2011 Keynote Video
2011 Oral Presentations
Oral Presentation & Poster Abstracts
Oral Presentation Abstracts
1) Dawn M. Makley, "Umpolung Amide Synthesis: Application to the Enantioselective Preparation of Peptide." The application of a novel amide synthesis developed in the Johnston lab, Umpolung Amide Synthesis (UmAS), to the enantioselective preparation of peptides will be presented. In this coupling reaction, α-bromo nitroalkanes are used as nucleophilic carbonyl surrogates, reacting with electrophilically activated amines to form the desired amide bond. Notably, the polarities of these two reactants are reversed (umpolung) from those seen in traditional amide coupling. In addition to being mechanistically interesting, the fact that the carbonyl surrogate is nucleophilic, as opposed to electrophilic, in nature means that epimerization at the α-carbon, an unsolved problem in traditional amide coupling, is mechanistically prohibited. Additionally, we are able to synthesize chiral α-bromo nitroamines, with high levels of enantioselection, using the chiral proton-catalyzed enantioselective addition of bromonitromethane to imines. By combining these two methodologies, we are able to enantioselectively synthesize, and incorporate into peptides, a number of arylglycine residues in a highly stereocontrolled manner. We demonstrate the utility of this methodology in our efforts towards an enantioselective synthesis of the naturally occurring, arylglycine-rich peptide feglymycin.
2) Will Birmingham, "Engineering a Biosynthetic Pathway for the Nucleoside Analog Drug Didanosine." Dideoxyinosine, also called didanosine, is a generic second line treatment for HIV as well as hepatitis and other viral infections. Access to this drug is greatly limited due to the significant expense for continued therapy—a cost that is primarily the result of a lengthy and wasteful chemical synthesis procedure. Our lab is proposing the synthesis of ddI through a chemo-enzymatic pathway. The pathway begins with a three step chemical synthesis of dideoxyribose from the cheap starting material glutamic acid, and then catalysis by three enzymes completes the formation of ddI. A combination of site-directed saturation mutagenesis as well as multiple rounds of error prone PCR will be used to augment activity of enzymes for non-natural substrates. Completion and implementation of the intended pathway may allow new means for efficient large-scale production of this life saving drug using renewable resources and green methods. This, in turn, may greatly reduce the cost of treatment, in addition to decreasing chemical waste, and would enable a vastly larger population to afford therapy for a life threatening condition.
3) Laura L. Anzaldi, "A Small Molecule that Modulates Staphylococcus aureus Respiration to Activate the Heme Stress Response." Staphylococcus aureus is a Gram positive pathogen that causes significant morbidity and mortality worldwide. This combined with the continuing rise of antibiotic resistance among clinical isolates highlights the need for innovative therapeutics. Most available therapeutics target the machinery required for bacterial replication in culture and none target energy generation via respiration. This is due to the fact that S. aureus can generate energy three ways: aerobic respiration, anaerobic respiration, and fermentation. The metabolic flexibility of this pathogen suggests that each strategy for energy production provides the bacteria advantages during infection. Aerobic and anaerobic respiration both require heme in order to generate energy. To fuel respiration, S. aureus can synthesize heme de novo under iron replete conditions orsteal host heme during infection when iron is scarce. Paradoxically, too much heme is toxic. We have previously identified a bacterial signaling system, named the heme sensor system (HssRS) that senses heme and protects the bacteria from heme toxicity. In an effort to elucidate the mechanism by which HssRS is stimulated we screened the VICB small molecule library to identify compounds that activate of HssRS. We have found that our most potent primary hit VU0038882 (‘882) causes heme stress by increasing endogenous levels of heme. By manipulating S. aureus genetically, pharmacologically, and nutritionally we have also established that ‘882 is particularly toxic to bacteria that are forced to ferment. Based on these data, we propose a model that ‘882 inhibits fermentation forcing the bacteria to respire which increases endogenous heme biosynthesis and activates HssRS. The metabolic strategies utilized by invading pathogens remain ill-defined though it is likely that many pathogens rely on fermentation at stages of infection. Thus ‘882 has great potential as a biological probe to elucidate bacterial respiratory pathways and may have clinical relevance in treatment of bacterial infections.
4) Sean DeGuire, "Synthesis of a Bicyclobutane Fatty Acid Identified from the Cyanobacterium Anabaena PCC 7120." Bicyclo[1.1.0]butylanabaenic acid methyl ester was isolated in 2007 from the cyanobacterium Anabaena sp. PCC 7120. It is the only natural product isolated to date containing the highly strained bicyclyobutane ring system. The potential for the identification of interesting biological roles that this novel oxylipin metabolite may have and the unique structure of the molecule attracted our lab to pursue a synthesis. Studies towards this synthesis have lead to the development of a vinylogous epoxide opening cyclization method for the formation of substituted vinyl bicyclobutanes. This reaction has being further implemented in a cascade reaction to form the natural product.
5) Ewa A. Kowal, "Structure and Stability of DNA Duplex Containing Exocyclic Deoxyadenosine Adducts Induced by 1,2,3,4-Diepoxybutane." 1,3-Butadiene is produced in large volumes for use in the manufacture of synthetic rubber and of thermoplastic resins. It is listed as a known human carcinogen. It is associated with A →T transversion mutations. Four exocyclic deoxyadenosine adducts of diepoxybutane (DEB), a carcinogenic metabolite, were identified: N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (1) (R,R-N6,N6-DHB-dA), (2) (S,S-N6,N6-DHB-dA), 1,N6-(2-hydroxy-3-hydroxymethylpropan-1,3-diyl)-2′-deoxyadenosine (3) (1,N6γ-HMHP-dA), and 1,N6-(1-hydroxymethyl-2-hydroxypropan-1,3-diyl)-2′-deoxy-adenosine (4) (1,N6-α-HMHP-dA)1,2. None of these adducts form Watson-Crick base pairs, and they are all anticipated to be pro-mutagenic lesions. We are utilizing NMR spectroscopy to determine how these adducts alter the structure and dynamics of DNA, in an effort to understand their mutagenic properties. The imino proton of the thymine which was complementary to either adducts 1 or 2 was severely exchange broadened. Thermal melting studies indicated reduced stabilities for duplexes containing either adducts 1 or 2. The H6/H8-H1’ region of the NOESY spectra of duplexes containing adducts 1 or 2 were similar as compared to the unmodified duplex, suggesting localized structural changes at the lesion sites. The structure of the duplex containing adduct 1 shows that the T6 base, which is complementary to the adduct, flips out from the duplex into major groove; the structure of the duplex containing adduct 2 appears to be similar. At nucleoside level adduct 3 undergoes the Dimroth-like rearrangement to form adduct 41. NOESY experiments were performed for adduct 3. The spectrum shows break in the connectivity between H8 of the adduct and bases adjacent to the lesion. Modification also affected a chemical shift of the T6 which is complementary to the adduct. The sample was heated and cooled to generate adduct 4 and NMR experiments were repeated.
6) Suraj Adhikary, "Structural Basis of DNA Damage Recognition and Repair by Schizosaccharomyces pombe 3-methyladenine DNA Glycosylase (Mag1)." Maintenance of genomic integrity depends on the ability to properly repair DNA damage. Alkylpurine DNA glycosylases initiate the base excision repair (BER) pathway by recognizing and removing a broad spectrum of alkylated nucleobases from DNA that arise from exposure to endogenous methyl donors and metabolites, environmental toxins, and chemotherapeutic agents. Glycosylases catalyze the hydrolysis of the base-sugar glycosylic bond, although the precise catalytic mechanism as well as the mechanism of detection and discrimination of damaged bases from normal and other damaged bases by DNA glycosylases, is still largely unknown. Lesion detection by DNA glycosylases is presumed to arise from both active site complementarity with the target nucleotide as well as intercalating residues that probe the minor groove of DNA during a processive search. Mag1 shares extensive sequence similarity with 3-methyladenine glycosylases from S. cerevisiae and E. coli (MAG and AlkA, respectively). However, unlike MAG and AlkA, which recognize and excise a wide array of damaged nucleobases including 1-N6-ethenoadenine (εA), Mag1 has a relatively limited number of substrates that include 3-methyladenine, 7-methylguanine, and 3-methylguanine. Here we present structural and biochemical evidence that a residue outside of the active site can confer substrate specificity in DNA glycosylases. Coupled with mutational analysis of spMag1, scMAG, and the Mag homolog from Bacillus halodurans (bhMag), this study offers a new model of substrate recognition among structurally homologous enzymes with disparate specificities.
7) Armand Guiguemde, "Screening for New Antimalarials." There has historically been a dearth of chemotypes that can be used to drive drug development campaigns for malaria. We utilized a phenotypic whole cell screen of erythrocytic co-cultures of malaria to identify novel chemical compounds that suppressed growth of or killed malaria. Representative members of the hit set were carefully profiled in a number of assays to understand selectivity, mechanism of action, and potential for development. From these studies, three series were selected for optimization as preclinical candidates. The process, findings and and resulting high priority leads will be discussed.
8) Paige Selvy, "Molecular Mechanism of Phospholipase D Inhibitors." Increasing evidence supports an integral role for the lipid second messenger phosphatidic acid (PtdOH) in cell signaling. In addition to altering curvature of biological membranes, PtdOH lies at the intersection between metabolism and signaling pathways and is involved in cell survival as well as proliferation pathways. Phospholipase D (PLD), an evolutionarily conserved phosphodiesterase that hydrolyzes phosphatidylcholine, is involved in generating signal-mediated PtdOH molecular species. Aberrant PLD expression and activity have been implicated in metastatic cancers, neurological disorders, and microbial pathogenesis, making this enzyme a potential therapeutic target for which we recently developed potent and isoform-selective pharmacological inhibitors. Here we define the mechanism of action for these novel small molecule inhibitors. Using backscattering interferometry, a novel method for measuring protein-lipid binding, we demonstrate that these compounds directly interact with PLD to allosterically block interfacial lipid binding and catalytic activity. Subcellular localization studies confirm these findings and demonstrate these compounds block signal-mediated translocation but not basal localization of the enzyme. This unanticipated finding represents a unique mechanism of phospholipase inhibition and a new paradigm for modulation of signaling pathways.
Poster Presentation Abstracts
(Poster presentation abstracts will be posted upon submisson.)
1) Lilu Guo, "Phosphatidylethanolamines modified by γ-ketoaldehyde Induce endoplasmic recticulum stress responses and endothelial activation," Lilu Guo, Zhongyi Chen, Brian E. Cox, Venkataraman Amarnath, Raquel F. Epand, Richard M. Epand, and Sean S. Davies. Peroxidation of plasma lipoproteins has been implicated in the endothelial cell activation and monocyte adhesion that initiates atherosclerosis, but the exact mechanisms underlying this activation remain unclear. Lipid peroxidation generates lipid aldehydes, including the γ-ketoaldehydes (γKA)† also termed isoketals or isolevuglandins, that readily modify the amine headgroup of phosphatidylethanolamine (PE). We hypothesized that aldehyde modification of PE could mediate some of the proinflammatory effects of lipid peroxidation. We found that PE modified by γKA (γKA-PE) induced THP-1 monocyte adhesion to human umbilical cord endothelial cells (HUVEC). γKA-PE also induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in HUVEC. To determine the structural requirements for γKA-PE activity, we tested several related compounds. PE modified by 4-oxo-pentanal (OPA-PE) induced THP-1 adhesion, but N-glutaroyl-PE and C18:0N-acyl-PE did not, suggesting that an N-pyrrole moiety was essential for cellular activity. As the N-pyrrole headgroup might distort the membrane, we tested the effect of the pyrrole-PEs on membrane parameters. γKA-PE and OPA-PE significantly reduced the temperature for the liquid crystalline to hexagonal phase transition (TH) in artificial bilayers, suggesting that these pyrrole-PE markedly altered membrane curvature. Additionally, fluorescently labeled γKA-PE rapidly internalized to the endoplasmic reticulum (ER), γKA-PE induced CHOP and BiP expression and p38MAPK activity, and inhibitors of ER stress reduced γKA-PE induced CHOP and BiP expression as well as EC activation, consistent with γKA-PE inducing ER stress responses that have been previously linked to inflammatory chemokine expression. Thus, γKA-PE is a potential mediator of the inflammation induced by lipid peroxidation.
2) Suraj Adhikary, "Structural basis of DNA damage recognition and repair by Schizosaccharomyces pombe 3-methyladenine DNA glycosylase (Mag1)," Suraj Adhikary, Brandt F. Eichman. Maintenance of genomic integrity depends on the ability to properly repair DNA damage. Alkylpurine DNA glycosylases initiate the base excision repair (BER) pathway by recognizing and removing a broad spectrum of alkylated nucleobases from DNA that arise from exposure to endogenous methyl donors and metabolites, environmental toxins, and chemotherapeutic agents. Glycosylases catalyze the hydrolysis of the base-sugar glycosylic bond, although the precise catalytic mechanism as well as the mechanism of detection and discrimination of damaged bases from normal and other damaged bases by DNA glycosylases, is still largely unknown. Lesion detection by DNA glycosylases is presumed to arise from both active site complementarity with the target nucleotide as well as intercalating residues that probe the minor groove of DNA during a processive search. Mag1 shares extensive sequence similarity with 3-methyladenine glycosylases from S. cerevisiae and E. coli (MAG and AlkA, respectively). However, unlike MAG and AlkA, which recognize and excise a wide array of damaged nucleobases including 1-N6-ethenoadenine (εA), Mag1 has a relatively limited number of substrates that include 3-methyladenine, 7-methylguanine, and 3-methylguanine. Here we present structural and biochemical evidence that a residue outside of the active site can confer substrate specificity in DNA glycosylases. Coupled with mutational analysis of spMag1, scMAG, and the Mag homolog from Bacillus halodurans (bhMag), this study offers a new model of substrate recognition among structurally homologous enzymes with disparate specificities.
