Vanderbilt Institute of Chemical Biology


VICB Student
Research Symposium






2016 VICB Student Research Symposium

Wednesday, August 17, 8:00 am to 5:00 pm

Vanderbilt Student Life Center


Guest Speaker:


Squire Booker
Penn State University





Richard N. Armstrong Prize in Chemical Biology Award Winner:

Pedro Garcia


Oral Presentation Award Winners:

Kyle Floyd

Michelle Mitchener

Bart Roland


Poster Presentation Winners:

Synthetic Chemistry: Jamin Keith, Chris Fullenkamp

Thearpeutics and Translation: Linda Zhang, Westley Baurer

Molecular Discovery: Lisa Lojek, Orrette Wauchope

Systems Analysis: Alexandra Trevisan, Allison Galassie


iPad Raffle Winner




For additional information, contact the VICB office at 615-322-0907.



Oral Presentation & Poster Abstracts


Oral Presentation Abstracts


1.  "MALDI-TOF imaging mass spectrometry reveals the regulation of building blocks for bacterial cities," Kyle A. Floyd, Allison R. Eberly, Jessica L. Moore, Richard M. Caprioli, Maria Hadjifrangiskou


Most bacterial species have evolved to exhibit multicellular behavior, forming large multicellular communities, known as biofilms. Life in a biofilm confers many "amenities" to the bacteria, including protection from environmental stressors, the ability to exchange genetic material with neighbors, and the acquisition of nutrients under limiting conditions. Biofilm formation also allows for diversification of a genetically homogenous population, through the development of phenotypically distinct bacterial subpopulations resulting from variations in gene expression. Select subpopulations are impervious to antimicrobial treatment, and as a result biofilms are difficult to eradicate. Therefore, biofilm-associated infections pose a significant public health threat. An excellent example of a bacterial pathogen that forms biofilms as part of its pathogenic strategy is uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections (UTIs). UPEC can form biofilms on biotic surfaces within the urinary tract, as well as on abiotic surfaces such as indwelling urinary catheters. Catheter-associated UTIs (CAUTIs) have increased in incidence rate, and now account for ~30% of all hospital-acquired infections. Determining factors that are critical for biofilm formation and the development of subpopulations may provide insights into novel anti-biofilm strategies. In previous studies we used a MALDI-TOF imaging mass spectrometry (IMS) approach to dissect the spatial proteome of intact surface-associated UPEC biofilms, for the identification of subpopulations that contribute to biofilm formation and development. These studies were the first to demonstrate that MALDI-TOF IMS could distinguish biofilm subpopulations based upon protein expression. The same studies also revealed that expression of type 1 pili (fim) was unique to subpopulations comprising the air-exposed region of the biofilm. Type 1 pili mediate UPEC adherence to bladder epithelial cells, are key factors of biofilm infrastructure, and are essential for establishment of acute UTI. To test the hypothesis that oxygen regulates the expression of fim in UPEC, we analyzed fim expression under oxygen-deplete (anoxic) conditions and discovered that the absence of oxygen represses production of type 1 pili at the transcriptional and post-translational levels. Reduced ATP production during fermentation is not the likely reason of reduced fim transcription, given that other extracellular appendages that depend on energy for transport and assembly were produced during anoxic growth. Investigation of known transcriptional regulators acting at the level of the fim promoter indicated the fim recombinase FimE and the transcriptional regulator FNR both regulate transcription of fim during anoxic growth. Deletion of fimE altered the orientation of the phase-variable fim promoter to the transcription-competent state and restored FimA protein abundance under anoxic conditions. Deletion of the transcriptional regulator FNR led to up-regulation of the fimB recombinase, also re-positioning the fim promoter in a transcription-competent state. However, FNR also directly repressed fim operon transcription, as its deletion in a phase-locked strain led to high levels of fim transcription. These data suggest a model where under anoxic conditions FNR suppresses expression of the ON switching recombinase FimB, to allow the OFF switching recombinase FimE to function, thereby reducing fim expression. These data also suggest direct interactions of FNR with the fimS promoter under anoxic conditions, which perhaps serves to suppress fim expression in bacteria that have yet to switch OFF in order to prevent type 1 piliation in the absence of oxygen. 


2.  Synthesis of human milk trisaccharides and their effect on Group B Streptococcus," Dorothy L. Ackerman, Ryan S. Doster, Jennifer A. Gaddy, David M. Aronoff, Steven D. Townsend


Group B Streptococcus (GBS) is a leading cause of infant illness and mortality. As such, this pathogen serves as a point of interest in reducing the occurrence of both late and early onset infections, such as neonatal sepsis. Because GBS is a bacterial pathogen, its ability to form biofilms impacts its level of virulence. The mechanism of GBS acquisition is poorly understood, but transmission is known to occur in utero, during labor and delivery, or from other environmental sources. Some case reports suggest GBS can be transmitted from mother to infant via contaminated breast milk. This postulation poses a largely unexplored dichotomy between the protective benefits of breast milk and its potential as a vehicle of GBS transmission. Human milk oligosaccharides (HMOs), the third largest component of breast milk, are thought to have antiadhesive antimicrobial and immunomodulatory properties. We found that using a breast milk media shows promise for decreasing the growth of GBS. However, due to composition diversity across breast milk samples, it seemed most beneficial to study the effect of individual compounds. In order to probe what effects individual HMOs might have on GBS, we developed short, scalable syntheses of human milk trisaccharides. With these molecules in hand, we have explored their ability to significantly inhibit or promote GBS biofilm growth.


3.  William N. Beavers, Jessica L. Moore, Jeffrey M. Spraggins, Venkataraman Amarnath, Neal D. Hammer, Raymond L. Mernaugh, Sean S. Davies, L. Jackson Roberts II, Richard M. Caprioli, Eric P. Skaar


This abstract will be included in the "VICB Student Research Symposium Abstracts" hand-out on the day of the symposium.


4. "The nucleoside prodrug GS-5734 inhibits murine and human coronaviruses with intact proofreading and selects for resistance mutations in the nsp12 RNA-dependent RNA polymerase," Maria L. Agostini, Erica L. Andres, Everett Clinton Smith, Michelle M. Becker, James Brett Case, Joy Y. Feng, Robert Jordan, Adrian S. Ray, Tomas Cihlar, Richard L. Mackman, Ralph S. Baric, Mark R. Denison


The emergence of SARS-coronavirus (CoV) in 2002 and the continued circulation of MERS-CoV emphasize the capacity of CoVs to cause severe human disease and new zoonotic infections with pandemic potential. Despite the high mortality rates of these infections, no therapeutics or vaccines against any CoVs are currently available. GS-5734, a prodrug of an adenine C-nucleoside analog GS-441524 with known activity against filoviruses, is currently undergoing clinical testing for the treatment of Ebola virus infection. Here we demonstrate that GS-5734 is also highly active against -CoVs including wild-type murine hepatitis virus (MHV) (-2a), SARS-CoV (-2b), and MERS-CoV (-2c), and exhibits minimal cytotoxicity in vitro. Parent nucleoside GS-441524 also exhibits anti-CoV activity, but is less potent. In contrast to other nucleoside analogs and mutagens to which CoVs are resistant via the nsp14-ExoN proofreading exoribonuclease activity, GS-5734 and GS-441524 are active both against WT and ExoN(-) CoVs. MHV resistance to GS-441524 emerged slowly over 23 passages in cell culture but resulted in little to no fitness change relative to the WT virus. Resistant MHV population expressed two mutations in the "finger" domain of the MHV nsp12-RdRp at residues highly conserved across CoVs (F476, V553). When engineered into the isogenic MHV-A59 background, the mutations conferred resistance to both GS-441524 and GS-5734 at levels similar to the selected MHV population. Our results indicate that the anti-CoV mechanism of action of GS-5734 is RdRp-dependent, potentially through the incorporation of active nucleoside triphosphate into viral RNA. In summary, GS-5734 is broadly active against diverse CoVs irrespective of their ExoN proofreading activity and exhibits high in vitro barrier to resistance. These features suggest the potential of GS-5734 to act as an effective antiviral therapeutics against known and emerging coronaviruses.


5.  Kir4.1 (KCNJ10) inhibition with a newly developed antagonist induces diuresis in rats," Sujay K. Kharade, Haruto Kurata, Corey R. Hopkins and Jerod S. Denton


Inward rectifier potassium (Kir) channels play fundamental roles in diverse physiological processes, and in some cases, represent novel drug targets for human diseases. Outside of the nervous system, Kir4.1 (encoded by KCNJ10) is prominently expressed in the basolateral membrane of the thick ascending limb of Henle, distal convoluted tubule, and collecting duct of nephron. Kir4.1 can form homotetrameric channels or heterotetrameric channels with Kir5.1, although the most common channel subtype in the kidney is the heteromeric Kir4.1/5.1 form. Genetic ablation of Kir4.1 in mice or humans causes severe salt and water wasting resembling clinical phenotypes associated with diuretic use, suggesting that Kir4.1 might represent a novel diuretic target. Therefore, the aim of this study was to develop a selective small-molecule inhibitor of Kir4.1 with which to evaluate its therapeutic potential. From a high-throughput screen of ~ 75,000 compounds from the Vanderbilt Institute of Chemical Biology library, we identified an inhibitor, termed VU992, which inhibits homotetrameric Kir4.1 with an IC50 of 760 nM and heterotetrameric Kir4.1/5.1 with an IC50 of approximately 25 mM (33-fold selectivity). In addition, VU992 is at least 65-fold selective for Kir4.1 over other renal Kir channels Kir1.1 and Kir7.1. Lead optimization failed to identify analogs with improved potency toward Kir4.1. Finally, oral administration of VU992 dose-dependently increases urine output in volume-loaded, freely behaving rats. This study shows proof-of-concept that homotetrameric Kir4.1 might represent a molecular target for a novel class of diuretic for the treatment of hypertension. Its basolateral localization might offer advantages over other diuretic targets that are located on the luminal membrane of the nephron. Funding NIH 5R21 NS073097-01S1.


6.  "Directed evolution of a phospholipid flippase reveals mechanism of substrate backbone discrimination and a novel class of P4-ATPase sphingolipid substrates," Bartholomew P. Roland, Todd R. Graham


Phospholipid flippases of the type IV P-type ATPase family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known.  We have used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. Further, our examination of the glycerophospholipid/sphingolipid enzyme complex revealed the unexpected capacity for plasma membrane P4-ATPases in the yeast Saccharomyces cerevisiae to transport glycosphingolipids. This is the first demonstration of a glycosphingolipid P4-ATPase, and may have broad implications on cellular membrane biology and lipid homeostasis.


7.  "Nuclear Extracts Enzymatically Convert M1dG to 6-oxo-M1dG in Duplex DNA," Michelle M. Mitchener, Orrette R. Wauchope, Lawrence J. Marnett


Numerous studies correlate oxidative stress and inflammation with tumorigenesis. While the mechanism linking these processes remains to be fully elucidated, endogenously generated electrophiles, which can react with cellular macromolecules and alter their functions, are posited to play a role. Oxidative damage to polyunsaturated fatty acids and the deoxyribose ring of DNA gives rise to malondialdehyde (MDA) and base propenals, respectively. Both MDA and base propenals react with DNA in vitro to give primarily 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG). M1dG is present in significant amounts both in rodent and human genomic DNA isolated from various tissues, and adduct levels have been directly associated with exposure to oxidative stress.


M1dG results in base-pair substitutions and frameshift mutations, unless it is first removed from DNA. M1dG is repaired by nucleotide excision repair (NER) resulting in the release of a short fragment of single-stranded DNA that is subsequently digested to single nucleosides. Previous studies tracking the in vivo fate of free M1dG revealed that the adducted nucleoside is subject to oxidative metabolism by cytosolic xanthine oxidoreductase and aldehyde oxidase to 6-oxo-M1dG. Alternatively, we recently reported that M1dG adducts can be oxidized to 6-oxo-M1dG in nuclear DNA, at a rate faster than that of M1dG repair. In RAW264.7 macrophages, which have particularly high endogenous levels of M1dG, 6-oxo-M1dG was detected in nuclear but not mitochondrial DNA. The absence of 6-oxo-M1dG in mitochondria, despite higher M1dG levels in the mitochondria than in the nucleus, suggests this oxidative reaction is not likely the result of coincidental reaction of M1dG with reactive oxygen species.


In order to interrogate this observed activity, nuclear extracts from RAW264.7 macrophages were prepared and incubated with double-stranded oligonucleotides containing M1dG. Following incubation, the oligonucleotides were digested and analyzed via LC-MS/MS for the presence of 6-oxo-M1dG. Heat-denaturation or Proteinase K-treatment of the nuclear lysates ablated conversion, indicating that the oxidation is an enzyme-mediated process. We hypothesized that this enzyme might be an α-ketoglutarate-dependent dioxygenase, a class of iron-dependent enzymes that is responsible for the oxidation of various alkylated nucleic acids and/or amino acids. Indeed, we found that preincubation of nuclear lysates with EDTA or metals known to displace ferrous ion from proteins, resulted in a decrease in 6-oxo-M1dG generation. Preincubation with ferrous ion, but not ferric ion, or α-ketoglutarate increased the amount of 6-oxo-M1dG formed. Furthermore, nuclear lysates preincubated with either N-oxalylglycine, IOX1, or JIB-04, three structurally distinct α-ketoglutarate-dependent dioxygenase inhibitors, showed reduced 6-oxo-M1dG formation compared to their control counterparts.


These findings indicate the presence of a nuclear enzyme, conceivably a α-ketoglutarate-dependent dioxygenase, capable of converting M1dG to 6-oxo-M1dG in double-stranded DNA. Currently we are pursuing a variety of chemical biology techniques, including photoaffinity inhibitor probe capture and candidate siRNA knockdown, in attempt to identify the enzyme responsible for the generation of this novel adduct.


