Louesa Akin Title: "Improving Protein Structure Prediction by Incorporating Sparse NMR Restraints" Mentor: Jens Meiler Department: Chemistry Home Institution: Centre College
Research Abstract: Computational modeling can provide a way for structural biologists to predict protein structures when traditional experimental methods alone, such as X-ray crystallography and NMR, fail. A template-free, de novo protein structure prediction algorithm is currently under development within the Biochemical Library (BCL) suite of applications. This application, BCL::Fold, is capable of predicting protein structures for both soluble and membrane proteins with or without experimental restraints. While the current BCL::Fold algorithm can incorporate cryo-EM and EPR restraints, it cannot yet incorporate NMR restraints, which is the focus of this project. In order to incorporate these restraints into the current BCL::Fold framework, separate scoring functions for NOEs, a distance restraint, and RDCs, an orientational restraint, were introduced to evaluate predicted models versus the experimentally determined restraints. The effect of utilizing NMR restraints on the accuracy of the BCL::Fold algorithm was then assessed by predicting the structures of a set of benchmark proteins with and without restraints. More native-like structures (as determined by having an RMSD100 of less than eight angstroms) were generated when using NOE restraints and RDC restraints. This suggests that BCL::Fold will be a useful tool for predicting protein structures when NMR restraints are too sparse for traditional structure determination methods.
Timothy Boswell Title: "Quantifying PGE-M Levels in Urine by MALDI-MS" Mentor: Lawrence Marnett Department: Biochemistry Home Institution: Grove City College
Research Abstract: A number of cancers, including non-small cell lung cancer and colorectal carcinoma, demonstrate up-regulation of the cyclooxygenase-2 (COX-2) enzyme. Prostaglandin E2 (PGE2), a product of COX-2 catalysis, appears at elevated levels in cells of these cancers where it contributes to a number of oncogenic effects including immune evasion, angiogenesis, and proliferation. The major metabolite of PGE2, 11α-hydroxy-9,15-dioxo-2,3,4,5-tetranor-prostane-1,20-dioic acid (PGE-M), is eliminated in urine. Current studies, namely those concerning COX-2 inhibition in cancer treatment, require accurate quantification of PGE-M in urine samples. Current liquid chromatography coupled with mass spectrometry methods require time-consuming chromatographic separation and sequential analysis. We have been developing a method based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) which promises greater efficiency and speed of analysis. MALDI-MS also has a higher tolerance for impure samples and can easily be adapted to a high-throughput platform for parallel analysis. Analysis of PGE-M requires derivatization in order to gain properties that make ionization and detection by MALDI-MS favorable. We have tested various hydrazine and hydroxylamine reagents that derivatize ketones to hydrazones and oximes, respectively. Reagents tested included Girard’s T reagent, Girard’s P reagent, hydroxylamine-o-sulfonic acid, and Lucifer Yellow with favorable results from both Girard’s reagents. We have been seeking to optimize sample preparation, derivatization, and MS analysis for the detection of small concentrations of PGE-M in urine so that future studies may employ our method.
Zeljka Anna Burchill Title: Metabolomic Analysis of the Immunomodulatory Effects of Tropane Alkaloids" Mentor: John McLean Department: Chemistry Home Institution: Edinboro University of Pennsylvania
Research Abstract: Cocaine is a crystalline tropane alkaloid derived from the leaves of the coca plant. This commonly abused drug has been correlated with an increased susceptibility to various infectious diseases including HIV. While the specifics remain to be discovered, cocaine, in addition to its modulation of the central nervous system, has been shown to act directly on immune cells and indirectly by covalent modification of serum proteins. Other tropane alkaloids derived from the plant family solanaceae (such as scopolamine, hyoscyamine and atropine) are known to be competitive antagonists for muscarinic acetylcholine receptors found on numerous cell types including immune cells. In this research we focus on in vitro T-cell metabolic response to the four aforementioned compounds using nanoflow ultra performance liquid chromatography-nanoelectrospray ionization-ion-mobility-mass spectrometry (nUPLC-nESI-IM-MS) in an attempt to discover perturbations that are general to tropane alkaloid exposure, as well as chemical markers specific to cocaine exposure. Multivariate statistical analysis is used to focus on both common features that are indicative of the overlap in T-cell response to the four tropane alkaloids, as well unique features that are specific to the differential response. With this information one can begin to understand the T-cell response specific to each tropane alkaloid as well as the similarities in the response, allowing for greater appreciation of the deficits cocaine abuse may cause the immune system.
