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2010 Student Research Symposium

When:  Thursday, August 12, 8:00 am to 5:00 pm

Where: Vanderbilt Student Life Center:
Ballrooms A, B, C

Due Dates: 
+  May 15, online registration begins
+  July 1, oral presentation abstracts due
+  July 19, poster abstracts due
+  August 6, last day to register for attendance only

Who Can Attend? VICB lab member faculty, students, and staff.   All presentations will be made by students and postdoctorals.

Guest speaker

  Dr. Craig Crews - Yale University
  "Chemical Genetics: Exploring Cell Biology with Small Molecules"

raffle Prize

Apple iPad Winner:  Jonathan Hempel

cash Prizes
Three $500 cash awards for orals.  One $500 cash award for best poster, plus two $250 and five $100 awards for posters.

Morning Session
Continental Breakfast
8:00 A.M. – 8:45 A.M.

Welcome: Larry Marnett, Director, VICB
8:45 A.M. – 9:00 A.M.

Guest Speaker: Craig Crews, Yale University: “Chemical Genetics: Exploring Cell Biology with Small Molecules"
9:00 A.M. – 10:00 A.M.

Poster Session
10:00 A.M. – 11:30 A.M.

Oral Presentations
11:30 A.M. – 1:00 P.M.

Lunch / Poster Viewing

1:00 P.M. - 2:00 P.M.

Afternoon Session
Oral Presentations
2:00 P.M. – 4:00 P.M.

Student Awards & Raffle Prizes
(must be present to win)
4:00 P.M. – 5:00 P.M.

For additional information, contact the VICB office at 322-0907.



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Oral Presentation Awards:

Kelsey Duggan, “Molecular Basis for Cyclooxygenase Inhibition by the NonSselective Non-Steroidal Anti
                               Inflammatory Drug Naproxen"
Klarissa Hardy, "Cyclopentenone Prostaglandin 15-Deoxy-Δ12,14-Prostaglandin J2 is Metabolized In Vitroand In Viv
                              via Conjugation with Glutathione"
Emily Rubinson, "Crystal Structures of DNA Repair Protein AlkD Reveal a Novel Capture Mechanism for Excision of
                                DNA Damage"

Poster Presentation Awards:

1st Prize:  
Laura Anzaldi, "A Small Molecule Activator of Heme Biosynthesis Triggers the Heme Stress Response of
                           Staphylococcus aureus"

2nd Prize:  
Suraj Adhikary, "Structural basis of DNA damage recognition and repair by Schizosaccharomyces pombe 3
                             methyladenine DNA glycosylase (Mag1)"
Paige Selvy, "Characterization of the Mechanism of Action for Novel Isoform-Selective Small Molecule Inhibitors of
                            Mammalian Phospholipase D"
Carol Bansbach, "Phosphorylation Regulates SMARCAL1 Activity at Stalled Replication Forks"

3rd Prize:
Tyler Davis, "Chiral Proton Catalysis: Guidelines for the Development of More Reactive Bifunctional Catalysts, and
                       the First Enantioselective Synthesis of Chiral cis-4,5-Disubstituted Imidazolines"
Odaine Gordon, "Mechanistic Studies into the Formation of Epoxyketo-linoleic Acid"
Joel Musee, "Determinants of Prostaglandin H Synthase-2-Catalyzed Oxygenation of Endocannabinoid
                       Arachidonoylglycerol and the Development of a Novel Substrate Selective Inhibitor"
Jonathan Hempel, "Progress Towards the Total Synthesis of HMP-Y1 and Hibarimicinone"
Will Proffitt, "Improving the Design of a Chimeric (βα)8-Barrel with Rosetta"


Oral Presentation Abstracts

1) Ralf Mueller, "Novel Schizophrenia Therapeutics by Virtual High-Throughput Screening"
Selective potentiators of glutamate response at metabotropic glutamate receptor subtype 5 (mGluR5) have exciting potential for the development of novel treatment strategies for schizophrenia.  A total of 1,382 compounds with positive allosteric modulation (PAM) of the mGluR5 glutamate response were identified by Alice Rodriguez et al. through high-throughput screening (HTS) of a diverse library of 144,475. EC50 values were determined for all 1,382 hits. The present ChemInformatics project utilized these potency data for training artificial neural network (ANN) quantitative structure-activity relationship (QSAR) models that predict biological potency from the chemical structure. Numerical descriptors of chemical structure were computed as input for the machine learning procedure and optimized in an iterative protocol. The descriptors stem from 35 categories including scalar descriptors, 2D and 3D autocorrelation, radial distribution, and surface-autocorrelation functions. The ANN models achieved theoretical enrichment ratios of up to 38 for an independent data set not employed in training the model.  A database of 450,000 commercially available drug-like compounds was targeted in a virtual screen.  A  set of 824 compounds was obtained for testing based on the highest predicted potency values. Biological testing at Vanderbilt's HTS found 28.2% (232/824) of these compounds with various activities at mGluR5 including 177 pure potentiators and 55 partial agonists. These results represent an enrichment factor of 23 for pure potentiation of the mGluR5 glutamate response and 30 for overall mGluR5 modulation activity when compared with those of the original mGluR5 experimental screening data (0.94% hit rate).

2) Jerry Chang, "Membrane Dynamics and Regulation of Serotonin Transporter Revealed by Single Quantum Dot Tracking"
The serotonin transporter (SERT), plays a central role in terminating serotonergic neurotransmission, is the most well-known therapeutic target for the treatment of depression and anxiety disorders. Conventional biochemical studies indicate that SERTs localize to cholesterol-rich membrane microdomains; however, the underlying mechanisms are unknown. Here we report an innovative quantum dot (Qdot)-based single molecule analysis for real-time dynamic tracking of natively expressed single SERTs in living, neuronal cells. Step displacement-generated in-motion trajectories exhibit two pools of SERTs with distinct populations: slow-diffusive and confined movement. Gentle cholesterol depletion effectively decreased the confined movement population. Further utilizing dual-color imaging of Qdot-labeled SERT and dye-labeled membrane subcompartments, we establish that SERT surface mobility is biphasic, with a subpopulation constrained by membrane microdomains. Stimuli that act throughp38 MAPK-dependent signaling pathways to activate SERTs trigger rapid SERT movements within these microdomains. Our results strongly suggest that membrane raft microdomains modulate SERT regulation. Current studies are underway to see if SERT motilities are altered by genetic variants, such as that linked autism.

3) Will Proffitt, "Improving the Design of a Chimeric (βα)8-Barrel with Rosetta"
In 2008, Tanmay A. M. Bharat and Simone Eisenbeis constructed what should have been a (βα)8-barrel protein from structurally similar fragments of a (βα)8-barrel enzyme from the histidine biosynthesis pathway (HisF) and a protein of the (βα)5-flavodoxin-like fold (CheY) from Thermotoga Maritima. While the original design model predicted a (βα)8-barrel, the X-ray crystal structure revealed a ninth beta strand. Using Rosetta and other computational methods, this project discovered mutations to the CheYHisF chimera that created a more stable protein exhibiting a true (βα)8-barrel fold.

4) Qian Cheng, "An Unusual Cyclization of a Cellular Dipentaenone by Streptomyces coelicolor Cytochrome P450 154A1 without Oxidation-Reduction"
To investigate the physiological role and catalytic function of Streptomyces coelicolor P450 154A1, a combined genetic, biochemical and metabolomics approach was employed. The vability of spores was greatly compromised in P450 154A1 knock-out mutant. A novel dicyclopentaenone was identified as its endogenous substrate and an unexpected intramolecular cyclization was found without the involvement of oxidation-reduction.

5) Emily Rubinson, "Crystal Structures of DNA Repair Protein AlkD Reveal a Novel Capture Mechanism for Excision of DNA Damage," Emily H. Rubinson, Barry Gold, Thomas E. Spratt, and Brandt F. Eichman. DNA glycosylases safeguard the genome by locating and excising chemically modified bases from DNA through a base flipping mechanism, where modified nucleobases are rotated out of the DNA helix and into a complementary shaped active site.  AlkD is a recently discovered bacterial DNA glycosylase specific for positively charged alkylpurine nucleobases 3-methyladenine and 7-methylguanine.  Left alone, these cytotoxic (3mA) and mutagenic (7mG) lesions can have deleterious effects on cellular metabolism and can lead to cancer.  The crystal structure of Bacillus cereus AlkD revealed that the protein represents a new superfamily of DNA repair proteins, and is composed exclusively of helical HEAT-like repeats that form a solenoid perfectly shaped to accommodate a DNA duplex on the concave surface.  Here, we present the crystal structures of AlkD in complex with DNAs containing alkylated, mismatched, and abasic nucleotides.  These structures reveal unexpectedly that AlkD’s tandem HEAT-like repeats form complexes with destabilized base pairs without contacting the modified base.  Rather, AlkD senses the structural destabilization of the lesion through electrostatic interactions with the DNA backbone.  Unlike other glycosylases, AlkD recognizes DNA damage by capturing the damaged base in a solvent-exposed, extrahelical conformation, and not by flipping the lesion inside the active site.  Comparison of AlkD to existing DNA glycosylase structures, along with mutational studies of 7mG excision and DNA binding activities, provides important insight into the requirements for alkylation repair within DNA.  These results demonstrate for the first time how excision of nucleobases may be facilitated by being held captive in a solvent exposed environment.

6) Yuxiang Zheng, "Lipoxygenases, Essential Fatty Acids, and Epidermal Barrier Formation," Yuxiang Zheng, William E. Boeglin and Alan R. Brash, Department of Pharmacology. 12R-LOX and eLOX3 are the only human lipoxygenases that, if mutated to inactive forms, will cause a disease. The disease, autosomal recessive congenitial ichthyosis (ARCI), is characterized by dry, thickened, scaly skin and reflects a defective skin barrier function. Both genetic and biochemical studies suggest that the two enzymes act in tandem in the same pathway. How the two enzymes function in the skin, however, remains to be established.  Linoleate is the most abundant essential fatty acid in the skin. It is mainly esterified to the omega hydroxyl of the very long chain FA in a class of ceramides unique to the skin. For most of these ceramides, linoleic acid will be cleaved off, exposing the omega-hydroxyl which is then covalently attached to the cross-linked protein envelope, a requirement for structural integrity of the skin. We will present a hypothesis that connects lipoxygenases, essential fatty acids and the skin barrier function. We propose that the two lipoxygenases oxygenate the linoleate/ceramides. This oxygenation facilitates hydrolysis of the (oxidized) linoleate moiety and exposes the omega hydroxyl for coupling to proteins. We will show that the linoleate/ceramides are substrates for 12R-LOX, forming specifically the 9R-hydroperoxide, which will be further metabolized by eLOX3 to a specific epoxyalcohol. We will also present evidence for the natural occurrence of these specific products in pig and mouse epidermis and their absence in 12R-LOX-/- mouse epidermis. The data strongly support this new concept on the role of EFA and LOX in skin barrier formation.

7) Klarissa Hardy, "Cyclopentenone Prostaglandin 15-Deoxy-Δ12,14-Prostaglandin J2 is Metabolized In Vitroand In Vivo via Conjugation with Glutathione," Klarissa D. Hardy, Ginger L. Milne, Huiyong Yin, Jason D. Morrow, and L. Jackson Roberts II.  15-deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2)  is a highly reactive cyclopentenone prostaglandin  generated from dehydration of PGD2.  It possesses an α,β-unsaturated carbonyl moiety that can readily adduct cellular thiols, such as glutathione (GSH) and cysteine residues of proteins, via Michael addition.  Due to its reactivity, 15-d-PGJ2 is thought to modulate protein function and exert potent anti-inflammatory, anti-proliferative, and pro-apoptotic activity.  However, despite the large number of publications ascribing bioactivity to 15-d-PGJ2, evidence for its biosynthesis in vivo is lacking.  Here, we report studies to determine to what extent 15-d-PGJ2 is formed in vivo and the mechanisms that regulate its formation.  It has been previously determined that measurement of urinary metabolites is the best method to assess systemic prostaglandin formation in vivo.  To this end, we have undertaken studies to identify the major metabolite of 15-d-PGJ2 in rats and humans to use as a biomarker of its endogenous production.  In rats 15-d-PGJ2 was excreted into urine as a mercapturic acid conjugate (N-acetyl-cysteinyl-15-d-PGJ2).  Additionally, 15-d-PGJ2 was incubated with primary human hepatocytes for 24-hr, and the cell media was subjected to liquid chromatography/tandem mass spectrometry (LC/MS/MS) for metabolite purification and identification.  Twelve metabolites of 15-d-PGJ2 were identified from this analysis; the most abundant metabolites included 9-cysteinyl-sulfoxide-11-hydroxy-12,13-dihydro-15-d-PGJ2 (9-Cys-SO-11-hydroxy-12,13-dihydro-15-d-PGJ2), 9-Cys-SO-11-hydroxy-12,13-dihydro-20-hydroxy-15-d-PGJ2, and their corresponding glucuronide conjugates. These findings indicate that 15-d-PGJ2 is predominately metabolized via conjugation with GSH and is excreted as metabolized GSH-adducts.  It is possible that one or more of these metabolites could be used to assess 15-d-PGJ2 biosynthesis in vivo.

