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Guest speaker


Dr. Jon Clardy
Harvard Medical School
Visit Dr. Clardy's web site.

 
 
Awards & Photos

Click here to view photographs of award recipients and student organizers!

Click here to view poster session and reception photographs.

VICB Prize in Chemical Biology Award Recpient:
Laura Anzaldi Mike (Skaar Lab)

Certificate in Chemical Biology Recipients:
Brittany Allison (Meiler Lab), William Beavers (Marnett Lab), Emilianne McCranie (Bachmann Lab), Will Birmingham (Bachmann Lab), Glenna Kramer (Bachmann Lab), Brendan Dutter (Sulikowski Lab), Marta Wenzler (Sulikowski Lab), Matt O'Reilly (Lindsley Lab)

Oral Presentation Award Recipients:
Carrie Shaffer (Hadjifrangiskou Lab), Kerrie Grove (Caprioli Lab), Chris Gulka (Wright Lab)

Poster Award Recipients:
Christopher Aluise (Marnett Lab), Frank Couch (Cortez Lab), Marta Wenzler (Sulikowski Lab), Daniel Sprague (Johnston Lab), Nichole Lareau (McLean Lab), Ghazai Hariri (Harth Lab), Kyle Floyd (Hadjifrangiskou Lab), Odaine Gordon (Schneider Lab)

 
Agenda

MORNING SESSION

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: John Clardy, Harvard Medical School
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.
Nicole Chumber, Lacy Lab
David Nanneman, Meiler Lab
Christopher Gulka, Wright Lab

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

AFTERNOON SESSION

Oral Presentations
1:00 P.M. - 2:00 P.M.
Daniel Hermanson, Marnett Lab
Carrie Shaffer, Hadjifrangiskou Lab
Kevin Kumar, Bowman Lab

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

Oral Presentations
3:00 P.M. - 3:50 P.M.
Kerri Grove, Caprioli Lab
Laura Anzaldi Mike, Skaar Lab (VICB Excellence in Chemical Biology Award Recipient)


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



 
Contact
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)  Nicole M Chumbler, " Targeting the Mechanism of C. difficile Toxin B-Induced Necrosis with Small Molecule Inhibitors," Melissa A Farrow, D Borden Lacy

Clostridium difficile is the most common cause of antibiotic-associated nosocomial infection in the United States. C. difficile secretes two homologous toxins, TcdA and TcdB, which are responsible for the symptoms of C. difficile associated disease. The accepted dogma of the mechanism of toxin action has included an autoprocessing event where a cysteine protease domain (CPD) releases a glucosyltransferase domain (GTD) into the cytosol. The GTD can modify and inactivate Rho-family GTPases. We have shown that these activities are not necessary for TcdB induced necrosis in both colonic epithelial cells and porcine colonic explants. We have recently shown that TcdB initiates the assembly of the NADPH oxidase (NOX) complex, leading to aberrant reactive oxygen species (ROS) production and ultimately, necrosis. We have performed a high throughput screen and identified 176 compounds that confer protection against TcdB induced necrosis. I will describe on-going efforts to identify the mechanistic steps affected by our lead compounds and our plans to evaluate efficacy in animal models of C. difficile infection.

2)  Kerri Grove. "High Spatial Resolution Molecular Imaging for Efficacy of Drug Treatment in Diabetic Nephropathy," Paul Voziyan, Jeffrey Spraggins, Billy Hudson, Richard Caprioli

Diabetic nephropathy (DN) leads to progressive decline in renal function and is the leading cause of end-stage renal disease. Much still remains unknown about the pathogenesis of this disease, however, one of the mechanisms underlying diabetic nephropathy is thought to be renal accumulation of oxidative advanced glycation end products (AGEs). This project investigates the molecular changes that take place in the kidney glomerulus to explore the pathogenic mechanisms of this disease by use of matrix assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS). Pyridoxame (PM) is a promising drug candidate for the treatment of diabetic nephropathy and has been shown to have a renal protective effect in Phase II clinical trials, in part by inhibiting formation of AGEs. For this study, a robust mouse model of diabetes (eNOS -/- db/db) with and without pyridoxamine treatment is investigated. In this work, changes in different classes of biomolecules including proteins and lipids have been found in diseased and healthy kidneys. MALDI IMS profiling measurements of individual glomeruli have detected a significant increase in fibronectin deposition in eNOS-/- db/db mice compared to wild-type mice. Treatment of eNOS-/- db/db mice with PM significantly inhibited this glomerular fibronectin deposition. High spatial resolution IMS has shown other protein changes in the experimental groups, including a peak at m/z 4415 in the DN glomeruli that has a mass shift of +72 Da from a glomerular protein found in the wild type kidney. This mass shift is consistent with the AGE modification carboxyethyl lysine and its presence is reduced significantly with PM treatment. Measurements of single glomeruli such as this are not attainable with other proteomic methods. Additionally, high mass accuracy measurements indicate early glycation product modifications (Amadori, +162.0528 Da) on specific phosphatidylethanolamine (PE) lipids such as PE (18:0p/20:4) in the diabetic kidney tissue. High resolution IMS provides a powerful tool to detect protein and lipid modifications associated with diabetic nephropathy from specific structures within the renal cortex. Determining proteins that undergo change in disease conditions and evaluating response to treatment may lead to new molecular markers of disease, provide insight into disease pathogenesis, and characterize treatment response.

3)  Christopher P. Gulka, "Coffee Rings as Low-Resource Diagnostics: Detection of the Malaria Biomarker Plasmodium falciparum Histidine-Rich Protein-II Using a Surface-Coupled Ring of Ni(II)NTA Gold Particles," Joshua D. Swartz, Joshua R. Trantum, Frederick R. Haselton, and David W. Wright

Independent of antibodies and thermally sensitive materials, we report a novel, low-resource malaria diagnostic platform selective for pfHRP-II, a biomarker indicative of the Plasmodium falciparum parasite strain. The assay is inspired by the phenomenon that forms coffee ring stains on a kitchen counter, where a recombinant Histidine-Rich Protein 2 (rcHRP-II) biomarker is sandwiched between a ring of Ni(II)NTA particles and Ni(II)NTA-functionalized glass. Upon deposition of a particle-protein conjugate solution onto the functionalized glass slide, evaporation elicits the radial flow of suspended particles towards the drop edge, forming a ring adsorbed to the glass. A simple wash with deionized water after evaporation removes non-specifically bound materials, while maintaining the integrity of the surface-coupled ring in the presence of protein. The ring assay for rcHRP-II detection was explored with two separate Ni(II)NTA particle substrates: a 15 nm gold nanoparticle platform and a 1 μm gold-plated polystyrene sphere platform. The Ni(II)NTA gold nanoparticle ring assay exhibits a 14 nM rcHRP-II detection limit, despite possessing a narrow dynamic range. In order to improve upon the assay dynamic range and to achieve the 100 pM pfHRP-II limit of detection recommended by the World Health Organization, Ni(II)NTA gold-plated polystyrene microspheres were investigated as a particle platform because of their low density and large surface area. The high surface area of the 1 μm microspheres projects a larger two-dimensional viewable area for ring formation on glass in comparison to 15 nm gold nanoparticles, while the low density favors the particle-protein conjugates to flow radially towards the drop edge versus settling vertically to the center of the drop. In the presence of rcHRP-II, ring formation is visible between 10 pM-10 nM concentrations without additional signal enhancement methods, so that the assay has the potential to diagnose both asymptomatic and symptomatic individuals infected with malaria.

4)  Daniel Hermanson, "Substrate-Selective Inhibition of COX-2 as a Novel Strategy for In Vivo Endocannabinoid Augmentation," Nolan Hartley, Sachin Patel, and Lawrence Marnett

Over the past two decades the role of endogenous cannabinoids in the regulation of physiological and pathophysiological processes has grown exponentially. The two primary endocannabinoid ligands, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), activate the cannabinoid receptors, CB1 and CB2. Importantly, pharmacological strategies that augment endocannabinoid signaling have been suggested to be viable treatments for several disease states. In particular, inhibition of the AEA degrading enzyme fatty-acid amice hydrolase (FAAH) and the 2-AG degrading enzyme monoacylglycerol lipase (MAGL) have been extensively validated in preclinical studies for mood and anxiety disorders. In addition to FAAH and MAGL, in vitro studies suggest that cyclooxygenase-2 (COX-2) can degrade both AEA and 2-AG, however the role of COX-2 in the physiological regulation of endocannabinoid signaling in vivo has not been demonstrated to date. Moreover, all current inhibitors of COX-2 have profound effects on prostaglandin synthesis in vivo, which contributes to the cardio/cerebro-vascular adverse effects of COX-2 inhibitors, thus limiting pharmacological approaches to modulate endocannabinoid signaling via COX-2 inhibition.

We developed and screened novel substrate-selective inhibitors of COX-2 in vitro using purified murine COX-2. Compounds that demonstrated inhibition of COX-2 activity only when the endocannabinoids AEA and 2-AG were used as substrates, but not when arachidonic acid is used, were developed and optimized. Here we describe the in vivo effects of LM-4131, our most selective and potent inhibitor. We used LC/MS/MS to determine the effects of LM-4131 on brain endocannabinoids, related-lipids, and prostaglandins in the brain, liver and lung. We used behavioral assays of anxiety to determine the preclinical efficacy of LM-4131 for anxiety disorders.

Our data show that COX-2 regulates central endocannabinoid signaling under physiological conditions in vivo. Specifically, inhibition of COX-2 increased brain AEA levels in wild-type, but not COX-2 knock out mice. We show that substrate-selective pinhibition of COX-2 can selectively increase brain endocannabinoids with a high degree of specificity over related non-cannabinoid lipids, without affecting central or peripheral prostaglandin synthesis. We also demonstrate the biophysical determinants of substrate-selective pharmacology using structural studies and site-directed mutagenesis of purified COX-2. Lastly, we show that substrate-selective inhibition of COX-2 exerts anti-anxiety effects via activation of endocannabinoid signaling and CB1 cannabinoid receptors in multiple behavioral assays. Moreover, substrate-selective inhibition of COX-2 does not cause gastric bleeding or overt cannabimimetic behavioral effects in mice suggesting a low propensity for GI and psychotomimetic side-effects of LM-4131. These findings solidify COX-2 as a major in vivo regulator of endocannabinoid signaling and validate substrate-selective pharmacology as a viable approach to selectively enhance endocannabinoid signaling for therapeutic gain.

Overall, our findings provide a new conceptualization of central endocannabinoid metabolism to prominently include COX-2, in addition to FAAH and MAGL, and provide a viable pharmacological strategy to capitalize on the therapeutic potential of COX-2 regulation of endocannabinoid signaling. Given that endocannabinoid signaling is involved in a broad range of pathological and physiological processes, and that COX-2 is induced in response to many stimuli, our findings could have far reaching applicability to fields including metabolic regulation, neuro-inflammation, and other neuropsychiatric disorders.

5)  Kevin K. Kumar, "A High Throughput Screen for Modulators of Neuronal Manganese Status," Asad A. Aboud, M. Diana Neely, Andrew Tidball, C. David Weaver, Michael Aschner, Aaron B. Bowman

There is strong evidence that both genetic and environmental factors play a critical role in the pathogenesis of many neurodegenerative diseases. Exposure to the heavy metal, manganese (Mn), shows a strong correlation with incidence of Parkinson’s disease (PD) and modulation of neuronal Mn status has been seen in the pathogenesis of Huntington’s disease (HD). This study presents the results of a high-throughput screen (HTS) of small molecules to identify novel modulators of neuronal Mn accumulation. Specifically, we have begun screening multiple chemical libraries including: the SPECTRUM Collection, NIH Clinical Compound Collection, Chembridge, Chemdiv, and kinase inhibitors from GlaxoSmithKline, Roche, and Enzo. Our ultimate goal is to characterize identified compounds’ clinical efficacy in the treatment and prevention of these progressive neurodegenerative disorders. We hypothesize that pharmacological reduction of Mn accumulation in neurons will have therapeutic efficacy in patients at known genetic or environmental risk for PD or HD.

Despite the potential of HTS technology in neurotoxicology, screening for small molecule chemical modifiers of neurotoxicants has been limited by the scalability of existing phenotyping assays. Here we describe the successful optimization of the Cellular Fura-2 Manganese Extraction Assay (CFMEA) for HTS in an immortalized murine striatal cell line (STHdhQ7/Q7). By optimizing cellular density, Mn exposure conditions, and extraction parameters, the sensitivity and dynamic range of the fura-2 Mn response was enhanced to permit detection of positive and negative modulators of cellular Mn status. In addition, experimental and analytical strategies were developed to perform quality control monitoring during the screen including the use of an independent dextran-coupled fluorophore to assess liquid delivery volume. The bidirectional optimization of CFMEA was both highly reliable and accurate yielding a minimum Z-factor of 0.50.

Our current efforts seek to characterize HTS-identified pharmacological modulators of neuronal Mn status by using human induced pluripotent stem cell (hiPSC)-derived neural progenitors. Specifically, both midbrain dopaminergic and forebrain striatal progenitors, lineages affected by PD and HD, respectively, are utilized for this purpose.

