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2012 Student Research Symposium

When:  Thursday, August 9, 8:00 am to 5:00 pm

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


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

Guest speaker


Dr. James Chen, Stanford University, Department of Chemical and Systems Biology
"Chemical Probes of Embryonic Signaling and Patterning"

 
raffle Prize: 

Winner: Daniel Putman (view photos)

 
Awards

VICB Prize in Chemical Biology: (view photos)
Paul Barrett

Certificate in Chemical Biology Recipients:
(view photos)
Joseph Manna
Sean DeGuire
Adam Ketron

Oral Presentation Awards: (view photos)
Jamie Wenke
Neal Hammer
Mark Dobish

Poster Awards: (view photos)
Renee Raphemont
Sarah Schuck
Will Birmingham
Mike Danneman
Rebecca Sandlin
Marta Szulik
Kersten Davis
Lynn Samuleson

 
Agenda

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: James Chen, Stanford University: "Chemical Probes of Embryonic Signaling and Patterning"
9:00 A.M. – 10:00 A.M.

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

Oral Presentations
11:00 A.M. – 12:00 P.M.

Lunch/Poster Viewing
12:00 P.M. – 1:00 P.M.

AFTERNOON SESSION

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

Poster Session B
2:00 P.M. – 3:00 P.M.

Oral Presentations
3:00 P.M. - 4:10 P.M.


Student Awards & Raffle Prizes
(must be present to win)
4:10 P.M. – 5:10 P.M.



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

 


 

2012 Keynote Video
2012 Presentations
2012 Poster Session

Oral Presentation & Poster Abstracts

Oral Presentation Abstracts

1) Greg Pask, “Exploring Insect Odorant Receptor Function Through the Use of Novel Chemical Modulators,” Gregory M. Pask and Laurence J. Zwiebel

The detection of olfactory cues from the environment is largely mediated through large families of odorant receptors (ORs). While ORs in many animals act as GPCRs, insect ORs function as odorant-gated ion channels. Here, a functional OR complex consists of a conventional OR and an extraordinarily conserved co-receptor, Orco, in an unknown stoichiometry. A novel Orco agonist, VUAA1, has recently been identified and utilized to investigate the mechanistic properties of insect OR function and demonstrate that Orco subunits from various insect orders can form functional homomeric channels. VUAA1 was also used to examine the influence of conventional ORs on the ion channel pore, where apparent differences in ion permeability and susceptibility to channel blockade were observed. The exploration of the chemical space around VUAA1 yielded several analogs, and one of which not only acts as a competitive antagonist to VUAA1, but also as an allosteric, noncompetitive antagonist to odorant-mediated activation. Through the continued use and advancement of this novel class of modulators, we can continue to gain more insight into the structure-function relationship of insect ORs, as well as potentially develop chemical deterrents against disease vectors and agricultural pests.

2) Jamie Wenke, “Deciphering the Spatial Distribution of the Lens Membrane Protein AQP0 Modifications,” Jamie L. Wenke, David B. Friedman, Kevin L. Schey

Aquaporin 0 (AQP0) is a lens membrane protein that is known to undergo a high degree of modification with lens age. Some of these modifications are known to alter the permeability of the water channel or to target AQP0 to different regions of the cell membrane. The overall goal is to learn where the modified forms of AQP0 are spatially localized within the lens in order to understand how these modifications contribute to normal lens aging or cataract formation. To accomplish this task, we demonstrate two complementary techniques for quantitative spatial analysis of AQP0. MALDI imaging is employed to examine the distribution of modified AQP0. These ion images show a unique localization for different forms of AQP0 and demonstrate the feasibility of imaging intact and modified membrane proteins in the human lens. We also used morphology-directed Capture/LC-MS/MS for quantitative, site-specific analysis of AQP0 modifications. The MS-compatible staining used with this technique allows for microdissection of lens regions with a specific morphology in order to correlate AQP0 modifications to changes in membrane morphology. These complementary techniques shed light on the distribution of AQP0 modifications across the lens.

3) Elizabeth Dong, “Structural studies of the interaction between mGlu5 and allosteric modulators,” Elizabeth Nguyen Dong, Karen J. Gregory, Christoffer Norn, Kristian W. Kaufmann, P. Jeff Conn and Jens Meiler

The metabotropic glutamate receptor subtype 5 (mGlu5), a class C G-protein coupled receptor (GPCR), is involved in mammalian cognitive function through diverse signaling pathways that modulate synaptic plasticity. Selective modulators of mGlu5 have exciting potential for the development of novel treatment strategies for disorders that disrupt cognitive function. Because the orthosteric binding site is well conserved between all subtypes of mGlus relative to other regions on the receptor, an approach to selectively target mGlu5 is to identify ligands with allosteric binding sites. Positive allosteric modulation of mGlu5 activity could provide a novel treatment strategy for schizophrenia, while negative allosteric modulation could lead to improved cognitive function for patients with Fragile X Syndrome. Identifying specific residues on mGlu5 that contact these allosteric modulators would provide a deeper understanding of the binding interaction and aid in the development of such therapeutic compounds. While there is no high resolution structure of the transmembrane region of mGlu5, comparative structural models of GPCRs built with Rosetta are shown to model ligand binding poses within atomic resolution for all 14 GPCRs in the Protein Data Bank. A comparative model of mGlu5 using GPCR crystal structures as templates have been shown to accurately predict critical residues for allosteric modulation. The alignment between mGlu5 and 14 GPCRs was used to model the 7 transmembrane helices. Side chain and loop optimization was then carried out with Rosetta Membrane. Residues of mGlu5 critical for the binding of allosteric modulators were predicted using Rosetta Ligand docking studies and validated by experimental functional data. Selective targeting of mGlu5 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 mGlu5 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.

4) Neal Hammer, “Heme biosynthesis and pigment production enable metabolic flexibility required for Staphylococcus aureus pathogenesis,” Neal D. Hammer, Michelle L. Reniere, Amanda O. Hirsch James E. Cassat, M. Indriati Hood, and Eric P. Skaar

Two defining features of Staphylococcus aureus are the production of the pigment staphyloxanthin, which gives the bacteria its characteristic golden color, and the ability to form respiratory-arrested small colony variants (SCVs). SCVs arise through inactivation of respiratory pathways including defects in heme biosynthesis. We show that S. aureus heme biosynthesis powers a branched aerobic respiratory chain that is required for full virulence. The requirement for aerobic respiration during infection can be exploited through non-iron metalloporphyrins which act as small molecule respiratory inhibitors that induce the SCV phenotype. Finally, we demonstrate that treatment of S. aureus with both non-iron metalloporphyrins and a staphyloxanthin inhibitor severely impairs bacterial growth. These data support the conclusion that the metabolic plasticity afforded by both heme biosynthesis and pigmentation allows S. aureus to persist within the host.

5) Nick Adams, “Oligonucleotide probe design for label-free RNA biomarker detection using backscattering interferometry”

Backscattering interferometry has been shown to successfully monitor binding interactions of a variety of biological molecules with high sensitivity. The method works by analyzing high contrast interference fringes of light refracted from a microfluidic channel containing an analyte solution. When a binding event occurs between a molecule and a target analyte, the refractive index of the solution in the channel changes, causing the fringes to shift in a manner that is proportional to the concentration of the analyte. Backscattering interferometry has been used to successfully perform binding studies for a variety of molecular interactions but has never before been used for molecular detection studies. We have investigated the sensitivity of the method for detecting a specific RNA biomarker sequence using unique nucleic acid probes. Shifts in the refractive index of an analyte solution are generated by changes in molecular conformation, hydration, and charge density upon hybridization of oligonucleotide probes to RNA targets. To maximizing the sensitivity of backscattering interferometry for RNA detection, we have investigated how oligonucleotide probe design characteristics affect the refractive index upon binding an RNA target. We found that the oligonucleotide length, the number of distinct sequences, and the nucleoside conformation of the probes have substantial effects on the signal generated upon binding the RNA target. We have also observed that the hybridization of different sequences of oligonucleotide probes that are of the same length and GC content produce very different signal as detected by backscattering interferometry. We hypothesize that the shift in refractive index is proportional the degree of structural perturbation of the RNA target molecule upon binding the oligonucleotide probe. Future studies aimed to elucidate this sequence-dependent basis of signal generation will include nucleic acid structure analysis and thermodynamic studies of the RNA-probe hybrids.

6) Mark Dobish, "Strategies in Asymmetric Organocatalysis: Applications to Small Molecule Therapeutics and Enantioselective Iodolactonizations"

Herein, we report the use of enantioselective bromonitromethane additions to arylaldimines towards the pharmaceutical target (+)-VNI. A seven step, scalable synthesis, has produced gram quantities of the drug for testing, with the key step producing up to 20 grams of enantiopure product. Preliminary mouse data shows that (+)-VNI treats acute and chronic Chagas disease, by inhibiting CYP51 in Trypanosoma cruzi. The proposed route offers multiple points for diversification towards more effective drug candidates, which has produced a variety of small molecules for testing. Additionally, we report the development of highly enantioselective iodolactonizations that employ a chiral proton catalyst–N-iodosuccinimide (NIS) reagent. An approach that modulates the achiral counterion to optimize the enantioselection is documented for the first time in this transformation. In this way, unsaturated carboxylic acids are converted to γ-lactones in high yields and up to 98% ee.

VICB Prize Winner
7) Paul Barrett, "Structural and Cholesterol Binding Properties of the Amyloid Precursor Protein and the Etiology of Alzheimer's Disease," Paul J. Barrett, Yuanli Song, Wade D. Van Horn, Eric J. Hustedt, Johanna M. Schafer, Arina Hadziselimovic, Andrew J. Beel, and Charles R. Sanders

The amyloid precursor protein (APP) is a transmembrane protein that is implicated in the onset of Alzheimer's disease (AD). The biological role of APP is not well understood, leading to many questions concerning its role in AD pathogenesis. One potential interaction that could impact AD onset is the binding of cholesterol by APP, as it has been shown that there is an epidemiological correlation between elevated levels of cholesterol and occurrence of AD. It is known that APP is subject to alternative pathways of proteolytic processing; one pathway involves the enzymes α and γ secretase and produces benign fragments. The other pathway involves the enzymes β and γ secretase and the resulting fragment, commonly known as the Aβ peptide, is believed to cause Alzheimer’s disease. Cleavage of APP by β-secretase results in the formation of the transmembrane fragment referred to as C99 (or CTF-β), which is the subject of these studies. We not only present the novel structure of C99 determined by Nuclear Magnetic Resonance (NMR) spectroscopy, but we also show a novel mechanism for binding cholesterol via an unanticipated cholesterol binding motif. NMR and EPR spectroscopy demonstrated that the extracellular N-terminus of C99 includes a surface-embedded “N-helix” followed by a short “N-loop” connecting to the transmembrane domain (TMD). The TMD is a flexibly-curved 24 residue α-helix, making it well-suited for processive cleavage by γ-secretase. A combination of cholesterol titration and point mutagenesis studies revealed a binding site for this lipid, which offers a mechanistic explanation for the promotion of Aβ production by cholesterol. Membrane-buried GxxxG motifs were shown to play key roles in cholesterol binding via mutagenesis studies, revealing an alternative function for some of these motifs beyond their well-established role in membrane protein dimerization. The 3D structure determination and cholesterol studies have allowed for the formulation for new hypotheses as to how the cleavage of APP is regulated. Future studies involving confocal fluorescence microscopy and in vitro γ-secretase assays will test these hypotheses and ultimately generate new information on the cascade of events that lead to the development of AD.



Poster Presentation Abstracts

Poster presentation abstracts will be posted as they become available.

1) Dan Liu. Target LZAP for the Treatment of HNSC, Dan Liu, Natalia Issaeva, and Wendell G. Yarbrough
Head and Neck Squamous Cell Carcinoma (HNSCC) accounts for more than 90% of all head and neck cancers and is the sixth most common cancer worldwide. Exploring molecular contributors to HNSCC, our lab have previously shown that LZAP protein expression is decreased or lost in approximately 30% of HNSCC. Loss of LZAP has been associated with increased anchorage independent growth, cellular invasion, and xenograft tumor growth, suggesting that LZAP is a putative tumor suppressor. However the mechanism(s) of LZAP tumor related activities require further exploration.
We previously observed that knockdown of LZAP results in downregulation of p53, one of the most important known tumor suppressors and mutated in about 50% of all human cancers. Demonstration of functional effects of LZAP on p53, would further link the newly discovered tumor suppressor, LZAP, to the well-established p53 pathway, and will provide new insights into LZAP tumor suppressive functions while establishing a new mechanism of p53 regulation. We have shown that LZAP depletion significantly attenuates p53 protein expression, accompanied by decreased p53 phosphorylation. We are currently investigating molecular mechanisms of p53 protein destabilization upon LZAP depletion.
Chemo- and radio-therapies are two most common treatments to cancer patients by introducing DNA damage to tumor cells. Thus, we are investigating how LZAP downregulation affects cellular response to DNA damage, in particular to radiation and chemotherapeutic drugs. Clonogenic survival and proliferation assays revealed that depletion of LZAP in cells with wild-type p53 expression resulted in increased resistance to radiation and several DNA damaging drugs. However, downregulation of LZAP in cells lacking p53 or expressing mutant p53 sensitized cells to DNA damage.
To examine the role of LZAP in a more physiologically intact environment, we turned to a genetically engineered mouse model. Due to the embryonic lethality of homozygous loss of LZAP, the role of LZAP in regulating radiation response was tested using LZAP heterozygous mice (LZAP+/-). Bone marrow cells were isolated from LZAP+/- mice following irradiation and grown ex vivo. Bone marrow derived cells from irradiated LZAP+/- mice formed significantly higher number of colonies when compared to bone marrow cells from irradiated LZAP+/+littermates. These data are in agreement with in vitro clonogenic survival data and suggest that loss of even one allele of LZAP protects cells from DNA damage.
Together, these data suggest that loss of LZAP results in inhibition of cellular response to DNA damage that is, at least partially, mediated through destabilization of p53 with resultant decreased p53 activity. These studies describe a potential new regulatory mechanism altering p53 stability and activity. LZAP depletion protects cells expressing wild-type p53 from radiation and chemotherapeutic drugs, while sensitizing cells without p53 expression or expressing mutant p53 to the same treatment. This remarkably combination of activities suggest that methods or drugs with LZAP inhibitory properties may be useful for treating p53 mutant or null cancers while simultaneously protecting normal tissues to DNA damaging therapeutic agents.

