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2010 Trainees

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2010 Testimonials

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2010 REU Students

     
 

Mary Boyd
Massachusetts Institute of Technology 

Mentor(s):  Brian Bachmann
Research Project:  "Targeting the P450-mediated methylene oxidation of Apoptolidin A" 

Research Abstract:  Apoptolidin A is a 20-membered macrolactone polyketide that triggers apoptosis in several cancer cell lines. This compound is a natural product of Nocardiopsis sp. FU40, a Japanese soil bacterium. Targeted disruption of a hypothetical P450 gene called ApoP within the Apoptolidin gene cluster suggested that ApoP is responsible for the stereo-selective methylene oxidation in the macrolide ring of Apoptolidin A. The ApoP gene was cloned into vector pET28a(+) for expression as an N-terminal hexahistidine fusion protein, and the resulting plasmid was introduced into E.coli BL21(DE3) as the expression host. Enzyme expression was induced by the addition of IPTG and supplemented by aminolevulinic acid and Fe2+. The over-expressed protein was affinity-purified via a HisTrap FF column on an ÄKTA chromatography system. The purified enzyme was shown to be active in the presence of NADPH+, spinach ferredoxin, and spinach ferredoxin-NADP+-oxidoreductase. Further study into the detailed mechanism of oxidation and structure of this P450 will be pursued. This procedure, in addition to the established knowledge of other key enzymes in the gene cluster, has initiated the biochemical basis for describing and manipulating the biosynthesis of Apoptolidin A for the generation of analogs as potential probes of apoptosis with therapeutic potential.

     
 

Amalchi Castillo-Rodriguez
University of Puerto Rico 

Mentor(s):  Walter Chazin
Research Project:  "Biochemical analysis of the p48 subunit of human DNA primase''

Research Abstract:  DNA Primase is an essential component of the complex multi-protein replisome machinery. They are of the sole polymerases capable of de novo synthesis of RNA primers on the parental DNA strand, which is the required first step for DNA replication. The human DNA primase is a heterodimer composed of the large (p58) and small (p48) subunits, of which the latter contains the RNA polymerase catalytic site. Physical interactions between human DNA primase and replication protein A, a heterotrimeric single-stranded DNA binding protein, are essential to the recruitment of DNA primase to template DNA and initiation of priming. Although direct association between  RPA and p58 has been reported, specific domain-domain interactions between RPA and p49 remain poorly understood. To assess this problem, a combination of proteolysis protection and pull-down assays  were performed. Our results demonstrate direct physical interaction between p49 and the large 70kDa subunit of RPA, with similar binding to both RPA70N and the RPA70AB tandem domains. These findings provide the basis for future ITC and NMR studies to characterize the affinity of and structural mechanisms for DNA primase-RPA interactions.

     
 

Emily Donahue
Western State College of Colorado

Mentor(s):  Prasad Polavarapu
Research Project:  "Absolute configuration and conformational analysis of hibiscus acid derivatives using chiroptical spectroscopic methods"

Research Abstract:  Within drug discovery, compounds like (2S,3R)-tetrahydro-3-hydroxy-5-oxo-2,3-furandicarboxylic acid (hibiscus acid) are vital for progression because they are easily extracted from common plants and have pharmaceutical relevance.  The multitude of possible hibiscus acid conformers and hydrogen bonding interactions between the sample and solvent make computational and experimental measurements costly.  Thus, hibiscus acid dimethyl ester and hibiscus acid disodium salt are the hibiscus acid derivatives that were chosen for study.  Chiroptical spectroscopic methods and quantum mechanical calculations were used to identify the absolute configuration and all possible conformations for these compounds.  Results from these measurements confirm that the absolute configuration of both compounds is (2S,3R).  Comparison between experimental and computational results also reveals that there are four hibiscus acid dimethyl ester conformers that predominate the population, but only one viable hibiscus acid disodium salt conformer.  The inequality in possible conformers for each compound is notable because studying a compound that has fewer viable conformers decreases calculation time and eases the analysis process.  These results indicate a possible trend; examining a salt derivative of a compound is more advantageous than investigating a dimethyl ester compound.  Further testing will need to be completed to qualify this result.