8) Kelsey Duggan, “Molecular Basis for Cyclooxygenase Inhibition by the NonSselective Non-Steroidal Anti-Inflammatory Drug Naproxen,” Kelsey C. Duggan, Matthew J. Walters, Joel Musee, Joel M. Harp, James R. Kiefer, John A. Oates, Lawrence J. Marnett.  Naproxen ((S)-6-methoxy-α-methyl-2-naphthaleneacetic acid) is a powerful non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. The pharmacological effects of NSAIDs, including naproxen, arise from the inhibition of prostaglandin biosynthesis by inhibiting the cyclooxygenase enzymes, COX-1 and COX-2. Naproxen exhibits gastrointestinal toxicity but its cardiovascular toxicity may be reduced compared to other drugs in its class. Despite long-term use in the clinic, the molecular basis for COX inhibition by naproxen is not well defined. We performed a detailed study of naproxen-COX-2 interactions using site-directed mutagenesis, structure-activity analysis and X-ray crystallography. The results indicate that each of the pendant groups of the naphthyl scaffold are essential for COX inhibition and only minimal substitutions are tolerated. Mutation of Trp-387 to Phe significantly reduced inhibition by naproxen, a result that appears unique to this inhibitor. Substitution of S or CH2 for the O atom of the p-methoxy group yielded analogs that were not affected by the W387F substitution and that exhibited increased COX-2 selectivity relative to naproxen. Crystallization and X-ray analysis yielded structures of COX-2 complexed to naproxen and its methylthio analog at 1.73 and 2.20 Å resolution, respectively. These structures were consistent with the binding model predicted from site-directed mutagenesis and structure-activity analysis. The elucidation of critical interactions between naproxen and COX can be used in the specific design of more potent or selective naproxen analogs, which may be useful in dissecting the importance of isoform selectivity in cardiovascular toxicity or in the generation of gastrointestinal-sparing chemopreventive agents.

9) Mert Karakas, "BCL::Fold, A Novel De Novo Protein Structure Prediction Method," Mert Karakaş, Nils Woetzel, Nathan Alexander, Steffen Lindert, Rene Staritzbichler, Jens Meiler.  The objective of this study is to introduce a novel de novo computational protein structure prediction algorithm suitable for large and/or membrane proteins. BCL::Fold assembles proteins from secondary structure elements (SSEs) as determined by secondary structure prediction methods. Initially loop regions and side chain coordinates are omitted and the SSEs are represented as idealized geometries to reduce the conformational search space. The algorithm uses Monte Carlo sampling combined with knowledge-based potentials to guide the search. Another feature of BCL::Fold is the integration of available sparse and low-resolution experimental structural data generated by methods like cryo-Electron Microscopy, NMR-, and EPR-Spectroscopy. BCL::Fold addresses critical limitations of current de novo protein structure prediction methods in their applicability to large proteins, protein complexes, and membrane proteins. BCL::Fold was benchmarked on a set of 54 proteins of lengths ranging from 75 to 300 residues and of varying topologies with <30% sequence similarity.  95% of native SSEs were correctly identified by secondary structure prediction with an average deviation in length of ~1.5 residues. 10,000 models were generated by BCL::Fold for each protein, requiring 2 to 5 minutes on a single CPU per model depending on the length of the sequence. BCL::Fold was able to produce models of comparable accuracies with other de novo method: up to ~3.0Å RMSD for α-helical proteins, ~4.0Å RMSD for α-helices/β-sheet proteins and ~4.5Å RMSD for β-sheet proteins.



Poster Presentation Abstracts
1) Patrick Halvey, "Phenotypic discrimination of mutant adenomatous polyposis coli (APC) expressing cells and APC corrected cells by shotgun proteomics,"  Halvey PJ, Zhang B, Coffey RJ, Slebos RJ and Liebler, DC.
Recently, shotgun proteomic approaches, which incorporate improved peptide separation techniques and enhanced sensitivity, have been used to investigate the molecular events associated with subtle phenotypic variation.  Adenomatous polyposis coli (APC) mutations occur in 60-70% of colorectal cancer cases and play a central role in early adenoma formation.  Here we use shotgun proteomics on a standardized liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform to assess proteomic differences between mutant APC-expressing SW480 cells (nonsense mutation at codon 1338) and APC-corrected SW480 cells. Tryptic peptides from whole cell lysates were separated by isoelectric focusing (IEF) and analyzed by LC-MS/MS on a LTQ mass spectrometer.  Database searches with the resulting MS/MS spectra identified 4930 proteins across all groups (6 replicates per cell type, 4.2% protein FDR).  Of the identified proteins, 93% were observed in both mutant and corrected cell lines, indicating that a relatively small fraction of the proteome is affected by correction of the APC mutation.  We performed hierarchical clustering on differentially expressed proteins, calculating pairwise similarity based on the Pearson’s correlation coefficient and using the average linkage. We found strong proteomic clustering of mutant APC replicates and APC corrected replicates, thus demonstrating the ability of shotgun proteomics to differentiate between subtle biological phenotypes.  We also carried out parallel measurements of transcript levels using microarray analyses.  Combining  proteomic and microarray data in a single clustering analysis showed high concordance of the results obtained by the two orthogonal methods.  As expected, several members of the canonical Wnt signaling pathway were found to be differentially expressed between mutant corrected cells; these include Dickkopf-related protein 4 (DKK4), a known inhibitor of the signaling pathway.  In addition to known APC-related proteins, we identified a set of differentially expressed proteins that had not previously been linked to the Wnt pathway.  These proteins are involved in Ca2+ homeostasis, guanine nucleotide exchange apoptosis, and other functions.  Shotgun proteomics can detect proteomic differences between subtly different phenotypes that differ at a single genetic locus.  In this example, the approach provides new insights into the mechanism of mutant APC-associated tumorigenesis.   

2) Gordon Lemmon, "Small molecule docking applications predict the interactions and binding energy of protein/small molecule complexes."  These predictions can inform mutagenesis studies, filter high-throughput screening libraries for likely hits, or guide de novo drug design. In order to make accurate predictions, correctly modeling the flexibility of both the protein and the small molecule is necessary. Rosetta excels in modeling protein flexibility. We have adapted Rosetta to model ligand flexibility equally well. In addition to backbone and sidechain flexibility, and conformer sampling, RosettaLigand now allows (1) docking multiple ligands simultaneously, and (2) representing ligands as fragments, with fragment rotamers generated from a search of the Cambridge Structural Database. The addition of ligand fragment docking is a starting point for a fragment-based small molecule design application. Using a growing approach, a new Rosetta based protocol excels in designing flexible ligands into flexible proteins.

3) Steffen Lindert, "BCL::EM-Fold: Protein Folding Tool For Medium Resolution Density Maps." Both cryo-electron microscopy (cryoEM) and X-ray crystallography frequently yield medium resolution (6 – 10 Å) density maps of large macromolecular assemblies. Generally no atomic detail is resolved in these density maps, making it impossible to deduce the protein structure from the density map alone. We present a novel computational protein folding algorithm that aids in the interpretation of medium resolution density maps from cryoEM and X-ray crystallography. BCL::EM-Fold folds the amino acid chain de novo into density rods observed for α-helices at sub-nanometer resolution. This is done by incorporating the experimental data as restraints. The placement of helices is restricted to regions where density rods are observed in the density map. BCL::EM-Fold has been benchmarked with ten highly α-helical proteins of known structure that have between 250 and 350 residues. Starting with knowledge of the true secondary structure for these ten proteins, the method can identify the correct topology within the top scoring ten models. With more realistic secondary structure prediction information, the correct topology is found within the top scoring five models for seven of the ten proteins. Subsequent high-resolution refinement for the successful proteins using the density as a restraint in Rosetta creates models with RMSDs as low as 2.5 Å. The algorithm has been used to build an atomic model for large parts of human adenovirus protein IIIa. This protein, for which there is no high resolution structure, is predicted to be highly α-helical. It is resolved in a 6.8 Å resolution cryoEM adenovirus structure as a bundle of 14 α-helical density rods.

4) Rory Pruitt, "Structural analysis of Clostridium difficile Toxin A." The pathogenesis of Clostridium difficile is dependent on the secretion of two large homologous toxins, TcdA and TcdB. TcdA and TcdB action involves four major steps: receptor-mediated endocytosis, translocation of the catalytic domain across the membrane, release of the catalytic domain by autoproteolytic processing, and inactivation of Rho proteins by glucosylation. Four domains are responsible for these steps: receptor-binding, putative pore-forming, autoprotease, and glucosyltransferase domains. Crystal structures of a portion of the TcdA receptor-binding domain, the TcdA autoprotease domain, and the TcdB glucosyltransferase domain exist, but there is no structural information for the putative pore-forming domain or how the four domains interact with each other. We have visualized the native TcdA holotoxin by negative stain electron microscopy, determined its three-dimensional structure by random conical tilt, and mapped the organization of the TcdA functional domains within this structure. We further show that exposure to acidic pH induces a dramatic conformational change in the toxin. These data provide a structural framework for understanding TcdA receptor-binding, pore-formation, and autoprocessing.

5) Elizabeth Dong, "Structural Studies of the Interaction between mGluR5 and Allosteric Modulators."  The metabotropic glutamate receptor subtype 5 (mGluR5), a class C G-protein coupled receptor, is involved in mammalian cognitive function through diverse signaling pathways that modulate synaptic plasticity. Selective modulators of mGluR5 have exciting potential for the development of novel treatment strategies for disorders that disrupt cognitive function such as schizophrenia. Because the orthosteric binding site is well conserved between all subtypes of mGluRs relative to other regions on the receptor, an approach to selectively target mGluR5 is to identify ligands with allosteric binding sites. Identifying specific residues on mGluR5 that contact these allosteric modulators would provide a deeper understanding of the binding interaction and aid in the development of such therapeutic compounds. Comparative structural models of mGluR5 using the three mammalian GPCR crystal structures as templates have been shown to accurately predict critical residues for allosteric modulation. The alignment between mGluR5 and GPCR templates was used to model the 7 transmembrane helices. Side chain and loop optimization was then carried out with Rosetta Membrane. Residues of mGluR5 critical for the binding of allosteric modulators were predicted using Rosetta Ligand docking studies and validated by experimental functional data. Selective targeting of mGluR5 receptors with allosteric modulators can provide a novel approach to the development of therapeutic agents for disorders causing cognitive impairment. The development of a computational comparative structural model of mGluR5 together with ligand docking studies using Rosetta has been combined with experimental validation to allow for a deeper understanding of the residues important to allosteric ligand binding.

6) Will Proffitt, "Improving the Design of a Chimeric (βα)8-Barrel with Rosetta."  In 2008, Tanmay A. M. Bharat and Simone Eisenbeis constructed what should have been a (βα)8-barrel protein from structurally similar fragments of a (βα)8-barrel enzyme from the histidine biosynthesis pathway (HisF) and a protein of the (βα)5-flavodoxin-like fold (CheY) from Thermotoga Maritima. While the original design model predicted a (βα)8-barrel, the X-ray crystal structure revealed a ninth beta strand. Using Rosetta and other computational methods, this project discovered mutations to the CheYHisF chimera that created a more stable protein exhibiting a true (βα)8-barrel fold.

7) Steven Combs, "Characterization of a Computationally Designed Sequence and Fold Symmetric TIM Barrel Enzyme."  The (βα)8 barrel proteins are one of the most common folds seen in nature.  Their structural symmetry is believed to have arisen due to gene duplication events. Gene duplication events are responsible for the propagation and diversity of enzymes that perform biological functions however, an enzyme that is symmetric in both fold and in sequence has not yet been identified.  HisF, a (βα)8 barrel enzyme in the histidine biosynthesis pathway, has an internal two-fold symmetry. We present a computationally designed variant of HisF that is two-fold symmetric in both fold and in sequence that represents the “missing link” in enzyme evolution.  The catalytic activity, stability, and crystal structure are presented in this study.

8) Nathan Alexander, "Spin Label Sructure and Dynamics Determined by Rosetta Rotamer Libraries."  Electron paramagnetic resonance in conjunction with site directed spin labeling can provide structural information for important biological targets unable to be investigated by classic structural biology techniques such as X-ray crystallography and nuclear magnetic resonance. EPR can routinely provide intra-protein distances of 50Ã.  The limitation with EPR is that the spin label projects into an unknown position in space, which creates the need to interpret measured spin label distances relative to the protein backbone. A full atom explicit representation of the methanethiosulfonate spin label (MTSSL) has been introduced into Rosetta as a rotamer library. During full atom structural refinement, this allows comparison between the experimental and model spin label distances at atomic detail. Using singly labeled T4-lysozyme structures available in the protein data bank, Rosetta has been shown to be able to recover experimentally observed spin label conformations. The MTSSL rotamer library also allows Rosetta to accurately recover experimental spin label distances measured in doubly labeled T4-lysozyme.

9) Jing Jin, "A Novel Route to HNE-related Aldehydes: Lipoxygenase-catalyzed Formation via a bis-Allylic Dihydroperoxide." The aldehyde 4-hydroxynonenal (HNE) is a major cytotoxic product of lipid peroxidation. Although physiological actions of HNE have been studied quite well, its mechanism of formation is largely uncharacterized. The major unknown is the mechanism of the carbon chain cleavage reaction leading to the aldehyde fragment. Here, we show that 8R-lipoxygenase (8R-LOX) catalyzes the enzymatic production of an HNE-like enone structure via a bis-allylic dihydroperoxide intermediate. Incubation of arachidonic acid (50 μM) with 8R-LOX (10 μg/ml) formed initially 8R-hydroperoxy-eicosatetraenoic acid (8R-HPETE) which was further converted to a mixture of products including a prominent HPNE-like enone identified as 8-hydroperoxy-11-oxo-undeca-5Z,9E-dienoic acid. To identify the precursor of this aldehyde, we repeated the incubations on ice, and subsequent HPLC analysis revealed a prominent new product with 235 nm absorbance, the bis-allylic 8,11-dihydroperoxy-5,9,12,14-20:4ω6. Re-incubation of this bis-allylic dihydroperoxide with 8R-LOX successfully demonstrated its conversion to the enone products. Reaction was greatly accelerated by co-incubation with NDGA, a reductant of the LOX iron. Apparently, after formation of the bis-allylic dihydroperoxide by the LOX ferric iron, the ferrous form catalyzes homolytic cleavage of the 11-hydroperoxide and the resulting alkoxyl radical undergoes β-scission to give the 4-hydroxy-alkenal product. This study identifies a novel route for HNE formation that could contribute to the production of 4-hydroxy-alkenals in vivo.

