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Current Trainees

 

Brittany Allison (Mentor:  Jens Meiler, Chemistry)

“Computational Design of Protein-Small Molecule Interfaces”

Proteins that bind small molecules can act as therapeutics by sequestering ligands, stimulating signaling pathways, delivering other molecules to sites of action, and serving as in vivo diagnostics. Computational design of proteins that can bind any ligand would hold great value, but is not yet possible. Computational methods for design can search more sequences and a larger sampling space than more traditional experimental methods. (β/α)8 barrels, also known as “TIM” barrels, will be used as a scaffold because of their widely seen fold in nature. Using ROSETTALIGAND, a component of the ROSETTA modeling suite that enables modeling of protein-small molecule interactions, small molecules can be docked into the binding pocket while simultaneously designing the protein-small molecule interface. The resulting models with the best scores are expressed, characterized, and tested for binding.

Awards:
2011 National Science Foundation (NSF) Graduate Research Fellowship in Chemistry (GRF)

Publications:
Allison, B.
; Combs, S.; DeLuca, S.; Lemmon, G.; Mizoue.; Meiler, J., “Computational Design of Protein- Small Molecule Interfaces". Journal of Structural Biology 2013, Accepted, In press.

     
 

Cynthia Berry (Mentor: Craig Lindsley, Chemistry)

"Total Synthesis of Marineosin A"

Dopamine receptors are involved in many important central nervous system (CNS) processes and are indicated in disease such as schizophrenia, attention deficit hyperactivity disorder, Parkinson’s disease, and drug addiction. Since the discovery of the five subtypes of dopamine receptors, great effort has been taken to synthesize highly selective ligands in order to study each receptor’s involvement in disease. Our lab has developed an enantioselective synthesis to a morpholine-based D4 antagonist. This compound binds D4 with a Ki of 70 nM, is highly selective over the other dopamine receptors, and does not bind any of the 68 GPCRs, ion channels, and transporters in the Lead Profiling Screen at Eurofins. Through iterative, parallel synthesis, we plan to make a library around our lead to improve the binding affinity to less than 10 nM. With such a compound, we will have an ideal molecule for a PET tracer that can be used in vivo to study two disease states that we believe are closely associated with D4 receptors, L-DOPA-induced dyskinesia and cocaine addiction.

Publications:
Panarese, J. D., Konkol, L. C., Berry, C. B., Bates, B. S., Aldrich, L. N., and Lindsley, C. W. (2013) Spiroaminal Model Systems of the Marineosins with Final Step Pyrrole Incorporation, Tetrahedron Lett., 54, 2231–2234.

Aldrich, L. N., Berry, C. B., Bates, B. S., Konkol, L. C., So, M., and Lindsley, C. W. (2013) Towards the Total Synthesis of Marineosin A: Construction of the Macrocyclic Pyrrole and an Advanced, Functionalized Spiroaminal Model, European J. Org. Chem. 4215–4218.

     
 

Matthew Bryant (Mentor: David Wright)

Porphyrins and metalloporphyrins are essential bioinorganic cofactors for life. Heme, an example of a metal porphyrin (Iron Protoporphyrin IX) is the cofactor molecule for the protein hemoglobin, which is responsible for binding and transport oxygen in red blood cells. Metalloporphyrins are versatile molecules that can be used in optical detection, protein purification, electrochemistry, and novel materials. The Wright lab has always been focused on heme¹s properties and involvement with disease such as malaria and formation of hemozoin.

Using bioavailable and versatile molecules for bioinorganic chemistry research is my focus for my projects. Metal porphyrins provide a multifunctional molecule with useful optical properties. These colored materials can be used in a variety of applications for molecular sensing and detection through fluorescence techniques. The charged metal center can be used for binding to histidine rich proteins, an important biomarker of malaria. The propionate groups can be functionalized easily to introduce new qualities to the porphyrin molecule. This approach will span the gap between biochemistry, inorganic chemistry, and materials chemistry.

     
 

Nicole Chumbler (Mentor: Borden Lacy)

We have developed a high throughput screen for small molecule inhibitors of Toxin B (TcdB) from the gram-positive bacterium, Clostridium difficile.  This screen is luminescent, using ATP as a metabolic indicator to measure cell death.  We are currently screening a 160,000 compound library available through the Vanderbilt High Throughput Screening facility.  To date, we have screened 16,000 compounds and identified 176 hits with two common chemical scaffolds. 