     
 

Austin Eckhoffa
Brown University

Mentor(s):  Ned Porter
Research Project:  "Synthesis of the Pyridinol Antioxidants and their Effect on Oxysterol Levels in Simith-Lemi-Opitz Syndrome"

Research Abstract:  Oxidative stress is thought to play a role in Alzheimer's disease, Parkinson's disease, and Smith-Lemli-Opitz Syndrome (SLOS). SLOS is characterized by abnormally high levels of  7-Dehydrocholesterol (7-DHC), an intermediate in the biosynthesis of cholesterol. Under free radical conditions, 7-DHC oxidizes to form oxysterols which are potentially toxic. Recently, pyridinol antioxidant analogues have proven effective in inhibiting free radical oxidation. The pyridinol antioxidant, 6-hydroxy-3,4-dihydropyrido[2,3-d]pyrimidin-2(1H)-one (HDPO)  has proven to act more effectively than acetaminophen in reducing free radical oxidation of arachidonic acid. Furthermore, in vitro testing has proven HDPO to be nontoxic up to a concentration of 2 mM.  SLOS human fibroblasts treated with HDPO enhanced media displayed decreased levels of 3β-5α-dihydroxycholest-7-ene-6-one, a product of free radical oxidation of 7-DHC, as compared to an untreated control. HDPO itself was synthesized via a bromination, reduction, carbonylation, Boc protection, oxidation, and deprotection.  Because of the success of HDPO in inhibiting 7-DHC oxidation, analogues of HDPO were synthesized in which the amide nitrogens were mono- and di-alkylated.   The effects of alkylated analogues of HDPO will be analyzed through pharmacokinetic and pharacodynamic testing. In order to evaluate these methylated compounds' inhibition of oxidation, the compounds will be introduced into SLOS cells through media and analyzed for  levels of 3β-5α-dihydroxycholest-7-ene-6-one as a marker for oxidation of 7-DHC.

     
 

Aaron Edwards
Centre College

Mentor(s):  Eva Harth
Research Project:  "Evaluation of Polyester ‘Nanosponges’ for Therapy using the Chorioallantoic Membrane (CAM) and Murine Tumor Models with an Emphasis on Tumor Cell and Vascular Targeting"

Research Abstract:  We report the successful systematic preparation of a series of targeted, biodegradable nanoparticles in a variety of distinct nanoscopic size dimensions using an intermolecular chain cross-linking reaction. The particles were characterized by dynamic light scattering (DLS) and nuclear magnetic resonance (NMR) spectroscopy. These three different particles were then labeled with either Alexa Fluor 546 or 647 dyes to give a series of six different particles. To identify the most potent nanocarrier for therapy, we sought to utilize the chorioallantoic membrane (CAM) model as a preclinical evaluation tool, testing the targeting efficiency of the variously sized carriers. In order to assess the gathered data from the CAM model, we compared and validated the findings to similar studies in mammalian mouse models. Ongoing tumor growth delay studies using the targeted particles in single and combination therapies with paclitaxel and camptothecin have shown superior effects in comparison to systemic chemotherapy.

     
 

Laura Hench
Centre College

Mentor(s):  Walter Chazin
Research Project:  "Calcium-induced Conformational Change and the Metal Chelation and Antimicrobial Properties of Calprotectin"

Research Abstract:  Recent work demonstrating that the vertebrate host sequesters zinc and manganese has reinforced the importance of metal sequestration as a defense against bacterial infection. Previously we determined that human calprotectin plays a critical role in defense against Staphylococcus aureus infectionby chelating the essential nutrients zinc and manganese. Calprotectin is a component of the body’s innate immune response to S. aureus infection, making up 50% of the protein constituent of neutrophil cytoplasm. Neutrophils transport the protein to the infection site, resulting in calprotectin concentrations exceeding 1 mg/mL within abscessed tissue. These abscesses have very low concentrations of zinc and manganese, but are rich in calprotectin and calcium. Calprotectin is a heterodimer of EF-hand proteins S100A8 and S100A9, which together have four calcium binding sites. Given the calcium-induced conformational changes in S100 proteins, we hypothesized that calcium may influence the antimicrobial activity of calprotectin. To investigate this hypothesis we examined the effect of calcium availability on the ability of calprotectin to inhibit the growth of S. aureus and Acinetobacter baumannii. Both species are of substantial medical concern due to their increasing antibiotic resistance. Our results indicate that the calcium-induced conformational change in calprotectin increases antimicrobial activity. To examine the contribution of calcium binding to antimicrobial activity and zinc and manganese binding, we are utilizing site-directed mutagenesis to disrupt the ability to bind calcium. Further investigations of the zinc and manganese binding properties and antimicrobial activities of these calcium binding mutants will elucidate the modulatory effect of calcium on the antimicrobial activity of calprotectin.

