Vanderbilt University

home research discoveries core facilities training & research opportunities seminars & events news giving contact


2013 Student Research Symposium

When:  Thursday, August 14, 8:00 am to 5:00 pm

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

Registration:  All attendees (students, faculty, and presenters) please REGISTER ONLINE HERE.
Oral and poster presenters must submit abstracts via the online registration form.  Thank you!

Due Dates: 
+  May 30, online registration begins
+  July 1, oral presentation abstracts due
+  August 1, poster abstracts due
+  August 1, last day to register for attendance only

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. Rachelle Gaudet
Harvard University,
Department of Molecular And   Cellular Biology

  Visit Dr. Gaudet's web site.

raffle Prize: 


VICB Prize in Chemical Biology

Certificate in Chemical Biology Recipients

Oral Presentation Awards:
$100 honorarium for all 7 presenters.
$250 for top 3 presentations.

Poster Awards:
$200 to top 2 posters in four categories (Synthetic Chemistry, Therapeutics and Translation, Systems Analysis, Molecular Discovery) for a total of 8 prizes.



Coffee, Poster Hanging
8:00 A.M. – 8:45 A.M.

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

Guest Speaker: Rachel Gaudet, Harvard University
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.


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. - 3:50 P.M.

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

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



2013 Keynote Video
2013 Presentations
2013 Poster Session

Oral Presentation & Poster Abstracts

Oral Presentation Abstracts

1.  Scott McCall (VICB Prize), "The Chemistry and Biology of Bromine's New Role as an Essential Trace Element," A. Scott McCall, Christopher F. Cummings, Gautam Bhave, Roberto Vanacore, Andrea Page-McCaw, and Billy G. Hudson

Bromine is ubiquitously present in animals as ionic bromide (Br-) yet has previously had no known essential function. In an effort to understand the required cofactor for peroxidasin-catalyzed formation of sulfilimine crosslinks (S=N), a post-translational crosslink between Methionine and Lysine which is essential for tissue development and architecture within the collagen IV scaffold of basement membranes (BMs), we uncovered a unique and essential role for Br¬omide (Br-) within this process. Bromide, converted to hypobromous acid through the action of peroxidasin, forms a bromosulfonium-ion intermediate within collagen IV that energetically selects for sulfilimine formation. To validate this chemistry and understand its potential tissue level effects in vivo, we developed a model of dietary Br-deficiency in Drosophila. Dietary Br-deficiency is lethal in Drosophila, while Br-replenishment restores viability, demonstrating its physiologic requirement. Importantly, Br-deficient flies phenocopy the developmental and BM defects observed in peroxidasin mutants and indicate a functional connection between Br-, collagen IV, and peroxidasin. This work establishes that Br- is required for sulfilimine formation within collagen IV, an event critical for BM assembly and tissue development. Because of the universality of basement membranes within epithelial architecture, bromine is an essential trace element for animals and its deficiency within humans may be relevant to BM alterations observed in nutritional and smoking related disease.

2.  Will Beavers, "Mitochondrial Proteins are Highly Adducted Targets of Endogenously Generated Lipid Electrophiles in LPS-Activated RAW264.7 Macrophages," William N. Beavers, Kristie L. Rose, Keri A. Tallman, James J. Galligan, Stephen B. Milne, David A. Myers, Pavlina Ivanova, Xiaojing Wang, Jing Zhu, Alex Walsh, Melissa Skala, Bing Zhang, H. Alex Brown, Ned A. Porter, Lawrence J. Marnett

Linoleic acid (LA) is oxidized both enzymatically and by autoxidation to many inflammatory signaling molecules, and high concentrations of LA derived metabolites have been detected in many disease states. Oxidized LA can further react to form a series of electrophiles including unsaturated ketones and aldehydes. These electrophiles react with nucleophilic amino acid side chains to form protein adducts, potentially altering the function of cellular proteins. Much work has been done looking at the effects of protein adducts from the bolus addition of preformed electrophiles, but questions persist as to the biological relevance of these studies. We have developed ω-alkynyl linoleic acid (aLA) as a probe to study endogenous lipid electrophile generation and the resulting protein adduction via click chemistry. The addition of an alkynyl group to LA does not affect its autoxidation, oxygenation by lipoxygenases, or incorporation into lipid components of RAW264.7 macrophages. When oxidation is induced by lipopolysaccharide treatment of aLA-enriched RAW264.7 macrophages, we observed a robust adduction across the entire proteome by aLA metabolites. We employed Stable Isotope Labeling of Amino acids in cell Culture (SILAC) as a way of quantifying differences in lipid electrophile protein adduction before and after the introduction of an inflammatory stimulus. Our results show that during the inflammatory response, adduction occurs most heavily on mitochondrial proteins. Targets of adduction include proteins important for antioxidant defense and energy generation including superoxide dismutase 2 (Sod2), peroxiredoxin 5, TCA cycle proteins, and proteins in complexes III and V of the electron transport chain. We have profiled the activity of Sod2, and see a significant reduction in activity that correlates to an increase in adduction by aLA metabolites. Additionally, we have seen a reduction in cellular ATP levels corresponding to adduction of electron transport chain proteins. Modification of these proteins, which are vital for mitochondrial function, and the resulting change in activity, may contribute to disease susceptibility.

3.  Keertson Davis, "A Simple Sample Preparation Device for Improvement in RDT Performance for the Detection of Malaria," Keersten Davis, Lauren Gibson, David Wright

The advent of point of care (POC) diagnostic tools has changed the face of healthcare in nations affected by the ongoing spread of infectious diseases. These underdeveloped, remote areas are often characterized by poverty, absent or intermittent electricity, hot and humid environmental conditions as well as a lack of skilled clinicians. Rapid diagnostic tests (RDTs) were developed to circumvent these challenges in the form of a low-cost, rapid, easy to use POC test. Despite the many advantages of RDTs, the changing climate of infectious disease education, prevention and treatment has brought to light the areas in which these tests can be improved. The World Health Organization (WHO) annually reviews all malaria RDTs manufactured for diagnostic use, and sets the limit of detection for these tests at 100 parasites/µL. While a patient exhibits symptoms of malaria infection above 200 parasites/µL, the asymptomatic patient (≤200 parasites/µL) serves as transmission reservoir. There are an estimated 200 types of tests manufactured and less than 10% of those tests are effective at detecting 200 parasites/µL parasite densities. Poor manufacturing standards and storage conditions render many brands inoperable and unreliable. We have recently reported the creation of a low-resource extraction cassette that can extract, purify and concentrate the most common malarial biomarker, Plasmodium falciparum Histidine Rich Protein II (pfHRPII), from a blood sample, in less than 30 minutes. By preloading a series of aqueous buffer solutions separated by oil surface tension valves into a single length of tubing, we were able to purify the protein biomarker from asymptomatic blood samples with greater than 50% efficiency by processing biomarker bound magnetic particles through the cassette using a handheld magnet. Additionally, we observed the application of the extracted protein to commercially available RDTs afforded a marked increase in performance of the tests. Regardless of the WHO detection score, we found all brands to be improved within asymptomatic levels of infection--a regime of diagnosis that RDTs have traditionally been unable to detect. Both the visual signal and limit of detection were enhanced at least 4-fold, with two brands having a limit of detection of 3 parasites/µL. The ability to transform RDTs from a confirmative test to a quantitative one would be invaluable for malaria eradication campaigns.

4.  Quiyun Chen, "Arrestin shape-shifting: NMR study of free and receptor-bound arrestin," Qiuyan Chen, Tiandi Zhuang, Min-Kyu Cho, Sergey A. Vishnivetskiy, Tina M. Iverson, Vsevolod V. Gurevich, and Charles R. Sanders

G-protein-coupled receptors (GPCRs) are essential mediators of information transfer in eukaryotic cells. Interactions between GPCRs and their binding partners modulate the signaling process. For example, the interaction between GPCR and cognate G protein initiates the signal, while the interaction with cognate arrestin terminates G protein-mediated signaling. To fully understand receptor signaling it is important to determine the affinities of these interactions and to identify the residues involved. In visual signal transduction, arrestin-1 selectively binds to the phosphorylated light-activated GPCR rhodopsin to terminate rhodopsin signaling. Solution NMR spectroscopy of labeled arrestin-1 was used to explore its interactions with dark-state phosphorylated rhodopsin (P-Rh), phosphorylated opsin (P-opsin), unphosphorylated light-activated rhodopsin (Rh*), and phosphorylated light-activated rhodopsin (P-Rh*). Distinct sets of arrestin-1 elements were seen to be engaged by Rh* and inactive P-Rh, which induced conformational changes that differed from those triggered by binding of P-Rh*. Importantly, we observed widespread NMR peak disappearance of arrestin-1 in the presence of P-*Rh. This led to the hypothesis that a transition from a flexible but well-ordered structure to a partially disordered ensemble of conformations, which might be the central mechanism underlying the “activation” of other arrestins.

5.  Timothy Senter, "Development of ML399: A small molecule inhibitor of the menin-mixed lineage leukemia interaction," Timothy Senter, Craig W. Lindsley, Shaun R. Stauffer

The development of a novel small molecule protein-protein interaction (PPI) inhibitor as a potential theurapeutic approach for mixed lineage leukemia (MLL) will be discussed. The protein-protein interaction between menin and MLL is critical for oncogenic activity of MLL fusion proteins, resulting from translocations of the MLL gene. Patients with MLL-associated leukemias have very poor prognoses, with an estimated 35% overall five-year survival rate. Inhibition of this interaction with small molecule PPI inhibitors represents a possible therapeutic target for mixed lineage leukemia. Optimization of a hydroxymethyl piperidine series identified from a high-throughput screen of ~288000 compounds resulted in ML227, which closely mimics many key interactions of MLL with menin. Extensive crystallography studies enabled a structure-guided design approach for the optimization of this series towards ML399, a viable candidate for in vivo proof-of-concept studies. Improvements in potency and physiochemical properties described herein will allow for evaluation in animal models of mixed lineage leukemia and provides an example of inhibition of PPIs in the context of epigenetic regulation.

6.  Thomas Mathews, "Phospholipase D regulates deoxy-ribonucleotide biosynthesis through pyrimidine metabolic intermediates," Thomas P. Mathews, Kristie L. Rose, Craig W. Lindsley, H. Alex Brown

Cancer is widely recognized as a systematic redistribution of metabolic and signaling pathways contributing to rapid cellular replication and survival. To sustain this higher rate of growth, cancer cells often upregulate the biosynthesis of central cellular building blocks necessary for replication. In recent years, these biosynthetic pathways have been found lying downstream of discrete oncogenic targets. Phospholipase D (PLD) hydrolyzes membrane-bound phospholipids to produce phosphatidic acid (PtdOH), which can in turn activate oncogenic targets, some of which directly influence metabolic regulation. In an effort to define the signaling pathways associated with PtdOH production and cancer progression, our labs have designed, developed, and characterized the most potent and subtype selective small-molecule PLD inhibitors reported in the literature to date. Using these inhibitors in a glioblastoma model cell line, U87MG cells, we found that inhibition of PLD enzymatic activity reliably decreases deoxy-ribonucleotide biosynthesis. Further analysis of metabolomic and proteomic readouts revealed that PtdOH signaling through mTOR decreases the biosynthesis of metabolites upstream of pyrimidine ring structures – one of core dNTP subunits. We found that these decreases in pyrimidine biosynthesis, from impaired mTOR signaling, are responsible for the observed reduction in dNTP levels. In this way, these studies establish a novel, cell-specific regulatory mechanism for PLD in pyrimidine biosynthesis in malignant gliomas.

7.  Robert Boer, "Progress Toward the Total Synthesis of Arachidonic Acid Metabolites HKD2 and HKE2," Robert E. Boer, Claus Schneider, and Gary A. Sulikowski

Hemiketal D2 and E2 are eicosanoids identified by Schneider and coworkers in 2011. These novel arachidonic acid metabolites are cross-over products of the enzymes 5-LOX and COX-2 previously assumed to be operating independently in the biosynthesis of leukotrienes and prostaglandins, respectively. It was shown that hemiketals stimulate angiogenesis of vascular endothelial cells, suggesting these compounds may be involved in inflammation and tissue repair. As the enzymatic production of these compounds is low yielding, total synthesis provides an attractive option for their production for further study. Presented herein is the total synthesis of HKE2 and efforts toward HKD2, featuring a metal promoted cycloisomerization-oxidation sequence to form the unique keto hemiketal moiety.