8.  Pedro M. Garcia-Barrantes – Awardee for Prize in Chemical Biology


"Development of novel mGlu1 positive allosteric modulators as chemical tools for target validation in schizophrenia," Pedro M. Garcia-Barrantes, Hyekyung P. Cho, Anna L. Blobaum, Colleen M. Niswender, P. Jeffrey Conn, Craig W. Lindsley


Schizophrenia is a highly prevalent neuropsychiatric disorder and a public health challenge due to its severity and early onset. Current available therapies alleviate positive symptoms, but not symptoms in other domains of the disease. Metabotropic glutamate receptor type 1 (mGlu1) is a G-protein coupled receptor from the group I of mGlus and is encoded by the GRM1 gene. Stimulation of the receptor leads to an increase in activity of phospholipase C, with subsequent mobilization of calcium and increase of intracellular calcium concentrations, favoring the activation of the NMDA receptor. mGlu1 is expressed predominantly in post-synaptic neurons from the hippocampus, hypothalamus, thalamus, amygdala, cerebellum, basal ganglia and spinal cord. The function of mGlu1 has been implicated in several neural processes such as nociception, synaptic plasticity, learning and memory. Hypofunction and deregulation of glutamate signaling has been established as a key feature in schizophrenia pathogenesis. Moreover, recent genetic studies showed that mutations in the GRM1 gene are enriched in patients with schizophrenia and bipolar disorders, and we have confirmed that a subset of these mutations decreased receptor response; supporting its modulatory role in neuropsychiatric disorders and suggesting that therapies targeting the receptor's function recovery might be beneficial in these patients. In order to test this hypothesis, it is necessary to have suitable chemical tools to achieve the activation of the receptor and perform target validation studies. Our first efforts in the development of novel mGlu1 positive allosteric modulators (PAM) started by repurposing a known mGlu4 PAM chemotype through a multidimensional iterative parallel synthesis approach and lead to the discovery of VU0483605, a novel, selective and submicromolar mGlu1 PAM.  However, this compound was not stable in plasma and displayed a very short half-life. After several iterations, we arrived to the more potent and plasma stable VU0486321, a compound with an interesting ago-PAM pharmacodynamic profile and halflife under one hour. Finally, efforts have been made towards the optimization of VU0486321 pharmacokinetic profile in order to increase its half-life. This campaign led to the discovery of VU6004909, an analog with a fluoro substitution that confers great selectivity for mGlu1 and VU0487351, an isoindolinone analog, with excellent plasma stability. Overall, these new compounds represent potent and selective chemical tools, with good CNS penetration and a balanced DMPK profile. We expect that these new chemicals probes will open a door to interrogate the biology of the receptor in in vitro and in vivo target validation studies, and allow the exploration of the role of the mGlu1 receptor in schizophrenia and other neuropsychiatric disorders.   



Poster Presentation Abstracts


1. "Directed evolution of a phospholipid flippase reveals mechanism of substrate backbone discrimination and a novel class of P4-ATPase sphingolipid substrates," Bartholomew P. Roland, Todd R. Graham


Phospholipid flippases of the type IV P-type ATPase family establish membrane asymmetry and play critical roles in vesicular transport, cell polarity, signal transduction, and neurologic development. All characterized P4-ATPases flip glycerophospholipids across the bilayer to the cytosolic leaflet of the membrane, but how these enzymes distinguish glycerophospholipids from sphingolipids is not known.  We have used a directed evolution approach to examine the molecular mechanisms through which P4-ATPases discriminate substrate backbone. A mutagenesis screen in the yeast Saccharomyces cerevisiae has identified several gain-of-function mutations in the P4-ATPase Dnf1 that facilitate the transport of a novel lipid substrate, sphingomyelin. Further, our examination of the glycerophospholipid/sphingolipid enzyme complex revealed the unexpected capacity for plasma membrane P4-ATPases in the yeast Saccharomyces cerevisiae to transport glycosphingolipids. This is the first demonstration of a glycosphingolipid P4-ATPase, and may have broad implications on cellular membrane biology and lipid homeostasis.


2. "The nucleoside prodrug GS-5734 inhibits murine and human coronaviruses with intact proofreading and selects for resistance mutations in the nsp12 RNA-dependent RNA polymerase," Maria L. Agostini, Erica L. Andres, Everett Clinton Smith, Michelle M. Becker, James Brett Case, Joy Y. Feng, Robert Jordan, Adrian S. Ray, Tomas Cihlar, Richard L. Mackman, Ralph S. Baric, Mark R. Denison


The emergence of SARS-coronavirus (CoV) in 2002 and the continued circulation of MERS-CoV emphasize the capacity of CoVs to cause severe human disease and new zoonotic infections with pandemic potential. Despite the high mortality rates of these infections, no therapeutics or vaccines against any CoVs are currently available. GS-5734, a prodrug of an adenine C-nucleoside analog GS-441524 with known activity against filoviruses, is currently undergoing clinical testing for the treatment of Ebola virus infection. Here we demonstrate that GS-5734 is also highly active against β-CoVs including wild-type murine hepatitis virus (MHV) (β-2a), SARS-CoV (β-2b), and MERS-CoV (β-2c), and exhibits minimal cytotoxicity in vitro. Parent nucleoside GS-441524 also exhibits anti-CoV activity, but is less potent. In contrast to other nucleoside analogs and mutagens to which CoVs are resistant via the nsp14-ExoN proofreading exoribonuclease activity, GS-5734 and GS-441524 are active both against WT and ExoN(-) CoVs. MHV resistance to GS-441524 emerged slowly over 23 passages in cell culture but resulted in little to no fitness change relative to the WT virus. Resistant MHV population expressed two mutations in the “finger” domain of the MHV nsp12-RdRp at residues highly conserved across CoVs (F476, V553). When engineered into the isogenic MHV-A59 background, the mutations conferred resistance to both GS-441524 and GS-5734 at levels similar to the selected MHV population. Our results indicate that the anti-CoV mechanism of action of GS-5734 is RdRp-dependent, potentially through the incorporation of active nucleoside triphosphate into viral RNA. In summary, GS-5734 is broadly active against diverse CoVs irrespective of their ExoN proofreading activity and exhibits high in vitro barrier to resistance. These features suggest the potential of GS-5734 to act as an effective antiviral therapeutics against known and emerging coronaviruses. 


3. "Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection," Aimee D. Wilde, Nicole E. Putnam, Michael D. Valentino, Zachery R. Lonergan, Scott A. Hinger, Michael S. Gilmore, Eric P. Skaar, James E. Cassat

Staphylococcus aureus is a remarkably flexible pathogen capable of infecting a wide range of organ systems in the human body. This flexibility requires an extensive range of metabolic and virulence programs in order to invade and replicate within diverse environments. The bone serves as one of the most common sites of invasive staphylococcal infection.  In this study, we characterize the genetic programs required to sustain invasive infection in a murine osteomyelitis model using transposon sequencing (TnSeq) analysis. TnSeq identified more than 200 genes important for invasive S. aureus osteomyelitis, two of which encode the bacterial two-component system, SrrAB. SrrAB is known to affect survival of S. aureus during hypoxic and nitrosative stress. Consistent with this finding, healthy bone is intrinsically hypoxic, and we discovered that oxygen levels in bone decrease during osteomyelitis. Moreover, the fitness of an srrAB mutant during osteomyelitis was significantly improved by depletion of neutrophils, whereas a defect of the host oxidative burst only partially restored fitness, suggesting that neutrophils contribute hypoxic stress in addition to nitrosative stress in vivo.   Furthermore, we discovered changes in staphylococcal virulence programs corresponding with oxygen limitation in vitro. Hypoxic growth results in significant increases in quorum sensing-dependent toxin production and associated increases in cytotoxicity towards mammalian cells. Conversely, aerobic growth limited quorum sensing and toxin production in an SrrAB dependent manner, suggesting a mechanism by which S. aureus can sense and respond to the variety of host organ systems it encounters. Our data elucidate the genetic programs required for invasive osteomyelitis and demonstrate bacterial hypoxic responses as critical determinants of the host-pathogen interaction. Future work will determine the mechanism of SrrAB signaling that leads to its downstream regulatory effects.


4. "Administration Of Gut Bacteria Expressing N-acyl Phosphatidylethanolamine Reduces Steatohepatitis In LDLR-/- Mice Fed A Western Die," Linda S. Zhang, Zhongyi Chen, Youmin Zhang, Lei Ding, Patricia G. Yancey, Arion Kennedy, MacRae F. Linton, Alyssa Hasty, Sean S. Davies

Background: The rise in obesity in the United States has led to a concomitant rise in prevalence of non-alcoholic fatty liver disease (NAFLD). The four stages of NAFLD include accumulation of triglyceride (hepatosteatosis), development of chronic inflammation (non-alcoholic steatohepatitis, NASH), fibrosis, and finally cirrhosis. Unlike wildtype C57BL6 mice, low density lipoprotein receptor (LDLR) -/- mouse fed a diet enriched in fat and cholesterol (Western Diet) progress to NASH and fibrotic stages of NAFLD. We showed that incorporating engineered bacteria expressing N-acyl phosphatidylethanolamine (NAPE) into the gut microbiota can inhibit development of obesity. NAPE is a precursor of N-acylethanolamines, which are bioactive lipids with anti-inflammatory functions. Here, we test the hypothesis that administering these NAPE-expressing bacteria inhibits development of NASH and fibrosis. Methods: NAPE-expressing E. coli Nissle 1917 (pNAPE-EcN, n=10), control Nissle 1917 (pEcN, n=10), or vehicle (veh, n=10) were given via drinking water to LDLR-/- mice fed a Western diet for 12 weeks. LDLR-/- mice fed a low fat diet (LFD) (n=10) were included for comparison. Results: pNAPE-EcN reduced adiposity by 26% compared with pEcN and veh (P<0.05). pNAPE-EcN also dramatically reduced hepatic triglyceride levels by 45% (p<0.05) and lipid droplet size, as well as the hepatic expressions of tissue necrosis factor α (TNFα, p<0.05), chemokine receptor 2 (CCR2, p<0.01), and tissue inhibitor of matrix metalloproteinase (TIMP1, p<0.05), consistent with reduced inflammation and fibrosis. Trichrome blue staining of liver sections further demonstrated reduced fibrosis. Because fatty liver is associated with atherosclerosis, we checked to see if pNAPE-EcN was able to reduce the development of atherosclerotic lesions. While serum cholesterol was reduced by 23% with pNAPE-EcN treatment (p<0.05), atherosclerotic lesion size in proximal or en face aortas only tended to be reduced (20%, 18.6%) but was not statistically significant. Conclusions: Our results demonstrate that incorporating therapeutically modified bacteria into the gut microbiota has potential to inhibit the development of NAFLD.


5. William N. Beavers, Jessica L. Moore, Jeffrey M. Spraggins, Venkataraman Amarnath, Neal D. Hammer, Raymond L. Mernaugh, Sean S. Davies, L. Jackson Roberts II, Richard M. Caprioli, Eric P. Skaar


This abstract will be included in the "VICB Student Research Symposium Abstracts" hand-out on the day of the symposium.


6. "Activation Of G Protein-Gated Inwardly-Rectifying Potassium (Girk) Channels As A Novel Therapeutic Target For Pain," Kristopher K. Abney, Carrie Jones, Sukhbir Mokha, Jerri Rook, Corey Hopkins, Dave Weaver

In the United States approximately 100 million Americans suffer from chronic or acute pain. Symptoms can manifest from a myriad of circumstances including post-operative surgical pain, sports injuries, chemotherapy-induced neuropathy, and diabetic neuropathy. The most common clinically prescribed way to treat pain involves the use of opioid analgesics.  However, side effects associated with opioids are detrimental; involving a decrease in quality of life resulting from opioid induced constipation, drug tolerance, and most notably, addiction. Opioid receptors (ORs) are G Protein-Coupled Receptors (GPCR) that signal through Gαi/o heterotrimeric G proteins. ORs couple to a variety of downstream effectors that mediate their analgesic efficacy and side effects. G protein-gated inwardly-rectifying potassium (GIRK) channels are one such effector that couple to ORs and produce analgesia. These GIRK channels play an important role in controlling resting membrane potential and cellular excitability. GIRK channels form tetrameric complexes that are comprised of four closely related subunits: GIRK1-GIRK4. Neurons along the main pain pathway, the spinothalamic tract, express GIRK1/2 in the same regions as ORs. Furthermore, OR-mediated activation GIRKs decreases the excitability, which ultimately inhibits excitatory activities in both afferent neurons and second order neurons that carry noxious stimuli to higher cortical areas of the brain. GIRK’s contribution to nociception is demonstrated in GIRK1 and GIRK2 knock-out mice, where both mice show attenuated responses to morphine, an OR agonist. Recently, our lab developed a series of potent and selective modulators of GIRK1-containing channels. Previous studies have shown in vivo administration of some of these novel compounds decreases seizure-like activity in murine models of epilepsy, and also reduces anxiety-like behavior in murine models of anxiety. We hypothesize that further development of potent and selective GIRK1/2 activators may provide analgesia through a novel mechanism and that GIRK-induced analgesia may be free from the side effects associated with opioids.