 
     
 

Gregory Kurkis
Washington and Lee University

Mentor(s):  Gary Sulikowski
Research Project:  "Small molecule activators of the Staphylococcus aureus heme sensing system"

Research Abstract:  Staphylococcus aureus, a common cause of bacterial infection, is becoming increasingly dangerous to human health with the growing emergence of antibiotic resistant strains.  A potential area for new antibiotic development is the S. aureus heme sensing system, as previous research has identified a small molecule that affects heme metabolism and results in less virulent bacteria that are more sensitive to antibiotics.  A small molecule probe is needed to identify the biological target responsible for affecting heme metabolism.  As part of this probe development, the primary objective of our work is to optimize potency of small molecule activators of the S. aureus heme sensing system through analogs based on the previously identified lead compound, C3/‘8882.  Analogs were synthesized from linking selected acid chlorides and ethynyl anisole via a Sonogashira coupling, followed with internal cyclization by hydrazine hydrate and subsequent demethylation via BBr3.  The potencies of the synthesized compounds were then quantified through a XylE assay.  Results from this XylE assay will be obtained in the near future.  Our ultimate goal is for potency of the synthesized analogs to be improved over the original ‘8882 lead compound, preferably at an analog concentration less than 10 µM.  This would allow for probe development to continue through future attachment of a tool or linker, such as biotin, which would result in an effective probe that could be used to identify and pinpoint the location of the biological target responsible for affecting heme metabolism.

     
 

Nichole Lareau
The College of New Jersey

Mentor(s): John McLean
Research Project:  "Exploring Conformation Space for Natural Product Discovery" 

Research Abstract:  The discovery and analysis of natural products presents severalobstaclesdue to their structural complexity i.e. macrocyclic rings, numerous chiral centers, atypical amino acids and other features.  The utilization of structural analysis techniques such as X-ray crystallography and NMR requires the removal of impurities in a time consuming process.  However, exploration of conformation space using ion mobility-mass spectrometry (IM-MS) alleviates several of these analytical limitations.  This report presents the development of a linear peptide database in traveling wave ion mobility conformation space.  In order to produce a large set of peptides, seven proteins were digested with trypsin, chymotrypsin and lys-C.  All data was analyzed by electrospray ionization- and matrix assisted laser desorption/ionization-ion mobility-time of flight mass spectrometry.  The m/z values of linear peptides were identified by PeptideMass characterization tool on the ExPASY Proteomics Server.  The linear peptides of interest produced a trend line in conformation space due to similarity in structure.  Natural products will be compared to the linear peptide trend line in order to determine unique structures in conformation space.  This method offers rapid detection of possible natural product drug candidates based on their structural complexity compared to linear peptides.

     
 

Eleanor Powell
Xavier University (Ohio)

Mentor(s):  Jens Meiler
Research Project:  "Quantitative Structure-Activity Relationship Studies on Inhibitors of 17-beta Hydrosteroid Dehydrogenase Type 10, a Potential Alzheimer's Disease Drug Target"

Research Abstract: 17-beta hydroxysteroid dehydrogenase type 10  (17-β HSD 10)is an enzyme that has been found in elevated concentrations in the hippocampi of Alzheimer’s disease patients. 17-β HSD 10 may be responsible for the degradation of neuroprotective agents such as estradiol and allopregnanolone. Thus inhibition of 17-β HSD 10 has been indicated as a possible mean’s of treating Alzhemers disease.  A confirmatory high throughput screen (AID 893) found 5570 compounds which inhibited the enzyme, as determined by dose-response curves. The data set of inhibitors was clustered via common structure and aligned by root mean square alignment. Comparative molecular field analysis (CoMFA) and comparative molecular similarities indices analysis were performed on four of these clusters. Cluster A contained 19 molecules with q2=0.906 and r2=0.975 using CoMFA and q220.586 and r2=0.744 using CoMSIA. Cluster B contained 26 molecules with q2=0.691 and r2=0.967 using CoMFA and q2=0.618 and r2=0.947 using CoMSIA. Cluster C contained 37 molecules with q2=0.745 and r2=0.999 using CoMFA and q2=0.579 and r2=0.947 using CoMSIA. Cluster D contained 60 molecules with q2=0.510 and r2=0.976 using CoMFA and q2=0.485 and r2=0.864 using CoMSIA. To our knowledge, this is the first 3D - quantitative structure activity relationship analysis (3D-QSAR) on this important Alzheimer's disease target which yields insight into the structural features pertinent to bioactivity.

     
 

Andrew Reidenbach
University of Wisconson, Madison

Mentor(s):  Larry Marnett
Research Project:  "Synthesis of COX-2 Targeted Chemotherapeutic Agents"

Research Abstract:  Cyclooxygenase-2 (COX-2) enzyme is expressed in many malignant tumor types, even in very early stage pre-adenomas. Therapeutic targeting of cancer is still limited by the non-selective accumulation of chemotherapeutic agents in normal tissues. In this regard, we have developed a series of novel COX-2-targeted chemotherapeutic pro-drug conjugates where the nonsteroidal anti-inflammatory drug (NSAID) indomethacin was linked with uracil or 5-fluorouracil via an alkylated phenylene moiety containing amido or ether linkages. SAR-studies will be performed to identify potent and selective COX-2 ligands that can be used as radiosensitizers for radiation oncology.