6)  David P. Nannemann, "Human Neutralizing Monoclonal Antibodies that Recognize Respiratory Droplet Transmissible H5N1 Influenza Viruses," Natalie J. Thornburg, David L. Blum, Jessica A. Belser, Terrence M. Tumpey, Shyam Deshpande, Gloria A. Fritz, Jens C. Krause, Jeong Hyun Lee, Andrew B. Ward, David E. Lee, Sheng Li, Katie L. Winarski, Benjamin W. Spiller, Jens Meiler, and James E. Crowe Jr.

Recent research described the experimental adaptation of influenza H5 hemagglutinins (HAs) that confers respiratory droplet transmission to influenza virus in ferrets, which may mimic the future development of a highly pathogenic pandemic H5 “bird flu” virus in nature. This research set off a national debate on publication of research with potential dual use in bioterrorism. Human hybridomas from peripheral blood mononuclear cells from patients who had participated previously in clinical trials for an H5N1 vaccine were generated and characterized for antigen binding and neutralization of avian influenza viruses. Antibodies that bind the HA head domain and recognize both circulating and respiratory droplet transmissible H5 HAs have been identified. Using a hybrid methods approach to synthesize a number of structural techniques, including X-ray crystallography, electron microscopy, hydrogen/deuterium exchange mass spectrometry, and binding affinity assay, we defined a mechanism of antibody binding to the head of HA in a manner that retains molecular recognition of HAs with mutations that render H5N1 viruses transmissible by respiratory droplet in mammalian models. The model was validated through characterization of HA mutations predicted to confer antibody escape and explains naturally-occurring escape mutations. Rational design of novel H5 HA immunogens to protect against mammalian adapted mutants should consider incorporation or retention of this critical antigenic site. Furthermore, engineering of these antibodies may allow for therapeutic application in the event of avian influenza pandemic.

7)  Carrie L. Shaffer, "Synthetic Small Molecules Diminish the Oncogenic Potential of Helicobacter pylori by Inhibiting Type IV Secretion," James A. Good, Kyle A. Floyd, Jennifer A. Gaddy, John T. Loh, Fredrick Almqvist, Timothy L. Cover, Maria Hadjifrangiskou

Bacterial pathogens use extracellular adhesive organelles termed pili to interact with host cell surfaces and initiate events that are required to establish infection and persist within the host. Therefore, disruption of interactions between bacteria and the host represents an attractive strategy for the development of anti-virulence therapeutics. Helicobacter pylori is a ubiquitous Gram-negative pathogen that persistently colonizes the gastric mucosa of approximately 50% of the global population. Colonization with H. pylori that encode a functional cag type IV secretion system (T4SS) can lead to serious disease outcomes including gastric adenocarcinoma, which is the second leading cause of cancer-related deaths world-wide. The cag T4SS is utilized by H. pylori to inject the oncogenic protein CagA into gastric epithelial cells and induce robust inflammation by stimulating production of the pro-inflammatory cytokine IL-8. Once translocated into the gastric epithelium, CagA becomes phosphorylated on conserved tyrosine residues by host cell kinases and subsequently hijacks host cell signaling pathways, eliciting oncogenic phenotypes. Small, heterocyclic ring-fused 2-pyridone molecules were used previously to disrupt the biogenesis of type 1 pili adhesive organelles in uropathogenic E. coli. In this study, we analyzed a library of these compounds for the ability to block interactions between H. pylori and host cells. We found that four of the compounds reproducibly blocked CagA translocation, IL-8 induction, and activation of NFκB in a dose-dependent manner without perturbing bacterial or host cell viability. Two of the most potent compounds (CS10 and CS85), which differ in structure by a single methoxy group, had differential effects on components of T4SS pilus appendages. Exposure to CS85 significantly impaired the ability of H. pylori to form T4SS pili at the bacteria-host cell interface, an observation that may partially explain the reduced levels of CagA translocation and IL-8 induction. However, bacteria treated with CS10, which lacks the additional methoxy group, impacted CagA translocation but did not interfere with T4SS pilus formation or localization. Interestingly, the region encompassing one CagA tyrosine phosphorylation motif exhibits sequence homology to PapD, the E. coli chaperone that was utilized as a molecular scaffold for development of CS10 and CS85. We hypothesize that CS10 inhibits CagA phosphorylation by binding this motif, rendering the site inaccessible to host kinases. In summary, we propose to utilize these lead compounds as molecular scalpels to further dissect assembly and architecture of the H. pylori T4SS apparatus, and to exploit the ring-fused 2-pyridone backbone as a scaffold for the development of broad spectrum therapeutics that target type IV secretion effectors in multiple bacterial pathogens.

2013 VICB Excellence in Chemical Biology Award Recipient
8)  Laura A. Mike, "A Small Molecule that Stimulates Endogenous Heme Biosynthesis is Toxic to Fermenting Staphylococcus aureus," Brendan F. Dutter, Devin L. Stauff, Jessica L. Moore, Nicholas P. Vitko, Olusegun Aranmolate, Thomas E. Kehl-Fie, Sarah Sullivan, Paul R. Reid, Jennifer L. DuBois, Anthony R. Richardson, Richard M. Caprioli, Gary A. Sulikowski, and Eric P. Skaar

Staphylococcus aureus is a Gram-positive pathogen that causes a wide range of pathologies ranging from superficial skin infections to deep seeded osteomyelitis and endocarditis. In order to be a successful pathogen in diverse niches S. aureus senses and adapts to environmental changes using two-component signaling systems (TCSs). Upon entry into the host S. aureus encounters an environment devoid of free iron. S. aureus satisfies its iron and heme requirements by scavenging host heme from hemoglobin. While heme is a valuable iron source and co-factor, the reactivity of the molecule is a liability to the cell. The S. aureus TCS heme sensor system (HssRS) is activated in the presence of heme and up-regulates the heme regulated transporter (HrtAB), which protects the bacteria from heme toxicity. In order to understand the mechanism by which S. aureus senses and responds to heme toxicity during infection, a high-throughput screen for small molecule activators of HssRS was performed. The most potent HssRS activator, VU0038882 (‘882), was found to stimulate endogenous heme biosynthesis by perturbing central metabolism. Furthermore, the ‘882-mediated alterations to central metabolism are toxic to fermenting S. aureus, including clinically relevant small colony variants (SCVs). Targeting fermentation in a facultative anaerobe has therapeutic utility as ‘882 prevents the emergence of antibiotic resistance, enhances phagocyte killing and reduces S. aureus pathogenesis. This small molecule has revealed fundamental insights into staphylococcal heme homeostasis as well as provided a novel therapeutic strategy for combating this aggressive pathogen.


Poster Presentation Abstracts

Poster presentation abstracts will be posted as they are submitted.

1) Katherine Amato, "Targeting EphA2 in Tyrosine Kinase Inhibitor Resistant Lung Cancer," Nathanael Gray, Jin Chen

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

2) Stephanie H. DeLuca, "Rigid Body Sampling with Sparse EPR Data to Improve Membrane Protein Structure Determination Using Rosetta," Nathan S. Alexander, Hassane S. Mchaourab, Jens Meiler

Membrane proteins remain a particular challenge in structural biology. Only about 1.5% of reported tertiary structures and sixty unique membrane protein topologies consisting of more than one transmembrane span are represented in the PDB. However, these proteins make up over 30% of the entire proteome, and over half of all therapeutics target this group. Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is often used for the structural characterization of proteins that elude X-ray crystallography and NMR, but high-resolution structures are difficult to obtain via EPR due to uncertainty in the spin label location and sparseness of experimental data. RosettaEPR has been designed to combine the strengths of both computation and EPR spectroscopy. We have demonstrated the feasibility of using RosettaEPR with soluble proteins by testing the method on T4-lysozyme; after full-atom refinement, a 1.7Å model of the protein was produced (Hirst, et al., 2011). RosettaEPR was also benchmarked on eleven membrane proteins of known structure in combination with simulated EPR distance data. While it was generally observed that de novo folding in the presence of EPR restraints enriched the recovery of the proteins’ correct topology compared to when folding with no restraints, it was clear that an innovative conformational sampling strategy was still needed. The preliminary work presented here demonstrates how a new membrane protein sampling protocol, in which transmembrane helices are moved as rigid bodies, can further enhance RosettaEPR’s performance. By moving entire helices in relation to one another, Rosetta is able to form more long-range, high contact order interactions, which are extremely under-sampled when using the traditional fragment assembly folding protocol. Folding with rigid body sampling increased the percentage of correctly folded models for 10 of 12 helical membrane proteins, including rhodopsin. Rosetta was able to fold rhodopsin, a GPCR, to 4.5Å RMSD100 in the transmembrane regions. This newly implemented membrane protein folding algorithm can be combined with EPR data to enhance larger and more complex topologies that are consistent with experimental information.

3) Myriam Díaz Martínez, "Imaging Characterization of Adipose Tissue Before and After Roux-en-Y Gastric Bypass Surgery," E. Brian Welch

Background and Specific Aims: The aim of this study is to characterize adipose tissue (AT) in an obese rat model before and after gastric bypass surgery, utilizing MRI, PET imaging and molecular/cellular analysis. Morbid obesity is an increasing epidemic in the US and is the leading factor in the development of cardiovascular diseases such as coronary atherosclerosis, hypertension, dyslipidemia, and Type 2 diabetes (T2DM). Currently, gastrointestinal bypass surgery, specifically Roux-en-Y bypass (RGBP) surgery, is the most established and effective treatment for substantial and sustained weight loss in morbidly obese subjects. However, the mechanism in which the tissue of the adipose organ changes is not well understood. Strategies have been developed to understand the role and changes of this endocrine organ before, during and after the RGBP surgery, particularly its spatial distribution, triglyceride composition, and mitochondrial density along with molecular and cellular approaches.
Approach: We will investigate AT using MRI and PET imaging on obese rats before and after RGBP surgery. A number of control obese rats will undergo a sham RGBP surgery. MRI-based characterization of AT will include analysis of: (a) spatial distribution (visceral and subcutaneous) using fat-water MRI, (b) triglyceride chain length, unsaturation degree and polyunsaturation degree composition, and (c) R2* relaxation time within AT to detect iron concentration increases associated with inflammation. Also we will perform PET/CT scans using 18F-FDG and TSPO contrast agents to respectively determine glucose uptake and mitochondrial density within brown adipose tissue (BAT) that is distinct from white adipose tissue (WAT). We will compare our results with metabolic blood panels and molecular biology techniques to validate and understand AT changes before, during and after RGBP surgery.
Results & Significance: Many advances have been made to understand the underlying mechanism of prolonged weight loss after Roux-en-Y, but characterization of AT, specifically spatial distribution, triglyceride composition, inflammation status and mitochondrial density before and after surgery, has not been previously measured in a single imaging-based study. Investigating the role played by AT in combination with hormone and cellular signaling will provide a better understanding of weight loss mechanisms after gastrointestinal (GI) bypass intervention.

4) Laura A. Mike, "A Small Molecule that Stimulates Endogenous Heme Biosynthesis is Toxic to Fermenting Staphylococcus aureus," Brendan F. Dutter, Devin L. Stauff, Jessica L. Moore, Nicholas P. Vitko, Olusegun Aranmolate, Thomas E. Kehl-Fie, Sarah Sullivan, Paul R. Reid, Jennifer L. DuBois, Anthony R. Richardson, Richard M. Caprioli, Gary A. Sulikowski, and Eric P. Skaar

Staphylococcus aureus is a Gram-positive pathogen that causes a wide range of pathologies ranging from superficial skin infections to deep seeded osteomyelitis and endocarditis. In order to be a successful pathogen in diverse niches S. aureus senses and adapts to environmental changes using two-component signaling systems (TCSs). Upon entry into the host S. aureus encounters an environment devoid of free iron. S. aureus satisfies its iron and heme requirements by scavenging host heme from hemoglobin. While heme is a valuable iron source and co-factor, the reactivity of the molecule is a liability to the cell. The S. aureus TCS heme sensor system (HssRS) is activated in the presence of heme and up-regulates the heme regulated transporter (HrtAB), which protects the bacteria from heme toxicity. In order to understand the mechanism by which S. aureus senses and responds to heme toxicity during infection, a high-throughput screen for small molecule activators of HssRS was performed. The most potent HssRS activator, VU0038882 (‘882), was found to stimulate endogenous heme biosynthesis by perturbing central metabolism. Furthermore, the ‘882-mediated alterations to central metabolism are toxic to fermenting S. aureus, including clinically relevant small colony variants (SCVs). Targeting fermentation in a facultative anaerobe has therapeutic utility as ‘882 prevents the emergence of antibiotic resistance, enhances phagocyte killing and reduces S. aureus pathogenesis. This small molecule has revealed fundamental insights into staphylococcal heme homeostasis as well as provided a novel therapeutic strategy for combating this aggressive pathogen.