7. The IsdG family of heme oxygenases is conserved across Kingdoms, Lisa J. Lojek, Allison J. Farrand, Jennifer H. Wisecaver, Crysten Blaby-Haas, Sabeeha Merchant, Antonis Rokas, Eric P. Skaar


Heme degradation is a process that is conserved in all Kingdoms and facilitates the catabolism of heme to unique small molecule products and free iron. Two primary families of heme oxygenases have been identified; the HO-1 family present in eukaryotes and bacteria, and the IsdG family identified only in bacteria. Enzymes from both families are required for full virulence in pathogenic species of bacteria and heme oxygenases are nearly ubiquitously found in eukaryotes. Though IsdG family enzymes have been found primarily in Gram-positive bacteria, there is a growing body of literature reporting these enzymes in Gram-negative bacteria as well.  Based on phylogenetic analysis, we identified IsdG family members in over 800 bacterial genomes, as well as 12 eukaryotes and 8 archaea. Additionally, our analysis identified IsdG family enzymes in HO-1 family containing organisms. This is intriguing since no organism has been previously shown to contain multiple families of heme degrading enzymes. To confirm the assignment of the identified IsdG family heme oxygenases, we characterized a predicted eukaryotic IsdG family protein from the single celled alga Chlamydomonas reinhardtii. The expression of this protein, which we have named cMO, is upregulated under iron deplete conditions, similarly to many other heme oxygenases. Interestingly, cMO transcript expression is also regulated by circadian rhythms. In vitro assays show that cMO has the ability to both bind and degrade heme. Additionally, cMO contains the catalytic triad necessary for heme catabolism in IsdG family members, and mutation of this triad significantly abrogates heme degradation by cMO. Finally, the cMO heme catabolite has a retention time and absorbance spectrum distinct from previously identified heme degradation products. This study displays the breadth of IsdG family conservation and functionally describes the first eukaryotic IsdG family heme oxygenase.


8. Lillian J. Juttukonda, Jessica L. Moore, Yaofang Zhang, Christiaan Wijers, Matthew T. Stier, James Atkinson, R. Stokes Peebles, Richard M. Caprioli, and Eric P. Skaar


This abstract will be included in the "VICB Student Research Symposium Abstracts" hand-out on the day of the symposium.


9. Lauren Palmer, Kelli Boyd, Eric Skaar


This abstract will be included in the "VICB Student Research Symposium Abstracts" hand-out on the day of the symposium.



10. "Isolation and Characterization of S. aureus monoclonal antibodies using surface proteins as novel targets," Monique Bennett, Jim Crowe, and Eric Skaar

The iron-regulated surface determinant (isd) system has evolved in S. aureus to steal iron from the human host. S. aureus utilizes the hemoglobin receptor, IsdB, to bind hemoglobin and subsequently transfer heme to other Isd proteins for utilization in iron-metabolism. Because iron is an important metabolic requirement for both the human host and the bacterial pathogen, the development of a bacterial system to sequester it is unsurprising.  In vitro studies have shown that inactivation of isdB decreases hemoglobin binding and utilization of hemoglobin as a sole iron source. Furthermore, in vivo studies using isdB mutants show a reduction in S. aureus virulence in murine models. Previous work has also shown that an antibody to IsdB can protect from S. aureus infection in mice, although the mechanism of action is unclear. These data indicate the importance of heme-iron in the ability of S. aureus to grow and cause infection. These data also indicate that other metal-acquisition systems may be necessary for S. aureus to successfully grow and cause infection. We propose to isolate fully monoclonal antibodies (mAbs) specific to the surface exposed receptors of the iron-regulated surface determinant (isd) system. We hypothesize that these mAbs will be successful at blocking the ability of S. aureus to transport iron, thereby reducing bacterial burden during staphylococcal infection. Overall, the development of mAbs specific to IsdB and other metal receptors will help highlight the importance of metal acquisition in pathogen growth, metabolism, signaling and virulence. Moreover, a deeper understanding of the location of the Isd system and its function as a molecular machine may serve to not only elucidate Isd metal-acquisition and signaling but may also lead to the discovery of other transport systems in Gram-positive pathogens that can be targeted therapeutically.


11. "Mass Spectrometry Based Metabolomics Targeting Changes in Fecal Bile Salt Concentration," James Poland, Alexandra C. Schrimpe-Rutledge, Charles Robb Flynn, and John A. McLean

Over the past decade there has been growing interest in the bacteria that make up the gut microbiome. This is due in part to research suggesting that the gut bacterial community has an effect on human physiology, nutrition, and susceptibility to disease. Alterations to this community may be the cause of disorders such as diabetes and obesity. Many of these studies involve metagenomic studies that answer the question as to what bacteria are present in the microbiota. However, there is a lack of research into how bacteria are affecting these distant systems. One of the challenges to approaching this question is that the microbiota is composed of many different types of bacteria with different biological processes, complicating the contribution each bacteria has on the whole system. Metabolomics seeks to study endogenous cellular metabolites. Due to the dynamic nature of the metabolome, studying it requires instrumentation that is both fast and reproducible. Mass spectrometry has emerged as one of the most powerful analytical techniques to study changes in metabolites. Endogenous molecules are extracted from cells with simple sample preparation techniques minimizing alterations to the set of molecules. Advances in ion mobility mass spectrometry are used to identify these small molecules. It has been demonstrated that biological classes can be separated using ion mobility mass spectrometry. Taking advantage of this aspect, it is possible to analyze all molecules in various biological classes simultaneously. New methodologies have been developed using ion mobility coupled to mass spectrometry to analyze metabolites extracted from feces to address changes that result in the metabolomic profile after bile diversion surgery. This procedure relocates where bile acids are released as a way to minimize the absorption of lipid in the small intestines. This procedure provides an alternative to gastric bypass and insight on the effect of alterations to the gut have on overall health. Utilizing both separation features (retention time and drift time) and identification features (mass to charge ratio and fragmentation) metabolites of interest were detected and identified. Compounds that have significant fold changes in abundance across multiple experimental samples are prioritized. Preliminary results suggest that along with changes in the relative concentration of bile acids, there is also perturbation in fatty acid concentration. While we observed a decrease in bile acids after bile diversion surgery, there is a significant increase in the concentration of fatty acids. This is consistent with the hypothesis that bile diversion surgery results in malabsorption of fatty acids. In addition to bile acid and fatty acid changes, numerous additional small molecules exhibited variations in their intensity and fold changes, such as choline containing molecules. Further alterations to the absorption and conversion of metabolites are currently being investigated. The overall changes in the metabolite profiles suggest significant biological processes are affected as a result of changes to the microbiota as the result of bile diversion surgery.


12. "Cytoskeletal Control of Quantum Dot-Labeled Dopamine Transporter Diffusion Dynamics in Membrane Filopodia," Oleg Kovtun, Lucas B. Thal, Michael P. Frankland, Ian D. Tomlinson, and Sandra J. Rosenthal


The presynaptic dopamine transporter (DAT) is the primary regulator of the spatial and temporal dynamics of dopamine-mediated signaling. DAT is a transmembrane sodium-coupled symporter (SLC6A3) that actively clears extracellular dopamine into the presynaptic neuron. DAT is exclusively synthesized in the somatodendritic compartments of dopamine neurons in the midbrain and subsequently targeted to the dopaminergic axons. Since the cell surface presence of DAT is required for dopamine reuptake, proper membrane targeting and trafficking of DAT are essential components of dopaminergic neurotransmission. Recent studies demonstrated that a significant fraction of DAT molecules localize in filopodia-like membrane protrusions in a variety of cell hosts. Here, we sought to take advantage of our ligand-conjugated quantum dot (QD) DAT labeling strategy and characterize the diffusion dynamics of the filopodial pool of QD-labeled DAT proteins. We showed that DAT-QDs were retained by filopodia for the entire trajectory duration and displayed frequent bidirectional movement switching with a slow and fast velocity components. Furthermore, we demonstrated that actin cytoskeleton, but not microtubule, disruption significantly elevated the rate of lateral diffusion of filopodial DAT-QDs, which was accompanied by greater instantaneous velocity and increased 5-second net displacement. It appears that the integrity of the actin cytoskeleton network serves as an important determinant of filopodial DAT diffusion dynamics. In contrast to DAT, the majority of QD-wheat germ agglutinin conjugates were targeted away from filopodia to flat membrane regions, indicating a DAT-specific protein accumulation in membrane filopodia. Our future studies will be aimed at elucidating the role of DAT filopodial targeting and retention in dopamine synapse formation as well as shedding light at the molecular mechanisms underlying DAT axonal targeting and its potential contribution to dopaminergic axon guidance.


13. "Aptamer characterization and utilization for enhanced detection of malarial biomarker Plasmodium lactate dehydrogenase," Andrew G. Kantor, David W. Wright


Rapid diagnostic tests (RDTs) are widely utilized for diagnosis of malaria due to their facile administration at the point-of-care compared to more resource dependent techniques (i.e. microscopy and PCR). However, RDTs are limited by poor sensitivity, preventing early diagnosis of asymptomatic individuals. Early diagnosis followed by treatment at the point-of-care is critical to disease intervention, as asymptomatic individuals with malaria can serve as transmission reservoirs for the disease. Currently, commercial RDTs for malaria employ monoclonal antibodies (mAbs) for biomarker capture and detection. However, over the past twenty years aptamers have emerged as an alternative to mAbs for molecular recognition. Aptamers are synthetic nucleic acid sequences selected from a large combinatorial library that bind to target molecules with high (~nM) affinity. Aptamers offer several advantages over antibodies, namely decreased cost via automated chemical synthesis, increased thermal stability, and structural manipulation capabilities. Our lab has characterized the kinetics of binding for a multitude of mAbs and aptamers specific for malarial biomarker Plasmodium lactate dehydrogenase (pLDH). Moreover, we have developed a strategy that improves the detection of pLDH in several commercial RDTs. We employ aptamer-functionalized magnetic beads to capture native P. falciparum LDH from whole blood samples and elute the captured pfLDH using complementary DNA to enhance the signal on an RDT.


14. "Implications for 4-oxo-2-nonenal-derived histone adducts in mediating gene expression," Jeannie M. Camarillo, James J. Galligan, Lawrence J. Marnett


Histone modifications play a critical role in the maintenance of chromatin structure and the regulation of gene expression. For example, Lys acetylation to histone tails results in chromatin remodeling to allow transcriptional activation of targeted genes. Recent work has identified core histones as targets for adduction by the oxidative stress-derived lipid electrophile, 4-oxo-2-nonenal (4-ONE). Here, we investigate the potential implications of these modifications on gene expression. Using an alkyne analog of 4-ONE, a4-ONE, we employed click chemistry to selectively tag modified proteins from RKO cells treated with electrophile. Our data reveal that a4-ONE is a long-lived histone modification, present up to 24 hours after treatment. Using pharmacological inhibitors of histone acetylation and deacetylation, we observe alterations in the acetylation of core histones when cells are co-treated with a4-ONE. These data suggest that a4-ONE modifications occur at physiologically relevant sites that regulate chromatin structure and gene expression. Further, utilizing a combination of chromatin immunoprecipitation and click chemistry, we can selectively pull out DNA associated with adducted histones, further suggesting a putative role for these modifications in the regulation of gene expression. Together, our data suggest that histone adducts may play a role in chromatin structure and the mediation of gene expression.


15. "Hairpin DNA Functionalized Gold Nanoparticles as Molecular Probes for Disease," Alexis C. Wong and David W. Wright


The extensive use of gold nanoparticles (AuNPs) in nanomedicine, especially for intracellular imaging, photothermal therapy, and drug delivery, has necessitated the study of how functionalized AuNPs engage with living biological interfaces like the mammalian cell. We have demonstrated that functionalization of AuNPs with hairpin DNA results in a molecular probe that is efficiently transported into cells, where the nanoparticle can report on the expression of a specific target RNA associated with a disease state. Nanoparticle characteristics like size, shape, surface charge, and surface functionality can affect the accumulation of functionalized AuNPs in cells. Using confocal microscopy, flow cytometry, and inductively coupled plasma mass spectrometry we demonstrate that CaSki cells, a human cervical cancer cell line, internalize AuNPs functionalized with hairpin, single stranded, and double stranded DNA differently. Surface charge and DNA conformation are shown to have no effect on the cell-nanoparticle interaction. Using DNA-functionalized AuNPs of a defined size range, we establish that cells accumulate small DNA-AuNPs in greater quantities than large DNA-AuNPs, indicating that size is the major contributor to cellular uptake properties. These data suggest that DNA-AuNPs can be easily tailored through modulation of size to design functional AuNPs with optimal cellular uptake properties and enhanced performance in nanomedicine applications.


16. "Investigation of Molecular Interactions Between Lysophospholipase A2 and its Diverse Bioactive Lipid Substrates," James A. Wepy, Shu Xu, Shalley Kudalkar, Lawrence J. Marnett


Lysophospholipase A2 (LYPLA2) is a serine hydrolase responsible for the hydrolysis of lysophospholipids, including lysophosphatidic acids, lysophosphatidylcholines and lysophosphatidylserines. These lysophospholipids are known to elicit a variety of physiological effects, such as the activation and recruitment of phagocytes, induction of cytokine expression, aggregation of platelets, calcium mobilization, and proliferation of vascular smooth muscle cells. We have also recently identified LYPLA2 as the major prostaglandin glyceryl ester (PGG) hydrolase in human cancer cells. These metabolites of the endocannabinoid species, 2-arachidonoylglycerol (2-AG), are precursors to the bioactive prostaglandin species, which also elicit a variety of effects, including vasoregulation, hyperalgesia, and inflammatory responses.