We are also investigating the mechanism of the cysteine protease domain in these toxins.  In the field, it is accepted that cysteine protease autoprocessing is required for toxin activity.  We have interesting toxicity assay data that suggests the current dogma is not as straight forward as accepted.  We are investigating this data further with more toxicity and cell rounding assays. 

Additionally, we are investigating the differences in autoprocessing efficiency and reducing agent requirements between TcdA and TcdB.  TcdA requires 100 times more reducing agent and 1000 times more InsP6 than TcdB in in vitro cleavage assays.  We are continuing to follow up this observation with mutagenesis experiments to find a disulfide bond in TcdA.

     
 

Brett Covington (Mentor: Brian Bachmann)

“Metabolomics for Antibiotic Discovery and Mechanistic Characterization”

More than 85 years after Alexander Flemming’s serendipitous discovery of penicillin the mechanisms by which antibiotics lead to cell death are still debated, the antibiotic pipeline has nearly dried up, and antibiotic resistant pathogens dominate our hospitals. These seemingly insurmountable challenges in the fight against infectious disease have caused many to fear modern medicine is moving into a post antibiotic era. However, using metabolomics analyses we may be able to access a hidden antibiotic vault and elucidate the underlying mechanisms by which antibiotics kill microorganisms. The metabolome is the end point of the central dogma and thereby contains valuable information representing the phenotype of a cell.

When exogenous compounds like antibiotics perturb a biological system they alter the metabolic profile of the system in a manner specific to the antibiotics mechanism of action. My research addresses the problems of antibiotic discovery and mechanistic elucidation by employing metabolomics methods to determine the phenotypic consequences of antibiosis and various derepression techniques to unlock a new antibiotic vault.

     
 

Brendan Dutter (Mentor: Gary Sulikowski)

"Development of Chemical Probes for the Study of Heme Sensing Mechanisms in Gram Positive Pathogens"

The acquisition and regulation of heme is of critical importance to the pathogenesis of Staphylococcus aureus and Bacillus anthracis, bacteria of interest to public health and biodefense, respectively. The systems by which heme homeostasis is controlled are not well understood and identification of the proteins involved may present targets for the development of novel antimicrobials. A high throughput screen for activators of the HssRS heme sensing system in S. aureus yielded several small molecules with diverse structures. Using the lead compounds VU0038882 and VU0120205 from the screen, we are developing chemical probes to elucidate heme sensing mechanisms in these bacteria. Libraries of compounds were generated around the main scaffolds of these molecules to determine structure activity relationships.

These data have led to the development of probes for target identification using two primary strategies. The first strategy is aimed at affinity purification of the target from whole cell lysates using a probe immobilized on a solid support. The second strategy is focused on incorporation of a photoaffinity label and clickable linker into the probe where the target can be tagged in vivo and captured after cell lysis. Proteins identified by these methods will be evaluated by mutagenesis and subsequent biochemical assays. We hope to ultimately validate the target(s) of these molecules, understand their role in bacterial metabolism and evaluate them for potential pharmaceutical development.

     
 

David Earl (Mentor:  Brian Bachmann)

"Biosynthetic Engineering of Apoptolidin Analogs"

For millions of years, Mother Nature has been pursuing a drug discovery campaign of unparalleled scope and diversity. Utilizing an army of microorganisms as medicinal chemists, a vast array of bioactive compounds have been generated that modulate all parts of the cell. Mankind has fortuitously benefited from a number of these compounds for thousands of years and continues to rely on natural products in his own pharmaceutical efforts. However most of these compounds are intractable to modification by traditional synthetic methods, severely limiting their usefulness as therapeutics and biological probes.

Natural products are constructed in an assembly line fashion by biosynthetic enzymes which are organized into discrete clusters in bacterial genomes. By genetic manipulation of these enzymes, changes can readily be made to the oxidation, methylation, and glycosylation reactions normally undergone by a compound. Further, synthetic precursors can be processed by the biosynthetic machinery allowing for the incorporation of novel functional groups which can serve as amenable synthetic handles for probe development and compound optimization. By combining chemical and biological methods, biosynthetic engineering offers unprecedented access to Mother Nature’s chemical library.