2) Li Liang. Sequence-Dependent Structural Perturbations Induced by the b-Anomer of the Aflatoxin B1 Formamidopyrimidine (FAPY) Adduct in DNA, Li Liang, Kyle L. Brown, Michael P. Stone
Aflatoxin B1 (AFB1) is the predominant mutagenic fungal metabolite produced by Aspergillus flavus. The  genotoxic metabolic product, AFB1 epoxide, alkylates DNA regioselectively at N7-dG.  The initially formed N7-dG adduct may subsequently rearrange to a N7-dG formamidopyrimidine (FAPY) derivative. In DNA, the AFB1-FAPY derivative equilibrates between α and β deoxyribose anomers.  Using HPLC, we examined the hypothesis that the equilibrium of the α and β deoxyribose anomers of the AFB1-FAPY modified dG is dependent on the 5′- and 3′-neighbor nucleotides. We compared the 5′-AXC-3′, 5′-AXT-3′ and  5′-AXA-3′ sequences (X= AFB1-FAPY lesion), as well as the 5′-AXdeazaA-3′ and 5′-TXdeazaA-3′ sequences. The 5′-AXA-3′ sequence significantly shows a slower equilibrium rate between the α and β anomers, whereas the 5′-AXC-3′ and 5′-AXT-3′sequences show faster rate. Changing the dA to deazaA at the 3′-neighbor nucleotide didn’t change the equilibrium rate. This might be attributed to the sterical effect on the neighbor base that relatively bigger purine can decrease the equilibrium rate between the α and β deoxyribose anomers.  Supported by NIH grant R01 CA-55678 (M.P.S.).

3) Keersten Davis. Development of a “Switch-On” Iridium (III) Probe for the Detection of the Malarial Biomarker Plasmodium Falciparum Histidine Rich Protein II, Keersten M. Davis, Anna L. Bitting, David W. Wright
Delivery of diagnostic tools to low resource settings faces numerous challenges in areas where it is difficult to reach populations that are distributed sparsely over rural areas. Health workers dispatched from centralized facilities face difficult terrain, intermittent/lack of electricity, poorly equipped facilities, 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 affordable. In the case of malaria, blood smear microscopy is often impractical, while recent evaluations of current rapid diagnostic tests (RDTs) highlight significant variability in their efficacy. To counter these challenges we have begun to develop a highly selective iridium(III) “switch-on” probe for the detection of the malarial protein biomarker Plasmodium falciparum Histidine Rich Protein II (pfHRPII). We report a non-emissive, cyclometalated Ir(III) complex, Ir(ppy)2(H2O)2+ (Ir1), which upon binding histidine/histidine containing peptides, elicits a fluorescent signal in aqueous buffer.  The activity of Ir1 with numerous amino acids was examined; however, only the addition of histidine resulted in a 4 order of magnitude enhancement in intensity over that of the other amino acids.  Histidine containing peptides, such as BNT-II, a branched peptide mimic of pfHRPII, repeats, also triggered a fluorescent response.  By coupling this metal-based fluorescent probe into a previously reported self-contained pfHRPII extraction and purification device, we propose to create a “glowstick” diagnostic tool for pfHPRII detection.

4) Steven Coombs. Partial Covalent Interactions in Protein Design, Steven A. Combs, Jens Meiler
Partial covalent interactions (PCI) such as hydrogen bonds, salt bridges, cation-π, and π-π interactions contribute to protein thermostability. Algorithms that identify PCIs rely on pairwise atom-atom angles and distances, neglecting that PCIs occur between an electron deficient hydrogen and an electron orbital of the accepting atom. Accurate chemical representation of PCIs will improve protein thermostabilization via computational protein design. We have introduced orbital based chemical descriptors for PCIs into the Rosetta Suite of Protein Structure Prediction Algorithms. Native-like geometries of hydrogen bonds, salt bridges, cation-pi, and pi-pi interactions are recapitulated during minimization of protein conformation. Additionally, the accurate chemical definition of PCI increases sequence recovery by 9% in RosettaDesign benchmarks.

5) Ewa Kowal. Stabilization of Mutagenic O6-Benzyl-2’-deoxyguanosine Adduct by a Perimidinone Derived Synthetic Nucleoside by Unique Stacking Interaction, E. A. Kowal, R. R. Lad, P. S. Pallan, E. Muffly, Z. Wawrzak, M. Egli, S. J. Sturla, M. P. Stone.
O6-alkyl-2'-deoxyguanosine is an alkylation product of guanine by nitrosamines, that lead to G to A transition mutations during replication. In the crystal structure of the bulky O6-alkylguanine adduct O6-BnG with Dpo4 polymerase, when paired opposite cytosine in DNA duplex, forms a Wobble base pair, and thermal analysis suggests destabilization relative to GC base pair. However, it was shown that incorporation of the perimidinone derived synthetic nucleoside (dPer) opposite the O6-BnG adduct stabilizes the duplex, but no structural information has been available to elucidate the origin of stability. In this study, the crystal structure of a Dickerson-Drew dodecamer duplex containing the modified O6-BnG:dPer base pair is presented. The data show that O6-BnG does not form Watson-Crick hydrogen bonds with dPer, and exhibits a stacking interactions within the duplex. The presentation will discuss the implications for how alkylation and hydrophobic synthetic probes influence base pairing interactions in duplex DNA.

6) Lilu Guo. Lipid Peroxidation Generates Bioactive Aldehyde-modified Phosphatidylethanolamines, Lilu Guo, Zhongyi Chen, Venkataraman Amarnatm, Sean S. Davies
Oxidative stress has been implicated as a major mediator of chronic diseases including atherosclerosis, diabetes, neurodegenerative disorders, and various types of cancers. Although oxidative stress generates a wide range of downstream compounds, some of the most injurious are reactive lipid aldehydes formed during lipid peroxidation. These aldehydes are known to have pro-inflammatory and cytotoxic effects, but the mechanisms underlying these biological functions remain poorly understood. Protein and DNA modifications have been speculated as major pathways by which the damaging effects were exerted. However, our recent studies found that phosphatidylethanolamine (PE) is also a major target of these aldehydes. In this study, we demonstrated that oxidative stress-mediated generation of aldehyde-modified PEs (al-PEs) induced endothelial activation, expression of adhesion molecules and chemokine secretion, all of which are key steps in the initiation of inflammation. To systematically characterize lipid peroxidation products, we constituted oxidized liposome models and identified a novel family of al-PEs including N-hexanoyl PE, glutaryl-PE, oxidized HNE-PE, and IsoLG-PE. In addition, we found that modified al-PEs localized to the ER membranes to activate ER-stress signaling pathways. These results demonstrate that lipid peroxidation is an important cellular process under oxidative stress conditions and bioactive al-PEs are an entirely new class of proinflammatory lipid mediators via ER stress pathways. Such a finding will allow the development of targeted approaches to reduce their levels and thereby prevent the resulting disease.

7) Jamie Wenke, Deciphering the Spatial Distribution of the Lens Membrane Protein AQP0 Modifications, Jamie L. Wenke, David B. Friedman, Kevin L. Schey
Aquaporin 0 (AQP0) is a lens membrane protein that is known to undergo a high degree of modification with lens age. Some of these modifications are known to alter the permeability of the water channel or to target AQP0 to different regions of the cell membrane. The overall goal is to learn where the modified forms of AQP0 are spatially localized within the lens in order to understand how these modifications contribute to normal lens aging or cataract formation. To accomplish this task, we demonstrate two complementary techniques for quantitative spatial analysis of AQP0. MALDI imaging is employed to examine the distribution of modified AQP0. These ion images show a unique localization for different forms of AQP0 and demonstrate the feasibility of imaging intact and modified membrane proteins in the human lens. We also used morphology-directed Capture/LC-MS/MS for quantitative, site-specific analysis of AQP0 modifications. The MS-compatible staining used with this technique allows for microdissection of lens regions with a specific morphology in order to correlate AQP0 modifications to changes in membrane morphology. These complementary techniques shed light on the distribution of AQP0 modifications across the lens.

8) Janel McLean, Mining the Ubiquitinome for Endocytic DUB Substrates, Janel R. McLean, Ilektra Kouranti, Kathleen L. Gould
Deubiquitinating enzymes (DUBs) are ubiquitin (Ub) proteases that counteract E3 Ub ligases, resulting in modulation of cell signaling through changes in protein stability, localization, and/or binding partners and maintenance of Ub homeostasis. We recently reported a global study of fission yeast (Schizosaccharomyces pombe) DUBs, defining their intracellular localization, enzymatic activity/specificity, and protein binding partners (Kouranti et al. 2010 PLoS Biology 8(9)). Here we have taken a proteomics approach to DUB substrate discovery using a mutant strain containing multiple DUB deletions (5DUB delete= Δubp4Δubp5Δubp9Δubp15Δsst2) that accumulates Ub conjugates. We overexpressed His-biotin-His-Ub in wildtype and 5DUB delete fission yeast cells, performed two-step denaturing purifications in triplicate, and used 2D-LC-MS/MS to identify ubiquitinated proteins. Spectra with > 20 peaks were removed and files were converted to mzml format using Scansifter (Ma et al. 2011 Bioinformatics. 27(22)), and searched using TagRecon v1.2.32 (Dasari et al. 2010 J Proteome Res. 9(4)) and the S. pombe protein database from (http://www.pombase.org, May 2011) with decoy sequences (10354 entries). Peptide matches were assembled and filtered (FDR = 0%) in IDPicker v2.6.165 (Ma et al. 2009 J Proteome Res. 8(8)). Protein lists were further refined in Excel to include only proteins identified in at least 2 out of 3 biological replicates of the 5DUB delete strain with 2 unique peptides or more in each experiment. We then normalized spectral counts to the bait (Ub) and compared wildtype with the mutant DUB deletion strain to identify proteins enriched in the mutant DUB strain. Using the above criteria, we identified over 250 proteins that are enriched in the multiple DUB deletion strain (i.e. putative DUB substrates). Approximately half of all the Ub’d proteins identified using this scheme have not previously been identified as Ub-conjugates, highlighting the strength of this experimental strategy. Using annotations found in Pombase, we determined that these putative DUB substrates are localized to active sites of endocytosis and other cellular compartments including the ER, Golgi, nucleus, and mitochondria. Using manually curated GO annotations, we characterized the cellular functions of the enriched proteins and found over 30 transmembrane transporters as well as multiple unexpected endocytic players. We also discovered many of these putative substrates play roles in cell polarity, cytokinesis, lipid metabolism and secretion/vesicular traffic. To better define the regulatory circuits with which the putative DUB substrates are associated, we mapped their published physical interactions (BIOGRID) and found pathways involved in DNA replication, translation, cell polarity, actin regulation, cytokinesis and 14-3-3 proteins. We also used high resolution quantitative mass spectrometry to map Ub chain linkages in wildtype and the mutant strain. These results showed a significant enrichment of K11, K29, K48 and K63 Ub linkages, suggesting that these DUBs impact many modes of Ub signaling.

9) Lynn E. Samuelson, "Self-Reporting Dendritic Nanoparticles (Nanodendrons) for Drug Delivery Targeted to the Tumor Microenvironment and with Reduced Neurotoxicity," Lynn E. Samuelson, Randy L. Scherer, Kathy J. Carter, E. Ashley Dozier, Sheree Printz, Michael N. VanSaun, Paul E. Matrisian, Kang-Hsien Fan, Yu Shyr, Darryl J. Bornhop, Lynn M. Matrisian and J. Oliver McIntyre
Proteinases, including matrix metalloproteinases (MMPs), contribute to cancer progression and other pathologies. Selective MMP expression can be used to distinguish benign from malignant tumors and identify aggressive tumors associated with poor outcome. MMP9, a basement membrane-degrading type-IV collagenase/gelatinase, is associated with tumor invasion and metastasis. In this project, we describe a new class of dendritic nanoparticles, nanodendrons (NDs), with MMP molecular recognition and targeting capabilities. These NDs can be studied as individual dendrons tuned for specific functions such as enhanced imaging or targeted drug treatments. Additionally, the NDs can be coupled to facilitate multifunctional purposes such as in NDs that can self-report drug delivery to tumors. The prototypical system presented here describes NDs that are activated by MMP9: 1) NDPB, a near infrared imaging beacon; 2) NDPXL, a therapeutic that delivers paclitaxel (PXL) and 3) NDPB-NDPXL, a bi-functional agent. In vivo studies in two orthotopic models of breast cancer demonstrate efficacy of these NDs to image and treat breast cancer. The proteinase-activated prodrug, NDPXL, delivers PXL to breast cancer through release of the drug in the tumor microenvironment and increases therapeutic efficacy while reducing systemic toxicity (including peripheral neuropathy). The delivery of PXL using the proteolytically activated NDPXL is effective in inhibiting tumor growth in two orthotopic models of breast cancer (PyVT-R221A and MDA-MB231). Daily treatment of MDA-MB231 tumors with 12.5 mg/kg PXL as either NDPXL or Abraxane® (Abx), showed similar reduction in tumor growth as compared with vehicle-treated animals. Further investigation of the NDPXL in a fully immunocompetent mouse model (PyVT-R221A) with treatments given on alternate days at a dose of 12.5 mg/kg (NDPXL or Abx) yielded similar results: an average reduction in tumor growth of 58% and 53% in NDPXL and Abx cohorts, respectively. Peripheral nerve toxicity, a debilitating, long term side effect of Abx therapy, was assessed in both tumor and non-tumor mice through monitoring behavior indicative of peripheral nerve damage before, during and after administration of each drug. Peripheral neurotoxicity is markedly reduced in the NDPXL-treated versus Abraxane®-treated mice as evident in a number of behavioral assessments. The development of this novel class of NDs expands upon the current capabilities of modern proteinase-based optical beacons and prodrugs and is a step forward in treatment of both primary and metastatic cancer. [Supported in part by Susan G. Komen for the Cure®]