     
 

Ken Schwieter
Xavier University (Ohio)

Mentor(s):  Jeffrey Johnston
Research Project:  "The Synthesis of Chiral Proton Catalysts: A Multi-gram Scale Preparation of 4Pyrrolidine-Bisamidine and Advances Toward 4Pyrrolidine-azaquinBisamidine"

Research Abstract:  The Johnston group has demonstrated a variety of aza-Henry type additions to N-Boc-arylaldimines and similar electrophiles with high enantioselection using bifunctional Bronsted acid catalysts.  The synthesis of H,4PyrrolidineQuin-BAM (PBAM), the most widely used catalyst in the group, was optimized for large scale preparation.  This included, but was not limited to: the removal of column chromatography, decreasing the reaction times, and limiting the amount of harmful reagents.  In addition, advances were made towards a new bifunctional Bronsted acid catalyst bearing 1,8-naphthyridines instead of the standard quinoline ring systems. 

 
     
 

Eva Weiss
Jacobs University

Mentor(s):  Craig Lindsley 
Research Project:  “Synthesis of Positive Allosteric Modulators of the Metabotropic Glutamate Receptor Subtype 4”

Research Abstract: Metabotropic glutamate receptor 4 (mGluR4) plays an important role in the indirect pathway of basal ganglia circuitry. The basal ganglia circuitry was found to be imbalanced in Parkinson’s patients due to the decline in dopaminergic neurons. Rodent models have shown that the activation of mGluR4 has antiparkinsonian effects making the receptor a viable target for Parkinson’s treatment. High-throughput screening revealed a dihydroquinoline scaffold to be active as positive allosteric modulator (PAM) for mGluR4. In an attempt to study the Structure Activity Relationship (SAR) around the mGluR4 activity of this novel scaffold more than 20 analogues have been synthesized and more than 10 different compounds including diasteriomers were submitted to be tested for mGluR4 PAM activity. They were synthesized by a condensation reaction between an isatoic anhydride, an aldehyde and an aniline in a microwave whereby substituents on all three reagents were varied. Afterwards they were purified by reverse-phase high-performance liquid chromatography which often made it possible to resolve two diasteriomers. Although these compounds were active against mGluR4 in the micromolar range, they showed to be chemically unstable under various conditions. Due to this instability the dihydroquinoline series was abandoned and will not be further investigated as possible drug lead. In addition a novel synthesis of a hydantoin scaffold was investigated. These compounds were successfully synthesized in a one-step procedure in good overall yield which is an improvement over previously published procedures.

     
 

George Yanez
Northern Arizona University

Mentor(s):  Richard Armstrong, Gary Sulikowski
Research Project:  "Progress toward the Total Synthesis of Bacillithiol"

Research Abstract:  The antioxidant bacillithiol influences the fosfomycin resistance of FosA and FosB operations which form a part of RNA protein transcription1. Bacillithiol is a “low molecular weight thiol and antioxidant” that contains the gram positive bacteria formerly known as bacilli2. The synthesis of bacillithiol features a Schmidt glycosylation.

     
 

Grant Zimmerman
University of Illinois Urbana-Champaign

Mentor(s):  David Wright
Research Project: "Functionalized Gold Nanoparticles for Improved Immuno-PCR Detection of Respiratory Syncytial Virus"

Research Abstract:  Respiratory syncytial virus (RSV) is a major cause of hospitalization in American infants. Additionally, it can be fatal in immuno-compromised patients, such as the elderly. No proven vaccine has been developed, and treatment with antivirals would be greatly improved if better methods of virus detection were available. Immuno-polymerase chain reaction (I-PCR) is a tremendously sensitive technique for detecting viral proteins or other antigens in solution. By utilizing the specificity provided by monoclonal antibodies combined with the signal amplification of PCR, proteins can be detected from human serum down to the low picogram per milliliter (Adler, M et alAnalyst. 2008: 133, 702-18.) In the Wright group the I-PCR method has been implemented for RSV detection, utilizing gold nanoparticles as both an antibody and DNA carrier. Magnetic particles were conjugated to specific antibodies complementary to an RSV-antigen to facilitate washing and extraction of the virions from solution. By creating a “sandwich” of the virus between the antibodies conjugated to magnetic particles and gold nanoparticles, unbound solution elements could be washed away, allowing for a quantification of virus present based on the remaining DNA-tagged gold nanoparticles. In addition, gold nanoparticle functionalization was verified using a quartz crystal microbalance.