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VICB RESEARCH

The VICB boasts over 70 faculty members, with appointments in 18 departments located in the School of Medicine and the College of Arts and Sciences. Consequently, VICB research spans a broad range of interests and is characterized by interdisciplinary and cross-disciplinary approaches. Many VICB members have affiliations with other key institutes and centers at Vanderbilt, including the Vanderbilt Ingram Cancer Center (VICC) the Institute of Imaging Science (VUIIS), the Center for Structural Biology (CSB), the Mass Spectrometry Research Center (MSRC) and the Institute of Nanoscale Science and Engineering (VINSE). All investigators enjoy access to VICB core facilities that provide chemical synthesis, high throughput screening, antibody production, and small molecule NMR services.

 

Here we provide a brief summary of major themes encompassed by VICB research efforts:

Cancer Biology and Medicine

Neurodegenerative and Neuropsychiatric Diseases

Infectious Diseases and Immunity

Enzymatic and Nonenzymatic Lipid Oxidation in Human Health and Disease

Natural Products

Synthetic and Medicinal Chemistry

New Technologies

Chemical Approaches to Understanding Biological Processes

 

 

 



VICB research spans a broad range of interests and is characterized by interdisciplinary and     cross-disciplinary approaches.

 

     

 

Cancer Biology and Medicine

Approximately half of VICB members belong to the Vanderbilt Ingram Cancer Center, so it is not surprising that cancer is a major focus of investigation. VICB researchers address every aspect of cancer biology and medicine from understanding the underlying genetic mutations that lead to malignancy, to developing new methods for cancer prevention, detection, and therapy.

A major new VICB research thrust is in cancer drug discovery, first with the establishment of the Vanderbilt Chemical Diversity Center (VCDC). Co-directed by Alex Waterson and Gary Sulikowski, the VCDC is one of four such centers that participates in the newly established National Cancer Institute’s Chemical Biology Consortium. NCI-CBC membership unites Vanderbilt with Comprehensive Chemical Biology Screening Centers, Specialized Application Centers, and Chemical Diversity Centers at nine other institutions across the United States in the aggressive pursuit of new, high risk, but promising targets for anti-cancer therapy. Also contributing to the work of the center are VICB members Brian Bachmann, Jeff Johnston, Larry Marnett, and Jens Meilerw. The VICB’s strong programs in synthetic, medicinal, and natural products chemistry will be key to its successful participation in the NCI-CBC.

Soon after the establishment of the VCDC, Steve Fesik joined the faculty at Vanderbilt and initiated a vibrant in-house Cancer Drug discovery program. Dr. Fesik comes to the VICB with over 25 years of experience in cancer drug discovery efforts at Abbott laboratories. While at Abbott, he developed novel structure-based approaches to drug design and led a highly productive group that sent multiple new drug candidates into clinical trials. Since coming to Vanderbilt, Dr. Fesik worked in collaboration with Jennifer Pietenpol, David Cortez, Larry Marnett, and Carlos Arteaga to discover new therapeutics for the highly aggressive triple negative breast cancer. A recent Pioneer Award from the National Institutes of Health has enabled Dr. Fesik to further broaden his search for novel anti-cancer agents by focusing primarily on those that are considered too risky for industrial drug development programs.

Successful drug discovery requires a strong foundation in the basic sciences, and Vanderbilt is home to many exciting research programs that employ the chemical biology approach to understand the fundamentals of malignant disease. For example, VICB members Tom Harris, Carmello Rizzo, Mike Stone, Fred Guengerich, and Larry Marnett, investigate the effects of exogenous and endogenous DNA-damaging chemicals to learn how they cause mutations that contribute to the abnormal growth and invasiveness of cancer cells. Equally important is the work of Walter Chazin, Martin Egli, Brandt Eichman, and Niel Osheroff, who study the function of DNA replication and repair enzymes and how they prevent or contribute to the mutation events that lead to malignancy. Joseph Parello focuses on the process of histone acetylation and how inhibitors of this process sensitize cancer cells to other chemotherapeutic agents. Patrick Grohar is searching for small molecule inhibitors of the EWS-FLI1 transcription factor, an abnormal fusion protein that mediates the pathogenesis of Ewing’s sarcoma. Other researchers, including David Cortez, Jennifer Pietenpol, Ethan Lee, Alex Brown, and Al Reynolds study the abnormal biochemical and signaling processes expressed by tumor cells in order to understand how derangements of specific pathways lead to malignant growth and metastasis. Jonathan Irish takes the study of cell signaling to a new level, using flow cytometry and mass cytometry to study responses at the individual cell level. Efforts in the laboratories of Charles Manning, Wellington Pham, Richard Caprioli, and Larry Marnett are directed towards developing new methods to image cancer in vivo, leading to earlier and more accurate diagnosis, and better monitoring of therapeutic responses. Dan Liebler, as director of the Ayers Institute, takes a different approach to early diagnosis by using proteomics to study changes in serum or tissue proteins that are associated with malignant disease.