10) Elizabeth Dong, "Structural studies of the interaction between mGlu5 and allosteric modulators," Elizabeth Nguyen Dong, Karen J. Gregory, Christoffer Norn, Kristian W. Kaufmann, P. Jeff Conn and Jens Meiler

The metabotropic glutamate receptor subtype 5 (mGlu5), a class C G-protein coupled receptor (GPCR), is involved in mammalian cognitive function through diverse signaling pathways that modulate synaptic plasticity. Selective modulators of mGlu5 have exciting potential for the development of novel treatment strategies for disorders that disrupt cognitive function. Because the orthosteric binding site is well conserved between all subtypes of mGlus relative to other regions on the receptor, an approach to selectively target mGlu5 is to identify ligands with allosteric binding sites. Positive allosteric modulation of mGlu5 activity could provide a novel treatment strategy for schizophrenia, while negative allosteric modulation could lead to improved cognitive function for patients with Fragile X Syndrome. Identifying specific residues on mGlu5 that contact these allosteric modulators would provide a deeper understanding of the binding interaction and aid in the development of such therapeutic compounds. While there is no high resolution structure of the transmembrane region of mGlu5, comparative structural models of GPCRs built with Rosetta are shown to model ligand binding poses within atomic resolution for all 14 GPCRs in the Protein Data Bank. A comparative model of mGlu5 using GPCR crystal structures as templates have been shown to accurately predict critical residues for allosteric modulation. The alignment between mGlu5 and 14 GPCRs was used to model the 7 transmembrane helices. Side chain and loop optimization was then carried out with Rosetta Membrane. Residues of mGlu5 critical for the binding of allosteric modulators were predicted using Rosetta Ligand docking studies and validated by experimental functional data. Selective targeting of mGlu5 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 mGlu5 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.

11) William Birmingham, "Directed evolution of non-natural nucleoside analog drug biosynthesis from a dideoxyribose precursor," William R. Birmingham, David P. Nannemann, Timothy D. Panosian, Michelle M. Mitchener, Tina M. Iverson, Brian O. Bachmann.

Nucleoside analog drugs are mainstays in the treatment of viral infections but they are also expensive to manufacture, with up to 99% of treatment costs stemming from direct costs. Herein we describe a strategy to produce dideoxynucleosides using E. coli via a bioretrosynthetically engineered three step pathway. Having completed evolution of the product forming enzyme, purine nucleoside phosphorylase, we are currently engineering phosphopentomutase from Bacillus cereus, the penultimate enzyme, to continue the bioretrosynthesis paradigm. Phosphopentomutase catalyzes the interconversion of α-D-ribose 5-phosphate and α-D-ribose 1-phosphate. This simple transformation that activates ribose for nucleoside biosynthesis via a nucleoside phosphorylase can also be utilized to activate the non-natural sugar 2,3-dideoxyribose 5-phosphate for dideoxynucleoside biosynthesis. Analysis of phosphopentomutase substrate co-crystal structures suggested multiple residues as potential modulators of substrate binding. Targeted saturation mutagenesis of these residues generated variants with up to >500-fold change in substrate specificity compared to the wild-type enzyme. Subsequent evolution by random mutagenesis has increased phosphopentomutase activity in cell free extract. The engineered phosphopentomutase, combined with the previously engineered purine nucleoside phosphorylase in vitro, shows capable production of the nucleoside analog reverse transcriptase inhibitor 2’,3’-dideoxyinosine (ddI, Videx®), a nucleoside analog currently prescribed as treatment for HIV. Furthermore, we report the in vitro production of dideoxyinosine using the complete three step biosynthetic pathway beginning from the precursor 2,3-dideoxyribose. Once fully optimized, this biosynthetic pathway may offer a complementary or possibly an entirely alternative route for production of dideoxynucleosides, with the potential to be additionally tailored to generate other clinically relevant nucleoside analogs.

12) Daniel Putnam, "SMALL ANGLE X-RAY SCATTERING RESTRAINTS TO PROMOTE PROTEIN FOLDING," Daniel Putnam, Brian Weiner, Jens Meiler

Small angle x-ray scattering (SAXS) is an experimental technique used to create low resolution (50 Å to 10 Å) representations of protein structures in solution. Using BCL::Fold, a protein folding algorithm based on the placement of secondary structure elements (SSEs), I created an algorithm BCL::SAXS to convert x-ray crystallographic atomic coordinates to a SAXS scattering profile. The purpose of this research is to develop a method to 1) efficiently calculate SAXS curves from rigid body protein models and 2) calculate saxs curves for proteins containing idealized secondary structure elements, such as those produced by BCL::Fold. The combination of SAXS information with computational methods may be used as a restraint during minimization to enhance native like sampling. First I present a method for the calculation of accurate SAXS curves based on the Debye formula and a set of scattering form factors. The resulting curves are on par with CRYSOL, the gold standard for SAXS curve calculations, and can recognize native structures among native-like decoys. Then I present a method for generating SAXS profiles for protein models without explicit side chains, by placing the combined form factor calculation of the side chains at the C-β position of the residue. The curves generated with missing side chains are very similar in morphology to the full atom curves and can filter native-like protein models.

13) Parimal Samir, "Carbon Source Alters the Protein Composition of Yeast Ribosomes," Parimal Samir, Christopher Browne, Andrew J Link

The ribosome is the catalytic engine that drives translation. It has been assumed to have limited regulatory function or mRNA specificity. Recent studies have suggested that there are, in fact, classes of ribosomes that differ in their ribosomal protein (RP) composition. We hypothesized that sub-populations of ribosomes exist in a state of dynamic equilibrium. This equilibrium would shift towards a new state upon environmental or growth cues. We reasoned that the equilibrium of ribosome populations would be reflected in the RP composition in the purified ribosomes. To test this hypothesis, we quantitated the RP composition of S. cerevisiae in both purified ribosomes and whole cell extracts (WCE) grown in either glucose or glycerol. iTRAQ labeling followed by mass spectrometry was used to precisely quantify the RP. We identified five RPs that are differentially present in the ribosomes depending on the carbon source. Interestingly, these RPs have paralogs. To study the dynamic changes in ribosome composition, we shifted yeast growing in glucose to glycerol and quantified the changes in the RP over time. To test if distinct RP paralogs are required for the translation of specific mRNAs, we quantified the proteome of the null mutants for paralog genes that change in the ribosome population. Our data suggest that changing the equilibrium of ribosome populations may serve as a translational control mechanism.

14) Pedro Teixeira, "Elucidation of Membrane Protein Structure and Analysis of SNP Effects Using Correlation Analyses"

Protein structure classically consists of four divisions, from primary to quaternary. These classifications only take into account the static 3-dimensional structure of a protein. However, it is possible with mathematical analysis of multiple sequence alignments to detect correlations between amino acid sites and compile those into pairwise amino acid site couplings. Further analysis of these couplings elucidates discrete and nearly independent correlation networks comprised of contiguous amino acids in a protein’s 3-dimensional structure. Pairwise couplings and correlation networks provide valuable spatial information that simplifies the de novo protein folding problem. Initial results using our folding program BCL::Fold and the calculated pairwise couplings show an enrichment of native-like topologies within the set of generated models. In addition, recent literature has shown such information to be highly beneficial for membrane structure prediction. Membrane proteins structures are especially difficult to determine with traditional methods but nearly half of pharmaceutical targets include a membrane domain. Furthermore, these correlation networks or “protein sectors” imply strong functional significance for the contained amino acid sites. We believe it is possible to apply this knowledge of evolutionary entanglement between sites and the solved structure to improve current methods that evaluate a SNP’s impact on protein function.

15) Qi Sun, "Discovery of inhibitors of the Sos-mediated activation of K-Ras," Qi Sun, Jason Phan, Jason P. Burke, Michael C. Burns, Edward T. Olejniczak, Alex G. Waterson, Taekyu Lee, Olivia W. Rossanese, and Stephen W. Fesik

K-Ras is a highly validated cancer target due to its importance in tumor initiation and tumor maintenance. Traditionally it is thought to be an undruggable target. However, using a fragment-based screen, we have identified small molecules that bind to K-Ras in a hydrophobic pocket that is occupied by Tyr-71 in the apo-Ras crystal structure. Using structure-based design, we obtained analogs of the fragment hits with improved binding affinity as well as functional activity in a Sos-catalyzed nucleotide exchange assay. These compounds bind to K-Ras and block binding to Sos, thereby causes the inhibition of Sos-mediated nucleotide exchange.

16) Matthew Pence “Conformational Changes in the Human Y-family DNA Polymerase Kappa during Correct Nucleotide Incorporation,” Matthew G. Pence, Robert L. Eoff, Catinca Fercu, and F. Peter Guengerich

Protein conformational changes are observed in replicative polymerases upon binding the correct nucleotide substrates and contribute to nucleotide selectivity and overall polymerase fidelity. Y-family polymerases function in the bypass of bulky DNA adducts and contribute to the overall fidelity of DNA replication. Substantial work has been done investigating the biological roles and bypass mechanisms of these specialized polymerases. Structural studies show that the active site of Y-family polymerases remains open and solvent exposed during polymerization emphasizing their ability to accommodate bulky lesions in the active site. Large conformational changes are not observed. Yet, localized conformational changes may be important for catalysis. Changes in tryptophan fluorescence are observed with S. solfataricus Dpo4 upon correct nucleotide incorporation suggesting a conformational change. Individual members of the Y-family do not proficiently replicate past all types of DNA lesions. Biochemical and structural analyses demonstrate that unique structural features and base-pairing mechanisms employed by individual Y-family members lead to selectivity for the types of damage that is efficiently bypassed. Though not large, conformational changes may be vital to the reaction mechanism of Y-family polymerases. Tryptophan fluorescence was used to observe conformational changes in human DNA polymerase kappa.

17) Gina Kavanaugh, "High-throughput Whole Genome siRNA Screen to Identify Novel Replication Stress Response Proteins," Gina Kavanaugh, Fei Ye, Gloria Glick, Yu Shyr, and David Cortez

Proper replication of the DNA is crucial for the maintenance of the genetic information during cell division. Alterations to the DNA code, which result from both intrinsic and extrinsic DNA damage, can result in gene mutations leading to alterations in protein function and expression. These mutations may ultimately lead to disease states, including cancer. The replication stress response, which occurs when damaged DNA is encountered during replication, is complex and requires the coordination of a number of cellular pathways, including cell cycle checkpoint, replication fork and replisome stabilization, prevention and restart of replication fork re-firing, DNA damage repair, and restart of DNA replication. Due to the intricacy of these pathways, understanding of these responses is an ongoing process in which new players are being discovered regularly. We believe a number of novel proteins associated with the replication stress response are yet to be identified. Consequently, we have developed a whole genome siRNA approach in order to identify proteins that play a role in these responses. An immunofluorescent assay utilizing thymidine analog incorporation and the replication stress inducer hydroxyurea was designed to uncover proteins that are necessary for replication stress repair and replication restart. This assay takes advantage of the 384-well plate format and high-throughput analysis of immunofluorescent data in order to quickly identify proteins of interest. Here we outline our technique and demonstrate that our assay is successful, as we have detected known replication stress response proteins. The identification of known players serves as both a positive control and confirmation that our assay is successful for detection of such proteins. This assay will allow for the discovery of novel replication stress response proteins that will deepen our understanding of DNA replication and repair and how these pathways contribute to genomic stability.

18) Michael Danneman, "Chiral Proton Catalysis: BAM-Catalyzed Enantioselective Synthesis of a Potent GlyT1 Inhibitor and the Guided Development of Asymmetric Mono(Amidine) Organocatalysts for the Enantioselective Development of Nutlin Analogs

A potent GlyT1 inhibitor, discovered by Lindsley & Williams [U.S. Patent WO 2010/114907 A1], has been synthesized via the use of chiral Pyrrolidine Bis(AMidine) [PBAM] organocatalysis. A 3-nitroazetidine nucleophile is used in an asymmetric aza-Henry addition reaction, catalyzed by a PBAM-triflic acid salt, to furnish the expected adduct in high yield and enantioselectivity (93% yield, 92% ee). Subsequent denitration of this adduct affords a scalemic scaffold which, through a short sequence, can be converted to the pharmacologically more potent enantiomer. Additionally, highly efficacious mono(amidine) organocatalysts have been synthesized in order to facilitate the development of Nutlin analogs. These catalysts allow for the addition of aryl nitroalkane nucleophiles into aryl imine centers to furnish key cis-stilbene diamine backbones in high levels of diastereo- and enantioselectivity (up to 200:1 dr and 99% ee). These diamines can then be converted into a series of Nutlin derivatives.

19) Rene Raphemot, "Development of chemical probes for exploring renal inward rectifier potassium channels as novel mosquito insecticide targets to limit the transmission of vector-borne diseases," Rene Raphemot, Emily Days, Peter M. Piermarini, Klaus W. Beyenbach, C. David Weaver, Corey Hopkins, Craig W. Lindsley and Jerod S. Denton

Dengue fever, yellow fever, and malaria are vector-borne diseases that are transmitted through the bite of infected female mosquitoes during blood feeding. The excretion of water, salts, and nitrogenous wastes by the Malpighian tubules, which are functionally equivalent to the mammalian kidney, minimizes the physiological stresses brought on by blood feeding. Current means of reducing mosquito populations are becoming less effective because of the emergence of resistance to commonly used insecticidal control agents. Thus, the development of novel insecticides with new modes of action is essential for controlling the transmission of these devastating diseases. We therefore initiated a drug discovery campaign to identify small molecule inhibitors of inward rectifier potassium (Kir) channels to induce “kidney” failure in mosquitoes. A fluorescence-based, thallium (Tl+)-flux assay was developed to support a high-throughput screening (HTS) effort for discovery of small-molecule modulators of a barium-sensitive Kir channel, cloned from the Malpighian (renal) tubules of the yellow fever mosquito Aedes aegypti (AeKir1). From a screen of approximately 30,000 compounds from the VICB library, several moderately potent AeKir1 inhibitors have been discovered. High-throughput counterscreens against 8 human Kir channels revealed that several of these are selective for the mosquito channel. Preliminary studies indicate that pharmacological inhibition of AeKir1 dramatically reduces urine production and disrupts potassium homeostasis in intact mosquitoes. Lead optimization efforts have been initiated to improve the potency and selectivity of selected compounds. Funding is provided by a grant from the Foundation for the NIH, VCTR program.

