Center of Biomedical Research Excellence (COBRE) in Molecular Targets

Research Projects

COBRE in Molecular Targets – Project 1 - Glycolysis and Neoplastic Growth more

COBRE in Molecular Targets – Project 2 - Development and Testing of Small Molecular Antagonists of Monocyte/macrophage Migration Inhibitory Factor Recently Found to Play an Important Role in Carcinogenesis more

COBRE in Molecular Targets – Project 3 - Activation-Dependent Targeting of the Lipid Signalling Enzyme Sphingosine-Kinase more

COBRE in Molecular Targets - Project 5 - Structural Studies of TRAF6-mediated IL-1 and Toll-like Receptor Signalling Pathways more

COBRE in Molecular Targets – Project 4 - Development of a Sequence-specific Double-stranded DNA Cleaving Agent more

COBRE in Molecular Targets – Project 6 - The Programmed Death Pathway Initiated from the Endoplasmic Reticulum more

COBRE in Molecular Targets – Project 7 - Control of Tumour Growth by Ras-related Proteins more

COBRE in Molecular Targets - Project 9 - Molecular target discovery: Structure-activity analysis of tissue-specific carcinogens more

COBRE in Molecular Targets - Project 10 - VSEL and Brain Regeneration in a Murine Model of Acute Hypoxia more

COBRE in Molecular Targets - Project 8 - Roles of IKK1 in Skin Development and Dysplasia (October 2006 – present) more

Shared Core Facilities

Core A - Administration
Core B - Microarray Facility
Core C - Molecular Modeling Facility
Core D - Structural Biology [NMR] Facility
Core E - Proteomics Facility

Advisory Committees

Internal Advisory Committee
External Advisory Committee
Executive Committee

Publications since award

In September 2003 the James Graham Brown Cancer Center at the University of Louisville was awarded a five-year, $11 million Center of Biomedical Research Excellence (COBRE) grant in Molecular Targets by the National Center for Research Resources at the National Institutes of Health. This grant establishes the Molecular Targets Program at the Brown Cancer Center as a Center of Biomedical Research Excellence (COBRE) which allows them to continue to recruit and bring together the best researchers from a variety of disciplines to identify and develop new molecular 'targets' for anti-cancer drugs and therapies using the techniques of modern structural biology.

The two major objectives of this COBRE in Molecular Targets are to (1) discover new approaches to the therapy of cancer, and (2) ensure the success of junior investigators of the Molecular Targets program at the Brown Cancer Center. These objectives will be met through the following specific aims:

To foster successful independent research careers for the principal investigators of the five research projects, leading to independent R01 grants.
To further build the existing core facilities of the Brown Cancer Center which will enhance the research capability of the five principal investigators and other members of the program and the Center.
To continue to develop the Molecular Targets Program as a cohesive research program which promotes collaborative research projects and increases funding opportunities.
To develop an internationally and nationally recognized program which attracts the very best graduate students, postdoctoral fellows and additional faculty.
Most importantly, to make discoveries which lead to new and more effective treatments for patients with cancer.

The five related projects in this Center of Biomedical Research Excellence grant address a broad spectrum of molecular targets. The overarching theme is the use of structural biology to develop novel strategies for the therapy of neoplastic disease. We believe that these projects, along with those of the other members of the program, represent a coherent approach to the application of modern structural biology to translational cancer research.

As the Principal Investigators (P.I.'s) on these research projects obtain national funding for their research, they rotate off the COBRE in Molecular Targets and promising new investigators who fit the criteria for this grant are recruited. To date, three of the first five P.I.'s have obtained National Cancer Institute grants [Dr. Jason Chesney (Project 1), Dr. Robert A. Mitchell (Project 2) and Dr. Brian (Binks) Wattenberg (Project 3)] and new investigators have been/are being recruited to the program.

In addition to the five research projects, there are five support core facilities which are part of this program. In year 1, the COBRE in Molecular Targets also included funds to help support the renovation of Brown Cancer Center facilities to enhance the research setup for the COBRE investigators.

For information on the individual research projects (past and present) and the support core facilities which receive funding from this COBRE in Molecular Targets, please see the links in the right hand collumn on this page.