Apoptolidin, a unique glycosylated polyketide macrolide, is notable for its ability to selectively induce apoptosis in multiple cancer cell lines. Our hypothesis is that targeted genetic deletions combined with chemical complementation will afford useful apoptolidin derivatives for biological and/or therapeutic leads. In order to confirm this hypothesis we propose two specific aims 1) precursor directed biosynthesis of apoptolidin analogs and 2) genetic manipulation and characterization of post-polyketide synthase modifications. Together these studies will contribute to our lab’s long term goal of understanding complex biosynthetic pathways and applying that knowledge to the biosynthesis of non-natural compounds with both biologic and therapeutic value.

     
  Nichole Lareau (Mentor: John McLean)

"Structural Separations of Biological Classes by Ion Mobility-Mass Spectrometry Techniques"

The McLean lab focuses on developing and utilizing mass spectrometry techniques to study biological systems through class specific structural separations. Ion mobility-mass spectrometry (IM-MS) separates biological classes by their gas phase packing efficiencies and mass differences. IM and MS are readily integrated as both are separations in the gas phase. Ion mobility is used to measure the collision cross section or effective surface area, and subsequently, mass spectrometry is used to analyze the mass-to-charge ratio of ions. Recent collaborations provided access to a commercial prototype IM-MS instrument with improved resolution, measurement accuracy and sensitivity to current commercially available instruments. With this new platform, a database was compiled to define regions correlating to biological classes such as lipids, peptides, and carbohydrates. This conformational space map will guide future studies of complex biological samples as a tool to describe unknown molecular features by biological class.

Trends within biological classes have also been explored. In particular, trends in the carbohydrate database were studied leading to detailed fragmentation analyses. Fragmentation studies such as electron transfer dissociation (ETD) in combination with IM-MS provide detailed structural information about carbohydrates and glycoconjugates such as glycoproteins. The added dimension of mobility to traditional MS/MS techniques assists in the deconvolution of data and validation of analyte identification. Future studies will incorporate these various analytical tools to structurally probe glycoproteins.

Publications:
S. M. Stow, N.M. Lareau, K. M. Hines, C. R. McNees, C. R. Goodwin, B. O. Bachmann, J.A. McLean, Structural separations for natural product characterization by ion mobility-mass spectrometry: Fundamental theory to emerging applications. In Natural Products Analysis: Instrumentation, Methods, and Applications; V. Havlicek, J. Spizek, Ed. Wiley-Blackwell: New York. In Press.

Jody C. May, Cody R. Goodwin, Nichole M. Lareau, Katrina L. Leaptrot, Caleb B. Morris, Ruwan T. Kurulugama, Alex Mordehai Christian Klein, William Barry, Ed Darland, Gregor Overney, Kenneth Imatani, George C. Stafford, John C. Fjeldsted, and John A. McLean, Conformational Ordering of Biolmolecules in the Gas-Phase: Nitrogen Collision Cross-Sections Measured on a Prototype High Resolution Drift Tube Instrument, Analytical Chemistry, 2013.

     
 

Michelle Mitchener (Mentor:  Lawrence Marnett)

Cyclooxygenase-2 (COX-2) catalyzes the bis-dioxygenation of arachidonic acid (AA) to PGG2 and its reduction to PGH2, the committed steps in prostaglandin (PG) biosynthesis. COX-2 also oxygenates the endocannabinoid 2-arachidonoylglycerol (2-AG) leading to the formation of prostaglandin glycerols (PG-Gs). Purified mCOX-2 utilizes AA and 2-AG with approximately equivalent efficiencies as judged by kcat/Km values for oxygenation. However, cellular levels of PG-Gs are 2-3 orders of magnitude lower than PGs. The cause of this dramatic difference in PG-G levels produced by purified COX-2 versus cellular COX-2 has remained uncertain.

Studies with purified mCOX-2 revealed that AA can inhibit oxygenation of 2-AG, whereas 2-AG is a weak inhibitor of AA oxygenation. The effect of AA levels on 2-AG oxygenation in intact cells was evaluated in the RAW264.7 macrophage-like cell line. Reduction in AA levels, resulting from treatment with the cPLA2α inhibitor, giripladib, correlated with a >10-fold increase in 2-AG oxygenation as judged by PG-G production. Lipidomic studies, currently under way, are expected to elucidate both the nature of this substrate competition and also reveal the biosynthetic pathway by which PG-Gs are formed in this cell line. This research is important for understanding lipid-signaling pathways as well as for understanding how levels of PG-Gs, a poorly characterized lipid species, may be increased.