20) Emilianne McCranie, "Exploring the Unique Oxidation Chemistries of Orthosomycins"

Due to the increasing prevalence of multi-drug resistant bacteria in the clinical setting, new antibiotics are needed to treat patients with these types of bacterial infections. The orthosomycins are a class of polysaccharide antibiotics defined by an orthoester functional group that have activity against many drug-resistant strains. The orthoester is a non-canonical glycosidic linkage strategy unique to the orthosomycins. To understand the mechanisms by which these linkages are formed, the biosynthesis of two orthosomycins everninomicin and hygromycin B are being investigated. Genes implicated in orthoester formation have been disrupted resulting in abolished production and confirming their role in the biosynthesis of everninomicin.

21) Jenny Nesbitt, "Biologically Inspired Metal Oxide Formation," Jenny Nesbitt, Adam Travis, Nicholas Wright, David W. Wright

Biological systems are surprisingly adept at processing materials, ranging from the silk of spider webs to the intricate silica structures of diatoms and sponges. The sponges of the phylum Porifera, use enzymes to control the growth and patterning of silicon dioxide as part of their extracellular structure. The enzyme silicatein condenses the monomer silicic acid to deposit silica in the formation of its siliceous spicules. It is also homologous to the cysteine proteases cathepsin L1 and papain. Silicase hydrolyzes the O-Si-O bonds of silica to break down silica into silicic acid. It is highly homologous to carbonic anhydrase II. By investigating these enzymes, their homologies, and model compounds, we hope to better understand the mechanism of these sponges and obtain the same type of control and patterning of silica. When coupled to nano- and microscale deposition techniques, these enzymes will provide the basis for a new kind of soft lithography.

22) Brittany Allison, "VICB Symposium Abstract “Computational Design of Protein-Small Molecule Interfaces”

Proteins that bind small molecules can act as therapeutics by sequestering ligands, stimulating signaling pathways, delivering other molecules to sites of action, and serving as in vivo diagnostics. Computational design of proteins that can bind any ligand would hold great value, but is not yet possible. Computational methods for design can search more sequences and a larger sampling space than more traditional experimental methods. (β/α)8 barrels, also known as “TIM” barrels, will be used as a scaffold because of their widely seen fold in nature. Using ROSETTALIGAND, a component of the ROSETTA modeling suite that enables modeling of protein-small molecule interactions, small molecules can be docked into the binding pocket while simultaneously designing the protein-small molecule interface. The resulting models with the best scores are expressed, characterized, and tested for binding.

23) Charles Williams, "Eggmanone, a novel allosteric inhibitor of phosphodiesterase 4, modulates hedgehog signaling through targeted dysregulation of cAMP microdomain," Charles H. Williams, Jijun Hao, Audrey Y. Frist, Yanfeng Li, Michelle M. Williams, Li Zhou, Jonathan E. Hempel, Jonathan T. Fleming, Gary A. Sulikowski, Michael K. Cooper, Chin Chiang and Charles C. Hong

Cyclic AMP (cAMP) is a major secondary messenger for multiple signaling pathways. Its functional promiscuity within the cell is regulated by mechanisms of localized synthesis, degradation, and transport. Among the myriad roles cAMP plays in signaling pathways, cAMP is known to modulate hedgehog signaling by regulating activation of the hedgehog transcription factor Gli. However, the mechanisms underlying this regulation remain largely described. We describe a novel small molecule Eggmanone, an unusual allosteric phosphodiesterase 4 (PDE4) inhibitor; with a unique selectivity profile which disrupts cilium-to-nucleus Gli trafficking by perturbing local cAMP levels in the peri-cilium region around the basal body. Our chemical genetic study utilizing this novel pharmaceutical class to selectively modulate local cAMP levels in the basal body highlights the importance of this organelle in hedgehog regulation and provides a novel probe for the study of cAMP within the basal body.

24) Frank Couch, "Function of ATR in Genome Maintenance"

The Ataxia Telangiectasia and Rad3-related (ATR) protein kinase protects genome stability in response to replication stress. ATR phosphorylates numerous substrates at stalled replication forks, including the downstream effector kinase CHK1 and the DNA damage marker yH2AX. ATR signaling functions to prevent cell cycle progression in the presence of replication-associated DNA damage and coordinate DNA repair. ATR also promotes replication fork stabilization and restart during replication stress; however, the mechanism remains unclear. Recently, several companies have developed inhibitors to the kinase activity of ATR as a potential cancer therapy. Here, we investigate the function of ATR in promoting replication fork stability using inhibitors to the kinase activity of ATR. ATR inhibited cells exhibit rapid hypersensitivity to the replication stress agent hydroxyurea (HU). Furthermore, these cells accumulate pan-nuclear yH2AX phosphorylation which correlates with the formation of aberrant DNA structures at the replication fork. These aberrant structures involve the newly synthesized DNA strand becoming single-stranded – a structure not known to occur during normal replication. Our studies have focused on the functions of RECQ family helicases BLM and WRN as well as the resection scaffolding protein CTIP in the formation of nascent-strand ssDNA. This structure may result from replication fork reversal, a double strand break intermediate, or both. These studies reveal that during replication stress, one function of ATR is to prevent aberrant processing of the replication fork into nascent-strand ssDNA structures. Furthermore, the acute hypersensitivity to replication stress of ATR inhibited cells suggests a potential therapeutic window, as only an hour of treatment with HU and ATR inhibitor is required to kill virtually all S-phase cells.

25) William Beavers, "Endogenous generation of lipid metabolites and analysis of protein adduction from alkynylated fatty acids," William Beavers, Colleen McGrath-Lawrence, Keri Tallman, Stephen Milne, Michelle Armstrong, David Myers, Ned Porter, Alex Brown, Lawrence Marnett.

Arachidonic acid (AA) and linoleic acid (LA) are two of the most abundant ω-6 fatty acids found in the lipid bilayer, and are important precursors to many inflammatory signaling molecules. LA is metabolized to hydroxy-octadecadienoic acids by both the cyclooxygenase and lipoxygenase families of enzymes. Both metabolism pathways also produce a range of α,β-unsaturated carbonyls, which are capable of reacting with nucleophilic amino acid side chains, changing protein function. We have synthesized an ω-alkynylated arachidonic acid (aAA) and linoleic acid (aLA) to help us map endogenous protein adduction in RAW 264.7 mouse macrophages. aAA is incorporated and released from the lipid bilayer similarly to AA, but exhibits a different metabolite profile and kinetics than AA when reacted with COX-2. Here we present the comparison of aLA to LA as a physiological probe to measure protein adduction.

26) Marta Szulik, "Thermodynamic signature of DNA Damage: Characterization of 5-Hydroxy-2'-Deoxycytidine Based Paired With 2'-Deoxyguanosine In DNA," Marta W. Szulik, Manjori Ganguly, Boguslaw Nocek, Patrick S. Donahue, Kate Clancy, Barry Gold, and Michael P. Stone

Oxidation of DNA due to exposure to reactive oxygen species is a major source of DNA damage. Here we describe studies of the modified Dickerson-Drew Dodecamer 5’-CGC GAA TTX GCG-3’ (DDDOH), where X is 5-hydroxy-2'-deoxycytidine (5-OH-dC). It has been reported that 5-hydroxy-dC causes C to T transitions in E. coli at a rate of 2.5%. The lesion is repaired by DNA glycosylases that require the 5-hydroxycytidine to be extrahelical, in order to enter the enzyme’s active site where it is excised from the DNA backbone to afford an abasic site. The thermodynamic and high-resolution NMR results presented in these studies describe the effect of a 5-hydroxy-2'-deoxycytidine•2'-deoxyguanosine base pair on the stability of DNA. NMR spectroscopy as a function of temperature reveals an increased rate of exchange between the G4·X9, T8·A5 and G10·C3 base pairs and solvent, which suggests that base pairing is destabilized compared to the base pairs in the unmodified duplex. The presence of cross-peaks between the X9 amino and G10 and T8 imino protons suggests base stacking of 5-OH-dC with neighboring bases is conserved, which was also confirmed by 1.4 Å X-ray crystal structure. The results demonstrate that the lesion is highly destabilizing and that the energy barrier for the unstacking of 5-hydroxy-2’-deoxycytidine from the DNA duplex may be low. This could provide a thermodynamic mode of adduct identification by DNA glycosylases that require the lesion to be extrahelical.

27) Oleg Kovtun, "Single-Quantum Dot Tracking Reveals Altered Membrane Dynamics of an Attention Deficit/Hyperactivity Disorder-Derived Dopamine Transporter Coding Variant," Oleg Kovtun, Dhananjay Sakrikar, Ian D. Tomlinson, Randy D. Blakely and Sandra J. Rosenthal

The presynaptic, cocaine- and amphetamine-sensitive dopamine transporter (DAT) controls the intensity and duration of the synaptic dopamine signals by rapid clearance of dopamine into presynaptic nerve terminals. Abnormalities in DAT-mediated dopamine signaling have been linked to a variety of neurodegenerative and psychiatric disorders, including attention deficit/hyperactivity disorder, a brain disorder affecting over 5 million children in the U.S. Recently, we identified a rare DAT coding variant, R615C and demonstrated the molecule’s anomalous insensitivity to the endocytic effects of both amphetamine treatments and protein kinase C activation that is accompanied by disrupted association with GM1 ganglioside- and flotillin1-enriched membrane microdomains and a shift to a more rapidly cycling, endocytic pathway. To further investigate the effects of the R615C variant on DAT membrane localization, we implemented antagonist-conjugated quantum dots (QDs) to determine transporter dynamic behavior at the plasma membrane of living cells. Single QD trajectory analyses revealed that under basal conditions DAT 615C possesses a significantly greater membrane mobility as compared to the wildtype DAT 615R. Whereas cholesterol-rich membrane raft disruption and amphetamine treatments increased the mobility of QD-tagged DAT 615R, these manipulations failed to mobilize DAT 615C. Together, our data provide direct evidence that single particle tracking approaches can monitor differences in wildtype and mutant DAT plasma membrane dynamics and further elucidate properties of DAT 615C that may support its contribution to perturbed dopamine signaling in vivo.

28) Katherine Amato, "The Role of EphA2 in Tyrosine Kinase Inhibitor Resistant Lung Cancer," Katherine Amato and Jin Chen

Lung cancer is the leading cause of cancer-related deaths in the United States. Non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancers with an overall five-year survival rate of approximately 15%. However, NSCLC is not a single disease, but rather represents a composition of unique molecular aberrations. Identifying the relevant molecular subtypes of this heterogeneous disease and matching patients with appropriate targeted agents is of critical importance in the treatment of NSCLC. Although significant progress has been made over the past decade to develop new, molecularly-targeted therapeutics, challenges still remain in targeting the most commonly occurring mutations in lung cancer, KRAS and EGFR. Overcoming the large composite of challenges related to targeting KRAS and EGFR mutant lung tumors will require astute, robust strategies to identify and inhibit novel kinase targets required for cell viability in these subtypes of lung cancer. To this end, we have shown that the EphA2 receptor tyrosine kinase plays a unique role in the maintenance of cell viability in lung cancer, especially in lung cancers with KRAS and EGFR activating mutations. EphA2 is generally overexpressed in NSCLC, and it is often further overexpressed in KRAS and EGFR T790M mutant NSCLC. Preliminary data from our lab has shown that knockdown of EphA2 expression in a large number of human NSCLC cell lines inhibits tumor cell viability, most dramatically in cell lines bearing KRAS and EGFR mutations. To further assess the clinical relevance and utility of pharmacological inhibition of EphA2 to affect cell viability and tumor growth, we identified and characterized a novel, EphA2-specific kinase inhibitor, ALW-II-41-27. This EphA2 inhibitor has shown striking reduction of cell viability in vitro on an array of NSCLC cell lines including but not limited to KRAS and EGFR T790M mutant cell lines. ALW-II-41-27 has also induced tumor regression in KRAS and EGFR mutant NSCLC xenografts. Our preliminary data strongly suggests that targeting EphA2 holds unique promise for a difficult-to-treat subset of NSCLC. Based on our preliminary data, we hypothesize that EphA2 provides a novel target for modulating cell viability in lung cancers with KRAS or EGFR mutations associated with limited therapeutic options.

29) Joel Federspiel, "Determining the Composition of the ASK1 Signalosome," Joel Federspiel, Simona Codreanu, and Daniel C. Liebler

Apoptosis Signaling Kinase 1 (ASK1) is a mitogen activated protein kinase kinase kinase (MAP3K) that acts as a sensor for oxidative stress in the cell. ASK1 activation triggers the MAPK pathway to activate p38 and JNK downstream, which in turn determine cell death, survival, and differentiation. This activity has been reported to be important in several disease states, including cancer, diabetes, Alzheimer’s disease, and cardiac disease. ASK1 has been reported to interact with a host of other proteins in a high molecular weight complex termed the ASK1 Signalosome that serve to modulate the activity of ASK1. To determine the full composition of this signalosome and thereby gain some insight into triggering mechanisms for MAPK signaling, we performed an affinity purification of ASK1 and its associated proteins from HEK-293 cells stably expressing HA-tagged ASK1. Protein complexes were analyzed by shotgun proteomics coupled with western blot verification of selected targets. Comparison of ASK1 complex proteomes from differentially treated (non-treated, vehicle-treated, HNE-treated) ASK1-expressing cells identified members of the signalosome in both the steady-state and the activated state. These analyses identified previously reported ASK1-interacting proteins Thioredoxin, 14-3-3 family proteins, and HSP70, as well as the related kinases ASK2 and ASK3. These preliminary studies will be followed by confirmatory experiments in which the ASK2/3 proteins and major complex partners are targeted for capture and analysis. Definition of ASK signalosomes will provide a useful reference for understanding the mechanisms of stress responses to environmental factors.