COBRE in Molecular Targets - Core Groups (September 2003 - present)

Administrative Core

Dr. Donald Miller, Dr. John Eaton (co-Principal Investigators of this COBRE grant) and the Administrative Assistant, Nancy Roseberry, are responsible for all of the administrative aspects of this COBRE in Molecular Targets, including all of the scientific and administrative organization of the program. This core coordinates all regularly scheduled meetings of the Executive Committee, the Internal Advisory Committee and the External Advisory Committee as well as the annual Molecular Targets Program Research Retreat.

Microarray Core Facility

Wolfgang Zacharias, Ph.D., Facility Director

The Microarray Core Facility is located on the third floor of the Baxter II Biomedical Research Building. This facility contains an Affymetrix Genechip instrument which is available to all MT-COBRE members. Plans are underway for the development of a tissue array component of this facility as well. The Microarray Facility is under the direction of Dr. Wolfgang Zacharias and has been fully operational for the past five years. It contains a full-time staff member, Sabine Waigel, who provides technical assistance in sample processing, hybridization and data analysis. The services provided include genome-wide RNA expression analyss, complex SNP profiling, data analysis and mining, and RNA quantity/quality assays. The Microarray Core has a close relationships with the Department of Biostatistics & Bioinformatics, the Laser Capture Microscopy facility in the Department of Pathology, and a newly established Tumor Repository, located in the Brown Cancer Center, run by Dr. Richard Goldstein. This allows the preparation of samples with known pathologic characteristics and appropriate control specimens. Dr. Zacharias is available for assistance with experimental design and data analysis. The Microarray Core Facility provides important support for each COBRE P.I. during development of their project-specific microarray approaches.

Molecular Modelling Core Facility

John O. Trent, Ph.D., Facility Director

The Molecular Modeling Core Facility is a state-of-the-art molecular modeling facility which has the capability of providing models based on structural data obtained from either X-ray crystallographic or NMR studies. The Director, Dr. John Trent, is available on a collaborative basis to provide assistance and instruction for individual faculty members or students. This facility is located on the fourth floor of the James Graham Brown Cancer Center. The capabilities of the facility include techniques such as explicitly solvated molecular dynamics, homology modeling, docking, ESP calculations, and conformational searching. A significant capability of the core is in silico virtual screening drug discovery which has been proven to provide new or better inhibitors for a number of molecular targets. The Molecular Modeling Core is available to all COBRE in Molecular Targets investigators and provides important information on drug design and modification.

Structural Biology (NMR) Core Facility

Andrew N. Lane, Ph.D., Facility Director

The Structural Biology Core Facility is located in the Molecular Imaging Research Center, in the rear of the James Graham Brown Cancer Center. It contains both the capability for protein expression and structural analysis. This NMR portion of the facility contains two spectrometers, one operating at 600 MHz and the other at 800 MHz and is operated as part of the Structural Biology Program, under the direction of Professor Andrew Lane. Dr. Lane is accessible on a collaborative basis to assist with design of experiments and data interpretation. The laboratory manager, Dr. Sam Arumugam, provides assistance with the performance of experiments. The 600 MHz NMR instrument is a 4-channel Varian Inova 14.1 T system, equipped with a cold probe, 5 and 8 mm inverse probes, a 3 mm flow probe and flow system, and an automatic sample changer. This configuration enables a wide range of 3D experiments to be carried out on isotopically-labeled proteins including deuterium decoupling. The cold probe gives a welcome increase in sensitivity, which is especially valuable for less soluble proteins or complexes and, with the automation system, for high throughput metabolomics experiments. The Varian Inova 18.8 T system is particularly useful in the study of larger proteins and complexes using the TROSY technique. Additional probes have been purchased to increase the range of systems that can be studied, including molecules containing 31P (e.g., phosphopeptides, phosphorylated metabolites) or 77Se. A comprehensive protein expression laboratory is located within the Brown Cancer Center and is fully equipped to do large-scale protein preparations and purifications. Equipment for biophysical analysis includes an analytical ultracentrifuge recently purchased with an NCRR small instrumentation grant, and is now located in the new Biophysics Core facility in the Brown Cancer Center directed by Professor J. B. Chaires. The Structural Biology Core Facility is available to all members of the COBRE in Molecular Targets and provides important support for structural determinations, inhibitor design and target verification, as well as functional analysis of metabolism in response to novel anticancer agents.