 

 

 

Image Source: Model of DNA Bound to the Alkylation-Inducible DNA-Binding Protein (AidB), From the Brandt Eichman Lab, T. Bowles, et al. (2008) Proce. Natl. Acad. Sci. U.S.A., 105,15299

Neurodegenerative and Neuropsychiatric Diseases

Collaborative research in the Vanderbilt Center for Neuroscience Drug Discovery (VCNDD) has led to major advances in the discovery of promising new agents for the treatment of neurological disease. The VCNDD is pursuing neurotransmitter receptors as molecular targets. These membrane proteins receive the chemical signals that neurons use to communicate from cell to cell. The majority of current drugs available for treatment of neurological diseases modulate the function of these receptors. However most drugs also have unwanted side effects because they alter the function of receptors in the peripheral tissues of the body as well as the brain. To overcome this problem, Vanderbilt investigators have exploited the fact that most neurotransmitter receptors exist in multiple forms, or subtypes, and usually only one of the subtypes is involved in a particular disease process. By developing agents that can selectively modulate the activity of only the subtype responsible for aberrant brain function, drugs may be developed that will control the symptoms of the illness without unwanted side effects.

As Director of Medicinal Chemistry for the VCNDD, Craig Lindsley has synthesized numerous subtype-specific modulators of the muscarinic and glutaminergic receptors. These compounds show promise for the treatment of neurodegenerative diseases such as Parkinson’s Disease and Alzheimer’s Disease, and neuropsychiatric diseases, in particular, schizophrenia. The success of the program is exemplified by the partnerships that have been established with private foundations (e.g., Michael J. Fox Foundation, Alzheimer’s Association, The Stanley Foundation, PhRMA Foundation) and industry (e.g. Seaside Therapeutics, AstraZeneca, Bristol-Myers Squibb, Janssen Pharmaceuticals, and Johnson & Johnson) that will support further basic science and drug development efforts.

One of the major efforts in the VCNDD is led by Shaun Stauffer. This industry-supported project is seeking selective modulators of the metabotropic glutamate receptor 5 (mGluR5). Prior studies have shown that activation of this receptor may be beneficial as a treatment for schizophrenia, and probes previously discovered by the VCNDD support this hypothesis. Now, the Stauffer group is working to identify additional molecules that can serve as backup preclinical candidates as efforts are made to translate these discoveries to the clinic.

Scott Daniels serves as Director of Drug Metabolism and Pharmacokinetics for the VCNDD. His interest is to determine the pathways by which new allosteric modulators of neurotransmitter receptors are metabolized. By defining these pathways, the effects of metabolism on the in vivo pharmacology of these compounds can be understood, and structural changes can be made to optimize their drug-like properties.

The VICB backs up the VCNDD with strong basic science research. Examples include the investigations of Chuck Sanders into the structure and function of human peripheral myelin protein 22, an important component of myelin membranes in the peripheral nervous system. A second major interest in the Sanders lab is the amyloid precursor protein, which gives rise to the abnormal amyloid deposits found in Alzheimer’s disease brains. Qi Zhang studies the small vesicles within neurons that store neurotransmitters and release them by fusing with the plasma membrane in response to neural stimulation. The Zhang lab uses fluorescent quantum dots to label individual vesicles, allowing them to monitor vesicle movement and release of contents. While the Zhang lab studies the structures that store and release neurotransmitters, Randy Blakely and his laboratory investigate the molecular genetics and pharmacology of the proteins that transport neurotransmitters into the nerve terminal. On a very different theme, Carl Johnson focuses on understanding the neurological basis for circadian rhythms, Joseph Parello searches for natural products (terpenes) that have antidepressant activity, and Peter Martin takes neuropharmacology to the clinic with his efforts to identify the molecular basis of drug abuse.

 

 

Image Source: Binding of Cholesterol to the Amyloid Precursor Protein as Helps to Explain the Relationship of Cholesterol Levels to Alzheimer's disease. Chuck Sanders Laboratory. A.J. Beel, et al, Biochemistry (2008) 47, 9428.

 

Infectious Diseases and Immunity

Despite the success of antibiotics against many bacterial diseases, viral and parasitic illnesses remain a serious public health problem. In addition, the rise of antibiotic resistance has brought many previously controlled bacterial diseases back into play as major causes of morbidity and mortality. To address these challenges, VICB researchers are taking novel approaches to understand and combat infectious disease.