8.  Matthew Surdel, "A chemical genetic approach to interrogate the heme stress response of Staphylococcus aureus," Matthew C. Surdel, Brendan F. Dutter, Devin L. Stauff, Olusegun Aranmolate, Gary A. Sulikowski, and Eric P. Skaar

Staphylococcus aureus is a pathogen contributing to significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires heme as a nutrient iron source and as a cofactor for numerous critical processes. To satisfy these requirements, S. aureus imports host heme through dedicated systems, while retaining the ability to synthesize heme de novo. Although heme is an essential nutrient for growth, excess heme is toxic. S. aureus utilizes a two component system, the heme sensor system (HssRS), to sense and protect against heme toxicity. Upon activation, HssRS induces the expression of the heme-regulated transporter (HrtAB), an efflux pump that alleviates heme toxicity. The ability to sense and respond to heme is critical for pathogenesis, yet the mechanism of heme sensing remains unknown. Small molecules ‘8882 and ‘3981 were identified in a high-throughput screen as activators of staphylococcal HssRS. Importantly, the mechanisms through which these compounds activate HssRS differs. Whereas ‘8882 induces an increase in endogenous heme biosynthesis, ‘3981 induces HssRS independently of heme, suggesting heme is not the ligand for HssRS. Using multiple target identification strategies, we have uncovered numerous genetic requirements of the heme stress response. Notably, by utilizing a suicide strain containing a Phrt-driven relE construct, we have identified suppressor mutations preventing sensing of ‘8882 and heme. This has uncovered numerous residues within HssRS required for hrtAB activation, as well as additional genes required for sensing these molecules. In addition, a transposon screen for strains unresponsive to ‘3981 has uncovered bacterial nitric oxide synthase as crucial to the heme stress response, providing the first evidence that nitric oxide and staphylococcal heme sensing are linked. Ultimately, elucidating the mechanisms employed by S. aureus to overcome heme toxicity experienced in the host will provide a better understanding of the pathogenesis of this organism and may uncover potential therapeutic targets. Based upon the conservation of heme sensing systems across multiple medically relevant pathogens, this work may provide information applicable to heme sensing in a variety of infectious agents.

Poster Presentation Abstracts

Poster presentation abstracts will be posted as they are submitted.

1.  Thomas Mathews, "Phospholipase D regulates deoxy-ribonucleotide biosynthesis through pyrimidine metabolic intermediates," Thomas P. Mathews, Kristie L. Rose, Craig W. Lindsley, H. Alex Brown

Cancer is widely recognized as a systematic redistribution of metabolic and signaling pathways contributing to rapid cellular replication and survival. To sustain this higher rate of growth, cancer cells often upregulate the biosynthesis of central cellular building blocks necessary for replication. In recent years, these biosynthetic pathways have been found lying downstream of discrete oncogenic targets. Phospholipase D (PLD) hydrolyzes membrane-bound phospholipids to produce phosphatidic acid (PtdOH), which can in turn activate oncogenic targets, some of which directly influence metabolic regulation. In an effort to define the signaling pathways associated with PtdOH production and cancer progression, our labs have designed, developed, and characterized the most potent and subtype selective small-molecule PLD inhibitors reported in the literature to date. Using these inhibitors in a glioblastoma model cell line, U87MG cells, we found that inhibition of PLD enzymatic activity reliably decreases deoxy-ribonucleotide biosynthesis. Further analysis of metabolomic and proteomic readouts revealed that PtdOH signaling through mTOR decreases the biosynthesis of metabolites upstream of pyrimidine ring structures – one of core dNTP subunits. We found that these decreases in pyrimidine biosynthesis, from impaired mTOR signaling, are responsible for the observed reduction in dNTP levels. In this way, these studies establish a novel, cell-specific regulatory mechanism for PLD in pyrimidine biosynthesis in malignant gliomas.

2.  Katie Winarski, "The anti-influenza antibody H5.3 can neutralize different H5N1 variants," Katie L Winarski, Natalie J Thornburg, James E Crowe, Jr., Benjamin W. Spiller

The influenza virus H5N1, or avian flu, is not currently directly transmissible between humans, although recent laboratory-created, respiratory droplet transmissible H5N1 strains indicate that very few point mutations are necessary to allow direct transmission in the immunologically naïve human population. We are interested in the interaction between the influenza glycoprotein hemagglutinin 5 (H5) and the anti-influenza antibody, H5.3. H5.3 was isolated from a patient in an H5N1 vaccine trial and is capable of binding multiple H5N1 strains by interacting with the H5 head domain. We have determined the structure of H5.3 Fab in complex with the H5 head domain and found H5.3 neutralizes H5N1 by inserting its CDRH3 into the receptor binding site and mimicking interactions between H5 and its receptor, sialic acid. We have also determined the structure of H5.3 Fab in complex with respiratory droplet transmissible (rdt) H5 head domain and found H5.3 binds rdt H5 in the same manner as it binds wt H5, avoiding interactions with the rdt residues. This structure reveals how receptor binding site directed antibodies can bind hemagglutinins with different receptor specificity, indicating that receptor specificity may not be critically important for vaccine development.

3.  Cynthia Berry, "Optimization of a dopamine receptor 4 antagonist as a PET tracer and an in vivo tool to study cocaine addiction," Cynthia B. Berry, Michael Bubser, Carrie K. Jones, John P. Hayes, James A. Wepy, Charles W. Locuson, J. Scott Daniels, Craig W. Lindsley, and Corey R. Hopkins

Dopamine receptors are involved in many important central nervous system processes and are indicated in diseases such as schizophrenia, attention deficit hyperactivity disorder, Parkinson’s disease, and drug addiction. Since the discovery of the five subtypes of dopamine receptors, great effort has been taken to synthesize selective ligands in order to study each receptor’s involvement in disease. The Lindsley laboratory has developed an enantioselective synthesis of a morpholine-based dopamine receptor 4 (D4) antagonist. This compound binds D4 with a Ki of 70 nM, has an IC50 of 180 nM, and is highly selective over the remaining dopamine receptors and other GPCRs. Through iterative, parallel synthesis, a structure-activity relationship study was conducted around this lead to improve binding affinity and other pharmacological properties. We hope to develop a highly selective D4 antagonist for use as a PET tracer to image D4 receptors in the brain and an in vivo tool to study cocaine addiction, which has shown evidence of treatment by D4 antagonism.

4.  Orrette Wauchope, "Nuclear Metabolism of an Inflammation-Linked DNA Adduct in the Genome," Orrette R. Wauchope, James J. Galligan, Lawrence J. Marnett

The relationship between chronic inflammation and cancer has been well established in the literature. Inflammation, a normal metabolic process, has been shown to cause DNA damage, a major cause of genetic mutations. Inflammation is a complex biological process resulting in lipid, protein and/or DNA peroxidation. Lipid and DNA peroxidation generates highly reactive electrophiles that can form adducts with DNA bases. There has been an increased interest in the amount of DNA damage resulting from these electrophiles in healthy or disease-bearing individuals. In fact, understanding the metabolism of these DNA adducts is crucial as they can lead to increased mutagenesis and increased susceptibility to cancer development and progression. However, the metabolic fate of several DNA adducts has not been investigated or completely understood. Our laboratory has shown that 3-(2-deoxy-β-D-erythro pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) is the most abundant DNA adduct formed from the lipid or DNA oxidation product malondialdehyde or base propenal respectively. M1dG has been extensively studied in our laboratory and has been shown to be mutagenic in both mammalian and bacterial systems, inducing base-pair substitutions and frameshift mutations. M1dG has been detected in the genomic DNA of rodents and healthy human beings. It has also been detected in the urine of healthy individuals, which may make it a useful biomarker of DNA damage linked to oxidative stress. M1dG is rapidly removed from plasma after i.v. administration to rats (t1/2 = 10 min) and it is oxidized to 6-oxo-M1dG, which is also detectable in urine from rats. This discovery was the first report of an endogenous deoxynucleoside lesion being metabolized by oxidation in vivo. Our initial assumption was that 6-oxo-M1dG is generated from M1dG after it is removed from genomic DNA by nucleotide-excision repair. However, we recently made the exciting discovery that 6-oxo-M1dG is formed in single-stranded or double-stranded DNA when oligonucleotides containing M1dG are incubated with nuclear extracts. Although preliminary, this discovery leads us to hypothesize that 6-oxo-M1dG is formed in genomic DNA by enzymatic oxidation of M1dG. If correct, it has major implications for our understanding of the metabolism and mutagenicity of an important endogenous DNA adduct present in human genomic DNA. Consequently, the experiments presented herein highlight our initial investigations into the oxidation of M1dG in genomic DNA.

5.  Isi Ero-Tolliver, "The N-Terminal Immunoglobulin Domain of Peroxidasin Is Required to Cross-Link Collagen IV," Ero-Tolliver, I.A., Colon, S., Hudson, B.G., Bhave, G.

The collagen IV sulfilimine cross-link and its catalyzing enzyme, peroxidasin, represent a dyad, conserved throughout the animal kingdom, which is critical for tissue development. Peroxidasin forms novel sulfilimine bonds between apposing methionine and hydroxylysine residues to structurally reinforce the collagen IV scaffold, a function critical for basement membrane and tissue integrity. However, the molecular mechanism underlying the unique ability of peroxidasin to cross-link collagen IV remains unclear. In this work, we demonstrate that a combination of HOBr formation and direct binding to collagen IV allows peroxidasin to reinforce basement membranes. Thus, this molecular feature accounts for the evolutionarily conserved function of peroxidasin in tissue development and integrity.

6.  Carrie Shaffer, "Ring-fused 2-pyridones disrupt Helicobacter pylori type IV secretion," Carrie L. Shaffer, James A. D. Good, K. Syam Krishnan, Jennifer A. Gaddy, John T. Loh, Fredrik Almqvist, Timothy L. Cover, Maria Hadjifrangiskou

Helicobacter pylori utilize a type IV secretion system (cag T4SS) to inject the oncogenic effector protein CagA into host gastric epithelial cells. The mechanism by which T4SSs transport cargo into target cells remains poorly understood. Here, we describe two ring-fused 2-pyridones (C10 and KSK85) that significantly inhibit cag T4SS effector translocation and T4SS-dependent cellular alterations. KSK85 impedes biogenesis of T4SS-associated pili required for effector delivery, while C10 disrupts type IV secretion without perturbing pilus assembly. We provide evidence that 2-pyridones target CagA in a manner that is independent of their effects on T4SS function. In addition, we report that ring-fused 2-pyridones modulate DNA transfer through E. coli conjugative T4SSs. Thus, these chemical probes are molecular tools that enable mechanistic interrogation of diverse effector translocation processes and the unraveling of complex T4SS assembly dynamics.

7.  Michael Goodman, "The Investigation of the Chemical Mechanism and Inhibition of Microsomal Prostaglandin E2 Synthase 1 (MPGES1) Michael C. Goodman and Richard N. Armstrong

Prostaglandins function as signaling molecules involved in pain, fever, and many diseases associated with chronic inflammation. The most common therapeutic treatment of inflammation involves the inhibition of the COX enzymes by nonsteroidal anti-inflammatory drugs (NSAIDs) or COX-2 selective inhibitors (coxibs). The inhibition of the COX enzymes prevents the catalytic formation of the intermediate endoperoxide compound, PGH2. With the inhibition of PGH2, the production of other downstream prostaglandins is also inhibited. Therefore, COX inhibition can result in adverse gastrointestinal and cardiovascular side effects because of the subsequent low level of various prostanoids. Microsomal Prostaglandin E2 synthase 1 (MPGES1), a member of the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily, is the most prominent PGE synthase expressed during inflammation and is the terminal enzyme in the PGE2 synthesis pathway. It is a promising therapeutic target for the treatment of chronic inflammation and potential drug candidates have been pursued by the pharmaceutical industry in recent years. However, little is known about the actual chemical mechanism of the enzyme. Glutathione (GSH) appears to participate as a cofactor in the reaction, though there hasn’t been any evidence to support this. In order to investigate GSH as a cofactor in the reaction, it is necessary to detect the thiolate of GSH in the active site and determine its exchange rate off of the enzyme surface. One equivalent of the thiolate is observable by difference spectroscopy. However, simple difference spectroscopy isn’t possible since the enzyme is unstable in the absence of GSH. Therefore, a solution of the enzyme in complex with GSH can be rapidly mixed with the serine analog of GSH, γ-L-glutamyl-L-serylglycine (GOH), and the kinetics of the loss in absorbance from the replacement of GS- with GO- would confirm the presence of the thiolate in the complex and its rate of dissociation from the enzyme can be determined.