5) Md. Imam Uddin, "SLC7A11 and SLC1A5 Targeted Amino Acids− 18F-FP-GLU and 18F-GLN as Imaging-Biomarkers for Colon-Cancer," Yiu-Yin Cheung, Matthew R. Hight, Jason R. Buck, Sam A. Saleh, Mike Nickels, Eliot T. McKinley, George H. Wilson, Noor Tantawy, H. Charles Manning

Background and Specific Aim: [18F]-PET is one of the sensitive noninvasive modalities that can quantitatively analyze the tracer bio-distribution and tissue-uptake and could measure the activity of oncogenic events and have broad impact on clinical needs. Recent studies have shown that amino-acid transporter proteins SLC7A11 and SLC1A5 play an important role in transporting 18F-4FP-GLU and 18F-4F-GLN respectively, across the cell membrane. Though, 18F-FDG PET is used clinically for visualization of tumors in colon and other lesions, measures are more often confounded by glucose uptake in normal tissues that results in modest to poor tumor-to-background ratios. We envisioned to use18F-4FP-GLU and 18F-4F-GLN as new radiotracers for the detection of colon-cancer in xenograft models, with near term clinical applications.
Approach: Precursor was chemically synthesized that is structurally suitable for 18F-incorporation and PET-imaging studies. Human CRC cell line overexpressing SLC7A11 and SLC1A5 were used to generate tumor bearing athymic nude mice by implanting the cells. In vivo [18F]-PET imaging was performed by radiochemical synthesis and intravenous injection of the 18F-4FP-GLU and 18F-4F-GLN in tumor bearing xenograft models and PET-image analysis.
Results and Significance: Successful incorporation of 18F by radiochemical synthesis using a tosylate or a nosylate-precursor, and retro orbital injection followed by in vivo PET-imaging showed significant uptake of the radiotracer in tumor compared to normal tissues. These PET-radiotracers thus visualized and translated the proliferative tissues in xenograft model. In vivo stability of the probes and bio-distribution results reveled, these new tracers as promising PET ligand for evaluating proliferation in colon-cancers and potentially other cancer types.

 

6) Andrew Cognata, “Real-Time Electrochemical Monitoring of Metabolic Processes In Hollow Fiber Bioreactor Cellular Cultures,” David Cliffel

Two dimensional cell culturing are the primary means for investigation of animal tissue cells. The utility of this approach is limited by a number of constraints, most notably the limited lifespan of individual cultures and a failure to reproduce the in vivo conditions that cells and tissues would typically experience. Hollow Fiber Bioreactors (HFBx), a three dimensional culturing methodology that that simulates the physical stresses and selective behavior of biological membranes allowing for more realistic in vitro conditions when observing cell behavior, are an emerging solution to these limitations. Preliminary research using HFBx indicates that these conditions can dramatically affect the metabolic activity of cells, in many cases significantly increasing the yield of many biomolecules on a per cell basis. Awareness of the metabolic behavior of cells is essential both for the in-lab maintenance of cell cultures and observation of cellular behavior during experimental investigations. Current quantitative sampling techniques for HFBx center are predominately off-line, periodic methods, or else utilize expensive instrumentation. Such methods fail to provide a low-cost, real-time monitoring solution. To address this deficiency, a screen-printed electrode (SPE) array encased in a microfluidic flow chamber has been developed for use in electrochemical monitoring of cellular metabolites including glucose, lactate, oxygen, and extracellular acidification in the effluent released from the HFBx chamber. This approach, which utilizes a variety of modifiable electrodes, allows a selection of metabolic processes to be monitored in real time with excellent selectivity. The SPE is comprised of five platinum electrodes, which can be modified to electrochemically detect a wide number of analytes, including those mentioned above, through the application of a variety of selective enzyme and metal films. Individual sensors are inexpensive, reusable during their lifetime of several weeks, and easily configured using little or no modification to preexisting procedures. The capabilities of the monitoring method are demonstrated by real-time monitoring of both basal metabolic rates and responses to well-studied metabolic stimuli in immortalized cell lines and validated by comparison to similar experiments previously conducted using an established real-time detector for 2D cultures (such as the Multianalyte Microphysiometer) which is not compatible with the HFBx. This demonstrates the utility and validity of electrochemical detectors for the monitoring of HFBx, an important step in taking full advantage of the opportunities provided by bioreactors and over 2D culturing environments.

7) Amanda Duran, "Computational Design for a Symmetric Glycerol Facilitator Protein," Steven Combs, Jens Meiler

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 an inverted symmetric topology. The Glycerol Facilitator protein (GlpF) from Escherichia coli is a membrane protein that exhibits a two-fold inverted pseudo-symmetry. The crystal structure for GlpF (PDBID:1fx8) is of high resolution with a C α root mean square deviation (RMSD) of 1.8 Å between the inverted halves which initially made it ideal for this study. Using the Combinatorial Extension alignment server and the Rosetta Protein Modeling Suite, GlpF was computationally 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 and Design are currently being used to further re-design this protein.

8)  David P. Nannemann, "Human neutralizing monoclonal antibodies that recognize respiratory droplet transmissible H5N1 influenza viruses," Natalie J. Thornburg, David L. Blum, Jessica A. Belser, Terrence M. Tumpey, Shyam Deshpande, Gloria A. Fritz, Jens C. Krause, Jeong Hyun Lee, Andrew B. Ward, David E. Lee, Sheng Li, Katie L. Winarski, Benjamin W. Spiller, Jens Meiler, and James E. Crowe Jr.

Recent research described the experimental adaptation of influenza H5 hemagglutinins (HAs) that confers respiratory droplet transmission to influenza virus in ferrets, which may mimic the future development of a highly pathogenic pandemic H5 “bird flu” virus in nature. This research set off a national debate on publication of research with potential dual use in bioterrorism. Human hybridomas from peripheral blood mononuclear cells from patients who had participated previously in clinical trials for an H5N1 vaccine were generated and characterized for antigen binding and neutralization of avian influenza viruses. Antibodies that bind the HA head domain and recognize both circulating and respiratory droplet transmissible H5 HAs have been identified. Using a hybrid methods approach to synthesize a number of structural techniques, including X-ray crystallography, electron microscopy, hydrogen/deuterium exchange mass spectrometry, and binding affinity assay, we defined a mechanism of antibody binding to the head of HA in a manner that retains molecular recognition of HAs with mutations that render H5N1 viruses transmissible by respiratory droplet in mammalian models. The model was validated through characterization of HA mutations predicted to confer antibody escape and explains naturally-occurring escape mutations. Rational design of novel H5 HA immunogens to protect against mammalian adapted mutants should consider incorporation or retention of this critical antigenic site. Furthermore, engineering of these antibodies may allow for therapeutic application in the event of avian influenza pandemic.

9)  William R. Birmingham," Bioretrosynthetic Evolution of a Didanosine Biosynthetic Pathway," Timothy D. Panosian, David P. Nannemann, Chrystal A. Starbird, Tina M. Iverson, Brian O. Bachmann

The next logical step in the engineering of biocatalysts is their concatenation into multi-step non-natural biosynthetic pathways. We have combined the conceptual design paradigm of retrosynthesis and the theory of retrograde evolution in ‘bioretrosynthesis’, an applied method for constructing an in vitro biosynthetic pathway, and evaluate the strategy in vitro using the nucleoside analog didanosine as a model target. As a retrograde pathway extension from an engineered purine nucleoside phosphorylase, we have evolved phosphopentomutase activity toward the non-natural substrate 2,3-dideoxyribose 5-phosphate with a 700-fold change in substrate specificity and 3-fold increased activity in cell lysate. An additional retrograde pathway extension toward engineering a dideoxyribokinase provided a 50-fold total increase in didanosine production and ~9,500-fold change in nucleoside production specificity in a five step biosynthetic pathway beginning from a simple sugar analog 2,3-dideoxyribose and including an ATP regeneration cycle. Unexpectedly, the biosynthetic productivity of this engineered ribokinase is the result of a new phosphorylation regioselectivity, creating a pathway shortening bypass by directly forming a substrate for purine nucleoside phosphorylase. This previously uncharacterized activity was detected through testing for final product formation in the bioretrosynthesis evolution strategy rather than possibly having gone overlooked by screening for the anticipated dideoxyribose 5-phosphate intermediate.

10)  Sam DeLuca, "A Novel Method for Guiding High-Throughput Protein-ligand docking with QSAR-Derived Pharmacophore Maps," Jens Meiler

Currently, QSAR and computational ligand docking studies are valuable but independently used tools for drug design. Data from models produced by cheminformatics tools are typically used to filter results prior to computational ligand docking. ROSETTALigand has been previously successful at predicting binding poses with high resolution (Kaufmann, et. al, Proteins, 2009). We are developing ROSETTAHTS, an extension to ROSETTALigand which will integrate these two methods using cheminformatics descriptors in conjunction with descriptors derived from ligand docking. Discrete cartesian grids describing chemical features of the binding site are overlaid on the protein structure and score initial placement of the ligand prior to fine grained docking. As the scoring grids are precomputed, sampling of the binding site is rapid. The predicted binding pose is then used to generate interaction descriptors which are used in conjunction with ligand cheminformatics descriptors to train a neural network predicting ligand activity. The neural network is trained to predict ligand activity in general, rather than for a specific protein system. Rapid initial sampling, increases the practicality of structure based virtual High Throughput Screening (vHTS). The integration of structure and ligand based vHTS techniques allows the full range of pharmacological information to be considered at once. This technique can be used to rapidly develop small focused libraries for HTS, decreasing the number of compounds that need to be purchased for testing.

11)  Darwin Fu, "A Novel Method for Simultaneous Docking of Multiple Ligands Guided by Binding Affinity Data," Jens Meiler

SAR allows us to simultaneously examine multiple ligands sharing a common scaffold. We seek to use this information to guide RosettaLigand in identifying the correct binding pose. Current methods in RosettaLigand are capable of correlating with experimental affinities but can only bind a single ligand at a time. A new technique could take advantage of the fact that structurally similar active compounds should adopt a common pose in a binding pocket. The novel method would simultaneously dock a superimposed ensemble of ligands and use rank correlation to binding constants as an evaluative term. The new RosettaLigandEnsemble algorithm will build upon existing methods of conformational sampling and ligand docking to identify poses wherein active compounds score well and inactive compounds score poorly. Following benchmarking and scoring optimization, the method can then be applied to study membrane proteins important in CNS disorders in collaboration with wet lab work at Vanderbilt.

12)  Jessica L. Moore, "Imaging Mass Spectrometry for Co-Localization of Trace Metals and Proteins Staphylococcus aureus Abscesses," Yaofang Zhang, Thomas E. Kehl-Fie, Eric P. Skaar, Richard M. Caprioli

Bacteria require metals as cofactors for many processes, including energy generation, DNA replication, oxygen transport, and protection from oxidative stress. Pathogens must obtain metals from host organisms to survive. Host organisms have developed defense mechanisms to sequester essential metals from invading pathogens, a process known as nutritional immunity, which aids the host in controlling pathogen replication and dissemination. The study of nutritional immunity and the relevant metalloproteins is crucial to understand the pathogen-host interaction. Additionally, metal acquisition and homeostasis mechanisms have potential therapeutic promise. Imaging Mass Spectrometry [IMS] can be used to aid the discovery of potential metal-binding proteins involved in metal homeostasis and nutritional immunity by providing spatial localization of proteins and metals to areas of infection. Through co-registration of serial MALDI IMS protein images and LA-ICP IMS trace-metal images, proteins of interest can be connected spatially to metals of interest. The S100 proteins forming the calcium-binding heterodimer calprotectin were found to localize to regions of Staphylococcus aureus abscesses. Co-localization with LA-ICP IMS data show abundant signal for calcium in abscessed regions corresponds with the calcium-binding proteins. Additionally, abscessed regions were found to be void of nutrient manganese and zinc, consistent with previous findings. Using laser capture microdissection, areas of interest can be collected to further identify protein targets that localize to regions of interest.

13)  Charles Williams, "Antagonists of Acid-Sensing Receptor OGR1/GPR68 and Their Use in Modulating of Pulmonary Vascular Tone," Benjamin Mackowiak, Charles Hong

Failure of normal relaxation of pulmonary vascular tone at birth leads to abnormalities such as persistent pulmonary hypertension of the newborn (PPHN). Clinical and laboratory observations suggest that the interplay of acidosis and hypoxia contributes to pathologic states of persistent pulmonary vasoconstriction. However, the molecular mechanisms that underlie pH sensing are not fully understood. Using novel first in class inhibitors of OGR1/GPR68, we are studying the role of acid-sensing or proton-sensing G-protein coupled receptors in regulating the pulmonary vasculature. Since OGR1/GPR68 acts via Gq/11 and subsequent Ca+ release, we hypothesize that OGR1 plays a role in regulating pulmonary vascular tone, which is dependent on Calcium mediated contraction and that inhibition with our antagonist will reduce pulmonary vascular tone. Contrary to our hypothesis, initial calcium studies efflux studies using pulmonary smooth muscle and pulmonary endothelial cells suggests that OGR1 antagonists potentiate calcium efflux in response to acidosis.

14)  Joshua Bauer, "The Vanderbilt High-throughput Screening Facility: From Assay Conception to Lead Optimization"

The goal of the Vanderbilt High-Throughput Screening (HTS) facility is to provide screening-based services to help research investigators to identify novel drug targets and new compounds for basic research and pharmacological discovery. The HTS services include self-service instrumentation use and access, instrument training/assistance, assay design, assay optimization/development, pilot screening, liquid handling, plate reading, automation, data capture and analysis, compound distribution, and full high-throughput small molecule library screening, as well as informatics solutions to all assays and screens . New technologies and resources include an automated microscope imager for high content screening, a non-contact liquid handler for transferring biological reagents in nanoliter increments, human siRNA libraries for functional genomic screening, and small kinase inhibitor libraries for pilot screening. These resources along with knowledgeable HTS staff provide a highly dynamic environment that utilizes industry standard practices and novel technologies for biological screening. The HTS facility is well-equipped and staffed to guide investigators through the drug discovery process from assay conception to lead optimization.

