Higuchi Biosciences Center - University of Kansas

Fall 2006 Science Talks
November 30, 2006

Abstracts 1-15 | Abstracts 16-30 | Abstracts 31-45 | Abstracts 46-54

16. Covalent Modification of Microsomal Lipids by Thiobenzamide Metabolites in vivo
Tao Ji, Keisuke Ikehata, Yakov M. Koen, Steven W. Esch, Todd D. Williams, Robert P. Hanzlik
Department of Medicinal Chemistry, University of Kansas, Lawrence, KS
Kansas State University Lipidomics Research Center, Manhattan, KS

Purpose. Our purpose was to elucidate the chemistry of metabolic activation and protein covalent binding of the model hepatotoxin thiobenzamide (TB) in vivo. Surprisingly we also observed extensive adduction of hepatic microsomal lipids. Here, we identify the structure of these adducts.
Methods. An hepatotoxic dose (1 mmol/kg) of 14C-TB or a 1:1 mixture of unlabeled (d0) and deuterated (d5) TB (TB-d) was injected ip to phenobarbital pretreated male Sprague-Dawley rats. After five hr, liver microsomes were isolated and subjected to Folch lipid extraction. The extracts were analyzed by TLC, phosphorimaging and tandem MS.
Result. The amount of covalently bound radiolabel in the Folch extract was 1812 nmol-equiv. per mg of elemental phosphorus. TLC showed a single major radioactive spot well separated from normal lipids. MS examination of the corresponding TB-d material showed peaks at 871 and 876, suggestive of TB-d labeling. MS/MS analysis of these peaks showed fragments at 245/250. Parent ion scans for m/z 245/250 showed identical patterns of masses corresponding to benzimidoylated phosphatidylethanolamines (PE). The identity of theadduct was confirmed by analysis of synthesized N-benzoyl- and N-benzimidoyl-(dipalmitoyl-PE).
Conclusion. TB is converted by flavin-containing monooxygenase into the benzimidoylating agent PhC(NH)SO2H which reacts with the phosphatidylethanolamine (PE) fraction of rat liver microsomal lipid to the extent of 70.2 nmol-equiv./mg phospholipids (average MW 800 assumed). This is 1.9 times the extent of protein modification (36.8 nmol-equiv./mg protein).

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17. Kinetic and Structural Analysis of PvdJ, a Non-Ribosomal Peptide Synthetase in Pseudomonas AeruginosaJingping Lu, Audrey Lamb
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

The opportunistic human pathogen Pseudomonas aeruginosa is capable of causing infection in immunodeficient patients and in patients with cystic fibrosis. P. aeruginosa produces siderophores to scavenge metal ions and survive in the host enviroment. The siderophore pyoverdin is a low molecular weight high affinity ion chelator, which is synthesized in Pseudomonas using enzymes that belong to non-ribosomal peptidal synthetase class (NRPS). NRPS enzymes consist of functional cassettes, each of which contains a condensation domain (C), an adenylation domain (A) and a peptidyl carrier domain (P) and incorporate one amino acid residue into the siderophore peptide backbone. Adenylation domain activates the amino acid residue and is hypothesized to determine the specificity of each of the cassettes. Our goal is to decipher the role of a particular NRPS PvdJ in pyoverdin biosynthesis. PvdJ contains two NRPS cassettes, which we have labeled CAP1 and CAP2. Pyrophosphate production assay is being used to determine the amino acid specificity of each cassette and of the adenylation domain from each cassette. Based on the operon structure we hypothesize that CAP1 is responsible for lysine incorporation whereas CAP2 incorporates formyl-hydroxyornithine into the growing pyoverdin peptide. CAP1 and CAP2, adenylation domains of each have been cloned, overexpressed and purified.

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18. Investigations of the Expression and Localization of Phosphatase of the Regenerative Liver -1 (PRL-1) in Various Cell Lines
Lisa D. Crow, Trine Kvist, Jennifer S. Laurence
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS

Phosphatase of the Regenerative Liver-1 is a protein tyrosine phosphatase implicated in the invasiveness of some tumor cell lines. The protein has a bipartite polybasic nuclear localization signal and a C-terminal CaaX motif for farnesylation. Complex localization patterns are associated with PRL-1, which was originally discovered in the nucleus. The protein also associates with the centrosome during mitosis, early endosomes, and the plasma membrane. In this study, Western blots have been used to screen for PRL-1 in a number of cell line and tissue lysates. Some patterns indicative of posttranslational modifications are shown.