8.  Jessica Moore, "High Mass Resolution MALDI Protein Imaging reveals Oxidative Damage to Proteins During Staphylococcus aureus Infection," Jessica L. Moore, Jeffrey M. Spraggins, Neal D. Hammer, Kristie L. Rose, Eric P. Skaar, Richard M. Caprioli

Staphylococcus aureus is a cause of significant morbidity and mortality within the developed world. S. aureus infection causes the formation purulent inflammatory foci, called abscesses, characterized by the recruitment of neutrophils and an abundance of the host protein calprotectin. Calprotectin is composed of the calcium-binding S100 proteins S100A8 and S100A9, which are known to inhibit bacterial growth by restricting nutrient metals. Interestingly, S100 proteins are also damage-associated molecular pattern molecules (DAMPs), initiators of the inflammatory response and targets of oxidative damage. MALDI FTICR imaging mass spectrometry (IMS) allows oxidative modification of DAMPs to be regiospecifically characterized and strengthens comparisons to proteomics data using mass accuracy. This approach provides new molecular insights and oxidation-specific epitopes localized to the pathogen-host interface. Given that the abscess represents the complex nature of the host-pathogen interface, the identification of these molecules could reveal other inflammatory biomarkers.

9.  Norie Sugitani, "Re-defining the DNA-Binding Domain of Human XPA," Norie Sugitani, Steven M. Shell, Sarah E. Soss, and Walter J. Chazin

Xeroderma pigmentosum complementation group A (XPA) protein plays a critical role in the repair of DNA damage via the nucleotide excision repair (NER) pathway. XPA serves as a scaffold for NER, interacting with several other NER proteins as well as the DNA substrate. The critical importance of XPA is underscored by its association with the most severe clinical phenotypes of the genetic disorder Xeroderma pigmentosum. Many of these disease-associated mutations map to the XPA98-219 DNA-binding domain (DBD) first reported ~20 years ago. Although multiple solution NMR structures of XPA98-219 have been determined, the molecular basis for the interaction of this domain with DNA is only poorly characterized. In this report, we demonstrate using a fluorescence anisotropy (FA) DNA-binding assay that the previously reported XPA DBD binds DNA with substantially weaker affinity than the full-length protein. In-depth analysis of the XPA sequence suggested that the original DBD construct lacks critical basic charge and helical elements at its C-terminus. Generation and analysis of a series of C-terminal extensions beyond residue 219 yielded a stable, soluble human XPA98-239 contstruct that binds to a Y-shaped ssDNA-dsDNA junction and other substrates with the same affinity as the full-length protein. Two-dimensional 15N-1H NMR suggested XPA98-239 contains the same globular core as XPA98-219 and likely undergoes a conformational change upon binding DNA. Together, our results demonstrate that the XPA DBD should be redefined and that XPA98-239 is a suitable model to examine the DNA binding activity of human XPA.

10.  Kyle Floyd, "MALDI-TOF imaging mass spectrometry reveals oxygen-dependent regulation of adhesive fibers within uropathogenic Escherichia coli biofilms, Kyle A. Floyd, Jessica L. Moore, James A. D. Good, Carrie L. Shaffer, Eric P. Skaar, Fredrik Almqvist, Richard M. Caprioli, Maria Hadjifrangiskou

Bacterial biofilms are multi-cellular communities characterized by genetically heterogeneous populations that contribute to biomass resilience. The stratification and role of subpopulations within the biomass remains poorly defined. For uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, the ability to form intracellular and catheter-associated biofilms is paramount to their pathogenesis. Here, we investigated the spatial proteome of surface-associated UPEC biofilms using MALDI-TOF imaging mass spectrometry (IMS). We visualized 60 protein species between 2,000-25,000 m/z, which displayed one of four distinct biomass localization patterns. Subsequent identification revealed distinct localization of two adhesive fibers. The primary structural component of type 1 pili (FimA) localized specifically to the air-exposed (oxygen-rich) region, while the primary component of curli amyloid fibrils (CsgA) localized to the air-liquid interface (oxygen-limited). Type 1 pili (fim) mediate adherence to the bladder and facilitate biofilm cohesion. Given the FimA localization, we tested the hypothesis that fim expression is, at least in part, regulated by oxygen. Analysis of cells grown fermentatively in the absence of oxygen or other electron acceptors, revealed the phase-variable promoter of the fim gene cluster to be in the “OFF” orientation (fimOFF) preventing transcription, and showed a lack of type 1 pili production by anti-FimA immunoblot. In cells genetically tethered fimON, we still observed a significant reduction in FimA under fermentation. Consistent with the IMS observations at oxygen-limited biomass regions, transmission electron microscopy revealed production of amyloid fibers but no pili on the cell surface of cells grown fermentatively. Addition of an alternative electron acceptor partially restored pili production only in fimON cells. These data support two regulatory mechanisms for adhesive fiber expression in response to oxygen availability: one mechanism controls the fim promoter orientation, while another mechanism operates on the transcriptional/post-transcriptional level independent of promoter position. Such multi-level control may be critical in fine-tuning the stratification of adhesive fibers within the biomass. MALDI-TOF IMS has driven the discovery of these novel regulatory mechanisms, which could present new targets for anti-biofilm strategies.

11.  Brendan Dutter, "Probing Heme Biosynthesis and Homeostasis in Staphylococcus aureus," Brendan Dutter, Laura A. Mike, Matthew C. Surdel, Eric P. Skaar, Gary A. Sulikowski

The acquisition and metabolism of heme is of critical importance to the pathogenesis of Staphylococcus aureus, a bacterium of significant concern to public health. S. aureus acquires heme from its host to provide iron for cellular processes and to drive pathogenesis, but in high concentrations heme is toxic. The bacterium regulates heme levels through the HssRS two component system which upon activation induces expression of the efflux pump HrtAB which then alleviates heme toxicity. The mechanism by which HssRS senses heme is not well understood. A high throughput screen for activators of the HssRS system yielded the lead compounds, ‘8882 and ‘3981. We have demonstrated that ‘8882 activates heme sensing by increasing intracellular heme concentration and is dependent on the heme biosynthesis pathway while ‘3981 does not cause heme accumulation or require intact heme biosynthesis. Using a medicinal chemistry approach, we have determined structure activity relationships for both molecules and developed photoaffinity probes with clickable handles to identify the proteins to which these molecules interact. Coupled with parallel genetic approaches, we hope to elucidate the mechanism of HssRS heme sensing and understand the interplay and regulation of exogenous heme acquisition and endogenous heme biosynthesis.

12.  Li Liang, "Sequence-Specific Major Groove Hydrogen Bonding Patterns Control Aflatoxin B1 Formamidopyrimidine (FAPY) Adduct Structure in DNA," Li Liang, Kyle L. Brown, Ruidan Ma, and Michael P. Stone

Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus, is oxidized by cytochrome P450 enzymes to aflatoxin B1-8,9-epoxide, which alkylates DNA at N7-dG.  Under basic conditions, this N7-dG adduct rearranges to yield a N7-dG formamidopyrimidine (AFB1-FAPY) adduct. Structural analysis of the AFB1-FAPY adduct in the 5′-XA-3′ sequence indicated the preference of the (E) geometrical isomer of the formyl group, which was attributed to the potential for a hydrogen bond between the AFB1-FAPY formyl group and the N6 exocyclic amino group of the 3ʹ-neighbor adenine. Here, NMR analyses of duplex oligodeoxynucleotides containing the 5′-XA-3′, 5′-XC-3′, 5′-XT-3′ and 5′-XY-3′ sequences (X= AFB1-FAPY; Y=7-deaza-dG) demonstrate that the equilibrium between (E) and (Z) isomers of the AFB1-FAPY adduct is controlled by major groove hydrogen bonding interactions.  While the 5′-XA-3′ sequence exhibits the (E) isomer, the 5′-XC-3′ sequence exhibits a 7:3 (E):(Z) ratio at equilibrium at 283 K. The (E) isomer is stabilized by the formation of a hydrogen bond between the formyl group and the N4-dC exocyclic amino group of the 3'-neighbor cytosine.   In contrast, for the 5′-XT-3′ and 5′-XY-3′ sequences cannot form such a hydrogen bond between the formyl group and the 3′-neighbor T or Y, respectively, and the (Z) isomer is favored. In DNA, the AFB1-FAPY adduct also equilibrates between α and β anomers, with the β anomer being favored in duplex DNA.  The α:β anomeric equilibrium does not depend upon sequence, favoring the β anomer in each instance.

13.  Alexander Geanes, "BCL::EvoGen: An evolutionary algorithm for focused library design,” Alexander Geanes, Edward W. Lowe and Jens Meiler

In recent years, virtual high-throughput screening (vHTS) techniques have been successfully applied in the drug discovery process. In many cases, these vHTS techniques are leveraged to prioritize subsets of chemical libraries for acquisition and testing in physical screens. However, for computer-aided drug design (CADD), it is advantageous to have algorithms which are capable of designing new chemical entities for a specific biological target. An evolutionary algorithm was implemented as part of the BCL::CHEMINFO suite within the Biochemistry Library (BCL), a C++ library developed at Vanderbilt University, to iteratively generate chemical species with high predicted biological activities for use in focused library design for hit-to-lead optimization. Quantitative structure activity relationships based on machine learning techniques were used to predict the biological activity of compounds in each generation. The compounds with the highest predicted activity, as well as a smaller number of lower activity species, were subjected to combination, crossover, and mutation to form the subsequent generation. Termination criteria were based on whether a percentage of compounds in a single generation had achieved a predicted activity above a pre-determined cutoff, or after a pre-set number of generations had been reached. This method was benchmarked using a previously published set of 9 datasets designed for the validation of novel CADD methods. Each of the datasets was compiled from publicly available HTS data taken from PubChem, and contain a minimum of 150 active compounds each. In addition, the datasets span a range of protein targets including GPCRs, ion channels, and enzymes. Here we present the results of this focused library design application, BCL::EVOGEN.

14.  Katrina Leaptrot, "Theoretical Performance Assessment and Development of a Spatially Multiplexed 8-Channel Ion Mobility-Mass Spectrometer," Katrina L. Leaptrot, Jody C. May, John A. McLean

Ion mobility-mass spectrometry (IM-MS) is a two-dimensional separations technique which has demonstrated significant utility in the analysis of complex mixtures. Although a spatially multiplexed IM-MS has not yet been developed, such an instrument is expected to provide improved figures-of-merit in throughput, sensitivity, and versatility, among others. Here we discuss details from the ongoing development of an IM-MS with 8 discrete parallel ion channels. The instrument is being designed to perform with spectral reproducibility across each channel and figures-of-merit comparable to existing single channel IM-MS instruments in terms of resolving power and sensitivity. Development of the spatially multiplexed IM-MS has been organized into three phases. Phase one involves designing an 8-channel ion mobility spectrometer (IMS) with an electrospray ionization (ESI) source array. Phase two focuses on the development of a matrix assisted laser desorption/ionization (MALDI) source and adaptation of a commercial time-of-flight mass spectrometer (TOFMS) to simultaneously accept multiple ion beams. Phase three integrates the 8x IMS and the modified TOFMS and establishes data acquisition/visualization software. AutoCAD, SIMION 8.1, and COMSOL Multiphysics software are used to design instrument components, evaluate ion trajectories, and investigate flow dynamics, respectively. Successful designs are commissioned for fabrication and assembled in-house. Here we present the progress of constructing this instrument with emphasis on computer simulations for performance evaluation and modification of tandem ion funnels to facilitate pumping and low-powered electronics.