Kayla Fasano Title: "Optimization of a Novel Rapid Diagnostic Platform for the Purification and Detection of the P. falciparumMalarial Protein HRPII" Mentor: David Wright Department: Chemistry Home Institution: University of Massachusetts, Amherst
Research Abstract: Malaria is a life-threatening disease caused by four different species of the Plasmodium parasite, of whichPlasmodium falciparum is the most deadly. The disease is transmitted through the bite of an infected Anophelesmosquito, and the World Health Organization reports that approximately half of the world’s population is at risk for the disease. Rapid diagnostic tests (RDTs) have been developed to test for the presence of P.f. parasites by detection of the P.f. specific protein, Histidine Rich Protein II (HRPII). However, these RDTs can often give false positive or false negative results, greatly hindering the ability to properly and efficiently treat malarial patients. A self-contained extraction device, called the extractionator, has been developed to isolate HRPII using magnetic Co-NTA Dynabeads, which bind to the imidazole groups of the histidine residues in HRPII. The extractionator contains sample, wash, and elution chambers (separated by air or oil valves) within Tygon tubing. There are a variety of variables to take into account in order to optimize the performance of the device. Blood and plasma are rich in Histidine Rich Glycoprotein (HRG), which is capable of competing with HRPII for binding to the magnetic beads. Varying concentrations of imidazole in wash chambers were tested to determine the appropriate concentration needed to block HRG binding while still allowing HRPII to bind. Other variables such as changing the valve composition between chambers and the amount of magnetic beads to add to the samples were experimented with in order to enhance purity and yield. Also, an experiment using Ni-NTA conjugated Horseradish Peroxidase (HRPx) and recombinant HRPII was conducted to determine whether HRPx could be used to create the diagnostic aspect of the extractionator by generating a colorimetric reaction in the presence of HRPII. The optimization of the extractionator can lead to a sensitive, low-cost, easily operated RDT for malaria.
Matthew Fritz Title: "Determination of Three Dimensional Molecular Structures of Garcinia Acid Derivatives Using Chiroptical Spectroscopic Methods" Mentor: Prasad Polavarapu Department: Chemistry Home Institution: Shippensburg University
Research Abstract: Electronic circular dichroism (ECD), optical rotatory dispersion (ORD), and vibrational circular dichroism (VCD) spectra of two garcinia acid derivatives have been measured and analyzed in combination with quantum chemical calculations at multiple levels of theory. The absolute configurations of both methyl (2R)-2-[(3S)-1-benzyl-3-hydroxy-2,5-dioxo-pyrrolidin-3-yl]-2-hydroxy-acetate (GTMBA) and [(3aS,6aS)-5-benzyl-2,4,6-trioxo-3,6a-dihydrofuro[2,3-c]pyrrol-3a-yl] methyl carbonate (GAABA) are already noted. The objective of this investigation is to determine the predominant conformations of these compounds. Using the quantum chemical predictions of chiroptical spectra, GAABA was found to have two predominant conformations. The population weighted predicted chiroptical spectra are in good agreement with the corresponding experimental spectra indicating that the identified conformations are correct. The same calculation methods revealed GTMBA to have four conformations. The predicted population weighted ECD and VCD spectra for GTMBA are in good agreement with the corresponding experimental spectra, The same is not true for ORD spectra and the reasons for this discrepancy are to be resolved in the future.