10) Paige Selvy, "Characterization of the Mechanism of Action for Novel Isoform-Selective Small Molecule Inhibitors of Mammalian Phospholipase D," Paige E. Selvy, Craig W. Lindsley, Darryl Bornhop, H. Alex Brown.  PosterSessionAbstract: Phospholipase D (PLD) is an evolutionarily conserved phosphodiesterase that catalyzes the hydrolysis of phosphatidylcholine (PC) to generate the lipid second messenger phosphatidic acid (PA). PA lies at the intersection of critical signaling and metabolic pathways and serves as both a node for protein recruitment as well as the precursor to other lipid second messengers. Aberrant PLD activity and expression have been identified in a number of human cancers. As such, it is an attractive therapeutic target. Recently we identified a class of potent and isoform-selective small molecules that block PLD activity. These compounds were rigorously characterized both on purified protein to demonstrate direct inhibition, and in cultured cells to demonstrate in vivo efficaciousness. Current studies seek to define the molecular mechanism of small molecule-induced PLD inhibition in a reconstituted extruded vesicle system. Using backscattering interferometry and in vitro activity assays, changes in kinetic rate constants are measured for purified enzyme in the presence of small molecules and unilamellar lipid vesicles. We propose mammalian PLD specifically and directly binds the isoform-selective small molecule, thereby disrupting protein-lipid interface interaction and consequently inhibiting substrate binding and catalysis. In the absence of a mammalian PLD crystal structure, various full length and truncated PLD protein constructs are assessed to determine the approximate site of small molecule action. Characterization of the mechanism of small molecule-induced inhibition will facilitate future application of these compounds in cellular studies to interrogate the divergent roles for PLD isoforms in receptor-mediated signaling cascades.

11) Liping Du, "Synthesizing Self-Replication for Drug Discovery." We have developed a simplified, purified, bacterial translation system to facilitate studies of substrate recognition in protein synthesis and enable new applications. One envisioned application is directed evolution in vitro of small-molecule, peptidomimetic ligands and drug candidates. Towards this goal, we have redesigned the genetic code for the synthesis and display of polymers containing unnatural amino acids. Effects of individual domain changes in natural and unnatural aminoacyl-tRNAs on translation have been defined to facillitate engineering. Unexpectedly, the rate-limiting step in translation was found to be peptide bond formation, not accommodation, overturning dogma in translation. A long-term goal is synthesizing a minimal cell to better understand biological replication and advance biotechnology. Of the 151 genes we postulate to be necessary for self-replication from small molecules (synthetic life), protein synthesis constitutes 96%. “Biobrick”-based methods have been adapted to construct subsets of the proposed minimal genome useful for creating purified translation systems. Unexpectedly, class II terminators for T7 RNA polymerase were found to function only in vitro, not in vivo. These projects are pushing the boundaries in the new field of synthetic biology.

12) Eddie Ray Watts, "Chemical models of physiological peptide bond formation: importance of sterics versus pKa," Richard E. Watts and Anthony C. Forster.  In collaboration with the Ehrenberg lab, we have recently published the rates of ribosome-catalyzed peptide formation between certain pairs of amino acids. Using formyl-Met-tRNA as the electrophile, the order of nucleophile reactivity was Phe > Ala > Pro > N-methyl-Phe >> N-butyl-Phe, apparently independent of the adaptor tRNA. Because this order correlated roughly with the steric bulk of the amine nucleophile, we hypothesized that peptide bond formation is rate-limiting in these reactions. This is contrary to translation dogma which holds that accommodation is rate-limiting. To test this hypothesis, here we examine similar reactions under aqueous, uncatalyzed conditions. Amino acid amides and amino acids are used to model the nucleophiles, and highly activated formyl-Met-N-Hydroxy-Succinimide ester (fMet-NHS) at 4ºC is used to best model the electrophiles. The order of reactivity is Gly > Phe > Ala > Pro >> N-Me-Gly >> N-Me-Phe, with N-butyl-Phe being undetectable. This order correlated perfectly with that in the ribosome-catalyzed reactions. The results also correlated closely with the nucleophile’s steric bulk but not with its pKa. Only small differences in reactivity were observed between natural, non-N-alkyl amino acids (Ile, Val, Glu, Phe, Leu) as predicted. These data support our hypotheses that peptide bond formation is the rate-limiting step in translation, and that the rates of peptide-bond formation on the ribosome are heavily influenced by the sterics of the amino acid nucleophilic amine.

13) Aleksandra Baranczak, "Progress Toward the Total Synthesis of Dideoxy Lomaiviticinone," Aleksandra Baranczak, Gary A. Sulikowski.  In 2001, He and coworkers reported the isolation of the quinone-type natural product lomaiviticin A (1) from the marine actinomycete bacteria Micromonospora lomaivitiensis. Lomaiviticin A was identified as C2-symmetric glycoside possessing a unique diazobenzo[b]fluorene skeleton and a highly functionalized central core with a significantly hindered β-hydroxyketone unit. Its examination against a 24-cancer cell line panel revealed IC­50 values ranging from 0.007 to 72 nM. Our interest in diazoparaquinone family of antibiotics represented by 1 originated in the effort to prepare inaccessible from nature dimeric (dideoxy lomaiviticinone) and monomeric analogs of lomaiviticin A. The progress toward their syntheses will be presented.

14) Joshua Schwartz, "Malaria Ring Coffee Diagnostics," by Joshua D. Swartz, Joshua R. Trantum, Frederick R. Haselton, and David W. Wright.  In developing countries, infectious diseases are responsible for more than 50% of deaths. Prompt and accurate diagnosis of infectious diseases would lead to more effective treatment, and lower rates of mortality, and would also ultimately reduce the expense of treating patients and the economic burden of the illness in society. In the innovative developing countries (IDCs), it is often difficult to reach populations that are distributed sparsely over rural areas. Health workers dispatched from centralized hospitals or laboratory facilities to serve these areas face challenges such as difficult terrain, intermittent (or lack of) electricity, poorly-equipped facilities, an unskilled workforce, and limited financial resources. The consequences of these limitations are that diagnostic medical technologies common in industrialized regions are either not usable or not affordable. Thus, there is a need for new approaches to low resource diagnostics. We describe the creation of a low-cost diagnostic for the detection of malaria based on the phenomenon that forms coffee ring stains on a kitchen counter. The unique microfluidics present in an evaporating drop of colloidal solution produce a characteristic “coffee ring” stain of small particles visible with the naked eye. We propose a low resource device based on this phenomenon that, in the presence of a droplet of infected blood containing the histidine-rich protein II (HRP II) of P. falciparum, produces a readily visible colorimetric indicator of malarial infection.

15) Lilu Guo, "Phosphatidylethanolamine Modification Contributes to Levuglandin/Isoketal Induced Cytotoxicity,"
Lilu Guo, C. Blake Sullivan, Elena Matafonova, Zhongyi Chen, L. Jackson Roberts II, Venkataraman Amarnath, and Sean S. Davies.  Levuglandins and isoketals are g-ketoaldehyde isomers (gKA) formed by non-enzymatic rearrangement of prostaglandin H2 and H2-isoprostanes, respectively. gKA rapidly adduct to proteins and increases in gKA protein adduct levels occur in several inflammatory conditions. gKA induce cell death in cultured cells, potentially by adducting to cellular proteins. However, gKA also adduct to other primary amines including phosphatidylethanolamine (PE) in vitro. Whether gKA induce their biological activities by modifying PE has not been investigated. This study is to measure levels of gKA-PE in cells and determine whether gKA-PE mediates the endothelial cell death induced by gKA. There is ~40% gKA added to HEK cells bounding to phospholipids and only 8% with proteins. An LC/MS/MS assay was used to measure gKA-PE adducts in gKA treated endothelial cells (EC). gKA-PE added directly to EC dose-dependently induced cytotoxicity (LC50 2.2 μM) and cell adhesion.   Other oxidatively N-modified PEs could also induce cytotoxicity. Thus PE is a major cellular target of gKA and other reactive a,b-unsaturated aldehydes, and N-modified PEs can mediate induced cytotoxicity.

16) Robert Sprung, "Tumorigenic Transcriptional Pathways Revealed in Proteomic Signature of Early Colon Neoplasia," Robert Sprung, Misti Martinez, Robbert Slebos, Dan Liebler.  Proteomic methods offer strategies for the discovery of protein signatures that correlate with a pathological phenotype and can inform the development of diagnostic biomarker assays. To determine the protein signatures that arise early in the development of colon cancer, we applied a standardized shotgun proteomics platform developed in our lab to the analysis of 6 large adenomas of the colon and 6 samples of normal colon tissue from healthy volunteers. More than 4300 protein groups were identified at a protein level FDR of 4.6%. Of these protein groups, 280 were determined to be differentially expressed by at least 2-fold between the adenoma and normal tissue, based on spectral counts. Consistent with current knowledge of colon tumor initiation, we observed increased expression of transcriptional targets of the Wnt signaling pathway in adenoma tissue, including the ephrin receptors EPHB2 and EPHB3. Activation of the Notch signaling pathway is required for colon tumorigenesis and we found the Notch target gene olfactomedin 4 more frequently in adenoma tissue. Olfactomedin 4 has also been suggested as a putative marker for intestinal stem cells. Our data reveal increased expression of transcriptional targets of NFAT in adenomas, suggesting a novel role for NFAT in colon tumorigenesis. In addition, we obtained proteomic evidence for the aberrant presence of Paneth cells specifically in the adenomas. These cells are typically expressed in the small intestine, but have been reported in the context of gastrointestinal neoplasia as well. Finally, we present the first evidence for protein expression from open reading frame 1 of the LINE-1 transposon in human tissue, suggesting a potential role for transposon activation in tumor initiation. These results support existing models of tumorigenic pathways in the colon and suggest additional transcriptional pathways involved early in colon tumorigenesis.

17) Jonathan Hempel, "Progress Towards the Total Synthesis of HMP-Y1 and Hibarimicinone," Jonathan E. Hempel, Gary A. Sulikowski.  In 1998, Hori and co-workers reported the isolation of a family of complex natural products produced by a subspecies of the bacterium Microbispora named the hibarimicins. The family of greater than ten components is characterized by a semi-C2 symmetric conserved aglycon with variation only in the oligosaccharide moieties which decorate the outer rings. The hibarimicins exhibited growth inhibition of oncogene-transformed cells, as well as of myeloid leukemia cells, and selective inhibition of protein tyrosine kinase was observed at 1.2 mM. Due to their intriguing biological activities and relative inaccessibility in nature, we are currently working towards the total synthesis of a biosynthetic intermediate of the hibarimicins, HMP-Y1, and the aglycon termed hibarimicinone. Our progress in this endeavor will be presented.

18) Colleen McGrath, "SAR of Diffusible Lipid Electrophiles Associated with Phospholipid Peroxidation: 4-Hydroxynonenal and 4-Oxononenal Analogs," Colleen E. McGrath, Keri A. Tallman, Ned A. Porter, and Lawrence J. Marnett.  Diffusible and membrane bound lipid electrophiles are known to modify DNA and protein substrates and modulate cellular pathways including ER stress, antioxidant response, DNA damage, heat shock pathways, and apoptosis.  Electrophile-mediated disruption of cell signaling is involved in the pathogenesis of several diseases including atherosclerosis and cancer.  Of particular interest to our laboratory are derivatives of the reactive electrophiles, 4-hydroxynonenal (HNE) and 4-oxononenal (ONE), because of their immuno-modulatory capabilities as they pertain to inflammation and disease progression.  Herein we report on a structure activity relationship (SAR) for several analogs of HNE and ONE.  We examined the effects of electrophile analogs on toxicity in both human colorectal carcinoma (RKO) cells and a human monocytic leukemia cell line (THP-1).  Further analysis was performed in differentiated THP-1 macrophages to assess changes in macrophage activation and pro-inflammatory signaling in response to each analog.  Changes in levels of pro-inflammatory cytokines in response to lipid electrophiles were assayed and it was shown that these analogs inhibit THP-1 macrophage production of the pro-inflammatory cytokines, IL-6, IL-1b, and TNFa after LPS/IFNg activation.  We observed 45-75% inhibition of cytokine production with several analogs at submicromolar concentrations with as little as 30min of exposure.  Macrophage activation was assayed by using fluorescence microscopy to visualize phagocytosis of fluorescent beads by differentiated THP-1 macrophages following lipid electrophile treatment.  HNE and ONE inhibited phagocytosis by 20-40% under similar conditions as those found to inhibit cytokine production.