15.  Rafael Montenegro, "Real Time Metabolomic Analysis of Saccharomyces cerevisiae," J. Rafael Montenegro-Burke, Jeffrey R. Enders, Kevin T. Seale, John P. Wikswo,John A. McLean Systems biology focuses on the study of biochemical interactions in biological networks. The study of dynamic measurements of the metabolome and cell signaling provides vital information, however, characterization of these networks is extremely difficult due to the lack of comprehensive time-dependent experimental data and the highly transient nature of the analytes. Historically, sample preparation is necessary to remove abundant non-volatile salts that cause ion-suppression effects in the ionization process. This involves sample fractionation, thus losing temporal information. Metabolite concentrations in living organisms can change in seconds to minutes. Therefore, collecting a sample for minutes provides only the average concentration during that timeframe. Here, we describe a platform capable of online sample preparation for ESI-MS without sample fractionation for metabolome analysis. The platform consists of a Multitrap Nanophysiometer (MTNP), which sustains large arrays of individual cells or cell colonies. Subsequently, chemical stimuli are perfused through the MTNP at flows of 500 nL/min to stimulate various biochemical processes within the cells. The resulting cellular secretions (metabolites) are extracted and desalted online using a prototype microdialysis device designed and fabricated in-house. The desalted effluent is then directed to the nESI input of an ion mobility mass spectrometer for analysis.

16.  Brett C. Covington, "Unlocking Natures Chemical Arsenal," Brett C. Covington, Cody R. Goodwin, Dagmara K. Derewacz, John A. McLean, and Brian O. Bachmann

More than 85 years after Alexander Flemming’s serendipitous discovery of penicillin, the mechanisms by which antibiotics lead to cell death are still debated, the antibiotic pipeline has nearly dried up, and antibiotic resistant pathogens dominate our hospitals. These seemingly insurmountable challenges in the fight against infectious disease have caused many to fear modern medicine is moving towards a post-antibiotic era. However, recent high-throughput genome screening has identified a hidden arsenal of natural products locked within the genomes of producing organisms. We hypothesize these compounds, which are not produced under laboratory conditions, are used by producing organisms to respond to environmental stimuli from competing organisms and other physical factors, and using metabolomics analyses we have shown that by applying the right stimulus we can activate the production of hundreds of compounds from a single organism. This work has led to the discovery of several new natural products, ciromicins and mutaxanthenes, and has demonstrated how we may be able to unlock nature’s chemical arsenal to resupply the stifled antibiotic pipeline and postpone or even prevent a post-antibiotic era.

17.  John Hayes, "Progress Towards the Synthesis and Biological Evaluation of Lacto-N-neotetraose," John P. Hayes, Steven D. Townsend

The benefits of breastfeeding, such as lowering infants’ risk of infections and diarrhea, are now widely recognized. Many of these benefits are attributed to the presence of human milk oligosaccharides (HMO), which are known to stimulate growth of beneficial bifidobacteria in the infant gut. Additionally, some compounds in this class serve as antibiotics, providing protection against harmful bacteria such as Escherichia coli. Unfortunately, many infants are unable to breastfeed for the recommended six month timeframe and often suffer from illness as a result. To address this gap in infant wellness, it is important to develop viable synthetic routes to HMOs in order to develop infant formulas that provide nutritional benefits similar to human milk. This work aims to synthesize lacto-N-neotetraose in an orthogonally protected manner in order to access a small pool of human milk pentasaccharides. The synthetic molecules will then be tested for their ability to stimulate growth in bifidobacteria or inhibit growth in pathogenic strains.

18.   Soumya Ganguly, "Structural and Functional Characterization of the Disordered N- Terminal Region of HOX A11," Soumya Ganguly, Mauris C. Nnamani, Laura Mizoue, Carie Fortenberry, Heather Darling, Günter P. Wagner, Jens Meiler

Transcription is mediated by a large number of proteins that interact with DNA and with each other. The Hox family of transcription factors plays critical roles in embryonic development and morphogenesis. All HOX proteins bind to DNA with a highly conserved homeobox domain located at the C terminus and several crystal structures exist of HOX homeodomains bound to DNA. Although there have been many functional relevance associating gene regulation with the N-terminal region of HOX proteins, very little structural information is available. Here we present evidence that this region of HOX-A11 is intrinsically disordered. We have investigated binding of HOX-A11 to the KIX domain of the transcriptional coactivator CREB-binding protein (CBP) and determined the minimal amino acid sequence of N terminus HOX-A11 responsible for interaction. A model has been developed based on NMR titration of KIX with different HOX-A11 N-terminal mutants and gene deletion studies to explain the role of disordered region. The proposed model provides insight into the molecular basis of transcriptional regulation.

19.  Alex Brown, "Phospholipase D facilitates efficient entry of influenza virus allowing escape from innate immune inhibition," Thomas H. Oguin III, Shalini Sharma, Amanda D. Stuart, Susu Duan, Sarah A. Scott, Carrie K. Jones, J. Scott Daniels, Craig W. Lindsley, Paul G. Thomas, H. Alex Brown

Lipid metabolism plays a fundamental role during influenza virus replication, although key regulators of lipid-dependent trafficking and virus production remain inadequately defined. This report demonstrates that infection by influenza virus stimulates phospholipase D (PLD) activity and PLD co-localizes with influenza during infection. Both chemical inhibition and RNA interference of PLD delayed viral entry and reduced viral titers in vitro. Although there may be contributions by both major isoenzymes, the effects on viral infectivity appear more dependent on the PLD2 isoenzyme. In vivo, PLD inhibition reduced virus titer and correlated with significant increases in transcription of innate antiviral effectors. In vitro the reduction in viral titer downstream of PLD2 inhibition was dependent on RIG-I, IRF3, and MxA, but not IRF7. Inhibition of PLD2 accelerated the accumulation of MxA in foci as early as 30 minutes post-infection. Together these data suggest that PLD facilitates the rapid endocytosis of influenza virus, permitting viral escape from innate immune detection and effectors that are capable of limiting lethal infection.

20.  Sarah Scott, "Identification of Desketoraloxifene analogs as inhibitors of mammalian and Pseudomonas aeruginosa PLD," Sarah A. Scott, Cierra T. Spencer, Matthew O’Reilly, Kyle A. Brown, Robert R. Lavieri, Benjamin Neuenswander, H. Alex Brown, Craig W. Lindsley

Phospholipase D (PLD) hydrolyses cellular lipids to produce the important lipid second messenger phosphatidic acid. A PLD enzyme has recently been identified in Pseudomonas aeruginosa (PldA) leading to the hypothesis that PldA may serve as a virulence factor in bacterial infection. The importance of PLD in mammalian signaling and the potential of bacterial PldA in infection, makes inhibitors of these enzymes very desirable. Selective estrogen receptor modulators (SERMs) raloxifene and 4-hydroxytamoxifen, have been previously shown to inhibit mammalian PLD. A recent analog library of desketoraloxifene was published as a new class of SERMs and herein we identified some modest PLD inhibitors from that library and have designed novel desketoraloxifene based PLD inhibitors which inhibit both PLD and PldA. This study represents a novel lead structural class for further inhibitor development against mammalian and bacterial PLDs.

21.   Jeannie Camarillo, "CDK2 inhibition and cell cycle arrest through 4-hydroxy-2-nonenal modification," Jeannie M. Camarillo, Kristie L. Rose, Lawrence J. Marnett

Oxidative stress is a contributing factor to a number of diseases, including cancer, atherosclerosis, asthma, and neurodegenerative disease. 4-Hydroxy-2-nonenal (HNE), a lipid aldehyde generated from lipid peroxidation during periods of increased oxidative stress, has been shown to covalently modify cellular proteins, in many cases altering their activities. Previous proteomic studies have shown that CDK2, a cell cycle kinase responsible mainly for the G1/S transition, is modified by HNE. Here, we demonstrate CDK2 modification by HNE and show the functional alterations that occur as a result of modification. Tandem mass spectrometry revealed that HNE covalently adducts a number of residues on CDK2, most significantly His71 and His161, by Michael addition. Treatment of pure CDK2/cyclin E complexes with HNE led to a loss of kinase activity. HNE treatment delayed cell cycle progression into S-phase in G1-arrested cells. The delay was not due to changes in the levels of CDK2 and PARP cleavage, an indicator of apoptosis, was not observed. Most importantly, the delay in S-phase entry was independent of p53 and p21. Click chemistry with alkynyl-HNE revealed that progression of electrophile-treated cells into S-phase corresponded to loss of CDK2 modification, possibly through protein turnover. These data suggest that modification of CDK2 inhibits its kinase activity and may alter its turnover, thereby inhibiting cell cycle progression.

22.  Amanda Duran, "Designing a Super-Stable, Symmetric Membrane Protein," Amanda M. Duran, Jens Meiler

Protein design studies give insight for relationships between protein sequence, structure, and function. Computational protein design has the potential to contribute to various fields including drug design, protein therapeutics, and materials science. Stable scaffolds are needed to design a protein-ligand interface or as units to build a larger, stable protein. Symmetry will be used to create stable, scaffolds. Proteins that exhibit structural symmetry (despite divergent amino acid sequences) are proposed to be the result of gene duplication, fusion, and diversification events originating from a monomeric gene. Several membrane protein structures exhibit pseudo-symmetry. The Glycerol Facilitator protein (GlpF) from Escherichia coli is a membrane protein that exhibits a two-fold inverted pseudo-symmetry. Using Combinatorial Extension structural alignment and the protein structure prediction software suite, Rosetta, GlpF was engineered to be perfectly symmetric in sequence and presumably in structure. The symmetric gene was assembled, cloned, and expressed; however, many challenges have prevented structural characterization. In an attempt to better understand this poorly behaving engineered protein, Rosetta Membrane, Symmetry, and Design were implemented for in silico thermostabilization. To ensure optimal design, Rosetta Membrane is currently being benchmarked with a set of high-resolution membrane protein structures. Additionally, evolutionary information is being incorporated to further re-design the symmetric protein towards a super-stable scaffold.

23.  Caleb Morris, "Mathematical Descriptors of Biological Class Trends in Ion Mobility-Mass Spectrometry for Classification of Unknowns," Caleb B. Morris, Jody C. May, John A. McLean

In advancing strategies towards integrated omics, ion mobility has found great utility in the rapid separation of biological classes. Due to similar structural features within biomolecular classes, comparable gas phase packing efficiencies are observed within a class. This results in unique biomolecular class trends within 2-D IM-MS spectra. For analytes of 200 m/z or higher, these trends can be modeled using power functions. Below this approximate cut-off, the simple power function no longer sufficiently describes the data, and a more complicated formula must be used. Various mathematical descriptors were investigated for the best fit to the biomolecular trends in 2D IM-MS. The best fit, based on the coefficient of determination (R2), was found with the logistic function using a dataset of tetra-alkylammonium salts (N > 60) which exhibit a well defined IM-MS trend. This dataset also included low mass values which adjusted the required function from a power curve to a growth curve, with the specific function named the logistic function. This function has been applied to peptide, lipid, and carbohydrate trends gathered under uniform field conditions. Equations have been developed to classify unknowns and return confidence levels based on biomolecular class character. In certain instances, such as biomolecular class trends converging at low m/z, a clear biomolecular classification is impossible. In this case, a confidence level can be determined based on a percentage agreement with the nearest biomolecular class trendlines. This allows simplification of the possible classes in which an unknown may be a member.