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19. Synthesis of Flavone Scaffolds for Diversification and Library Development
Chris Knudtson, Chunjing Liu, Mianji Zhang, Jennifer Guerra, Paul R. Hanson
Department of Chemistry, University of Kansas, Lawrence, KS
Chemical Methodologies and Library Development Center, University of Kansas, Lawrence, KS

The synthesis and diversification of a library of flavone scaffolds using oligomeric alkyl cyclohexylcarbodiimide (OACC) is reported. Scaffolds with potential as cyclin-dependent kinase inhibitors were targeted. Flavones have demonstrated antiprolific activity and are novel anti-cancer agents with possible selective cytotoxicity. Synthesis of 6- and 8-amino substituted scaffolds capable of coupling with a carboxcylic acid was performed. Amination of carboxcylic acids with flavone scaffolds was performed using OACC as a coupling reagent in a homogenous solution and polystyrene-bound carbonate as a scavenger. The use of SPOC bypassed the need for chromatography and yielded products through simple filtration in good purity and high yields.

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20. Cloning, Expression, Purification and Characterization of SARS Coronavirus Nucleocapsid Protein
B.A. Kornilayev1, J.B. Patton2, R.R.R.Rowland2
1Structural Biology Center, University of Kansas, Lawrence, KS
2Department of Diagnostic Medical Pathobiology, Kansas State University, Manhattan, KS

The severe acute respiratory syndrome coronavirus (SARS-CoV) is identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). The nucleocapsid protein (N protein) in association with the viral RNA genome is the sole protein component of the helical nucleocapsid. The protein is believed to consist of two non-interacting structural domains, the N-terminal RNA-binding domain (RBD) (residues 45-181) and the C-terminal dimerization domain (residues 248-365) (DD). The C-terminal domain exists exclusively as a dimer in solution. SARS-CoV N protein gene from the Urbani isolate was cloned into pRSET and expressed in E. coli as a His-tagged fusion protein. An additional Xpress epitope was added to the N terminus. Purification of N protein consisted of Ni-NTA affinity chromatography at denaturing conditions with consequent refolding of the protein on the column by replacing 6 M Guanidinium Chloride with 8M Urea, followed by the gradient of Imidazole from 20 mM to 500 mM in 50 mM KPO4 buffer. Size-exclusion chromatography of Ni-purified protein in 0.2 M Imidazole-citrate, containing 0.4 M NaCl revealed three populations of the protein consisting of oligomeric structures of different sizes. The presence of N protein in all three populations was confirmed by peptide-mass fingerprint analysis and by Western-blot. Two major problems concerning rapid degradation and irreversible precipitation of the protein were encountered during purification. The problem with rapid degradation of the protein encountered during purification appeared to be Ni2+-dependent. Commonly used buffer systems, however, were not suitable for crystallization of the protein due to its tendency to precipitation. The most suitable buffer system – 0.2 M Imidazole-citrate (pH 8.0), containing 0.4 M NaCl – was found to be the only buffer system in which the protein stays soluble at high concentrations.

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21. Evaluation of Capillary Electrophoresis for the Separation of Dynorphin A (1-17) From Its Metabolites
Courtney D. Kuhnline, Susan M. Lunte
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS
Ralph N. Adams Institute of Bioanalytical Chemistry, University of Kansas, Lawrence, KS

Dynorphin A exhibits unique pharmacological properties that can lead to both beneficial and toxic effects. Dyn A and several of its N-terminal metabolites (specifically 1-11) are natural ligands for kappa (k)-opioid receptors. Opioid-based pain medications currently on the market target the mu (m)-receptors, and can lead to addiction and respiratory depression. The development of dynorphin based k-opioid receptor agonists as alternatives to current opioid drugs for the treatment of pain and addiction is currently an important area of research in the drug industry.
Along with its analgesic properties, dynorphin 1-17 and its C-terminal metabolites (specifically 1-13, 2-17, 2-13 & 13-17) have been shown to be neurotoxic through the activation of NMDA receptors. Elevated concentrations of dynorphin A 1-17 have been detected in the spinal cord following peripheral nerve damage. This peptide is believed to play a major role in the production of neuropathic pain. Neuropathic pain is associated with chronic, spontaneous pain, allodynia, and hyperalgesia, and thus far there are no viable treatment options. Endogenous metabolism of Dyn A 1-17 generates smaller peptide fragments that exhibit unique properties in vivo. Each fragment can traverse the BBB by different methods (carrier-mediated versus active transport) and exhibit unique pharmacological effects both via the blood stream and the CNS.
In order to better understand the pharmacology of Dyn A, it is important to have analytical methods that are capable of separating the parent compound (Dyn A 1-17) from its metabolites. In this work the separation of Dynorphin A 1-17 from Dyn A 1-6, 1-8, 1-13 and 2-17 was accomplished by conventional capillary electrophoresis with UV detection. Dynorphin peptides are very basic (pI=11.8) and adsorb strongly to the silica surfaces of the capillary. Phytic acid was used as a buffer additive to eliminate this interaction. In order to improve the sensitivity and selectivity of the method for in vitro studies using bovine brain microvessel endothelial cells (BBMEC), the effect of copper complexation on the separation of the dynorphin peptides was also evaluated by CE-UV. This approach has been shown previously to enhance both the sensitivity and selectivity of capillary electrophoresis with UV and electrochemical detection for peptides.
The methods developed here will be used to monitor the metabolism of dynorphin A in BBMEC and whole animal studies in both conventional and microchip format.