19) Yu Du, "Characterization of Apoptolidin Gene Cluster and Production of New Analogs," Yu Du, Sean M. Deguire, Dagmara K. Derewacz, Gary A. Sulikowski and Brian O. Bachmann.  Apoptolidin A is a natural product produced by Nocardiopsis sp. that selectively induces apoptosis in several cancer cell lines via a putative mitochondrial target. The compound is a complex polyketide containing a 20 membered macrolide. The polyketide is further oxidized at C16 and C20 positions as well as glycosylated at C9 and C27. The genome of the apoptolidin producer, Nocardiopsis sp. FU40, was sequenced and a 116 kb area containing 38 open reading frames was identified as the apoptolidin biosynthesis gene cluster and was shown to possess a type I polyketide synthase (PKS) at its core. A cosmid library with 3000 members was subsequently created and 7 cosmids that cover the complete cluster were identified by colony hybridization. The identity of this gene cluster was confirmed by analysis of mutants generated by targeted disruption of a PKS gene, a P450 gene and a glycosyl transferase gene. Remarkably, apoptolidin analogs were isolated from these mutants that did not have the C27 sugar as well as various reduced analogs of apoptolidin A. Identification of the gene cluster and characterization of the polyketides produced by mutants has provided valuable insight into the details of the biosynthesis of this therapeutically significant natural product and provides a basis for future mutasynthetic methods for the generation of non-natural apopotolidins.

20) Edward Nam, "A Novel Region of the DNA Damage Kinase ATR Regulates Viability and the Replication Stress Response." Phosphorylation is a primary mechanism of regulating the DNA damage responsive kinases ATM (Ataxia-telangiectasia Mutated) and DNA-PK (DNA-dependent Protein Kinase). Whether phosphorylation regulates the related kinase ATR (ATM and RAD3-related) is unknown. ATR contains 16 evolutionarily conserved candidate autophosphorylation sites. We confirmed that at least one of these, SQ944, is phosphorylated in cells although functional analysis failed to detect a critical activity for this single site. To determine if multiple sites might be important we mutated the 16 conserved candidate ATR autophosphorylation sites to alanine, creating a 16A-ATR mutant. The 16A-ATR mutant maintained G2 checkpoint activity. However, it failed to rescue the essential function of ATR for cell viability and failed to promote recovery of replication from a transient exposure to replication stress. Further analysis identified TQ1566A/TQ1578A/TQ1589A (3A-ATR) as critical mutations causing this separation of function activity. Secondary structure predictions indicate these sites are within an extended region between ATR HEAT repeats 31R and 32R. This region aligns with regions of ATM and DNA-PK containing regulatory autophosphorylation sites. Thus, our analysis identified a new regulatory region in ATR that is shared with the DNA damage kinases ATM and DNA-PK. Mutations in this region create a separation of function mutant in human ATR that suggests the essential function of ATR is independent of its G2 checkpoint activity and is linked to its function in promoting proper replication in the context of replication stress.

21) Dawn Makley, “Umpolung Reactivity in Amide Coupling: Applications in Enantioselective Peptide Synthesis,” Dawn M. Makley, Bo Shen, Jeffrey N. Johnston. Amide bonds, which serve as the essential linkage between amino acids, are widely found in peptides and proteins as well as in pharmacologically active natural products and small molecule therapeutics. Nature constructs these essential bonds through the simple condensation of amines and carboxylic acids. Most synthetic methods in use today also utilize dehydrative approaches, with the help of coupling reagents. We have recently discovered a novel amide bond coupling reaction in which the polarities of the reactants are reversed (umpolung) to those employed in condensative methods. The inherent reversal in reactivity makes this novel coupling an attractive supplement to traditional methods. While in condensative couplings activated carboxylic acids act as the electrophilic coupling partner, and the amine the nucleophilic, our method employs α-bromo nitroalkanes which act as nucleophilic carbonyl surrogates once deprotonated. These can then be coupled to amines, which are converted to electrophiles in situ by N-iodination. One major advantage of this procedure is that racemization at the α-stereocenter of the carboxylic acid surrogate is not mechanistically possible. In addition, by utilizing the asymmetric aza-Henry reaction, chiral α-bromo nitroamines can be synthesized enantioselectively, providing for the facile enantioselective synthesis of aryl glycine amino acids.

22) Tyler Davis, "Chiral Proton Catalysis: Guidelines for the Development of More Reactive Bifunctional Catalysts, and the First Enantioselective Synthesis of Chiral cis-4,5-Disubstituted Imidazolines." BisAMidine chiral proton catalysts are a class of mixed Brønsted acid/Brønsted base reagents amenable to both electronic and steric modification. A counterintuitive approach in which we created more Brønsted basic versions of these Brønsted acid catalysts led to more reactive catalysts that delivered highly diastereo- and enantioselective aza-Henry reactions. This development has allowed us to considerably expand the nitroalkane scope, including secondary nitroalkanes. Enantiomerically enriched compounds of medicinal importance are now easily accessible through this system. In one example, we overcame the problems associated with the use of aryl nitromethane pronucleophiles in the aza-Henry reaction (low dr/ee), and have converted their addition products to the corresponding cis-imidazoline. This strategy has been successfully applied to the first enantioselective synthesis of (–)-Nutlin-3, a p53-MDM2 inhibitor currently in development by Hoffman-LaRoche.

23) Julia Koehler, "Paramagnetic Restraints as Novel NMR restraints for Membrane Proteins." The structures of membrane proteins are very difficult to determine by solution NMR since the slow tumbling of the protein-micelle complex results in severe line-broadening of the peaks in the spectrum. In addition, conventional NMR restraints for structure determination, such as NOEs, are extremely difficult to measure, especially for alpha-helical membrane proteins. To alleviate this problem we aim to introduce the measurement of paramagnetic restraints on membrane proteins. Paramagnetic restraints have been established for structure determination of soluble proteins but their use for membrane proteins is lacking behind. The measurement of these restraints such as Paramagnetic Relaxation Enhancements (PREs), Residual Dipolar Couplings (RDCs), and Pseudo-Contact-Shifts (PCSs) requires the introduction of a paramagnetic center into the protein. This will be achieved by attaching an EDTA-derived chelating agent onto a Cysteine residue, which will coordinate a paramagnetic metal ion, preferably of the lanthanide group. The proteins chosen for this study are Diacylglycerolkinase (40 kDa) and KCNE3 (14 kDa).

24) Priya Mathew, "Progress Toward the Total Synthesis of Mitomycin C," Priya A. Mathew, Jayasree M. Srinivasan, Amie L. Williams and Jeffrey N. Johnston. Mitomycin C is a natural product isolated from Streptomyces caespitosus that is clinically used as an anticancer drug.  The biological activity arises from the key aziridine functionality which is responsible for DNA alkylation and crosslinking.  Despite numerous synthetic approaches, its total chemical synthesis has been achieved only twice since its isolation in 1956.  In our synthetic approach, the aziridine is installed using a Brønsted acid-catalyzed aza-Darzens reaction.  The convergent synthesis of an advanced intermediate is performed using an aminomercuration followed by a quinone coupling.  Methods to install C(10) on the advanced intermediate will be presented.

25) Sam DeLuca, "Guiding Ligand Docking with Parmacophore Maps." Currently, QSAR and computational ligand docking studies are valuable but independently used tools for drug design. Data from Pharmacophore maps produced by tools such as COMFA are typically compared to the results of docking simulations by hand in a qualitative manner. We are developing an extension to RosettaLigand which will use information from QSAR derived pharmacophore maps to guide the low resolution phase of ligand docking. This will allow successful docking to be performed on lower resolution structures and will increase the speed at which high throughput docking of large numbers of structures sharing a common scaffold can be performed.

26) Matthew Spellings, "Machine Learning Methods for Structure-Property Relationships of LogP." In the drug design process, properties of the molecules in consideration must be carefully moderated. For a potential drug to be safe and effective, factors such as metabolism (how long it lasts inside the body and what happens to it as it is broken down), toxicity (whether the drug itself or any of its metabolites could be harmful inside the body), and activity (how effective the drug is for its intended purpose and how much of it actually reaches the target) should be accounted for. Experimental determination of properties related to these on the scale used in modern drug screenings is quite costly, both in terms of time and money; therefore, it is desirable to be able to predict relevant properties of potential drugs computationally. LogP, the logarithm of the equilibrium octanol-water partition coefficient for a given substance, is a familiar, readily-available metric of the hydrophobicity or hydrophilicity of compounds relevant to how drugs are absorbed, distributed, and excreted from the body. Currently, the industry-standard method of predicting logP is XlogP: each atom of the molecule is assigned a contribution according to its type and the final prediction for logP is the sum of these contributions. In this project, models were built using three machine learning methods: artificial neural networks, support vector machines, and kappa nearest neighbors. The inputs and extra parameters for each model were systematically optimized to produce predictors that performed better than XlogP for compounds excluded from the building of the models.

27) Odaine Gordon, "Mechanistic Studies into the Formation of Epoxyketo-linoleic Acids," Odaine Gordon, Claus Schneider. Epoxyketo-linoleic acids (Epoxyketooctadecenoic acids, EKODEs) are products of enzymatic and non-enzymatic oxidation of linoleic acid in vivo and in vitro. Six EKODE isomers containing cis and trans epoxides have been reported, with at least one isomer having potent biological activity, stimulating biosynthesis of aldosterone in adrenal glomerulosa cells. In our studies of the autoxidation of the trans-trans form of linoleic acid, we identified four EKODE isomers with trans epoxides as abundant products. The isomers were separated and purified via HPLC and the structures confirmed using NMR and GC-MS analysis. The EKODEs were more abundant products in the autoxidation of trans,trans than cis,cis linoleic acid, and also with increasing concentration of alpha-tocopherol. Since both conditions are known to favor the formation of the bis-allylic hydroperoxide (11-HPODE), we hypothesized that the EKODEs are transformation products of 11-HPODE. Autoxidation of HPLC-isolated trans,trans 11-HPODE in the presence of alpha-tocopherol gave all four EKODEs, and in addition, the bis-allylic ketone (trans,trans 11-KODE), a dehydration product of the bis-allylic hydroperoxide. The identification of 11-HPODE as an intermediate lends itself to a novel mechanism of formation of the EKODEs with potential relevance to their formation in vivo.

28) Mark Dobish, “Strategies in Asymmetric Organo-Catalysis: Reaction Development for the Functionalization of the 3’ Carbon of Indoles and Current Pharmaceutical Applications of aza-Henry Type Additions,” Mark Dobish and Jeffrey N. Johnston. The Johnston group has demonstrated a variety of aza-Henry type additions to N-Boc-arylaldimines with high enantioselection using a bifunctional Bronsted acid catalyst. Departing from our normal mode of reactivity, herein we highlight the development of an asymmetric functionalization of the indole 3’ carbon using the same class of catalysts, a formal Michael type addition. A variety of nitroalkanes have been added to an in situ generated alkylideneindolenine intermediates with good levels of enantioselection (up to 90% ee) and modest d.r. (up to 4:1). We also report on the scalable use of the enantioselective bromonitromethane additions to arylaldimines towards the pharmaceutical target SDZ-284692. Preliminary data shows that SDZ-284692 inhibits CYP51 in Trypanosoma brucei and Trypanosoma cruzi and suggests it may be an effective treatment for human trypanosomiases. Here we report on a six step, scalable synthesis, with an overall yield of 24%. The proposed route offers multiple points for diversification towards more effective drug candidates.

29) Matthew Bryant, "Gold Nanoparticle Technology to Probe Heme-Binding Proteins." Emerging applications of nanotechnology to diagnostic techniques is beginning to make a significant impact today. An important platform for biomedical applications of nanotechnology is the functionalized gold nanoparticle (AuNPs). The use of AuNPs brings together attractive optical properties, convenient functionalization chemistry, and well documented biological inertness. We have developed a heme-based ligand containing an esterified Lipoic Acid (thiol) Tri-Ethylene Glycol (spacer) [LPAPH] to understand heme-protein binding and interactions. One application in which a heme binding protein plays an important role is in heme detoxification during malarial infection. The parasite releases the histidine rich protein II (pfHRPII) into the host blood stream to protect itself from heme released during RBC lysis. pfHRP2 contains numerous repeats of AHH, and is a main target for rapid diagnostic tests. We hypothesize that coordination of HRP II to heme-functionalized AuNPs will result in aggregation of the AuNPs, resulting in a colorimetric change which can be used for assay signal.

30) Noemi Tejera Hernandez, "Studies on the ex vivo formation of novel eicosanoids derived from crossover of the 5-LOX and COX-2 pathways," Noemi Tejera Hernandez, Takashi Suzuki, Claus Schneider. Prostaglandins and leukotrienes are potent eicosanoid lipid mediators involved in homeostatic functions, and in inflammation, cancer, and other diseases. The leukotriene and prostaglandin biosynthetic pathways are initiated from the common substrate arachidonic acid by the action of 5-lipoxygenase (5-LOX) or cyclooxygenase (COX), respectively. Research carried out in our lab has provided evidence for an unexpected biosynthetic crossover of the 5-LOX and COX-2 pathways: the 5-LOX product 5-HETE is transformed by COX-2 to a di-endoperoxide that has structural similarities to the prostaglandin endoperoxide (PGH2) derived from arachidonic acid. The unstable PGH2 is transformed enzymatically to prostaglandins like PGE2 and PGD2, and the di-endoperoxide is transformed by the enzyme PGD synthase to a novel eicosanoid containing a hemiketal structural element that we named Hemiketal D2 (HKD2). We also have shown that HKD2 and the isomer HKE2 are formed non enzymatically from the di-endoperoxide. We are using a model of human blood ex vivo to characterize the formation of the hemiketal products. Blood samples are stimulated with LPS to induce COX 2 activity followed by treatment with calcium ionophore A23187 (to activate 5-LOX), 5-HETE, or both, to provide the substrate for the COX 2 reaction. Following extraction samples are analyzed using LC-MS in the SRM (selected reaction monitoring) mode. To improve detection sensitivity and stabilize the hemiketals, we have developed a derivatization strategy using pentafluorphenylhydrazine (PFPH). Preliminary results show formation of both hemiketals and thus provide initial evidence of a functional crossover of the 5-LOX and COX-2 pathways in vivo.