24.   Susan Ramos-Hunter, "Design and Synthesis of Small Molecule Probes to Support Studies of GIRK1/2 Channels," Susan Ramos-Hunter, Wandong Wen, Ian Romaine, Craig Lindsley, C. David Weaver, Gary Sulikowski

G-protein activated inward rectifying potassium (K+) channels, or GIRKs are a part of a larger family of potassium channels that regulate potassium concentration and control excitability in cells. GIRK channels are found in various tissues including the heart and in the brain as hetero and homotetramers (G1-4) and have potential links to atrial fibrillation schizophrenia, epilepsy, pain perception and addiction and withdrawals. Research into GIRK channels has been limited due to lack of selective and potent small molecule probes. Recently, a high throughput screen followed by SAR studies has led to the identification of a GIRK activator, VU810 (aka ML297) found to selectively activate GIRK1 containing channels. During the course of SAR studies a highly GIRK1/2 selective enantiomer (of unknown configuration) of a trans-methylcyclopropane was discovered following chiral LC resolution. This compound’s ability to discriminate between the major brain GIRK, GIRK1/2 and the major cardiac GIRK, GIRK1/4 is very significant. The preparation of single enantiomers of known configuration will provide insight into the GIRK1/2 over GIRK1/4 selective activation.

25.  Kim Fong, "Exploring Heme Distribution as a Validation Method for the Hemozoin Drug Target Pathway in Plasmodium falciparum," Kim Y. Fong, Rebecca D. Sandlin, David W. Wright

Even though malaria is a preventable and treatable disease, it remains widespread among tropical regions and is the cause of more than 600,000 deaths each year. One of the most important drug targets for this disease is the hemozoin formation pathway, a heme detoxification process found in Plasmodium falciparum, the most virulent form of malaria. In the search for novel compounds that can inhibit this target pathway, a high throughput in vitro assay was developed in our laboratory. Using this model, the GlaxoSmithKline (GSK) library, consisting of 13,229 in vitro antimalarial lead compounds, was screened for activity against β-hematin formation, the synthetic analogue of hemozoin. Despite both possessing in vitro antimalarial activity and inhibiting β-hematin formation, it is vital that these results are also validated within the phenotypic target to ensure that leads are in fact acting upon the expected pathway. The top 17 GSK β-hematin inhibitors were subsequently tested using the previously published heme speciation assay within a parasite culture. Of these, 15 compounds were confirmed to produce a rise in free intravacuolar heme levels with increasing drug concentration. The rise in free heme correlated with a decrease in hemozoin and parasite survival. The high validation rate is indicative of the robustness of our in vitro assay design. We hypothesize that by more faithfully recapitulating the biological environment of the parasite where hemozoin is formed compared to other assays developed, potential leads capture more of the requirements of an active antimalarial drug.

26.  Pedro Garcia-Barrantes, "Development of novel mGlu1 PAMs as tools to improve functionality of mutant receptor isoforms found in a schizophrenic population," Pedro M. Garcia-Barrantes, Hyekyung P. Cho, John T. Brogan, Corey R. Hopkins, Colleen M. Niswender, Ryan D. Morrison, J. Scott Daniels, P. Jeffrey Conn, Craig W. Lindsley

Metabotropic glutamate receptor type 1 (mGlu1) is a G-protein coupled receptor from the group I of mGlus and is encoded by the GRM1 gene. Stimulation of the receptor leads to an increase in activity of phospholipase C, with subsequent mobilization of calcium and increase of intracellular calcium concentrations. mGlu1 is expressed predominantly in post-synaptic neurons from the hippocampus, hypothalamus, thalamus, amygdala, cerebellum, basal ganglia and spinal cord. The function of mGlu1 has been implicated in several neural processes such as nociception, synaptic plasticity, learning and memory. Hypofunction and deregulation of glutamate signaling has been established as a key concept in schizophrenia pathogenesis. Moreover, mutations in GRM1 gene that lead to hypofunction of the receptor have been observed in patients with schizophrenic and bipolar disorders, supporting the modulatory role of the receptor in neuropsychiatric disorders and suggesting that therapies targeting the receptor’s function recovery might be beneficial. In order to test this hypothesis, it is necessary to have suitable chemical tools to achieve the activation of the receptor and perform target validation studies. This work present the development of novel mGlu1 positive allosteric modulators (PAM) by repurposing a known mGlu4 PAM chemotype through a multidimensional iterative parallel synthesis approach. The compounds developed represent a new chemotype for potent and preferential mGlu1 PAM activity. The efficacy of these probes was evaluated in constructs of the mGlu1 receptor with mutations that decrease the functionality of the receptor and that have been identified in the schizophrenia population.

27.  Lauren Gibson, "Chromogenic, Fluorescent, and Catalytic Signal Amplification with Porphyrins," Lauren E. Gibson, David W. Wright

Signal amplification is an essential component of assays used for the detection of low abundance analytes. Signal amplification occurs when multiple signal producing molecules result per analyte molecule. Enzyme-linked immunosorbent assays (ELISAs) are a common assay format that employs signal amplification. Typically, the amplification in this assay is based upon the enzyme horseradish peroxidase (HRP), which in the presence of peroxide turns over a substrate producing a colorimetric change. Recently, a new chromogenic signal amplification method based upon metal oxide nanoparticles has been reported. In this method, nanoparticles are conjugated to detection antibodies and signal amplification is achieved through dissolution of metal nanoparticles and subsequent detection of the metal ions. Thus, thousands of metal ions result for every analyte molecule attached to a detection antibody. We have applied this method of signal amplification to porphyrin nanoparticles. Dissolution of these nanoparticles into individual porphyrins allows the unique absorbance, fluorescent or catalytic properties of the various porphyrin molecules to be used for amplification of the assay signal. In this work, tetra (4-carboxyphenyl) porphyrin nanoparticles have been used to explore assays based upon the strong and inherent absorbance and fluorescent properties of this porphyrin. It was found that although the absorbance properties were not sufficient to produce an improved signal for the developed assay, employing the fluorescent properties resulted in an assay with a limit of detection in the pM range. Additionally, nanoparticles were synthesized from microperoxidase-11 (MP-11). This porphyrin molecule has catalytic properties similar to HRP, so upon the dissolution of these particles, enzymatic turnover of a substrate, in the presence of peroxide results in a colorimetric change. Thus, in assays utilizing detection antibodies conjugated to MP-11 particles, signal amplification is achieved not only through nanoparticle dissolution but also catalytically. This work has given a novel application to porphyrin nanoparticles by using them in unique signal amplification assays for the detection of low abundance analytes.

28.  Kasia Derewacz, "Microbial interactions as a tool for inducing secondary metabolism in Nocardiopsis sp.," Kasia Derewacz, Brett C. Covington, Cody R. Goodwin, John A. McLean, Brian O. Bachmann

Secondary metabolites isolated from microorganisms have been one of the major sources of leads in the drug discovery pipeline since the 1930’s. Though extensively mined, recent developments of rapid genome sequencing techniques has revealed that microbial secondary metabolism is still greatly underexplored, with the majority of gene clusters remaining unexpressed. Interactions between competing microorganisms may be the key driving force for secondary metabolite expression in nature, and culturing bacteria and fungi with competing organisms has been shown to activate secondary metabolism. Therefore, we decided to utilize these methods in attempts to express “silent” genes in a genomically characterized Nocardiopsis sp. In this work we present the outcome of using the microbial interactions as a tool for inducing secondary metabolism in Nocardiopsis sp. When cultured with E. coli, Bacillus, Tsukamurella and Rhodococcus, Nocardiopsis produces a previously unreported polyene natural product ciromicin A, which undergoes a conversion to its structural isomer ciromicin B when exposed to ambient sunlight. Both ciromicins possess moderate cytotoxic activity.

29.  Nichole Lareau, "Enhancing Glycoprotein Characterization with Ion Mobility-Mass Spectrometry Techniques," Nichole M. Lareau and John A. McLean

Glycoproteins are highly implicated in biological signaling for cellular recognition and regulation. The characterization of glycoproteins is challenging due to the biological complexity and corresponding difficulty of separation. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) techniques play a critical role in the study of post translational modifications of proteins such as glycosylation and phosphorylation. Traditional MS/MS fragmentation studies such as vibrational activated collision-induced dissociation (CID) do not retain labile glycosylation post-translational modifications (PTMs). Alternatively electron transfer dissociation (ETD) is a radical driven fragmentation technique which preserves PTMs in a complementary manner to CID. Although ETD is a more comprehensive technique for glycoprotein analysis, data analysis can be difficult due to interfering background signals. Ion-mobility mass spectrometry (IM-MS) shows promise towards mitigating these challenges in glycomics. Analytical limitations resulting from sample complexity are addressed by performing rapid separations based on structure (IM) and mass (MS). Additionally, the IM-MS configuration allows ETD and subsequent CID fragmentation as complementary techniques in support of structural assignment. This report presents the development of methodologies for the characterization of proteins, glycans, and glycoproteins by ETD-IM-CID-MS.

30.  Jacob Choby, "S. aureus SaeRS facilitates toxicity of a small molecule inhibitor of fermentation," Laura A. Mike, Jacob E. Choby, Brendan F. Dutter, Shawn Barton, Paul M. Dunman, Gary A. Sulikowski, Eric P. Skaar

Staphylococcus aureus is a Gram positive pathogen responsible for extensive morbidity and mortality worldwide. S. aureus causes a variety of infections including soft tissue abscesses and osteomyelitis. At these foci of infection, oxygen levels drop creating a hypoxic or anaerobic local environment. In the absence of oxygen, S. aureus can produce energy by fermentation instead of aerobic respiration. The metabolic switch to fermentation also produces the clinically relevant small colony variant phenotype which is recalcitrant to antibiotic treatment and can be the etiological agent of persistent infections. Our previous work has shown that a small molecule from a High-Throughput Screen of the VICB compound library (‘8882) which stimulates endogenous heme biosynthesis, also diminishes fermentative activity and is bacteriostatic to fermenting S. aureus. In order to understand the mechanism of toxicity, wildtype S. aureus strain Newman was screened for spontaneously 8882-resistant isolates; whole genome sequencing yielded seven isolates all with lesions in the saePQRS locus. SaeRS is a staphylococcal two-component system (TCS) which regulates toxin production and other critical cellular processes. The Newman SaeRS is hyperactive because of a point mutation in the histidine kinase SaeS. This point mutation is necessary and sufficient for SaeRS-facilitated 8882 toxicity in anaerobiosis. Adding to our understanding of the connection between SaeRS and toxicity, a reverse genetics approach has identified two genes that are SaeRS regulated and required for 8882 toxicity. Taken together, the complementary strategies of forward and reverse genetics have identified numerous genes which will reveal underlying principles of S. aureus metabolism and toxicity associated with perturbing fermentation using chemical biology.

31.  Shu Xu, "The structural basis of conjugated fluorescent inhibitor binding in the active site of cyclooxygenase-2," Shu Xu, Md Jashim Uddin, Surajit Banerjee, Kelsey C. Duggan, James R. Kiefer, and Lawrence J. Marnett

Cyclooxygenase-2, an inducible enzyme expressed in inflammation, premalignant and malignant lesions, is an attractive target for molecular imaging of COX-2 in inflammation and cancer. Fluorocoxibs A and B, are potent and selective COX-2 inhibitors, synthesized by tethering bulky fluorescent functional groups onto indomethacin. They are extensively used as COX-2-targeted molecular imaging agents. Our early efforts to determine the structural and functional basis of fluorocoxib A for selective COX-2 inhibition by crystallography were unsuccessful due to the binding flexibility of the bulky fluorescent carboxy-X-rhodamine moiety with amino acid residues located at the COX-2 channel termed as lobby. Here, we report the first crystal structure description of murine cyclooxygenase-2 in complexes with LM-4702 and LM-4712 at resolutions of 2.2 Å and 2.5 Å, respectively. Both LM-4702 and LM-4712 are fluorescent COX-2 selective optical probes containing a relatively smaller fluorophore group than those of fluorocoxibs A and B. The electron density maps in the active site were of excellent quality to fit not only the indomethacin moiety but also conjugated fluorescent groups of these two fluorescent probes. In the cyclooxygenase-2 channel, the indomethacin moieties of LM-4702 and LM-4712 exhibited similar interactions as indomethacin in the active site of the enzyme. The conjugated fluorescent groups were projected toward the membrane-binding domain mainly by hydrophobic interactions. The linker breeched the constriction site at the base of the cyclooxygenase active site. Comparing the two complexes, it is quite interesting that although the linker of LM-4702 is two carbons shorter than that of LM-4712, the two probes shared similar interactions at the lobby of the enzyme. Thus, these crystal complexes provided for the first time insights into the molecular basis of COX-2 inhibition by LM-4702 and LM-4712. This information will be useful for the design of fluorescent- or toxin-conjugated COX-2-selective inhibitors.