3) Marta W. Szulik, "Structural studies of DNA containing a 7-aminomethyl-7-deaza-2'-deoxyguanosine adduct," Marta W. Szulik, Manjori Ganguly, Barry Gold, and Michael P. Stone. Incorporation of 7-aminomethyl-7-deaza-2'-deoxyguanosine (7amG) into the self-complementary dodecamer d(GAGAXCGCTCTC)2, X = 7amG, tethered a site-specific ammonium ion into the major groove of DNA at neutral pH. Thermodynamic data revealed that the tethered ammonium ion stabilized the duplex. NMR spectroscopy as a function of temperature revealed that the two 5' neighboring base pairs A4⋅T9 and G3⋅C10 were stabilized with respect exchange of their imino protons with solvent. Molecular dynamics calculations restrained by experimental NMR data revealed that the tethered ammonium ion was approximately in plane with the modified base pair. Base stacking and Watson-Crick base pairing was maintained throughout the duplex. The tethered ammonium ion was >5Å from the 5'-phosphate oxygen, indicating that it did not form a salt bridge to its 5'-phosphate. In contrast, placing a neutral hydroxymethyl tether at the same site destabilized this DNA duplex. The results confirm the role of major groove cation binding in stabilizing duplex DNA structure. Supported by NIH grants R01 CA-76049 (B.G.) and R01 CA-55678 (M.P.S).
4) Laura L. Anzaldi, "A small molecule that modulates Staphylococcus aureus respiration to activate the heme stress response," Laura L. Anzaldi, Paul F. Reid, Brendan F. Dutter, Nicholas Vitko, Anthony R. Richardson, Gary A. Sulikowski, & Eric P. Skaar. Staphylococcus aureus is a Gram positive pathogen that causes significant morbidity and mortality worldwide. This combined with the continuing rise of antibiotic resistance among clinical isolates highlights the need for innovative therapeutics. Most available therapeutics target the machinery required for bacterial replication in culture and none target energy generation via respiration. This is due to the fact that S. aureus can generate energy three ways: aerobic respiration, anaerobic respiration, and fermentation. The metabolic flexibility of this pathogen suggests that each strategy for energy production provides the bacteria advantages during infection. Aerobic and anaerobic respiration both require heme in order to generate energy. To fuel respiration, S. aureus can synthesize heme de novo under iron replete conditions orsteal host heme during infection when iron is scarce. Paradoxically, too much heme is toxic. We have previously identified a bacterial signaling system, named the heme sensor system (HssRS) that senses heme and protects the bacteria from heme toxicity. In an effort to elucidate the mechanism by which HssRS is stimulated we screened the VICB small molecule library to identify compounds that activate of HssRS. We have found that our most potent primary hit VU0038882 (‘882) causes heme stress by increasing endogenous levels of heme. By manipulating S. aureus genetically, pharmacologically, and nutritionally we have also established that ‘882 is particularly toxic to bacteria that are forced to ferment. Based on these data, we propose a model that ‘882 inhibits fermentation forcing the bacteria to respire which increases endogenous heme biosynthesis and activates HssRS. The metabolic strategies utilized by invading pathogens remain ill-defined though it is likely that many pathogens rely on fermentation at stages of infection. Thus ‘882 has great potential as a biological probe to elucidate bacterial respiratory pathways and may have clinical relevance in treatment of bacterial infections.
5) Bianca Sirbu, "Analysis of Protein Dynamics at Active, Stalled and Collapsed Replication Forks," Bianca M. Sirbu, Frank B. Couch, Jordan T. Feigerle, Srividya Bhaskara, Scott W. Hiebert, David Cortez. Successful DNA replication and packaging of newly synthesized DNA into chromatin are essential to maintain genome integrity. Defects in the DNA template challenge genetic and epigenetic inheritance. Unfortunately, tracking DNA damage responses (DDRs), histone deposition, and chromatin maturation at replication forks is difficult in mammalian cells. Here we describe a technology called iPOND (isolation of proteins on nascent DNA) to analyze proteins at active and damaged replication forks with high spatil and temporal resolution. iPOND uses click chemistry to biotin-tag nascent DNA and facilitate single-step streptavidin purification of proteins bound to nascent DNA. Combining iPOND with pulse-chase methodologies, we define the timing of histone deposition and chromatin maturation. Furthermore, we show that fork stalling with hydroxyurea causes changes in the recruitment and phosphorylation of proteins at the damaged fork. Checkpoint kinases catalyze H2AX phosphorylation, which spreads from the stalled fork to include a large chromatin domain even prior to double-strand break formation. We demonstrate a switch in the DDR at persistently stalled forks that includes MRE11 nuclease-dependent RAD51 assembly. These data reveal a dynamic recruitment of proteins and post-translational modifications at damaged forks and surrounding chromatin. Furthermore, our studies establish iPOND as a useful methodology to study DNA replication and chromatin maturation.
6) Zachary Glaser, "Docking and Synthesis of Novel Ligands for Labeling hSERT via Quantum Dots," Zachary Glaser, Steven Combs, Ian Tomlinson, Jens Meiler, Sandra Rosenthal. The human serotonin transporter (hSERT) is implicated in several brain disorders and the monoamine theory postulates that disruption or malfunction of the central nervous system’s serotonergic system increases the risk of developing depression. High affinity ligands conjugated to quantum dots (qdots) may be used to image the hSERT protein in live cell cultures. Qdot labeling of hSERT has been achieved using a ligand composed of a hSERT antagonist attached to an alkyl spacer. This is then coupled to a biotinylated polydispersed polyethylene glycol (PEG) 5000 linker. The terminating biotin forms the point of attachment to streptavidin-coated quantum dots. It has been demonstrated that the length of the alkyl spacer is important for biological activity, and compounds with a longer alkyl spacer have a higher affinity than those with a short spacer. Additionally, increasing the alkyl spacer increases nonspecific binding to the membrane. Computational docking of candidate parent drugs with different alkyl spacer lengths using the hSERT homology model was performed to predict the optimal ligand for quantum dot labeling of the protein.
7) Oleg Kovtun, "Single-Molecule Analysis of Dopamine Transporter Trafficking Using Ligand-Conjugated Quantum Dots," O. Kovtun, I. D. Tomlinson, D. Sakrikar, J. C. Chang, R. D. Blakely, and S. J. Rosenthal. The presynaptic dopamine (DA) transporter (DAT) is responsible for DA inactivation following release and is a major target for the psychostimulants cocaine and amphetamine. Dysfunction and/or polymorphisms in human DAT (SLC6A3) have been associated with schizophrenia, bipolar disorder, Parkinson's disease, and attention-deficit hyperactivity disorder (ADHD). Despite the clinical importance of DAT, many uncertainties remain regarding the transporter regulation, in part due to the poor spatiotemporal resolution of conventional methodologies and the relative lack of DAT-specific fluorescent probes suitable for single-molecule analyses. We developed a quantum dot (QD)-based labeling approach that uses a DAT-specific, biotinylated ligand, 2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (IDT444), that can be captured by streptavidin-conjugated QDs. Flow cytometry and high-speed, line scanning confocal microscopy were used to detect DAT in stably- and transiently-transfected mammalian cells. Single DAT molecules labeled with IDT444-QD complexes were demonstrated to undergo acute redistribution from the plasma membrane to endosomal compartments following phorbol ester-mediated PKC activation. Using this approach, we also detected and quantified, for the first time, the lateral diffusion of single, cell surface DAT proteins. Ongoing studies seek to determine the impact of signal transduction pathways, protein-protein interactions, and disease-associated DAT mutants on single-molecule diffusion kinetics. Our methods provide new opportunities to elucidate the molecular mechanisms supporting DAT regulation.
8) Robert Lavieri, "Defining the Roles of Phospholipase D Isoforms in Cancer via Isoform-selective Inhibitors," Robert R. Lavieri, Sarah A. Scott, Paige E. Selvy, Kwangho Kim, J. Scott Daniels, H. Alex Brown, and Craig W. Lindsley. Pospholipase D (PLD) catalyzes the production of the lipid second messenger phosphatidic acid (PA). PLD expression and/or enzymatic activity are both elevated in a variety of human cancers. Inhibition of PLD enzymatic activity, via genetic or biochemical methods, leads to decreased cancer cell invasion and decreased cancer cell survival. The aforementioned evidence provided the impetus for our medicinal chemistry project focused on the development of isoform-selective PLD inhibitors. The development of such inhibitors is an essential step in advancing the study of PLD as a potential cancer drug target. A group from Novartis published a report in 2007 disclosing halopemide as a hit from a high throughput screen for PLD inhibitors. While we initiated our iterative analog synthesis with halopemide we have explored a broad chemical space. We utilized technology-enabled synthesis to develop a library of approximately 600 compounds. This effort has yielded the most potent, isoform-selective PLD inhibitors described to date. While halopemide inhibits both PLD isoforms relatively equally VU0359595 inhibits PLD1 1,700 times more potently than PLD2, and VU0364739 inhibits PLD2 75 times more potently than PLD1. In a triple negative breast cancer cell line, MDA-MB-231, VU0364739 significantly decreases cell viability whereas VU0359595 does not affect cell viability; indicating that a PLD2 selective inhibitor may be the optimal therapeutic agent in this particular malignancy. We are currently utilizing our novel, isoform-selective PLD inhibitors in order to more clearly characterize the role of PLD signaling in oncogenic processes such as the suppression of apoptosis, cell invasion and metastasis.
9) Edward Prage, "Identification of Inhibitor Binding Sites in the Inducible Prostaglandin E Synthase, MPGES1," Edward B. Prage, Sven-Christian Pawelzik, Laura S. Busenlehner, Kwangho Kim, Ralf Morgenstern, Per-Johan Jakobsson, and Richard N. Armstrong. The inducible microsomal prostaglandin E synthase 1 (MPGES1) is an integral membrane protein co-expressed with, and functionally coupled to, cyclooxygenase-2 (COX-2) generating the pro-inflammatory lipid mediator PGE2. MPGES1 holds promise as a therapeutic target, as an alternative to COX inhibition, to treat inflammatory diseases. Here we describe the use of backbone amide hydrogen/deuterium (H/D) exchange mass spectrometry (MS) to map the binding sites of different types of MPGES1 inhibitors. The results reveal the locations of spatially distinct binding sites including the glutathione (GSH) cofactor site and the putative substrate binding site. Since there are no crystal structures of the inhibitor-bound enzyme, these results provide the only physical evidence that three pharmacologically active inhibitors bind within a hydrophobic cleft composed of transmembrane helices at the interface of subunits in the homotrimer. In addition, a comparison of the structures and H/D exchange behavior of MPGES1 and the closely related microsomal GSH transferase 1 (MGST1), complexed with either GSH or the inhibitor glutathione sulfonate (GSO3¯), confirms the unusual observation that two proteins from the same superfamily bind GSH differently from one another.
10) Jay Forsythe, "Nanostructured Films for Cellular Analysis: Integrating MS Imaging with Optical Microscopy," Jay G. Forsythe, Joshua A. Broussard, Jenifer L. Lawrie, Michal Kliman, Yang Jiao, Sharon M. Weiss, Donna J. Webb, and John A. McLean. Nanostructure-initiator mass spectrometry (NIMS) imaging is a powerful, emerging technique which employs porous silicon substrates to analyze biological molecules with superior sensitivity, high spatial resolution and a substantial mass dynamic range. However, current NIMS substrates are not transparent, limiting their compatibility with modern optical cellular imaging methods. Recently, we developed semi-transparent porous silicon substrates for use in pairing high-spatial-resolution NIMS imaging with traditional light-based microscopy. Using these novel substrates, we have obtained both phase-contrast and NIMS images of a wounded cell monolayer. Moreover, single-cell membrane components were observed using a sub-cellular spatial resolution, indicating the potential capability to image sub-cellular compartments specifically identified using optical microscopy.
11) Hubert Muchalski, "Stereospecific Reactions of a-Amino-b-Diazonium Intermediates: Mechanistic Studies, New Reaction Discovery and Application to a Bidirectional Synthesis of (+)-Zwittermicin A," Hubert Muchalski and Jeffrey N. Johnston. The mechanism of the Brønsted acid-catalyzed aza-Darzens reaction is explored by charting the stereochemical outcome of the triflic acid-promoted conversion of trans-triazolines to cis-aziridines. These experiments are consistent with the intermediacy of an a-diazonium-b-amino ester intermediate. However, the behavior of an a-diazo imide is used to demonstrate that this intermediate, commonly invoked in reactions of diazoalkane addition reactions to imines, may not be as significant as previously believed. Our mechanistic investigations led to development of a new approach to syn-1,2-amino alcohol motif. We will demonstrate a conceptually new approach to syn-1,2-aminoalcohols that involves Brønsted acid activation of an imine and novel a-diazo imide. After initial C–C bond formation between azomethine and diazoalkane, a latent nucleophilic oxygen terminates the addition reaction by cyclization to the diazo carbon. The net result is a highly diastereoselective and efficient equivalent to a glycolate Mannich reaction. An efficient and diastereoselective formal anti-aminohydroxylation of a,b-unsaturated imines and investigations of the mechanism of trans-triazoline fragmentation was recently published by our group. Here we report the first study of substrate-controlled diastereoselection in a double [3+2] dipolar cycloaddition of benzyl azide with a,b-unsaturated imides. Using a strong Brønsted acid (triflic acid) to activate the electron deficient imide p-bond, high diastereoselection was observed provided that a 1,1,3,3-tetraisopropoxydisiloxanylidene group (TIPDS) is used to restrict the conformation of the central 1,3-anti diol. This development provides a basis for a stereocontrolled approach to the aminopolyol core of (+)-zwittermicin A using a bidirectional synthesis strategy.