31) Larissa Fenn, "Structural Characterization of Natural Products from Complex Biological Mixtures Using Ion Mobility-Mass Spectrometry," Larissa S. Fenn, Cody Goodwin, Brian O. Bachmann, John A. McLean. Natural products are reemerging as an important component of drug discovery efforts due to their unique biochemical properties. Most of the exceptional properties of natural products are due to their functional complexity and well ordered structural conformation. It is known that natural products occupy a unique area of chemical space defined by their structural and chemical diversity, which is due to the biologically active natural products often containing non-standard amino acids, macrocyclic structures, high degrees of ring fusion, chiral centers, as well as other unique properties that set them apart from standard peptides and drug molecules. However, since these species are typically synthesized biologically by various bacteria, extensive purification is necessary to acquire a sample suitable for further structural studies (i.e. NMR or X-ray crystallography). In this work, we aim to mitigate this challenge through the use of structural separations provided by ion mobility-mass spectrometry (IM-MS). The additional dimension of separation provided by ion mobility creates the ability to rapidly distinguish natural products from other species present in a complex mixture. Absolute collision cross sections, or apparent surface areas, for a number of natural products from different structural classes were determined using IM-MS. A case study of cyclic peptides interpreted the collision cross section values using molecular modeling to determine representative structures. These findings were then used to access natural products of interest in complex cellular extracts.

32)  Andy Liedtke, "Development of Cyclooxygenase(COX)-1-Selective Inhibitors Derived from 2’-Des-Methyl Sulindac Sulfide," Liedtke, Andy J., Blobaum, Anna L., Felts, Andrew S. and Marnett, Lawrence J.  Cyclooxygenase(COX)-1, but not COX-2 is highly expressed in human malignant ovarian cells and represents a potential target for the prevention and treatment of ovarian cancer. The recent discovery that (E)-2'-des-methyl sulindac sulfide (LM-4503) is a weak (IC50= 1.8 microM) but selective COX-1 inhibitor represents the first report of selective COX-1 inhibition by a member of the arylacetic acid class of inhibitors. Against this background and the possible non-COX related therapeutic potential of the des-methyl sulindac analogues, the objective of our study is to optimize the anti-COX-1 potency and at the same time completely eliminate the COX-2 inhibitory activity. By following a concrete derivatization strategy, several new compounds are being generated and evaluated for their COX-1/-2 inhibitory potential in a routine COX assay. The most promising compounds are intended for additional experiments, e. g. in intact cells, and/or state-of-the-art animal models. The biochemical screening of a first series of free acid analogues of (E)-2’-des-methyl sulindac sulfide already led to the identification of a promising inhibitor candidate (LM-4624) bearing a biphenylidene substituent instead of the benzylidene residue in LM-4503, which was selected for follow-up trials and currently serves as our lead structure. The COX-1-IC50 value of LM-4624 was found to be as low as 565 nanoM and this compound did not markedly inhibit COX-2 under the test conditions used. Furthermore, we presume that the E-isomer of 2’-des-methyl sulindac sulfide and its benzylidene analogues bind in a completely different orientation within the active site of COX, compared to their origin sulindac sulfide (Z-form), which is presently under investigation by X-ray crystallography.

33) Mariana Boiani, "Stabilization of Antiapoptotic Mcl-1 by the Hsp70 Co-Chaperone BAG3 in Cancer Cell Lines," Mariana Boiani, Michael D. Hogarty, and Lawrence J. Marnett. Defective apoptosis is a hallmark in cancer, which point to members of the apoptotic pathway as candidates for drug development. The intrinsic or mitochondrial pathway of apoptosis is dependent upon the Bcl-2 family of proteins that comprise pro- and anti-apoptotic members. The antiapoptotic Bcl-2 family proteins (e.g. Bcl-2, Bcl-XL, Bcl-w and Mcl-1) function by directly binding and inhibiting pro-apoptotic Bcl-2 proteins. Overexpression of anti-apoptotic Bcl-2 proteins is frequently observed in cancer cells and is associated with poor prognosis and chemoresistance. This have led to the development of Bcl-2 antagonists, such ABT-737 that shows single-agent activity against some cancer cell lines and increase sensitivity to chemotherapeutics. However, ABT-737 does not bind Mcl-1 and Mcl-1 expression led to ABT-737 resistance. Since Mcl-1 is a short-lived protein, stabilization/destabilization of Mcl-1 protein can have a marked impact in its level. Previous work from our group has identified BAG3, a co-chaperone of Hsp70, as an important factor in stabilizing Bcl-2 proteins, mainly Mcl-1. We are currently studying the effect of BAG3 in Bcl-2 proteins levels, apoptosis and ABT-737 sensitivity in cancer cell lines. Among the six BAG family members, BAG3 was the only one that showed a correlation with Mcl-1 expression. Silencing BAG3 led to a reduction in Mcl-1 protein level, induction of apoptosis and increase in ABT-737 sensitivity, in some cell lines to the same extent observed under Mcl-1 knockdown. The cell response to other chemotherapeutics (Doxorubicin, Geldanamycin) is similar for BAG3 or Mcl-1 silenced cells, further supporting that the effects observed for BAG3 knockdown are linked to its effect on Mcl-1.

34) Nicole Chumbler, "Inhibition of Clostridium difficile Toxin B." Clostridium difficile is a major cause of antibiotic-associated intestinal disease with symptoms ranging from uncomplicated diarrhea to life-threatening pseudomembranous colitis. The pathogenic effects of C. difficile infection have been attributed to a potent exotoxin, Toxin B (TcdB). Cell toxicity depends on receptor binding, translocation across the endosomal membrane, and a cysteine protease-mediated autocatalytic cleavage event within the toxin. Upon cleavage, an N-terminal glucosyltransferase is released into the cell where it inactivates small GTPases, resulting in cell rounding and death. We have developed a high throughput screen for small molecule inhibitors of TcdB cytotoxicity. We present our progress in completing a screen of a 160,000 compound library and validating candidate inhibitors. Identifying small molecule inhibitors of TcdB will enhance our understanding of the pathogenic mechanism and could lead to the development of novel therapeutics to combat C. difficile-associated disease.

35) John Stone, "Gold Nanorod-Protein Composites as a Novel Material for Viral Vaccine and Therapeutic Development," John W. Stone, Natalie Thornburg, Jonathan Walker, Stephen R. Jackson, James E. Crowe Jr., and David W. Wright. Human respiratory syncytial virus (RSV) is a paramoxyvirus and the primary cause of lower respiratory track infections in infants, the elderly, and immune compromised individuals resulting in approximately 1 million pediatric deaths annually worldwide. Early attempts at vaccine development, i.e., live attenuated virus vaccinations, failed resulting in enhanced secondary infections and death. Presently, there is no licensed vaccine for RSV resulting in the need for new, innovative approaches toward drug/vaccine development. Gold nanoparticles are stable, chemically inert, and non-cytotoxic materials making them attractive as templates for novel drug design. In current work, the fusion protein from RSV was covalently attached to the surface of gold nanorods via standard carbodiimide chemistry. Conjugated particles were sufficiently purified as measured by ELISA and characterized to confirm particle integrity and stability. A significant increase in particle stability in high salt buffers was observed as monitored by UV-vis. Circular dichroism measurements were carried out on both free and nanorod-bound protein to confirm the retention of secondary structure post conjugation. In dendritic cell/T cell assays, protein coated particles were found to activate T cells with a magnitude similar to that of free protein confirming activity of protein-bound composites. Additional studies have included the prophylactic addition of gold nanorod protein conjugates to HEp 2 cells followed by infection with RSV. Earlier results indicate a significant inhibitory effect as measured by a 93% decrease in optical density when compared to infected cells that remained untreated.

36) Takashi Suzuki, "Oxidative transformation of curcumin – a key to its cancer chemopreventive activity?" Takashi Suzuki, Noemi Tejera, Christopher S. Williams, and Claus Schneider. Curcumin, a polyphenol isolated from the plant turmeric, is recognized for its anti-inflammatory and anti-tumorigenic bioactivities. It is currently being evaluated in multiple clinical trials for the prevention or treatment of cancers of the colon, rectum, pancreas, multiple myeloma, and also for neurodegenerative diseases. Although a plethora of in vitro targets of curcumin have been identified, the precise molecular mechanism(s) of its biological activities have not been elucidated. We have recently discoverd a novel, previously unrecognized oxidative transformation of curcumin. In preliminary experiments we have detected the major oxidative metabolite of curcumin in plasma and intestinal mucosa after oral administration of curcumin to mice. The reaction of curcumin with molecular oxygen is rapid, prominent, and gives rise to novel and reactive metabolites that could potentially be the mediators of some of the pharmacological effects of curcumin. The oxidative transformation proceeds through a quinone methide intermediate and the final product is a bicyclic cyclopentadione derivative of curcumin. We propose that the electrophilic quinone methide exerts biological effects through reaction with glutathione and protein thiols whereas the cyclopentadione is a carbon nucleophile with potential selectivity for reaction with protein sulfenic acids. The concept of oxidative activation of curcumin could result in a new paradigm of understanding the structure and function of the dietary cancer chemopreventive agent curcumin.

37) Libin Xu, “Oxysterols Formed from the Most Oxidizable Lipid, 7-Dehydrocholesterol, in vitro and in vivo: Implications for Smith-Lemli-Opitz Syndrome." Smith-Lemli-Opitz syndrome (SLOS) is a devastating metabolic and developmental disorder that is characterized by multiple congenital malformation and mental retardation. 7-DHC is accumulated in SLOS patients due to mutations in the gene encoding 7-DHC reductase (Dhcr7), the enzyme that catalyzes the reduction of 7-DHC to cholesterol. In our previous studies on the reactivity of lipids toward free radical-mediated peroxidation reactions, 7-dehydrocholesterol (7-DHC) was found to be the most oxidizable lipid ever known. In such peroxidation reactions, 7-DHC is over 10 times more reactive than arachidonic acid and about 200 times more reactive than cholesterol. Over a dozen 7-DHC oxidation products, i.e., oxysterols, with novel structures were discovered in a 7-DHC peroxidation reaction in vitro. A free radical mechanism was proposed to account for the formation of all of the oxysterols. More significantly, multiple new oxysterols were identified by HPLC-MS and 2-D NMR in Dhcr7-deficient Neuro2a cells, SLOS human fibroblasts, mutant mouse SLOS model, and drug-induced rat SLOS model, while they are not present in the corresponding control samples. The 7-DHC-derived oxysterols were found to reduce cell viability in a dose- and time-dependent manner, some of the compounds showing activity at sub-μM concentrations. The complex 7-DHC oxysterol mixture added to control Neuro2a cells also triggers the gene expression changes that were previously identified in Dhcr7-deficient Neuro2a cells. Thus, we propose that the 7-DHC-derived oxysterols may play important roles in the pathogenesis of SLOS.

38) Kellen Harkness, “Analyzing Supramolecular Assemblies on Gold Surfaces," Supramolecular assemblies, ubiquitous products of evolution, exhibit unique emergent chemical and biological activity. Using a biomimetic approach, small molecules can be self-assembled through non-covalent interactions in order to produce a precise chemical interaction which is not inherent to any component of the assembly. One pathway to the formation of stable, highly tunable supramolecular assemblies is the use of gold nanoparticles (AuNPs) as a scaffold for a mixture of thiol-bearing molecules. The binding of the sulfur head group to the gold surface orients each molecule, allowing for controlled intermolecular interactions. These interactions, along with entropic energy gains, drive the molecules toward a highly reproducible conformation on the gold surface, forming unique anisotropic materials which can be used for in vivo chemistry. Unfortunately, explorations of novel molecular mixtures are limited by an inability to experimentally observe the final conformation of molecules on the gold surface. We have developed a structural mass spectrometry methodology in order to better understand the structure and function of these novel supramolecular assemblies. By liberating portions of the gold-thiol interface, we are able to investigate the presence or absence of precise supramolecular assemblies on the AuNP scaffold. Here we demonstrate the use of this technique to analyze assemblies with a mixture of non-drug-like, drug-like, and peptide molecules.

39) Laura Anzaldi, "A Small Molecule Activator of Heme Biosynthesis Triggers the Heme Stress Response of Staphylococcus aureus," Laura L. Anzaldi, Devin L. Stauff, Paul F. Reid, Olusegun Aranmolate, Susan L. Mercer, Gregory M. Kurkis, Alex G. Waterson, Gary A. Sulikowski, Eric P. Skaar. Staphylococcus aureus is a Gram positive pathogen that causes significant morbidity and mortality. In order to successfully infect its vertebrate host, S. aureus requires nutrient iron. In the host environment, most iron is bound to a protoporphyrin ring in the form of heme and it is this form of iron that S. aureus preferentially utilizes during infection. However, the reactive nature of heme causes toxicity to the bacteria. To cope with this paradox, S. aureus uses a two-component system (TCS) to sense and respond to heme toxicity. This heme sensor system (hssRS) is composed of a membrane bound sensor histidine kinase HssS and a cytoplasmic response regulator HssR. TCSs are the primary signaling system that bacteria use to sense and respond to their environment and are a prime target for novel therapeutics. In order to further characterize the mechanism by which HssS recognizes its ligand we performed a high-throughput screen to identify small molecule activators of HssS. The most potent activator of HssS is ‘8882. We have determined that activation of HssS by ‘8882 depends on endogenous heme biosynthesis and increases intracellular levels of heme. This indicates that the S. aureus heme stress response is activated by both endogenous and exogenous heme. A small molecule that stimulates endogenous heme synthesis may have broad utility as a biological probe and lays the groundwork for the generation of novel therapeutics that target bacterial heme biosynthesis.