With purified recombinant LYPLA2, we have used mass spectrometric approaches to evaluate and compare the kinetic rates of hydrolysis of its various substrates, including PGGs, lysophospholipids, and the prostanoid precursors, 2-AG and arachidonoylethanolamide. With these data, we have shown that LYPLA2 preferentially hydrolyzes 1-glyceryl esters of esterified lipids, and selectively metabolizes PGGs over their endocannabinoid precursor, 2-AG. We have recently solved the first crystal structure of LYPLA2 and used this data to develop site-directed mutants, providing new insights into the binding site of this enzyme. Structural investigations of LYPLA2 reveal a large channel at the surface of the protein, as well as a mobile loop near its catalytic triad. To further investigate the binding site interactions of LYPLA2 with its structurally diverse substrates, site-directed mutagenesis was employed to express, purify and assess the hydrolytic activity of mutants at these hypothesized binding sites. Mutations of amino acids located in the surface channel were mutated to large tryptophan residues to sterically hinder binding of lipid substrates in the channel, and have shown to significantly inhibit the enzyme's hydrolytic activity. Here we describe the first kinetic analyses of this enzyme's ability to hydrolyze a wide range of PGG, endocannabinoid, and lysophospholipid substrates. Collectively, these data provide the first structural interpretation of the interactions of LYPLA2 with its bioactive lipid substrates.


17. "Analysis of everninomicin biosynthesis in pursuit of novel analogs," Audrey E. Yñigez-Gutierrez, Emilianne M. Limbrick, and Brian O. Bachmann


The growing threat from antibiotic resistant illnesses has renewed interest in revitalizing previously described natural products with known antimicrobial properties. One such class of molecules are the everninomicins, complex polysaccharides produced by Micromonospora carbonacea var. aurantiaca. Previously, the everninomicins showed strong activity against antibiotic resistant strains of bacteria via inhibition of protein translation. However, the everninomicins' development was hindered by an inability to access analogs via total synthesis or semi-synthetic methods. We propose to derivatize the everninomicins using the natural biosynthetic machinery to develop novel analogs with increased antibacterial potency. All natural everninomicin analogs contain at least one iterative type I polyketide synthase (iPKS)-derived dichloroisoeverninic acid (DCE) moiety. However, this conserved functionality's biosynthesis and role in activity are poorly understood. In order to understand the temporal ordering of DCE biosynthesis, we successfully deleted the genes encoding for the DCE-associated O-methyltransferase and halogenase to provide novel everninomicin analogs. As well, these enzymes are currently being evaluated in vitro to identify their exact substrates and to evaluate their ability to incorporate non-natural substrates. Genomic editing and incorporation of non-natural substrates will allow for investigations into the ability of M. carbonacea var. aurantiaca to produce everninomicin analogs with diversified everninic acid moieties. Analogs properly designed with useful synthetic handles could eventually be utilized for coupling reactions to increase everninomicin potency and study ribosomal inhibition. This work will further elucidate the biosynthesis of everninomicins and provide novel analogs in an attempt to revitalize this potent class of antibiotics.


18. "Characterization of the 2,6-Diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine DNA Lesion," Stephanie N. Bamberger, Ryan S. Bowen, Chanchal K. Malik, Tracy L. Johnson-Salyard, Carmelo J. Rizzo, and Michael P. Stone

The N7 imidazole nitrogen of guanine is the most susceptible site for DNA methylation, resulting in the formation of the cationic adduct 7-methylguanine (7MeG).  Opening of the imidazole ring occurs upon exposure to hydroxide ion, producing the 2,6-diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine (MeFapy-dG) lesion. This lesion is known to convert between α and β anomers, but the possibility of additional conformational isomers cannot be ruled out.  We report progress towards characterizing this DNA adduct, including the synthesis and purification of a 12mer oligodeoxynucleotide in which the CH3 group of the MeFapy-dG is 13C-labeled in the sequence 5'-CATXATGACGCT-3'; X=13C-MeFapy-dG.  The reverse phase HPLC chromatogram showed two equilibrating chromatographic peaks that were verified as the 13C-MeFapy-dG 12mer by MALDI mass spectrometry and capillary zone electrophoresis (CZE), indicating that the two peaks were equilibrating isomers.  It is hypothesized that the two equilibrating peaks are a result of anomerization of the MeFapy-dG adduct; such epimerization has been found to occur in other Fapy lesions.  To investigate whether this held true for MeFapy-dG the unlabeled MeFapy-dG phosphoramidite was studied by NMR spectroscopy.  By utilizing dichloromethane (DCM) as the solvent potentially water exchangeable protons could be observed in spin systems using a TOCSY experiment; eight unique spin systems of the deoxyribose ring were observed.  Further characterization through NOE cross-peaks between the H3', H2', H2'', and H1' protons of the deoxyribose ring revealed that four of the species were the α anomer while four were the β anomer; the α anomer was found to be the major component.   In the NMR spectra of the duplex the C1H1'-A2H8, A2H1'-A2H8, and A2H1'-T3H6 NOE cross-peaks exhibited doubling, as did corresponding NOE cross-peaks in the deoxyribose H2' and H2'' region of the spectrum.  Tripling of the T3H1'-T3H6 and C21H5-C21H6 NOE cross-peaks and corresponding NOE cross-peaks in the deoxyribose H2' and H2'' region of the spectrum was also observed.   These NMR data indicated the presence of two to three distinct chemical environments for nucleotides near the lesion.  No NOE cross-peaks were observed for the deoxyribose H1', H2', or H2'' protons of the MeFapy-dG lesion.  A HSQC experiment of the 13C-labeled MeFapy-dG 12mer indicated that the methyl group of MeFapy-dG is in ten unique chemical environments, which was reduced to six in duplex.  The peaks were spilt into two distinct groups separated by 3-4 ppm on the carbon axis.  A series of temperature dependent HSQC experiments indicated that the two groups are caused by anomers rather than rotamers.    Supported by NIH Grant P01 CA-160032 (M.P.S. and C.J.R.) and the NIH Training Grant in Molecular Toxicology, T32 ES-007028 (S.N.B.).


19. "Investigating the Metabolism of a Major Peroxidation DNA adduct, M1dG, in the Genome," Orrette R. Wauchope, William N. Beavers, James J. Galligan, Michelle M. Mitchener, Philip J. Kingsley, and Lawrence J. Marnett


Chronic inflammation results in increased production of reactive oxygen species (ROS), which can oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacterial and mammalian cells and is repaired via the nucleotide excision repair system. Here, we report that M1dG levels in intact nuclear DNA were increased from basal levels of 1 adduct per 108 nucleotides to 2 adducts per 106 nucleotides following adenine propenal treatment of RKO, HEK293 or HepG2 cells. We also found that M1dG in genomic DNA was oxidized in a time-dependent fashion to a single product, 6-oxo-M1dG, (to ~ 5 adducts per 107 nucleotides) and that this oxidation correlated with a decline in M1dG levels. Investigations in RAW264.7 macrophages indicate the presence of high basal levels of M1dG (1 adduct per 106 nucleotides) and the endogenous formation of 6-oxo-M1dG. Interestingly, in all cell lines investigated, 6-oxo-M1dG was not observed in mitochondrial DNA. Basal levels of M1dG in the mitochondria were found to be approximately two orders of magnitude higher (~ 1 adduct per 106 nucleotides) than those observed in nuclear DNA and were increased with electrophilic stimulation. Further studies, in all the investigated cell lines, indicated a correlation between M1dG levels and oxidative stress in the mitochondrion. This is the first report of M1dG in mitochondrial DNA in intact cells and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis and repair.


20. "Progress Towards the Total Synthesis of Chrysophaentin A," Christopher Fullenkamp, Somnath Jana, Kwangho Kim, Carole A. Bewley, Gary A. Sulikowski


Antibiotic resistance is now known for every clinically prescribed antibiotic, which means new antibiotics with novel mechanisms of action are needed. Chrysophaentin A, a marine natural product isolated from the alga Chrysophaeum taylori, exhibits micromolar activity against gram positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The mechanism of action of this natural antimicrobial agent is believed to be the inhibition of FtsZ, which results in the direct inhibition of cellular division. Unfortunately further study of Chrysophaentin A has been inhibited due to its limited supply by isolation from its marine source. Total synthesis of Chrysophaentin A will not only allow further study of its novel mechanism of action but also access to other analogs for further development. To date our lab has been able to synthesize a derivative of Chrysophaentin A, which showed significant antimicrobial activity. Our immediate goal remains the total synthesis of Chrysophaentin A.


21. "Structure Refinement of α-helical Membrane Protein by Paramagnetic LAN-thanide Ion tagging," Julian Gräb, Soumya Ganguly, Heather Darling and 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.


22. "Cyclooxygenase-2 (COX-2) Dimer Interface Kinetics and Inhibition," Michael C. Goodman, Shu Xu, Carol Rouzer, and Lawrence Marnett


Cyclooxygenase-2 (COX-2) is one of two isoforms of the prostaglandin endoperoxide H synthases (PGHSs) that catalyze the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2) in the presence of two molecules of oxygen and two electrons. The enzymes initially oxygenate the substrate AA at the cyclooxygenase (COX) active site, forming the unstable intermediate compound prostaglandin G2 (PGG2), which is subsequently reduced at a separate peroxidase (POX) site, forming the product PGH2. PGH2 is a substrate for various terminal prostanoid synthases that catalyze the formation of prostaglandins (PGs), as well as thromboxanes. PGs are short-lived lipid compounds that exert various biological effects, including platelet aggregation, as well as gastrointestinal and cardiovascular regulation.


Although COX enzymes are structural homodimers, they behave as functional heterodimers. One subunit of the enzyme contains a heme cofactor and functions as the catalytic site. The second subunit lacks this heme cofactor and functions as an allosteric site to regulate substrate oxygenation. Evidence of the allosteric regulation of COX is proven by the ability of nonsubstrate fatty acids to exclusively affect the oxygenation of one substrate, 2-AG, in comparison to AA. An example of a positive allosteric modulator is the substrate-selective compound, 13-methylarachidonic acid (AM-8138), which increases 2-AG oxygenation up to 3.5-fold while having no effect on AA oxygenation.


The activity of COX enzymes is inhibited by non-steroidal anti-inflammatory drugs (NSAIDs). These drugs have distinct chemical characteristics and can also have different inhibitory kinetics. Two inhibitors, ibuprofen and mefenamic acid, are weak, competitive inhibitors of COX-2 in the presence of AA, but act as potent inhibitors in the presence of 2-AG. These data perpetuate the hypothesis that some NSAIDs like mefenamic acid can be substrate-selective by binding in one subunit and affecting the oxygenation of 2-AG in the catalytic subunit. However, the inhibitor must bind in both subunits to effectively inhibit AA oxygenation in a competitive manner.


Previous X-ray crystal structures of the enzyme have revealed amino acid residues that interact with ligands and also form various contact points between the two protein subunits through hydrogen bonding with one another. These residues at the subunit interfaces could potentially affect ligand-dependent communication or conformational changes between the subunits, leading to altered enzyme kinetics and inhibitor potency.

The overall goal of this research is to investigate how the interface between subunits of COX-2 dictates substrate selective inhibition and potential conformational changes that occur upon the presence of particular substrates, inhibitors, or positive modulators. Site-directed mutagenesis of amino acid residues at the dimer interface can potentially reveal important sites of communication between the catalytic and allosteric sites of the enzyme.


23. "Mapping Microbial Natural Product Responses to Activation Stimuli," Brett Covington, John McLean, and Brian Bachmann


Over the past 30 years the incidence of antibiotic resistant infections has steadily increased, and at the same time it is becoming more and more difficult to discover new antibiotics. The combination of these problems is leading us towards what many medical professionals are calling a "post-antibiotic" era in which antibiotics will no longer be effective at treating bacterial infection. But it may be possible to postpone or even prevent this era. Microbial natural products are an important reservoir of therapeutic molecules. These compounds have been the source of the majority of modern antibiotic and chemotherapeutic drugs, and it is estimated that we have accessed less than 1 % of these natural resources. This is largely because these diverse compounds are often not produced when grown under laboratory conditions. We hypothesize that producing organisms use their natural products to respond to environmental cues from competing organisms, to nutrients and other physical factors, and when grown in isolation in the lab the organisms no longer receive these cues and subsequently do not produce their compounds of interest. Here we demonstrate how self-organizing map analytics can be used to interpret metabolomic changes engendered by discrete activation stimuli in actinomycetes. This process can prioritize secondary metabolites for isolation in a discovery pipeline and may additionally enable the connection between activation stimuli and genetic regulatory elements for natural product gene clusters.


24. Jacob E. Choby and Eric P. Skaar

This abstract will be included in the "VICB Student Research Symposium Abstracts" hand-out on the day of the symposium.


25. "Acinetobacter baumannii employs a zinc-binding GTPase to overcome host-mediated metal sequestration," Zachery R. Lonergan, Brittany L. Nairn, Jiefei Wang, Joseph J. Braymer, Yaofang Zhang, M. Wade Calcutt, John P. Lisher, Walter J. Chazin, Valerie de Crécy-Lagard, David P. Giedroc, and Eric P. Skaar


Acinetobacter baumannii is an important nosocomial pathogen causing a range of diseases including pneumonia and bacteremia. During infection, A. baumannii must acquire nutrient metals in order to survive and colonize the host. Vertebrates elaborate mechanisms to sequester these metals from invading pathogens through a process termed nutritional immunity. For example, the metal binding properties of the host protein calprotectin inhibit bacterial growth in vitro. However, A. baumannii combats metal limitation by inducing metal acquisition genes. Specifically, during zinc starvation, the most highly induced gene encodes a hypothetical protein within the COG0523 subfamily of G3E GTPases which we have named zigA (Zur-induced GTPase A). Genetic and protein function analyses demonstrate that ZigA is homologous to metallochaperones that aid in metal cofactor delivery during metal-containing enzyme maturation. Inactivation of zigA results in increased sensitivity to zinc starvation and a growth deficiency during calprotectin-mediated zinc sequestration. This inactivation also causes accumulation of zinc within the bacterial cell and inappropriate induction of metal acquisition systems, demonstrating that zigA is critical for properly regulating intracellular zinc concentrations. As a predicted metallochaperone, we hypothesize that ZigA delivers a metal cofactor to a cognate target protein. Two potential targets include a predicted D-ala-D-ala carboxypeptidase which we have named zrlA (Zur-regulated lipoprotein A) as well as an enzyme in the histidine utilization (Hut) operon named hutH, both of which bind zinc. Genetic inactivation of zrlA or hutH decreases growth during zinc sequestration. Furthermore, both zrlA and hutH are necessary for full colonization of the lungs in mice, and zigA is necessary for complete dissemination to the liver, demonstrating the importance of zinc homeostasis during infection. Taken together, these findings reveal a multi-faceted system within A. baumannii to overcome host-mediated zinc sequestration and provide novel targets for antimicrobial drug development.