Marilyn Holt Title: "Identification of the Zinc and Manganese Binding Sites in Calprotectin and Their Role in Host Defense against Staphylococcusaureus" Mentor: Walter Chazin and Eric Skaar Department: Chemistry and Microbiology Home Institution: Houghton College
Research Abstract: The neutrophil protein calprotectin is an important factor in vertebrate innate immune response to infection, comprising 40-50% of the protein component of the neutrophil cytoplasm. During infection, calprotectin inhibits bacterial and fungal growth via the sequestration of the essential nutrient metals manganese and zinc, reaching concentrations of 1 mg/ml in infected tissues. Calprotectin is an S100A8/S100A9 heterodimer with two distinct transition metal binding sites. Site I is composed of four histidine residues, and Site II is composed of one aspartate and three histidine residues. Zinc and manganese bind to calprotectin with high affinity in 2:1 and 1:1 metal to protein ratios, respectively, and calprotectin is necessary for manganese sequestration in vivo. To fully elucidate the relationship between metal chelation and bacterial growth suppression, the structural basis for transition metal chelation by calprotectin was examined and correlated with the effect of calprotectin on S. aureus metabolism. Site-directed mutagenesis was employed to create two variants of calprotectin, each with a single disrupted metal-binding site. Isothermal titration calorimetry (ITC) analysis revealed that, while both sites bind to zinc, only Site I binds manganese with high affinity. The Site I and Site II variants were also tested for anti-staphylococcal activity. Disruption of either Site I or Site II increased the IC50 against S. aureus by twofold as compared to wild type calprotectin. The Site II variant exhibited increased antimicrobial activity as compared to that with the Site I variant, revealing a critical role for manganese sequestration in this protein’s antimicrobial activity. These results indicate that combined zinc and manganese chelation significantly enhances the antimicrobial activity of calprotectin. This data also shows that the four histidine site is capable of binding both manganese and zinc, but the three histidine, one aspartate site is not. These results provide a basis for the development of novel therapeutics utilizing nutrient metal chelation.
Ismail Kassim Title: "Analysis of Jurkat Cell Immune Response to Chemical Biological Warfare Agents" Mentor: John McLean Department: Chemistry Home Institution: North Carolina State University
Research Abstract: Chemical Biological Warfare Agents (CBW) represent a significant threat to the modern world as they are able to cause biological damage in small dose. The success of treatment after CBW toxin exposure depends on the ability to quickly confirm that an individual is exposed as well as being able to identify the specific toxin used. In this study, we are interested in characterizing the chemical response of cells exposed to a variety of CBW toxins, with the goal of establishing a chemical profile that can be used to identify the toxin. Thus, we hope to be able to determine what toxin a person is infected with by the metabolic chemical traces left in the immune system as well as the change in motion of immune response cells as a result of changes in their environment. Three common CBW toxins examined in this experiment include: Staphylococcal Enterotoxin A (SEA), T-2, and Aflotoxin. This project utilizes Jurkat cells—a model system for activation of T-cells—to better understand the cellular immune response to toxins. To test the immune response incited by T-2, SEA, and Aflotoxin, cell mobility and exposure experiments were conducted. Initial results indicate the Jurkat cells do elicit a physical response to toxin exposure and mass spectrometry analysis shows chemical signatures which are likely toxin-specific. Through study of these cells, we can better understand how toxins metabolize in the body. Further studies include the study of the toxins Anthrax, Ricin, Tetrodoxin and Staphylococcal Enterotoxin B (SEB) by the techniques discussed above as well as characterization of their chemical profiles.
Carl Sedgeman Title: "Optimization of Nanosponge Delivery System to Further Control Drug Release Profiles, Increase Loading Capacities and Solubility" Mentor: Eva Harth Department: Chemistry Home Institution: University of North Dakota
Research Abstract: Today’s technology to treat cancer includes treating the body with drugs using small molecule delivery. However these techniques have many challenges, such as low bioavailability, nonspecific delivery, inconsistent drug release profiles, and high toxicity with low solubility. In order to improve such deliveries, the development of novel chemotherapeutic drug delivery systems using nanoparticles has been made to address these issues. These nanoparticle delivery systems are made to be water soluble, non-toxic, can target specific tumor sites within the body, and can be made to slowly release the encapsulated drug over a long period of time. The nanoparticles are formed through the cross linking of linear polyester polymers. Various polymers have been used for the formation of the highly functional nanoparticles, such as poly (valeractone-allyvalerolactone). The nanoparticles are then modified with the targeting peptides and loaded with chemotherapeutics.