     
 

Joseph Manna (Mentor:  Lawrence Marnett)

"Identification of a hydrolase responsible for the metabolism of prostaglandin glycerol esters to prostaglandins and its involvement in lipid metabolism and cellular function"

Work performed in order to determine the identity of the enzyme responsible for hydrolysis of glycerol prostaglandin E2 to prostaglandin E2. We have demonstrated in MDA-MB-231 breast cancer cells that the enzyme is a serine hydrolase because of hydrolytic inactivation with irreversible serine hydrolase inhibitor fluorophosphonate, (FP). Cell lysate from MDA-MB-231 were chromatographically separated and proteomics was conducted on fractions that displayed hydrolytic activity to identify serine hydrolases.

Additionally, competitive FP-TAMRA tagging of the fractions against excess PGE2-G and anti-TAMRA immunoprecipitations (IP) were also conducted on the samples to pull down serine hydrolases within the fractions. Because of the excess PGE2-G present during tagging, serine hydrolases with activity against PGE2-G were not tagged by FP-TAMRA. Knockdowns of the identified serine hydrolases were done to further validate the hits by assessing the knockdown samples ability to hydrolyze PGE2-G to PGE2.

Publications:
Manna JD
, Reyzer ML, Latham JC, Weaver CD, Marnett LJ, Caprioli RM. High-throughput quantification of bioactive lipids by MALDI mass spectrometry: application to prostaglandins. Anal Chem. 2011 Sep 1;83(17):6683-8. doi: 10.1021/ac201224n.

Aluise CD, Rose K, Boiani M, Reyzer ML, Manna JD, Tallman K, Porter NA, Marnett LJ. Peptidyl-prolyl cis/trans-isomerase A1 (Pin1) is a target for modification by lipid electrophiles. Chem Res Toxicol. 2013 Feb 18;26(2):270-9. doi: 10.1021/tx300449g.

     
 

Jessica Moore (Mentors: Eric Skaar & Richard Caprioli)

"Imaging Mass Spectrometry as a tool to Study Bacterial Infections"


Jessica L. Moore uses MALDI Imaging Mass Spectrometry [IMS] to study the pathogen-host interaction. IMS is used to obtain spatial information about analytes of interest. When the technique is applied to animal models that have been presented with bacterial challenge, spatial information can be obtained to provide insight on pathogenesis of bacteria and host response. Moore applies this technology to a broad range of analytes, including antimicrobial agents, novel pharmaceuticals, host proteins, and lipids.

Publications:
M. Indriati Hood, Brittany L. Mortensen, Jessica L. Moore, Yaofang Zhang, Thomas E. Kehl-Fie, Norie Sugitani, Walter J. Chazin, Richard M. Caprioli, Eric P. Skaar. Identification of an Acinetobacter baumannii Zinc Acquisition System that Facilitates Resistance to Calprotectin-mediated Zinc Sequestration. PLoS Pathogens 8(12): e1003068. doi:10.1371/journal.ppat.1003068

Laura A. Mike, Brendan F. Dutter, Devin L. Stauff, Jessica L. Moore, Nicholas P. Vitko, Olusegun Aranmolate, Thomas E. Kehl-Fie, Sarah Sullivan, Paul R. Reid, Jennifer L. DuBois, Anthony R. Richardson, Richard M. Caprioli, Gary A. Sulikowski, and Eric P. Skaar. Activation of heme biosynthesis by a small molecule that is toxic to fermenting Staphylococcus aureus. Proceedings of the National Academy of Sciences of the United States of America. 110(20): 8206-8211

Thomas E. Kehl-Fie, Yaofang Zhang, Jessica L. Moore, Allison J. Farrand, M. Indriati Hood, Subodh Rathi, Walter J. Chazin, Richard M. Caprioli, and Eric P. Skaar. MntABC and MntH contribute to systemic Staphylococcus aureus infection by competing with calprotectin for nutrient manganese. Infection and Immunity. 2013 Jul 1. [Epub ahead of print]

Jessica L. Moore, Kyle W. Becker, Joshua J. Nicklay, Kelli L. Boyd, Eric P. Skaar*, Richard M. Caprioli*. Imaging mass spectrometry for assessing temporal proteomics: Analysis of calprotectin in Acinetobacter baumannii pulmonary infection. Article first published online: 24 JUL 2013. DOI: 10.1002/pmic.201300046