40) Jody C. Ullery, “4-Hydroxynonenal Mediated Activation of Heat Shock Transcription Factor-1 Induces the Expression of Prolyl-4-Hydroxylase, a Key Regulatory Molecule in the Cellular Response to Hypoxia,” Jody C. Ullery, Aaron T. Jacobs, Lawrence J. Marnett. Abnormalities in the ability of cells to respond to oxygen lead to oxidative stress and hypoxia. Exposure of cells to high levels of oxygen results in the generation of reactive oxygen species, such as 4-hydroxynonenal (4-HNE), which can covalently modify DNA and proteins. Previous studies show that treatment of RKO colon cancer cells with 4-HNE results in the activation of heat shock transcription factor 1 (HSF-1), a transcription factor that mediates the cellular response to heat shock. Microarray analysis shows that when HSF-1 is silenced in RKO cells, there are a number of concomitant changes in the expression of downstream genes including a fourfold decrease in the expression of prolyl-4-hydroxylase (P4HA2), a critical regulator of hypoxic cell signaling. To analyze this observation further, changes in P4HA2 gene and protein expression in HSF-1 silenced cells were assayed via RT-PCR and Western blot, respectively. A significant decrease in both the gene and protein expression of P4HA2 was detected upon HSF1 silencing, in the presence or absence of HNE. Furthermore, treatment of RKO cells or hypoxia-sensitive cell lines (including human umbilical vein endothelial cells, MCF-7 breast adenocarcinoma cells and A549 lung adenocarcinoma cells) with HNE activated HSF1 in an HNE-concentration dependent manner and resulted in corresponding increases in P4HA2 expression, suggesting that HSF-1 could regulate P4HA2 expression, potentially at the level of transcription. The results of these studies provide significant information on the mechanisms controlling the cellular response to oxygen.

41) Robert Lavieri, "Defining the Roles of Phospholipase D Isoforms in Cancer via Isoform-selective Inhibitors," Robert R. Lavieri, Sarah A. Scott, Paige E. Selvy, Kwangho Kim, J. Scott Daniels, H. Alex Brown and Craig W. Lindsley. Phospholipase D (PLD) catalyzes the production of the lipid second messenger phosphatidic acid (PA). PLD expression and/or enzymatic activity are both elevated in a variety of human cancers. Inhibition of PLD enzymatic activity, via genetic or biochemical methods, leads to decreased cancer cell invasion and decreased cancer cell survival. The aforementioned evidence provided the impetus for our medicinal chemistry project focused on the development of isoform-selective PLD inhibitors. The development of such inhibitors is an essential step in advancing the study of PLD as a potential cancer drug target. A group from Novartis published a report in 2007 disclosing halopemide as a hit from a high throughput screen for PLD inhibitors. While we initiated our iterative analog synthesis with halopemide we have explored a broad chemical space. We utilized technology-enabled synthesis to develop a library of approximately 600 compounds. This effort has yielded the most potent, isoform-selective PLD inhibitors described to date. While halopemide inhibits both PLD isoforms relatively equally VU0359595 inhibits PLD1 1,700 times more potently than PLD2, and VU0364739 inhibits PLD2 75 times more potently than PLD1. In a triple negative breast cancer cell line, MDA-MB-231, VU0364739 significantly decreases cell viability whereas VU0359595 does not affect cell viability; indicating that a PLD2 selective inhibitor may be the optimal therapeutic agent in this particular malignancy. We are currently utilizing our novel, isoform-selective PLD inhibitors in order to more clearly characterize the role of PLD signaling in oncogenic processes such as the suppression of apoptosis, cell invasion and metastasis.

42) Ran Ye, "Molecular and Phenotypic Correlates of Brain Serotonin Transporter Expression Identified via Ex Vivo and In Silico Analysis of BXD Recombinant Inbred Mice." Serotonin transporter (5HTT, SERT) regulates extracellular 5-HT levels via high-affinity, antidepressant and cocaine-sensitive, 5-HT reuptake. Alterations in SERT function have been associated with many neurobehavioral disorders including anxiety, depression, obsessive-compulsive disorder (OCD) and autism. As disease-associated SERT protein variants are generally rare, we have proposed that the presence of as yet unidentified disease-associated genes that contribute at a network level to SERT expression and regulation. Previously, we have characterized of SERT-dependent gene and phenotypic associations using combined biochemical and in silico analyses of recombinant inbred (RI) mouse lines (Carneiro et al. PNAS, 2009). Here we report an initial analysis of SERT protein expression levels in the midbrain and hippocampus of 34 BXD (C57BL/6J x DBA/2J) RI mouse lines, obtained via quantitative western blotting of midbrain and hippocampal extracts with SERT-specific antibodies. In silico, genome-wide quantitative trait loci (QTL) mapping identifies multiple genomic loci to be significantly associated with SERT protein expression, that appear to be gender-dependent, that include monoaminergic synaptic proteins, and where associations lead to hypotheses of shared regulatory mechanisms. At a phenotype level, we identify correlates of SERT protein expression that match phenotypes identified in a previous examination of traits associated with functional SERT coding variation, including, surprisingly, CNS iron levels. Our work demonstrates the power of convergent use of biochemical and in silico approaches based on RI line phenotyping and supports the importance of SERT expression and activity, and presumably 5-HT signaling, for control of brain iron-modulated phenotypes.

43) Kathryn Haley, "Function and Regulation of a Heme Oxygenase in the Human Pathogen Staphylococcus Lugdunensis." Staphylococcus lugdenesis is a coagulase negative bacterium that is often found as part of normal human skin flora but has the potential to cause invasive infections. Like nearly all bacteria, S. lugdunensis requires iron to successfully infect its vertebrate host. However, the concentration of free iron within host tissues is far too low to support the level of growth required to establish infection. The most abundant source of iron within humans is the metalloporhphyrin heme that is bound by hemoproteins such as hemoglobin. Recently, we have identified a heme oxygenase belonging to the IsdG family within S. lugdunensis and have shown that this protein degrades heme, releasing nutrient iron. Additionally, it was demonstrated that the product of this degradation is the chromophore staphylobillin, identifying another species in addition to Staphylococcus aureus capable of degrading heme to this tetrapyrrole. Sequence analysis suggests that transcription of this gene is regulated by iron through the ferric uptake regulator (Fur). This was confirmed by immunoblot analysis where it was shown that S. lugdunensis produces more IsdG when grown in iron deplete conditions. This research identifies the first gene product in S. lugdunensis involved in nutrient acquisition. Importantly, research in other bacteria species, specifically S. aureus has shown that proteins involved in iron acquisition are critical for full virulence to be achieved and are considered potential drug targets. Furthermore, this research establishes the evolutionary conservation of the IsdG family of heme oxygenases within the Staphylococcal species.

44) Vanessa Bright, "“Development of Functionalized Iron Oxide Nanoparticles for Molecular Imaging,” Vanessa Bright, Michael Nickels, Jingping Xie, Jared Cobb, John C. Gore and Wellington Pham. Nanoparticles have shown great potential for imaging and drug delivery within in vivo systems. Specifically Iron oxide nanoparticles, which are ideal since they are non-toxic, biocompatible, biodegradable, can dictate size, and are paramagnetic. Superparamagnetic iron oxide (SPIO) nanoparticles can be used alone as imaging agents, for MR imaging, and they can be conjugated with bioactive ligands for targeted imaging applications. Labeling SPIO nanoparticles with a biological agent is efficient when they are functionalized with amine groups on the surface. Dextran-coated SPIO nanoparticles are not ideal in this case due to the presence of hydroxyl groups on dextran. To enhance the productivity of bioconjugation, a linker containing epoxide and amine termini was synthesized. The epoxide easily reacts with the alcohol to produce surface amines, which prepare the particles for conjugation. To demonstrate the utility of aminated SPIO for biological study, we labeled the nanoparticles with fluorescein isothiocyanate and folic acid. These labeled particles demonstrated preferential accumulation in folate receptor-expressed cells and could be imaged using optical and MR imaging.

45) Kelly Hines, "Analysis of Wound Fluid by IM-MS for Protein Signatures of Wound Healing," Kelly M. Hines, Sameer Ashfaq, Jeffrey M. Davidson, Susan R. Opalenik, John A. McLean. Poorly healing wounds are a chronic symptom for diabetes patients. A deeper understanding of the proteins involved in the wound healing process may lead to advances in medical treatment for slowly healing wounds. For the purpose of this study, 10 rats were treated with streptozotocin to induce diabetes. In each of 10 diabetic and 6 control rats, PVA sponges were inserted subcutaneously through an incision to collect acute wound fluid. The sponges were harvested at 2 and 5 days, after which the fluid was spun out and collected. IM-MS is a two-dimensional gas phase separation based on mass-to-charge (m/z) and collision cross section, which allows for rapid separation of the classes of biomolecules in complex biological samples. The correlation between collision cross section and m/z yields characteristic trendlines for each class of biomolecules, from which signals corresponding to proteins and peptides can be extracted. The wound fluid samples were analyzed by IM-MS to identify potential protein signatures in the wound healing process. Statistical analysis of the IM-MS data reveals signals which vary amongst the diabetic and control groups. A proteomic analysis of the wound fluid, including tryptic digestion, collision induced dissociation (CID), and database searching, was performed in the attempt to identify protein signatures that distinguish diabetic from control wound fluids.

46) Nicholas Adams, “Low Resource Extraction and Processing of Biological Samples Using Surface Tension Valves," Nicholas Adams, Hali Bordelon, Rick Haselton and David Wright. For illnesses that advance as a result of rapid doubling times or tissue degeneration it is critical that time-to-diagnosis be minimized. However, because blood often interferes with sample detection methods, receiving the results from blood tests for specific biomarkers can take several days, especially in low resource settings such as rural hospitals and small clinics where samples are sent out for laboratory testing. Simple blood-borne biomarker extraction methods that remove contaminants coupled with simple detection methods are likely to improve the limits of detection and thus enable earlier detection. I have developed and tested a self-contained device that uses the properties of surface tension valves to extract specific nucleic acids or proteins from biological samples, removing contaminants contained in the samples that interfere with detection methods, and concentrate the target biomolecules in an elution buffer that could be integrated with a detection platform. The technology could provide an effective point-of-care diagnosis for a variety of diseases.

47) Matias Moller, "Characterization of Novel Adducts from Lipid Peroxyl Radicals andTtyrosyl Radicals Coupling," Matias Moller, Duane Hatch, Hye-Young Kim, Roman Shchepin & Ned Porter. The covalent modification of proteins is an important mechanism of regulation of enzymatic reactions and cell signaling. Reactive species produced during oxidative stress can also modify proteins in a less controlled manner, and sometimes it is observed a dramatic effect on the activity of the modified protein. We have recently found that lipid peroxyl radicals can form covalent adducts with tyrosine, through the intermediacy of the tyrosyl radical. The main products were isolated and studied by 2D-NMR, which showed that the peroxide bond is linking the lipid to the para position of the tyrosine. This finding was unexpected because most of the known tyrosine modifications by reactive species occur at the ortho position. We have studied this reaction in different phenol/tyrosine-lipid systems including a peptide with methyl linoleate and found that the main products are para-peroxide adducts, while no ortho-peroxide adducts were found. However, these adducts are not stable and cyclize via an intramolecular Diels-Alder reaction to yield tricyclic compounds. The rate of cyclization is solvent dependent and has a half-life of approximately two hours in ethanol at 37°C. An alternative route for the acyclic adduct is reduction. Cuprous ion reduction of the adduct yielded the corresponding hydroxy-lipid and the para-hydroxy derivative of the tyrosine. Synthesized para- and ortho-hydroxy derivatives of the tyrosine were used to demonstrate that the reduction product was unequivocally the para-hydroxy derivative. Given the ease with which these adducts were formed, the same adducts should be expected in proteins, and studies are under way to prove this.

48) Rebecca Sandlin, "Hemozoin Formation: Basic Science to Drug Discovery," Rebecca D. Sandlin. Anh Hoang, Timothy J. Egan, and David W. Wright. Each year, over one million lives are lost due to infection by the protozoan parasite, malaria.  Drug-resistant strains of malaria have now emerged in most endemic regions and no affordable alternative has been offered.  Chloroquine, the former gold standard in malaria treatment, is thought to act by inhibiting the formation of hemozoin, a by-product of heme detoxification necessary for parasite survival.  Here, the mechanism of formation of β-hematin, synthetic hemozoin, has been investigated.  The in vivo environment of hemozoin formation has been imitated using an emulsion of synthetic lipid bodies in acetate buffer (pH4.8, 37°C).  This system produces β-hematin crystals with an average half life of <1 minute.  TEM images then revealed the alignment of crystals at the lipid-water interface, suggesting epitaxial nucleation occurs at this interface.  This knowledge has recently allowed the development of a high-throughput screening assay.  To date, 32,400 compounds have been screened with a hit rate of 0.42%. These active compounds have been further tested for activity against the most virulent strain of malaria, Plasmodium falciparum.  Fifty compounds from the original screen show inhibitory activity against the parasite, with several of these hits boasting concentration response curves in the nanomolar range.  As a result of this pilot screen, four primary chemotypes, previously unreported in the malaria literature, have been identified for further study and optimization.