15)  Marta W. Szulik, "Structural Studies of 5-Hydroxycytosine Opposite Guanine and Adenine in DNA.," Patrick S. Donahue, Michael P. Stone

Incorporation of 5-hydroxycytosine (5OHdC) into DNA leads to C to T transition mutations. Crystal structure of 5OHdC in DNA with polymerase revealed that the lesion stabilizes the incoming dGMP via canonical base pairing. However the incorporation of dAMP opposite the lesion leads to destabilizing unstacking. Here we describe studies of the modified DNA, where 5OHdC is base base paired with dG and dA. High resolution NMR spectroscopy reveals that the 5OHdC•dG is less stable than its Watson-Crick partner dC•dG, although it does not differ significantly in structure. When mismatched with dA, 5OHdC is displaced into the major groove. The structural and thermodynamic properties of the modified base pair and mismatch could provide for a mechanism of recognition by DNA polymerases and repair enzymes.

16)  Emilianne McCranie, "Investigating the Biosynthesis of Unique Features of Orthosomycin Antibiotics," Kathryn M. McCulloch, Tina M. Iverson, Brian O. Bachmann

Due to the increasing prevalence of multiply resistant bacteria in the clinical setting, there is a dire need for new antibiotics with novel bacterial targets. The orthosomycins are polysaccharides defined by the presence of an orthoester linkage and possess activity against a variety of resistant Gram-positive bacteria. We hypothesize that the orthosomycins are uniquely tailored for their bacterial targets; however the structural features contributing to this specificity are unknown. We are especially interested in understanding the contributions of the orthoester linkages and methylenedioxy bridge to orthosomycin activity. Analysis of the gene clusters of five orthosomycins revealed a conserved group of non-heme iron, α-ketoglutarate dependent oxidases which we hypothesize are responsible for orthoester and methylenedioxy bridge formation. Disruption of the three putative oxidases of the everninomicin producer resulted in abolished production confirming their role in orthosomycin biosynthesis. Optimization of culture conditions resulted in a 47-fold increase in everninomicin production providing the levels necessary to begin the process of identifying analogs which may accumulate in the mutant strains. In addition, crystal structures of one oxidase, EvdO2, revealed the expected double-stranded β-helix fold along with an unusually large binding cleft. Due to their activity against a variety of drug-resistant Gram-positive bacteria as well as their novel bacterial targets, the orthosomycins have the potential to become clinically useful drugs. Understanding the biosynthesis of these unique compounds will provide tools to generate new analogs with increased antibacterial activity and efficacy.

17)  Soumya Ganguly, "Structure Refinement of α-Helical Membrane Protein by Paramagnetic Lanthanide Ion Tagging," Jens Meiler

Structure determination of integral membrane proteins is hindered due to challenges associated with protein expression, purification and folding. For α-helical membrane proteins further challenge awaits while using NMR to collect structural restrain because of narrow chemical shift dispersion in the proton dimension and lack of long range distance restrain from traditional NOE based measurements. Here we explain a novel method of labeling helical membrane protein with paramagnetic lanthanide ion tags to collect long range distance and angular information of the protein. Long range distance restraints and angular restraints were measured from paramagnetic relaxation enhancement (PRE), pseudo-contact shift (PCS) and residual dipolar coupling (RDC) data collected from a single set of 1H, 15N IPAP TROSY. By carefully tagging strategic sites at different transmembrane regions of the helical membrane protein sufficient number of restraints can be collected for atomic level refinement of alpha helical membrane protein structures.

18)  Matthew C. Surdel, "Identification of a Small Molecule that Activates the Staphylococcal Heme Stress Response Independently of Heme," Brendan F. Dutter, Devin L. Stauff, Olusegun Aranmolate, Gary A. Sulikowski, Eric P. Skaar

Staphylococcus aureus is a major human pathogen contributing to significant morbidity and mortality worldwide. Within the vertebrate host, S. aureus requires the acquisition of nutrients, such as heme and iron. To satisfy these requirements, S. aureus imports host heme through dedicated transport systems, while also retaining the ability to synthesize heme endogenously. Although heme acquisition is required for pathogenesis, excess heme is toxic. S. aureus utilizes a two component system, the heme sensor system (Hss), to sense and protect against heme toxicity. Upon stimulation, the sensor histidine kinase (HssS) activates the response regulator (HssR) to induce the expression of the heme-regulated transporter (HrtAB), an efflux pump that reduces heme toxicity. To further our understanding of how HssRS senses heme toxicity, we performed a high throughput screen to identify small molecule activators of HssRS. Previously studied activators of HssRS have been shown to be dependent upon heme accumulation within the cell. Interestingly, one of the most potent compounds from our screen, ‘3981, activates HssRS independent of heme accumulation, thereby providing a unique probe to interrogate this two component system. Preliminary structure-activity relationship (SAR) studies have provided insights into the structural determinants crucial to this phenotype. Although the parent compound had significant toxicity against S. aureus, we have identified derivatives of ‘3981 with increased potency in activating HssRS, while lacking toxicity. Ultimately, a thorough understanding of the mechanisms employed by S. aureus to overcome the toxicities experienced in the host will lead to a better understanding of the pathogenesis of this organism and may uncover potential therapeutic targets. Due to the conservation of heme sensing systems across numerous medically relevant pathogens, this work will provide a broader understanding of bacterial heme sensing during a number of infectious diseases.

19)  Elwood A. Mullins, "An HPLC-Tandem Mass Spectrometry Method for Simultaneous Detection of Alkylated Base Excision Repair Products, Emily H. Rubinson, Kevin N. Pereira, M. Wade Calcutt, Plamen P. Christov, and Brandt F. Eichman

DNA glycosylases excise a broad spectrum of alkylated, oxidized, and deaminated nucleobases from DNA as the initial step in base excision repair. Substrate specificity and base excision activity are typically characterized by monitoring the release of modified nucleobases either from a genomic DNA substrate that has been treated with a modifying agent or from a synthetic oligonucleotide containing a defined lesion of interest. Detection of nucleobases from genomic DNA has traditionally involved HPLC separation and scintillation detection of radiolabeled nucleobases, which in the case of alkylation adducts can be laborious and costly. Here, we describe a mass spectrometry method to simultaneously detect and quantify multiple alkylpurine adducts released from genomic DNA that has been treated with N-methyl-N-nitrosourea (MNU). We illustrate the utility of this method by monitoring the excision of N3-methyladenine (3mA) and N7-methylguanine (7mG) by a panel of previously characterized prokaryotic and eukaryotic alkylpurine DNA glycosylases, enabling a comparison of substrate specificity and enzyme activity by various methods. Detailed protocols for these methods, along with preparation of genomic and oligonucleotide alkyl-DNA substrates, are also described.

20)  Sonja C. Brooks, "Structural and Biochemical Characterization of DEMETER, a Multidomain 5mC Glycosylase," Brandt F. Eichman

The methylation state of DNA is important for gene expression, gene imprinting, X-chromosome inactivation, and transposon silencing in mammals and plants. DNA methylation is established by methyltransferases to mark a silenced gene in the form of 5-methylcytosine (5mC), but the mechanism of 5mC removal in mammals remains poorly understood. In Arabidopsis thaliana, DEMETER (DME) is a 5mC DNA glycosylase that activates expression of the maternally imprinted MEDEA gene. Thus, DME has evolved what is normally a DNA repair function to remove the non-toxic 5mC. We are carrying out a structure-function analysis of DME to understand the basis for this unique activity. DME contains a conserved iron-sulfur cluster (Fe4S4)-containing DNA glycosylase domain, as well as two flanking domains (A and B) necessary for base excision activity but whose structures and functions are unknown. A homology model of DME constructed from EndoIII, a DNA glycosylase specific for oxidized pyrimidines, reveals that the A-domain may be integral to the glycosylase fold. Using this homology model as a guide for mutational analysis of base excision and DNA binding, we have identified several residues important for DME activity. For example, Asn778 and Met1238 significantly reduce base excision, while still retaining DNA binding ability, and are predicted to intercalate the DNA to stabilize an extrahelical 5mC in the active site. In addition, recent literature indicates that removal of 5mC from the genome may proceed by removal of oxidation derivatives of 5mC by thymine DNA glycosylase (TDG). We find that DME has reduced activity for 5-hydroxymethylcytosine (5hmC), limited activity for 5-carboxyctyosine (5caC), and no activity for 5-formylcytosine (5fC). Finally, we have compared DME’s activity against two Arabidopsis DME paralogs, ROS1 and DML3, in order to understand the rationale for high redundancy of 5mC excision in plants.

21)  Gordon Lemmon, "Towards Ligand Docking Including Explicit Interface Water Molecule," Jens Meiler

Small molecule docking predicts the interaction of a small molecule ligand with a protein at atomic-detail accuracy including position and conformation of the ligand as well as conformational changes of the protein upon ligand binding. While successful in the majority of cases, docking algorithms including RosettaLigand fail in some cases to predict the correct protein/ligand complex structure. In this study we show that simultaneous docking of explicit interface water molecules greatly improves Rosetta’s ability to distinguish correct from incorrect ligand poses. This result holds true for both protein-centric water docking wherein waters are located relative to the protein binding site and ligand-centric water docking wherein waters move with the ligand during docking. Protein-centric docking is used to model 99 HIV-1 protease/protease inhibitor structures. We find protease inhibitor placement improving at a ratio of 9:1 when one critical interface water molecule is included in the docking simulation. Ligand-centric docking is applied to 341 structures from the CSAR benchmark of diverse protein/ligand complexes. Across this diverse dataset we see up to 56% recovery of failed docking studies, when waters are included in the docking simulation.

22)  Glenna Kramer, "Structural Activity Relationship Studies of K-26, a Microbially Biosynthesized Angiotensin-I Converting Enzyme Inhibitor," Akif Mohd, David Nannemann, J. Albert Abrie, Sylva L. U. Schwager, Geoffrey Masuyer, K. Ravi Acharya, Edward. D. Sturrock, Jens Meiler, and Brian O. Bachmann

The tripeptide K-26 typifies a class of secondary metabolites produced by soil bacteria which incorporates an unusual phosphonate analog of tyrosine (AHEP). This class of natural products is of pharmacological relevance due to potent inhibitory activity against human angiotensin-I converting enzyme (ACE), an enzyme central to blood pressure and fluid balance regulation. Co-crystallization of K-26 in both physiologically relevant domains of human ACE (nACE and tACE) and a Drosophila melanogaster ACE homolog (AnCE) have shown an unprecedented inhibitor binding motif. SAR studies of the naturally occurring analogs with ACE has confirmed the class-wide potency, but has revealed minimal selectivity between the domains of human ACE. However, a computational study has presented the potential for development of AHEP containing tripeptides with increased ACE potency or domain selectivity. Furthermore, K-26 has been developed into a solid-phase probe capable of capturing human ACE with potential applications in target identification and the study of the biosynthesis of this class of secondary metabolites.

23)  Jing Yang, "Site-Specific Mapping of Protein S-Sulfenylation," Hye-young Kim, De Lin, Keri Tallman, Ned Porter, Kate Carroll, Daniel Liebler

Reversible formation of sulfenic acid from cysteinyl thiols in proteins, a post-translational modification termed protein sulfenylation, has emerged as a potential mechanism for protein functions. However, the substrates and specific sites of sulfenylation in thiol proteome remain largely unknown, mainly because of the low abundance and inherent liability of this transient modification, as well as the difficulty of selective capture of sulfenyl peptides for analysis. Here we utilized a bioorthogonal dimedone-based probe, DYn-2, for labeling, capture by Click chemistry, and identification of protein sulfenylation by liquid chromatography-tandem mass spectrometry. We report the first global survey of site-specific protein sulfenylation in human tumor cell lines. Captured sulfenyl peptides were analyzed on a high-performance Thermo QExactive instrument. We identified >1000 cysteine sulfenylation sites on >700 proteins. Preliminary inventories include, in addition to regulators of cellular redox homeostasis, proteins involved in diverse processes, including intermediary metabolism, cytoskeleton, RNA splicing, and nuclear transport. This approach should enable targeted, quantitative assessment of protein sulfenylation as a putative regulator of cellular functions.

24)  Katrina L. Leaptrot, "Development and Theoretical Evaluation of a Spatially Multiplexed Ion Mobility Spectrometer," Jody C. May, and John A. McLean

Ion mobility-mass spectrometry (IM-MS) is a developing technology that has significant utility in the analysis of complex mixtures. A need for improved figures-of-merit in IM-MS has been recognized. Although, to date, no one has built a spatially multiplexed IM-MS, such an instrument could provide benefits in throughput, sensitivity, and versatility, among others. Here we discuss details from the ongoing development of an IM-MS with 8 discrete parallel channels, a step toward a 96-channel IM-MS for 96-well plate analyses. 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 8-channel IM-MS has been compartmentalized into three stages. Stage one focuses on design of an 8-channel ion mobility spectrometer (IMS) with an electrospray ionization (ESI) source array. Stage two involves developing a matrix assisted laser desorption/ionization (MALDI) source and adapting a commercial time-of-flight mass spectrometer (TOFMS) to accept multiple ion beams simultaneously. Stage three will integrate the 8x IMS and modified TOFMS and establish data acquisition and 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. Though initial progress has occurred in all stages, the instrument is considered in the first development stage. We present the progress of constructing this instrument with emphasis on the vacuum system, ion source, and interfacing ion optics.