30) Mary Keithly, "Structural and Functional Investigation of the Fosfomycin Resistance Protein FosB," Mary E. Keithly, Matthew K. Thompson, Don Stec, and Richard N. Armstrong

Fosfomycin is a broad-spectrum antibiotic that is clinically used in the treatment of lower urinary tract infections (UTI), gastrointestinal infections, and has been suggested for use in treating multi-drug resistant bacterial infections. However, the presence of fosfomycin resistance enzymes can limit the use and efficacy of fosfomycin in the clinical setting. FosB is a thiol-transferase found in Gram positive bacteria and inactivates fosfomycin by conjugation with a thiol. FosB is described as a Mg2+-dependent enzyme that utilizes cysteine for inactivation of fosfomycin. Recently, bacillithiol (BSH), was discovered in Gram positive bacteria and preliminary data suggest that BSH may be a better thiol substrate in vivo for FosB inactivation of fosfomycin. Using 31P with 1H decoupled NMR, we have completed time course kinetic analysis of FosB from four different bacterial strains (Staphylococcus aureus, Bacillus subtilis, Bacillus anthracis, and Bacillus cereus) using both cysteine and BSH. The results indicate that FosB from S. aureus and B. subtilis has a significant increase in reactivity with BSH compared to cysteine, but FosB from B. cereus and B. anthracis has only a moderate increase in reactivity with BSH compared to cysteine. We also investigated the metal dependence of the reaction using both Ni2+ and Mg2+. The data shows that the activation of the enzyme with Ni2+ or Mg2+ is similar in B. subtilis, B. anthracis, and B. cereus, but is significantly higher with Ni2+ for S. aureus. In combination, the results indicate that FosB may not be a Mg2+-dependent cysteine transferase. Further analysis of metal activation and enzymatic activity with BSH in conjunction with structural studies may yield insight into the mechanism of FosB resistance. A better understanding of the enzymatic mechanism of fosfomycin resistance in these pathogenic bacteria species could increase the efficacy and uses for fosfomycin in the clinical setting.

31) Brandon Vara, "Synthesis of cis-Imidazolines (Nutlin Derivatives) via Catalytic Asymmetric aza-Henry Addition Reactions to Disrupt p53-MDMX," Brandon A. Vara and Jeffrey N. Johnston

The Nutlin class of small molecules are cis-imidazolines that have been shown to inhibit the apoptosis-related protein-protein interaction (PPI) involving p53 and MDM2. The more potent enantiomer of Nutlin-3, (–)-Nutlin-3, has been used extensively since its disclosure in 2004 to probe the role of p53 in cancer cell biology. One mechanism for oncogenesis is the overexpression of MDM2 to inhibit p53 signaling. (–)-Nutlin-3 restores apoptosis through competitive binding to MDM2. The MDM2 homolog MDMX, binds to p53 and is overexpressed in a range of cancers, some common to pediatric retinoblastomas. Selective inhibition of MDMX by a small molecule therapeutic remains an unsolved problem. In prior work we developed the first enantioselective preparation of (–)-Nutlin-3 []. Adaptation of this approach to analogs often delivered the key intermediate with suboptimal enantioselection. To solve this problem, a series of mono and bis(amidine) catalysts were developed to catalyze the addition of aryl nitromethanes to aryl aldimines (aza-Henry reaction) with high diastereoselection (dr) and enantiomeric excess (ee) (>200:1 dr and up to 99% ee). This superior selectivity has allowed for inclusion of various substituted aryl groups, leading to diversification of the cis-imidazoline core substituents found in the Nutlins. Some of these small molecules show promising levels of inhibition of p53-MDMX.

32) Josiah Hutton, "Metabolic Protein Expression Changes due to Oncogenic KRAS Signaling," Josiah E. Hutton, Patrick J. Halvey, Lisa Zimmerman, Robbert J. Slebos, Kevin Haigis, Robert J. Coffey, and Daniel C. Liebler

The KRAS gene is mutated in about 40% of all colorectal cancers. In this study, we have used shotgun proteomics and multiple reaction monitoring (MRM) to identify metabolic protein expression changes that result from oncogenic KRAS signaling in the isogenic colorectal cancer cell lines DKO-1 and DKs-8. The DKO-1 cells contain a single mutant KRAS G13D allele, whereas the DKs-8 cells contain a single wild-type (WT) KRAS allele. Shotgun proteomics was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) on a Thermo LTQ after isoelectric focusing of trypsin digested cell lysates. From this shotgun data, 12 glycolytic and 13 Krebbs Cycle enzymes were found to be differentially regulated due to oncogenic KRAS signaling. We then expanded this list of 25 proteins to include an additional 3 glycolytic, 1 Krebbs Cycle, 8 Pentose Phosphate Pathway, and 11 metabolism altering proteins and developed a 48 metabolic protein panel for MRM analysis by LC-MS/MS on a Thermo TSQ Vantage. DKO-1 cells produced lactate at a faster rate than the DKs-8 cells, thus confirming a KRAS-driven Warburg effect in this model. We next used this 48 metabolic protein MRM panel to analyze 16 microdissected formalin-fixed, paraffin-embedded (FFPE) human stage II colorectal cancers genotyped for KRAS. From the 9 KRAS mutant stage II tumors, 7 of the 50 metabolic proteins, including SLC2A1, ALDOA, GAPDH, PGAM1, PKL/R, CS, and MDH2, were expressed at significantly higher levels in comparison to the 7 KRAS WT stage II tumors. Further work is being performed to validate the metabolic protein expression changes due to oncogenic KRAS signaling using a mouse model for colorectal cancer. (Supported by NIH grant U24CA159988.)

33) Rebecca Sandlin, "Finding the Pathway: New Machine Learning Paradigms for the Identification of Lead Compound Targets from Phenotypic Antimalarial Assays," Rebecca Sandlin, Mariusz Butkiewicz, Jens Meiler, David Wright

Recent screening efforts from GlaxoSmithKline (GSK) and Novartis have identified thousands of chemical starting points for new antimalarial lead compounds.1 However, due to the phenotypic nature of the assay used the biochemical targets of these compounds are unknown. The appropriate identification of the biochemical pathway is a critical component to successfully achieving lead optimization. But how does one take a compound that kills P. falciparum and identify a potential biochemical target pathway? The objective of the present research is to employ machine learning paradigms, such as artificial neural networks (ANNs),2 to develop QSAR models for hemozoin inhibition activity. Activity data for inhibitors obtained from an in vitro hemozoin formation high-throughput screen of ∼150,000 compounds was used to develop the QSAR model.3 A set of fragment-independent and transformation-invariant chemical descriptors serves as input for the ANN. A novel strategy for the selection of an optimal descriptor subset yields QSAR models that enrich active compounds by a factor of up to 65% in independent data sets. The method is applied to a virtual screen of the ~13,500 GSK Tres Cantos antimalarial compound set (TCAMS). Extrapolation of data to date predicts over 180 new hemozoin inhibitors within the TCAMS data set. Such an integrated approach to virtual screening could have important ramifications for the discovery of new lead compounds for tropical diseases. Having an approach to predict target pathway activity of a compound allows the researcher to translate phenotypic HTS data into a target pathway rapidly. This identification of target pathway is a key component to designing synthetic strategies for lead compound optimization through medicinal chemistry. Finally, this generalized approach allows investigators to optimize the focus of target identification and could be applied to diseases beyond malaria.

34) Lindsey Morris, "Allosteric Modulation of the Glucagon-Like Peptide-1 Receptor Induces Signaling Bias through β-Arrestin," Lindsey Morris, Emily Days, David Weaver, and Kevin Niswender

Hormones secreted post-prandially from specialized cells in the intestine, termed “incretins,” have distinct roles in enhancing insulin secretion, protection of β-cell mass, and promoting satiety (among many other effects). Glucagon-like peptide-1 (GLP-1) is one such hormone, and GLP-1 peptide analogues have been successful in treating type 2 diabetes and obesity. With multiple effects in several target tissues, GLP-1 analogues largely act by stimulating GLP-1 receptor (GLP-1R) signaling in the pancreas leading to potentiation of glucose-dependent insulin secretion. Despite successes, these peptides are limited by undesirable side effects and failure to selectively target therapeutically discrete pathways. The GLP-1R belongs to the family B G-coupled protein receptor family, generally characterized by large and structurally complex orthosteric binding sites. Thus, few small molecule agonists have been identified, making allosteric modulation of receptor function an attractive strategy. We previously screened a library of small molecules using parallel calcium mobilization and cAMP production assays and identified a number of selective GLP-1R positive allosteric modulators (PAMs). Capitalizing on these hits, we further hypothesized that allosteric modulators may be utilized to induce signaling bias that may result in context-dependent therapeutic effects, including enhanced insulin secretion, decreased β-cell apoptosis, and increased β-cell proliferation. Induction of bias either toward or away from β-arrestin recruitment and signaling may yield desirable effects in a variety of tissue targets and systems. Fine tuning intracellular coupling with allosteric modulators would thus potentially allow both unprecedented control over the pharmacologic outcomes of GLP-1R activation and a plethora of new hypotheses and biology to be addressed. We demonstrate that among our primary hits, we have compounds that differentially activate β-arrestin recruitment and receptor internalization. Finally, we present critical proof-of-principle data that GLP-1R PAMs potentiate glucose- and GLP-1-dependent insulin secretion from primary mouse islets.

35) Daniel J. Hermanson," DEVELOPMENT OF LM-4131, A SUBSTRATE-SELECTIVE INHIBITOR OF CYCLOOXYGENASE-2," Daniel J. Hermanson, Nolan D. Hartley, Sachin Patel, and Lawrence J. Marnett

We recently reported that rapid-reversible inhibitors of cyclooxygenase-2 (COX-2) are substrate-selective inhibitors, while slow, tight-binding inhibitors of COX-2 are non-substrate-selective inhibitors. In particular, we found that (R)-profens, which had previously been classified as inactive toward COX-2, are actually substrate-selective inhibitors of COX-2. We also found that (R)-profens increased the levels of anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) while decreasing the levels of prostaglandin ethanolamides (PG-EAs) and prostaglandin glycerol esters (PG-Gs) in primary murine dorsal root ganglia cells stimulated to express COX-2. However, in vivo, (R)-profens undergo a one way stereoisomerization to (S)-profens, which are non-substrate-selective inhibitors of COX-2. In the present study, we sought to develop new substrate-selective inhibitors of COX-2 for use in vivo. Site-directed mutagenesis indicated that disruption of the hydrogen bonding and ion-pairing network at the base of the active site can cause slow, tight-binding, non-substrate-selective inhibitors to become rapid-reversible, substrate-selective inhibitors. As suggested by the site-directed mutagenesis data, we found that tertiary amides of indomethacin are potent inhibitors of endocannabinoid oxygenation by COX-2. In particular, the morpholino amide of indomethacin, LM-4131, is a potent substrate-selective inhibitor with an IC50 of 620 nM for inhibition of 2-AG oxygenation by COX-2. LM-4131 also inhibited PG-G production in stimulated RAW 264.7 macrophages with an IC50 of 660 nM while increasing the levels of 2-AG. LM-4131 did not inhibit fatty acid amide hydrolase activity (FAAH) or monoacylglycerol lipase (MAGL) in vitro.

36) Sarah Shuck, "Covalent protein modification by an electrophilic DNA Adduct, N6-(3-oxo-1-propenyl)-2′deoxyadenosine," Sarah C. Shuck, Kristie L. Rose and Lawrence J. Marnett

Reactive oxidants produced following oxidative stress chemically modify lipids, proteins, and DNA leading to chemical transformation and alteration of function. Oxyradical-induced lipid and DNA peroxidation leads to the production of malondialdehyde and base propenal, which modify DNA to produce mutagenic and electrophilic DNA adducts including N6-(3-oxo-1-propenyl)-2′-deoxyadenosine (OPdA). OPdA forms covalent cross-links with N-α-acetyllysine, an observation that indicates the potential for this DNA adduct to modify protein. In this study, we describe covalent amino acid modification by OPdA using bovine serum albumin (BSA) as a model protein. Data from mass spectrometry-based proteomics establish that stable, covalent cross-links are formed at several solvent exposed lysine residues but not at histidine, arginine or cysteine residues. This work represents the first demonstration of protein modification by OPdA and establishes that it selectively forms stable adducts to lysine residues.

37) Kallie Stavros, "Structural Investigation of the Sequence Dependence for the Food Mutagen 2-amino-3-methylimidazo[4,5-f]quinolone in the NarI Recognition Sequence by NMR," Kallie M. Stavros, Edward K. Hawkins, Carmelo J. Rizzo, Michael P. Stone

2-amino-3-methylimidazo[4,5-f]quinolone is a potent food mutagen, possible human carcinogen and one of the most genotoxic compounds of the heterocyclic amines. Structural characterization of the IQ adduct in the NarI DNA sequence, a hotspot for IQ-induced mutations, provides insight as to the genotoxicity of the adduct. Using high field NMR, we have determined the structure of the IQ adduct at the G1 and G3 positions of the NarI sequence. Results show that both adducts adopt a base-displaced intercalated structure, where the opposite base is flipped into the major groove and is no longer hydrogen bonding with the modified base. However, the loss of Watson-Crick hydrogen bonding does not change the thermal stability of the duplex, presumably due to base-stacking interactions from the intercalated IQ ring.

