William Birmingham (Mentor: Brian Bachmann, Biochemistry)

Matthew Bryant (Mentor:  David Wright, Chemistry)

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,Microbiology and Immunology)

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.

Sean DeGuire (Mentor: Gary Sulikowski, Chemistry)

"Total synthesis of a bicyclobutane fatty acid natural product and biosynthetically empowered investigation of the biological activity of apoptolidin"

My graduate research has been focused on two projects focused on the chemical synthesis of complex natural products. In the first project, I completed the total synthesis of a unique bicyclobutane natural product produced by the cyanobacterium Anabaena sp. PCC7120. In this effort I investigated many synthetic transformations toward key intermediates, optimized routes, designed and implemented a novel cascade cyclization/epoxidation reaction to provide access to a unique enzymatically synthesized natural product of unknown function. An ongoing project studies the mechanism of selective cytotoxicity of the apoptolidin natural products.

Upon isolation from Nocardiopsis sp. FU40, apoptolidin was reported to selectively induce apoptosis in cancer cell models and was subsequently ranked among the top 0.1% most cell line selective cytotoxins screened against the NCI’s 60 cell line panel. While initial biological activity investigations identified it as an inhibitor of the FoF1ATPase (Ki= 5 μM), this weak inhibition fails to fully account for the observed potent and selective cytotoxicity. Equipped with our high yielding fermentation protocol, knowledge of the biosynthetic genes, novel mutant producers yielding diverse apoptolidins and structure-activity relationships, I have synthesized apoptolidin derivatives with fluorescent and affinity handles that retain cytotoxicity. With fluorescent derivatives, we are measuring the sub-cellular localization of apoptolidin and differences in cellular transport due to sugar moieties important for cytotoxicity. With biotinylated derivatives we are searching for protein targets that reveal the mechanism of the selective cytotoxicity.

Awards
ACS Division of Organic Chemistry Travel Grant Fellowship to attend the 2013 National Organic Symposium.

Publications for Sean DeGuire

B. O. Bachmann, R. McNees, B. J. Melancon, V. P. Ghidu, R. Clark, B. C. Crews, S. M. DeGuire, L. J. Marnett, G. A. Sulikowski. 2010, “Light-Induced Isomerization of Apoptolidin A leads to Inversion of C2−C3 Double Bond Geometry”  Org. Lett. 2010, 12, 2944-2947. PMC3079550

Y. Du, D. K. Derewacz, S. M. Deguire, J. Teske, J. Ravel, G. A. Sulikowski, B. O. Bachmann. 2011, “Biosynthesis of the apoptolidins in Nocardiopsis sp. FU 40”  Tetrahedron 2011, 67, 35, 6568-6575. PMC31359176

S. M. DeGuire, S. Ma, G. A. Sulikowski. 2011, Synthesis of a Bicyclobutane Fatty Acid Identified from the Cyanobacterium Anabaena PCC 7120.” Angewandte Chemie Int. Ed. 2011, 50, in press. PMID: 21898738.

Brendan Dutter

"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

"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.

Adam Ketron (Mentor: Neil Osheroff, Biochemistry)

"Poisons of Human DNA Topoisomerase II"

Topoisomerases are essential enzymes that regulate DNA supercoiling in cells and remove tangles and knots from the genome. However, because they generate DNA strand breaks as requisite intermediates in their catalytic reactions, they also have the capacity to fragment the genome. This potentially lethal feature of human topoisomerases IIa and IIb has been exploited to treat a variety of human cancers. Anticancer drugs that target these enzymes kill cells by a unique mechanism. Rather than depriving cells of the essential functions of topoisomerases, these agents “poison” the enzymes and convert them to potent cellular toxins. Thus, they are called topoisomerase poisons to distinguish them from classic catalytic inhibitors. Topoisomerase II-targeted drugs include some of the most widely prescribed chemotherapeutics currently in clinical use.

These drugs are used to treat a variety of systemic cancers and solid tumors. In addition, a number of natural products recently have been shown to target human type II topoisomerases. Many of these act through a mechanism that is fundamentally different from that of traditional topoisomerase II-targeted anticancer drugs. The aim of my research is to investigate the mechanistic basis for the actions of different classes of topoisomerase II poisons, and to assess their relative contributions to preventing, as well as causing, human cancers.

Publications for Adam Ketron

A.C. Ketron, W.A. Denny, D.E. Graves, and N. Osheroff (2011) Biochemistry, submitted. “Amsacrine as a Topoisomerase II Poison: Importance of Drug-DNA Interactions.”

Joseph Manna (Mentor:  Lawrence Marnett, Biochemistry)

"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 for Joseph Manna

Manna, J.D.,Reyzer, M.L. Latham, J.C., Weaver, C.D., Marnett, L.J., and Caprioli, R.M., 2011, “High-Throughput Quantification of Bioactive Lipids by MALDI Mass Specrometry: Application to Prostaglandins.” Anal. Chem. 83:6683-6688. [PMCID: PMC3165080]