26. "Total synthesis of hemiketals D2 and E2 in support of biological studies," Robert Davis, Zachary Austin, Alex Allweil, Robert Boer, Somnath Jana, Juan Antionio Gimenez Bastida, Claus Schneider, Gary Sulikowski


Inflammation is a complex biological response through which the body can defend against infections and heal from injuries. Lipid mediators have been the focus of much attention in this area due to their important role in the inflammatory response. In 2011, the Schneider group reported the isolation of two new cyclic hemiketal eicosanoid metabolites: HKD2 and HKE2. Initial biological studies showed that both HKD2 and HKE2 stimulated migration and branching of vascular endothelial cells, implicating a proangiogenic role of the hemiketals in inflammatory sites. These results, while sparking great interest in further study, could not be followed due to lack of available material. In order to access useable quantities of material, our group has completed the first total synthesis of HKE2 and is currently exploring routes to access HKD2.


27. "Synthetic and Biological Study of Bielschowskysin and Pleiogenone A," Jason Hudlicky, Jordan Froese, Cameron Overbeeke, Tomas Hudlicky, and Gary Sulikowski


Natural products have a long history of serving as therapeutic leads (e.g. taxol and compactin) and biological tools (e.g. rapamycin and oligomcyin). Our lab seeks to develop synthetic routes to rare natural products to provide material for biological study as well as provide significant training and education in the practice of chemical synthesis. Bielschowskysin, a rare and structurally complex marine diterpene, was isolated in 2004 from the extracts of the gorgonian coral, Pseudopterogorgia kallos. Initial biological assays indicated moderate antiplasmodial activity against Plasmodium falciparum (IC50 = 10 µg/mL) and cytotoxicity against EKVC non-small cell lung cancer (GI50 < 0.01 µM) and CAKI-1 renal cancer (GI50 = 0.51 µM). Presented here are the recent efforts toward the total synthesis of. Another molecule that has piqued our interest due to its biological activity is pleiogenone A, recently isolated by Kingston and synthesized by Hudlicky. In a collaboration with the Hudlicky lab at Brock University (St. Catharines, Ontario, Canada) the synthetic sample of pleiogenone A has demonstrated activity against MV411 cells (GI50 = 310 nM) and further studies are ongoing. Future work will focus on preparing synthetic analogs of pleiogenone A to improve activity.


28. "Binding of Arrestin-3 to SH2-SH3 of HRK," Jakob Gasse, Sandra Berndt, Vsevolod V. Gurevich


Signal transduction within a cell is diverse and so far not fully understood. One big group of receptors which mediate the signal transfer from the extracellular space to the cytoplasm are G protein-coupled receptors (GPCRs). After signal transduction the receptor gets phosphorylated by G protein-coupled receptor kinases (GRK), which results in arrestin binding to GPCRs, suppressing the G-protein dependent pathway. Additionally to the desensitization of the GPCR many possible G-protein-independent arrestin-mediated signaling can follow. Preferential engagement of G protein or arrestin-dependent pathways is called biased signaling.


Mammals have four different arrestins. Two are in the visual system (arrestin-1 and arrestin-4) and two non-visual ones (arrestin-2 and arrestin-3, also called β-arrstin1 and β-arrestin2).


Arrestin-1, arrestin-2 and arrestin-3 bind many down stream effectors. Especially arrestin-3 can bind more than 200 different effectors, including c-Src family kinases. C-Src is a proto-oncogene which can cause cancer once permanently activated. The elucidation oft he process of Src activation has high pharmacological importance.

The c-Src family kinases have 4 homologous sequences (SH1 – SH4) throughout the family which play a crucial role in binding, auto-phosphorylation and kinase activity.

Although interactions between arrestin-1 and arrestin-2 and the SH1, SH2 and SH3 domains (sequence homology) of c-Src or c-Src family kinases have been studied there are still a lot of uncertainties about the detailed understanding of this interaction. Here, we want to analyze the specific binding for arrestin-3 to c-Src. First, we focus on the SH2-SH3 domain of HRK, a c-Src family kinase. For this characterization we use pull-down assays.


Our results show that the presence of inositol hexakisphosphate (IP6) enhances arrestin-3 binding to SH2-SH3. This might be due to  the coverage of negatively charged lysine residues that favor the binding. To elucidate the impact of arrestin conformation on the binding to these downstream effectors we used different arrestin-3 constructs. Furthermore, we want to use specific structural motives of arrestin-3 to define the binding sites responsible for this interaction.


29. "Synthesis of human milk tetraoses," Kelly M. Craft, John P. Hayes, Steven D. Townsend


Human milk oligosaccharides (HMOs) are the third largest macromolecular component of breast milk and offer infants numerous health benefits ranging from healthy gut development to infection prevention. However, as HMOs are not present in infant formula, formula-fed infants do not receive the same health benefits. To overcome this, carbohydrate formula additives have been developed.  Unfortunately, currently approved additives do not feature the structural complexity of HMOs. As the benefits derived from HMOs are known to be dependent on oligosaccharide structure, the need remains to develop additional infant formula additives. To do this, it is imperative to better understand specific HMO structure-activity relationships. As it is difficult to isolate appreciable amounts of homogenous HMOs from breast milk, chemical synthesis provides the best method to obtain sufficient quantities of HMOs for biological testing. Therefore, we first aim to synthesize lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT), two core tetrasaccharides common to longer-chain HMOs. Functionalization of these core structures will allow access to additional HMOs. We next propose to evaluate the growth of bifidobacteria on the synthetic HMOs. A dominant presence of bifidobacteria in the gut is one of the hallmarks of a healthy infant microbiome. We hypothesize these results will improve our understanding of which HMO structural features are important for healthy gut development in infants.


30. "Bridging in Silico and in Vitro Methodologies to Predict the Chemical Induction of Intracellular 7-Dehydrocholesterol Levels," Phillip Wages, Alexander Geanes, Jens Meiler, Ned Porter


The genetic disorder Smith-Lemli-Opitz Syndrome results from a defect in DHCR7, the protein that converts 7-Dehydrocholesterol (7DHC) to cholesterol. The accumulation of 7DHC is one of the key factors leading to a variety of phenotypic outcomes including both behavioral and physical defects. This is in part supported by 7DHC's ability to undergo free radical oxidation to form oxysterols that cause oxidative and electrophilic stress. Recently it was reported that a number of pharmaceutical agents elevate intracellular 7DHC levels, which could account for unwanted off target side effects. To elucidate the impact of chemical-induced 7DHC levels, this study endeavors to elucidate the characteristics of a small chemical that inhibits 7DHCR in order to predict other chemicals with similar biological effects. Using a collection of 30 known compounds that elevate 7DHC levels, a predictive model based on the Surflex-Sim technique, which utilizes 3-D molecular alignment, was developed. The resulting pharmacophore model had three key characteristics: 1) a halogenated aromatic group, 2) a centrally located nucleophilic group, and 3) a heterocyclic group. This predictive model along with a quantitative-structure-activity relation (QSAR) model was applied to the Vanderbilt Discover Collection chemical library. The top 50 consensus hits from both predictive models were selected for experimental testing to validate the accuracy of the predictions. Each chemical was exposed to the neuroblastoma cell line, Neuro-2A, at 1 µM for 24 hours in triplicate and then assayed for 7DHC and cholesterol levels. Compared to a vehicle exposure no chemical altered cholesterol levels; however, 62% of the tested chemicals were demonstrated to elevate 7DHC levels significantly. These results provide a foundation to understand how chemicals can impact DHCR7 activity by expanding the known library of chemicals able to increase intracellular 7DHC levels, an important step towards developing an accurate predictive model of chemical inhibitors of DHCR7 that could be utilized to address concerns of pharmacologic side effects.


31. "Development of rhodol-based thallium sensing probes for the study of potassium ion channels," Anna Ender, Brendan Dutter, Gary A. Sulikowski, C. David Weaver


Ion channels are pore-forming proteins that promote conduction of ions across cellular membranes. These channels are found in every cell in the body and can be localized in the plasma membrane, as well as in intracellular compartments such as the nucleus, mitochondria, and lysosomes. Ion channels serve many functions in organisms including electrical signal transduction, chemical signaling, regulation of cell volume, regulation of cytoplasmic and vesicular ion concentration, and regulation of pH. Ion channels are also implicated in many human diseases, including epilepsy, cardiac arrhythmia, chronic pain, and cancer. There are numerous channels types which are selective for specific ions: sodium, calcium, potassium and others. The largest and most diverse class of ion channels is potassium channels. Although potassium channels have been targeted from treatment of disease, e.g. retigabine (epilepsy), glibenclamide (diabetes), most potassium channels do not have potent and selective pharmacological tools with which to investigate their physiological role and therapeutic potential.


In order to discover new pharmacological tools for potassium channels using high-throughput screening, fluorescent metal-sensing dyes are utilized. Because intracellular potassium concentrations are very high, >100 mM, potassium-sensitive dyes have proven largely unsuccessful. Thus, thallium is used as a surrogate for potassium because it exhibits similar flux through potassium channels, it is not found in cells, and it can be added exogenously. Current fluorescent dyes such as Thallos are based on fluorescein conjugated to a thallium binding moiety. Upon binding thallium, an increase in fluorescence is observed. Despite their utility, fluorescein-based sensors show some disadvantages, particularly a high sensitivity to intracellular pH changes due to the pKa of a critical phenolic hydrogen. Rhodols are hybrids of fluorescein and rhodamine that exhibit greater tolerance to lower pH. In addition, the emission and absorption maxima of rhodols are at longer wavelengths than fluorescein which may lead to decreased cell damage during the excitation of the probe. The red-shifted spectral properties of rhodols may also decrease interference from fluorescent compounds found in high-throughput screen libraries, the spectral distribution of which is biased toward shorter wavelengths. The goal of this project is to prepare numerous rhodol-based thallium sensors by chemical synthesis and evaluate their utility in vitro and cell-based potassium channel assays.


32. "All-in-one, multiplexed on-bead ELISA for detection of two malarial biomarkers," Christine F. Markwalter, Keersten M. Ricks, Anna L. Bitting, Lwiindi Mudenda, David W. Wright


As malaria transmission declines, accurate diagnosis becomes increasingly important for defining disease prevalence and distribution, as well as monitoring impact of interventions. Further, in low-transmission settings, identification and treatment of asymptomatic carriers are critical for eliminating the disease. Current antigen-detecting rapid diagnostic tests (RDTs) for malaria are unreliable in the asymptomatic regime (< 200 parasites/µl), and laboratory protein-based detection strategies, such as well-plate ELISAs, can require 5 – 8 hours of incubation time and are limited to one analyte.  To address this, we have developed a multiplexed, magnetic bead-based ELISA with incubation times totaling less than 1 hour and single parasite/µl detection limits, rivaling those of well-plate ELISAs. This multiplexed assay detects two malarial biomarkers: (1) Plasmodium lactate dehydrogenase (pLDH), which is detectable for all five species of malaria, and (2) histidine-rich protein II (HRPII), present only in P. falciparum infections. In this assay, magnetic particles functionalized with antibodies specific for pLDH and HRPII are added directly to lysed whole blood samples along with detection antibodies with distinct enzymes for each biomarker. Sandwich complexes for pLDH and HRPII form on the surfaces of the magnetic beads, which are washed and sequentially re-suspended in detection enzyme substrate for each antigen. Detection of both biomarkers is advantageous because it avoids false-positives due to slow HRPII clearance and allows for differentiation between falciparum and non-falciparum infections, ultimately informing treatment. With these advantages, as well as high sensitivity and detection limits in the asymptomatic regime, the developed multiplexed assay for pLDH and HRPII is an attractive alternative to well-plate ELISAs and a promising detection strategy for an elimination setting. Further, the modularity of the multiplexed on-bead ELISA makes it applicable to any series of infectious disease biomarkers for which there are antibody pairs available.


33. "Structural Consequences of the C8-Guanine DNA Adduct Formed by
3-Nitrobenzanthrone; an Environmental Carcinogen," Dustin Politica, Amritraj Patra, Chanchal Malik, Arindom Chaterjee, John Tokarsky, Zucai Suo, Ashis Basu, Martin Egli, Michael P. Stone


Damage to DNA results from many endogenous and exogenous sources and plays a significant role in cancer etiology. 3-Nitrobenzathrone (3-NBA) is a component of diesel exhaust that is known to form DNA adducts. Aminobenzanthrone (ABA) DNA adducts result through the reaction of electrophilic nitrenium ions formed during 3-NBA's metabolic reduction in vivo. Furthermore, 3-NBA has been reported as being carcinogenic in animal model systems and human exposure has been demonstrated. The structural impacts of the C8-guanine-aminobenzanthrone (C8-dG-ABA) adduct, a major DNA adduct of 3-NBA, which occurs at the C8 position of guanine are examined. This adduct produced a base-displaced intercalated structure within a DNA duplex. The ABA moiety intercalated into the duplex displacing the cytosine opposite the lesion. The adducted guanine rotated about the glycosidic bond into the syn conformation. A ternary structure of C8-dG-ABA in complex with a trans-lesion synthesis polymerase, human polymerase eta (hPol η) indicated the conformation of the C8-dG-ABA adduct within the active site of hPol η was markedly different from the structure observed in duplex DNA. The complex was captured in a non-mutagenic insertion state which is favored for bypass of C8-dG-ABA by hPol η as determined by in vitro bypass data. The complex structure revealed that adducted guanine was in the anti conformation and was observed to form a Watson-Crick basepair with an incoming cytosine triphosphate (dCTP). The ABA moiety was rotated out of the active site of hPol η and resided in a hydrophobic pocket. The position of the ABA moiety did not allow for a clear path for the next template base to be translocated into the active site, and may reflect a means by which the adduct blocks replication. An overlay of the duplex and ternary complex structures reveals a potential mechanism for mutagenic bypass.