38) Kyle Brown, "Synthesis of a Desketoraloxifene Library," Kyle A. Brown, Matt O’ Reilly, Sarah A. Scott, H. Alex Brown, Craig W. Lindsley

The desketoraloxifene series are analogues of compounds currently used in the clinic to treatment osteoporosis and breast cancer. This project focuses on modifying the structure of a known anti-cancer raloxifene drug that is known to inhibit phospholipase D (PLD) activity in addition to its better characterized effects as selective estrogen receptor modulators (SERMs). By excluding a CO and making additional manipulations to the structure, the estrogen receptor effects will be minimized (or removed entirely), which allowed us to look at the effects on PLD isoenzymes. Using a previously described synthesis, selected members of a desketoraloxifen library were created. The synthesis began with an electrophilic aromatic substitution, then proceeded with a regioselective Songashir reaction, a methylthioaltion, an idocyclization, and finally a Suzuki- Miyaur coupling reaction. Thin layer chromatography (TLC) and liquid chromatography and mass spectrometry (LCMS) were used to monitor the progress of each reaction along while preparative scale high performance liquid chromatography (both regular and reverse phase) for purification. Finally, NMR was used to verify that the intended compound was synthesized after each step and final characterization is high-resolution mass spectrometry. After the compound structure is confirmed it is tested in enzymatic assays to measure effects on PLD activity. This includes measurements of the consumption of phosphatidylcholine (PC) and the production of choline and phosphatidic acid. In cell-based assays the production of a transphosphatidylation product, phosphatidylbutanol, is measured instead to provide a more metabolic stable measurement of the reaction in cells. In conclusion, this library will lead to a better understanding of where these compounds bind on the PLD isoenzymes. In turn this may lead to a more effective PLD inhibitor that can be used as a better therapeutic treatment of human glioblastomas and infectious diseases.

39) Cierra Spencer, "Biochemical characterization of a phospholipase D enzyme expressed by Pseudomonas aeruginosa," Cierra Spencer and H. Alex Brown

Pseudomonas aeruginosa is an important human pathogen that is the major cause of mortality and morbidity of cystic fibrosis (CF) patients. The emergence of multi-drug resistant strains prompts the investigation into the pathogenesis of P. aeruginosa for the development of novel therapeutics. P. aeruginosa expresses a phospholipase D (PLD) enzyme, PaPLD, that hydrolyzes phospholipids to generate phosphatidic acid, a critical lipid signaling molecule in human cells. The plda gene was found to be important for persistence in a rat pulmonary infection model. Chronic infections of P. aeruginosa lead to lung disease and eventually respiratory failure in CF patients. This information coupled with the known function of other bacterial and host phospholipase enzymes makes PaPLD an attractive candidate to study. In this project, the activity of PaPLD is characterized with monomeric lipids and reconstituted vesicles using a variety of techniques including a head group release assay and mass spectroscopy. Successful expression and purification of PaPLD using an E. coli expression system was attained. PaPLD was found to utilize a wide array of phospholipid substrates with varying efficiency and acyl chain specificity. The enzyme requires the presence of a divalent cation, but interestingly only under certain circumstances. Surprisingly PaPLD was also stimulated by phosphatidylinositol-4,5-bisphosphate (PIP2), a stimulator of human PLD. Further evaluation into the role of PaPLD for infectivity is planned using parallel genetic and chemical approaches. To complement studies utilizing plda wt and knockout P. aeruginosa strains, chemical probes to inhibit the activity of PaPLD have been identified. This work serves as a foundation to accomplish the overall goal to assess the function of PaPLD in chronic infections and evaluate it as a novel therapeutic target for P. aeruginosa.

40) Adam Ketron, "Oxidative Activation Converts Curcumin to a Poison of Human Topoisomerase II," Adam C. Ketron, Odaine N. Gordon, Claus Schneider, and Neil Osheroff

Type II DNA topoisomerases are essential enzymes that modulate DNA topology, thus facilitating a number of critical cellular processes including replication, transcription, and mitosis. By passing an intact double helix through a transient, covalently stabilized double-strand break in another helix, topoisomerase II effectively removes knots and tangles from DNA and relieves torsional stress caused by DNA over- or underwinding. Topoisomerase II also is an important target for a variety of chemotherapeutic agents. These compounds take advantage of the ability of the enzyme to cleave DNA by elevating the number of topoisomerase II-generated DNA strand breaks to a cytotoxic level. Despite their clinical utility, the process by which these agents (called topoisomerase II poisons) alter enzyme activity is not well understood. Curcumin, a polyphenolic component of the spice turmeric, is being studied extensively for antioxidant, anti-inflammatory, chemopreventive, and chemotherapeutic potential. Curcumin appears to induce topoisomerase II-mediated DNA strand breaks in treated cells, but in vitro studies have been unable to identify topoisomerase II as a direct target of the compound. Previous work suggests that the health-promoting properties of curcumin may be attributed to either degradative or oxidative cellular metabolites of the parent compound. Thus, we investigated the potential of curcumin to act as a redox-dependent poison of topoisomerase IIα. DNA cleavage assays performed under oxidizing conditions demonstrated that oxidative intermediates of curcumin act as potent topoisomerase II poisons, while degradation products of curcumin have little effect on enzyme activity. The topoisomerase II-reactive metabolites of curcumin displayed hallmarks of redox-dependent topoisomerase II poisons, and chemical modifications that prevent autoxidation abrogated curcumin’s ability to enhance enzyme-mediated DNA cleavage.

41) Jessica Moore, "Imaging Mass Spectrometry as an Analytical Tool to Study Immune Response to Bacterial Challenge," Jessica L. Moore, Eric P. Skaar, Richard M. Caprioli

Imaging Mass Spectrometry (IMS) is a powerful analytical tool used to obtain spatial information about analytes of interest. When applied to animal models that have been presented with bacterial challenge, spatial information can be obtained to provide insight on the pathogen host interaction. Calprotectin is an S100 family protein that is present in high concentration in neutrophils. This important inflammatory marker has been previously shown to have antimicrobial properties through the chelation of manganese and zinc. Using IMS, Calgranulin A, a subunit of the Calprotectin heterodimer, can be monitored to visualize inflammatory response. This robust signal has been well characterized for murine models systemically infected with Staphylococus aureus, specifically in large abscesses that form in the kidneys of the animals. In addition to the systemic infection model, IMS of Calgranulin A is also shown to visualize inflammatory response in pulmonary infection. Acinetobacter baumannii is an opportunistic pathogen that is the common cause of hospital acquired pulmonary infections. Using a murine pulmonary infection model, IMS shows robust signal for Calgranulin A. This supports the idea that zinc and manganese metal sequestration by the host is an important defense mechanism in lung tissue infected with A. baumannii.

42) Yi Xiao, "Cytochrome P450 2W1 oxidizes both lysophopholipids and free fatty acids Names," Yi Xiao, F. Peter Guengerich

Fifty-seven cytochrome P450 genes have been identified upon the completion of human genome project, of which ~13 members are termed “orphans” because their physiological and xenobiotic functions are not established. One of the orphan human P450 enzymes, P450 2W1, is over-expressed in colorectal cancer tissues and no expression has been reported in normal tissues. In order to elucidate the physiological function and substrate specificity of P450 2W1, the purified recombinant enzyme was incubated with colorectal cancer extract and equal amounts of 18-O and 16-O molecular oxygen. Isotopically labeled monooxygenation products were identified by searching for M/M+2 doublets in the LC/MS data. In addition to previously reported fatty acids, oleyl (18:1) lysophosphatidylcholine (lysoPC, lysolecithin) was identified as a substrate of P450 2W1; other human P450 enzymes tested showed little activity. In addition to lysoPCs, P450 2W1 acted on a series of other lysophospholipids, including lysophosphatidylinositol, lysophosphatidylserine, lysophosphatidylglycerol, lysophosphatidylethanolamine, and lysophosphatidic acid but not the diacylphospholipids. P450 2W1 preferred sn-1 18:1 lysoPC as substrate over the sn-2 isomer, and we conclude that the sn-1 isomers of lysophospholipids are preferred substrates. The kinetics and position specificities of 2W1 catalyzed oxygenation of lysophospholipids (16:0 lysoPC and 18:1 lysoPC) and fatty acids (16:0 and 18:1) were also determined. The epoxidation and hydroxylation of 18:1 lysoPC is considerably more efficient than the free 18:1 fatty acid. Oxidized lysophospholipids are under investigation as ligands of nuclear receptors.

43) Amanda Duran, "Computational Design for a Symmetric Glycerol Facilitator Protein"

Using ROSETTA, a perfectly symmetric membrane protein was designed based on the wild type 2-fold inverted structurally symmetric glycerol facilitator protein (GlpF) from E. coli (PDBID:1fx8). GlpF consists of six trans-membrane spanning helices and two broken helices that meet in the center of the membrane. The variant of GlpF was designed to be symmetric in sequence and structure while conserving all functionally important sites. Structural comparison of the symmetric variant to the wild type protein using super secondary structural elements can evaluate the success of the design. We plan to structurally characterize using x-ray crystallography and NMR spectroscopy. Dynamic light scattering will be used for functional assays to determine the conductance of glycerol through the membrane based on swelling of the liposome. Assuming a less functional variant, the design will be modified in an attempt to rescue the activity. This project will test the gene duplication and fusion hypothesis for inverted membrane protein topologies.

44) Dewei Tang, "Positron emission tomography (PET) of TSPO expression in preclinical models of human cancer," Dewei Tang, Matthew R. Hight, Eliot T. McKinley, Allie Fu, Jason R. Buck, R. Adam Smith, Mohammed Noor Tantawy,Todd E. Peterson, Daniel Colvin, M. Sib Ansari, Mike Nickels, and H. Charles Manning

There is a critical need to develop and rigorously validate molecular imaging biomarkers to aid diagnosis and characterization of human cancer. Elevated expression of translocator protein (TSPO) has been shown to predict disease progression and aggressive, invasive behavior in a variety of solid tumors. Thus, noninvasive molecular imaging of TSPO expression could form the basis of a novel, predictive cancer imaging biomarker. In quantitative preclinical PET studies, we have evaluated a high-affinity pyrazolopyrimidinyl TSPO imaging ligand ([18F]DPA-714) and an aryloxyanilide-based TSPO imaging ligand ([18F]PBR06), as translational probes for quantification of TSPO levels in glioma, breast cancer, and colorectal cancer. In these studies, glioma-bearing rats or genetically engineered mice bearing spontaneously arrising breast or colorectal cancers were imaged with TSPO PET ligands in a microPET system. Dynamic images were acquired simultaneously upon injection of radioactive TSPO PET ligands. Arterial blood was collected to derive the input function (AIF), with HPLC radiometabolite analysis performed upon select samples for AIF correction. Compartmental modeling was performed using the corrected AIF. Immediately following imaging, tumor and healthy surrounding tissues were harvested for validation by western blotting and immunohistochemistry. Our results illustrate the feasibility of using TSPO PET ligands for visualization of TSPO-expressing tumors of the brain, breast and colon. Importantly, both [18F]DPA-714 and [18F]PBR06 appear suitable for quantitative assay of tumor TSPO levels in vivo. Given the relationship between elevated TSPO levels and poor outcome in oncology, these studies suggest the potential of these imaging agents to serve as a novel predictive cancer imaging biomarkers.

45) Matthew Thompson, "Structural insights into fosfomycin resistance by the metallothiol transferase, FosB," Matthew K. Thompson, Mary E. Keithly, Joel Harp, and Richard N. Armstrong

The fosfomycin resistance protein, FosB, is a metallothiol tranferase that catalyzes nucleophillic addition of either L-cysteine or bacillithiol to the antibiotic, fosfomycin [(1R,2S)-epoxypropylphosphonic acid], resulting in a modified compound with no bacteriacidal properties. The x-ray crystal structures of FosB from Bacillus cereus, FosBBc, and FosB from Staphylococcus aureus, FosBSa, have been determined to 1.27 and 1.42 Å resolution, respectively. Both structures reveal that FosB belongs to the vicinal oxygen chelate (VOC) superfamily of enzymes maintaining the three-dimensional domain-swapped arrangement of paired βαβββ-motifs. Additional crystal structures of FosBBc co-crystallized with fosfomycin and a variety of divalent metals, including Ni2+, Mn2+, Co2+, and Zn2+, for SAD phasing of the data reveal that the antibiotic coordinates to the metal center in an orientation similar to that found in the related Mn2+ dependent fosfomycin resistance enzyme, FosA. Surface analysis of the FosBBc structures exposed a well-defined binding pocket and solvent access channel to C1 of fosfomycin, the carbon to which nucleophillic addition of the thiol occurs. The pocket and access channel are occupied by glycerol and water, respectively, in the structures and are appropriate in size and shape to accommodate L-cysteine or bacillithiol. Further investigation of the structures revealed that the fosfomycin molecule, anchored by the metal, is surrounded by a cage of amino acids that hold the antibiotic in an orientation such that C1 is centered at the end of the solvent channel positioning the compound for direct nucleophillic attack by the thiol substrate. The amino acids that construct the fosmomycin cage are conserved throughout all currently reported FosB enzymes. Finally, the structure of FosBBc in complex with the L-cysteine-fosfomycin product coordinated to a Mn2+ metal in the active site has been determined to 1.55 Å resolution. As anticipated from the structural analysis, the L-cysteine moiety is located in the solvent channel, and the L-cysteine thiol has added to the backside of fosfomycin C1 located at the end of the channel. These structural insights provide a foundation for the development of inhibitors of the FosB enzymes potentially leading to an expanded use of fosfomycin as an effective antimicrobial agent.