A major goal in many laboratories is developing a better understanding of pathogen-host interactions. Paul Bock, for example, has directed his long-time interest in the biochemistry of blood coagulation towards an investigation of how streptococcal and staphylococcal proteins serve as virulence factors by interfering with the function of critical coagulation and fibrinolysis enzymes. Borden Lacy uses advanced structural biology techniques to understand how clostridial neurotoxins and the vacuolating toxin of Helicobacter pylori damage the integrity of host cell membranes and disrupt cell function. Realizing that pathogenic bacteria compete with the host for critical nutrients, Eric Skaar investigates the mechanisms used by invading organisms to obtain iron from host hemoglobin reserves, and how host cells fight back by producing iron binding proteins.  John Williams is interested in the immunology and pathogenesis of the human metapneumovirus, a newly discovered pathogen that causes respiratory illness in young children.  His laboratory is attempting to understand how the virus enters cells, how the immune system responds to the infection, and how a critical viral fusion protein functions.  Gerald Stubbs’s interests are in the structure of prion proteins, the agents responsible for devastating neurological disorders such as “mad-cow” disease in cattle and Jacob-Creutzfeld disease in humans. Although prions are not viruses or cells, but rather simple proteins, their transmission through ingesting “infected” tissue bears many of the hallmarks of infectious disease. Finally, Gerald Stubbs’s interests are in the structure of prion proteins, the agents responsible for devastating neurological disorders such as “mad-cow” disease in cattle and Jacob-Creutzfeld disease in humans. Although prions are not viruses or cells, but rather simple proteins, their transmission through ingesting “infected” tissue bears many of the hallmarks of infectious disease.

While the above projects are directed primarily at understanding the mechanisms of interaction between host and infectious agent, VICB members are also working to develop new means to combat infection. For example, the Eric Skaar lab has identified a small molecule inhibitor of bacterial glycolysis that markedly suppresses microbial growth under anaerobic conditions. This inhibitor exhibits impressive in vivo antibacterial activity under conditions, such as in abscesses, where oxygen supplies are low. Maria Hadjifrangiskou and her laboratory are seeking new treatments for bacterial urinary tract infections. They are targeting the ability of uropathogenic E.coli, a major cause of bladder infections, to form biofilms within bladder cells and on surfaces in the urinary tract. They have identified multiple genes that regulate biofilm formation in E. coli, and are currently focusing on the role of the QseBC signaling system as a potential antimicrobial target. In contrast, Kip Guy takes a direct medicinal chemistry approach in his search for agents to combat two parasitic diseases, malaria, caused by Plasmodium species, and sleeping sickness resulting from infection with Trypanosoma brucei. Developing anti-malarial agents is also a focus for Brian Bachmann, whose natural products discovery program searches for active compounds among the natural products of cave Actinomycetes (see Natural Products).

Larry Zwiebel's research focuses not on the pathogen, but on its mechanism of transmission. The Anopheles gambiense mosquito is the primary carrier of Plasmodium falciparum, the unicellular parasite that causes malaria. The Zwiebel lab studies the biochemistry and molecular biology of odorant receptors in the host mosquitoes. Since the insect uses these receptors to find its blood meals, the goal is to develop methods to interfere with the feeding process and eliminate the Anopheles as an important vector for malaria in humans.

 

 

 

Image Source: Structure of the Staphylococcus aureus IsdG-family heme oxygenase, a heme degrading enzyme. From the Eric Skaar Lab. M.R. Reniere et al. (2010) Molecular Microbiology, 75, Cover..

Enzymatic and Nonenzymatic Lipid Oxidation in Human Health and Disease

Polyunsaturated fatty acids are a key component of cell membranes. These lipids are subject to free radical peroxidation reactions, which occur nonenzymatically, leading to the formation of reactive electrophiles that can damage membranes, proteins, and DNA. These processes are increased under states of “oxidative stress” and contribute to the pathogenesis of a range of neurological, cardiovascular, and inflammatory diseases, obesity, and cancer. Ironically, nature has also harnessed these reactions through the function of lipoxygenase and cyclooxygenase enzymes that catalyze the controlled stereospecific peroxidation of polyunsaturated fatty acids to form a series of lipid signaling molecules. These lipid oxidation products regulate multiple immune, cardiovascular, neurological, and reproductive processes. The role of enzymatic and nonenzymatic lipid peroxidation in health and disease is a focus of research in a number of VICB member laboratories.

Enzymatic lipid peroxidation is a major focus of Larry Marnett’s in depth studies of cyclooxygenase structure, function, and inhibition. The cyclooxygenase pathway is also the subject of Rich Breyer’s investigations on the signaling role of prostaglandins. These products of the cyclooxygenase pathway of arachidonic acid metabolism, exert their effects through specific G-protein coupled receptors. The Breyer lab seeks to understand every aspect of the molecular biology, biochemistry, and pharmacology of these receptors. In contrast, the lipoxygenase pathways are the primary focus for Alan Brash and Claus Schneider, who strive to characterize the enzymes involved and their chemical mechanisms.

Nonenzymatic lipid peroxidation is the primary focus for Ned Porter, Dan Liebler, Jack Roberts, and Sean Davies, and an ongoing interest for Larry Marnett. The Porter lab strives to understand the chemical mechanisms of lipid peroxidation and how antioxidants can be used to prevent damaging oxidation reactions that contribute to disease. The Liebler laboratory is interested in the identity and chemistry of protein modifications by lipid-derived electrophiles. The Roberts lab focuses on specific classes of lipid peroxidation products, the isoprostanes and neuroprostanes. They use isoprostane levels as a reliable marker for oxidative stress, and attempt to understand the biologic roles of both isoprostanes and neuroprostanes in various disease states. The Davies lab concentrates their efforts on the highly reactive isoketal products of lipid peroxidation, with a major interest in their role in obesity and aging. The Marnett lab works to characterize the protein- and DNA-damaging effects of the bis-electrophile products of nonenzymatic lipid peroxidation reactions.