34. "Total synthesis of the Morganella morganii Zwitterionic polysaccharide repeating unit," D. Jamin Keith, Steven D. Townsend


The immune system is capable of fighting off bacterial, fungal, pathogens, cancer, etc. Over the past decade it has been realized that carbohydrates can induce an immune response. In general carbohydrates are poorly immunogenic in comparison to protein-specific antibodies. However, a particular class of carbohydrates called zwitterionic polysaccharides (ZPSs) elicit a much more prominent T cell response resulting in immunologic memory. ZPSs bear a zwitterionic charge motif of alternating positive and negative charged monosaccharides. Development of ZPSs as tools to stimulate the immune system can be used in cancer immunotherapy. In theory, vaccine constructs can be developed by attaching poorly immunogenic tumor associated antigens to ZPS carriers. In order to pursue such studies sufficient synthesis of ZPSs in appreciable quantities is necessary. Total synthesis of a ZPS isolated from the biofilm of the gram negative bacteria, Morganella morganii, is currently our area of focus.


35. "Galactose improves lipid metabolism and redox status in cultured SLOS fibroblasts and Dhcr7-deficient Neuro2a cells," Thiago C. Genaro-Mattos, Wei Liu, Keri Tallman, Zeljka Korade, Ned A. Porter.


Human dermal fibroblasts cultured in either reduced-lipid or galactose-containing media present increased metabolic stress. In response to this stress, dermal fibroblast taken from healthy individuals change their mRNA and miRNA gene expression profile, and upregulate many transcripts critical for lipid metabolism. Noteworthy, the same transcripts show a baseline expression difference in experimental models of Smith-Lemli-Opitz Syndrome (SLOS), a neurodevelopmental disorder caused by mutations in dehydrocholestrol reductase 7 (DHCR7) gene, an enzyme that converts 7-dehydrocholesterol (7DHC) into cholesterol. Based on these findings, we hypothesized that metabolizing galactose (GAL) might improve the disrupted cholesterol metabolism seen in SLOS. We tested our hypothesis across two in vitro SLOS models:  fibroblasts harvested from SLOS patients and Dhcr7-deficient Neuro2a cells. We found that replacement of glucose (GLU) with GAL in the culturing media resulted in improved lipid profile in both SLOS fibroblast and Dhcr7-deficient Neuro2a cells. In addition to decreased 7DHC levels, there was significant reduction in 7DHC-derived toxic oxysterol production (DHCEO) in both SLOS models. SLOS cells were shown to present increased levels of protein adduction promoted by 7DHC-derived oxysterols. We then speculated that GAL could decrease protein modification promoted by oxysterols. To address that, we incubated control and SLOS cells with an alkynyl surrogate of 7DHC and analyzed protein adduction using click chemistry and streptavidin Western blotting. These analyses revealed that GAL treatment increases protein adduction levels in control cells, while it decreases the levels in SLOS cells, suggesting that GAL is improving the redox status of SLOS cells. Altogether, our results suggest that GAL consumption is preferentially changing cellular metabolism in cells with Dhcr7 mutations, and might normalize some of the critical molecular deficits related to SLOS pathophysiology.


36. "Hemin Nanoparticle Signal Amplification for Detection of Biomolecules in Low-Resource Settings," Lauren E. Gibson and David W. Wright


Methods for signal amplification are driven by the need for more sensitive diagnostics. Not only do diagnostics need to be sensitive, but ideally they should also be environmentally stable and cost effective. The prevalently used horseradish peroxidase amplification system employed by enzyme-linked immunosorbent assays (ELISA), is an example of a sensitive method that lacks stability. This has led to the development of a variety of enzyme mimics that attempt to achieve signal amplification and maintain the sensitivity of an ELISA, but with more stable and inexpensive materials. Our work explores this area through the development of a dual mode amplification system based upon hemin nanoparticles. In this system, the first type of amplification achieved is nanoparticle-based amplification. This occurs when a hemin nanoparticle, which is bound to a biomarker of interest, is broken apart by pyridine into individual hemin molecules. Thus, instead of one nanoparticle, there are thousands of signal-producing hemin molecules. The presence of pyridine in this solution allows each hemin molecule to mimic the active site of the horseradish peroxidase enzyme. As a result, the second form of amplification occurs through the catalytic turnover of a substrate, by hemin, in the presence of hydrogen peroxide. This dual amplification strategy produces a sensitive assay, resulting in low picomolar limits of detection for the model biomarker, IgG. Thus, the hemin nanoparticle detection method is sensitive as well as stable, a combination of characteristics not commonly found in ELISAs. As the hemin nanoparticle method is a platform technology, it can be applied to the detection of many diseases. The assay has been optimized for the detection of the malarial biomarker Plasmodium lactate dehydrogenase (pLDH) in lysed whole blood. In the future, this detection strategy will be used in a paper diagnostic format resulting in the development of a stable and sensitive diagnostic for the accurate detection of disease in low-resource areas.


37. "Untargeted Metabolomic Analysis of Cyanobacteria With Varying Circadian Rhythms," Berkley Ellis, Chi Zhao, Carl Johnson, John A McLean


Mass spectrometry is emerging as the premier platform for metabolomics due to its exceedingly low limits of detection and its compatibility with high-throughput workflows. The chemical diversity of the metabolome and its artifacts make this a unique challenge necessitating multi-dimensional analysis. To address this issue, we use Liquid Chromatography Ion Mobility Mass Spectrometry (LC-IM-MS) as a means to analyze and separate molecules from all chemical classes simultaneously. With this platform, we acquire both separation features (retention time and drift time) and identification features (mass to charge ratio and fragmentation pattern). This technology has been used for untargeted metabolomics studies in order to examine the entire metabolome without bias. Specifically, we use this analytical technique to characterize cyanobacteria with various modifications to the genes responsible for the circadian rhythm to better understand its intricacies as well as its roles in metabolism. Several sample preparation procedures have been tested on cyanobacteria subjected to normal and altered light cycles in order to optimize the pool of extracted metabolites. These experiments suggest that suspending cells in 80:20 methanol: water (v/v) overnight is an efficient method with 840 metabolites of 2000 exhibiting a max fold change ≥ 2 between varying light cycles. Current efforts focus on generating putative identifications for these molecules.


38. "Magnetically assisted biomarker capture-and-release reagents to enhance malaria rapid diagnostic tests," Westley S. Bauer, Gulka C. P., Adams N. M., Haselton F. R., Wright D. W.


At a time when resistance to the most efficacious antimalarial drugs is rising, a critical window is open for malaria eradication campaigns. The need for rapid and accurate diagnosis is essential so only infected patients are treated, mitigating liberal administration of antimalarial compounds, which leads to drug resistance. Current healthcare infrastructures in rural settings are ill-equipped to handle modern diagnostic tools such as PCR, microscopy, and ELISA. Instead, lateral-flow rapid diagnostic tests (RDTs) offer the best diagnostic solution. Commercial species specific malaria RDTs detect both Plasmodium falciparum Histidine-Rich Protein-II (HRPII) and Plasmodium Lactate dehydrogenase (pLDH). Unfortunately, current dual RDTs are limited by their sensitivity. To improve test sensitivity, we have developed a strategy employing a multiplex magnetic bead, that captures and concentrates the HRPII and pLDH available in a 200 mL blood sample and delivers them onto an RDT without exceeding the tests capacity. This is achieved by using an immobilized metal affinity magnetic bead and a pLDH "capture and release" antibody reagent. The "capture and release" antibody reagent consists of a pLDH capture antibody functionalized with hexahistidine peptides. The intrinsic histidine content of HRPII and hexahistidine peptide functionalization enables HRPII and the antibody reagent to be reversibly bound to the magnetic bead utilizing metal coordination chemistry. Thus, the optimal loading density of the "capture and release" antibody reagent equips the bead with the ability to simultaneously capture pLDH and HRPII. Subsequent to capture, the multiplex bead acts as a vehicle to magnetically concentrate and release high copies of biomarkers into a small volume, employing a common high-salt elution strategy, that is then added to the RDT. The multiplex bead can capture and release greater than 92% of the total pLDH and HRPII available in a sample with an additional assay time of 8 minutes. Performing this process on a 200 mL blood sample delivers increased copies of HRPII and pLDH to a commercially available malaria dual RDT for a 10-fold increase in the limit of detection compared to the manufacture suggested protocol. The result of this 10-fold increase in limit of detection is the ability to detect single digit parasitemias.  


39. "Role of oxidative transformation in the inhibition of NF-kB by curcumin," Rebecca L. Edwards, Paula B. Luis, Paolo V. Varuzza, Akil I. Joseph, Sai Han Presley, Rupesh Chaturvedi, and Claus Schneider


Curcumin, the major diphenol of dietary turmeric, exerts anti-inflammatory effects by targeting the NF-κB pathway. Inhibition is thought to involve covalent adduction of curcumin via its electrophilic enone moiety to NF-κB directly as well as to the upstream kinase, IKKβ. Since curcumin rapidly degrades under physiological conditions we hypothesized that its electrophilic degradation products rather than the parent compound are the ultimate mediators of the anti-inflammatory effects. We synthesized structural analogs of curcumin that retain the enone moiety but have varying stability toward degradation. Inhibition of NF-κB was quantified in LPS-activated RAW264.7 cells stably transfected with an NF-κB response-element driving expression of luciferase. We observed a correlation between the rate of degradation of the analogs and the IC50 for inhibition of luciferase. Depletion of the cellular defense against electrophiles using the glutathione synthesis inhibitor buthionine sulphoximine (BSO) decreased the IC50 for curcumin. Inhibition of NF-κB is associated with reduced phosphorylation of IKKβ and IκBα in the HeLa cells. An unstable, alkynyl-tagged curcumin analog showed abundant adduction to cellular protein visualized by gel electrophoresis. Changes in adduction abundance were monitored after cellular pre-treatment with curcumin or its stable analogs. We conclude that the enone moiety of curcumin is not a functional electrophile mediating the inhibition of NF-κB. Rather, curcumin is a pro-drug, and oxidative activation into reactive electrophiles mediates its anti-inflammatory effects.


40. "Parsing the conformational and configurational equilibria of a 2´-deoxyribosylurea DNA adduct," Andrew H. Kellum Jr., Vijay Jasti, Ashis Basu, and Michael P. Stone


2´-deoxyribosylurea (urea) lesions form within DNA as a result of the cleavage of thymine from hydroxyl radicals resulting from ionizing radiation.  In addition, urea lesions are able to form from 8-oxoguanine. Previous NMR studies of the urea lesion on the nucleoside level showed that the 2´-deoxyribose ring equilibrated between the alpha (α) and beta (β) configurations.  In this study we examined the urea lesion in the single strand oligodeoxyribonucleotide 5´-(CTXA)-3´(X=urea).  Reverse phased HPLC revealed a ratio of 1:1 between two equilibrating species.  In addition, trapping the different species in basic environment slowed the equilibration.  NMR spectroscopy was used to definitely identify the two different species.  NOESY and TOCSY spectra were used to assign the resonances of the different nucleotides.  NOESY spectra revealed the identity of the two different species as α and β anomers of the urea lesion.  TOCSY and one-dimensional NMR spectroscopy showed that the urea lesion was in the anti conformation regardless of the configuration the 2´-deoxyribose ring.  The presence of the α anomer is a possible explanation as to why the lesion acts a strong block to replication. 


41. "Chemical genetic approach identifies role of proton sensing GPR68 in modulation of migration in melanoma," Charles H. Williams, H. Russel Day, Charles C. Hong


Increased glycolysis resulting in local acidification is a hallmark of cancer. However, the mechanisms by which this acidification affects cellular behaviors such as migration are not understood. We report the discovery of Ogremorphin (OGM) a first in class inhibitor of GPR68 in a phenotypic zebrafish screen. The target of OGM was identified through a cheminformatics and receptor profiling, and validated genetically with knock down technology. GPR68 plays a critical role in neural crest development during zebrafish development. Furthermore, hiPSC derived Neural crest stem cell migration is inhibited by OGM.  GPR68 is proton sensitive GPCR that is maximally active at ~pH6.6, and is upregulated in melanoma cell lines. We show that melanoma are more motile when acutely exposed to acidic media. Acidification promotes both increased formation of focal adhesion complexes and contractile strength of the melanoma. Furthermore, the OGM attenuates the formation of focal adhesions complexes, contractile capacity, and increase in migratory capacity. Taken together, the data suggests that pH mediated signaling is a critical component during embryonic development, and that GPR68 represents a possible novel pharmacological target for melanoma metastasis.