12) Elizabeth Dong, "Structural Studies of the Interaction Between mGluR5 and Allosteric Modulators," E.N. Dong, K.J. Gregory, K.W. Kaufmann, P.J. Conn and J. Meiler. The metabotropic glutamate receptor subtype 5 (mGluR5), a class C G-protein coupled receptor, is involved in mammalian cognitive function through diverse signaling pathways that modulate synaptic plasticity. Selective modulators of mGluR5 have exciting potential for the development of novel treatment strategies for disorders that disrupt cognitive function. Because the orthosteric binding site is well conserved between all subtypes of mGluRs relative to other regions on the receptor, an approach to selectively target mGluR5 is to identify ligands with allosteric binding sites. Positive allosteric modulation of mGluR5 activity could provide a novel treatment strategy for schizophrenia, while negative allosteric modulation could lead to improved cognitive function for patients with Fragile X Syndrome. Identifying specific residues on mGluR5 that contact these allosteric modulators would provide a deeper understanding of the binding interaction and aid in the development of such therapeutic compounds. While there is no high resolution structure of the transmembrane region of mGluR5, comparative structural models of mGluR5 using mammalian GPCR crystal structures as templates have been shown to accurately predict critical residues for allosteric modulation. The alignment between mGluR5, bovine rhodopsin, human beta-2-adrenergic receptor and human A2a adenosine receptor was used to model the 7 transmembrane helices. Side chain and loop optimization was then carried out with Rosetta Membrane. Residues of mGluR5 critical for the binding of allosteric modulators were predicted using Rosetta Ligand docking studies and validated by experimental functional data. Selective targeting of mGluR5 receptors with allosteric modulators can provide a novel approach to the development of therapeutic agents for disorders causing cognitive impairment. The development of a computational comparative structural model of mGluR5 together with ligand docking studies using Rosetta has been combined with experimental validation to allow for a deeper understanding of the residues important to allosteric ligand binding.
13) Uyen Le, "Progress Towards Understanding the Role of M4 Muscarinic Acetylcholine Receptor in Schizophrenia via Allosteric Modulation." Acetycholine is one of the major neurotransmitters in the CNS that regulates various functions ranging from cognition to motor. Muscarinic acetylcholine receptor M4 is highly expressed in the brain, and it has been implicated to be involved in mediating the actions of acetylcholine. Therefore, it is an appealing target for the regulation of acetylcholine functions. We are interested in looking at the positive allosteric modulation (PAM) of M4 to enhance the basal response of acetylcholine at M4. However, because of its high structural homology among its receptor subtypes, it is difficult to target M4 selectively. Here we establish that specificity, potency, and the pharmacology of our recently published M4 PAM can be improved. Using iterative parallel library synthesis, we develop a set of compounds based on VU0152100 that displays better pharmacology profile.
14) Mathew Sijo, "Kidney Developmental Phenotypes associated with the Integrin b1 NPxY motifs are due to structure alterations to Cytopasmic Domain." Integrins are the principal cell surface receptors responsible for modulating cell extracellular matrix interactions. These receptors exist as heterodimers of a and b subunits. b1 is the major beta subunit present in kidney and is important for its development and structural integrity. It is hypothesized that the two NPxY motifs in the b1 cytosolic domain are important for integrin activation and integrin-mediated functions. Here we defined the mechanisms by which the tyrosine residues in this motif regulate kidney development ultrastructure. We also show that alterations of structure in cytosolic tail of the integrin b1 protein results in aberrant functional and development phenotypes. The mice with the double Tyr-to-Ala mutation in the integrin b1 tail failed to develop the kidneys normally. Double Tyr-to-Phe mice developed normally but were more susceptible to injury. IMCD cells expressing the mutant proteins showed defects in cell adhesion, cell migration and cell proliferation on various integrin b1 dependent matrices. These mutant proteins also showed variations in response to growth factors in vitro assays. Since collagen IV is the major basement membrane protein present in kidney glomerulus, we investigated the structural changes to Integrin a1b1, the major receptor for collagen IV, to understand the mechanism using NMR spectroscopy. The heterodimer complex formed between integrin a1 and b1 was achieved under NMR conditions in DMPC/DHPC bicelles. Mutations of the tyrosine residues in NPxY motifs of integrin b1 to either alanine or phenylalanine did not alter heterodimerization but were seen by NMR to induce structural changes, which we propose may be the basis for the alterations in integrin function and associated mouse kidney phenotypes. These structural changes may disrupt b1’s association with various cytoplasmic proteins too. This study shows that the NPxY motifs of integrin b1 as part of the a1b1 complex play critical roles in kidney development and maintenance and that structural disruption of these motifs leads to kidney abnormalities.
15) Andy Liedtke, "Development of a Novel Class of Cyclooxygenase(COX)-1-Selective Inhibitors," Liedtke, Andy J., Crews, Brenda C., Daniel, Cristina M., Blobaum, Anna L., Kingsley, Philip J., Ghebreselasie, Kebreab, and Marnett, Lawrence J. Cyclooxygenase(COX) isozymes, namely COX-1 and COX-2, are critical membrane proteins that catalyze the double dioxygenation of free fatty acids, e. g. arachidonic acid (AA) to prostaglandin endoperoxides. The endoperoxides are converted to prostaglandines, which are powerful lipid mediators in many physiological and pathophysiological responses. Their biosynthesis is effectively inhibited by non-steroidal anti-inflammatory drugs (NSAIDs), which interact with both COX forms to varying extents and block AA entry to the COX binding sites. While specific inhibition of COX-2 has been extensively investigated, relatively few COX-1-selective inhibitors have been described. However, several reports about a possible contribution of COX-1 in analgesia, neuroinflammation or carcinogenesis have kindled interest in this field and imply that COX-1 might represent a viable therapeutic target . One accepted approach for the development of isoform-selective COX inhibitors is structural re-design of established NSAIDs, e. g. sulindac. Our laboratory recently identified (E)-2’-des-methyl sulindac sulfide (E-DMSS) as a selective but weak inactivator of COX-1 (IC50: ~2 µM) [2, 3]. For the present study we designed, synthesized and evaluated 80 follow-up E-DMSS-based COX inhibitors. Here we will explicitly review the inhibitory effects of these structurally interrelated derivatives on recombinant COX enzymes and discuss the structural requirements for discriminating COX-1 and/or COX-2 inactivation. The biological applications of exemplified derivatives in a range of cultured human cell lines will be discussed as well. Our most promising development candidate, a biphenylmethylidene indene derivative bearing an acidic trifluoromethyl sulfonimide functionality as COOH bioisoster inhibited COX-1 at 470 nM (no COX-2 inhibition @ 4 µM). Using a human ovarian epithelial cancer cell line OVCAR-3, which constitutively expresses COX-1, we showed that in vitro incubation with optimized COX-1-selective inhibitors resulted in a significant reduction of intracellular COX-1-mediated AA metabolism . These inhibitors did not affect prostaglandin formation in COX-2-expressing human head and neck squamous cell carcinoma (HNSCC 1483) cells, whereas celecoxib, a recognized COX-2 inhibitor did. OVCAR-3 cell viability was reduced in a dose-dependent manner upon 2-day treatment with selected compounds (EC50s as low as 130 µM vs. sulindac sulfide as control, EC50: 210 µM). Although, this extended anti-proliferative potential appeared to be, at least in vitro, largely independent from COX inhibition, the reported molecules represent useful tools in biomedical research on the role of COX-1 in human health and disease.
16) Daniel Putnam, "Analysis of Small Angle X-Ray Scattering (SAXS) experimental restraints for protein folding in BCL::Fold," Daniel K. Putnam, Brian Weiner, Nils Woetzel, and Jens Meiler. SAXS is a technique used to create low resolution representations of protein structures in solution. To obtain these representations, a protein sample is irradiated by x-rays at a constant wavelength. Some x-rays collide with the sample and scatter, while other x-rays pass through the sample undeterred. The scattered x-rays are recorded on a detector and the corresponding momentum transfer (q) values are calculated. The intensity I(q) of the signal is calculated as a function of the momentum transfer (q value). The q-value is then plotted against I(q) to create a scattering curve profile for a given protein. Here we describe an algorithm in BCL::Fold to fold proteins in silico using SAXS restraints. BCL::Fold is a Monte Carlo folding algorithm based on the placement of discrete secondary structure elements. The SAXS experimental data improves the folding algorithm by limiting the search space of secondary structure element positions. This restraint was incorporated into a scoring function used during protein folding. A scattering curve is simulated for the protein model and directly compared with the experimental curve. The method was benchmarked by comparing SAXS curves with those generated by the gold-standard program, CRYSOL. Finally, the method will be benchmarked by assessing the accuracy of models generated with and without experimental SAXS data.
17) Hai-Young Kim, "Fragment-based screening for small molecule modulator of Oncogenic Ras activation," Hai-Young Kim, Michael Burns, Edward Olejniczak, Olivia Rossanese, Stephen Fesik. Multiple studies indicate that dysregulation of guanine nucleotide exchange factors (GEFs) contribute to the oncogenic activity of Ras, suggest that GEFs may be attractive cancer drug targets. Son of sevenless homologue 1 (SOS1) is among the GEFs that stimulate the exchange of GDP for GTP to generate the active form of Ras. Aberrantly activated Ras triggers downstream signaling cascades that promote several different cancers. Thus, inhibition of SOS-mediated Ras activation could offer broad therapeutic applications against Ras-driven tumorigenesis. High resolution X-ray structures and relevant biological information of SOS1 enabled us to design protein constructs comprising the SOS catalytic domain: Rem-Cdc25 (SOScat). We are using this construct to discover potent and drug-like small molecule inhibitors of SOS1. Following the optimization of SOScat for NMR spectroscopy, we tested the binding of 12,672 fragments using saturation transfer difference (STD) NMR. The hits in this screening were further tested using surface plasmon resonance (SPR) and 2-dimensional heteronuclear single-quantum correlation (HSQC) NMR spectroscopy. Currently, we are trying to determine the 3-dimensional structure of SOScat-fragment complexes using NMR spectroscopy and X-ray crystallography to guide the optimization of these molecules. In addition, we plan to screen our fragment library for compounds that could bind to a second nearby site that could be linked to our first site ligands to obtain a potent lead molecule.
18) Charles Williams, "Phenotypic Chemical Genetic Screen Identifies Incaskin: A Novel Exquisitely Selective Inhibitor of CK2α that Promotes Cardiomyogenesis," Charles H. Williams, Jijun Hao, T.K. Feaster, Audrey Frist, Charles C Hong. Zebrafish is quickly becoming a choice model for invivo chemical genetic screening. In a phenotypic screen for modulators of dorsoventral axis formation in zebrafish we identified a novel small molecule (incaskin) that selectively inhibits the ubiquitous serine/thrionine protein kinase Casein kinase 2α. (CK2α). Using incaskin we have shown a novel role for CK2α in modulating dorsoventral patterning via the Wnt signaling pathway in zebrafish. Furthermore, we show the potential therapeutic applications for this molecule as a cancer treatment. Additionally, incaskin robustly and reproducibly induces cardiomyocytes from murine ES cells, which could be used as an agent in regenerative medicine.
19) Michael Schulte, "General Access to Pharmaceutically Relevant Scaffolds via Organocatalysis," Michael Schulte, Craig Lindsley. In recent years, organocatalysis has had a significant impact on chemical synthesis providing a mild and practical alternative to the more conventional metal-based catalysis. Organocatalysis has recently been applied towards several asymmetric transformations, including the α-functionalization of aldehydes. Previous work in our lab led to a one-pot protocol to provide pharmaceutically relevant chiral secondary and tertiary β-fluoroamines via organocatalysis. On the basis of our previous work, we envisioned a general and straightforward procedure for the diastereoselective preparation of primary β-fluoroamines. This approach is based on enantioselective α-fluorination of aldehydes, condensation with enantiopure N-tert-butylsulfinamines, followed by addition of various organometallic reagents and deprotection of the amine to afford chiral β-fluoramines. We have also employed organocatalysis in the development of routes to chiral aziridines. The first route makes use of enantioselective α-chlorination of aldehydes, subsequent reductive amination with a primary amine, and SN2 displacement to afford previously unattainable chiral N-alkyl terminal aziridines with a wide range of N -substituents. Finally, based on our previous aziridine work and primary β-fluoroamine studies, we developed a diastereoselective route to N-(tert-butanesulfinyl)-aziridines. This method utilizes enantioselective α-chlorination of aldehydes followed by condensation with enantiopure N-tert-butylsulfinamines to afford α-chlorinated N-(tert-Butanesulfinyl)-imines. Addition of excess Grignard reagent results in addition to the imine and subsequent cyclization provides the corresponding chiral aziridines.
