Minu Bidzimou

Mentor: Gary Sulikowski
Home Institution: Grinnell College
Research Abstract:
Bacterial antibiotic resistance is a global health problem that requires discovery of preventative methods and treatments. To this end, marine natural products are readily exploited and implemented as potent treatments against resistant bacteria. Chrysophaentin A is an antimicrobial natural product isolated from the marine chrysophyte alga Chrysophaeum taylori. This natural product has potent activity against resilient gram positive bacteria such as methicillin-resistant Staphylococcus aureus by inhibiting the bacterial cytoskeleton protein FtsZ. Although multiple kinetic and binding studies have been published on this interaction, Chrysophaentin A has not faced extensive clinical and in vivo studies due to its low isolation levels. We address this problem by investigating a synthetic route to produce Chrysophaentin A from readily available starting material. Chrysophaentin A has two diarylbutene units connected by two ether bonds, and decorated with multiple chlorines. Our envisioned synthetic route includes a regioselective stannyl cupration to synthesize a vinyl chloride, a ring closing metathesis reaction to install an olefin, and a key alkyl migration catalyzed by the Montmorillonite K10 clay. We herein report optimization of our synthetic route using a simplified model system.

Lee Cantrell

Inuloxin A is a phytotoxin obtained from Inula viscosa, a widespread Mediterranean plant well known for its therapeutic metabolites. Specifically, Inuloxin A displays inhibition towards Leishmania donovani: a parasite responsible for visceral leishmaniasis. The absolute configuration (AC) of chiral molecules like Inuloxin A is critical to their bioactivities. Therefore, we used chiroptical spectroscopic measurements and predictions to assign the absolute configuration of Inuloxin A. Using Gaussian09 programs, we optimized the geometry of various enantiomeric conformers of Inuloxin A and then isolated the lowest energy conformers for each enantiomer. After applying a Boltzmann distribution, we interpreted the theoretical spectra against experimental spectrum by visual and quantitative analysis in CDSpecTech programs. No chiroptical spectra could facilitate definitive AC assignment; however, the AC (7R,8R,10S) is suggested.

Amanda Cao

Mentor: Lauren Buchanan
Home Institution: Cal Poly Pomona
Research Abstract:
Misfolded proteins are gaining interest in both the scientific and medical fields due to their propensity for forming cytotoxic amyloids, which are associated with a number of diseases such as type II diabetes, Alzheimer’s, Huntington’s, and Parkinson’s. On the other hand functional amyloids can be seen in human hormone storage in secretory granules. Amyloid fibrils, which can be functional or cytotoxic, are peptide or protein aggregates that organize into long, unbranched fibrils with a cross β-sheet structure. The structural mechanism of how proteins can self-assemble is currently unknown. Our research is focused on functional amyloids as a model for the aggregation process for cytotoxic amyloids. Specifically, two human hormones, cholecystokinin (CCK) and gastrin, that share the C-terminal sequence: WMDF. This is the minimal sequence needed for biological activity and has demonstrated the ability to convert between amyloid and crystalline structures by varying pH levels. In the body CCK and gastrin exist as varying short peptide fragments of the full length hormone. The short fragments are easily synthesized; thus, they are excellent starting points for studying amyloid reversibility. 2D infrared spectroscopy, using ultrafast lasers, has the temporal resolution necessary to observe real time structural changes, and when implemented with isotope-labeled samples can monitor protein structure with single-residue resolution. This can be used to determine a precise aggregation mechanism. Understanding the structural mechanistic properties of functional amyloids can provide insight on disease-linked amyloids and future therapeutics or reversal of diseases associated with amyloid fibrils.

Gabriel D'Agistino

Mentor: Brian Bachmann
Home Institution: University of South Carolina, Columbia
Research Abstract:
Microbial natural products are diverse molecules that perform many biologically important tasks. Many products are thought to provide a survival advantage by limiting competitor growth; additionally, over 80% of current anti-infective agents are based on natural product scaffolds. Nevertheless, new natural products are urgently needed due to the emergence of diseases such as multi-drug resistant bacteria. Genomic sequencing of select microorganisms has revealed multiple biosynthetic gene clusters capable of synthesizing diverse natural products. However, under standard laboratory conditions most encoded natural products are not synthesized and are thus unknown and considered unknown. Understanding ways to activate these biosynthetic gene clusters is a promising approach to isolate novel natural products. The primary goal of our study is to identify conditions that stimulate the production of natural products in an underexplored genera of microorganisms.