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22. Copper-Catalyzed Multicomponent Cascade Process for the Synthesis of Hexahydro-1H-Isoindolones
Lei Zhang and Helena C. Malinakova
Department of Chemistry, University of Kansas, Lawrence, KS
Chemical Methodologies and Library Development Center, University of Kansas, Lawrence, KS

Copper-catalyzed coupling of imines, dienylstannanes and acryloyl chlorides followed by a Diels-Alder reaction allowed for a rapid construction of substituted hexahydro-1H-isoindolones. The methodology demonstrates a new concept in utilizing a-substituted amides as templates for diversity oriented synthesis, and provides a new uniquely useful tool for a future construction of isoindolone combinatorial libraries. The strategy provides five points of diversity, four of which arise from the selection of building blocks. Furthermore, Pd-catalyzed C-C bond forming reactions open up multiple avenues for a final structural diversification.

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23. Combinatorial Synthesis of Libraries via Hydroarylations
Kelin Li and Jon A. Tunge
Department of Chemistry, University of Kansas, Lawrence, KS
Chemical Methodologies and Library Development Center, University of Kansas, Lawrence, KS

Coumarin derivatives are one of the most important metabolites in the plant world and show anticancer, antitumor and antibiotic properties. The parallel synthesis of libraries including amidophenol, aminophenol, amidocoumarin, aminocoumarin and aminodihydrocoumarin was performed and 174 compounds were obtained in ≥90% purity. The synthesis of dihydrochalcones and flavanoids by using dihydrocoumarins as versatile synthons will also be presented.

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24. Needle protein MxiH interacts with IpaD in Shigella flexneri
Lingling Zhang, Roberto De Guzman, Wendy L. Picking, William D. Picking
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

Many bacterial gram-negative human pathogens possessing type III secretion systems (T3SSs) as their key virulence elements. The TTSS is a multifunctional structure that participates in the secretion and translocation of virulence proteins of type III secretion. T3SS extracellular domain of S. flexneri is a hollow needle that is composed of a single type of protein MxiH. Invasion plasmid antigen D (IpaD) of was recently demonstrated that it’s localized at the needle tip of S. flexneri. Our goal is to determine the interaction between MxiH and IpaD and to identify the residues involved in the binding using biophysical methods. We have demonstrated that there is interaction between the two proteins by fluorescence polarization (FP) and NMR (nuclear magnetic resonance) spectroscopy. Residues clustered within the core domain of MxiH are perturbed upon MxiH-IpaD complex formation based on NMR chemical shift mapping. We therefore propose that MxiH core domain might be involved in its binding interaction with IpaD.

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25. Capture, ROMP, Cuprate Release: A New Method for Library Development
Toby Long1, Josh Waetzig1, Maria Jimenez1, Paul R. Hanson1, Daniel Flynn2
1Department of Chemistry, University of Kansas, Lawrence, KS
Chemical Methodologies and Library Development Center, University of Kansas, Lawrence, KS
2Deciphera Pharmaceuticals LLC, Lawrence, KS

A new method employing norbornenyl-tagging of various novel scaffolds using a norbornenyl monochlorophosphate tag as a means of facilitating phase-trafficking events is reported. Subsequent ring-opening metathesis polymerization utilizing either Grubbs ruthenium benzylidene catalyst or a catalyst-armed resin employing the Roberts’ protocol produces oligomeric phosphate scaffolds that are either soluble or resin-bound. Facile cleavage via an array of cuprates cleanly releases various diversified products.