20) John Brogan, "Total synthesis and biological evaluation of marine alkaloid natural product (+)-7-Bromotrypargine." (+)-7-Bromotrypargine 1 is a recently isolated tetrahydro-β-carboline alkaloid from the Australian marine sponge Ancorina sp. (Ancorinidae). We originally undertook the total synthesis of this natural product in order to show the utility of recently published methodology to furnish the tetrahydro-β-carboline core through an asymmetric Pictet-Spengler reaction. However, in the event, it was found that the electronics of the bromine-substituted tryptamine precursor prevented enantioselective ring formation through a variety of catalyst systems and reaction conditions. With these data in hand, we moved forward to complete the core structure using a Bischler-Naperalski reaction followed by asymmetric transfer hydrogenation. Herein we report the enantioselective total synthesis of (+)-7-Bromotrypargine in 37% overall yield across eight steps. Upon completion of the natural product, (+)-7-bromotrypargine was subjected to a number of assays in order to determine its cytotoxicity. While (-)-trypargine has been shown to exhibit high cytotoxicity, (+)-7-bromotrypargine showed no effect on cell viability and proliferation in assays using both normal and tumor cell lines (SW620, H520, and HCT116). Once it was known that the natural product possessed no inherent cytotoxicity, (+)-7-bromotrypargine was subjected to a wide range of radioligand binding as well as functional assays in order to elucidate its discrete molecular targets with therapeutic potential. (+)-7-Bromotrypargine was found to be an effective inhibitor of the dopamine transporter (DAT IC50=3.83 uM, Ki=3.04 uM) and norepinephrine transporter (NET IC50=1.96 uM, Ki=1.95 uM), however it showed no appreciable inhibition of the serotonin transporter at concentrations up to 10 uM. Additionally, (+)-7-bromotrypargine showed significant inhibition of the histamine 3 receptor (H3 IC50=3.63 uM, Ki=1.77 uM ). Combined, these data present a unique pharmacological profile for (+)-7-bromotrypargine, making it a potential lead compound toward the development of therapeutics in a range of disease states from attention deficit disorders to depression, anxiety, and schizophrenia. In an effort to improve this profile, libraries of derivatives were synthesized taking advantage of the variety of functional groups presented by the natural product and its precursors. This derivitization was conducted in an effort to determine the structure activity relationship that (+)-7-bromotrypargine has with its various molecular targets and improve its potentially positive therapeutic properties.
21) Noemi Tejera, "Cross-Over of the 5-LOX and COX-2 Pathways: Biosynthesis of Novel Hemiketal Eicosanoids in Activated Human Leukocytes," Noemi Tejera, Takashi Suzuki, William E. Boeglin, and Claus Schneider. Research carried out in our lab has provided evidence in vitro for an unexpected biosynthetic cross-over of the 5-LOX and COX-2 pathways: the 5-LOX product 5S-hydroxyeicosatetraenoic acid (5S-HETE) can serve as an efficient substrate for COX-2. The major stable products are two hemiketal (HK) eicosanoids, HKD2 and HKE2, together with 5,15-diHETE and 5,11-diHETE as minor by-products. Here we provide the first evidence that cross-over of the 5-LOX and COX-2 pathways is functional in humans by showing formation of hemiketals HKD2 and HKE2 in activated human leukocytes. Furthermore, we provide evidence for COX-2 dependent biosynthesis of 5,15-diHETE as an additional marker of the novel pathway. Human peripheral blood leukocytes were isolated using a dextran gradient and treated with lipopolysaccharide (LPS) to induce expression of COX-2, and with calcium ionophore A23187 to stimulate 5-LOX activity. Biosynthesis of HKD2 and HKE2 (about 0.2-1 ng/106 cells) from endogenous substrate was detected using LC-MS in the selected reaction monitoring mode, enhanced twofold when exogenous 5S-HETE was added, and abrogated when the activated leukocytes were pre-treated with aspirin, the COX-2 inhibitor NS-398, the 5-LOX inhibitor AA-861, or the FLAP inhibitor MK-886. The levels of 5,15-diHETE ranged between 0.3 and 0.4 ng/106 cells in different donors, reaching about 25-50% of the levels of LTB4. Aspirin-treatment of the cells abolished formation of 5,11-diHETE and, as expected, spared 5,15-diHETE. NS-398 reduced the levels of 5,15-diHETE and 5,11-diHETE to less than 0.1 ng/106 cells, and similar reduction was achieved by MK-886. Thus, human leukocytes are able to biosynthesize both hemiketals and the di-HETEs upon activation of 5-LOX and COX-2. These results and parallel studies showing stimulation of tubulogenesis of pulmonary endothelial cells implicate a physiological role of the hemiketals in inflammatory sites that involve expression of 5-LOX and COX-2.
22) William Beavers, "Endogenous Metabolism and Protein Adduction of #omega#-Alkynyl Arachidonic Acid in RAW 264.7 Macrophages," Beavers Jr., William N., McGrath-Lawrence, Colleen, Tallman, Keri A., Milne, Stephen B., Armstrong, Michelle D., Porter, Ned A., Brown, H. Alex, and Marnett, Lawrence J. Arachidonic acid is metabolized to prostaglandins (PG) through the cyclooxygenase pathway, and hydroxyeicosatetraenoic acids (HETE) through the lipoxygenase pathway. In addition to the major metabolites, reactive electrophiles are formed, including 4-hydroxynonenal (HNE) and 4-oxo-2-nonenal (ONE), which can adduct nucleophilic groups of proteins and DNA. Previous work in our laboratory has focused on treating cells with HNE or ONE, and measuring RNA and protein expression, as well as protein adduction. In this project, we will be comparing arachidonic acid (AA) and #omega#-alkynyl arachidonic acid incorporation and metabolism in RAW 264.7 murine macrophages. We also tested stimulation conditions using Kdo2 lipid A and ATP. Finally, protein adduction resulting from endogenously produced reactive electrophiles of #omega#-alkynyl arachidonic acid (aAA) was measured. Proteins with alkynyl adducts were attached to an azido-biotin linker by Huisgen 1,3 dipolar cycloaddition or “click chemistry”, and visualized using streptavidin conjugated horseradish peroxidase. Both AA and aAA were seen to be incorporated and released in a similar manner. Enzymatic metabolism of aAA was seen to be significantly lower than that of AA. However, aAA was shown to give increased protein adduction with increased stimulation and release, making aAA a viable surrogate for AA to identify adducted proteins.
23) Julia Koehler, "Novel experimental and computational tools developed for Membrane Proteins: Application to KCNE3." The structures of membrane proteins are very difficult to determine by solution NMR since the slow tumbling of the protein-micelle complex results in severe line-broadening of the peaks in the spectrum. In addition, conventional NMR restraints for structure determination, such as NOEs, are extremely difficult to measure, especially for alpha-helical membrane proteins. To alleviate this problem we aim to introduce the measurement of paramagnetic restraints on membrane proteins. Paramagnetic restraints have been established for structure determination of soluble proteins but their use for membrane proteins is lacking behind. The measurement of these restraints such as Paramagnetic Relaxation Enhancements (PREs), Residual Dipolar Couplings (RDCs), and Pseudo-Contact-Shifts (PCSs) requires the introduction of a paramagnetic center into the protein. This will be achieved by attaching an EDTA-derived chelating agent onto a Cysteine residue, which will coordinate a paramagnetic metal ion, preferably of the lanthanide group. The protein chosen for this study is the 12 kDa protein KCNE3 that modulates potassium channel function. On a different note, we introduce the first prediction tool that is able to simultaneously predict the secondary structure as well as identify trans-membrane spans from a protein sequence. The novelty of the approach is the application to both α-helical proteins as well as β-barrels. An artificial neural network was trained on databases of 102 membrane proteins and 3499 soluble proteins. The output is a nine-dimensional probability vector for each residue in the sequence which is the combination of three secondary structure types (helix, strand, coil) with three environment types (trans-membrane, interface, solution). The prediction accuracy of simultaneously identifying secondary structure and environment correctly is 71% for nine possible states. Secondary structure prediction (three states) yields accuracies of up to 75% and trans-membrane span prediction (three states) correctly identifies 94% of the residues.
24) Louesa Akin, "Improving Protein Structure Prediction by Incorporating Sparse NMR Restraints." Computational modeling can provide a way for structural biologists to predict protein structures when traditional experimental methods alone, such as X-ray crystallography and NMR, fail. A template-free, de novo protein structure prediction algorithm is currently under development within the Biochemical Library (BCL) suite of applications. This application, BCL::Fold, is capable of predicting protein structures for both soluble and membrane proteins with or without experimental restraints. While the current BCL::Fold algorithm can incorporate cryo-EM and EPR restraints, it cannot yet incorporate NMR restraints, which is the focus of this project. In order to incorporate these restraints into the current BCL::Fold framework, separate scoring functions for NOEs, a distance restraint, and RDCs, an orientational restraint, were introduced to evaluate predicted models versus the experimentally determined restraints. The effect of utilizing NMR restraints on the accuracy of the BCL::Fold algorithm was then assessed by predicting the structures of a set of benchmark proteins with and without restraints. More native-like structures (as determined by having an RMSD100 of less than eight angstroms) were generated when using NOE restraints and RDC restraints. This suggests that BCL::Fold will be a useful tool for predicting protein structures when NMR restraints are too sparse for traditional structure determination methods.
25) Michael Turo, "Structural Determination of N6-(2-hydroxy-3-buten-1-yl)-2’-deoxyadenosine Adduct Induced by 3,4-epoxy-1-butene," Michael Turo, Ewa A. Kowal, Srikanth Kotapati, Natalia Tretyakova, Micheal. P. Stone. 1,3-Butadiene is an important industrial and environmental chemical present in urban air and cigarette smoke, and classified as a human carcinogen. It can be metabolized by cytochrome P450 to 3,4-epoxy-1-butene. 3,4-Epoxy-1-butene has been known to cause point mutations in DNA, and while it prefers to attack the N7 nitrogen in guanine it also can mutate adenine, most notably at the N6 position. The N6 adenine adducts of 3,4-epoxy-1-butene are particularly stable causing a potential accumulation in vivo. In the present work, structural analysis was performed on N6-(2-hydroxy-3-buten-1-yl)-2’-deoxyadenosine adduct1 ((2S)-N6-HB-dA) in DNA duplex using NMR spectroscopy. The sequence being used is 5’-d(C1T2T3C4T5T6G7T8C9C10G11)-3’5’-d(C12G13G14A15C16Y17A18G19A20A21G22)-3’ (Y = N6-HB-dA). In the NOESY spectrum cross peaks between the adduct and C16 and T5 were observed, which orients the adduct towards 3’ direction. In the NOESY spectrum collected in H20 imino proton of T6 was broaden which suggests that Y17T6 base pair is less stable compared to the unmodified duplex. Analysis of the duplex reveals that the N6-HB-dA maintains Watson-Crick base pair with the opposite T6 and does not disrupt the structure of DNA duplex.
26) Matthew Leighty, "Enantioselective Synthesis of a-Hydroxy Amides via Umpolung Amide Synthesis," Matthew W. Leighty, Bo Shen, and Jeffrey N. Johnston. The a-hydroxy amide is present within many natural products and medicinal agents. These motifs are commonly prepared asymmetrically through the corresponding carboxylic acids which can be accessed through enantioselective cyanohydrin or enzymatic methods. Alternatively, the Passerini reaction allows direct access the desired amides. However, enantioselective variants of this reaction are scarce in the literature. We sought a complementary route to these motifs based on our recently reported Umpolung Amide Synthesis. In this approach, an asymmetric Henry reaction with bromonitromethane is added to aldehydes employing a readily available catalyst system to afford the desired bromonitroalkanes with high levels of enantioselection. After MOM protection of these adducts, UmAS affords the desired a-hydroxy amides in good yields. The reaction optimization and substrate scope for both the Henry reaction and UmAS will be discussed.
27) Jing Jin, "Isolation and Structural Analysis of Leukotriene A Epoxides: Insights into the Mechanism of the Lipoxygenase-catalyzed Transformation," Jing Jin, Yuxiang Zheng, William E. Boeglin and Alan R. Brash. A pivotal role of Leukotriene A (LTA) epoxides is established in formation of bioactive mediators, including the leukotrienes, eoxins, lipoxins, resolvins, maresins and (neuro)protectins. LTA biosynthesis is attributed to a lipoxygenase (LOX)-catalyzed reaction with fatty acid hydroperoxides (HPETEs). Due to their extreme instability, LTA-type intermediates of LOX catalysis have not been isolated and their structure has not been analyzed directly. We hypothesize that transformation of the fatty acid hydroperoxide to LTA epoxide depends on participation of the lipoxygenase non-heme iron in catalyzing both the initial hydrogen abstraction and in facilitating cleavage of the hydroperoxide moiety. This postulate implies that the hydrogen abstracted and the hydroperoxide lie in suprafacial relationship, which in turn, dictates that the cis or trans epoxide configuration of the LTA product depends on the pro-R or pro-S chirality of the H-abstraction (an inherent property of the specific lipoxygenase) and the R or S chirality of the HPETE substrate. To test this hypothesis, we have developed methods for isolation and direct structural analysis of LTA epoxides. We expressed human 15-LOX-1 in E. coli and purified the protein by nickel affinity chromatography by using an N-terminal His-tag. Reaction of 15S-HPETE with purified human 15-LOX-1 was performed in a biphasic hexane/pH 7.5 aqueous system. After 1.5 min of vortex mixing at 0 °C, UV spectroscopy of the hexane phase showed a decrease of substrate and appearance of a new chromophore with lmax at 280 nm characteristic of the LTA epoxide. Rapid esterification (diazomethane) and RP-HPLC (pH 8) or SP-HPLC (with 0.5% TEA) produced 14,15-LTA4 methyl ester in 10-20 microgram quantities. Subsequent NMR analysis showed that the epoxy configuration of the generated 14,15-LTA4 is trans, which is consistent with our hypothesis. The results demonstrate the feasibility of enzymatic synthesis, isolation and characterization of LTA epoxides and provide the experimental basis for analysis of LTA epoxide intermediates in formation of eoxins, lipoxins, resolvins, and other novel products.