46) Stephanie DeLuca, "RosettaEPR: An Integrated Tool for Protein Structure Determination from Sparse EPR Data," Stephanie DeLuca, Nathan Alexander, Hassane Mchaourab, Jens Meiler

Membrane proteins remain a particular challenge in structural biology. Only about 1.5% of reported tertiary structures and 60 unique membrane protein topologies consisting of more than one transmembrane span are represented in the PDB. However, these proteins make up an estimated 30-40% of the entire proteome, and over half of all therapeutics target this group. Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is often used for the structural characterization of proteins that elude other techniques, such as X-ray crystallography and NMR. However, high-resolution structures are difficult to obtain due to uncertainty in the spin label location and sparseness of experimental data. RosettaEPR has been designed to improve high-resolution protein structure prediction using sparse SDSL-EPR distance data. The “motion-on-a-cone” spin label model was converted into a knowledge-based potential, which was implemented as a scoring term in Rosetta. We have demonstrated the feasibility of using RosettaEPR with soluble proteins by benchmarking the method on T4-lysozyme. RosettaEPR increased the fractions of correctly folded models (RMSDCa < 7.5Å) and models accurate at medium resolution (RMSDCa < 3.5Å) by 25%. After full-atom refinement, RosettaEPR yielded a 1.7Å model of T4-lysozyme, thus indicating that atomic detail models can be achieved by combining sparse EPR data with Rosetta (Hirst et al., 2011). RosettaEPR was also benchmarked on a set of membrane proteins of known structure. Because EPR experimental data were not available, simulated data were derived from the existing structures. It was generally observed that de novo folding in the presence of EPR restraints enriched the recovery of the proteins’ correct topology compared to when folding with no restraints.

47) Kai Wang, "The Roles of Human Cytochrome P450s 1A1, 2W1, and 2S1 in the Anti-tumor Processes of Fluorinated 2-Aryl-benzothiazoles: Bioactivation and Deactivation"

Both 5-fluoro-2-(3,4-dimethoxyphenyl)-benzothiazole (GW 610) and 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), sharing a benzothiazole pharmacophore, are lead compounds currently under development for cancer treatment (Aiello, S. et al. 2008, J. Med. Chem. 51, 5135-5139). Particularly, GW 610 possesses potent and selective in vitro growth inhibition properties in human colon cell lines (Mortimer, C. G. et al. 2006, J. Med. Chem. 49, 179-185), and P450 2W1 is overexpressed in human colon cancer tissue selectively (Gomez, A. et al. Mol. Pharmacol. 2010, 78, 1004-1011). Prior studies on cancer cell lines suggested P450 1A1 and 2W1-mediated bioactivations participate in the antitumor activities of 5F 203 and GW 610, whereas P450 2S1, on the contrary, reduces their efficacies (Tan, B. S. et al. Mol. Cancer Ther. 2011, 10, 1982-1992). However, P450 2W1 and 2S1 are “orphan” P450s, whose functions are poorly understood. In this study, the biotransformation pathways of GW 610 and 5F 203 were investigated, and the structures of the oxidation products of GW 610 and 5F 203 catalyzed by P450 1A1 and 2W1 were identified by comparing LC-MS and UV spectra with synthesized standards or elucidated by 1D and 2D NMR spectroscopy. GW 610 has been found to undergo a two-step oxidation process catalyzed by P450 1A1 or 2W1 in a similar manner: a regio-specific demethylation and a further ortho-hydroxylation. Our steady-state kinetic results showed the P450 2W1-catalyzed oxidation of GW 610 is 5-fold more efficient than the P450 1A1-catalyzed reaction. The oxidations of 5F 203 catalyzed by P450 1A1 and 2W1 yielded different products, among which a hydroxylamine, formed in the latter process, could be converted to a nitrenium ion and further lead to a dGuo adduct as observed in LC-MS. Formation of glutathione conjugates of GW 610 and 5F 203, presumably through a 1,2-quinone and a quninoneimine intermediate, respectively, was also detected by LC-MS. These results indicate that human P450 1A1 and 2W1 mediate GW 610 and 5F 203 bioactivation to form reactive intermediates leading to glutathione conjugates and DNA adducts that may account for the antitumor activities of GW 610 and 5F 203 and also be involved in cell toxicity. P450 2S1 catalyzes the reduction of the reactive intermediate, hydroxylamine, to the amine, 5F 203, under anaerobic conditions and, to a lesser extent, under aerobic conditions, thus attenuating the anticancer activity.

48) Jennifer Baker, "HIGH-THROUGHPUT SCREEN FOR INHIBITORS OF HUMAN METAPNEUMOVIRUS," JENNIFER BECKER, MICHELLE LEWIS, JOHN V. WILLIAMS

Human metapneumovirus (HMPV) is a paramyxovirus that is a leading cause of lower respiratory infection in children worldwide. There are no licensed vaccines or therapeutics for HMPV, and mechanisms of pathogenesis are poorly understood. A high-throughput screen (HTS) was developed using physiologically relevant human bronchial epithelial cells (BEAS-2B) cells. Several conditions were optimized to increase the dynamic range of the assay. Cell seeding density was optimized to maximize viability and cell confluence. Infectious dose was increased to the maximum dose that yielded a high degree of infection but retained cell viability. A monoclonal antibody to the HMPV fusion (F) protein was used to identify infected cells. In order to ensure that cell viability was not reduced, a lipophilic dye was used to stain all cells. The Z’ of the assay was >0.5. The compounds that inhibit viral infection >50%, but are not toxic to cells, were scored as hits. The HTS was used to screen the Spectrum Collection (~2000 biologically active and structurally diverse compounds from libraries of known drugs, experimental bio-actives, and natural products), the NIH Clinical Collections (~730 small molecules previously used in human clinical trials), and the Bio-active Lipid Screening Library (~850 bio-active lipids). Several compounds were identified as hits that inhibited viral infectivity without reducing cell viability. The screen identified compounds that are known to inhibit other RNA viruses. In addition, a few compounds were identified that inhibited HMPV entry through an unknown mechanism. The mechanism of inhibition of these compounds will be further evaluated to determine how they inhibit HMPV. In conclusion, we developed a new HTS for inhibitors of HMPV, an important human pathogen. The assay discovered several potential new inhibitors of HMPV whose mechanism of action will be further characterized. These experiments will likely lead to novel antivirals and uncover unknown aspects of viral entry, replication, and pathogenesis.

49) Joseph Manna, "Identification of a Hydrolase Responsible for the Metabolism of Prostaglandin Glycerol Esters," Joseph D. Manna, Matthew J. Walters, Kristie L. Rose, Ku-Lung Hsu, Benjamin F. Cravatt, Lawrence J. Marnett

Prostaglandin glycerol esters (PG-G’s) are COX-2-dependent metabolites of the endocannabinoid, 2-arachidonoylglycerol. The identity of the enzyme responsible for hydrolysis of PGE2-G to PGE2 is unknown so we investigated PGE2-G hydrolysis in the breast cancer cell line, MDA-MB231. Inactivation of the hydrolase by a fluorophosphonate (FP) indicated the enzyme is a serine hydrolase. Cell lysates were subjected to ion-exchange chromatography and proteomic analysis of fractions which showed hydrolytic activity identified serine hydrolases. siRNA knockdown of the identified serine hydrolases verified the identity of proteins hydrolyzing PGE2-G to PGE2.

50) Odaine Gordon, "Discovery of Novel Curcumin Metabolites and their Biological Activity," Odaine Gordon, Claus Schneider

The dietary polyphenol, curcumin, shows great promise as an anti-cancer and anti-inflammatory agent. While many cellular targets of curcumin have been identified, the chemical mechanisms whereby these targets are affected remain unclear. Curcumin undergoes extensive metabolism in the body, and the resulting metabolites have been considered as direct mediators of curcumin's effects. Orally administered curcumin undergoes metabolic glucuronidation and sulfation, as well as reduction of its dienone double bonds. We have recently discovered a separate, oxidative metabolism of curcumin in vitro, occurring spontaneously at physiological pH. The oxidative transformation of curcumin generates a di-oxygenated bicyclopentadione derivative as the final, major metabolite. In this study we set out to determine the complete profile of curcumin oxidative metabolites as an initial step towards assessing their bioactivity and in vivo formation. We used 14C-curcumin to detect and isolate nine novel oxidized metabolites, including three intermediates of the reaction, using high-performance liquid chromatography. These metabolites were identified by UV spectroscopy, mass spectrometry, and a combination of 1D and 2D NMR methods, such as 1H, H,H-COSY, HSQC, and HMBC. Using the structural data from these molecules, combined with H218O isotopic studies, we propose a mechanism of formation of the oxidative metabolites through reactive quinone methide and epoxy intermediates. The final products all incorporate two oxygen atoms and possess a common nucleophilic β-diketo moiety. In biological studies, a crude mixture of these metabolites was more potent than curcumin at inhibiting Parathyroid-Hormone-related-Peptide (PTHrP) secretion in MDA-MB-231 breast cancer cells. Curcumin has been shown to prevent lytic bone destruction relating to breast cancer bone metastasis through inhibition of PTHrP, and our studies implicate that this effect may be mediated by its oxidative metabolites. In a separate study, we show that autoxidative activation of curcumin is required for its topoisomerase poisoning activity. Further, we detected the final bicyclopentadione product in human and mouse plasma after oral administration of curcumin, indicating that this reaction is prominent in vivo. Altogether, our studies for the first time detail the major products of curcumin oxidative transformation and their mechanisms of formation. Our studies also support the hypothesis that oxidative metabolites of curcumin are direct mediators of some of its bioactivity. Further exploration of the cellular targets of the oxidative metabolites will increase our understanding of the mechanism of action of the cancer chemopreventive agent curcumin.

51) Kathryn Haley, "Staphylococcus lugdunensis degrades heme to release nutrient iron," Kathryn P. Haley and Eric P. Skaar

Staphylococcus lugdunensis is frequently found as part of the normal skin flora but has the potential to cause invasive infections even in healthy individuals however; the molecular mechanisms that enable S. lugdunensis to transition from a skin commensal to an invasive pathogen remain poorly defined. Analysis of the complete S. lugdunensis genome reveals a putative iron-regulated surface determinant (Isd) system. The Isd system has been identified in several species of bacteria and has been shown to allow for the use of host hemoglobin as a source of nutrient iron to facilitate bacterial growth during infection. We have established that S. lugdunensis expresses an iron-regulated IsdG family heme oxygenase that binds and degrades heme releasing free iron. Using mass spectrometry we determined that S. lugdunensis IsdG degrades heme to the chromophore staphylobilin. Furthermore, we have shown that S. lugdunensis IsdG-mediated heme catabolism permits the use of heme as an iron source, establishing IsdG as a pathophysiologically relevant heme oxygenase. This research establishes the S. lugdunensis Isd system as being involved in heme-iron utilization and offers insight into how S. lugdunensis fulfills its nutritional requirements during infection. Importantly, proteins involved in iron acquisition in other bacterial species are required for full virulence and are considered potential drug targets.

52) Matthew Hight, "Advances in Microfluidic Radiochemistry – A Means of Discovery," Matthew R. Hight, Mike L. Nickels, Jashim Uddin, Sam Saleh, Jason R. Buck, and H. Charles Manning

Background and Specific Aims:
The development and optimization of novel radiochemistries can be a bottleneck within the molecular imaging probe development paradigm. Traditional radiochemical preparations are inherently low throughput and limit the ease and efficiency at which protocol optimization and utilization can be achieved. Microfluidic radiochemistry offers a high throughput alternative approach to classical, box-based radiosyntheses and enables rapid optimization and evaluation of various radiochemical conditions.
Approach:
Here we have utilized the Advion NanoTekTM LF system and microfluidic radiochemistry as a means for rapidly developing and optimizing novel and unconventional radiochemistries. This system possesses several characteristics that set it apart from traditional radiochemistry modules, such as: precise delivery of small isotope/precursor quantities, laminar flow mixing of reagents, solvent backpressure control, and rapid/sequential production of multiple tracers in a matter of hours.
Results:
Recent developments have shown that one of the best uses for the NanoTek system is in the optimization and evaluation of novel radiochemical strategies. For example, in collaboration with Dr. Larry Marnett and colleagues, we evaluated strategies for labeling a novel indomethicine derivative with [18F] for PET imaging of COX2 expression. Through use of the NanoTek, it was possible to triage multiple variations of the indomethicine precursor and effectively eliminate compounds which due to instability were difficult to label. Similarly, the NanoTek has been instrumental towards optimizing the radiochemical production of [18F]4-fluoro-glutamine (a complex three-step radiochemical production) due to the system’s through-put, versatility, and capabilities of performing successful labelings with low tracer levels (<100mCi). These improvements have accelerated the development of novel probes that are currently being explored in programs such as the Vanderbilt ICMIC program, Digestive Disease Research Center (DDRC) and GI SPORE Program.
Significance :
The NanoTek system has demonstrated great utility towards determining labeling feasibility, probe stability, and optimal reaction conditions prior to transitioning to full-scale hot-cell based production. This pre-production optimization has begun to streamline probe development capabilities achievable by traditional radiochemistries.

53) Mariusz Butkiewicz, "GPU-Accelerated Machine Learning Techniques Enable QSAR Modeling Of Large HTS Data"

Quantitative structure activity relationship (QSAR) modeling using high-throughput screening (HTS) data is a powerful technique which enables the construction of predictive models. These models are utilized for the in silico screening of libraries of molecules for which experimental screening methods are both cost- and time-expensive. Machine learning techniques excel in QSAR modeling where the relationship between structure and activity is often complex and non-linear. As these HTS data sets continue to increase in number of compounds screened, extensive feature selection and cross validation becomes computationally expensive. Leveraging massively parallel architectures such as graphics processing units (GPUs) to accelerate the training algorithms for these machine learning techniques is a cost-efficient manner in which to combat this problem. In this work, several machine learning techniques are ported in OpenCL for GPU-acceleration to enable construction of QSAR ensemble models using HTS data. We report computational performance numbers using several HTS data sets freely available from PubChem database. We also report results of a case study using HTS data for a target of pharmacological and pharmaceutical relevance, cytochrome P450 3A4, for which an enrichment of 94% of the theoretical maximum is achieved.