 

 

 


Image Source: Jack Roberts Laboratory, Vanderbilt University, "Phosphatidylcholine Containing an F2 -Isoprostane."

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Natural Products

Nature offers a marvelous diversity of chemical structures, and many of our valuable therapeutic agents have been derived from natural products. Thus, there is a growing interest in mining these resources for new drug discovery efforts. The chemistry of natural products is often complex and requires specialized synthetic and analytic techniques. VICB chemists are ready to meet this challenge.

Gary Sulikowski specializes in tackling difficult natural product syntheses. He chooses his targets on the basis of structural complexity, novel biologic activity, and the opportunity to explore new chemical reactions. Among the successes enjoyed by the Sulikowski lab are the syntheses of apoptolidins, natural products noted for their ability to induce apoptosis in cultured cells, and antitumor agents of the landomycin and mitomycin classes.

A characteristic of most natural products is the presence of multiple chiral centers that exist in a single conformation. Stereoselective synthesis of these centers is one of the greatest challenges in natural products chemistry. Jeff Johnston and his lab focus primarily on techniques to meet this challenge. Their novel application of chiral proton catalysis is a particularly promising approach to amine- and peptide-based natural products.

Brian Bachmann takes a different path to natural products, harnessing biosynthetic methods in combination with chemical approaches to achieve his goals. His Natural Products Discovery Program aims to identify new natural products sources with a current focus on cave Actinomycetes. State-of-the-art high throughput fractionation and screening methodologies are then used to identify promising lead compounds for the battle against malaria.

 

 

 


Image Source: Gary Sulikowski Laboratory, Vanderbilt University, "Total Synthesis of Complex Natural Products."

 

Synthetic and Medicinal Chemistry

A successful chemical biology program requires a strong foundation in synthetic chemistry. The VICB’s team of chemists provides that support for institute-wide research efforts. But equally important, these researchers are actively discovering new reactions and reaction mechanisms that broaden our fundamental understanding of chemistry.

As part of the Neuroscience Drug Discovery Program, Craig Lindsley’s group provides the chemistry support that has led to the discovery of promising subtype-selective modulators of muscarinic and glutaminergic receptors. Natural products synthesis, isolation, and characterization are the major interests of Gary Sulikowski, Brian Bachmann, and Jeff Johnston, as described in greater detail elsewher (see Natural Products).

For many VICB labs, chemical synthesis is used to support the overall research program. Complex dendrimer nanoparticles are the synthetic focus in Eva Harth’s lab, while Sandy Rosenthal’s interests lead her to synthesize drug-conjugated fluorescent nanocrystals (for more, see Nanoscience). Larry Marnett, Charles Manning, and Wellington Pham synthesize novel molecules to be used as imaging probes (see Imaging), and Kip Guy’s synthetic interests are focused on the discovery of new agents for the treatment of malaria and sleeping sickness (see Infectious Diseases). Carmello Rizzo and Tom Harris synthesize oligonucleotides bearing damaged bases to study the impact of specific DNA adducts on replication, repair, and mutagenicity.

 

 

mage Source: Brian Bachmann Laboratory, Vanderbilt University, "Biosynthetic Approaches to Novel Antibiotics."

New Technologies

Chemical biology research strives to apply the latest advances in chemical synthesis and analysis to complex biological systems. VICB researchers are at the forefront of developing and applying the latest approaches to the chemistry-biology interface, such as:

 

Nanoscience

Imaging

High-Throughput Synthesis and Screening

Nuclear Magnetic Resonance (NMR) and Other Spectroscopic Approaches

X-ray Crystallography

Mass Spectrometry, Proteomics, and Lipidomics

Computational Approaches and Bioinformatics

Antibody and Protein Expression

 

 

   

Nanoscience: Nanotechnology is an exciting new field that interfaces chemistry, biology, and engineering to develop novel particulate materials of less than 100 nm diameter. The huge diversity of substances that can be produced through this new technology holds promise for revolutionizing multiple aspects of biology and medicine including in vivo and ex vivo imaging, drug delivery, protein and peptide delivery, and cancer diagnostics and therapeutics.

VICB investigator Eva Harth is capitalizing on this new technology to synthesize dendritic molecular transporters, which are particulate polymeric structures that can be used to carry bioactive small molecules across cell membranes and deliver them selectively to desired subcellular compartments.

Sandy Rosenthal focuses her work on the synthesis of fluorescent nanocrystals, which can be specifically targeted to membrane receptor proteins. In collaboration with Randy Blakely, she uses these to monitor the activities of neurotransmitter receptors and transporters in functioning neurons.

In addition to his interest in electrochemistry on the nanoscale, Dave Cliffel studies the lock and key interactions of proteins through the design of targeted linear and loop peptide structures deposited on the surface of gold nanoparticles. He has found that these particles are particularly useful for calibrating immunoassays.