28) Carol Bansbach, "Phosphorylation Regulates SMARCAL1 Activity at Stalled Replication Forks," Carol Bansbach, Remy Betous, Courtney Lovejoy and David Cortez. The replication stress response (RSR) is a genome maintenance program that acts during every cell cycle to deal with genotoxic insults that challenge DNA replication. The RSR promotes accurate replication of the genome, which is essential for cell survival and the avoidance of disease. Mutations in SMARCAL1 (HARP) cause Schimke immuno-osseous dysplasia (SIOD). SIOD is a multisystem disorder characterized by growth defects, immune deficiencies, and other complex phenotypes. The mechanistic basis for this disease is unknown. Using functional genomic screens, we identified SMARCAL1 as a replication stress protein. We have determined that SMARCAL1 localization and enzymatic activity at sites of stalled replication are important for genome integrity during S phase. Our data suggest that defects in the genome maintenance activities of SMARCAL1 are partly responsible for SIOD. To further understand the regulation of SMARCAL1 genome maintenance activities, we are currently investigating the role of phosphorylation. Components of the RSR are commonly targets of the DNA damage kinases. We determined that SMARCAL1 is a substrate for the DNA damage kinases Ataxia-telangiectasia mutated (ATM), ATM and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK). SMARCAL1 is hyperphosphorylated in cells following exposure to replication stress agents. Using targeted and unbiased approaches, we have identified critical phosphorylation sites in SMARCAL1. The consequences of this phosphorylation on ATPase activity and in vivo activities will be presented.
29) Danielle Kimmel, "Multianalyte Microphysiometry of Macrophage Metabolism Triggered by Oxidized Low Density Lipoprotein," Danielle W. Kimmel, William P. Dole, and David E. Cliffel. Oxidized low-density lipoprotein (oxLDL) uptake by macrophages has been regarded as the pivotal component toward the pathogenesis of atherosclerosis. Here, we are using the multianalyte microphysiometer (MAMP) to electrochemically detect extracellular glucose, oxygen, lactate, and acidification during macrophage oxidative burst, which has led to valuable knowledge of the mechanism of action for oxLDL. RAW-264.7 macrophages exposed to 50 μg/mL oxLDL for six minutes had an immediate increase in glucose and oxygen consumption, lactate production, and extracellular acidification. This demonstrates the onset of oxidative burst, leading to foam cell formation. The metabolic response of macrophages to L-4F and a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/L-4F mixture was also explored. L-4F is a drug candidate that potentially prevents and reverses atherosclerosis due to oxidative burst. DMPC is a lipid thought to alter the binding activity of oxLDL to macrophage receptors. Preincubation of macrophages with both solutions prior to oxLDL exposure resulted in an attenuation of oxidative burst and led to a decrease in consumption of glucose and oxygen. Premixture of the L-4F and DMPC/L-4F with oxLDL 120 min prior to exposure also produced an attenuation of the expected oxLDL response. These studies present a novel approach to the study of macrophage metabolic response of oxLDL and the attenuating effects of L-4F and DMPC/L-4F, giving insight to the mechanistic pathway of atherogenic oxidative burst.
30) Matthew Pence, "Measuring Conformational Changes in Y-family DNA Polymerases using Fluorescence Spectroscopy,” Matthew G. Pence, Robert L. Eoff, Catinca Fercu, and F. Peter Guengerich. Protein conformational changes are observed in replicative polymerases upon binding the correct nucleotide substrates and contribute to nucleotide selectivity and overall polymerase fidelity. Y-family polymerases function in the bypass of bulky DNA adducts and contribute to the overall fidelity of DNA replication. Substantial work has been done investigating the biological roles and bypass mechanisms of these specialized polymerases. Structural studies show that the active site of Y-family polymerases remains open and solvent exposed during polymerization emphasizing their ability to accommodate bulky lesions in the active site. No large conformational changes are observed as a basis for nucleotide selectivity. Yet, localized conformational changes may be important for catalysis. Changes in tryptophan fluorescence are observed with S. solfataricus Dpo4 upon correct nucleotide incorporation suggesting a conformational change. Individual members of the Y-family do not proficiently replicate past all types of DNA lesions. Biochemical and structural analyses demonstrate that unique structural features and base-pairing mechanisms employed by individual Y-family members lead to selectivity for the types of damage that is efficiently bypassed. Though not large, conformational changes may be vital to the reaction mechanism of Y-family polymerases. The project aims to observe experimentally, using tryptophan fluorescence, conformational changes that occur in human Y-family polymerases and describe a mechanism for nucleotide selectivity.
31) Christopher Thomas, "The Oxidation and Subsequent Hydrolysis of Linoleoyl-w-hydroxyceramide is Fundamental in Epidermal Barrier Formation," Christopher P. Thomas, Yuxiang Zheng, William E. Boeglin, Valerie B. O’Donnell, and Alan R. Brash. The outer epidermis of mammalian skin functions as a barrier vital to maintaining life on dry land. Our group has shown that linoleoyl-w-hydroxyceramide is oxygenated by the consecutive actions of 12R-lipoxygenase and epidermal lipoxygenase-3 forming 9R-HPODE and its derivatives that occur naturally in pig and mouse epidermis. These products are absent in 12R-lipoxygenase null mice, which die shortly after birth from transepidermal water loss. We propose that oxygenation of linoleoyl-w-hydroxyceramide is required to facilitate ester hydrolysis of the oxidized linoleate, allowing covalent bonding of the ceramide to protein via the free w-hydroxyl, helping to seal the barrier. Mouse 12R-LOX only reacts with linoleate esters although free 9R-HODE is found in murine epidermis, indicating release by a specific hydrolase. One candidate is sPLA2-IIF, a secretory phospholipase, in which the mouse knockout is associated with reduced free 9-HODE in the epidermis and impaired barrier function (K. Yamamoto et al, Keystone Conference Abstract 337, June 2010). We have expressed catalytically active sPLA2-IIF in E.coli and the enzyme is now being screened for activity with a range of oxidized phospholipids and ceramides possibly involved in barrier function. Phospholipid substrates have been synthesized via several routes including auto-oxidation yielding the racemic linoleic acid phosphatidylcholine and phosphatidylethanolamine derivatives, 9 and 13-HPODE. The use of the rat liver micosomal acyl-transferase enzyme has allowed the coupling of 5S, 12R,S and 15S HETE acids to a lyso-PC substrate of choice and the oxidation of linoleic acid in SLPE and SLPC by linoleate 9R-LOX from Anabaena sp. PCC 7120, has been utilized to generate phospholipids containing 9R-HODE. Additionally, the analysis of epidermal lipids is being extended to human skin in which genetic studies strongly implicate the actions of 12R-lipoxygenase and epidermal lipoxygenase-3 in barrier function, and the structures of the oxidized ceramides and lipids in human epidermis will be reported.
32) Gabriel LeBlanc, "Photoreduction of Platinum Particles onto Multilayer Films of Photosystem I." Using the abundance of available electrons generated by immobilized multilayers of the photoactive protein complex Photosystem I (PSI), we have photoreduced platinum particles which are catalytically active for the H2/H+ redox couple. The resulting platinized PSI films were optimized using electrochemical measurements, and then characterized using X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), and scanning electrochemical microscopy (SECM). These results demonstrate a novel method for generating immobilized platinum catalysts which are readily available on the surface of a photoactive PSI multilayer.
33) Patrick Halvey, "Proteomic Analysis of Phenotypes Produced by Cancer-Related Mutations," Patrick J. Halvey, Bing Zhang, Haixia Zhang, Natasha G. Deane, Ajaz Bhat, Punita Dhawan, Lisa J. Zimmerman, R. Daniel Beauchamp, Robert J. Coffey, Daniel C. Liebler, Robbert J.C. Slebos. The last two decades have seen major advances in our understanding of the genomic variations which underlie cancer phenotypes. However, genes and transcripts execute most of their functions through the proteins they encode. Systematic characterization of cancer proteomes thus provides a means to understand the translation of genomic variation to cancer phenotypes. Here we use liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics and LC-multiple reaction monitoring (MRM) mass spectrometry (LC-MRM-MS) to determine the proteomic characteristics of colon tumor cell lines containing genomic alterations associated with colorectal cancer (CRC). We examined the proteomic consequences of a single gene change by using two transgenic cell models; the SW480APC model (altered adenomatous polyposis coli, APC) and the DKO-1/DKS-8 model (altered kirsten-RAS-2, KRAS). Analysis of SW480APCNull (mutant) and SW480APC (APC restored) cell lines identified 4,588 proteins at 1% peptide FDR, of which 155 proteins showed significantly different expression (adjusted qausi p-value < 0.1). The negative Wnt regulators DKK4 and CLU were decreased, while CACYBP, which participates in CTNNB1 degradation, and USP15, a deubiquitylation enzyme which prevents APC degradation, both were increased in APCnull cells. We observed proteins upregulated in APCnull cells that mapped to cell cycle regulation (SMARCA4, CENPF), RNA processing (DDX23, RBM10, SART1) and nuclear transport (NUP50, NUP155). These findings suggest that enhancement of RNA processing and transport is essential to supporting rapid proliferation, a hallmark of the APCnull phenotype. In the DKO-1/DKS-8 model we detected 2,893 proteins at 1.0% protein FDR, of which 74 protein differences reached statistical significance (adjusted quasi-p value < 0.1). DKO-1 cells (KRAS mutant) displayed elevated expression of EGFR and several associated proteins, including EGFR kinase substrate ANXA1 and the Sh2 domain protein TNS4. We also observed expression differences in some proteins involved in glucose metabolism and bioenergetics (e.g. NDUFS1, MDH1, IDH2, PHGDH), which we investigated further by LC-MRM analysis of 28 metabolic proteins. These analyses suggested KRAS-driven Warburg effect. We next asked whether a set of different mutations that affect a common pathway could yield consistent proteomic signatures. We analyzed a collection of 10 CRC cell lines that differ in their DNA mismatch repair (MMR) status. Proteomic differences reflected known molecular defects related to MMR deficiency, e.g. absence of MLH1, MSH2 and MSH6 and decreases in frameshifted proteins RAD50 and LMAN1. A distinct co-expression cluster revealed dramatically reduced expression of cytoskeletal/adhesion proteins in the RKO cell line, suggesting an underlying epithelial to mesenchymal transition phenotype (EMT), which was confirmed by migration/invasion assays. Additionally, network analysis revealed differentially expressed clusters relating to protein synthesis, folding and turnover, protein and vesicle trafficking and transport, GTPase and Ras regulation, and intermediary metabolism. The functions represented in these co-expression clusters suggest a coordinated program of adaptation to the translation of misfolded, variant polypeptides in MMR- cells. Thus, the data provide evidence for new and more complex mechanisms underlying cancer phenotypes.
34) Stacy Sherrod, "Efficiency and Reproducibility of Phosphotyrosine Peptide Immunoaffinity Capture for Quantitative Mass Spectrometry," Stacy D. Sherrod, Amy-Joan L. Ham and Daniel C. Liebler. Protein phosphotyrosine (pTyr) is an important, cancer-relevant post-translational modification, which regulates signaling networks that control cell growth and differentiation and that are targets of anticancer drugs. Quantitative analysis of pTyr proteins is vital to understand signaling mechanisms and to improve treatment. Analysis of pTyr proteins is challenging because these modifications are present at low abundance (≤0.05% of protein phosphorylation) and are highly dynamic. MS-based analysis methods employ selective enrichment techniques, such as immunoaffinity purification of pTyr peptides, prior to MS analysis. However, variation in performance of affinity enrichment is a major potential source of variation. Here we evaluate the efficiency and reproducibility of phosphotyrosine peptide capture using a quantitative MS approach. Studies were performed using 20 mg of total protein digest/experiment from a human epithelial carcinoma cell line, A431, stimulated with epidermal growth factor (EGF) (2 μg) for 30 minutes. Prior to pTyr peptide enrichment, varying concentrations of isotopically labeled phosphopeptides were spiked into the protein digest. Peptides were incubated at 4°C overnight with either of two anti-pTyr antibodies, pY100(240μg) or 4G10(40μg), and the supernatant was removed and resin washed with MOPS buffer and H2O. The peptides were eluted with 0.15% trifluoroacetic acid, the solutions were evaporated to near dryness, and the samples were redissolved in 0.1% formic acid for MS analysis. Reverse phase LC-MS analysis was performed by nanoelectrospray in positive ion mode on a ThermoFisher LTQ Velos instrument. We have initiated studies aimed at evaluating the capture efficiency (percent recovery) and reproducibility of two mouse monoclonal anti-pTyr antibodies (pY100 and 4G10), both of which have been previously used for pTyr peptide and protein enrichment. We spiked four non-human (bacterial protein) pTyr peptides at varying concentrations (0 to 5000 fmol) into a complex background (A431 peptide digest) matrix. After immunoaffinity purification, isotope-labeled peptides for the same sequences were spiked in at a constant concentration and analyzed by targeting both unlabeled and isotopically-labeled peptides. The percent peptide recoveries for each peptide at each concentration were calculated from the amount of peptide recovered (using peak area ratios) divided by the amount of peptide spiked in prior to immunoaffinity enrichment. Peptide recoveries ranged from 0-9%. Generally, 4G10 outperformed pY100, with peptide recoveries between 2-5%, whereas pY100 typically yielded recoveries of <1% for the same peptides. Interestingly, the recovery for individual peptides did not increase significantly at higher spike-in amounts. Recovery for non-human phosphopeptide GpYNGLAEVGK using the 4G10 antibody was 1.9% for the 25 fmol spike, yet was only 1.6% for the 5000 fmol spike. These results suggest that recovery of peptides in spiked samples is independent of peptide concentration. The reproducibility of immunoaffinity capture was evaluated by spiking 1000 fmol of three isotopically-labeled human epidermal growth factor receptor pTyr peptides into a complex background. After immunoaffinity purification using either pY100 or 4G10, a second set of differently isotope-labeled peptides for the same sequences were spiked in at a constant concentration. Targeted MS analysis for all peptides (unlabeled, isotopically labeled and differently isotopically labeled) was performed to measure the immunoaffinity capture reproducibility among three biological replicates. These experiments provide a better understanding of the immunoaffinity purification methods that are typically used to enrich pTyr peptides prior to MS analysis.