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26. Parallel Synthesis Using Dipolar Cycloadditions: Pyrazolines and Isoxazolidines
Shankar Manyem, Mukund P. Sibi, Apurba Dutta
Chemical Methodologies and Library Development Center, University of Kansas, Lawrence, KS

A parallel synthesis of a library (80 members) of 2-pyrazolines in solution-phase is described. The 2-pyrazoline core was accessed through the [3+2] cycloaddition of nitrilimines with enoyl oxazolidinones. The cycloaddition provided two regioisomers, the major product being the C regioisomer. The oxazolidinone moiety was further reduced to the primary alcohol producing another library of 5-hydroxymethyl-2-pyrazolines. The Lipinski profiles of the compounds produced are also reported. Progress towards accessing a library of bicyclic isoxazolidines from a chiral aminobutenolide is also presented.

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27. Biochemical Characterization of the Ornithine Hydroxylase from Pseudomonas Aeruginosa
Kathy Meneely, Audrey Lamb
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

The cause of death for most cystic fibrosis patients is not due to the genetic disorder itself, but to chronic infections by Pseudomonas aeruginosa leading to debilitating lung damage. One of the virulence factors of P. aeruginosa is siderophore production, allowing the bacteria to colonize iron-poor environments such as the lungs.
The secreted siderophores form a complex with iron and are imported into the bacterium through specific membrane receptors. After import, the iron is released for use by the bacterium. The focus of our work is the biosynthetic enzymes of the siderophore pyoverdin, which are targets for new anti-microbial drug design. An important early step in the biosynthesis is the production of ornithine derivatives that serve as iron chelators in all pyoverdins. PvdA is an L-ornithine hydroxylase that converts L-ornithine to hydroxyornithine for inclusion into pyoverdin. PvdA was cloned from PA01 P. aeruginosa genomic DNA, overexpressed in Escherichia coli, and purified using nickel chelating chromatography. PvdA activity was optimal at pH 7.5 and low ionic strength. Characterization of PvdA shows strict cofactor specificity and substrate requirements include the correct isomer, proper side chain length, and inclusion of the peptide amine and carboxyl group. Binding affinities for the cofactors FAD and NADPH have been determined, as well as a turnover number of 0.213 ± 0.010 sec-1. Inhibition studies indicate a cysteine may be involved in catalysis, as was also seen for the PvdA homologs, the lysine hydroxylase from E. coli and p-hydroxybenzoate hydroxylase from P. fluorescens.

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28. Prolyl-4- Hydroxylase from Bacillus Anthracis
Megen Miller, Julian Limburg
Department of Chemisty, University of Kansas, Lawrence, KS

Bacillus anthracis, the causative agent of anthrax, is spore forming gram positive bacteria. The spore coat has three layers including an outer layer known as the exosporium. The immunodominant antigen in the exopsorium is a glycine-rich glycoprotein whose protein sequence contains collagen-like Gly-Pro-Thr repeats (Bacillus collagen-like protein of anthracis, or BclA), where the Thr are the sites of glycosylation. Collagen found in higher organisms contains Gly-Xaa-Yaa repeats where Xaa is often Pro and Yaa is often trans-4-hydroxyproline (Hyp), where Hyp adds stability to the collagen triple helix by providing hydrogen bonding interactions. It has been reported that BclA forms a hair-like nap on the surface of the exosporium and electron diffraction data are consistent with the formation of a collagen-like triple helix. It is not known whether BclA contains hydroxyproline, but studies by Bann and Bachinger (J. Biol. Chem. 2000 275 24466 – 24469) have shown that replacing Pro with Hyp in Ac(Gly-Pro-Thr(b-D-Gal))10-NH2 increases Tm of the triple helix by 15.7˚C. A BLAST search of the anthrax genome against the alpha-subunit of type I collagen prolyl-4-hydroxylase (P4H) from human revealed a sequence with 30 % identity with the C-terminal end of the human protein and a 40 % identity with P4H from Arabidopsis thaliana. P4H is a member of the a-ketoglutarate (a-KG) dependent mononuclear non-heme iron dioxygenases family and oxidizes Pro to Hyp in procollagen. The His-Xaa-Asp-(Xaa)n-His Fe(II)-binding domain along with Lys involved in a-KG binding are conserved in the sequence of B. anthracis P4H (anthrax-P4H). We have overexpressed a recombinant form of anthrax-P4H as a soluble protein in E. coli. Gel filration chromatography indicates that anthrax-P4H is a homodimer of ~ 24 kDa subunits. Collagen-P4H from mammals is known to catalyze the oxidative decarboxylation of aKG in the absence of collagen substrate (uncoupled oxidation). We have shown that anthrax-P4H can catalyze the same uncoupled reaction in an Fe(II)-dependent manner. This is consistent with the assignment of anthrax-P4H as an aKG dependent non-heme iron enzyme. Work is currently underway to characterize the product of the enzymatic reaction between anthrax-P4H and BclA.