Qi Zhang uses quantum dots bearing fluorescent labels to monitor the movement of neurotransmitter vesicles in living neurons (see Neuroscience). This innovative approach allows his lab to distinguish between neurotransmitter release by conventional fusion of the vesicle with the cell membrane and a newly discovered “kiss-and-run” mechanism.

Darryl Bornhop is interested in analytical methods on the nanoscale. His back scattering interferometer allows detection of picomolar concentrations of an analyte in nanoliter quantities of sample. This novel technology also provides a means to monitor conformational changes that occur in biological macromolecules upon binding of ligand or a second macromolecule.

 

 

 

 

Image Source: Effective delivery of IgG-antibodies into infected cells via dendritic molecular transporter conjugate IgGMT. Eva Harth Laboratory. S.K. Hamilton, et al., Molecular BioSystems (2008) 4, 1209.

Imaging: A revolution in biological imaging at the cellular and whole animal level is taking place, and VICB researchers are partnering with the Vanderbilt Institute of Imaging Science to maximize opportunities in this emerging field.

Many imaging modalities require contrast agents or chemical labels to highlight the protein or structure of interest. Wellington Pham works with all imaging modalities in his goal to develop new molecular probes for cancer detection. Similarly, Charles Manning’s approach is broadly based, as he searches for novel imaging agents to observe each stage in cancer development, and to better assess tumor stage and responsiveness to therapy. Larry Marnett’s goal is also cancer detection and therapeutic response, but he focuses primarily on developing probes to detect the enzyme cyclooxygenase-2, which is expressed at unusually high levels in most tumor tissues.

Dave Piston has developed multiphoton excitation fluorescence imaging modalities, which he uses to study glucose metabolism and insulin secretion in beta cells of intact pancreatic islets. Richard Caprioli is revolutionizing the field of protein metabolism through his use of mass spectrometry to image proteins in situ in intact tissues.  Kevin Schey is also using mass spectrometry for imaging with a special focus on membrane proteins in the lens of the eye.  Sandy Rosenthal’s fluorescent nanocrystals are designed to monitor the expression and function of neurotransmitter receptor and transporter proteins, while Qi Zhang uses quantum dots to reveal the movement of neurotransmitter vesicles. These imaging molecules promise to help unlock key aspects of neuronal signaling that plays a role in such fundamental processes as mood, sleep, appetite, and aggression.

 

 

Image Source: Wellington Pham Laboratory, Vanderbilt University, "Tracking the Migration of Dendritic Cells In Vivo."

High-Throughput Synthesis and Screening: Key to the practice of chemical biology is the discovery of new molecular probes that can be used to investigate the role of specific biochemical and signaling pathways in cellular physiological or pathophysiological processes. In addition to providing new insights into critical questions in biology, molecular probes also often pave the way for the development of new therapeutic agents. Efficient molecular probe discovery requires high-throughput screening and chemical synthesis capabilities.

The VICB is at the cutting edge in both of these critical disciplines. The VICB High-Throughput Screening (HTS) Core Facility under the direction of Dave Weaver has been collaborating with Vanderbilt researchers since 2005. The facility has completed or is currently executing over 40 screens, most of which were developed on site. The HTS facility possesses a broad assortment of state-of-the-art instruments for multiple screening modalities, and the staff provides training and expertise in assay development and execution. Of equal importance to the success of HTS at Vanderbilt is the availability of a 190000 member compound library, which is expanding at a rate of 10000 compounds a year through acquisitions from commercial sources, collaborating labs, and the VICB’s own synthetic and natural products chemists.

Once HTS has identified a lead compound that possesses a desired biological activity, it is the role of synthetic chemists to use that initial discovery to create new molecules that have better potency, selectivity, and bioavailability with reduced toxicity. Technology- enabled synthesis (TES), introduced to the VICB by Craig Lindsley, combines a parallel synthesis approach with microwave technology and mass-directed LC-MS purification to achieve the goal of high-throughput chemical library production. Now Chemical Synthesis Core investigators under the direction of Gary Sulikowksi apply this approach as well as the full range of medicinal chemistry methodology to molecular probe discovery efforts in collaboration with other VICB investigators and the broader Vanderbilt community.

 

 

Image Source: Vanderbilt High-Throughput Screening Core Facility, Vanderbilt University.

Nuclear Magnetic Resonance (NMR) and Other Spectroscopic Approaches: Organic chemists have used NMR for the characterization of small molecules for decades, and the VICB’s Small Molecule NMR Core Facility, under the direction of Don Stec provides state-of-the-art instrumentation and expertise for just that purpose. However, the use of NMR has now expanded to include the characterization of protein and nucleic acid macromolecules. Many VICB members, some in collaboration with the Center for Structural Biology (CSB), use this approach in addition to other spectroscopic methods to study a wide array biomolecular structures and interactions.

Steve Fesik uses NMR techniques to detect the binding of small molecules to protein targets as part of his fragment-based approach to drug design. NMR also assists in determining how small molecules bind to the target protein, enabling researchers to design structural changes to strengthen key interactions. These methods are a major component of the approach taken by Vanderbilt’s cancer Drug Discovery Program (see Cancer).