35) Plamen Christov, "Synthesis and Characterization of Oligonucleotides Containing a Nitrogen Mustard Formamidopyrimidine (NM-Fapy-dGuo) Mono-adduct of Deoxyguanosine." Plamen P. Christo and Carmelo J. Rizzo. Nitrogen mustards are a family of bifunctional DNA alkylating agents that were first introduced into a clinical setting in 1942 for the treatment of non-Hodgkin lymphoma. These agents are still used today for the treatment of a variety of cancers. Nitrogen mustards react with DNA to give primarily a cationic N7-nitrogen mustard deoxyguanosine adduct, which can undergo base-induced opening of the imidazole ring to form 2,6-diamino-4-hydroxy-N5-(N-ethyl-N-(2-hydroxyethyl)-2-amonoethyl)-formamidopyrimidine (NM-FAPy-dGuo). We have developed chemistry for the site-specific synthesis of oligonucleotides containing the NM-Fapy-dGuo lesion (R=Et). These oligonucleotides were used to assess the incision of the NM-Fapy-dGuo lesion by E. coli endonuclease IV (Endo IV), E. coli formamidopyrimidine glycosylase (fpg) and human oxoguanine glycosylase (hOGG1).
36) Robert Sprung, "Multiple Reaction Monitoring MS Protein Analysis of Formalin-Fixed Paraffin-Embedded Tissue," Robert W. Sprung, Mary Kay Washington, Amy-Joan L. Ham, Daniel C. Liebler. Verification of candidate protein biomarkers requires their accurate and reproducible measurement. Access to sufficient quantities of clinically-characterized samples can be limiting for verification work when fresh frozen tissue is analyzed. Formalin-fixed paraffin-embedded (FFPE) tissue offers an alternative source of samples. Although recent work has demonstrated that FFPE tissue can be successfully analyzed by shotgun proteomics workflows, formalin fixation leads to chemical modification and crosslinking of proteins in the tissue, which may impact the suitability of these archival specimens for reproducible quantitative analysis by multiple reaction monitoring (MRM). Here we assess MRM analysis of proteins from FFPE tissue. Results We initially employed an empirical approach to select suitable MRM target peptides when working with an FFPE tissue digest. A list of candidate peptides was generated by shotgun proteomic analysis of isoelectric focusing (IEF)-fractionated tryptic digests from FFPE and frozen renal clear cell carcinoma (RCC) tissue. MRM analysis was conducted on unfractionated FFPE and frozen RCC digests, targeting 110 peptides selected from a range of peptide abundance based on spectral count data (between 2 and 272 observed shotgun spectra). Comparison of the coefficients of variance (CV) obtained from replicate analyses of FFPE and frozen samples revealed no significant difference in average CV, indicating that MRM measurement variation was similar for peptides derived from FFPE or frozen samples. Although the signal intensity was generally lower for peptides derived from FFPE tissues, the same peptide selection criteria could be applied to frozen and FFPE tissue. The criteria used selected for peptides between 7 and 20 amino acids in length lacking methionine, cystine and histidine residues. Application of MRM in FFPE specimens was demonstrated through the analysis of levels of Her2 receptor in mouse xenograft models of breast cancer. Using an MRM assay, we were able to unambiguously detect and quantify Her2 in 1μg of FFPE tissue tryptic digest from a Her2 overexpressing xenograft. Specificity was shown through the absence of Her2 signal when analyzing an FFPE mouse xenograft of triple negative breast cancer. The method was further extended to the quantification of Her2 receptor levels in human FFPE breast cancer. Receptor levels ranged from 110,000 to 468,000 receptors per cell in the Her2+ cases and 2,000 to 14,000 receptors per cell in the triple negative cases, clearly demarcating the positive and negative cases. This work demonstrates the reproducible quantitation of clinically-relevant biomarkers in FFPE tissue and suggests that FFPE tissues are amenable to reliable biomarker verification studies using MRM methods.
37) Priya Mathew, "Progress Toward the Total Synthesis of Mitomycin C," Priya A. Mathew, Jayasree M. Srinivasan, Amie L. Williams and Jeffrey N. Johnston. Mitomycin C is a natural product isolated from Streptomyces caespitosus that is clinically used as an anticancer drug. The biological activity arises from the key aziridine functionality which is responsible for DNA alkylation and crosslinking. Despite numerous synthetic approaches, its total chemical synthesis has been achieved only twice since its isolation in 1956. In our synthetic approach, the aziridine is installed using a Brønsted acid-catalyzed aza-Darzens reaction. The convergent synthesis of an advanced intermediate is performed using an alkynyl amination followed by a quinone coupling. Methods to install C(10) on the advanced intermediate obtained after alkynyl amination and quinone coupling, and methods to introduce C(10) prior to alkynyl amination will be presented.
38) De Lin, "Development of Immunoaffinity-Mass Spectrometry Method for Cancer Protein Biomarker Validation," De Lin and Daniel C. Liebler. A continuing challenge in cancer biomarker research is the development of sensitive, specific assays. We have employed an immunoaffinity-mass spectrometry-based approach, in which an antibody is used to enrich a target protein from plasma and then the captured protein is digested and analyzed by multiple reaction monitoring mass spectrometry (MRM-MS). Our studies have focused on the putative colon cancer biomarker SERPINB5 (maspin). An anti-maspin antibody was covalently immobilized to aldehyde-activated beaded agarose resin. Captured proteins were eluted, separated by SDS-PAGE and digested in gel with trypsin. The unique maspin peptides GQINNSIK, DVEDESTGLEK and NIIFFGK were analyzed by MRM with the stable isotope-labeled analogs added as internal standards. We obtained a linear response from 4 to 512 ng/mL for spiked recombinant maspin protein in normal human plasma. The same capture antibody was used for western blot analysis of maspin in large adenomas and colon carcinoma tissues. We noted that the maspin band (42KDa) detected in the tissues was electrophoretically resolved from recombinant human maspin expressed in E. coli, suggesting possible maspin modifications in the tissues. Preliminary tandem mass spectrometry of tissue-derived maspins suggested acetylation at Lys173 in tumor tissue. These studies are intended to optimize an immuno-MRM assay for maspin forms present in colon tumor tissues.
39) Narayan P. Niraula, "Novel Heme-dioxygenase and Catalase-related Hemoproteins from Nostoc punctiforme PCC 73102 Cooperate in Transformations of Oleic and Linoleic Acids," Narayan P. Niraula, William E. Boeglin, and Alan R. Brash. The enzymes involved in eicosanoid and oxylipin metabolism are diverse and exhibit remarkable and unusual catalytic properties. The novel enzymes identified in this study are from the photosynthetic cyanobacterium Nostoc punctiforme PCC 73102. The gene of a putative heme dioxygenase (542 amino acids, 63 kDa) annotated as heme peroxidase “cyclooxygenase-2” (NCBI, YP_001868719) lies directly next to the gene of a putative catalase-related hemoprotein (393 amino acids, 45 kDa), annotated as a hypothetical protein (NCBI, YP_001868718). The “cyclooxygenase-2” retains all the key catalytic features of the heme peroxidase-dioxygenase family. The mini-catalase has the sequence RDTH at the distal heme His, supporting a role in fatty acid hydroperoxide metabolism, while the conserved distal heme Asn is substituted with His, and the proximal heme ligand, Tyr373, appears as usual very near the C-terminus. We cloned these two genes from the genomic DNA and recombinants with His-tag for E.coli expression were prepared. Expression of N-terminal His-tag protein of the heme-peroxidase was achieved with approximately 7.5 mg protein/L of culture collected through nickel affinity columnn. Although expression of the mini-catalase with a C-terminal His-tag was weak, nickel affinity purification yielded ~1.34 mg protein/L of culture. Preliminary analyses of catalytic activity show that the heme peroxidase reacted weakly with arachidonic acid (O2 uptake, oxygen electrode). Linoleic acid, α-linolenic acid and oleic acid were good substrates and were transformed to their respective 10(S)-hydroperoxides. Reaction rates were monitored by oxygen uptake (oxygen electrode), and by UV spectroscopy. Products were analyzed by UV, HPLC and 1H-NMR. Notably, the mini-catalase reacts with the product of the peroxidase-dioxygenase, and the analyses reveal a novel hydroperoxide lyase activity that matches a long-known but uncharacterized biochemical activity in mushrooms, namely the transformation of 10(S)-hydroperoxy-linoleic acid to 10-oxo-8E-decenoic acid and 1-octen-3-ol. The latter is of commercial interest in the flavor industry as it imparts the characteristic aroma of mushrooms and is also used to attract biting insects such as mosquitos. The catalytic activities of each of the hemoproteins we identified add to the known diversity of oxylipin metabolism.
40) Narayan P. Niraula, "Novel Heme-dioxygenase and Catalase-related Hemoproteins from Nostoc punctiforme PCC 73102 Cooperate in Transformations of Oleic and Linoleic Acids," Narayan P. Niraula, William E. Boeglin, and Alan R. Brash. The enzymes involved in eicosanoid and oxylipin metabolism are diverse and exhibit remarkable and unusual catalytic properties. The novel enzymes identified in this study are from the photosynthetic cyanobacterium Nostoc punctiforme PCC 73102. The gene of a putative heme dioxygenase (542 amino acids, 63 kDa) annotated as heme peroxidase “cyclooxygenase-2” (NCBI, YP_001868719) lies directly next to the gene of a putative catalase-related hemoprotein (393 amino acids, 45 kDa), annotated as a hypothetical protein (NCBI, YP_001868718). The “cyclooxygenase-2” retains all the key catalytic features of the heme peroxidase-dioxygenase family. The mini-catalase has the sequence RDTH at the distal heme His, supporting a role in fatty acid hydroperoxide metabolism, while the conserved distal heme Asn is substituted with His, and the proximal heme ligand, Tyr373, appears as usual very near the C-terminus. We cloned these two genes from the genomic DNA and recombinants with His-tag for E.coli expression were prepared. Expression of N-terminal His-tag protein of the heme-peroxidase was achieved with approximately 7.5 mg protein/L of culture collected through nickel affinity columnn. Although expression of the mini-catalase with a C-terminal His-tag was weak, nickel affinity purification yielded ~1.34 mg protein/L of culture. Preliminary analyses of catalytic activity show that the heme peroxidase reacted weakly with arachidonic acid (O2 uptake, oxygen electrode). Linoleic acid, α-linolenic acid and oleic acid were good substrates and were transformed to their respective 10(S)-hydroperoxides. Reaction rates were monitored by oxygen uptake (oxygen electrode), and by UV spectroscopy. Products were analyzed by UV, HPLC and 1H-NMR. Notably, the mini-catalase reacts with the product of the peroxidase-dioxygenase, and the analyses reveal a novel hydroperoxide lyase activity that matches a long-known but uncharacterized biochemical activity in mushrooms, namely the transformation of 10(S)-hydroperoxy-linoleic acid to 10-oxo-8E-decenoic acid and 1-octen-3-ol. The latter is of commercial interest in the flavor industry as it imparts the characteristic aroma of mushrooms and is also used to attract biting insects such as mosquitos. The catalytic activities of each of the hemoproteins we identified add to the known diversity of oxylipin metabolism.