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29. Effect of Chronic Exposure of Bisphenol A on SULT1A1 Activity in the Human Placental Choriocarcinoma Cell Line BeWo
Pallabi Mitra, Kenneth L. Audus
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS

Purpose: The sulfotransferase enzyme SULT1A1 is widely expressed in many human tissues where it is involved in the sulfation of drugs, xenobiotics and endogenous compounds. It is also expressed in the human placenta but little is known about its placental function or how it is affected by factors such as maternal drug abuse, smoking, exposure to environmental chemicals etc. Bisphenol A, utilized in the manufacture of polycarbonate plastics, exhibits estrogenic properties and regulates the expression of many estrogen-responsive genes. Though primarily eliminated by glucuronidation, it is also a substrate of SULT1A1. Taking into account that its placental concentrations are five fold higher than maternal plasma concentrations, and that estrogenic compounds from different sources have been shown to inhibit sulfotransferase activity, the objective of this study was to evaluate the effect, if any, of bisphenol A on SULT1A1 activity.
Methods: BeWo cells (trophoblast cells of placental choriocarcinoma origin) were utilized as a model of the human placenta. Cultures were exposed to bisphenol A for 48 hours, followed by extraction of either the cytosolic protein or the mRNA. Enzyme activity studies were determined with 4µM 4-nitrophenol (marker substrate for SULT1A1).
Results: Chronic exposure of BeWo cells to bisphenol A (100nM, 1µM, and 50µM) produced a significant decrease (p<0.05) in SULT1A1 activity at all three concentrations tested relative to the control (0.1% ethanol). At these concentrations, bisphenol A was not cytotoxic to the BeWo cells as assessed by the WST assay. Reverse-transcription polymerase chain reaction however did not indicate any change in mRNA levels in the bisphenol A treated cells relative to the control.
Conclusion: Though chronic exposure of BeWo cells to bisphenol A produced a significant decrease in enzyme activity, the lack of any effect on the mRNA levels seems to indicate that the regulatory effect is post-transcriptional in nature. Future experiments will focus on elucidating the mechanisms of this regulation.

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30. Trapping of a Dopaquinone Intermediate in the TPQ Cofactor Biogenesis in a Copper-containing Amine Oxidase from Arthrobacter globiformis
1Robyn Moore, 1Minae Mure, 2M. Ashley Spies, 2Shun Hirota, 2Toshihide Okajima,
2Tanizawa Katsuyuki, 3Misa Kim, 3Hiroshi Yamaguchi
1Department of Chemistry, University of Kansas, Lawrence, KS
2Institute of Scientific and Industrial Research, Osaka University, Japan
3Faculty of Science, Kwansei Gakuin University

The biogenesis of the topaquinone (TPQ) cofactor of copper amine oxidases (CAOs) is self-catalyzed and requires Cu2+ and O2. A dopaquinone intermediate has been proposed to undergo 1,4-addition of a water molecule to form the reduced form of TPQ, followed by facile oxidation by O2 to yield the mature TPQ. In this study, we have incorporated a lysine residue in the active site of a CAO from Arthrobacter globiformis (AGAO) by site-directed mutagenesis. As a result, we obtained a mature protein that does not exhibit a characteristic UV-vis absorbance of TPQ (480 nm) but shows an absorbance maximum at 454 nm. The X-ray crystal structure of D298K-AGAO (2.5 Å) revealed that the covalent linkage has been formed between e-amino side chain of Lys298 and the C2 position of a dopaquinone derived from Tyr382, a precursor to TPQ. We assigned this species as an iminoquinone tautomer (LTI) of the lysine tyrosylquinone (LTQ), the organic cofactor of lysyl oxidase (LOX). The LTI in D298K-AGAO is stabilized by the hydrogen bonding interaction (2.4 Å) between O4 and the conserved tyrosine (Tyr284). To support this idea, a quinone absorbing at 504 nm, similar to LTQ was generated when the hydrogen bonding interaction was disrupted by site-directed mutation (Y284FD298K).

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