-crystal structure revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in CSR35 and Lys169. Chapter 4 presents a hybridization strategy between ALK2 type I inhibitor LDN-214117 and B-Raf type I½ inhibitor PLX4032 as an approach to conformational distinct αC-helix-displacing RIPK2 inhibitors. Potent and selective RIPK2 inhibitors (CSLP43 and CSLP37) were achieved through modifications of the trimethoxyphenyl in LDN-214117 that occupies the hydrophobic pocket in RIPK2 next to the αC-helix. RIPK2inhibitor co-crystal structures of several derivatives suggested that they bind in a type I mode. However, further analysis of CSLP43 and CSLP37 is warranted since they display greater cellular potency.Item The Endogenous Metabolite 5-Methoxytryptophan as a Potential Novel Therapy for Pancreatic Cancer(2017-08) Murdoch, Emma E.; Ruan, Ke-He; Dixon, Richard A. F.; Williams, Louis; Das, Joydip; Gao, XiaolianPancreatic adenocarcinoma is a highly aggressive cancer, and due to a lack of early detection methods, is often diagnosed in the late stages of the disease. One reason for the poor prognosis in pancreatic cancer is the repopulation of tumors, which is driven by pathological cyclooxygenase-2 production. The L-tryptophan metabolite 5-MTP has previously been shown to reduce COX-2 transcription via the p300 histone acetyltransferase. In a scratch test assay using the PANC-28 cell line, we demonstrated that PANC-28 cell invasion could be reduced by treatment with 5-MTP at the micromolar level. This project included molecular modeling of 5-MTP at the PGE2 receptor EP3. 5-MTP showed a strong binding score, in a similar location to that of the endogenous prostaglandins. A cell counting kit was used to measure cell survival following 5-MTP administration, and showed that 5-MTP was non-toxic to cells. Given our results, it is possible that 5-MTP may have a beneficial role in pancreatic cancer. Future studies will include biochemical and ligand binding assays to build upon these findings.Item The Role of Pre-Synaptic Munc 13-1 in Voluntary Ethanol Consumption and Tolerance(2018-08) Wooden, Jessica I.; Leasure, J. Leigh; Roman, Gregg; Das, Joydip; Kosten, Therese A.In the United States alone, approximately 18 million people suffer from alcohol use disorders. The role of the munc 13-1 pre-synaptic protein in alcohol-related behaviors has been little-studied, despite being a known site for ethanol binding. Munc 13-1 is an active zone protein that is vital for vesicle maturation at the synapse. Mice that are heterozygous for the gene regulating munc 13-1 produce synapses that, while structurally sound, show an approximate 50% decrease in this protein. Ethanol binds munc 13-1, decreasing its functional ability and likely antagonizing glutamatergic targets. Exposure to NMDA receptor antagonists has long been known to increase hippocampal neurogenesis, and it is likely that mice with inherently deficient NMDA stimulation would show the same pro-neurogenic outcomes. The current study aimed to perform a thorough behavioral analysis of mice heterozygous for munc 13-1. Few phenotypes have been identified in this specific type of mouse, and genetically engineered mice may express very different phenotypes from mutation to mutation. This project also endeavored to assess how loss of munc 13-1 affects voluntary binge and chronic alcohol consumption. Separately, munc 13-1 heterozygotes were evaluated for changes to acute functional tolerance following an injection of a standardized dose alcohol, using an accelerating rotarod as a measure of motor coordination. The last goal of the current project was to compare levels of doublecortin (DCX)-positive cells in the dentate gyrus between alcohol-exposed and alcohol-naïve mice, and also compare DCX levels across wildtype and heterozygous mice. Taken together, these results will give future researchers a better understanding of the role that pre-synaptic protein munc 13-1 plays in alcohol consumption and tolerance.