41) Matthew Myers, "Protein Expression Signatures of Epidermal Growth Factor Receptor Inhibition," Matthew V. Myers and Daniel C. Liebler. A major problem in cancer therapeutics is differential responsiveness of tumors to inhibitors of epidermal growth factor receptor (EGFR) signaling. Activation of the EGFR axis is typically measured via the phosphorylation status of EGFR tyrosine kinase substrates and downstream targets, but these measurements are difficult to achieve in tissues due to the low abundance of phosphorylated protein forms and the challenge of preserving phosphorylation status during sample preparation. Although EGFR signaling is known to induce changes in protein phosphorylation in downstream signaling networks, we hypothesized that accompanying protein expression changes would also indicate EGFR stimulation and therapeutic inhibition. Global protein analyses offer the opportunity to identify mechanisms of cellular response to therapies and to identify biomarkers of therapeutic efficacy. A mass spectrometry-based, global proteomic (shotgun) approach was used to detect protein expression changes associated with EGFR stimulation and inhibition in A431 carcinoma cells. Protein inventories (≥2 peptides per protein at 5% peptide FDR) for each treatment were approximately 3800 proteins at < 3% protein FDR. Differential protein expression was estimated by comparison of spectral count data with a generalized linear modeling (GLM) approach, such that differential proteins had a fold change ≥ 2.0 at a quasi-p-value of ≤ 0.2. EGF stimulation induced significant expression changes in 148 proteins compared to proliferating cells. Effects of the inhibitors on this “stimulation signature” were then determined by GLM comparisons of spectral count data for cetuximab- and gefitinib-treated cell proteomes. These analyses identified 13 proteins whose EGF-induced expression was reversed by both drugs; 15 proteins were reversed only by cetuximab and 57 proteins reversed only by gefitinib. Targeted multiple reaction monitoring (MRM) analysis verified differential expression of 12 of 13 proteins reversed by both drugs, which comprise an “EGFR inhibition signature”. These include the transcription factors c-Jun and JunD and the cell cycle inhibitor Cip1, which were upregulated by EGF and the protein CCDC50, which was decreased by EGF. Coefficient of variation (CV) values for all protein measurements verified using targeted MRM methods range from 3-27% across replicate analyses. Expression of the “EGFR inhibition signature” proteins is currently being assessed in a variety of biological systems (colon cancer cells lines, mouse xenografts, and human tissue). Protein expression changes directed by EGFR activation may provide more robust measures of EGFR activation and inhibition than phosphorylated protein forms alone. Ongoing studies will test the hypothesis that an EGF inhibition signature provides an early index of therapeutic responses to EGFR-targeted cancer therapies.
42) Matthew Windsor, "Synthesis and Evaluation of Achiral Substrate-Selective COX-2 Inhibitor," Matthew A. Windsor and Lawrence J. Marnett. COX-2 is an inducible cyclooxygenase enzyme known to oxygenate the substrates arachidonic acid (AA), 2-arachidonylglycerol (2-AG), and arachidonylethanolamide (AEA). 2-AG and AEA are endocannabinoids that exert analgesic and anti-inflammatory effects in vivo; oxygenation of these endocannabinoids by COX-2 is believed to reduce their therapeutic effects at sites of inflammation and tumorigenesis. Unfortunately, the lack of substrate-selective inhibitors, molecules that prevent the oxygenation of 2-AG and AEA but not AA, makes this hypothesis difficult to test. Our lab has demonstrated that several (R)-profens, which are weak inhibitors of AA oxygenation by COX-2, are potent inhibitors of 2-AG oxygenation in vitro and in intact cells. A complication of the use of (R)-profens as in vivo probes of endocannabinoid oxygenation is their conversion to the (S)-enantiomers, which are potent inhibitors of AA oxygenation. Herein we describe the synthesis of achiral profen molecules that cannot interconvert in vivo and the evaluation of their ability to selectively inhibit COX-2 induced oxygenation of 2-AG compared to AA.
43) Christopher Gulka, "Biomimetically Inspired Recognition of 2,4,6-Trinitrotoluene," Christopher Gulka, Matthew Bryant, David Wright. Research efforts to detect the nitroaromatic explosive, 2,4,6-trinitrotoluene (TNT), have expanded as a result of rising environmental pollution and terrorist threats. Although many TNT sensors function effectively in a laboratory setting with low limits of detection, specific field diagnostics have yet to be developed. One approach to explosives detection that has gained recent attention is the utilization of molecular recognition elements derived from proteins that specifically interact with TNT through hydrophobic pi-pi electron stacking interactions. It has been previously demonstrated that bombolitin II, an ampiphilic peptide found in bumblebee venom, displays TNT binding activity, while porphyrin ring systems, which constitute the backbone of our oxygen transport protein, hemoglobin, possess an affinity towards TNT and 2,4-dinitrotoluene (DNT). In addition, the Enterobacter cloacae PB2 bacteria species is able to bind and utilize TNT as a nitrogen fuel source via the aromatic Trp-His-Tyr-Thr active site of its pentaerythritol tetranitrate (PETN) reductase enzyme. By utilizing this information, biomimetic recognition peptides have been identified that resemble the active site of PETN reductase to create future scaffolds for the specific detection of nitroaromatic explosive molecules. In this investigation, we report a novel scaffold where biomimetic peptides containing molecular recognition motifs anchored to gold substrates serve as platforms for specific TNT biosensor development. The peptides are designed with a cysteine at the C-terminus to promote gold-thiol covalent bonding to the substrate, an octaethylene glycol spacer to limit interactions between the recognition sequence and substrate, and a 12-mer recognition sequence to facilitate hydrophobic pi-pi interactions. Binding events are observed using a quartz crystal microbalance (QCM), so that mass loading for each target signifies molecular recognition. The sensitivity and specificity of assorted peptide scaffolds are assessed with the nitroaromatic targets, TNT and DNT. Using our system, we report that peptide sequences containing a terminal Trp-His-Trp recognition motif can detect TNT as low as 14 µM in the liquid phase, while exhibiting minimal DNT binding. The terminal motif of this peptide not only provides specificity for TNT recognition, but its biological framework offers potential applications in fabricating specific biomimetic field diagnostics.
44) Tyler Davis, "Chiral Proton Catalysis: The Development of More Reactive Catalysts Enables their Application to the Synthesis of Therapeutics," The emergence of BisAMidine chiral proton catalysts with increased Brønsted basicity has led to high levels of reactivity in the aza-Henry reaction. This has allowed us to approach complex molecules using our catalytic system. A potent GlyT1 inhibitor developed by the Lindsley group has been synthesized in high enantiomeric excess using the aza-Henry addition of a secondary nitroalkane and subsequent denitration. The asymmetric addition of aryl nitroalkanes to aryl imines provides chiral non-racemic masked cis-stilbene diamines. This strategy has allowed us to synthesize a potent p53-MDM2 inhibitor (-)-Nutlin-3 in an enantioselective fashion. Recent improvements on this synthesis have allowed us to make over 500 mg of material for animal studies. A synthesis capable of producing a large library of these more basic BisAMidine catalysts, one amenable to multi-gram scale has been developed.
45) Karen Gregory, "Application of an Operational Model of Allosterism to Investigate the Structural Determinants of Positive Allosteric Modulation of Metabotropic Glutamate Receptor 5," Karen J. Gregory, E.N. Dong, S.D. Reiff, J.M. Rook, M.J. Noetzel, H.P. Cho, K.W. Kaufmann, J.T. Manka, Y.S. Zhou, P.N. Vinson, S.R. Stauffer, C.M. Niswender, C.W. Lindsley, J. Meiler, and P.J. Conn. Metabotropic glutamate receptor subtype 5 (mGlu5) is found in areas of the brain that are implicated in the pathology of schizophrenia. Numerous lines of evidence suggest that enhancement of mGlu5 activity is a viable approach for therapeutic intervention for this disorder. Furthermore, mGlu5 is known to be involved in learning and memory and enhancement of mGlu5 activity may also have efficacy in improving the cognitive deficits associated with schizophrenia, offering a significant improvement over existing antipsychotics. Our approach to identify selective mGlu5 ligands has been to target allosteric sites that are topographically distinct from the endogenous (orthosteric) ligand binding site. These so-called allosteric modulators offer significant advantages, including the potential for maintenance of spatial and temporal aspects of neurotransmission. Positive allosteric modulators (PAMs) enhance, whilst negative allosteric modulators (NAMs) inhibit, the affinity and/or efficacy of an orthosteric ligand. Herein, we validate application of an operational model of allosterism to quantify mGlu allosteric modulator pharmacology estimating modulator affinity and cooperativity from modulation of glutamate agonism. Significant correlation was observed between affinity estimates derived from functional assays compared with those from radioligand binding-based experiments for both PAMs and NAMs encompassing from diverse chemical scaffolds. Multiple allosteric binding sites have been predicted for mGlu5; therefore, we were interested in applying this analysis method to quantify the impact of previously reported and novel point mutations introduced into mGlu5 on allosteric modulator pharmacology. We have generated a homology model of the transmembrane-spanning region of mGlu5 that has served as a tool to predict and interpret the impact of mutations. Mutations were introduced into the mGlu5 protein and were stably expressed in HEK-293A cells and the consequences were assessed using both radioligand binding and intracellular Ca++ mobilization assays. Application of the operational model allowed for determination of PAM and NAM affinity estimates at receptor constructs that possessed no detectable radioligand binding as well as delineation of effects on affinity versus cooperativity. Novel mutations within the transmembrane domain regions were identified that had opposite effects on PAMs versus MPEP, a prototypical NAM. Combining site-directed mutagenesis with homology modeling and quantitative pharmacology provides insights into how allosteric modulators exert their effects, with the ultimate goal of aiding rational drug discovery efforts. Supported by grants from the NIH, NH&MRC (Australia) and NARSAD. Vanderbilt is a Specialized Chemistry Center in the Molecular Libraries Probe Centers Network.
46) Mark Dobish, "Strategies in Asymmetric Organocatalysis: The Large-Scale Synthesis of (+)-VNI as a Treatment for Chagas Disease and the BAM-Catalyzed Enantioselective Construction of sec-Alkyl-3-Substituted Indoles," Mark C. Dobish and Jeffrey N. Johnston. The Johnston group has reported a variety of aza-Henry type additions to N-Boc-arylaldimines with high enantioselection using a bifunctional bisamidine (BAM) Brønsted acid catalyst. Herein we report on the use of enantioselective bromonitromethane additions to arylaldimines towards the pharmaceutical target (+)-VNI. A seven step, scalable synthesis, has produced gram quantities of drug for testing. Preliminary mouse data shows that (+)-VNI treats acute Chagas, by inhibiting CYP51 in Trypanosoma cruzi, and suggesting it may be an effective treatment for human trypanosomiases. The proposed route offers multiple points for diversification towards more effective drug candidates, which has produced a variety of small molecules for testing. Additionally, we present the development of an asymmetric functionalization of the indole 3’ carbon using the same class of catalysts, a formal Michael type addition. A variety of nitroalkanes have been added to in situ generated alkylidene intermediates with good levels of enantioselection (up to 90% ee) and modest d.r. (up to 4:1).
47) Nicholas Adams, "Development of a Low-Resource RNA Extraction Cassette Based on Surface Tension Valves." Nucleic acid-based diagnostics are highly sensitive and specific, but are easily disrupted by the presence of interferents in biological samples. In a laboratory or hospital setting, the influence of these interferents can be minimized using an RNA or DNA extraction procedure prior to analysis. However, in low resource settings, limited access to specialized instrumentation and trained personnel presents challenges that impede sample preparation. We have developed a self-contained nucleic acid extraction cassette suitable for operation in a low-resource setting. This simple design contains processing solutions preloaded within a continuous length of 1.6 mm inner diameter Tygon tubing. Processing solutions are separated by air gaps and held in place during processing by the surface tension forces at the liquid-air interface, viz. surface tension valves. Nucleic acids preferentially adsorbed to silica-coated magnetic particles are separated from sample interferents using an external magnet to transfer the nucleic acid biomarker through successive solutions to precipitate, wash and elute in the final cassette solution. The efficiency of the extraction cassette was evaluated using quantitative reverse transcriptase PCR (qRT-PCR) following extraction of respiratory syncytial virus (RSV) RNA. RNA was recovered from TE buffer or from lysates of RSV infected HEp-2 cells with 55 and 33% efficiency, respectively, of the Qiagen RNeasy kit. Recovery of RSV RNA from RSV infected HEp-2 cells was similar at 30% of the RNeasy kit. An overall limit of detection after extraction was determined to be nearly identical (97.5%) to a laboratory-based commercially available kit. These results indicate that this extraction cassette design has the potential to be an effective sample preparation device suitable for use in a low-resource setting.
48) Edward Hawkins, "DNA Damage Recognition by the Nucleotide Excision Repair Pathway." The xeroderma pigmentosum complementation group C protein (XPC) is believed to bind to damaged DNA in the initial recognition step of the nucleotide excision repair (NER) pathway. A high-throughput fluorescence anisotropy screening assay has been developed to test the binding proficiency of XPC•HR23b to a series of oligonucleotides incorporating a broad spectrum of DNA damage. Preliminary results suggest that there is little discrimination among the various types of DNA damage. Investigations are underway to determine if additional binding factors, such as centrin, may be necessary for damage recognition.