Walter Chazin uses NMR to probe the structure and dynamics of large proteins and multi-protein machines. His goal is to understand DNA replication and repair in addition to calcium and ubiquitination signaling in cells. Chuck Sanders is pushing the envelop for NMR-based protein structure determination and recently solved the structure of the 42 kD diacylglycerol kinase enzyme. The lab will now use their structural data to determine how mutations of the enzyme result in misfolding and lead to aberrant function.

Al Beth capitalizes on the ability of EPR (electron paramagnetic resonance) to probe protein structure in solution or in a membrane bilayer. His interest is in the structure and function of the erythrocyte anion exchange protein, and in the epidermal growth factor and serotonin receptors. His studies are focused on understanding the dynamics of each protein upon ligand binding and signal transduction processes.

Mike Stone’s primary interest is on nucleic acids rather than proteins. His work has led to increased understanding of the effect of damaged bases on DNA structure and on the interaction of the DNA with replication and/or repair enzymes.

 

 



Image Source: The first 3D structure determiend for an E3 ubiquitin ligase U-box domain showing the location of two key hydrogen-bonding networks that stabilize the structure. Water Chazin laboratory. M. D. Ohi et al, Nature Structural Biology (2003) 10, 250..

 

X-ray Crystallography: X-ray crystallography is not a new technique, but recent advances have resulted in improvements in protein crystallization, data acquisition, and data analysis that have kept this approach at the forefront of protein and nucleic acid structure determination. VICB investigators have taken advantage of the latest technology in X-ray crystallography to gain insight into the structure and function of key macromolecules.

Brandt Eichman’s primary interest is in understanding the process of DNA replication. He uses X-ray crystallography to explore the structure of key proteins that are responsible for unwinding DNA and assembling DNA polymerases at the replication fork. Tina Iverson’s focus is on mechanisms of cell signaling. She explores the structures of G-proteins, which transduce the signals of many membrane receptors, and investigates how a key bacterial respiratory protein responds to anoxic conditions. Borden Lacy, Mike Waterman, and Gerald Stubbs are applying detailed structural studies of pathogen macromolecules to develop new ways to fight infection (see Infectious Diseases and Immunity). Martin Egli’s interests are quite diverse, with goals to determine the structures of proteins involved in processes such as regulation of biological clocks and DNA-protein complexes.

 

 

Image Source: Brandt Eichman Laboratory, Vanderbilt University, "Protein Crystal for Crystallography."

Mass Spectrometry, Proteomics, and Lipidomics: The past 20 years have seen an explosion in mass spectrometry technology, greatly facilitating the analysis of small molecules and macromolecules in a diverse range of contexts, including pure samples, complex mixtures, and even intact tissues. The Mass Spectrometry Research Center, brings all of these new analytical modalities to Vanderbilt, and VICB investigators have not hesitated to capitalize on the advantages that state-of-the-art mass spectrometry has to offer.

Dan Liebler applies the latest techniques of mass spectrometry-based proteomics to the analysis of complex protein mixtures in the search for new biomarkers of malignancy. A second goal of his lab is to characterize the protein damage caused by reactive lipid-derived electrophiles. Proteomics is also a focus in Andy Link’s work. He uses this approach to identify novel proteins and post-translational modifications associated with yeast ribosomes during the process of protein synthesis.  Kevin Schey’s focus is on membrane proteins.  His lab uses top down proteomics analysis on a MALDI-TOF-TOF platform to monitor changes in ocular lens membrane proteins during development and aging.  His lab also uses laser capture microdissection and MALDI tissue imaging (see Imaging) to explore protein structure in the intact tissue environment.  Alex Brown uses mass spectrometry to analyze the complex array of lipids in cellular membranes in an effort to dissect precursor-product relationships in lipid signaling networks. His work represents a major contribution to the new discipline of lipidomics. Richard Caprioli’s ground-breaking work in the study of in vivo metabolism couples microdialysis sampling with mass spectrometric identification and quantification of key metabolites. This novel methodology has allowed the Caprioli lab to track the metabolism of neurotransmitters in the brain. His new approach to mass spectrometry-based imaging of proteins in intact tissue is discussed elsewhere (see Imaging).

John McLean’s interest is in expanding the horizon for mass spectrometric analysis of complex biologic mixtures. His lab is exploring the application of two-dimensional gas-phase separations using ion mobility-mass spectrometry. This approach separates molecules on the basis of surface area in one dimension and mass-to-charge ratio in the second dimension. Data are analyzed with the help of predictions made by molecular dynamics simulations. This method offers the advantage of separating molecules by major class (lipids versus peptides, versus carbohydrates, versus nucleotides) due to distinct surface area characteristics. Each class of molecule is then further separated on the basis of charge-to-mass ratio. Ion mobility-mass spectrometry offers great promise for biomarker discovery, metabolomics analysis, and detection of post-translational modifications and conformational changes in proteins.