25)  James Galligan, "Alkylation of Histones by 4-oxo-2-nonenal as a Novel Modification Linked to Oxidative Stress," Rose, KL, Beavers, WN, Aluise CD, Shuck, SC, Marnett, LJ

Sustained oxidative stress leads to the generation of toxic concentrations of the lipid aldehydes 4-hydroxy-2-nonenal (4-HNE) and 4-oxononenal (4-ONE), which are capable of covalently modifying the side-chains of Cys, His, and Lys residues. These protein modifications have been identified as a contributing factor in numerous disease states, including cancer, cardiovascular disease, neurodegeneration and diabetes. Here, we describe a novel class of histone modifications resulting from lipid electrophile adduction of His and Lys residues. Utilizing click chemistry with ω-alkynyl-4-ONE (a4-ONE), we have identified all-four core histones as targets for modification in RKO cells treated with physiologically relevant concentrations of electrophile. Isolation of chromatin from these cells reveals H2B as a highly susceptible target to modification by 4-ONE, while H2A, H3 and H4 show minimal reactivity. Interestingly, similar experiments with a4-HNE failed to identify any of the core histones as targets for modification. 4-HNE reacts heavily with Cys residues, which histones lack, whereas 4-ONE reacts primarily with the ε-amine of Lys residues. A proteomic investigation of site-specific modifications in RKO cells treated with 4-ONE reveals H2B Lys117 as a major target for modification. The predominant stable species detected on H2BK117 results from a 1,2-addition and net acylation analogous to Lys acetylation. H2BK117 is a surface exposed Lys residing on the face of the nucleosome core particle. Treatment of RKO cells with physiologically relevant doses of 4-ONE results in the altered expression of 40 genes. Electrophile adduction of histones provides a previously unexplored link between oxidative stress and gene regulation.

26)  Aaron C. Mason, "Structural Basis for DNA Junction Preference by a Conserved Nucleic Acid Binding Domain," Robert P. Rambo, Michael Pritchett, Akosua Badu-Nkansah, John A. Tainer, David Cortez, Brandt F. Eichman

SMARCAL1 (SWI/SNF-related, matrix associated, actin-dependent regulator of chromatin, subfamily a-like 1), also known as HARP (HepA-related protein), is a recently identified DNA damage response protein found at stalled replication forks. Mutations in SMARCAL1 result in the developmental disorder Schimke Immunoosseous Dysplasia (SIOD). SMARCAL1 travels with elongating replication forks and its absence leads to MUS81-dependent double-strand breaks and cellular sensitivity to replication stress agents. SMARCAL1 is a distant SNF2 family member of ATP-dependent DNA translocases, has a binding preference for branched DNA structures, and promotes branch migration of Holiday junctions and fork regression of model replication forks. The substrate specificity and branch migration activity is dependent on a unique “HARP” domain N-terminal to the conserved ATPase motor. Although the ATPase domain is homologous to other SNF2 motors, including recombination-repair protein Rad54, virtually nothing is known about the HARP domain. Here, we describe the crystal structure of the HARP domain, together with small angle X-ray scattering (SAXS) analysis of the catalytic HARP-ATPase core domain. The HARP domain is structurally homologous to the mismatch and damage recognition domains of MutS and XPB, and the HARP-ATPase catalytic core as modelled by SAXS bears a strong resemblance to the T4 recombinase UvsW. SAXS analysis of the protein-DNA complex, together with mutational analysis of DNA binding and fork regression activities, illustrates how the HARP-ATPase catalytic core binds DNA junctions to promote stabilization of stalled replication forks in a manner reminiscent of E. coli RecG.

27)  Myriam Díaz Martínez, "Imaging Characterization of Adipose Tissue Before and After Rouxen- Y Gastric Bypass Surgery," E. Brian Welch

Background and Specific Aims: The aim of this study is to characterize adipose tissue (AT) in an obese rat model before and after gastric bypass surgery, utilizing MRI, PET imaging and molecular/cellular analysis. Morbid obesity is an increasing epidemic in the US and is the leading factor in the development of cardiovascular diseases such as coronary atherosclerosis, hypertension, dyslipidemia, and Type 2 diabetes (T2DM). Currently, gastrointestinal bypass surgery, specifically Rouxen- Y bypass (RGBP) surgery, is the most established and effective treatment for substantial and sustained weight loss in morbidly obese subjects. However, the mechanism in which the tissue of the adipose organ changes is not well understood. Strategies have been developed to understand the role and changes of this endocrine organ before, during and after the RGBP surgery, particularly its spatial distribution, triglyceride composition, and mitochondrial density along with molecular and cellular approaches.
Approach:
  We will investigate AT using MRI and PET imaging on obese rats before and after RGBP surgery. A number of control obese rats will undergo a sham RGBP surgery. MRI-based characterization of AT will include analysis of: (a) spatial distribution (visceral and subcutaneous) using fat-water MRI, (b) triglyceride chain length, unsaturation degree and polyunsaturation degree composition, and (c) R2* relaxation time within AT to detect iron concentration increases associated with inflammation. Also we will perform PET/CT scans using 18F-FDG and TSPO contrast agents to respectively determine glucose uptake and mitochondrial density within brown adipose tissue (BAT) that is distinct from white adipose tissue (WAT). We will compare our results with metabolic blood panels and molecular biology techniques to validate and understand AT changes before, during and after RGBP surgery.
Results & Significance: Many advances have been made to understand the underlying mechanism of prolonged weight loss after Roux-en-Y, but characterization of AT, specifically spatial distribution, triglyceride composition, inflammation status and mitochondrial density before and after surgery, has not been previously measured in a single imaging-based study. Investigating the role played by AT in combination with hormone and cellular signaling will provide a better understanding of weight loss mechanisms after gastrointestinal (GI) bypass intervention.

28)  Tiffany Onifer, "The Structural Characterization of Polyurethane Precursors: Methylenedianiline Trimer and Tetramers," Sarah Stow, Jay Forsythe, David Hercules, John McLean

Polyurethanes (PUR) are one of the most versatile substances, contributing to the development of our society through the creation of medical devices and other consumer products. Studying PUR precursors has become of great interest to polymer scientists. A limitation is the ability to fully characterize a mixture of polymer compounds owing to the wide dispersity of different species/distribution of species present. Mass Spectrometry is the best method to analyze these polymers but it alone cannot distinguish between the isobaric (i.e. same mass) isomers. This study offers a method to determine the isomeric conformations of multimers of methylenedianiline (MDA). MDA is a precursor to methylene diphenyl diisocyante, which is the major hard block component in many PUR. The MDA trimer and tetramer conformations have been analyzed using matrix assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) and electrospray ionization - ion mobility mass spectrometry (ESI-IM-MS). These species were evaluated in an assortment of matrices (e.g. a-cyano-4-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, & dithranol, etc.) and with various cations (e.g. Ag+, Li+, & Na+), to probe potential underlying trimer and tetramer isomeric conformations. The collision cross-sections of the parent ions were calculated to identify respective gas-phase ion structures. The tetramer compound was found to have two or more isomeric conformations, and the trimer was identified to have only one conformation. Characterizing MDA multimeric structures using IM-MS provides a method to further evaluate complex mixtures that contain various isomeric conformations.

29)  Kyle A. Floyd, "Proteomic Analysis of Biofilm Development by Uropathogenic Bacteria: Location, Location, Location," Jessica L. Moore, Catherine A. Wakeman, Carrie L. Shaffer, Eric P. Skaar, Richard M. Caprioli, Maria Hadjifrangiskou

Urinary tract infections (UTIs) are among the most common infections afflicting humans. Community acquired UTIs are most prevalent among women, with approximately 50% of women experiencing at least one infection during their lifetime. In the hospital setting, catheter-associated UTIs are the most common nosocomial infections and can lead to serious complications if resistant to treatment. UTIs are caused by several uropathogenic bacteria, with uropathogenic Escherichia coli (UPEC) being the predominant causative agent. Besides UPEC, Pseudomonas aeruginosa is a leading cause of catheter-associated UTIs and Klebsiella pneumoniae is prevalent among diabetic patients. Uropathogen virulence relies heavily on the ability of the bacteria to express extracellular adhesive organelles and form multi-cellular communities known as biofilms. Biofilm-associated bacteria are encapsulated by a self-produced extracellular matrix that provides protection from the host immune response as well as antibiotics. Therefore, the capacity to form biofilms offers the bacteria the ability to persist under a wide range of environmental conditions on either biotic or abiotic surfaces and, in the case of UPEC, within cells. A significant amount of work has established that biofilm communities are composed of heterogeneous populations. Although biofilm architecture has been relatively well-studied, the role and stratification of proteins within a given biomass remains largely unexplored. MALDI Imaging Mass Spectrometry (IMS) allows for the analysis of the localization/distribution and relative abundance of distinct protein species within a given biomass. In this study we used MALDI IMS to examine the localization/distribution of proteins within in vitro biofilms of UPEC, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We have defined a partial proteomic profile for 48 hour biofilms formed by each of these uropathogens, over a range of m/z 2,000 – 20,000. These profiles highlight four distinct protein species localizations within the biomass: species specific to the air-associated (oxygen-rich) interface, species specific to the liquid-associated (oxygen-poor) interface, species found only at the air-liquid junction (site of primary bacterial attachment), and species distributed throughout the overall biomass. Utilizing proteomics-based techniques we were able to putatively identify six of the observed protein species from the 48 hour UPEC biomass. Putative protein identifications include adhesion/attachment-related proteins such as the major curlin subunit CsgA (UniProtKB QIRDB7) and DNA-protective proteins such as DNA-binding protein HU (UniProtKB Q1R5W6, Q1RF95). We were able to follow the distribution of these identified protein species over the course of UPEC biofilm development from 24 to 96 hours using MALDI IMS. The ability to monitor temporal protein localization and abundance within biofilms will provide insight into bacterial multicellular behavior, and will identify novel biofilm effector proteins that can be targeted by therapeutic strategies designed to inhibit or disassemble pathogenic biofilms.

30)  Brendan Dutter, "Medicinal Chemistry and Target Identification of Small Molecule Activators of Heme Sensing in Staphylococcus aureus," Laura A. Mike, Matthew C. Surdel, Katherine M. Chong, Paul R. Reid, Susan Ramos-Hunter, Eric P. Skaar, Gary A. Sulikowski

The acquisition and regulation of heme is of critical importance to the pathogenesis of Staphylococcus aureus, a bacterium of significant concern to public health. The systems by which heme homeostasis is controlled are not well understood and identification of the proteins involved may present targets for the development of novel antimicrobials. A high throughput screen for activators of the HssRS heme sensing system in S. aureus yielded several small molecules with diverse structures. Using the lead compounds VU0038882 and VU0043981 from the screen, we are developing chemical probes to elucidate heme sensing mechanisms in S. aureus. Compound libraries were generated around these scaffolds to determine structure activity relationships. These data are being used to develop probes for target identification using two primary strategies. The first strategy involves affinity purification of the target from whole cell lysates using probes immobilized on a solid support. The second strategy is focused on incorporation of a photoaffinity label and clickable handle into the probe where the target can be tagged in vivo and captured after cell lysis. We aim to validate the target(s) of these molecules, understand their role in bacterial metabolism and evaluate them for potential antimicrobial development.

31)  Shalley Kudalkar, "Direct Binding Analysis of Cyclooxygenase-2 to Heme and Inhibitors by Back-Scattering Interferometry Supports the Heterogeneous Behavior of a Homodimeric Protein," Frank Moss, Amanda Kussrow, Darryl Bornhop, Lawrence J. Marnett

Cyclooxygenases (COX-1 and COX-2) are enzymes that convert arachidonic acid (AA) to prostaglandin H2 (PGH2), which is further converted to bioactive prostaglandins and thromboxane by specific synthases. Cyclooxygenases are targets for various therapeutic non-steroid anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors called coxibs. COX-2 can also efficiently oxygenates endocannabinoids, 2-arachidonylglycerol (2-AG) and arachidonylethanolamide (AEA), to their respective prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs). Cyclooxygenases are homodimeric enzymes and each monomer has a heme-containing peroxidase active site and a cyclooxygenase active site. Previous studies have shown that homodimeric COX enzymes function as conformational heterodimers during catalysis and inhibition. The functionally heterodimer COX enzymes are also suggested to have two independent subunits- allosteric and catalytic. The nature of the ligand bound to the allosteric subunit dictates the catalytic efficiency of the catalytic subunit. Recently, our lab has proposed a model based on the inhibitor studies with substrate-selective inhibitors (SSIss), which suggests that binding of competitive reversible inhibitors to both the subunits is required for the inhibition of arachidonic acid oxygenation by mouse COX-2 (mCOX-2) whereas, binding of these inhibitors to only one subunit is required for blocking the 2-AG oxygenation by mCOX-2. We employed back scattering interferometry to explore the heterodimeric nature of homodimeric mCOX-2 with respect to heme and NSAIDs binding. We found that binding of heme to the peroxidase active site or (R)-profens (SSI) to cyclooxygenase active site of mCOX-2 produced a single binding isotherm indicating binding of one molecule of heme or inhibitors to the COX-2 dimer. The competitive reversible and time-dependent inhibitors produced two-binding isotherms with variable affinities suggesting binding of two molecules of these inhibitors to COX-2 dimer. Changes in binding affinities and isotherms were also noticed when these inhibitors were incubated with the site-directed mutants of mCOX-2 (S530A and R120Q). We suggested that binding of the inhibitors to one or both the subunits is dependent on the identity of the inhibitors and nature of their binding determinants in the cyclooxygenase active site. The differences in binding isotherms and affinities correlated well with the functional differences of these inhibitors suggesting that the nature of the ligand bound to the allosteric subunit does dictate the catalytic efficiency of the catalytic subunit. The differences in the binding nature and binding affinities of various ligands to mCOX-2 dimer provide additional evidence to support the fact that cyclooxygenases are structurally homodimers but functionally heterodimers.