49) Stephanie DeLuca, "Ligand Mimicking Receptor Variant Discloses Binding and Activation Mode of Prolactin Releasing Peptide," Daniel Rathmann, Diana Lindner, Stephanie H. DeLuca, Kristian Kaufmann, Jens Meiler, and Annette G. Beck-Sickinger. The prolactin-releasing peptide receptor (PrRPR) and its bioactive RF-amide peptides, PrRP20 and PrRP31, mediate physiological processes, including energy metabolism, anorexic effects, and circadian rhythm. Using rational receptor modeling-assisted double mutagenesis, the binding crevice could be identified on a molecular level by a novel approach leveraging a constitutively active mutant (CAM) of the receptor. The direct interaction of the conserved receptor residue D6.59 to the R19 of PrRP20 was identified by double cycle mutagenesis of both residues to alanine. The ionic nature was confirmed by permutation to D6.59R and D19PrRP20. Interestingly, the mutation to D6.59R reveals constitutive activity, suggesting that the mutated residue at the top of transmemberane helix 6 (TMH6) mimics R19 in PrRP20 by engaging additional binding partners. In search of these binding partners, a comparative model of the receptor was generated and revealed a set of five spatially proximal residues. Double mutants, including D6.59R/E5.26A, D6.59R/W5.28A, and D6.59R/Y5.38A, eliminated basal activity, leading to the hypothesis that these residues form the ligand binding site. D6.59A and E5.26A revealed a loss in potency, and further double mutants of E5.26 reveal this residue as second binding partner for R19. Y5.38A and W5.28A receptor mutants unveil significantly impact on receptor activation/signal transduction, exposing reduced efficacy of 35-41%. Using these experimental data as restraints, a model of the PrRP8-20/PrRPR complex was constructed to interrogate the structural determinants of ligand recognition. Our approach to investigate intramolecular double mutants in CAMs can contribute to the development of future therapeutics for human diseases related to CAMs.
50) Katya V. Petrova, "Characterization of Deoxyguanosine-Lysine Cross-link Induced by Methylglyoxal," Katya V. Petrova, Amy Millsap, Emilianne McCranie, Edward Hawkins, Amy-Joan Hamm, and Carmelo J. Rizzo. ethylglyoxal has the ability to covalently modify both DNA and proteins. A 2001 study offered evidence that methylglyoxal, a known endogenous and environmental mutagen, caused a stable DNA-protein cross-link. Model studies suggested that the cross-link involved the sidechain of lysine; however, the structure of the cross-link was not determined. We have reexamined the reaction between dGuo, Nα-acetyllysine and methylglyoxal and determined the structure of cross-link by NMR, mass spectrometry, and chemical synthesis. In order to quantitate the low level of the cross-link, we developed a sensitive liquid chromatography electrospray ionization tandem mass spectrometric assay using the stable isotope dilution method with a 13C315N1-cross-link standard. A mechanism for the formation of this cross-link has been proposed, and NMR and mass spectroscopic analyses are being performed to confirm our hypothesis. We also examined the reaction of dGuo, methylglyoxal and the model peptide Ac-AVAGKAGAR. Cross-linking between dGuo, methylglyoxal and the lysine residue was confirmed by mass spectrometry. Methylglyoxal adducts of lysine and arginine were also characterized
51) Odaine Gordon, "Are Oxidative Metabolites of Curcumin Novel Anti-Cancer Agents?" Odaine Gordon, Claus Schneider. Curcumin, the polyphenolic yellow pigment from the turmeric plant, is the subject of several dozen ongoing clinical trials for its efficacy in inflammatory diseases including cancers and diabetes. Curcumin has been shown to be effective in animal cancer models, in part by targeting the pro-inflammatory NF-κB and cyclooxygenase-2 pathways, although the molecular-chemical mechanism of action of curcumin is largely unclear. Here we present evidence that curcumin undergoes a previously unrecognized (aut-)oxidative transformation. The novel products include reactive electrophilic intermediates and a dioxygenated bicyclopentadione as the final stable metabolite. We isolated and identified one of the intermediates as a reactive epoxide and showed that it formed covalent adducts with cellular nucleophiles (GSH, N-acetylcysteine). We hypothesize that the products and intermediates of oxidative transformation are mediators of some of the biological effects of curcumin, and specifically, that their reactivity with activated thiols is the mechanistic basis for the inhibition of NF-κB and induction of antioxidant signaling by curcumin. We detected abundant levels of the bicyclopentadione in plasma and intestinal mucosa of mice after oral administration of curcumin suggesting that oxidative transformation is a prominent reaction in vivo. Further exploration of the cellular targets of the reactive intermediates and final bicyclopentadione will increase our understanding of the mechanism of action of the cancer chemopreventive agent curcumin.
52) Edward Nam, "T1989 Phosphorylation Is a Marker of Active Ataxia Telangiectasia-Mutated and Rad3-Related (ART) Kinase," Edward A. Nam, Runxiang Zhao, Gloria G. Glick, Carol E. Bansbach, David B. Friedman, David Cortez. The DNA damage response kinases ATM, DNA-PK, and ATR signal through multiple pathways to promote genome maintenance. These related kinases share similar methods of regulation, including recruitment to specific nucleic acid structures and association with protein activators. ATM and DNA-PK also are regulated via phosphorylation, which provides a convenient biomarker for their activity. Whether phosphorylation regulates ATR is unknown. Here we identify ATR T1989 as a DNA damage-regulated phosphorylation site. Selective inhibition of ATR prevents T1989 phosphorylation, and phosphorylation requires ATR activation. Cells engineered to express only a non-phosphorylatable T1989A mutant exhibit a modest ATR functional defect. Our results suggest that, like ATM and DNA-PK, phosphorylation regulates ATR, and phospho-peptide specific antibodies to T1989 provide a proximal marker of ATR activation. ATR T1989 phosphorylation may provide a useful biomarker to examine activation of the DNA damage response in tumors and may predict treatment response to DNA damaging chemotherapies.
53) Mariana Boiani, "Heat Shock Response and Cancer: Role of Hsp70 Co-Chaperone BAG3 in the Stabilization of Anti-Apoptotic Mcl-1," Mariana Boiani, Michael D. Hogarty, and Lawrence J. Marnett. Lipid electrophiles such as 4-hydroxynonenal (HNE) elicit several protective cellular responses. Previous work from our group showed that the heat shock response, regulated by HSF1, plays an important role in the protection of the cell against HNE-induced apoptosis. Among many genes regulated in an HSF1-dependent manner, BAG3 a co-chaperone of Hsp70 was found to be central for HSF1 anti-apoptotic effect. BAG3 was shown to stabilize anti-apoptotic Bcl-2 proteins, such as Bcl-xL and Mcl-1, in RKO cells, preventing apoptosis. We are currently interested in the role of BAG3 in Mcl-1 protection in cancer cells. Defective apoptosis is a hallmark in cancer, and overexpression of anti-apoptotic Bcl-2 proteins is frequently observed, associated with poor prognosis and chemoresistance. Mcl-1 is a highly regulated protein, and unlike Bcl-2 many apoptotic stimulus promote its degradation by both the proteosome and caspases. Recently, it has been pointed out that proteins able to stabilize this otherwise labile protein, could play a role in Mcl-1 driven cancers. By using a panel of neuroblastoma cell lines with a defined Bcl-2 profile, we found that BAG3 is exclusively expressed in cells that depend on Mcl-1 for survival. Further, BAG3 co-immunoprecipitate (co-IP) with Mcl-1 in Mcl-1 dependent cells, and silencing BAG3 using siRNA downmodulated Mcl-1 protein levels. Consistent with a mechanism of post-translational regulation, BAG3 silencing had no effect on Mcl-1 mRNA levels. In addition, Mcl-1 protein levels were recovered after short treatment with the proteosome inhibitor MG132, confirming that BAG3 was not affecting Mcl-1 synthesis, but preventing its proteosomal degradation. This effect was shown to be specific of BAG3 knockdown because siRNA targeting the BAG3 3’ unstranslated region (UTR) had no effect in cells expressing ectopic BAG3 without its 3’ UTR. BAG3 interaction with Hsp70 may be required for Mcl-1 stabilization, since a BAG3 mutant with a reduced binding to Hsp70 did not protect Mcl-1. Finally, downmodulation of BAG3 by decreasing Mcl-1 induced apoptosis in Mcl-1 dependent cell lines and increased sensitivity to ABT737, a Bcl-2 antagonist whose activity is inhibited by high Mcl-1 expression. The results find so far point to BAG3 as an attractive target for drug development.
54) Katherine Windsor, "Capture-and-Release of Enediynes and Alkynyl Peptides," Katherine Windsor and Ned A. Porter. Through a recent collaboration, our laboratory developed a method to selectively isolate alkynyl-derivatized phospholipids from whole cell extracts. Treatment of an extract with dicobalt octacarbonyl facilitates the formation of cobalt-alkyne complexes, which can be removed from the phospholipid mixture via solid-phase immobilization. Subsequent decomplexation of the cobalt adducts releases the alkynes, completing the purification process. Current efforts are focused on expanding the scope of this methodology to the isolation of other types of alkyne-containing compounds and pursuing new practical applications for our cobalt “pulldown” method. To that end, we demonstrated proof-of-principle capture-and-release examples of enediynes, dipeptides, and longer (nine-residue) peptides. We aim to use the cobalt chemistry to identify sites of protein adduction by lipid-derived electrophiles (e.g. 4-hydroxynonenal (HNE)).
55) Uyen Le, "Progress Towards Understanding the Role of M4 Muscarinic Acetylcholine Receptor in Schizophrenia via Allosteric Modulation." Acetycholine is one of the major neurotransmitters in the CNS that regulates various functions ranging from cognition to motor. Muscarinic acetylcholine receptor M4 is highly expressed in the brain, and it has been implicated to be involved in mediating the actions of acetylcholine. Therefore, it is an appealing target for the regulation of acetylcholine functions. We are interested in looking at the positive allosteric modulation (PAM) of M4 to enhance the basal response of acetylcholine at M4. However, because of its high structural homology among its receptor subtypes, it is difficult to target M4 selectively. Here we establish that specificity, potency, and the pharmacology of our recently published M4 PAM can be improved. Using iterative parallel library synthesis, we develop a set of compounds based on VU0152100 that displays better pharmacology profile.
56) Matias Moller, "Detection of Tyrosine Oxidation-Derived Electrophiles," Matias Moller, Duane Hatch, Hye-Young Kim & Ned Porter. We have recently found that free radical oxidation of tyrosine-containing peptides in the presence of polyunsaturated lipids leads to the formation of tyrosine-lipid peroxide adducts. The phenol in tyrosine is oxidized to a cyclohexa-2,5-dien-4-one, with a peroxide bond in position 1 linked to the lipid. The peroxide bond can easily be reduced to yield the 1-hydroxy cyclohexadienone derivative. The alpha, beta-unsaturated carbonyl makes it a likely target for nucleophilic attack, suggesting interesting biological activities, including a role in protein aggregation. Herein we studied the reaction of different nucleophiles with 1-lipid peroxy- or 1-hydroxy- tyrosine derivatives and found that thiols like cysteine and glutathione make mono- and bis-adducts via a 1,4 addition. Very remarkably, we found that TCEP (tris carboxyethyl phosphine), a common reagent in biochemistry used to reduce disulfides, reacted very rapidly with our tyrosine derivatives to yield the 1,4 addition product. Considering the high reactivity to these reagents, we synthesized thiol and phosphine derivatives with an incorporated alkyne group that could be used as a reporter. After “clicking” the alkyne with an azido-photocleavable-biotin, the modified peptides can be selectively enriched by streptavidin capture and photo-release. We have successfully applied these probes to model systems and have found evidence of electrophilic amino acid formation in a mitochondria-mimic system including cytochrome C with cardiolipin by streptavidin-western blot. We are currently working on the identification of the sites of modification in cytochrome C, to assess the role of tyrosines in the formation of protein oxidation derived electrophiles.
57) John Foley, "Partitioning of Transfusion-Transmitted Viruses in Whole Blood," John Foley, Caren Chancey, Maria Rios. Hepatitis C Virus, (HCV) is an RNA virus in the family Flaviviridae that has infected 180 million people worldwide and is the leading cause for liver transplant. Because it is transmissible by transfusion, all blood donations are screened for the virus by both antibody and nucleic acid tests (NAT) using plasma as the test sample. Consequently, if virions bind to blood cells of infected donors they will not be detected by NAT performed in the plasma sample. West Nile Virus, (WNV) is a Flavivirus that is primarily transmitted by mosquitoes and infects thousands of people per year. It is also transmissible by transfusion. Previous work in our group reported that in human WNV infection, virions bind to red blood cells (RBCs) in the presence and absence of antibodies in levels sometimes exceeding that of the corresponding plasma unit, and that WNV virions bound to RBCs remain infectious (Rios et al, Clin Infect Dis. 45:181-186; 2007). Viral load (VL) was quantified for five components: whole blood (WB), RBCs, buffy coat (BC), platelets (PLT), and plasma (PL) for 17 HCV specimens and two components: RBCs and PL for 8 WNV specimens to verify suitability of plasma as testing material. Viral RNA isolation was performed following Invitrogen’s Trizol protocol in all components except PL, where Qiagen’s spin protocol was used. VL was quantified using Taqman RT-PCR with HCV/WNV specific primers and probes. 12/15 (80%) of the HCV samples that tested positive for viral RNA in any component showed PL to have the highest VL. 3/15 (20%) showed buffy coat to have the highest VL. In the case of WNV, 4/5 qRT-PCR positive samples had the highest VL in PL. Variation in VL was observed among samples tested of the same collected specimen. Testing more than one sample of a given specimen may be more accurate to ensure proper diagnostics. Individual differences in the surface antigens of blood cells could explain some of the disparities in VL of various blood components. VL varies between individuals and it also may vary with the stage of infection. Those who are HCV antibody positive but were infected many years ago could have an extremely low VL that is too low to be detected by the assay (observed in two tested donor samples)