Myxobacteria is one such underexplored genus. With over 40 new products discovered in the last 8 years and some entering clinical trials for therapeutic use, they serve as a potential reservoir for new products. We use the model myxobacterium Myxococcus xanthus DK1622. DK1622 encodes 23 biosynthetic gene clusters but only 13 have known products. Previous studies in non-myxobacteria found that sub-lethal antibiotics or rare earth metals stimulate natural product synthesis. Here, we address how these stimuli affect natural product production in DK1622. We isolate natural products in the presence and absence of putative stimuli using liquid chromatography-mass spectrometry (LC-MS) and comparative metabolomics methodologies. The study serves as a basis for understanding how stimuli affect natural product production in mxyobacteria.

Lauren Harris

Mentor: Sandra Rosenthal
Home Institution: University of Maryland, Baltimore County
Research Abstract:
When dopamine levels are unregulated, there can be a drastic impact on the human mind and body. The dopamine transporter protein (DAT) regulates the amount of dopamine in a neuronal synapse by reuptake, which facilitates effective communication between neurons. Recently, the dopamine transporter protein A559V variant was discovered in patients diagnosed with disorders such as autism spectrum disorder (ASD), attention-deficit/hyperactive disorder (ADHD), and bipolar disorder. In this effort, we aim to investigate the internalization dynamics of the A559V variant protein. In order to study internalization, HEK293 cells are transiently transfected with two coding variant plasmids- one for the A559V variant and one for the wild type. When the cells express their respective dopamine transporter, the DAT proteins are tagged with DAT specific ligand-conjugated quantum dots. 3D single quantum-dot tracking experiments will be carried out by spinning disk confocal microscopy, which allows us to view the individual transporter proteins in living cells. We hypothesize that the internalization dynamics of the A559V variant protein will distinctly differ from those of the wild-type, which may indicate a correlation between internalization and dysfunction. This information could subsequently be implemented in quantitative diagnosis for behavior disorders.

Hyungyu Lee

Mentor: Jeffrey Johnston
Home Institution: Bowdoin College
Research Abstract:
The use of carboxylic acids is ubiquitous in nature and synthetic chemistry due to their symmetry and their ability to hydrogen bond. Carboxylic acids have two oxygen atoms with equal reactivity but there is not yet an established catalytic mode to differentiate between carboxylate oxygens. In nature, one mechanism of activation of a carbamic acid by dethiobiotin synthetase proceeds via oxygen atom differentiation. This is achieved by hydrogen bond donation from an amide N-H and water molecule to one oxygen, and ionic hydrogen bonding from a lysine residue to the other. This activation mode is hypothesized to cause selective phosphorylation of one oxygen atom over the other.  The Johnston group hypothesized that a similar three-point binding strategy could be employed to generate a chiral carboxylate with oxygen atoms differentiated. For high enantioselectivity to be achieved, two parameters must be accounted by the catalyst: the conformation of the central C-C bond and the nucleophilicity of each carboxylate oxygen. The Johnston group was able to optimize the organocatalyst through the enantioselective iodolactonization of cyclopentene carboxylic acid substrate. We are trying to extend this result to cyclohexadiene carboxylic acid substrate. While synthesizing the organocatalyst in two steps, different cyclohexadiene carboxylic acid substrates are synthesized through the Birch Reduction of benzoic acid. Catalytic iodolactonization of these substrates is achieved in subsequent reactions. Characterizing this enantioselective iodolactionization methodology could ultimately provide novel approaches towards synthetic chemistry and the derivatization of bicyclic lactone products could lead to new approaches to carbocyclic nucleoside based drugs.

Emily Lilla

Cardiac disease is the leading cause of death in the United States, and the current methods of treatment on the market are known to cause excess and sometimes fatal bleeding, such as Warfarin and Plavix. The Protease-Activated Receptors subtype 1 and 4, located on platelets, are responsible for the activation of clotting, the body’s response to excess bleeding. Targeting Protease-Activated Receptor-4 (PAR4) exclusively could allow for clotting while preventing thrombosis, or the formation of a blood clot. Because the protein crystal structure is not available, we use a homology modeling method in order to transition from a competitive molecular series to a noncompetitive molecular series. We intend to develop a series of molecules that have improved target binding to the PAR4 while maintaining potency and pharmacokinetic properties. We aim to use the homology model to drive structure activity relationship (SAR) with various organic chemistry methods, in order to develop a noncompetitive antagonist for PAR4. In developing this anti-platelet series of molecules the hope is that patients will no longer have to struggle with the negative side effects of the current therapeutics.