32.  Cierra Spencer, "Biochemical characterization of a secreted bacterial phospholipase," D Cierra Spencer and H. Alex Brown

Pseudomonas aeruginosa is an important opportunistic human pathogen that is a major cause of hospital acquired infections and for which antibiotic resistance is a growing problem. To address the emergence of antibiotic resistance associated with bacterial infections, one approach is to identify and target virulence mechanisms that are not critical for cell viability but do promote pathogenesis. P. aeruginosa expresses a phospholipase D enzyme, PldA, that hydrolyzes phospholipids to generate phosphatidic acid, a critical lipid signaling molecule in eukaryotic cells. PldA has recently been validated as a secreted effector of the Type Six Secretion System of P. aeruginosa that targets both bacterial and eukaryotic cells. PldA has been shown to mediate chronic infection and host cell invasion without being required for cell viability. Its potential to promote disease and its interactions with human cells makes PldA an attractive candidate to study. In this work, we have identified several likenesses in vitro between PldA and the human PLD isoforms, as well as several properties in which the enzymes diverge. Notable differences include the substrate preference. But despite this difference, in lung epithelial cells, the molecular species of phosphatidic acid generated by PldA mimicked those of the host PLD enzymes. The ability of PldA to target eukaryotic cells begs new questions of potential regulatory mechanisms of PldA within human cells. Of particular interest for future studies are the observed stimulation by phosphoinositides and calcium, which could potentially regulate activity and localization of PldA in eukaryotic cells. Human PLD1 localization and activity is heavily regulated by phosphoinositides in cells, and PldA may also be regulated in a similar manner. Its ability to modify host lipids may facilitate the stability of exogenous PldA and also promote vesicle secretion by host cells. As a bacterial effector that targets eukaryotic cells, PldA may serve as a viable therapeutic target for P. aeruginosa infections, however there is a lack of potent small-molecule inhibitors for PldA. Human PLD inhibitors of three different scaffold classes were screened for their potential to inhibit PldA, and in this work a novel structural class of inhibitors of PldA was identified.

33.  Dorothy Ackerman, "Progress toward the synthesis of a pool of human milk trisaccharides: en route to functionalized galactooligosaccharides," Dorothy Ackerman, Steve Townsend

As the most cost effective public health strategy used to combat infant illness and mortality, breastfeeding serves as the model for developing infant nutrition. In cases where breastfeeding is not a viable option, it is important that infant formula closely mimics the nutritional and prebiotic components of breast milk. Many of the health benefits are attributed to human milk oligosaccharides (HMOs) because they significantly regulate and direct the development of intestinal microflora in an infant’s gut. The goal of this project is to synthesize short chain HMOs and other glycoconjugates from a selectively protected lactose core. These oligosaccharides will then be biologically evaluated for their ability to aid or inhibit the growth of both beneficial and harmful bacterial strains. Additionally, we will study the chemoezymatic polymerization of these oligomers to generate functionalized galacto-oligosaccharides (GOS) that are structurally similar to large HMOs. Our goal is to measure whether it is possible to generate compounds that are superior to commercially available, unfunctionalized GOS in influencing the infant microbiota.

34.  Daniel J. Sprague, "Artificial Biocatalysis: Adaptation of a Small Molecule Catalyst to a Hetero-Diels-Alder Reaction Hypothesized for Brevianamide Biosynthesis," Daniel J. Sprague, Benjamin M. Nugent, Ryan A. Yoder, Brandon A. Vara, and Jeffrey N. Johnston

A collection of chiral diamine-derived small molecules were evaluated for their ability to effect stereocontrol during an intramolecular hetero-Diels-Alder (HDA) reaction using hydrogen bonding as the key directing functionality. The substrate is an intermediate in Williams’ total synthesis of brevianamides Α-B. These small molecules were chosen for their potential to chaperone an otherwise thermal HDA reaction, resulting in modulation of both diastereo- and enantioselection. The highest level of enantioselection achieved was modest, and might be attributed to the similar size of the chaperone and its substrate, combined with a significant background (non-catalyzed reaction) rate at ambient temperature. Collectively, these results provide compelling evidence that small molecule hydrogen bond catalysis, if even based on an artificial biosynthesis construct (artificial biocatalysis), holds significant potential within enantioselective natural product synthesis.

35.  Kathleen Mittendor, "Characterization of Human Peripheral Myelin Protein 22 using Electron Microscopy," Kathleen F. Mittendorf, Mariena Silvestry, Cheri M. Hampton, Elizabeth R. Wright, Melanie D. Ohi, and Charles R. Sanders

Peripheral myelin protein 22 (PMP22) is a 160-residue tetraspan integral membrane protein highly expressed in Schwann cells responsible for peripheral nervous system myelination. The importance of this protein to proper myelination is highlighted by its involvement in the human dysmyelinating disorder Charcot-Marie-Tooth disease. Deletion, gene duplication, and missense mutations of PMP22 all result in disease phenotypes. Despite our knowledge of the importance of PMP22 in myelination, the exact function of PMP22 has not yet been identified. Recent single particle electron microscopy (EM) experiments reveal that, like its distant paralog lens membrane protein 20, PMP22 can form a heterogeneous assortment of tetramers under low detergent concentration conditions. Refolding of PMP22 into vesicles of certain lipid compositions results in the formation of 2-dimensional arrays that may be suitable for higher resolution structural studies of this important tetraspan protein. Through the use of 2-D electron crystallography, we hope to obtain a medium-to-high (10-4 Å) resolution structure of PMP22, shedding light on its oligomeric state in a membrane bilayer and the packing of its helices. Additionally, EM experiments in which PMPM22 is reconstituted into vesicles of compositions approximating myelin indicate that one function of PMP22 may be to assist stacking of Schwann cell membranes by supporting membrane-membrane adhesion and/or encouraging membrane curvature and spiraling to form the multi-layered myelin. We have refolded purified recombinant PMP22 into vesicles composed of POPC:egg sphingomyelin mixtures by the dialysis method and observed that PMP22 facilitates the formation of myelin-like assemblies (MLAs). Electron tomography reveals that the MLAs are composed of compressed, wrapping vesicles. Future mutagenesis work will endeavor to determine the portions of PMP22 important for the formation of MLAs and the effect of disease-causing mutations in MLA formation. We expect this work will shed light on the function of WT-PMP22 and the effects of neuropathic mutations of PMP22 in Schwann cell physiology.

36.  Hye-Jung Kim, "Quantitative profiling of protein tyrosine kinases in human cancer cell lines by multiplexed parallel reaction monitoring assays," Hye-Jung Kim, Ming Li, Daniel C. Liebler

Protein tyrosine kinases (PTKs) play key roles in cellular signal transduction, cell cycle regulation, cell division, and cell differentiation. Dysregulation of PTK-activated pathways, often a consequence of receptor overexpression, gene amplification, or genetic mutation, is a causal factor underlying numerous cancers. RTKs have been targeted with inhibitor drugs in clinical trials. Thus, there is potential utility in quantifying the expression of RTKs to identify drug response signatures and reveal new biological characteristics. In this study, we have developed a parallel reaction monitoring-based assay for quantitation of 83 PTKs, which detects proteotypic peptides from 54 receptor tyrosine kinases and 29 nonreceptor tyrosine kinases in a single run. Quantitative comparisons were based on the labeled reference peptide method. We tested the assay in four cell model studies: 1) proliferating versus epidermal growth factor (EGF)-stimulated A431 cells, 2) SW480 (mutant APC) and SW480APC (APC restored) colon tumor cell lines, 3) 10 colorectal cancer cell lines with different genomic abnormalities, and 4) lung cancer cell lines with either susceptibility or acquired resistance to the epidermal growth factor receptor tyrosine kinase inhibitor erlotinib. In these models, we observed distinct PTK expression changes that differed with genomic features or treatments in each model system. For example, EGF-stimulation of A431 cells induced downregulation of EPHA1. APC deletion in the SW480 model was associated with upregulation of EGFR and down regulation of SYK. In the 10 colon tumor cell lines, MSI-high cells exhibited upregulation of UFO and downregulation of IGF1R. Some differentially-expressed proteins were further evaluated by western blot analysis. The immunoblot results were highly consistent with PRM data. Ongoing work is investigating the possible coordinated regulation of PTK expression. Multiplexed PRM assays provide a targeted, systems-level profiling approach to evaluate cancer-related proteotypes and adaptations.

37.  Bishal Paudel, "Linking Heterogeneous Drug Responses to Molecular Signatures in BRAF-mutated Melanomas Treated with Targeted Therapies," Bishal Paudel, Vito Quaranta

Understanding the drug response dynamics of cancer cells is a clinical need to assess and improve the efficacy of the inhibitors. Recently approved small molecule inhibitors of BRAF (e.g. Vemurafenib) are clinically effective for treating melanoma patients with activating BRAF-kinase mutations (BRAFV600E) with most patients achieving rapid initial response. However, tumors almost invariably recur within a few months, even with continued treatment. Treatment failure or recurrence post therapy has often been linked to clone-to-clone genetic and nongenetic variation that exists within a tumor. However, it has been challenging to predict recurrence prior to treatment based on existing heterogeneity. To explore this possibility, we aim to map existing variation to the drug responses at the clonal level, understand how it relates to recurrence and determine the molecular underpinnings of distinct drug responses. Our high throughput imaging approach allows quantification of drug response variation at the clonal level to identify distinct response phenotypes. Preliminary data suggest that BRAF-mutated melanoma cells exhibit multiple cellular fates in response to PLX4720 (Vemurafenib research-analog); these cellular fates are incorporated into steady-state responses at the population level. The steady-state responses are quantified by the linear fit to the data to obtain drug-induced proliferation (DIP) rates. Recent data on BRAF-mutated cultured patient-derived xenografts (PDXs) and melanoma cell lines suggest that each cell line continues to proliferate at a reduced drug-induced proliferation (DIP) rate, reduction of which is concentration dependent. To understand the extent of clonal drug response variability within the population level response, we used clonal fractional proliferation (cFP) assay. Results in both cell lines and PDXs show that there exist variable clonal responses, aggregate of which represents the mean population response at a particular concentration. These clonal behaviors could be isolated into stable sublines with varying degrees of drug sensitivities. Further investigations will explore the key molecular differences in these sublines using transcriptomic and proteomic approaches.

38.  Daniel Putnam, "Protein Structure Determination guided by Small Angle Scattering Data," Daniel K Putnam, Jens Meiler

Understanding protein structure is one critical goal in biomedical research as it allows understanding of protein function at atomic detail thereby enabling structure-based drug discovery. Over 50% of all therapeutics target membrane proteins, yet there is a dearth of knowledge on their three-dimensional structure. As many membrane proteins evade crystallization, protein structure determination is experimentally challenging and in some cases impossible. Therefore computational approaches are developed that use alternative and limited experimental data to determine the structure of membrane proteins. We develop a computer algorithm termed BCL::MP-Fold that computes the coordinates of a membrane protein given the sequence of amino acids as input. In a benchmark test of 40 proteins of known structure, this method sampled the correct topology in 34 cases. The BCL::MP-Fold MP algorithm was able to sample the native topology of a 12 TM, 488-residue protein (PDBID: 2XUT). The algorithm failed to predict the correct topology for two other large protein systems (PDBID: 3HFX and 2XQ2). To restrict the conformational search space of proteins we incorporate Small Angle X-Ray scattering (SAXS) experimental restraints to enrich our sampling of native-like topologies for proteins with non-globular conformations.