John Tellis Title: "Application of the Enantioselective Aza-Henry Reaction to the Synthesis of Unsymmetrical Nutlin Derivatives" Mentor: Jeff Johnston Department: Chemistry Home Institution: Elizabethtown College
Research Abstract: The aza-Henry reaction of aryl aldimines with aryl nitroalkanes provides an efficient means for the formation of masked stilbene diamines that can serve as the building blocks for the synthesis of biologically active compounds. Use of a chiral Pyrrolidine Bis(AMidine) (PBAM) proton catalyst in these reactions affords these 1,2-diamine precursors with high diastereo- and enantioselectivity. One such chiral vicinal diamine of medicinal importance is the Nutlin class of cis-imidazolines. These small molecules have shown potent anticancer activity through the orthosteric inhibition of MDMX/p53 interactions, inducing apoptosis in tumor cells. Presented here are the syntheses of two different Nutlin derivatives utilizing the enantioselective aza-Henry reaction. The versatility of this route to the synthesis of unsymmetrical Nutlin derivatives is demonstrated by the use of two separate synthetic schemes arriving at similar derivatives, but originating from dissimilar starting materials.
Amy Tresenrider Title: "Synthesis of Bacillus anthracis HssRS and BAS1816-17 activator library" Mentor: Gary Sulikowski and Eric Skaar Department: Chemistry and Microbiology Home Institution: Claremont McKenna College
Research Abstract: The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax infection. During anthrax pathogenesis, Bacillus anthracis obtains iron from host heme. However, at high concentrations, heme can be toxic. The Bacillus anthracis two component heme-sensing system HssRS activates transcription of hrtAB which encodes for a transporter responsible for alleviating heme toxicity. As such, a high throughput screen in S. aureus identified 1 as a small molecule activator of HssRS. In Bacillus anthracis, 1 activates hrtAB transcription through both HssRS dependant and independent pathways. This is due to the activation of a newly identified two component system BAS1816-17 by 1. To begin identifying structural determinants that contribute to the activity of 1, a compound library was synthesized by reacting various anilines with 9H-fluorene-2,7-disulfonyl dichloride to form sulfonamides similar to 1. The products were tested using a reporter assay that measures the activation of HssRS and BAS1816-17. Results from the assay reveal characteristics important in development of a probe targeting HssRS and BAS1816-17.
Wayne Tse Title: "Synthesis of a Clickable Probe for Electrophiles Formed in Sterol Biosynthesis" Mentor: Ned Porter Department: Chemistry Home Institution: Case Western Reserve University
Research Abstract: We report advances in the synthesis of a clickable probe to further the understanding of the pathogenesis of Smith-Lemili-Optiz Syndrome (SLOS). SLOS is a genetic disorder that causes an error in the last step of the cholesterol biosynthesis pathway, the reduction of 7-dehydrocholesterol (7DHC) to cholesterol. A mutation in the 3β-hydroxysterol-Δ7 reductase (DHCR7) gene leads to a deficiency in functional DHCR7. This causes the buildup of 7DHC, a compound that is prone to form toxic oxysterols upon reaction with oxygen. Studies suggests that these 7-DHC derived oxysterols, many of which are electrophiles, are present in tissues and fluids of SLOS patients. Nucleophilic amino acid residues of proteins are likely to react with these 7-DHC oxysterols by Michael addition, resulting in covalent protein modifications. The probe that is the target of our synthesis resembles 7DHC except for carbons 25 and 26. An alkyne will be placed on the terminal carbons of the ligand chain, allowing the prototypical azide-alkyne cycloaddition click reaction to occur. With the development of a clickable probe, the modified proteins can be isolated for analysis. This synthesis would develop an effective probe to identify the affected proteins of improper cholesterol metabolism.