Claire Mammoser

Mentor: David Wright
Home Institution: Valparaiso University
Research Abstract:
Schistosomiasis is a water-borne parasite disease found in tropical regions, especially those with diminished access to clean water. Symptoms, including abdominal pain and chronic diarrhea, affect the ability of infected people to work or learn. Traditionally, schistosomiasis is diagnosed by a fecal smear, but this method requires trained personnel and an advanced infection. To overcome these challenges, a lateral flow assay has been developed which tests for Circulating Anodic Antigen (CAA), a negatively-charged glycoprotein produced by the parasites. This assay is easy to use and has high sensitivity, but concentrating CAA in the urine samples used, would further improve it. Our objective was to use positively-charged dendrimers to concentrate CAA onto magnetic beads, which could then be deposited onto the assay. Using an ELISA, an antibody-based quantification method, we determined the most effective conditions for capture and elution of CAA from the beads. We found that larger (generation 4-6) dendrimers provided the best capture of CAA, at >80%. We have also determined that CAA capture stays constant across changes in volume when using 10 μL of functionalized beads, allowing us to use larger urine samples for CAA concentration. Following this optimization, we will incorporate our magnetic beads into a rapid assay, significantly increasing sensitivity for diagnosing schistosomiasis.

Edgar Marroquin

Mentor: Michael Stone
Home Institution: University of Texas, Austin
Research Abstract:
TBA

Audrey Thomas

Mentor: Eric Skaar and Borden Lacy
Home Institution: Kalamazoo College
Research Abstract:
Clostridium difficile infections in the colon cause severe gastritis and diarrhea, with pathology stemming from two secreted toxic proteins, TcdA and TcdB. With C. difficile's already high transmission and infection rates on the rise in the United States, hospitals are desperate for more effective treatments. Preliminary data show that C. difficile TcdA/TcdB knockout strains still produce proteins that cause toxicity in gut epithelial cells. Finding the structure of these proteins, in particular those with no known function or homologs, would allow for a deeper understanding of significant components that may play roles in C. difficile infections. Amino acid sequences of selected knockout-strain proteins were analyzed through bioinformatics tools to identify promising crystallization targets. Recombinant expression of these proteins was tested in E. coli.; those with good expression and solubility underwent large-scale purification. Once high-purity protein samples are obtained, broad crystallization screens will be conducted to test optimal crystallization conditions for subsequent x-ray crystallography analysis. By conducting large proteomic screens and purification, C. difficile targets of interest can quickly be chosen for crystallization later on. This pipeline method will help define structures of unknown proteins and characterize their roles within the infection pathways of C. difficile and other harmful bacteria, possibly leading to future drug targets.

Erin Uhleski

Healthcare-associated infections cause significant morbidity and mortality in the United States. The bacteria Staphylococcus aureus and Acinetobacter baumannii are both considered serious threats by the CDC due to multi-drug resistance, making any infection they cause difficult to remedy. These pathogens often infect immunosuppressed hospital patients which can result in pneumonia or bloodstream infections. Subsequently, it is vital that these organisms are studied to discover alternative treatments for resistant infections. By structurally analyzing proteins from these bacteria that may have a role in survival mechanisms, it is possible to establish important regions for protein function. To select our protein targets, we used multiple bioinformatic tools to analyze protein characteristics that could influence expression and crystallization. Initial recombinant protein expression trials identified two promising candidates for crystallization, HemA from S. aureus and HutC from A. baumannii. We have mutated several residues near the N-terminus of HemA, an enzyme involved in the heme biosynthesis pathway, to improve its stability. Additionally, we have optimized expression and purification of the transcriptional regulator HutC.  Examining these two proteins in broad crystallization screens will reveal favorable conditions for crystal formation. Ultimately the information gathered from these structures will inform functional studies that may uncover downstream effects that these proteins have within the cell. Such knowledge could give us a better understanding of their function and may reveal new targets for the next generation of antimicrobials.