39.  Nivriti Gahlaut, "Targeted Nanocarriers for Therapy of Retinal Vascular Diseases," Nivriti Gahlaut, Megan Capozzi, John S. Penn, Ashwath Jayagopal

Diabetic Retinopathy and Age Related Macular Degeneration are the leading causes of blindness in the working age and elderly, respectively. Targeted therapies are needed to improve clinical management of these diseases. Cell adhesion molecules (CAMs), particularly ICAM-1 and VCAM-1, are markers of inflammation expressed on retinal endothelial cell surfaces in a broad spectrum of ocular vascular diseases, including retinal neovascularization, and therefore constitute potential targets for promoting homing, binding, and internalization of nanoscale agents. We have developed a series of nanocarriers targeted against CAMs which can bear imaging or therapeutic payloads and deliver them to the cytoplasm of dysfunctional endothelial cells. The goal of this study was to demonstrate the utility of CAM targeted nanocarriers for intracellular delivery of antiangiogenic siRNA in two rat models of retinal neovascularization. CAM targeted nanocarriers bearing VEGFR2 and other siRNAs were characterized to determine optimal size, surface charge, and encapsulation efficiencies. Cytotoxicity, delivery efficiency, and functional knockdown of several molecular targets were determined in retinal microvascular endothelial cells. Biodistribution and efficacy of nanocarriers in animal models of neovascular age-related macular degeneration and oxygen-induced retinopathy were analyzed. CAM targeted nanocarriers were capable of specific targeting of the CAMs ICAM-1 and VCAM-1 on inflamed retinal endothelial cells in vitro and in vivo. Specific targeting of inflamed retinal endothelium was observed in both animal models of vascular disease, using CAMs on neovessel endothelial cells as a portal for delivery of therapies. Knockdown of several molecular targets via siRNAs was achieved in vitro and in vivo. CAM targeted nanocarriers are a promising framework for the delivery of diverse imaging and therapeutic payloads to the cytoplasm of diseased retinal endothelial cells in vivo.

40.  Darwin Fu, "Modeling Allosteric Modulators of Metabotropic Glutamate Receptor 3," Darwin Fu, Jens Meiler

G-Protein Coupled Receptors (GPCRs) are major targets of drug discovery efforts for neurological disorders. One important group is the Metabotropic glutamate receptors in family C. There are eight subtypes of glutamate receptors (mGlu1 – mGlu8). Allosteric modulation of these receptors has been implicated as potential treatments for major cognitive and behavioral symptoms of schizophrenia, depression, and Alzheimer’s disease. However, subtype selectivity remains a critical obstacle to efficacious and targeted therapies. In this study, we generated comparative models of mGlu3, and utilized molecular docking to elucidate the critical interactions in the protein-ligand binding. In collaboration with the Craig Lindsley lab, we employed virtual screening to identify novel binders of mGlu3.

41.  Josiah E. Hutton, "KRAS is a key driver of metabolic reprogramming in colorectal cancer cell lines and primary human tumors," Josiah E. Hutton, Lisa J. Zimmerman, Robbert J. Slebos, Ming Li, Daniel C. Liebler

KRAS is the third most frequently mutated gene in colorectal cancer and mutational activation of KRAS renders these tumors unresponsive to anti-EGFR treatments. Mutational activation of KRAS results in increased signaling through the phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) signaling pathways. Mutational activation of KRAS has originally been associated with increased cellular proliferation and increased tumorigenicity, but recent reports have linked KRAS mutations to altered metabolism, known as metabolic reprogramming. Previous studies have used either RNA-seq or Western blotting in combination with metabolite measurements to determine if cancer cells have undergone metabolic reprogramming. We have used a multiple reaction monitoring (MRM) protein-centric based method to quantitatively and reliably interrogate the expression of 62 metabolic proteins encompassing 21 glycolytic, 14 citric acid cycle (TCA), 10 pentose phosphate pathway (PPP), 4 serine biosynthesis, and 13 other metabolic proteins. MRM analysis was performed on a Thermo triple stage quadrupole (TSQ) Vantage instrument. We applied this MRM panel to an isogenic KRAS cell line, the DLD-1 parental cell line that expresses both KRAS G13D and wild-type KRAS, the DLD-1 KRAS mutant cell line that expresses only KRAS G13D, and the DLD-1 KRAS WT cell line that expresses only KRAS WT. DLD-1 KRAS mutant cells produce lactate at a significantly higher rate than the DLD-1 KRAS WT cell line, indicating that metabolic reprogramming has occurred in the KRAS mutant cells. MRM analysis of these cell lines revealed that KRAS mutation was mainly associated with the increased expression of glucose and glutamine transporters, glycolytic enzymes, enzymes involved in serine biosynthesis, and a few TCA enzymes. Furthermore, comparison of the isogenic KRAS mutant DLD-1 cells or the KRAS WT DLD-1 cells to the original DLD-1 parental cell line revealed distinct expression changes not revealed in the comparison of the DLD-1 KRAS mutant DLD-1 cells to the KRAS WT DLD-1 cells, signifying that WT KRAS and mutant KRAS play distinct roles in metabolic reprogramming. The MRM panel was also used to analyze 16 human formalin-fixed, paraffin-embedded (FFPE) Stage II colorectal cancers for metabolic reprogramming, and though the expression of the metabolic proteins varied from patient to patient, there was a consensus of 7 metabolic proteins that were significantly increased in KRAS mutant tumors. Lastly, this MRM panel was used to determine how signaling through the RAS-RAF-ERK signaling pathway with an isogenic RKO cell lines that either lack a BRAF mutation (BRAF (+, +)) or contain an activating BRAF mutation (BRAF (V600E, +)). Preliminary results with these two cell lines show that oncogenic BRAF signaling contributes to KRAS-dependent metabolic reprogramming, but the degree of metabolic reprogramming in BRAF mutant cells is not a pronounced as in KRAS mutant cells. These results demonstrate that mutational activation of KRAS induces metabolic reprogramming via signaling through both BRAF and PI3K signaling pathways in colorectal cancer cells by the altered expression of metabolic proteins.

42.  Jessica Finn, "De novo modeling of antibody CDRH3 loops with constraints," Jessica A. Finn, David P. Nannemann, Jordan R. Willis, James E. Crowe, Jr., Jens Meiler

Numerous advances have been made in the computational structural prediction of antibodies over the last 20 years. However it remains difficult to accurately model the heavy chain complementarity determining region 3 (CDRH3) loop, which often plays a key role in antibody/antigen interactions. I hypothesize that de novo modeling of this loop can be improved by adding knowledge-based constraints developed from the available antibody structures in the Protein Data Bank (PDB), which would allow for accurate modeling of longer CDRH3 loops than are currently available. These constraints restrict the sampling space of Rosetta LoopModel in regions of the CDRH3 loop known to maintain a canonical backbone structure, known as the CDRH3 torso, improving the overall RMSD of generated benchmark models in comparison to the native structure. In addition, the use of these constraints during scoring results in better discretion, observed as improved correlation between score and RMSD of benchmark models.

43.   Craig Goodwin, "Myeloid cell leukemia-1 (MCL-1) is an important apoptotic survival factor in Triple Negative Breast Cancer," Craig M. Goodwin, Olivia W. Rossanese, Edward T. Olejniczak, Stephen W. Fesik

Breast cancer is the second-most frequently diagnosed malignancy in U.S. women. The triple negative breast cancer (TNBC) subtype, which lacks expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor-2, afflicts 15% of patients and is refractory to current targeted therapies. Like many cancers, TNBC cells often deregulate apoptosis. In healthy cells, pro- and anti-apoptotic proteins are balanced; however in cancer cells anti-apoptotic proteins of the BCL-2 family are frequently upregulated to inhibit programmed cell death. One family member, MCL-1, is commonly amplified in TNBC and correlates with a poor clinical prognosis. Here we show the effect of silencing MCL-1 and BCL-XL expression on viability in a panel of seventeen TNBC cell lines. Cell death was observed in a subset of these lines upon MCL-1 knockdown. In contrast, BCL-XL knockdown only modestly reduced viability, indicating that MCL-1 is a more important survival factor for these cell lines. Confirming this observation, the selective BCL-XL inhibitor WEHI-539 was only weakly cytotoxic across the panel, but sensitization by MCL-1 knockdown dramatically improved its EC50. ABT-199, which selectively inhibits BCL-2, did not synergize with MCL-1 knockdown, indicating the low importance of BCL-2 in these lines. MCL-1 sensitivity is not predicted by mRNA or protein levels of single BCL-2 family members, except for a weak correlation to BAK protein expression. However, a more comprehensive index composed of MCL-1, BCL-XL, BIM, BAK, and NOXA protein expression correlates well with MCL-1 sensitivity and may be a useful diagnostic criterion to predict dependency.

44.  Alberto Cisneros, "The structural basis for antibody polyspecificity," Alberto Cisneros, James E. Crowe, Jens Meiler

Specific Aims: To identify and characterize novel human influenza antibodies. I hypothesize that novel, broadly binding (polyspecific) antibody sequences can be designed using computational methods and knowledge collected from available antibody/antigen co-crystal structures. Abstract: The continued threat of pathogenic influenza virus strains has driven investigators to determine many high-resolution structures of antibodies binding to influenza hemagglutinin (HA) molecules. These structures provide information about the interaction between the antibody and HA molecule. Of the available structures for influenza-neutralizing antibodies, many bind to the head region of HA, which is highly variable and often limits the recognition of the antibody to one HA subtype. However, motifs for increasing neutralization breadth can be found in antibodies that target a conserved region of the HA stem domain or the conserved receptor binding site. Identification of critical binding interactions through the aid of computational techniques will provide invaluable information in understanding the basis for specificity and breadth in antibody-antigen interactions. A collaboration between the Crowe and Meiler laboratories recently demonstrated that the ROSETTA multi-state design (MSD) protocol is capable of generating novel antibody sequences predicted to have polyspecific binding profiles by re-designing available antigen/antibody complexes. Starting from 20 co-crystal structures of antibodies that target the better conserved HA stem region and receptor binding domain, ROSETTA MSD will be employed to optimize an antibody sequence for affinity to multiple HAs. Where the standard ROSETTA design algorithm involves optimization of a single complex, in MSD multiple antibody/HA complexes are designed in parallel - one for each HA subtype that is to be considered. The resulting antibodies will be tested for HA binding by ELISA and through biosensor assays. Determination of binding affinity will be verified through isothermal titration calorimetry. In order to determine the neutralization activity of the designed antibodies, micro-neutralization assays will be performed against functional influenza virus or pseudotyped virus where necessary.

45.  Shalley Kudalkar, "13-Methyl-arachidonic Acid is a Powerful Substrate-Selective Activator of Endocannabinoid Oxygenation by Cyclooxygenase-2," Shalley N. Kudalkar, Spyridon Nikasa, Philip Kingsley, Shu Xu, Michelle Mitchener, Carol A. Rouzer, Alex Makriyannisa, Lawrence J. Marnett

Cyclooxygenase-2 (COX-2) is a homodimeric enzyme that catalyzes the oxidation of arachidonic acid (AA) in the committed step of prostaglandin (PG) biosynthesis. COX-2 also oxygenates the endocannabinoids, 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (AEA) to their respective prostaglandins. COX-2 acts as a conformational heterodimer with a catalytic monomer and an allosteric monomer. Binding of non-substrate fatty acids, like palmitic acid has been shown to increase oxygenation of AA in the catalytic site. Here we tested various AA, 2-AG, and AEA analogs as potential substrates, inhibitors, and potentiators of COX-2. None of the analogs was oxidized by COX-2; however a few weakly inhibited the ability of COX-2 to oxidize either AA or 2-AG. AM-8138, an AA analog with an (S)-methyl group at carbon-13, significantly increased the oxidation of 2-AG by increasing Vmax and reducing substrate inhibition with little to no effect on AA oxidation. The kinetic data suggests that AM-8138 might be potentiating 2-AG oxidation by allosteric regulation. This observation was further supported when the inhibition of 2-AG oxidation by weak reversible inhibitors, such as (R)-flurbiprofen, was reversed in the presence of AM-8138. Also, the presence of AM-8138 blocked the inhibition of AA or 2-AG oxidation by time-dependent inhibitors like indomethacin. Additionally, the activity of COX-2 variants that were unable to oxidize 2-AG was fully recovered in the presence of AM-8138. Thus, our study shows that binding of AM-8138 to the allosteric site causes conformational changes to COX-2, which makes the catalytic site more conducive for 2-AG binding and enhances its oxidation by COX-2.