42. "Druggability Assessment of the CTLA-4 and PD- 1 Immune Checkpoint Pathways by Small Molecules," Evan Perry, Feng Wang, Bin Zhao, Tyson Rietz, Taekyu Lee, Jason Phan, Edward T. Olejniczak, Stephen W. Fesik


Cancer cells display a highly mutated phenotype resulting from the accumulation of many genetic alterations that result in the loss of normal cell regulatory processes. Many of these mutated cellular proteins are expressed as neoantigens on the tumor cell surface and are able to be differentiated from healthy cells by the immune system. Cytotoxic T cells with T cell receptors (TCRs) that are specific for the mutated tumor antigen are able to recognize and eliminate the tumor cells in a safe and nontoxic manner. Effective tumor-specific T cell activation and killing of tumor cells requires T cells to undergo a series of checkpoints to avoid unwanted activity towards healthy cells. Many checkpoints of the T cell life cycle have been identified as co-receptors expressed on the surface of T cells that modulate T cell activity and function. Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed death-1 (PD-1) are the first two checkpoints that were discovered to inhibit T cell function at the stages of T cell activation and tumor cell recognition, respectively. In attempt to stimulate the immune system to more aggressively fight tumor cells, antibody based therapeutics have been developed that block these inhibitory checkpoints interactions with their respective ligands. These therapeutics have shown unprecedented success in human clinical trials and have revolutionized the field of immuno-oncology. The development of small molecule inhibitors of the T cell checkpoints, however, is greatly lagging behind that of the antibody based therapeutics. We hypothesize that small molecule inhibitors of these checkpoint pathways will have preferred pharmacokinetic properties over antibody therapeutics potentially leading to an alternative therapeutic that is more cost effective, safer and better suited for combination therapies. Our lab is currently screening our fragment library of 15,000 diverse low molecular weight fragments against CTLA-4, PD-1 and its ligand PD-L1 by 2D heteronuclear magnetic resonance (NMR) screening methodology to access the druggability of these targets by small molecules. Fragments that bind to these proteins will also be followed up by SAR and structure based design strategies to develop potent small molecule inhibitors of these pathways.


43. "Progress Toward the Chemical Synthesis of Prostaglandin Metabolite PGD2-M," Jennifer Benoy, Ginger Milne, and Gary Sulikowski


Prostaglandin D2 (PGD2) levels affect smooth muscle contraction, sleep, platelet aggregation, anaphylaxis and many diseases, such as mastocytosis.  Interestingly, PGD2 has three major metabolites: PGD-M, Tetranor PGDM, and 15-deoxyD 12, 14 – PGJ2. While their respective routes of metabolism are understood, their function in different disease states remains unknown.  To study the role of oxidative stress on PGD2, an isotopically labeled internal standard of each of the metabolites is required for accurate quantification. Though there has been one successful synthesis of PGD-M, there is a need for a more direct and economic route to continue studies on PGD2 metabolism.  We propose that PGD-M can be accessed via two different synthetic routes.  One approach involves a cross coupling and contrasteric allylation, while the other features a diastereoselective C-H insertion.


44. "Stoichiometric Analysis and Discovery of Lysine and Arginine Modifications (StARKMod) from Complex Biological Samples," James J. Galligan, Jeannie M. Camarillo, Philip J. Kingsley,Orrette R. Wauchope, Michelle M. Mitchener, James A. Wepy, Peter S. Harris, Kristofer S. Fritz, Lawrence J. Marnett


Posttranslational modifications (PTMs) affect a proteins function, localization, and stability, yet very little is known about the stoichiometry of these modifications. Here, we describe a novel method to quantitate and assess the relative stoichiometry of Lys and Arg modifications (StARKMod) in complex biological settings. In addition, this platform can be used with synthetic protein and peptide substrates to quantitate alterations in PTMs. This method was used to determine the stoichiometry of Lys and Arg PTMs in murine liver subcellular fractions, revealing me3Lys as the most abundant PTM tested. Lastly, we describe a product ion scanning technique that offers the potential to discover unexpected and possibly novel Lys and Arg modifications.  In summary, this approach offers accurate quantitation of protein PTMs in complex biological systems without the requirement of high mass accuracy instrumentation.


45. Joseph P. Zackular, Jessica L. Moore, Ashley T. Jordan, Lillian J. Juttukonda, Michael J. Noto, Yaofang Zhang, Lorraine B. Ware, M. Kay Washington, Walter J. Chazin, Richard M. Caprioli, and Eric P. Skaar


This abstract will be included in the "VICB Student Research Symposium Abstracts" hand-out on the day of the symposium.


46. Reece J. Knippel, Joseph P. Zackular, and Eric P. Skaar

This abstract will be included in the "VICB Student Research Symposium Abstracts" hand-out on the day of the symposium.


47. "The use of fluorescently-tagged apoptolidins in cellular uptake and response studies," Katherine M. Chong, Nalin Leelatian, Sean M Deguire, Asa A Brockman, David Earl, Rebecca A Ihrie, Jonathan M Irish, Brian O Bachmann, and Gary A Sulikowski


The apoptolidins are glycomacrolide microbial metabolites reported to be selectively cytotoxic against tumor cells. Using fluorescent apoptolidin derivatives, we observe selective uptake of these tagged glycomacrolides in cancer cells over healthy human blood cells. We also demonstrated the utility of these fluorescently tagged glycomacrolides in fluorescent flow cytometer as a method to monitor cellular uptake and response of the glycomacrolides.


48. "The role of the engulfment receptor Jedi1 in dorsal root ganglia development and function," Alexandra J. Trevisan, Chelsea S. Sullivan, Mary Beth Bauer, Kevin P.M. Currie, and Bruce D. Carter


In the developing dorsal root ganglia (DRG), approximately half of the primary afferent sensory neurons initially born undergo apoptosis, leaving behind thousands of cellular corpses that are subsequently engulfed and degraded by the specialized glia of the DRG called satellite glial cells (SGCs). Inefficient phagocytosis of cellular debris has been implicated in neurodegenerative diseases in the retina, where deficiencies in the engulfment of photoreceptor outer segments by the retinal pigment epithelium causes vision loss and retinitis pigmentosa, and in Alzheimer's disease, where the inability to phagocytose Aβ plaques leads to glial activation and inflammation. Our lab previously showed through DRG neuron/glia co-culture that SGC-mediated phagocytosis of dying DRG neurons was dependent on JEDI1 (PEAR1/MEGF12), a mammalian homolog of the Drosophila and C. elegans engulfment receptor Draper and CED1, respectively. To determine whether Jedi1 mediates clearance of neurons in vivo and the effect this might have on somatosensory processing, we are examining the DRGs in Jedi1 knock-out mice. Our data demonstrate that the lack of Jedi1 significantly alters DRG development and function. We observed that lack of Jedi1 causes a deficiency in dead cell clearance, and, as a result, accumulation of cellular corpses. We hypothesize that lack of SGC mediated engulfment causes neurodegeneration in the DRG similar to the mechanisms that contribute to photoreceptor cell death and Alzheimer's disease as described above. We found that Jedi1 knock-out animals show a 25% loss in total neuron cell number and that there is an altered distribution of DRG neuron subtypes, causing a 30% increase in the percentage of capsaicin-sensitive nociceptors. Electrophysiological experiments show that the remaining Jedi1-knock neurons fire more action potentials in response to depolarizing current compared to their wild type counter parts, which may reflect the change in distribution of DRG neuron modality or indicate general neuron hypersensitivity. SGCs in jedi1-/- animals are also activated, as measured by GFAP expression, which resembles the gliosis that occurs during neurodegeneration. Despite the cellular changes to the DRG, we show no behavioral changes in basal non-noxious mechanical stimulation or thermal sensitivity in jedi1-/- mice. Future studies will assess the thermal sensitivity of jedi1-/- mice following sensitization by intraplantar capsaicin injection. In summary, we propose that loss of Jedi1 in SGCs causes inefficient clearance of developing sensory neurons, leading to neurodegeneration and gliosis.


49. "Modeling Clonal Heterogeneity in BRAF-mutant Melanoma," Corey E. Hayford, Leonard A. Harris, B. Bishal Paudel and Vito Quaranta


Melanoma is a particularly combative skin cancer that corresponds to dysregulation of the Mitogen-Activated Protein Kinase (MAPK) pathway. A large portion of melanoma patients have a mutation in the BRAF gene, leading to expression of constitutively active BRAF. Activation of downstream components MEK and ERK prompt expanded cell proliferation and inhibition of apoptotic signals. Melanoma patients with the BRAF mutations typically undergo treatment with BRAF inhibitors, such as Vemurafenib. Typically, the patient tumor will initially regress in the presence of drug, followed by tumor recurrence. We suspect tumor clonal heterogeneity is the primary reason behind treatment failure. Using BRAF-mutant melanoma cell line SKMEL5, we seek to understand the response dynamics of melanoma tumor cells to Vemurafenib research analog, PLX 4720. Using population-level cell proliferation assays, we have discovered a novel phenotype of post-drug, nutrient-abundant non-growth, which we term 'idling.'  Clonal sublines have been isolated and reflect three key phenotypes with initial differential drug sensitivity, all of which transition to idling drug tolerance. To explain the cell population response in the context of the clonal behavior, we have developed a simple three-state mathematical model of clonal competition and phenotypic state transitions. We test the robustness of model predictions using clonal mixture experiments, which we then use to optimize model parameters. The model provides a basis for future exploration of melanoma drug response dynamics, and will continue to be updated with new experimental data.


50. "Benchmarking a New Topology Scoring Metric in BCL:Fold," Maximilian Fritz, Michaela Fooksa, and Jens Meiler.


De novo prediction of a protein's fold from its primary sequence is a daunting challenge in modern structural biology. Despite the efforts that were put into algorithms able to reveal the tertiary structure of a given amino acid chain, there is yet no such algorithm that reliably predicts the topology of proteins with several hundred residues, as was shown during the CASP11 experiment. One major problem that appeared in the BCL::Fold prediction pipeline was that the algorithm was unable to select the most accurate models which were sampled. After initial sampling, models are clustered based on the RMSD100 metric, which quantifies the similarity between the alpha carbon coordinates of models; cluster medioids are then selected for further refinement. However, this metric is rather stringent with regard to minute structural changes. To improve the clustering algorithm, a new metric was designed that is based on the distances between several parts of secondary structure elements. The aim of this work is to compare the RMSD100 metric, the GDT_TS metric – two widely-used metrics for assessment of prediction accuracy – and the newly created metric in terms of their appropriateness for model selection. A benchmark on a set of proteins will be conducted show whether this approach for representation of topology of proteins is able to overcome the limitations of clustering by more traditional protein quality metrics.     


51. "Isomer separations in Ion Mobility-Mass Spectrometry: Fundamentals and Future Directions," James Dodds, Jody C. May and John A. McLean


The separation of biological species of interest in complex samples is a key aim of bioanalytical chemistry.  In this work we examine the potential of coupling ion mobility, a gas phase separation based on molecular structure, to traditional mass spectrometry techniques as an orthogonal approach to aid in the separation of isomeric species. This study examines various ion mobility techniques in order to examine the relationship between ion size and shape (Collision Cross Section, CCS) and separation efficiency. Overall, using both mathematical and experimental approaches a relationship between ion mobility efficiency (resolving power) and separations was observed for uniform field instruments.


52. "Improving Prediction of Helix‒Helix Packing in Membrane Proteins Using Predicted Contact Numbers as Restraints," Bian Li, Jeffrey Mendenhall, Elizabeth Dong Nguyen, Brian E. Weiner, Axel W. Fischer, and Jens Meiler

One of the challenging problems in computational prediction of tertiary structure of helical membrane proteins (HMPs) is the determination of rotation of α-helices around the helix normal. Incorrect prediction of rotation of α-helices around the helix normal substantially disrupts native residue–residue contacts while inducing only a relatively small effect on the overall fold. To address this problem, we previously developed a predictor for residue contact numbers (CNs), which measure the local packing degree of residues within the protein tertiary structure. In this study, we tested the idea of incorporating predicted CNs as restraints to guide the sampling of helix rotation. For a benchmark set of 15 HMPs with simple to rather complicated folds, contact recovery (CR) was improved for all targets, the likelihood of sampling models with CR greater than 20% was increased for 13 targets, and RMSD100 was improved for 12 targets. This study demonstrated that explicitly incorporating CNs as restraints improved the prediction of helix‒helix packing.


53. "A Modular Framework to Extend Rosetta Protocols with Multistate Design," Patrick Löffler, Samuel Schmitz, and Rainer Merkl

Computational protein design (CPD) is a powerful technique to design novel proteins. Many CPD objectives such as design on backbone ensembles, multi-specificity design and the integration of negative design demand the simultaneous optimization of multiple design states. Rosetta is a popular software suite to study and design proteins. Rosetta's protocols consist of specific procedures and a fine-tuned set of parameters to carry out a given task. An example is the use of specific sequence design cycles and catalytic constraints in the enzyme design protocol. At present, the multistate design implementation of Rosetta is a generic approach lacking options to fine tune the calculations in the same manner as specialized single state protocols.

We have developed a framework for CPD that integrates multistate design in existing Rosetta protocols while preserving the protocol's original functionality. Our framework consists of two, easily exchangeable components: i) The optimizer searches the sequence space and ii) the evaluator scores the sequences according to the given design task. Currently, we utilize Rosetta's genetic algorithm as an optimizer; the protocols for enzyme design or AnchoredDesig  serve as evaluators. However, due to the modularity of both components, multistate functionality can be transferred to arbitrary Rosetta applications with little effort. We benchmark the above two applications on two datasets consisting of conformational ensembles.