32)  Joel Federspiel, "Analysis of the Apoptosis Signal-Regulating Kinase Signalosome," Simona Codreanu, Daniel C. Liebler

Apoptosis signal-regulating kinase 1 (ASK1) is a key sensor kinase in the mitogen-activated protein kinase (MAPK) pathway that determines cellular response to a variety of stressors and has been implicated in several disease states. ASK1 is regulated by a large protein complex, termed the ASK signalosome, which dynamically assembles around ASK1 and helps to either keep the kinase inactive in a non-stressed cell or to promote activity in response to oxidants and other stress factors. The responsiveness of ASK1 to oxidative stress has predominantly been studied using H2O2 as a model oxidant, but this provides an incomplete representation of MAPK activators formed during oxidative stress. We examined the effect of the prototypical lipid electrophile, 4-hydroxy-2-nonenal (HNE), on ASK1 activity and signalosome composition. We generated cell lines stably expressing tandem-affinity tagged ASK1 and its putative interacting partners ASK2 and ASK3. ASK2 is a reported component of ASK1 signalosomes, whereas the recently described ASK3 was identified in our initial shotgun proteomic screen of the complex. A preliminary study was done on ASK3 expressing cells treated with HNE, H2O2, ethanol (vehicle control), or left untreated. The treated cells were harvested and ASK3 was immunoprecipitated using the tandem-affinity tag and each immunoprecipitate (IP) was analyzed by SDS-PAGE fractionation and liquid chromatography-tandem mass spectrometry to determine the signalosome protein composition in each condition. The activation state of the ASK1 MAPK pathway was assessed by western blot analyses of phosphorylated JNK, p38 and MKK7 to compare HNE and H2O2 evoked responses. Future work will add more replicate analyses to the ASK3 data as well as ASK1 and ASK2 cell line IPs to generate a consensus signalosome.

33)  Marta Wenzler, "Progress Toward the Total Synthesis of Upenamide," Bruce Melancon, Steve Luo, Kurt Kiewel, Gary Sulikowski

Upenamide is a marine alkaloid natural product isolated in 2000 by Scheuer and coworkers from the marine sponge Echinochalina sp. Protolithospongia. The biological activity has not been established as only a small amount of Upenamide has been isolated. Upenamide features a highly functionalized 20-membered macrocycle containing a spirooxaquinolizidinone and a hemiaminal. Upenamide presents an interesting synthetic challenge as the absolute stereochemistry is unknown. We propose a racemic synthesis of spirooxaquinolizidinone ring system, a stereocontroled synthesis of the hemiaminal ring system, and final macrocyclization to establish the stereochemistry of Upenamide.

34)  Brittany Allison, “Computational Design of Protein Ligand Interfaces Using RosettaLigand,” Sam DeLuca, Gordon Lemmon, Jens Meiler

The computational design of proteins that bind small molecules is one of the unsolved challenges 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. For apolar, rigid small molecules the interactions are dominated by short-range van der Waals forces. As the number of polar groups in the ligand increases, hydrogen bonds, salt bridges, cation-π, and π-π interactions gain importance. 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. To assess the current state of protein-ligand interface design, we benchmarked the computer algorithm Rosetta on a diverse set of 43 protein-ligand complexes. On average, we achieve sequence recoveries in the binding site of 59% when the ligand is allowed limited reorientation, and 48% when the ligand is allowed full reorientation. When simulating the redesign of a protein binding site, sequence recovery among residues that contribute most to binding was 52% when slight ligand reorientation was allowed, and 27% when full ligand reorientation was allowed.

35)  Ronald Bruntz, "Phospholipase D2 Mediates Survival Signaling Through Direct Regulation of Akt in Glioblastoma Cells"

The median survival time following glioblastoma multiforme (GBM) diagnosis is around one year due, in part, to a lack of innovative therapeutic targets. Phospholipase D (PLD) enzymes mediate diverse cellular functions including promotion of cancer cell survival. We show a novel regulation of Akt, another critical mediator of cell survival, by PLD in GBM. Utilizing small molecule PLD inhibitors and siRNA, we demonstrate the requirement of phosphatidic acid (PtdOH), the product of the PLD reaction, for membrane recruitment and activation of Akt. Inhibition of PLD and subsequently Akt decreases GBM cell viability by inhibiting autophagic flux. We propose a mechanism where phosphorylation of Beclin1 by Akt prevents binding of Rubicon, an interaction known to inhibit autophagic flux. Our findings thus provide opportunities to promote GBM cell death through Akt inhibition without the limitations associated with previous Akt inhibition strategies.

36)  Sarah A. Scott, "Regulation of Phospholipase D Activity and Phosphatidic Acid Production Following Purinergic (P2Y6) Receptor Stimulation," Yun Xiang, Thomas P. Mathews, Hyekyung Cho Plumley, David S. Myers, Michelle D. Armstrong, Keri A. Tallman, Matthew C. O’Reilly, Craig W. Lindsley, H. Alex Brown

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor tyrosine kinase and G-protein coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y6 receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y6 receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation, however when PLD is blocked the cell compensates by increasing PA production from other sources. We further delineate the P2Y6 signaling pathway showing that PLCβ3, PLCδ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system which occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y6 receptor signaling pathway.

37)  Kelly M. Hines, "UPLC-IM-MS/MS Profiling of Metabolites in Human Breast Cancer Tissues," Billy R. Ballard, Dana R. Marshall, John A. McLean

Breast cancer is well-known to have broad impacts on cellular metabolism and aberrant metabolism in breast cancer tumors has been widely studied by both targeted and untargeted metabolomic analyses to characterize the affected metabolic pathways. Contemporary techniques for such studies include nuclear magnetic resonance (NMR) of tissue extracts or cell lysates, with magic angle spinning (MAS) becoming particularly beneficial to the direct analysis of whole tissues, as well as liquid and gas chromatography-mass spectrometry (LC-MS, GC-MS, respectively). In this work, we utilize ultra-performance liquid chromatography (UPLC) in tandem with ion mobility-mass spectrometry/mass spectrometry (IM-MS/MS), which provides chromatographic, structural, mass and fragmentation information, to characterize the differential metabolome associated with cancer, as demonstrated with human breast cancer tumor and normal breast tissues. Homogenized tumor (n=3) and grossly normal non-involved breast tissues (n=3) were extracted to isolate polar and nonpolar metabolites, and the respective extracts were subsequently analyzed by UPLC-IM-MS/MS. To identify differences in the metabolite profiles of normal and tumor breast tissues, orthogonal projection to latent structures-discriminant analysis (OPLS-DA) was performed on the data. Statistically significant features were validated with fragmentation data from the MS/MS portion of the analysis. Identified features included saccharides, endogenous antioxidants, and a metal-containing porphyrin.

38)  David S. Myers, "A Novel Form of Cross-Talk Between Glycerophosholipid and Sterol Pathways Identified in Primary Macrophages," Ronald C. Bruntz, Stephen B. Milne, Pavlina T. Ivanova, Sarah A. Scott, Darren Engers, Craig Lindsley, and H. Alex Brown

Primary bone marrow derived macrophages are used to elucidate lipid changes under physiologically relevant inflammatory conditions using a paradigm of TLR4 priming ahead of challenge with ATP to target P2X receptors. Responses were diverse across lipid classes but include both phosphatidic acid (PA) production under ATP stimulation and elevation of several cholesterol biosynthesis intermediates. Subsequently, a dual isoform inhibitor of phospholipase D (PLD) was used to specifically examine downstream effects on lipids, resulting in an unexpected increase in 7-dehydrocholesterol (7DHC) levels, but only when no stimulation was applied. Experiments employing PA rescue and silencing of PLD1 and PLD2 isoforms conducted in U87-MG cells, which are also monocyte derived, collectively suggest that increased 7DHC response specifically occurs due to inhibition of the production of PA by PLD. This novel crosstalk between glycerophospholipid and sterol pathways is interesting in light of the 7DHC’s role upstream of both cholesterol and vitamin D synthesis.

39)  Kristin Droege, “Characterization of 5-Lipoxygenase and 5-Lipoxygenase Activating Protein Complexes in Leukotriene Biosynthesis,” Mary Keithly, Richard Armstrong

Leukotrienes are potent lipid mediators of inflammation. The upregulation of leukotrienes has been associated with several chronic inflammatory diseases, such as asthma, atherosclerosis, and rhinosinusitis rhinitis. An essential step in leukotriene production is the oxidation of arachidonic acid by 5-lipoxygenase (5-LOX) in complex with 5-lipoxygenase activating protein (FLAP). Because of their essential role in leukotriene biosynthesis, these enzymes have been identified as therapeutic targets. A structural understanding of 5-LOX and FLAP interaction will provide information that is essential in the development of novel therapeutics that are specific for the 5-LOX/FLAP complex. This project will detect 5-LOX and FLAP binding sites via hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS is a highly sensitive method that utilizes the exchange rate of hydrogens on the amide backbone of the protein to detect conformational changes and interactions with substrates and other proteins. 5-LOX and FLAP were expressed and purified. The activity of 5-LOX was determined and the identification of peptides upon protein digestion is being optimized for both proteins. Currently, 91% of 5-LOX sequence and 51% of FLAP sequence have been identified. This information will be used in HDX-MS experiments to locate the binding interactions of 5-LOX and FLAP

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

HKD2 and HKE2 are two cyclic hemiketal eicosanoids, isolated by Schneider and coworkers in 2011. These compounds were the non-enzymatic rearrangement products of a diendoperoxide that results from the treatment of 5-LOX product 5(S)-HETE with COX-2. Furthermore, these hemiketals were shown to stimulate angiogenesis of vascular endothelial cells, providing evidence for biological significance of these compounds in inflammatory sites involving co-expression of COX-2 and 5-LOX. Unfortunately, isolation of these natural products is very low, and little else is known about their biological activities. Total synthesis provides an attractive option for further investigation into the interesting biological properties of the hemiketals. Presented herein is the progress towards the total synthesis of HKE2 and HKD2, including a metal-catalyzed oxidative cyclization to form the key complex hemiketal moiety.

41)  Ghazal Hariri, “Sequential Targeted Delivery of Nanoparticle Encapsulated Microtubule and Topoisomerase Inhibitors in a Murine Lung Cancer Model,” Aaron D. Edwards, Tyler B. Merrill, Joshua M. Greenbaum, Alice E. van der Ende, and Eva Harth

Many conventional chemotherapy protocols for the treatment of cancer produce side effects that limit biological efficacy and compromise patient outcomes. Development of targeted drug delivery systems have allowed cytotoxic chemotherapeutics to be administered selectively to malignant tumors while sparing healthy tissues from undesirable side effects, however, solubilization of hydrophobic drugs and tailoring of drug dosages remains a challenge. Consequently, ‘nanosponge’ drug carriers developed in our lab have been found suitable due to their degradable polyester-based nano-network, consisting of a cross-linked 3-dimensional scaffold, which can be synthesized in different sizes, network densities and can be functionalized with tumor targeting units and imaging reagents to enable targeting and visualization in vivo. Hydrophobic drugs can be loaded into these nanosponges using a nanosolublization method, producing readily injectable formulations with linear drug release profiles. In this study, we sought to use the Tax-interacting protein 1 (TIP-1) targeting peptide (HVGGSSV) to guide the nanosponge delivery system to lung tumors treated with ionizing radiation in a murine lung cancer model. The potential of sequential combination drug delivery using these targeted nanosponges is investigated using mitotic inhibitors and topoisomerase I inhibitors. Paclitaxel, a mitotic inhibitor, has shown promise as a chemotherapeutic alone and in combination with other drugs such as camptothecin, a topoisomerase inhibitor, in the treatment of a variety of cancers. Despite its potency, the use of camptothecin in the clinic has been limited greatly due to its poor water solubility, systemic toxicity and side effects. Drug delivery carriers that can release highly hydrophobic small molecules such as paclitaxel and camptothecin in a targeted and controlled fashion exhibit particular promise for combination and metronomic chemotherapy, as they enable greater tailoring of drug dosages, reduced systemic toxicity and enhanced bioavailability to tumor tissues. Results from this study show that sequential paclitaxel → camptothecin chemotherapy exhibited a ‘priming’ effect on lung cancer cells that enhanced camptothecin’s effect on cells when given second. Nanosponge functionalization with targeting and imaging agents, sustained drug release profiles, and improved water solubility make them ideal drug carriers for metronomic chemotherapy. Furthermore, tumor-specific targeting of nanosponges to radiation-induced receptors enhanced therapeutic efficacy and lowered systemic toxicity.