46.  Cody Covington, "Advances in Determining the Absolute Configurations of Natural Products: A focus on the Electronic and Vibrational Dissymmetry Factor," Cody Covington and Prasad Polavarapu

The absolute configurations of two natural products, namely a new S-bridged pyronaphthoquinone dimer from a cave-derived actinomycete, Nonomuraea specus and a rare sesquiterpene ,(+)-3-ishwarone, from Peperomia scandens are determined using chiroptical spectroscopy. To facilitate this structural determination, a new method for comparing predicted chiroptical spectra to the corresponding experimental spectra, using an in-house developed computer program CDSpecTech, has been developed. This method utilizes the comparison of dissymmetry factor spectra, rather than circular dichroism and absorption spectra separately, and appears to be more robust.

47.  Dennis Kuo

The goal of this project is to determine how the regio- and stereochemistry of deoxyinosine adducts resulting from the alkylation of adenine at the N1 position by 3,4-epoxy-1-butene (EB) affects the structure and downstream biological processing of dsDNA. 1,3-butadiene (BD) is an industrially important chemical obtained from petrochemical extraction. It is the 36th most produced chemical in the United States and is primarily used in the synthesis of styrene butadiene polymer, the material used in rubber tires. BD is also a byproduct of gasoline combustion and cigarette smoke. BD has also been classified as a human carcinogen by the Nationals Toxicology Program. EB, the primary metabolite of BD, has two reactive epoxide carbons (C’ and C’’) which can each react with the N1 of adenine to produce two stereoisomers, leading to a total of four distinct adducts. These adducts can spontaneously deaminate to form deoxyinosine (dI) adducts. The C’ adducts are observed to induce A to G mutations in COS-7 cells. Interestingly, the (R)- isomer is more mutagenic. It has been hypothesized that these A to G mutations are promoted by Hoogsteen base pairing between the modified base and an incoming protonated dCTP. Previously determined solution structures of the C’ adducts show the modified base in the syn conformation, supporting this hypothesis. Surprisingly, NMR data suggests that the modified bases of the C’’ adducts remain in the anti conformation, suggesting that epoxide regioselectivity has a significant effect on the modified DNA conformation. This observation may imply that C’ and C’’ adducts have different downstream biological processing and may lead to different mutagenesis products, and points to these C’’ adducts as key targets for future mutagenesis studies and translesion synthesis studies.

48.  Lisa Lojek, "Determining the role of heme catabolites in microbes," Lisa J. Lojek, Allison J. Farrand, Kate P. Haley, Iris Gao, Heather K. Kroh, D. Borden Lacy, Eric P. Skaar

In order to survive, bacteria must acquire essential nutrients. Iron is one such nutrient, which can be acquired from heme. Intracellular heme is degraded by heme oxygenases, resulting in the release of iron and the concomitant production of catabolite(s). Heme oxygenases are conserved in all kingdoms of life. There are three known classes of heme oxygenases, the HO-1 family, the IsdG family, and the MhuD family, each of which produces different catabolites upon heme degradation. The HO-1 family of heme oxygenases, which is the only class of heme oxygenases described in eukaryotic cells, degrades heme to biliverdin, carbon monoxide, and free iron. These small molecules can act as antioxidants and signaling molecules in eukaryotic cells, respectively. In the bacterium Staphylococcus aureus, heme degradation by the heme oxygenases IsdG and IsdI results in the formation of staphylobilin, formaldehyde, and free iron. However, the precise functions of these heme catabolites in microbes are unknown. We aim to determine the function of these heme catabolites. Microarray data show that inactivating IsdG and IsdI in S. aureus results in transcriptional changes in more than 100 genes. Moreover, replacing IsdG and IsdI in S. aureus with other heme oxygenase classes, which make unique products but still release iron, enables S. aureus to utilize heme as the sole iron source, indicating that these enzymes are functional. These results lay the foundation for experiments focused on determining the role of heme catabolites in microbes.

49.  Michelle Mitchener, "Lipid Electrophiles in Osteoclastogenesis," Michelle Mitchener, William Beavers, Alyssa Merkel, Shellese Cannonier, Julie Sterling, Lawrence Marnett

Bone erosion, occurring in diseases such as osteoporosis and rheumatoid arthritis, is primarily attributable to the action of osteoclasts, the body’s bone-resorbing cells. Recent studies have suggested that mitochondrial reactive oxygen species contribute to osteoclast formation. Thus we hypothesized that resulting lipid peroxidation products might contribute to osteoclast formation through adduction of mitochondrial proteins and alteration of their functions. To assess lipid electrophile protein adduction during osteoclastogenesis, RAW264.7 macrophage-like cells were enriched with alkynyl-linoleic acid and treated with recombinant receptor activator of NF-κB ligand (RANKL) to differentiate them into osteoclasts. Proteins adducted by alkynyl lipid electrophiles were conjugated to biotin using click chemistry and visualized with a streptavidin fluorophore. Osteoclast formation was monitored using tartrate-resistant acid phosphatase (TRAP) staining and microscopic visualization of multinucleated cells. Increased levels of lipid electrophile protein adduction were observed after 48 hours of cell treatment with RANKL, concomitant with cellular expression of TRAP. After 72 hours, TRAP-positive multinucleated cells (osteoclasts) were observed and increased protein adduction also persisted. This increase in protein adduction just prior to osteoclast formation suggests that lipid electrophiles may promote osteoclastogenesis through adduction of proteins and alteration of their functions.

50.  Brittany Allison, "Computational Design of Tighter Protein-Ligand Interfaces”

The computational design of proteins that bind small molecules remains a difficult challenge in protein engineering. The relatively small size of the ligand limits the number of intermolecular interactions, and near perfect geometries between interacting partners are required to achieve high binding affinities. Despite the challenges, the ability to computationally recapture and predict native-like interactions with high accuracy and efficiency would be an asset in biotechnology and medicine, such as for therapeutic development, enzyme design, and engineering functional proteins. Because protein-ligand interactions are difficult to model, we have developed a systematic approach to designing interfaces. We identify ligands with naive binding affinity to our protein scaffold HisF, then use RosettaLigand to dock/design this interface to create a tighter protein-ligand interaction. This way, we are taking a ‘shot in dim light’ as opposed to a ‘shot in the dark’, allowing us to more thoroughly investigate the successful and not-so-successful designs and improve the computational methods. NMR experiments allow us to identify hits as well as identify the residues involved in the protein-ligand interaction, crucial for computational designs and analysis. Of ~3500 ligands screened, we identified 25 weakly-binding ligands, and thus far have successfully designed a tighter interface for one of the compounds.

51.  Stephanie Evans, "Imaging of Retinal Vascular Disease Using Hypoxia-Sensitive Contrast Agents," Evans, Stephanie; Kim, Kwangho; Craft, Jason; Sulikowski, Gary; Jayagopal, Ashwath

Retinal vascular diseases, including diabetic retinopathy and age related macular degeneration, are often associated with retinal hypoxia. Therefore, the ability to image hypoxic retinal tissue in vivo would be beneficial for improved clinical management of these diseases. For this purpose, a hypoxia-sensitive fluorescent contrast agent was developed and characterized for imaging of hypoxia in retinal tissue using established cell culture and animal models of retinal vascular disease. Fluorescently-labeled, hypoxia-sensitive contrast agents were synthesized, purified chromatographically, and characterized by mass spectrometric and nuclear magnetic resonance analyses. To evaluate the utility of this contrast agent for imaging hypoxia, in vitro assays using Human Retinal Microvascular Endothelial Cells (HRMEC) and in vivo studies using mice with oxygen-induced retinopathy (OIR) or laser-induced choroidal neovascularization (LCNV) were performed to determine the hypoxia-associated sensitivity and specificity of this contrast agent. Fluorimetric assays were performed on normoxia- or hypoxia-conditioned HRMEC exposed to contrast agents to measure contrast agent binding and uptake. OIR and LCNV animal models were intravenously or intraocularly-injected with the contrast agents and analyzed by in vivo and ex vivo retinal fluorescence imaging to determine specificity and sensitivity of the contrast agent. Pimonidazole hydrochloride immunostaining was utilized to confirm specificity of the contrast agent for hypoxic tissue. HRMEC conditioned under hypoxia for varying durations of time up to 24 hrs. exhibited dose-dependent fluorescence enhancement due to hypoxia-selective uptake of the contrast agent. In animal models, regions of tissue hypoxia staining positive for pimonidazole hydrochloride were also colocalized with contrast agent uptake, as indicated by in vivo and ex vivo imaging. Contrast agent accumulation in hypoxic tissue was detectable within 2 hrs. post-injection. These studies support the feasibility of imaging hypoxic tissue in vivo using targeted contrast agents in conjunction with readily available retinal fluorescence imaging equipment. Hypoxia-sensitive contrast agents, if clinically translated, may be useful for early detection of retinal vascular diseases and monitoring of therapeutic response in patients.

52.  Tarvi Teder, "Formation of hydroxy-endoperoxides in the lipoxygenase catalyzed metabolism of fatty acid epoxides," Tarvi Teder, Alan R. Brash

Lipoxygenases (LOX) catalyze the dioxygenation of polyunsatured fatty acid to form corresponding hydroperoxides. In plants, fatty acid hydroperoxides derived from linole(n)ate are the substrates for plant allene oxide synthase, lyase and peroxygenase pathways which give rise to a variety of derivatives. For instance, the co-operation of LOX and peroxygenase enzymes result in the formation of epoxy and hydroxy fatty acids. In mammals, the cyclooxygenases (COX) are paired with cytochrome P450s in the transformation of arachidonic acid to prostaglandin endoperoxides and on to thromboxane A2 and prostacyclin. Alternatively, the metabolism of P450-derived epoxyeicosatrienoic acid (EET) by COX result in epoxy alcohol formation. The present work describes a new type of interaction to produce fatty acid hydroxy-endoperoxides from fatty acid epoxides by lipoxygenases. We demonstrated this transformation using soybean LOX-1 in the metabolism of 15,16-epoxy-α-linolenic acid, and murine platelet-type 12-LOX and human 15-LOX-1 in the metabolism of 14,15-epoxyeicosatrienoic acid (14,15-EET). A detailed examination of the transformation of the two enantiomers of 15,16-epoxy-α-linolenic acid by soybean LOX-1 revealed that the expected primary product, a 13S-hydroperoxy-15,16-epoxide, underwent a non-enzymatic transformation in buffer into a new derivative that was isolated by HPLC and identified by UV, LC-MS and 1H-NMR as a 13,15-endoperoxy-16-hydroxy-octadeca-9,11-dienoic acid. The formation of a 5-membered endoperoxide ring (a 1,2-dioxolane moiety) characteristic for monocyclic peroxides is previously described as a 5-exo cyclization via hydroperoxy radical. In this study, the reaction mechanism can be explained by the intramolecular nucleophilic substitution (SN2-type) between the hydroperoxy (nucleophile) and unhindered carbon of the epoxy-group (electrophile). The configuration of the endoperoxide (cis or trans side chains) is determined by the stereochemistries of the hydroperoxy- and epoxy-group. Equivalent transformations were documented in metabolism of the enantiomers of 14,15-EET by the two mammalian LOX enzymes. We conclude that this type of interactions could occur naturally with the co-occurrence of LOX and cytochrome P450 or peroxygenase enzymes, and it could also contribute to the complexity of products formed in the enzymatic and autoxidation reactions of polyunsaturated fatty acids.









Vanderbilt University School of Medicine | Vanderbilt University Medical Center | Vanderbilt University | Eskind Biomedical Library

The Vanderbilt Institute of Chemical Biology 896 Preston Building, Nashville, TN 37232-6304 866.303 VICB (8422) fax 615 936 3884
Vanderbilt University is committed to principles of equal opportunity and affirmative action. Copyright © 2013 by Vanderbilt University Medical Center