     
 

Susan Ramos Hunter (Mentor: Gary Sulikowski)

"Design and Synthesis of Small Molecule Probes to Support Studies of GIRK 1/2 Channels"

G-protein activated inward rectifying potassium (K+) channels, or GIRKs, are a part of a larger family of G-protein channel receptors that regulate cellular potassium concentration. GIRK channels are found primarily in the brain as hetero and homotetramers and have demonstrated links to schizophrenia, epilepsy, pain perception and addiction and withdrawals. Without the tools to activate and inhibit these channels, research into GIRK channels has been limited. Recently, a high throughput screen has successfully identified a GIRK activator that lead to the development of VU0456810. This molecule was found to have an EC50 of 160nM. Development of this scaffold has opened the door to further investigation of GIRK activators and inhibitors by preparation and evaluation of single isomer methylcyclopropanes and the characterization of small molecule binding sites using photo affinity labeled probes. This research offers key insights into a new territory of neurochemistry by the potential to understand GIRK targets and neuropathology.

Publications:

Ramos-Hunter, S., Engers, D., Lindsley, C., Weaver, D. & Sulikowski, G. Discovery and SAR of a novel series of GIRK ½ and GIRK ¼ Activators. Bioorganic & Medicinal Chemistry Letters. (2013) 23, 18, 5195-5198.

     
 

Carl Sedgeman (Mentor: Fred Guengerich)

The DNA of all living organisms is constantly exposed to chemicals that can cause diverse harmful lesions, one of which is DNA-peptide cross-links. The formation of DNA-peptide cross-links (DPCs) has been shown to occur from the conjunction of bis-electrophiles to DNA bases and concomitantly nuclear proteins such as O6-alkylguanine DNA alkyltransferase (AGT). These conjugates have been discovered in cells and can induce both DNA mutations and cell death. Whereas cytotoxicity would arise from DNA polymerases being unable to bypass this lesion, the formation of DNA mutations implies that certain DNA polymerases are able to replicate past the lesion with elevated miscoding. Formation of DNA mutations suggests that the AGT protein is being partially degraded in order for the replication to occur. Our hypothesis is that select translesion DNA polymerases are capable of replicating past DNA-peptide cross-links. A corollary of our central hypothesis is that AGT (25 kDa) cross-linked to DNA causes mutations after being partially degraded by proteases, because the protein is too large to be bypassed directly.

The proposed study will consist of three aims: 1) to differentiate the ability of several translesion DNA polymerases to bypass the cross-link, 2) to further study the interactions between the DNA lesion and polymerases, and 3) to determine the AGT fragments that are bound to DNA after protease digestion in vivo. To test our hypothesis, we will employ steady state kinetics to determine which of the DNA polymerases is effective at replicating through the DPC, followed by pre-steady state kinetics and X-ray crystallography to further elucidate the mechanism of the reaction. We will then perform proteomic analysis to determine the in vivo polypeptide mutagen from the active site of AGT that can be replicated past. This project will help further our knowledge of the mechanisms in which polymerases bypass DNA-peptide cross-links.

     
 

Erin Shockley (Mentor:  Carlos Lopez)

"Mass-Action Modeling of the ErbB Signaling Network"

The four ErbB receptors bind a diverse set of ligands and regulate multiple signaling pathways responsible for cell proliferation and survival. Disregulation of ErbB signaling frequently occurs in a wide variety of cancers. Due to this disregulation, the receptors themselves and their downstream targets are attractive pharmaceutical targets in the treatment of cancer. Effective manipulation of the signaling to produce therapeutic benefit requires understanding an extremely complex network of interactions, a task amenable to computational modeling of the network. To this end I use mass-action based modeling and the Python framework PySB to create expansive models consisting of systems of ordinary differential equations. Once calibrated to experimental data, these models allow me to simulate the dynamics of chemical species in the system and probe what species are key for generating a given phenotypic response (i.e. proliferation or apoptosis). Comparative analysis of data obtained from models of drug‐responsive and drug‐resistant cells will generate hypothesis regarding mechanisms of disease resistance and suggest methods to prevent the development of drug resistance through the combination of therapies.

     
     

 

 

 

 

 

 




 

 

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