42)  J. Rafael Montenegro-Burke, "Combined Utility of Ultra Performance Liquid Chromatography (UPLC) and Supercritical Fluid Chromatography (SFC) for Biological Samples," Cody R. Goodwin, Brian Bachmann, John A. McLean

Supercritical Fluid Chromatography (SFC) possesses some advantages over Ultra Performance Liquid Chromatography (UPLC), such as speed of analysis and the capability of reproducible normal-phase separations. However, SFC has been utilized mostly by the pharmaceutical industry with known compounds where screening of different mobile and stationary phase combinations is possible to achieve optimal separation and coverage. This is not the case with biological type samples, where the composition of analytes is largely unknown. Since reversed-phase (such as UPLC) separation is the gold-standard for complex sample analysis, an orthogonal separation technique such as SFC is promising. The use of multiple separation techniques in connection with one another is particularly attractive for discovery driven files, such as natural products discovery. In order to analyze very complex samples with a high diversity of molecular classes such as bacterial culture extracts, powerful analytical tools are required. The tunability of the stationary-mobile phase interactions in SFC allows for polar separations and the speed of analysis makes this technique very efficient, increasing throughput and duty cycle, as compared to UPLC. High-resolution mass spectrometry has the capability of accurate mass detection. Coupled to ion mobility (IM-MS), which separates ions in the gas-phase by their size-to-charge ratio, isobaric molecules may be separated by both size and mass. This added dimension of separation increases the peak capacity of the analysis allowing untargeted fragmentation for identification purposes. In this study a total of 1189 peaks were detected in a complex bacterial extract using both SFC-IM-MS and UPLC-IM-MS, at 15,000 mass resolution, where ~90% of those peaks were detected with SFC with different stationary phases. In this study, we demonstrate the versatility and high coverage of SFC when coupled to IM-MS (UPC2 and Synapt G2-S from Waters, Milford, MA) for comprehensive analysis of bacterial cocultures. We also demonstrate the orthogonality of classic reversed-phase chromatography and supercritical fluid chromatography.

43)  Jonathan E. Hempel, "A novel small molecule hedgehog inhibitor defines a new role of cAMP microdomain in Gli trafficking," Charles H. Williams, Jijun Hao, Audrey Y. Frist, Michelle M. Williams, Yanfeng Li, Li Zhao, Jonathan T. Fleming, Gary A. Sulikowski, Michael K. Cooper, Chin Chiang, Charles C. Hong

The ubiquitous secondary messenger cyclic adenosine monophosphate (cAMP) controls myriad cellular processes, and its functional specification within the cell requires the maintenance of subcellular signaling microdomains. Compartmentalization of cAMP signaling plays important roles in many species and its signaling has been implicated in numerous human diseases such as cancer, neurodegenerative disorders, COPD, heart failure, and autoimmune disorders. The hedgehog (Hh) pathway relies on cAMP concentrations within the primary cilium for signal transduction, and constitutive activation through oncogenic mutations in the transmembrane receptor Smoothened (Smo) have been implicated in basal cell carcinoma and medulloblastomas. Currently marketed therapeutics target Smo, but Hh oncogenic mutations further downstream can render these treatments ineffective. Therefore, identifying inhibitors of downstream Hh components remains essential for treatment of these deadly cancers. Despite much research focusing on further understanding compartmentalization of cAMP signaling, few reagents are available for microdomain-selective modulation. Further, the ability to minimize global cellular cAMP accumulation holds promise in therapeutic design considering undesirable side effects of current therapies are linked to unrestrained cAMP increases. To this end, we have discovered the first selective small molecule cAMP microdomain modulator termed eggmanone (EGM). Through a high-content screen for embryonic zebrafish developmental perturbation, we identified EGM as a highly selective and reproducible inducer of the Hh-null phenotype which was confirmed by its inhibition of Hh signaling in an Hh mouse reporter cell line. Utilizing a computational algorithm for small molecule target identification, we identified the phosphodiesterase family as EGM’s likely target and further narrowed this family to the cAMP-specific PDE4. Most importantly, EGM exerts its hedgehog inhibition by increasing cAMP concentration only in the primary cilium, in contrast to canonical PDE4 inhibitors which modulate global cellular cAMP. This fact presents the tantalizing application of cAMP microdomain-selective PDE4 inhibitors as important Hh-dependent cancer therapeutics with a minimized side-effect profile.

44)  David Earl, "Biosynthetic Engineering of Apoptoldin and Ammocidin, Selective Inducers of Cell Death," Gary Sulikowski, Brian Bachmman

Apoptolidin and ammocidin are selectively, cytotoxic macrolides produced by members of the gram-positive bacteria genus Amycolatopsis. Remarkably, these compounds are low nanomolar inhibitors of melanomas and gliablastomas, two cancers for which no viable therapeutic interventions exist. Further these natural products do not inhibit proliferation of non-transformed cells. By combining biosynthetic and synthetic approaches, we have been able to determine critical structure activity relationships and have developed a suite of probe compounds to study the molecular underpinnings of this remarkable selectivity. Currently, we are studying the mode of cell death induced by apoptoldin and ammocidin and undertaking a multi-pronged approach to identify the molecular target by chemical proteomics, gene knockout libraries, and evaluation of cell signaling node activation in primary tumor samples.

45)  Nichole M. Lareau, "Structural Analysis of Carbohydrates in the Gas Phase Using Ion Mobility-Mass Spectrometry (IM-MS)," Jody C. May, and John A. McLean

Carbohydrates are a structurally diverse class of biological molecules implicated with diseases such as Alzheimer’s Disease, obesity, and several forms of cancer. Analytical limitations in the analysis of carbohydrates arise from the low natural abundance and extensive level of structural heterogeneity within the biomolecular class. Specific challenges in studying carbohydrates by mass spectrometry (MS) stem from a preponderance of isobaric species including epimers, structural, and positional isomers. 2D structural separations based on size-to-charge (IM) and mass-to-charge (MS) ratios potentially differentiate isomers using the resulting collision cross sections (CCS) values obtained by IM-MS. CCS values describe the apparent surface area of the molecule in the gas phase, which in turn correlates to the conformation of the molecule. Here, a CCS database was created to describe the 2D-IM-MS space corresponding to the gas phase conformational occupancy of carbohydrate molecules in order to facilitate the structural interpretation of carbohydrates from IM-MS data. This report presents a compilation of over 100 individual carbohydrate CCS values resulting from more than 1100 measurements in nitrogen drift gas and just under 200 CCS values resulting from approximately 3500 measurements in helium drift gas covering a broad range of conformational space within the region corresponding to carbohydrate molecules.

46)  Sarah C. Shuck, "Chemical Characterization of Protein Modification by Base Propenal," James J. Galligan, Orrette R. Wauchope, Philip J. Kingsley, Kristie L. Rose, Lawrence J. Marnett

Exposure to endogenous and exogenous agents produces reactive oxidants that induce lipid and DNA peroxidation forming malondialdehyde and base propenal, respectively. These α,β unsaturated aldehydes modify protein and form DNA adducts including M1dG and OPdA. Nuclear formation of base propenal makes it a likely source for nuclear protein modification and a factor for influencing DNA replication and repair in response to oxidative stress. To investigate base propenal (specifically adenine propenal) modification of protein, we chemically characterized its reaction with N-α-acetyllysine and purified human serum albumin. N-α-acetyllysine reaction with base propenal formed two primary products, a lysine-lysine cross-link and an oxopropenyllysine product. HPLC and spectrometric analyses demonstrate as N-α-acetyllysine concentration increases, adenine propenal is depleted and the lysine-lysine cross-link is formed, representing 90% of the starting material. Oxopropenylation was less dependent on N-α-acetyllysine concentration and represented 10% of starting material. The formation of both the oxopropenyllysine product and the lysine-lysine cross-link is hypothesized to form through an amino-enemine intermediate, which we have characterized using mass spectrometry and NMR. Adenine propenal modification of human serum albumin was examined using proteomics-based mass spectrometry, revealing oxopropenylation on 32% of lysine residues. Protein cross-links were also detected. To study nuclear protein modification, colon carcinoma cells were treated with synthesized adenine propenal, nuclear proteins extracted, enriched with biotin hydrazide and analyzed with streptavidin detection. Adenine propenal was found to increase nuclear protein modification. These studies have comprehensively characterized the chemical modification of N-α-acetyllysine as well as human serum albumin by adenine propenal and have demonstrated nuclear protein modification by this reactive electrophile.

47)  Christopher D. Aluise, "Pin1 is a target for oxidative modification by the lipid electrophile 4-hydroxynonenal," Kristie Rose, Michelle D. Reyzer, Mariana Boiani, Keri A. Tallman, Ned Porter, Lawrence J. Marnett

Oxidation of membrane phospholipids is associated with inflammation and cancer. Phospholipid peroxidation results in the production of reactive electrophiles that adduct proteins and modulate their function. 4-Hydroxynonenal (HNE), a common product of oxidative damage to lipids, adducts proteins at exposed Cys, His, or Lys residues. Here, we provide evidence that peptidyl-prolyl cis/trans isomerase A1 (Pin1), an enzyme that catalyzes the conversion of pSer/pThr-Pro moieties in signaling proteins from cis to trans, is highly susceptible to HNE-modification. Incubation of purified Pin1 with HNE followed by MALDI-TOF/TOF mass spectrometry revealed that the most reactive amino acids are His-157 and Cys-113, both of which are active site residues. Pin1 was adducted in MDA-MB-231 breast cancer cells treated with 8-alkynyl-HNE as judged by click chemistry conjugation with biotin followed by streptavidin-based pulldown and western blotting with anti-Pin1 antibody. Furthermore, a Cys-113-HNE Michael adduct was detected in cells treated with HNE, while other modifications, including His-157 adduction, were not detected. siRNA knockdown of Pin1 in MDA-MB-231 cells partially protected the cells from HNE-induced toxicity. Recent studies indicate that Pin1 is an important molecular target for the chemopreventive effects of green tea polyphenols. The present study establishes that it is also a target for electrophilic modification by products of lipid peroxidation.

48)  Francisco G. Rodriguez, "Probing The Structural Dynamics Involved in Ca2+/Calmodulin-Dependent Protein Kinase II Activation by Spectroscopic Methods," Laurel Hoffman, Hassane S. Mchaourab

CaMKII is a ubiquitously expressed Ser/Thr kinase which is activated through binding of the Ca2+-sensing protein calmodulin (CaM). Being a regulator of highly diverse processes such as control of heart rate, memory formation, and energy expenditure, the development of CaMKII-specific drugs remains a challenge; however, rational design and optimization of small molecules that target CaMKII cannot ignore the highly dynamic nature of the protein. Although crystal structures of CaMKII to bound inhibitors and Ca2+/CaM are available, they are unable to define the amplitude of the dynamic equilibrium of the regulatory domain, which modulates ATP access to the enzyme active site and binds CaM. For robust analysis on the dynamic nature of CaMKII activation, we explored the hypothesis that binding of CaM and ATP to the regulatory domain of CaMKII disrupts the regulatory domain equilibrium and induces conformational rearrangements in the catalytic core of the kinase. Specifically, the active site is relieved of steric hindrance upon CaM binding by dislodging the auto-inhibitory helix from the kinase core and pulling the CaM-binding segment away from the active site. This rearrangement facilitates ATP binding, in turn permitting the lobes of the kinase core to rotate towards one another in an active conformation. Toward this end, spin probes were placed at specific positions in the regulatory domain and in the kinase core for distance measurements by Double Electron-Electron Resonance (DEER), a pulsed EPR technique. We measured distances between the regulatory domain and the kinase core and within the kinase core in apo, +ATP, and +CaM conditions. Our findings demonstrate 1) a restructuring of the autoinhibitory helix upon CaM binding, 2) a change in the dynamic equilibrium of the CaM-binding segment from a docked to a flexible conformation due to ATP binding, and 3) shorter distances between the catalytic lobes after addition of ATP, in concordance with a closed kinase conformation.

49)  Hubert Muchalski, "Kinetic Isotope Effect of Deuterium Reinforced 7-Dehydrocholesterol in Tocopherol Mediated Free Radical Chain Oxidation," Libin Xu, Ned Porter

Sterols are particularly prone to undergo radical chain oxidation, and evidence suggests that this process, known as lipid peroxidation, occurs in vivo under a variety of conditions that are the result of an oxidative stress. 7-Dehydrocholesterol (7-DHC), the immediate biosynthetic precursor of cholesterol, was found to be one of the most oxidizable lipids known. Also, preliminary computational data suggest that tunneling may be involved in the H-atom transfer reaction of 7-DHC. This work provides experimental support for this hypothesis through the total synthesis of deuterium-reinforced 7-dehydrocholesterol and examination of its kinetic isotope effect in the tocopherol-mediated for H-atom transfer reactions.