54) Marta Wenzler, “Synthesis of Oxotetrahydroquinolinone- and Phenazine-based Libraries for Potential Circadian Rhythm Modulators,” Marta Wenzler, Bianca Ramirez, Jing Xiong, Hugo M. Borsetti, Gary A. Sulikowski, Carl H. Johnson

Circadian rhythms are oscillations that occur in biological processes on a daily basis and these changes affect many biological processes like the sleep-wake cycle, the release of hormones, and other important cellular functions. Identification of lead compounds affecting the rhythm of the circadian clock will form a foundation for therapeutic strategies aimed at treating circadian disorders. A high throughput screen identified phenazines as a lead circadian rhythm period elongating scaffolds and oxotetrahydroquinolinone as a lead period shortening scaffold. Amide analogs of these compounds will be screened for activity using the rat1 Per2 promoter cell line and a luciferase expression reporter to determine their effects on the circadian rhythm period.

55) Nina Collins, "Progress Towards the Total Synthesis of Bielschowskysin," Nina R. Collins, Steven D. Townsend and Gary A. Sulikowski

The furanocembranoids are a family of natural products that demonstrate nature’s generation of complex molecular architecture and biological activity through oxidation of well-established terpene frameworks. The biosynthesis of these metabolites involves oxidative installation of butenolide and furan functionality from cembrane. Transannular cyclization produces complex heterocycles such as providencin, bielschowskysin, verrillin, and plumarelide. Bielschowskysin is a hexacyclic diterpene isolated from the Caribbean gorgonian octocoral Pseudopterogorgia kallos by Rodriguez and coworkers in 2004. Its novel tetradecane ring system incorporates eleven stereocenters (seven contiguous). Additionally, the metabolite was shown to exhibit antimalarial activity against Plasmodium falciparum (IC50= 10 µM); as well as strong anticancer activity against small cell lung cancer (GI50 < 0.01 µM), and renal cancer (GI50 < 0.51 µM). Owing to its intriguing architecture and therapeutic properties, we are currently exploring a route toward the synthesis of this diterpene.

56) Richard Daniels, "Discovery of interfacial binders to the K-Ras/SOS complex co-investigators," Daniels, R. N.; Burns, M. C.; Sun, Q.; Kim, H.-Y.; Lee, T.; Waterson, A. G.; Rossanese, O. W.; Phan, J.; Fesik, S. W.

K-Ras is a small GTPase that functions as a molecular switch cycling between inactive (GDP-bound) and active (GTP-bound) states. The conversion of K-Ras-GDP to K-Ras-GTP is the rate-limiting step in the activation of K-Ras and is catalyzed by guanine nucleotide exchange factors such as the son of sevenless (Sos). Mutations in K-Ras fix the protein in the active state and endow cells with capabilities that represent the hallmarks of cancer. These include the ability to proliferate, evade apoptosis, reprogram cell metabolism, induce angiogenesis, activate invasion and metastasis, and escape immune destruction. Indeed, aberrant K-Ras signaling plays a role in 30% of all human cancers, with the highest incidence of activating mutations found in pancreatic (70-90%), colon (30-50%), and lung (20-30%) carcinomas. Downregulation of activated Ras reverses the transformed phenotype of cells and results in the dramatic regression of tumors in murine xenograft models. Thus, K-Ras inhibition represents an attractive therapeutic strategy for many cancers. However, Ras activation and signaling is accomplished primarily through protein-protein interactions. Such protein interfaces typically lack well-defined binding pockets and have been difficult to target with small molecules. Multiple chemical series of compounds that bind weakly to K-Ras were found using a fragment-based screen of our 11,000-membered fragment library. Crystallographic studies indicate that binding of the hit compounds to K-Ras occurs at a novel site on the protein that is not present in the absence of compound. Through an iterative library approach, using fragment-growing techniques, the indole amino piperidines identified in the screen were modified and tested for their ability to bind to K-Ras. To our surprise, these analogs were found to activate, rather than inhibit, SOS-mediated nucleotide exchange. In an effort to understand how these compounds were causing this effect, we found that these compounds bind more tightly to the SOS:K-Ras complex than to either SOS or K-Ras alone. Compound binding to the complex occurs at the allosteric activation site of SOS, which increases the rate of nucleotide exchange at low micromolar concentrations. The cellular implications of this activation are currently under investigation in the Fesik laboratory.

57) Robert Boer, "Progress Toward the Total Synthesis of HKD2 and HKE2," Robert E. Boer, Gary A. Sulikowski, and Claus Schneider

HKD2 and HKE2 are two cyclic hemiketal eicosanoids, isolated by Schneider and coworkers in 2011, that result from the biosynthetic cross-over of the 5-LOX and COX-2 pathways. Furthermore, these hemiketals stimulate angiogenesis of vascular endothelial cells, providing evidence for biological significance of these compounds in inflammatory sites involving co-expression of COX-2 and 5-LOX. Unfortunately, isolation of these natural products is low and little else is known about their biological role. Total synthesis provides an attractive option for further investigation into the interesting biological properties of the hemiketals. The carbon framework of these compounds is achieved from Sonogashira coupling of enantiopure fragments. Methods to generate a 1,2-dicarbonyl as a platform for producing the cyclic hemiketal moiety will be presented.

58) Nichole Lareau, "Native Glycan Analysis by Structural Ultra Performance Liquid Chromatography-Ion Mobility-Mass Spectrometry (UPLC-IM-MS)," Nichole M. Lareau, Larissa S. Fenn, Cody R. Goodwin, Jody C. May and John A. McLean

Glycosylation, one of the least studied post translational modifications, is implicated in neurological diseases such as Alzheimer’s disease and several forms of cancer when dysregulated. Structural analysis of N-linked glycan and glycan conjugates is challenging due to the high level of heterogeneity of glycan isomers and the corresponding difficulty of separation. The study of native glycans poses additional challenges due to their low abundance and the inherent preference of sodiated glycans in endogenous biological matrices containing salts which contributes to interfering chemical noise. Liquid chromatography (LC) and mass spectrometry (MS) techniques are frequently used for rapid characterization of carbohydrate samples, but commonly require extensive sample preparation and purification as well as multi-stage fragmentation analysis in order to gleam structural information. Ion mobility-mass spectrometry (IM-MS) addresses many analytical limitations of the complex heterogeneity of glycan analysis through rapid mobility separations based on structural selection, which is complementary to MS. Quantitative structural information can also be derived from these mobility measurements that are specific to isomeric species. This report presents the development of methodologies for the analysis of native glycan isomers using ultra performance liquid chromatography-ion mobility-mass spectrometry (UPLC-IM-MS). With minimal sample preparation and no prior purification necessary, this robust methodology can be applied to various complex glycan samples.

59) Cynthia Berry, "Progress toward the Total Synthesis of Marineosin A Cynthia Bollinger Berry VICB Symposium"

In 2008, Fenical and co-workers reported the isolation, characterization, and biological evaluation of marineosin A from a marine actinomycete related to the genus Streptomyces. Marineosin A has a novel spiroiminal structure with two pyrrole functionalities. When tested in cancer assays, it showed inhibition of human colon carcinoma (HCT-116) with an IC50 = 0.5µM as well as activity in other cancer cell types. Reported here is the progress toward synthesizing marineosin A in the Lindsley lab.

60) Michael Schulte, "Progress Towards the Total Synthesis of Stemaphylline," Schulte, M.L.; Turlington, M.L.; Lindsley, C.W.

Stemona alkaloids represent a class of approximately 100 biogenetically intriguing and structurally unique natural products. They are isolated from plants of the Stemona genus (Stemonaceae family) and are known to contain chemically diverse alkaloids with a pyrrolo[1,2-a]azepine core. Recently in 2009, stemaphylline and stemaphylline-N-oxide were isolated from the root extracts of Stemona aphylla by Pitchaya and co-workers. Stemaphylline showed moderate acetylcholinesterase (AChE) inhibitory activity, pronounced insecticidal activity, and weak antimicrobial activity against Escherichia coli, Staphylococcus aureus, Pseudomonas auruginosa, and Candida albicans. To date, there have been no reported synthetic efforts towards a total synthesis of this natural product. Herein, we report our progress towards an asymmetric synthesis of stemaphylline using novel methodology that would allow for further biological evaluation and derivatization of this natural product.

61) Sarah Stow, "Exploring the Conformational Space of Natural Products Using Distance Geometry and Ion Mobility-Mass Spectrometry," Sarah M. Stow, Cody R. Goodwin, Michal Kliman, Brian O. Bachmann, Terry P. Lybrand, and John A. McLean

Ion mobility-mass spectrometry (IM-MS) allows the separation of ionized molecules based on their structural properties such as size and shape, in addition to their mass-to-charge ratio. The drift time data that is obtained from IM-MS is used to calculate the collision cross section of the ionized molecule, which is representative of the ion’s conformation. Studying the conformational landscape of these ionized molecules computationally provides further insight into the structures that these collision cross sections represent. Simulated annealing has often been paired with IM-MS data to describe the conformational space, but the high kinetic energies used can cause damaging distortions to the molecules and do not ensure sampling of all conformational space. Distance geometry creates conformations based on all the pair wise distances between the atoms within the molecule. Our preliminary results suggest that distance geometry is a better-suited computational approach due to its ability to sample all conformational space without subjecting the molecules to high kinetic energies. Once the initial structures are created with distance geometry, the Molecular Operating Environment (MOE) software developed by the Chemical Computing Group is used to create chemically feasible structures. The MOE software provides a force field suitable for these drug-like molecules, a set of descriptors, which allow easy elimination of chemically unreasonable structures, and the scientific vector language (SVL), which makes the large number of possible conformations much more manageable. Currently, we are using distance geometry to study a small set of natural products for which we have experimentally measured collision cross sections from MALDI-IM-MS.

62) Sandeepkumar Kothiwale, "Small Molecule Knowledge Based Conformational Sampling"

The three-dimensional conformation of small molecules is important for binding to the target protein. High throuput screening of target protein with small molecules require the determination of physiochemical interactions. Accurate estimation of these interactions depends heavily on conformation of small molecule. On the other hand, quantitative structure-activity relationship (QSAR) models require the 3D conformation of a molecule to describe the spatial relationship of atomic properties, such as hydrogen bond donors, that are potentially important for binding to the target protein. The two strategies are expected to produce the best prediction when given the conformation of a molecule that interacts with the active site of the protein. However, protein structure prediction and determination of the ligand-protein interface remains computationally prohibitive for virtual high throughput screening. We are investigating the use of conformational ensembles to describe individual small molecules conformations. To generate a conformational ensemble, a library of fragments, and their conformers are generated. Fragments are generated by fragmenting molecules found in the crystallographic structure database (CSD). Conformers for each such fragment is then searched from the database and a conformational ensemble is generated. These conformational ensembles are then used to sample conformations of small molecules.

63) Michael Burns, "Small Molecules Perturb Ras Protein–Protein Interactions and Signaling by Stabilizing an Intermediate Ras Conformation"

K-Ras is a small GTPase that functions as a molecular switch, cycling between inactive (GDP-bound) and active (GTP-bound) states. The conversion of K-Ras-GDP to K-Ras-GTP is the rate-limiting step in the activation of K-Ras and is catalyzed by guanine nucleotide exchange factors such as the Son of Sevenless (SOS). Oncogenic activation of K-Ras endows cells with the ability to proliferate, evade apoptosis, reprogram cell metabolism, induce angiogenesis, activate invasion and metastasis, and escape immune destruction. Indeed, aberrant K-Ras signaling plays a role in 30% of all human cancers, with the highest incidence of activating mutations found in pancreatic (70-90%), colon (30-50%), and lung (20-30%) carcinomas. Thus, K-Ras inhibition represents an attractive therapeutic strategy for many cancers. However, Ras exerts its oncogenic functions and signaling through protein-protein interactions, and affecting these interactions with small molecules has proven difficult in the past. Here we report the identification and characterization of cell-active small molecules which bind directly to K-Ras, alter SOS-mediated nucleotide exchange, and perturb Ras-signaling. An NMR-based screen of our 11,000-membered fragment library identified small molecules that bind directly to K-RasG12D and increase SOS-catalyzed nucleotide exchange. This activation of nucleotide exchange was shown to be SOS-dependent, and is mediated by an allosteric Ras-binding site on SOS. Crystallographic studies indicate that binding of the hit compounds to K-Ras occurs at a hydrophobic pocket located between the canonical switch regions on Ras. Ligand binding stabilizes an intermediate conformation of Ras, which is hypothesized to bind to the allosteric site of SOS in an analogous manner to Ras-GTP resulting in the observed increase in nucleotide exchange. Supporting this hypothesis we find that ligand binds free Ras as well as the SOS:Ras complex. These compounds disrupt Ras-mediated signaling by increasing MAPK signaling and decreasing PI3K signaling and are currently being investigated for their potential to abrogate Ras-driven oncogenesis.

64) Sam DeLuca

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. RosettaLigand has been previously successful at predicting binding poses with high resolution (Kaufmann, et. al, Proteins, 2009). We are developing RosettaHTS, an extension to RosettaLigand which will integrate these two methods by using information from QSAR derived pharmacophore maps to guide the low resolution phase of ligand docking. Discrete cartesian grids describing the hydrogen bonding ability, electrostatics and shape of the ligand binding site are overlaid on the protein structure, and these grids are used to score the initial placement of the ligand prior to fine grained docking. Sampling of the ligand within this grid is guided by the chemical information provided by the pharmacophore map. As the scoring grids are precomputed, ligand scoring is extremely fast, and thorough Monte Carlo sampling of the ligand binding site can be rapidly performed before fine grained ligand docking using the high resolution Rosetta scoring function. This efficient and fast initial sampling makes it possible to distinguish between active and inactive compounds with less fine grained sampling, decreasing the amount of CPU time necessary to predict a single binding interaction, and increasing the practicality of structure based virtual High Throughput Screening (vHTS). The integration of structure based and ligand based vHTS techniques allows the full range of pharmacological information surrounding a target and drug scaffold to be considered in a single approach. This technique can be used to rapidly develop small focused libraries for High Throughput Screening, increasing the hit rate and decreasing the number of compounds that need to be purchased for testing.

 

 

 

 

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