54. "Using quantitative proteomics to profile changes in individual immune cell types after an AS03-adjuvanted H5N1 vaccine," Allison C. Galassie, Johannes Goll, Parimal Samir, Travis Jensen, Kristen L. Hoek, Leigh M. Howard, Tara M. Allos, Xinnan Niu, Laura Gordy, C. Buddy Creech,  Heather Hill, Sebastian Joyce, Kathryn M. Edwards, Andrew J. Link


Vaccine adjuvants, particularly oil-in-water emulsion adjuvants, have been found to markedly enhance immune responses to pandemic influenza vaccines. However, the mechanisms by which adjuvants enhance immunogenicity are not well understood. We investigated global protein expression after either an Adjuvant System 03 (AS03)-adjuvanted or unadjuvanted split-virus H5N1 influenza vaccine in five primary human immune cell populations: neutrophils, monocytes, natural killer (NK) cells, T cells, and B cells. An immune cell common standard, prepared using cell lysates, normalized the 8-plex iTRAQ experiments, which were analyzed using MuDPIT on an LTQ Orbitrap XL. Peptides identified using Sequest were assembled into proteins and quantified using ProteoIQ. Protein level quantifications were obtained by calculating the median of log2 ratios compared to the standard for all assigned peptides. Permutation tests identified significantly different baseline fold changes for the AS03 vs. unadjuvanted group. The analysis set was comprised of quantifications for 3247 proteins (835 protein families) overall, with 533 (T-cells), 665 (NK-cells), 813 (B-cells), 860 (neutrophils), and 1,252 (monocytes) unique protein families. PCA showed that most of the total variation in protein ratios was due to cell type. Additionally, between-subject variability was significantly greater than within-subject variability. Distinct responses were observed in all cells, but monocytes demonstrated the strongest differential signal. Day 3 following the first AS03-adjuvanted vaccine dose, immunological pathways, including MHC class I-mediated antigen processing and presentation, were enriched in monocytes and neutrophils. Inflammation and oxidative stress proteins in monocytes (day 1) and immunoproteasome subunits (day 3) after the first adjuvanted vaccination predicted seroprotective antibody responses at day 56 (28 days post-second dose). While comparison between proteomic and transcriptomic results showed little overlap overall, enrichment of the Class I antigen processing and presentation pathway and increase in PSME2 (proteasome activator complex subunit 2) expression following AS03 adjuvanted vaccination was confirmed in monocytes by both approaches.


55. "Single Oligomer Polyurethane Synthesis: Characterization by Ion Mobility-Mass Spectrometry and Computational Strategies," Tiffany Crescentini, Sarah Stow, Robert Davis, Gary Sulikowski, David Hercules, John McLean


Polyurethanes (PURs) shape the world around through numerous consumer products. The unique properties associated with PURs are attributed to the number and orientation of hard and soft blocks within a chain. Methylenedianaline (MDA) is a precursor to 4,4-methylene diphenyl diisocyanate (MDI), which is the major hard block component in PURs. Soft blocks are long flexible segments such as an ester (E) group; in this work, E is composed of 1,4-butanediol (B) and adipic acid (A). Previously, we structurally characterized 2-ring MDA isomers (4,4-MDA, 2,4-MDA, and 2,2-MDA), 3-ring and 4-ring MDA regioisomers using a combination of electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) IM-MS, MS/MS and computational modeling. We observed significant differences between the mass spectra from ESI and MALDI. In our MALDI studies, the formation of a [M-H]+, [M.]+, and [M+H]+ ions were observed, whereas in ESI only [M+H]+ ions were seen.


Using these same mass spectrometry characterization techniques, PUR isomers (H-B-E-MDI-E-B-H and H-B-E-E-MDI-B-H) are being synthesized using a single oligomer stepwise synthesis. Initial computational modeling of the H-B-M-E-E-B-H and H-B-E-M-E-B-H compounds indicate that the distribution of collision cross section (CCS) theoretical values significantly differ between species. When the molecule is more symmetrical, trends indicate that the CCS value increases vs that of the nonsymmetrical molecule. Synthesizing these PUR isomers will allow us to better understand structural heterogeneity within a polymer. Proton addition and cation studies will elucidate an understanding of where fragmentation occurs within these species, leading to a 1,3- and 1,5-hydrogen shift. This study of isomeric PUR synthesis and characterization will aid in our understanding polymer heterogeneity and structural-performance between isomers.


56. "A New Convergent Synthesis of 1,3,4-Oxadiazoles from Acyl Hydrazides," Kazuyuki Tokumaru and Jeffrey N. Johnston


In medicinal chemistry, the 1,3,4-oxadiazole is useful as an aromatic ring spacer which can modulate molecular properties due to its relatively low lipophilicity among common monocyclic aromatic rings. In other cases, this heterocycle can be used as a metabolically stable bioisostere for carbonyl moieties, such as amide, ester, and carbamate. Although various methods for the construction of the 1,3,4-oxadiazole core are reported, most of them have limitations. For example, the dehydrative cyclization of 1,2-diacyl hydrazides often requires harsh conditions such as strong acid, high temperature, or a strong desiccant, that can limit chemoselectivity. We have developed a new method for the construction of substituted 1,3,4-oxadiazoles from monoacyl hydrazide under mild conditions. Umpolung Amide Synthesis (UmAS) enables amide bond construction from halonitroalkanes and aliphatic amines with the assistance of a halonium reagent. Application of this method to monoacyl hydrazides resulted in the formation of 1,3,4-oxadiazoles directly at ambient temperature. Mechanism-guided reaction analysis led to the finding that diacyl hydrazide, a possible intermediate in a dehydration-based mechanism, is not formed. This new reaction showed broad functional group tolerability on substrates and gave desired 1,3,4-oxadiazoles in moderate to good yield.


57. "Antibacterial photosensitization through activation of coproporphyrinogen oxidase," Matthew C. Surdel, Lisa J. Lojek, Pedro L. Teixeira, Brendan F. Dutter, Matthew Albertolle, Gary A. Sulikowski, D. Borden Lacy, Harry Dailey, & Eric P. Skaar


Gram-positive bacteria cause a majority of skin and soft tissue infections (SSTIs), resulting in the most common reason for clinic visits in the United States. Recently it was discovered that Gram-positive pathogens utilize a unique heme biosynthesis pathway, which implicates this pathway as a novel target for development of antibacterial therapies. Small molecule '882 was previously identified in a screen for activators of the Staphylococcus aureus heme sensing system (HssRS), which induces the expression of the heme-regulated transporter (HrtAB) to alleviate heme toxicity. HssRS activation is triggered by massive accumulation of heme in '882-exposed bacteria; however, the mechanism by which '882 activates heme biosynthesis has not been uncovered. To identify the target of '882, a suicide strain containing a Phrt-driven relE construct was employed enabling selection of S. aureus strains that are unresponsive to '882.  This uncovered numerous residues within HssRS and the hrt promoter required for hrtAB activation. In addition, a strain lacking mutations in this region was identified and was found to have a mutation in coproporphyrinogen oxidase (HemY).  In keeping with this, we have shown that '882 is a small molecule activator of HemY from Gram-positive bacteria, an enzyme essential for heme biosynthesis. Activation of HemY induces accumulation of coproporphyrin III and leads to photosensitization of Gram-positive pathogens. In combination with light, HemY activation reduces bacterial burden and tissue ulceration in murine models of SSTI, including S. aureus and Propionibacterium acnes. Thus, small molecule activation of HemY represents an effective strategy for the development of light-based antimicrobial therapies.


58. "Computational Design of Positive Allosteric Modulators of Metabotropic Glutamate Receptors," Benjamin K. Mueller, Pedro M. Garcia-Barrantes, Craig W. Lindsley, Jens Meiler


Metabotropic glutamate receptors (mGlu) are class C G-protein-coupled receptors (GPCRs) found throughout the mammalian central nervous system and are responsible for synaptic transmissions and modulating neuronal excitability. Separate from the extracellular orthosteric glutamate binding site, the transmembrane domain of mGlu proteins bind allosteric ligands. These ligands can up- or down-regulate the response to glutamate binding. These ligands are an attractive therapeutic target and studies have shown that these ligands have the potential to treat many neurological disorders such as: Alzheimer's disease, Parkinson's disease, epilepsy, schizophrenia, depression, and anxiety disorders, among others. However, while progress has been made in developing therapeutics, developing mGlu specific ligands remains a challenge.


Cocrystal structures of allosteric modulators bound to both mglu1 and mglu5 have been published paving the way for more detailed structure activity relationship (SAR) studies. In addition, recent algorithms have been developed that evaluates partial covalent interactions in proteins, currently this is being expanded for ligand / protein interactions. This will better describe the more nuanced ligand / protein interactions, while still allowing for fast sampling. These computationally designed ligands, using more detailed interaction descriptors, will allow for more precise chemical synthesis of mglu1 allosteric modulators.


59. "Structure-based Discovery of Selective DDR1 Receptor Kinase Inhibitors," Marcin J. Skwark, Nick Steyns, Sandeepkumar Kothiwale, Corina Borza, Ambra Pozzi, Jens Meiler


Receptor tyrosine kinases (RTKs) are cell-surface receptor proteins with high affinity for a variety of cytokines, growth factors and hormones, thus regulating vast array of cellular processes, including cell division. RTKs are also implicated in development and progression of many cancers. Discoidin domain receptor family (DDR) proteins differ from other RTKs, as they are activated by matrix collagen. DDRs binding collagen makes them unlike other RTKs, as collagen is neither small, scarce or soluble, which differs from the ligands of the rest of RTKs. Despite binding collagen, DDRs are unlike collagen receptors, as on contrary to e.g. integrins, they do not undergo major conformational changes upon activation. Dysregulation of DDR1 plays central role in breast, ovarian and esophageal cancers, as well as inflammatory conditions, such as pulmonary fibrosis and arteriosclerosis.

As tyrosine kinases are attractive targets for development of cancer therapeutics (e.g. imatinib/Gleevec), there are several classes of DDR1 kinase domain inhibitors already developed, some of which have been demonstrated to have a therapeutic potential. These drug-like compounds have rather poor target specificity and thus cannot be effectively used in clinical settings.

In this work we present an ongoing research aiming to discover a novel, selective DDR1 inhibitor. We developed an iterative protocol, which blends computational drug design and discovery, macromolecular modelling and experimental screening. The core of the method is a machine learning QSAR model aiming to discriminate between the compounds active and inactive for DDR1. We use this model to both inform the searches against the databases of known compounds and to assess the viability of the novel, designed ones. We use protein-ligand docking to elucidate the binding poses of candidate compounds to a vast array of tyrosine kinase domains. This enables us to construct an accurate pharmacophore of DDR1 inhibitors and find the characteristics distinguishing DDR1 kinase domain from the other tyrosine kinases. Compounds identified by computational methods are then subject to TR-FRET (time-resolved fluorescence energy transfer) assays to ascertain their binding.


Finding a high-affinity, selective DDR1 inhibitor would be instrumental in devising new therapy strategies for breast, ovarian and esophageal cancers, as well as pulmonary fibrosis.      


60. "A Critical Comparison of Collision Induced Dissociation and Surface Induced Dissociation for Metabolomics," Rachel A. Harris, Ewa Jurneczko, Sophie R. Harvey, Vicki H. Wysocki, John A. McLean


High-dimensional measurements using ion mobility-mass spectrometry (IM-MS) have found great utility in metabolite identification, in particular when combined with CID activation for IM-MS/MS [1]. Recently surface induced dissociation (SID) has emerged as an alternative to CID that has been demonstrated as an effective tool to probe protein quaternary structure [2]. In contrast to CID, SID imparts a large quantity of energy to analyte molecules in a single, rapid collision step so that dissociation proceeds under kinetic, rather than thermodynamic, control. In the analysis of proteins, the unique fragmentation pathways accessed by SID allow for minimal disruption of the protein complex native state [3].  This report examines the utility of SID for metabolomic analyses, specifically lipids, carbohydrates, and metabolically relevant small molecules. Comparisons of CID and SID for these systems are used to evaluate differences in fragmentation spectra for enhanced molecular characterization. 


61. "A Cyclooxygenase-related Fatty Acid Dioxygenase in Mycobacterium tuberculosis: Protein Expression, Purification, and Enzymatic Activity," Carsyn K. Snagg, William E. Boeglin, Zahra Mashhadi, and Alan R. Brash


Tuberculosis (TB) is a highly infectious airborne disease caused by a common pathogenic bacterium, Mycobacterium tuberculosis (Mt). Recent sequencing of a drug-resistant strain of Mt by the Sanger Institute revealed a putative cyclooxygenase-related fatty acid dioxygenase in the genome. Analysis of the Mt protein using Swiss-Model indicated an excellent structural homology to mammalian COX-1. This Mt dioxygenase gene sits in a context with adjacent fatty acid desaturase, a cytochrome P450, and an uncharacterized 76 kD protein. We cloned the Mt COX-related gene into a pET32a vector for overexpression in E. coli. Under at least twenty different conditions the protein expressed but was insoluble and with no catalytic activity. Successful expression of the 60 kD Mt dioxygenase was achieved at 14°C for 44 h as a thioredoxin fusion protein with added trace metals, hematin, δ-ALA, and with IPTG induction. In common with many other bacteria, M. tuberculosis does not contain polyunsaturated fatty acids which are the natural substrates for mammalian COX; monounsaturated fatty acids related to oleic acid (C18:1ω9) are present in Mt and oleate was tested as a potential substrate for the Mt dioxygenase.  Robust metabolism of [9,10-3H]oleic acid was demonstrated, the major more polar product having a UV chromophore with λmax at 226 nm, likely representing transformation of an initially formed fatty acid hydroperoxide. Further analyses of the activity will utilize UV-Vis spectrometry, oxygen uptake assays (O2 electrode), HPLC and LC-MS. A knowledge of the activity of this novel MT dioxygenase will represent an important step towards understanding its potential role in this strain of TB, as will characterization of the neighboring desaturase, P450 and the unknown gene product.




































































































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