49) Jay Forsythe, "Optimization of Porous Silicon Films for Nanostructure Initiator Mass Spectrometry Imaging," Jay Forsythe, Michal Kliman, Yang Jiao, Jenifer Lawrie, Sharon M. Weiss, John A. McLean. Matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) has led to significant findings in biological research. Nevertheless, two significant limitations of organic-acid based MALDI-IMS have emerged – questionable sensitivity below 500 m/z and spatial resolution dependent on the irregular co-crystallization of analyte and matrix. As a result, matrix-free techniques such as desorption ionization on porous silicon (DIOS) and more recently nanostructure initiator mass spectrometry (NIMS) have received interest. Developed by Siuzdak and colleagues in 2007, NIMS employs an etched nanoporous silicon scaffold filled with viscous siloxane molecules called “initiators.” Although it is a relatively new technique, NIMS has demonstrated impressive sensitivity when compared to MALDI in the literature. Here, we report the discovery and development of semi-transparent porous silicon films attached to glass slides for use in NIMS imaging. Films are characterized via scanning electron microscopy (SEM) and profilometry, and are optimized by varying porous silicon etching parameters and comparing signal intensity of biological molecules. In addition, due to film transparency, preliminary images of cell monolayers are corroborated with traditional microscopy.

50) Carol Bansbach, "Phosphorylation Regulates SMARCAL1 Activity at Stalled Replication Forks." The replication stress response (RSR) is a genome maintenance program that acts during every cell cycle to deal with genotoxic insults that challenge DNA replication. The RSR promotes accurate replication of the genome, which is essential for cell survival and the avoidance of disease. Mutations in SMARCAL1 (HARP) cause Schimke immuno-osseous dysplasia (SIOD). SIOD is a multisystem disorder characterized by growth defects, immune deficiencies, and other complex phenotypes. The mechanistic basis for this disease is unknown. Using functional genomic screens, we identified SMARCAL1 as a replication stress protein. We have determined that SMARCAL1 localization and enzymatic activity at sites of stalled replication are important for genome integrity during S phase. Our data suggest that defects in the genome maintenance activities of SMARCAL1 are partly responsible for SIOD. To further understand the regulation of SMARCAL1 genome maintenance activities, we are currently investigating the role of phosphorylation. Components of the RSR are commonly targets of the DNA damage kinases. We determined that SMARCAL1 is a substrate for the DNA damage kinases Ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK). SMARCAL1 is hyperphosphorylated in cells following exposure to replication stress agents. Using targeted and unbiased approaches, we have identified critical phosphorylation sites in SMARCAL1. Interestingly, phosphorylation in the C-terminus of SMARCAL1 enhances forked-DNA stimulated ATPase activity. The consequences of this phosphorylation on DNA binding and in vivo activities will be presented.

51) Eleanor Powell, "Quantitative Structure-Activity Relationship Studies on Inhibitors of 17-beta Hydrosteroid Dehydrogenase Type 10, a Potential Alzheimer's Disease Drug Target," Eleanor Powell, Edward W. Lowe Jr., Jens Melier. 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 inhbited 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. Comparitive 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 q2=0.586 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.

52) Edward Lowe, "PHARMMAP: A Novel Pharmacophore Mapping Algorithm." Three dimensional quantitative structure activity relationship (3D-QSAR) analysis is a powerful tool in drug design, particularly when little structural information about the target is available. Many of these techniques, such as comparative molecular field analysis (CoMFA) and comparative molecular similarity indexes analysis (CoMSIA), require data sets based around a similar scaffold such that the molecules can be superimposed. Proper superimposition is a time consuming step and often requires highly accurate geometries for the molecules of interest. This requirement essentially limits the applicability of these methods to scaffolds which have already been experimentally explored. We are developing a novel pharmacophore mapping algorithm, PHARMMAP, based on machine learning techniques which hopes to circumvent these limitations. This will allow for pharmacophore maps to be rapidly generated based on high-throughput screening data instead of on small, super-imposable data sets for any scaffold of interest. Thus, the pharmacophore maps are generated based on the experimental data of +100,000 molecules from a high-throughput screen whereas traditional pharmacophore methods rely on the bioactivity data of only those molecules which can be superimposed. Here, we present preliminary benchmarking results of this novel and exciting method.

53) Michal Kliman, "Mass Spectrometry Imaging and Relative Quantitation of Cholesterol and 7-Dehydrocholesterol Oxysterols in Smith-Lemli-Opitz Syndrome Mouse Model," Michal Kliman, Zeljka Korade, Libin Xu, John A. McLean, Ned A. Porter. In human Smith-Lemli-Opitz Syndrome (SLOS), mutations in the gene encoding 7-dehydrocholesterol reductase (Dhcr7) enzyme, which catalyzes the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol, lead to elevated levels of 7-DHC and reduced level of cholesterol in tissues and fluids. The symptoms of SLOS include incomplete myelination, developmental defects, and autism-like mental impairment. Recently, oxidative metabolites of 7-DHC, i.e., oxysterols, that were identified in the nervous tissue of SLOS mice and human fibroblasts, were shown to be potentially critical in the pathophysiological changes seen in SLOS. Using mass spectrometry imaging and relative quantitation, we confirmed drastic differences in cholesterol, 7-dehydrocholesterol and its oxysterols distributions between normal and knock-out mouse SLOS model brain tissues. We are developing sample preparation methodologies and high resolution imaging approaches to follow the spatial distributions of these metabolites in SLOS tissues.

54) Dale Rosado, "Evaluation of Pyridinol and Pyrimidinol Antioxidants for Suppression of Oxysterol Formation in Fibroblast Harvested from Smith-Lemli-Opitz Syndrome Patients." Dale Rosado, Eric Karrsen, Austin Eckhoff, Libin Xu, Zeljka Korade, Ned Porter. Smith-Lemli-Opitz syndrome (SLOS) is a genetic metabolic disorder that is characterized by abnormally high levels of 7-Dehydrocholesterol (7-DHC, an intermediate in cholesterol biosynthesis) and decreased levels of cholesterol. This is caused by a defect in the genes that code for the 7-dehydrocholesterol reductase enzyme, thus preventing the conversion of 7-DHC to cholesterol. Characteristics of SLOS include organ malformation, microcephaly, mental retardation, muscle weakness, and fusion of digits. Currently, the only treatment for SLOS is an increase in the consumption of dietary cholesterol.  Our lab has recently shown that 7-Dehydrocholesterol (7-DHC) is 200 times more oxidizable than cholesterol and 10 times more oxidizable that arachidonic acid under free radical conditions. The result of the auto-oxidation of 7-DHC is the formation of various oxysterols that are potentially toxic. To counter the auto oxidation, a variety of pyridinol and pyrimidinol antioxidants were synthesized. These antioxidants have proven to be potent in the prevention of free radical oxidation of arachidonic acid. It was hypothesized that the pyridinol and pyrimidinol antioxidants would be effective at the prevention of auto-oxidantion of 7-DHC in various SLOS cell lines. To test this hypothesis, SLOS human fibroblasts were treated with media containing antioxidants. The sterols were isolated and analyzed by LC-APCI-MS to determine the levels of 3β-5α-dihydroxycholest-7-ene-6-one (an oxysterol formed from 7-DHC auto-oxidation). It was found that treatment of the SLOS fibroblast with several of pyridinol and/or pyrimidinol antioxidants resulted in significant decreases in the amount of this oxysterol as compared to untreated fibroblasts.  

55) Eric Karssen, "Synthesis of Aminopyridine- and Aminopyrimidine- Analogues of Resveratrol as Potential Antioxidants in Reduction of Oxysterols in SLOS Fibroblast Cells," Eric Karssen, Dale Rosado, Ned Porter. Resveratrol (3,4’,5-trihydroxystilbene) is a natural product found in various plants (e.g. grapes, berries and peanuts). During the last decades this natural stilbene has been studied in numerous in-vitro and in-vivo test because of its interesting biological effects. These effects include mainly antioxidant- and anti-inflammatory- activities as well as its inhibitory effect in different stages of carcinogenesis. Despite its interesting pharmacological profile resveratrol itself has proven to have a very low bioavailability as well as a poor solubility. Furthermore, resveratrol seems to be toxic in in-vitro assays. Studies in the past have shown that incorporation of nitrogen into the aromatic rings of phenolic antioxidants increase the ionization potenial (IP) without substantially affecting the O-H bond dissociation energy (BDE). As a consequence these nitrogen containing phenolic antioxidants undergo a fast H-atom-transfer reaction with radicals without making them less stable to air. Hypothetically the incorporation of nitrogen will also influence the solubility as well as toxicity. Based on these hypotheses nitrogen-containing resveratrol analogues were synthesized via Heck- and Sonogashira- coupling reactions. The synthesized analogues were evaluated ­in-vitro and have proven effective in suppressing the auto-oxidation of 7-dehydrocholesterol in Smith Lemli Opitz Syndrome (SLOS) fibroblast cells.

56) Ewa Kowal, "Structural Studies of Exocyclic Deoxyadenosine Adducts of 1,2,3,4-diepoxybutane." Ewa A. Kowal, Uthpala Seneviratne, Natalia Tretyakova, Michael P. Stone. 1,2,3,4-Diepoxybutane (DEB), the metabolite of 1,3-butadiene induces A →T transversion mutations. Novel exocyclic S, S and R,R N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2’-deoxyadenosine (N6,N6-DHB-dA) adducts have been identified. Preliminary bypass studies have shown that R,R isomer is not a block to replication under normal or SOS-induced conditions, while the S,S isomer is a medium block. UV thermal studies have shown that both isomers destabilize DNA duplex, where oligonucleotide containing S,S isomer is less stable. Nuclear Magnetic Resonance (NMR) spectroscopy was utilized to determine how N6,N6-DHB-dA adducts alter the structure and dynamics of the DNA duplex. Both isomers lack the features to form Watson-Crick base pair and imino proton of thymine which was opposite the adduct was not observed in the NOESY spectrum in H2O. H6/H8-H1’ region of the NOESY spectrum for both isomers is similar compared to unmodified duplex suggesting a localized structural changes at the lesion site.

57) Glenna Kramer, "Structural Studies with K-26 as a Tool to Develop N-Terminal and C-Terminal ACE Selective Inhibitors and Probe for Microbial Targets of K-26." K-26, a natural product first isolated from the soil bacterium Astrosporangium hypotensionis, is a potent angiotensin converting enzyme (ACE) inhibitor (Ki = 15.1 nM). As a key component of the renin-angiotensin system, the dipeptidyl carboxypeptidase ACE is responsible for blood pressure homeostasis and is correspondingly a validated drug target of high clinical relevance. Human ACE possesses two homologous domains presenting the Zn2+ dependent peptidase activity, with differing specificity and physiological function. However, the functions of these domains have not been completely elucidated in part due to the lack of domain selective chemical probes. K-26, which is composed of isoleucine, tyrosine and a nonproteogenic phosphonate analog of tyrosine, (R)-1-amino-2-(4-hydroxyphenyl) ethylphosphonic acid (AHEP) differs structurally from archetypal ACE inhibitors used in the clinic. Through X-ray crystallographic investigations, we have illuminated the interactions of K-26 in the active site of two forms of ACE in Drosophila (AnCE) and human testis (tACE). The interactions of K-26 analogs in the active site of these enzymes will provide insight into the mode of action of this natural product and, based on its distinct binding mode, provides a basis for designing N-terminal and C terminal ACE active selective compounds. Additionally, these structural studies provide useful information for the development of K-26 conjugated probes for determining the potential microbial targets of K-26, which are presumably ACE-related enzymes in various phyla of ecological relevance to the producing organism.

58) Aroop Chandra, "Unified Synthetic Strategy towards the Indole Marine Alkaloids," Aroop Chandra and Jeffery N. Johnston. Our progress towards the total syntheses of (+)-Ambiguine G and other chlorinated hapalindoles is described. A Friedel-Crafts acylation/alkylation sequence is used in sequence with intermolecular Diels-Alder cycloaddition to ultimately arrive at the ambiguine G ABCD-tetracycle convergently. Synthesis of the hapalindole skeleton (ABCD ring system of ambiguine G), including all the requisite chiral centers, has been accomplished in 13 steps from 2,3,3-trimethylacrylic acid. The synthetic strategy demonstrated here for Ambiguine G was also extended to other members of marine indole alkaloid family. In summary, a uniform synthetic strategy for numerous members of the marine indole alkaloids has been developed.

59)  Ghazal Hariri, "Single and Combination Therapy with Polyester based ‘Nanosponges’ Investigated in Murine Lung Cancer Models," Ghazal Hariri, Aaron Edwards, Dennis E. Hallahan, and Eva Hartha. We present tumor growth delay studies of lung cancer in mouse models treated with polyester based ‘nanosponges’. The particles were functionalized with targeting peptides containing the recognition units HVGGSSV for treatment of irradiated tumors and the recognition unit cRGD for treatment of unirradiated tumors. The two different targeted nanosponges were turned into drug delivery systems, encapsulating paclitaxel and camptothecin. The studies included treatment of the tumors with single dose paclitaxel or camptothecin, or combination therapy. In vitro experiments indicated that the order of the combination treatments could play a role in the success of tumor treatment. Therefore, we studied both combinations of paclitaxel/camptothecin and the sequences of administration. The results of the tumor growth delay studies and the efficacy in single and combination therapy of the nanosponge-drug delivery systems will be discussed together with the biodistribution and the pharamacokinetics.