Jonathan Irish combines mass spectrometry with flow cytometry in his use of the new technique of mass cytometry. This approach replaces the fluorescent antibodies traditionally used to label cells for flow cytometry analysis. Instead, antibodies bearing a lanthanide metal tag are used to label cells. The cells are then introduced as single cell droplets into the mass cytometer where they are vaporized and analyzed by mass spectrometry. The mass spectrometer differentiates the cells on the basis of the pattern of transition metal tags identified. The advantage of mass cytometry over traditional flow cytometry is that a much larger number of different labeled antibodies can be used, providing the basis for monitoring a wider range of cell characteristics and signaling responses.

Dave Hachey is director of the Mass Spectrometry and Proteomics Core facilities of the Mass Spectrometry Research Center. In that position he collaborates with many other VICB members in the design and execution of mass spectrometry-based experiments and guarantees that the cores are current with the latest technology and methodology.

 

 

Using mass spectrometry to image the distribution of a drug and its metabolites in vivo. Shown are cross-sectional images of a rat 2 hours after dosing with olanzapine. From the Richard Caprioli Laboratory. Reproduced with permission from S. Khatib-Shahidi et al., (2006) Anal. Chem. 78, 6448. Copyright 2006 American Chemical Society.

Computational Approaches and Bioinformatics: As the sophistication of molecular modeling and informatics software rapidly increases, computational approaches are taking an increasingly important role in the solution of complex chemical and biological problems. Many VICB members are successfully using these new methodologies to address interesting research challenges, and some concentrate on improving and/or developing new computational methodology.

Terry Lybrand uses computational methods to develop models for protein ligand complexes in order to study the basis for binding and to design ligands with higher affinity. His studies encompass both proteins for which confirmatory structural data are available and those for which computational methods are the only avenue possible. The Lybrand lab is also working to develop new and better software for computational molecular modeling.

Jens Meiler is combining computational and experimental approaches to elucidate the structure of membrane proteins in order to understand protein folding pathways, and to explore the interactions of proteins with small molecule therapeutics. Another major focus of the Meiler lab is to develop and apply computational chemical biology tools such as cheminformatics to virtually screen compound libraries for novel drug and probe lead compounds. These efforts support the work of the medicinal and synthetic chemists who are engaged in the VICB's multiple drug and probe discovery programs (see Cancer Biology and Medicine, Neurodegenerative and Neuropsychiatric Diseases, and Synthetic and Medicinal Chemistry).

Dave Tabb’s interest is in developing and applying computational methods to mine information from the complex data sets obtained through proteomics analyses.  His work facilitates the identification of proteins in complex mixtures and also helps to reveal protein-protein and protein-lipid interactions.

Bing Zhang’s lab applies bioinformatics techniques to the analysis of complex proteomics and transcriptomics datasets in order to discover their biological meaning. A major focus in the lab is the combination of RNA-Seq and shotgun proteomics data to obtain a comprehensive understanding of gene expression. In the WebGestalt project, the Zhang lab is developing a user-friendly web interface to facilitate the analysis of large datasets using gene-centric databases and statistical methods also developed in the lab. A practical application of their work is to identify the specific interconnected gene networks that are deregulated in metastatic cancer. Their goal is to understand the genetic drivers of metastasis so that this major contributor to cancer morbidity and mortality can be addressed therapeutically.

Carlos Lopez’s lab uses a combination of statistical mechanics, molecular simulation, mesoscale modeling, reaction kinetics, and cell population modeling to develop theoretical models of complex cell signaling pathways. Application of their approach to the process of programmed cell death (apoptosis) has led to knew insights into the extrinsic regulation of the apoptotic pathway.

 

 

Image Source: Jens Meiler Laboratory, Vanderbilt University, "Computational Design of Novel Protein Antibiotics."

Antibody and Protein Expression: The ability to generate specific polyclonal and monoclonal antibodies directed against target antigens has long been an important biochemical and pharmacologic tool.  As Director of the Vanderbilt Antibody and Protein Resource Core Facility (VAPR), Rob Carnahan and his team not only produce high quality monoclonal antibodies, they work together with investigators to tailor those antibodies to best meet the intended purpose.  Recent expansion of the VAPR has provided the capability for high level protein expression in both bacteria and mammalian cell systems, and the constant search for innovative methodologies promises to provide continuing improvements in services in the future.

A slightly different approach to antibody production is the single chain antibody, which is a fusion protein of the variable regions of the heavy and light chains of immunoglobulins.  These molecules retain the specificity of the original antibodies, but can be expressed in high quantities in bacterial expression systems.  Ray Mernaugh is interested in the development of single chain antibodies to a variety of cancer- and inflammation-related targets.

 

          

The new ClonePix system has increased productivity and broadened available services at the Vanderbilt Antibody and Protein Resource. 

 

Chemical Approaches to Understanding Biological Processes

Although chemical biology approaches are often directed towards applied goals such as drug discovery, they are also highly useful in the quest to expand our knowledge of normal physiology at the cellular and whole organism level. Many VICB researchers have this as a primary goal.