50)  Daniel Sprague, "Highly Stereoselective Synthesis of anti α-Substituted α,β-Diamino Acid Derivatives via Chiral Proton Catalysis" Anand Singh, Jeffrey N. Johnston

α,β-Diamino acids are a nonproteinogenic group of chiral, non-racemic 1,2-diamines which exist in nature, either in their free form or as motifs within complex molecules. These atypical amino acids have garnered interest in biochemistry and drug discovery due to their ability to modify the physical and chemical properties of peptides upon incorporation. α,α-Disubstituted α-amino acid and α,α-disubstituted α,β-diamino acid residues, subsets of α,β-diamino acids, can impart resistance towards chemical and enzymatic degradation in peptides as well as induce helix formation. The synthesis of such residues is a sought after transformation due to the difficulty in the stereoselective production of a fully substituted (quaternary) carbon center. We hoped to utilize the asymmetric aza-Henry reaction to produce these products. Herein, we present a chiral Bis(AMidine) (BAM) catalyzed addition of α-substituted nitroacetates to aryl aldimines, resulting in differentially protected α,β-diamino acid derivatives, in good yields with excellent diastereo- and enantioselectivity (up to >20:1 d.r. and 99% e.e.). These products can be unmasked in a sequential fashion for further regioselective transformations. Additionally, we show that minor changes in the catalyst structure reverses diastereoselectivity while maintaining high levels of enantioselection.

51)  William Beavers, "Analysis of Protein Adduction by Lipid Electrophiles Generated Endogenously from ω-Alkynyl Linoleic Acid," Kristie Rose, Keri Tallman, Jing Zhu, Stephen Milne, Michelle Armstrong, David Myers, Bing Zhang, Ned Porter, Alex Brown, Lawrence Marnett

Linoleic acid (LA) is a precursor to many inflammatory signaling molecules. LA can be oxidized by both cyclooxygenases and lipoxygenases to hydroperoxy-octadecadienoic acids. Further metabolism can produce a range of α,β-unsaturated carbonyls capable of reacting with nucleophilic amino acids of proteins, resulting in a change of protein function. High levels of oxidized linoleic acid products have been measured in atherosclerotic lesions. To better understand LA metabolism in cells, we have synthesized ω-alkynyl linoleic acid (ωaLA), which was incorporated into the lipid bilayer of RAW264.7 mouse macrophages, and released following activation with ATP and Kdo2 lipid A. Click chemistry and protein enrichment enabled identification of proteins adducted by metabolism generated electrophiles. Stable isotope labeling of amino acids in cell culture (SILAC) was used to quantify differences in protein adduction between activated an unactivated cells.

52)  Cheryl Law, "Protein Stability of Voltage-gated Potassium Channel KCNQ1," Dungeng Peng, Charles R. Sanders

Quantitatively studying thermodynamics and folding of membrane proteins has been difficult over the years due to the inability to obtain sufficient quantities of pure folded protein, aggregation of the unfolded protein, reversibility, and other difficulties. However, difficulty is balanced by importance, as protein stability closely relates to physiological function, misfolding, and disease. In this study, we examine the unfolding of the voltage-sensing domain of a potassium-gated channel KCNQ1. In cardiac cells, KCNQ1 is responsible for the slow-delayed rectifying current of potassium ions and repolarization of the cell, leading to decreasing the action potential within the cardiac cell. Several mutations in KCNQ1 lead to changes in function of the channel and can cause Long QT syndrome (LQTS). Our lab has shown using NMR spectroscopy that the voltage-sensing domain of KCNQ1 is well folded when purified from E. coli cells and reconstituted into the LMPG micelles. In this study, we monitor unfolding using circular dichroism in the presence of the denaturant guanidine hydrochloride (GuHCl). Future direction of this study will be to determine reversibility of the unfolded KCNQ1 WT, as well as determine the stability of mutants known to be associated with LQTS.

53)  Sarah M. Stow, "Structure Prediction of Small Molecules From Ion Mobility Measurements Obtained Under Different Operating Conditions," Jay G. Forsythe, Cody R. Goodwin, Jody C. May, David M. Hercules, Brian O. Bachmann, Terry P. Lybrand, John A. McLean

Ion mobility-mass spectrometry (IM-MS) allows the separation of ionized molecules based on their structural properties such as size and shape, in addition to their mass-to-charge ratio. The drift time data that is obtained from the IM-MS experiment can be used to calculate the collision cross section (CCS) of the ionized molecule, which is an intrinsic ion property that is representative of the ion’s gas phase conformation. Studying the conformational landscape of these molecules computationally provides further insight into the possible structures that these CCS values represent. Differences in the experimental operation of different IM-MS instruments play a major role in the calculation of collision cross section values. For example, electrostatic vs. electrodynamic field drift tubes require different methods for CCS determination. Specifically, an electrostatic field instrument can use gas phase kinetic theory to determine CCS values, while electrodynamic drift tubes require calibration standards. Also, the use of helium vs. nitrogen as a drift gas results in molecules transversing the drift tube at different rates due to size and polarizability differences for those drift gases, which lead to different CCS values for the same molecule. Although theoretical methods that consider the drift gas do exist to calculate CCS values, they are very computationally expensive due to the consideration of electronic structure. In this report, polyurethane precursor: 4,4’-methylenedianiline and natural products: valiomycin (cyclic peptide) and erythromycin (macrolide) have been used to study the differences in structure prediction for these differing instrument parameters. CCS values were determined from three instrument platforms: helium electrostatic field, nitrogen electrostatic field, and nitrogen electrodynamic field. The conformational space of these compounds was also sampled computationally and a theoretical CCS was determined for each generated conformation. The experimental and computational overlap was then examined for each of the three platforms. Differences in predicted structure representatives between the three methods were also examined.

54)  Connor R. Lamberson, "Isotope Effects of Deuterium Reinforced Polyunsaturated Fatty Acids in Tocopherol Mediated Free Radical Chain Oxidations," Libin Xu, Hubert Muchalski, Catherine F. Clarke, Mikail S. Shchepinov, Ned A. Porter

Oxygen is one of the most essential elements for metabolism and energy production for the large majority of life forms on Earth. Despite the life-sustaining properties a so called ‘Oxygen Paradox’ exists, where some reactions involving oxygen can give rise to reactive oxygen species (ROS) which subsequently lead to substantial damages in cells, tissues, and organs. Autoxidation of polyunsaturated fatty acids (PUFAs) and sterols is one of the consequences of ROS generation. This work describes kinetic isotope effects (KIE) experienced by deuterium reinforced PUFAs during autoxidation in the presence of α-Tocopherol (α-TOH). The values reported herein exceed the “classical’ range (<7), suggesting that hydrogen atom tunneling may be occurring during peroxidation of PUFAs.

55)  Frank Couch, "ATR Phosphorylates SMARCAL1 During Replication Stress to Prevent Aberrant Fork Processing," Carol E. Bansbach, Jessica W. Luzwick, Gloria G. Glick, Rémy Bétous, Clinton M. Carroll, Sung Yun Jung, Jun Qin, David Cortez

The DNA damage response kinase Ataxia-telangiectasia and Rad3-related (ATR) coordinates much of the cellular response to replication stress. The exact mechanisms by which ATR regulates DNA synthesis in conditions of replication stress are largely unknown, but this activity is critical for the viability and proliferation of tumor cells making ATR a potential therapeutic target. Here we use a selective ATR inhibitor to demonstrate that acute inhibition of ATR kinase activity disrupts the timing of replication initiation, slows replication elongation rates, induces stalled forks to collapse, and yields rapid cell lethality. In the absence of ATR function, nucleases cleave the stalled fork yielding double-strand breaks (DSBs) and excess single-stranded template and newly synthesized DNA strands. The SMARCAL1 DNA translocase is required for this aberrant fork processing. SMARCAL1 catalyzes DNA annealing and fork regression at damaged forks, but too much of its activity interferes with DNA replication and causes fork damage. We identify a conserved SMARCAL1 residue as a damage-induced ATR phosphorylation site. Phosphorylation of this site limits its activity thereby preventing aberrant fork processing. Thus, phosphorylation of SMARCAL1 is one mechanism by which ATR promotes the completion of DNA replication and maintains genome integrity.

56)  Brittney S. Bates, "Marineosin A: Developing a Natural Product into a Tool Compound," Leslie N. Aldrich, Cynthia B. Berry, Leah C. Konkol, Joseph D. Panarese, Miranda So, Craig W. Lindsley

Cancer is a heterogenous disease that can rapidly mutate and become resistant to therapy; therefore, cancer drug discovery is difficult and complex. The development of probe molecules that can be used to validate druggable cancer targets is a critical part of the cancer drug discovery process. Because the majority of cancer drugs on the market today are derived from natural products, we propose a natural product medicinal chemistry plan to this end. First, we intend to complete the total synthesis of marineosin A and determine the minimal pharmacophore required for biological activity. We will next make unnatural analogs from this core structure and develop structure activity relationships (SAR) to create potent molecules. Finally, we will use the most promising tool compound in affinity purification to determine the target of marineosin A. When successfully completed, this work has the potential to elucidate a new target for cancer drug design. In the event that the marineosin A target is not a new target, useful correlations between these molecules and current cancer research will still be obtained. Additionally, this research will add to the current knowledge of the prodigiosins, a structurally related group of molecules, and has the potential to produce compounds that are antimalarial, immunosuppressive, and/or antibacterial, as have been found with the prodigiosins.

57)  Amy D. Millsap, "Translesion Synthesis Past C8 and N2 Deoxyguanosine Adducts of the Dietary Mutagen 2-amino-3-methylimidizo-[4,5-ʄ]-quinoline (IQ) by Human DNA Polymerases ζ and hRev1," Carmelo J. Rizzo

The 2-amino-3-methylimidizo-[4,5-ʄ]-quinoline (IQ) is a potent dietary mutagen which induces two-base deletions in the Ames assay. IQ and its analogs react with deoxyguanosine forming adducts at the C8 and N2 positions. To date, human and bacterial polymerases have shown differential processing abilities in bypass and extension studies of IQ at G1 and G3 of the Nar1 recognition site (5'G1G2CG3CC-3'), a hotspot for two base deletions. Human DNA polymerase ζ (pol ζ), an eukaryotic translesion synthesis (TLS) polymerase, is an error prone polymerase concerned with the bypass of replication fork blocking lesions. We hypothesize that the pol ζ and hRev1 are involved in the replication of the bulky IQ adduct and will differentially repair C8IQ and N2IQ adducts at the G1 versus G3 position of the Nar1 sequence. Preliminary results indicate pol ζ will not insert a base opposite the N2IQ or C8IQ-dG adducts at either the G1 or G3 positions. However, the human Y-family polymerase Rev1, a dCTP transferase, inserts dCTP opposite N2IQ adducts at the G1 and G3 positions. We found pol ζ extends with differing efficiencies from bases dT, dA, and dC opposite the C8IQ and N2IQ lesions at positions G1 and G3 in the Nar1 sequence. However, pol ζ extension is blocked when dG is opposite the IQ adduct. Finally, studies show that extension by pol ζ from both C8IQ and N2IQ adducts at the G3 position was more efficient in the presence of hRev1. Futher studies will examine extension by pol ζ and hRev1 for the IQ adducts at G1, as well as overall enzyme kinetic studies.

58)  Mariusz Butkiewicz, "Application of Quantitative Structure Activity Relationships Reveals Pathway Specific Inhibitors for ß-Hematin Crystallization in Plasmodium falciparum," Rebecca Sandlin, Kim Fong, David Wright, Jens Meiler

This research project is a collaborative effort between the laboratories of David Wright and Jens Meiler at Vanderbilt to guide experimental expertise with computational algorithms to find effective therapeutics against Malaria. GlaxoSmithKline and Novartis screened nearly 4 million compounds for activity against Plasmodium falciparum, a species of Plasmodium and the primary causative agent of human Malaria. The goal of this research project is an affordable in silico screening approach to sort and prioritize compounds according to their likelihood to interact with a specific target. An in-house diverse small molecule library (144,330 total compounds) with an initial hit rate of 0.4% (530/144,330) was used as a knowledge-base to establish Quantitative Structure Activity Relationship models. These models were applied to virtually screen the publically available GSK and Novartis databases of in vitro antimalarial compounds. The GSK in vitro antimalarials were obtained (13,229 compounds) and an experimental enrichment of ~40 was achieved for the compounds based on a concentration threshold of 70µM. These compounds were subsequently tested in the heme-speciation assay to confirm the perturbation of the hemozoin formation pathway. The scientific community now has access to an arsenal of thousands of compounds that are highly toxic to the parasite, but the process of developing these hits into robust lead compounds will be challenging. Identification of the molecular targets responsible for activity could aid in prioritization of these hits, while the lead optimization could be streamlined since the hit-target interactions could be directly studied, saving time, and money. While the experimental determination of targets is possible in some cases, it is an expensive and time-consuming process. An affordable in silico screening approach as proposed in this project would greatly benefit the efforts to find new chemo types and thus help mitigate drug resistance of the Malaria parasite.

 

 

 

 

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