60) Oleg Kovtun, "Visualization of The Dopamine Transporter Protein with Ligand-Conjugated Quantum Dots." The dopamine transporter protein (DAT) is of unique importance for its role as a target for the widespread psychostimulants cocaine and amphetamine as well as its recent implication in schizophrenia, bipolar disorder, Parkinson's disease, and attention-deficit hyperactivity disorder (ADHD). DAT terminates dopamine signaling in the brain by reuptake of dopamine from the synaptic space. Despite its increasing clinical importance, little is known about DAT dynamic regulation due to the poor spatiotemporal resolution of conventional methodologies and the lack of an efficient DAT-specific fluorescent probe. We developed a labeling approach for dynamic imaging and single quantum dot tracking in mammalian cell cultures that uses a high-affinity, DAT-specific, biotinylated 2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (IDT444) in conjunction with streptavidin-conjugated quantum dots. Flow cytometry and confocal microscopy were used to demonstrate our ability to visualize DAT transiently and stably expressed in mammalian cells. Specific labeling of DAT was apparent at low nanomolar concentrations, and its relative magnitude, as quantified by flow cytometry, increased in a dose-dependent manner. The IDT444 ligand was determined to have a binding affinity of 53.4 ± 1.0 nM, and its binding interactions with DAT were rapidly achieved and characterized by excellent stability. IDT444 can be immediately useful as a part of a quantum-dot based fluorescent assay to monitor DAT expression, function, and cellular localization and will ultimately be used to dynamically image DAT trafficking in living cells.

61) Bridget Anderson, "Design and Synthesis of Small Molecule Activators of Heme Sensing in S. aureus," A. Rose, B.R. Anderson, L.L. Anzaldi, O. Aranmolate, A.G. Waterson, P.R. Reid, E.P. Skaar and S.L. Mercer. Staphylococcus aureus is one of the most common causes of nosocomial infections and the leading cause of endocarditis in the United States. Bacterial resistance is on the rise; therefore, new therapies need to be developed in order to combat this threat to human health. Iron acquisition from heme is required for S. aureus proliferation within its host. Therefore, heme metabolism plays a major role in the progression of S. aureus infection. Staphylococcal virulence gene regulation is controlled by a signal transduction pathway named the heme sensor system (Hss). The HssRS system senses heme and activates expression of the HrtAB efflux pump, which maintains heme homeostasis. It has been shown that S. aureus is kept in a less virulent state when HssRS senses heme and up-regulates the expression of HrtAB. In keeping with this, small molecule modifiers of heme metabolism may be used as potential therapeutics against the bacterium. A high-throughput screen was performed to identify molecular activators of heme sensing. Of the lead compounds, VU0038882 is the most promising and is the current focus of our studies. Previous data support that a phenyl ring can be substituted for the napthyl ring and that a 2-hydroxyl is required for HssRS activation. Herein we describe the design and synthesis of ‘8882 analogs investigating hydroxyl group substitution around the phenyl ring. Pending biological results, these analogs will aid in target identification, providing a mechanistic explanation for how these small molecules activate heme sensing in S. aureus.

62) Laura Keigher, "Targeting iASPP: A New Strategy in the War on Cancer," Keigher, L, Rossanese, O. Phan, J. Olejniczek, E. Lee, T., Camper, D. and Fesik, S.  iASPP is the inhibitory member of the ASPP family.  iASPP binds to, and inactivates, p53 and p73, preventing transactivation of pro-apoptotic genes.  iASPP mRNA is known to be overexpressed in a number of cancers including breast, leukemia, lung, and pancreatic. One study has shown that releasing p73 from iASPP with a large peptide results in a significant increase in apoptosis. However, compelling data have not been produced to prove definitively that knocking out iASPP with a small molecule or by RNAi will result in a significant increase in apoptosis.  We are simultaneously exploring two aspects of iASPP’s relevance as an oncology target: 1) understanding the molecular mechanism of iASPP’s interaction with p53 and p73 and its role in regulation of apoptosis and 2) identification of small molecules that will bind to and inhibit iASPP. In the first objective, we will use RNAi to explore the consequences of removing iASPP from the p53 and p73 pathways and examine the resulting effects on cell growth, cell survival, and apoptosis.  The RNAi studies will first be conducted in breast cancer cell lines with wild type p53, p53 mutant, and p53 null statues to investigate the role of iASPP in the p53 and p73 pathways among breast cancers. For our second objective, we have cloned, expressed, and purified 15N and 13C labeled iASPP c-terminus. Currently, we are screening this protein by NMR against our fragment library and have identified 4 fragments that bind to iASPP in the same region that p53 binds iASPP.

63) Joel Musee, "Determinants of Prostaglandin H Synthase-2-Catalyzed Oxygenation of Endocannabinoid 2-Arachidonoylglycerol and the Development of a Novel Substrate Selective Inhibitor," Joel Musee, Andy Liedtke, Jashim M. Uddin, Kelsey C. Duggan, Daniel Hermanson, Kebreab Ghebreselasie and Lawrence J. Marnett. The endocannabinoid 2-arachidonoyl glycerol (2-AG) is a selective substrate for the inducible isoform of prostaglandin H synthase (PGHS), PGHS-2, in vitro. Its turnover leads to formation of glyceryl analogs of traditional prostaglandins (PG-Gs), a subset of which elicit agonism at unique receptors, at picomolar to nanomolar concentrations. Agonism by PG-Gs has been shown to counteract the analgesic effects of 2-AG, placing PGHS-2 at the pivot point of pain (action of PG-Gs) and analgesia (action of 2-AG at CB1 receptors). While both arachidonic acid (AA) and 2-AG are turned over comparably in vitro, it has become apparent that both substrates have differing requirements for the activation of their oxygenation by PGHS-2. The activation of oxygenase activity in COX is dependent on the turnover of the peroxide product of AA or 2-AG oxygenation (PGG2 and PGG2-G respectively). We hypothesized that PGG2-G is a less efficient POX substrate than PGG2 and therefore a poor activator of 2-AG turnover. We have previously demonstrated that PGG2 and PGG2-G surrogates (15-HpETE and 15-HpETE-G) are reduced comparably as peroxidase substrates. Here we extend this finding to in situ generated PGG2 and PGG2-G, demonstrating that both peroxidase substrates are reduced equally effectively by the peroxidase active site of hPGHS-2, only that PGG2-G is released poorly for reduction at the peroxidase active site, relative to PGG2. Translating these findings to the cellular milieu, stable knock-down of the phospholipid hydroperoxide glutathione peroxidase (PHGPx or GPx4) in 3T3 fibroblasts leads to a 2-4 fold increase in PG-G production in these cells, with little to no change in PG production. These findings have allowed us to develop a 2-AG specific inhibitor that acts on the peroxidase active site of PGHS-2 (the first of its kind), which could have enormous potential in tipping this balance toward analgesia and away from pain.

64) Katya V. Petrova, "Synthesis of the Four Stereoisomers of 2,3-Epoxy-4-hydroxynonanal, Potential Genotoxic Products of Lipid Peroxidation, and Their Reactivity with Deoxyguansoine." Katya V. Petrova, Ivan D. Kozekov, Donald F. Stec, Markus W. Voehler and Carmelo J. Rizzo. The reaction of 2,3-epoxy-4-hydroxynonanal (EHN), a proposed product of lipid peroxidation, with 2’-deoxyguanosine (dGuo) has been reported to give three types of exocyclic DNA adducts: unsubstituted 1,N2-e-dGuo, 7-(1,2-dihydroxyheptyl)-e-dGuo, and ring-closed derivative of  7-substituted e-dGuo  adduct. The epoxy aldehydes exists as a pair of diastereosomers and several studies have been carried out with partially separated, racemic syn and anti diastereoisomers of EHN. The absolute stereochemistry of these EHN-dGuo adducts was not determined. In the present work, we synthesized the four stereoisomers of EHN starting from (4R)- and (4S)-4-hydroxy-2-nonenal (HNE). The reaction of the individual EHN stereoisomers with dGuo afforded a single stereoisomers of 7-(1,2-dihydroxyheptyl)-e-dGuo and a small amount 1,N2-e-dGuo; the subsequent incubation of each 7-substituted e-dGuo adduct in buffer solution formed two of tetracyclic isomeric adducts. The relative stereochemistry of the tetracyclic adducts were consistent with selective NOE spectra and observed vicinal coupling constants.  The twelve stereiosomeric EHN-dGuo adducts were isolated and characterized by 1H NMR and circular dichroism spectroscopy. Reverse-phase HPLC conditions were developed that could separate most of the adduct mixture.

65) Suraj Adhikary, "Structural basis of DNA damage recognition and repair by Schizosaccharomyces pombe 3-methyladenine DNA glycosylase (Mag1)," Suraj Adhikary and Brandt F. Eichman. Maintenance of genomic integrity depends on the ability to properly repair DNA damage. Alkylpurine DNA glycosylases initiate the base excision repair (BER) pathway by recognizing and removing a broad spectrum of alkylated nucleobases from DNA that arise from exposure to endogenous methyl donors and metabolites, environmental toxins, and chemotherapeutic agents. Glycosylases catalyze the hydrolysis of the base-sugar glycosylic bond, although the precise catalytic mechanism as well as the mechanism of detection and discrimination of damaged bases from normal and other damaged bases by DNA glycosylases, is still largely unknown. Lesion detection by DNA glycosylases is presumed to arise from both active site complementarity with the target nucleotide as well as intercalating residues that probe the minor groove of DNA during a processive search. Mag1 shares extensive sequence similarity with 3-methyladenine glycosylases from S. cerevisiae and E. coli (MAG and AlkA, respectively). However, unlike MAG and AlkA, which recognize and excise a wide array of damaged nucleobases including 1-N6-ethenoadenine (εA), Mag1 has a relatively limited number of substrates that include 3-methyladenine, 7-methylguanine, and 3-methylguanine. Here we present structural and biochemical evidence that a residue outside of the active site can confer substrate specificity in DNA glycosylases. Coupled with mutational analysis of spMag1, scMAG, and the Mag homolog from Bacillus halodurans (bhMag), this study offers a new model of substrate recognition among structurally homologous enzymes with disparate specificities.

66)  Zachary Glaser, "Determination of High Affinity Ligand for Labeling hSERT Via Quantum Dots." The human serotonin transporter (hSERT) is implicated in several brain disorders. The monoamine theory postulates that disruption or malfunction of the central nervous system's serotonergic and noradrenergic systems attribute to the risk and/or symptoms of depression. hSERT has been linked to depression, which affects approximately 4.5% of the world's population. Rapid reuptake of serotonin (5-HT) from the synaptic cleft is speculated to result in symptoms of depression. The use of high affinity ligands conjugated to quantum dots (qdots) is one method used to label the hSERT protein, which could help evaluate the cause of depression. Qdot labeling of hSERT has had limited success due to the lack of an optimal ligand that accurately and specifically binds to hSERT4. RosettaLigand will be used to dock ligands that will be used for attachment of quantum dots into a hSERT homology model to improve qdot-conjugatd ligand affinity to the hSERT protein.

67) Stephen Chau, "Synthetic Studies on Cell-Selective Cytotoxic Macrolides." Ammocidin A, a 20-membered macrolide isolated from a soil microbe, is a potent inducer of apoptosis in Ras dependent hematopoietic cells. Ammocidins B-D were isolated in 2009 and shown to preserve much of the cell cytotoxicity of the parent macrolide (ammocidin A). Structurally, ammocidins B-D share the regions of unsaturation and the highly functionalized pyranose ring system. However, each differs modestly when compared to ammocidin A in glycosylation pattern at C24 and oxidation at C16. Currently, the absolute and relative stereochemistry for aglycone (ammocidinone) is unassigned but hypothesized by our group to be similar to apoptolidinone, a presumed biosynthetic relative.  Additional support for the proposed stereochemistry is provided in the forum of unpublished NMR data. With this background we propose a total synthesis of ammocidin D and completion of its structural assignment.

68) Jijun Hao, "WinD, a Bioactive and Selective Small Molecule Inhibitor of Wnt/β-catenin Pathway," Jijun Hao, Morgan Webb, Li Zhou, Kaleh Karim, Clare Murphy, Jessica Keel, Kelly Christian, Ethan Lee and Charles Hong. Canonical Wnt/β-catenin signaling pathway plays pivotal roles in regulating cell fate, tissue homeostasis and regeneration. Aberrant activation of Wnt signaling is implicated in variety of important diseases such as gastrointestinal cancers and cardiac hypertrophy.  Therefore, selective small molecule inhibitors of Wnt/β-catenin signaling have been highly sought-after as potential therapeutic leads. Using zebrafish as a screening platform, we recently discovered a bioactive and very selective small molecule inhibitor (WinD) of Wnt/β-catenin pathway, based on its ability to perturb embryonic dorsoventral axis. Our study demonstrates that WinD targets very distal component of Wnt/β-catenin cascade in the nuclei, possibly the C-terminus of β-catenin.

69)  Louesa Akin, "Scoring With NOE Restraints Improves Protein Structure Prediction Results."  Computational modeling can provide a way for structural biologists to predict protein structures when traditional experimental methods, such as X-ray crystallography and NMR, fail. A template-free 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. NOEs are a form of NMR data that provide distance restraints between two protons within a protein molecule. In order to incorporate these restraints into the current BCL::Fold framework, a scoring function was 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 structure of a benchmark protein 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. 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.







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