Understanding pathways for cell signal transduction is critical to nearly every discipline of biomedical research. Heidi Hamm’s work focuses on the molecular mechanisms by which G-proteins carry a signal from a specific receptor to effector molecules, such as adenylate cyclase or specific membrane channels. Her work has important implications for neuromodulation, exocytosis, and vascular physiology. Vsevolod Gurevich is interested in the receptors that use G-proteins to transduce their signals. These receptors are modulated by a group of proteins called arrestins, which usually inhibit receptor function after ligand binding. The Gurevich lab studies the interaction between the arrestin and the receptor protein in order to define the basis for the inhibitory action.

John York’s lab is also focused on major signaling pathways, particularly those related to inositol phosphates. Initially generated during phospholipase C-mediated lipid signaling, these molecules undergo a complex series of phosphorylations and dephosphorylations generating additional biochemical messengers. Inositol phosphates play a role in a widely diverse range of cellular functions that control cell growth, differentiation, migration, endocytosis, and apoptosis. The York lab is currently working to identify critical enzymes in these pathways and to discover small molecule modulators of the enzymes’ activities as probes to further delineate their role in health and disease.

Gregor Neuert investigates cellular signaling at the level of the individual cell. He uses a combination of approaches, including fluorescent in-situ hybridization with single cell resolution, live cell time-lapse microscopy, flow cytometry, and single molecule-based mathematical modeling to evaluate signaling events in minute detail. His work focuses on factors that regulate gene expression, including the role of gene expression “noise” which can play a major role in the variability that exists between genetically identical cells responding to the same environment.

Roger Cone’s interest is how the brain regulates energy homeostasis, a topic that encompasses two very common conditions in modern society, obesity and anorexia.  A primary focus of the lab is on pro-opiomelanocortin (POMC) a protein produced in the pituitary gland and hypothalamus that serves as a precursor for adrenocorticotropins (ACTH), melanotropins (MSH), and endorphins.  The Cone lab has cloned and characterized a family of five receptors for ACTH and MSH peptides and defined the roles of the melanocortin-3 and melanocortin-4 receptors in a mouse model of obesity.  Their ongoing interests are to understand the neural, hormonal, and nutritional signals that control the POMC system and to develop new models of energy homeostasis regulation.

David Wright works to understand the basic mechanisms of biomineralization – the process by which inorganic crystalline materials are deposited into a biological matrix. This process is critical for the formation of such basic structures as bone, teeth, and shells. He studies the scaffold biopolymers upon which the minerals are deposited in order to understand the structural factors that dictate the biomineralization process.

Billy Hudson’s main interest is in type IV collagen, which is the primary structural component of basement membrane. He strives to understand the basic structure of this fascinating material and how aberrations of that structure through mutation or environmental insult can lead to disease.

Protein dynamics are the primary interest of Hassane Mchaourab’s lab. They use electron spin resonance (ESR) spectroscopy of spin-labeled proteins as a major tool to explore the regulatory or functional motion of proteins. Examples of proteins that are of current interest in the lab include the bacterial lipid flippase, the human multidrug resistance protein, and a number of neurotransmitter transporters.

Richard Armstrong’s primary focus is on the mechanism of enzymes that are responsible for detoxication of xenobiotic compounds. Current interests in his lab include elucidation of the kinetic mechanisms of glutathione transferase, epoxide hydrolase, and UDP-glucuronyl transferases. The lab is also exploring the way by which some microorganisms use detoxicating enzymes to develop antibiotic resistance, a growing clinical problem.

Charles Hong’s lab uses the zebrafish model, both to study development, and as a model of specific human diseases. They develop high-throughput screens to identify small molecules that alter development or reverse the pathology in their disease models. Through this approach, the Hong lab has discovered important regulatory pathways for development of the cardiovascular and skeletal systems, and their small molecule inhibitors are serving as lead compounds for new drugs to treat cardiovascular diseases, anemia, and diseases of excessive bone formation.

Wenbiao Chen is another researcher capitalizing on the versatility of the zebrafish model. His interest focuses on the regulation of glucose and lipid metabolism by pancreatic alpha and beta cells, which secrete glucagon and insulin, respectively. In particular, he has found that metabolic stresses induce not only changes in hormone secretion by the cells, but there are also actual increases in the number of alpha and beta cells in the pancreas. These compensatory responses are conserved in zebrafish, and the Chen lab is working to understand the mechanism controlling them.

Jerod Denton’s focus is on understanding the biochemistry, physiology, and pharmacology of inward rectifying potassium channels. These important proteins, which promote the movement of potassium ions into the cell, play a key role in maintaining resting cell potential in neuronal, cardiac, endocrine, and epithelial tissues. The Denton lab is using high-throughput screening to identify small molecule inhibitors of individual members of this protein family, and using them in combination with genetic approaches and protein biochemistry to explore the function of these very interesting channels.

 

 

 

Subtle changes in compound structure creates a molecule that inhibits zebrafish bone formation or a molecule that inhibits vascular development. From the Charles C. Hong Laboratory. Hao et al. (2010) ACS Chemical Biology 5, 245

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