Zollie White Title: "Comparative Analysis of Machine Learning Techniques for the Prediction of DMPK Parameters" Mentor: Jens Meiler Department: Chemistry Home Institution: Morehouse College
Research Abstract: Several machine-learning techniques were evaluated for the prediction of intrinsic hepatic clearance (CLint) and plasma protein binding. The algorithms used include artificial neural networks (ANN), support vector regression (SVR), kappa nearest neighbor (k-NN), Kohonen networks, and decision trees. Molecules were described using optimized feature sets derived from a series of scalar, two- and three-dimensional descriptors including 2-D, and 3-D autocorrelation, and radial distribution function. Feature optimization was performed as sequential forward feature selection (SFFS). CLint refers to the ability of the liver to remove a compound from the blood in the absence of other confounding factors such as blood flow and protein binding. It is a function of enzyme activity or biliary excretion. Fu, the fraction unbound of a compound is a metric of that compound's efficiency, which correlates to the degree to which it binds to the proteins within blood plasma. The data set for CLint contains over 750 experimentally determined values while that of fu contains over 600 experimentally determined values. Both data sets were collected from the Vanderbilt Program in Drug Discovery DMPK division. The final models were 10-fold cross-validated. These fully in silico models will be useful in guiding initial stages of drug discovery, such as virtual library screening and analogue prioritization prior to synthesis and biological testing.
Tycho Mevissen Title: "Investigations into CHIP Oligomerization Requirements for Ubiquitination Activity" Mentor: Walter Chazin Department: Chemistry Home Institution: Freie Universitaet Berlin
Research Abstract: CHIP, carboxy-terminus of Hsc70-interacting protein, is a U-box containing E3 ubiquitin ligase that catalyzes the coordinated transfer of ubiquitin from E2 ubiquitin conjugating enzymes to cytosolic protein substrates. In many cases, this post-translational modification leads to proteasomal degradation of the substrate. CHIP also interacts with several chaperone proteins and thus is critically important for cellular protein homeostasis. The U-box domain is structurally conserved across a group of E3 ligases and across many species. This domain is the site of homodimerization for CHIP and is instrumental for recognition of and interaction with E2 enzymes. Other U-box E3 ligases, and the structurally homologous RING E3 ligases, have been shown to oligomerize through an interface similar to the CHIP dimer interface, suggesting that this is key to the mechanism of ubiquitin transfer. However, it was recently shown that the U-box containing E3 ligase E4B functions as a monomer.
What is the role for oligomerization in the function of E3 ubiquitin ligases? To address this question we first compared the structures of CHIP and E4B U-box domains. Interestingly, a hydrophobic pocket is present at the CHIP dimer interface, whereas E4B contains polar and charged amino acids at homologous sites. Using site-directed mutagenesis, the dimer interface of the CHIP U-box was mutated to resemble the corresponding region of E4B. Through the application of biophysical methods including multi-angle and dynamic light scattering (MALS) and nuclear magnetic resonance (NMR) spectroscopy, as well as biochemical in vitro ubiquitination assays, we structurally und functionally characterize the perturbed CHIP dimer interface. We demonstrate that while the mutant protein is monomeric, it is not uniquely folded or active in autoubiquitination. These investigations provide a foundation for future studies of the molecular basis for the function of U-box containing E3 ubiquitin ligases.
Christian Wallen Title: "Parallel Synthesis of Indomethacin Analogues" Mentor: Lawrence Marnett Department: Biochemistry Home Institution: Union University
Research Abstract: Indomethacin is a non-steroidal anti-inflammatory drug (NSAID) that is a potent cyclooxygenase (COX) inhibitor. It promotes anti-inflammatory and analgesic activity, but also contributes to gastrointestinal toxicity. Indomethacin and some of its derivatives have also been shown to have interesting and potentially useful off-target effects. Of these indomethacin derivatives, any which do not inhibit COX have greater potential to be useful drugs because of their presumably lower gastrointestinal toxicity. A parallel synthesis approach has been taken to produce a series of indomethacin analogues for the purpose of further investigating these off-target effects and gaining better insight into the COX-independent biology of these NSAIDs. This synthetic approach used a straightforward and flexible reaction sequence to form a number of differently substituted analogs of indomethacin. Various keto-acids or their respective esters were cyclized with a fluorinated phenyl-hydrazine hydrochloride in a Fischer-indole synthesis to form the indole intermediates in an initial step. Assorted acyl substituents were then introduced at the indole nitrogen atoms to form indole alkyl acid-esters. Hydrolysis of the esters was achieved in the last step to obtain the free acids of the compound, which will be evaluated in different biochemical and biological assays. State-of-the-art techniques such as microwave-assisted reactions and automated flash chromatography were utilized to enhance the compound production.
Vanderbilt Summer Undergraduate Research Program, Vanderbilt University, Nashville, TN