Proteomics Core Facility

Jon B. Klein, M.D., Ph.D., Facility Director

The Proteomics Core Facility began operation in 1998 and was one of the first laboratories in the nation equipped for high throughput proteomic analysis based on laboratory automation. It is directed by Dr. Jon Klein, who is also a member of the Internal Advisory Board of the COBRE in Molecular Targets. He is assisted by two laboratory personnel who have several years of experience in this area. Dr. Klein is available to all MT-COBRE investigators on a collaborative or consultative basis. The excellence of this laboratory is attested by the fact that it is used as a 'beta testing' site for a number of companies making proteomics-associated products. The Core Proteomics Laboratory is uniquely equipped with robotic workstations that increase gel throughput, image analysis, spot excision, in-gel peptide digestion, and MALDI-MS sample preparation. The automated workstations include a Propic gel spot picker that visualizes and excises spots from fluorescence-stained gels and a ProGest station that automates in-gel trypsin digestion in a 96 or 384 well format. The laboratory also contains a ProMS robot that automates the desalting, concentrating and spotting of peptide samples onto MALDI targets (Genomics Solutions, Inc.) thereby allowing fully integrated high-throughput analysis of protein samples. MALDI-MS analysis is performed using a Micromass, Inc. TOF-Spec 2E. Recently, the laboratory instituted automated analysis of high-resolution two-dimensional gels using Progenesis software from Nonlinear Dynamics, Inc. The Progenesis software significantly decreases analysis time of two-dimensional gels, thereby accelerating throughput. The Proteomics Core Facility plays an important role in several of the COBRE in Molecular Targets projects, particularly in looking at downstream effects of target inhibition.

COBRE in Molecular Targets Advisory Committees

Internal Advisory Committee

Scott R. Whittemore, Ph.D., Department of Neurological Surgery
David W. Hein, Ph.D., Chairman, Department of Pharmacology
Suzanne T. Ildstad, M.D., Director, Institute for Cellular Therapeutics
David Gozal, M.D., Director, Kosair Children's Hospital Reseach Institute
Jon B. Klein, M.D., Ph.D., Director, Proteomics Core Facility
Chair - John W. Eaton, Ph.D., Head, Molecular Targets Program; Co-PI, COBRE in Molecular Targets

External Advisory Committee [as of 7/06]

Richard Bucala, M.D., Ph.D., Yale University
Mitchell P. Fink M.D., University of Pittsburgh
Daniel M. Raben, Ph.D., Johns Hopkins University
John E. Repine, M.D., University of Colorado

Executive Committee

Donald M. Miller, M.D., Ph.D., co-PI
John W. Eaton, Ph.D., co-PI
Douglas C. Dean, Ph.D.
Haribabu Bodduluri, Ph.D.
Robert A. Mitchell, Ph.D.
Brian (Binks) W. Wattenberg, Ph.D.
Jason Chesney, M.D., Ph.D.

COBRE in Molecular Targets Publications

Ye H, Chen T, Xu X, Pennycooke M, Wu H, Steegborn C. Crystal structure of the putative adapter protein MTY1859. J Struct Biol 148:251-6, 2004

Chesney J, Telang S, Yalcin A, Clem A, Wallis N, Bucala R. Targeted disruption of inducible 6-phosphofructo-2-kinase results in embryonic lethality. Biochem Biophys Res Commun 331:139-46, 2005

Lane AN, Arumugam S. Improving NMR sensitivity in room temperature and cooled probes with dipolar ions. J Magn Reson 173:339-43, 2005

Pitson SM, Xia P, LeClercq TM; Moretti PA, Zebol JR; Lynn HE; Wattenberg BW; Vadas MA. Phosphorylation-dependent translocation of sphingosine kinase to the plasma. J Exp Med 201:49-54, 2005

Swant JD, Rendon BE, Symonc M, Mitchell RA. Rho GTPase-depencent signaling is required for MIF-mediated expression of cyclin D1. J Biol Chem 280(24):23066-72, 2005

Chesney J. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and tumor cell glycolysis. Curr Opin in Clin Nutrition and Metabolic Care, in press, 2006.

Klein JB, Barati MT, Wu R, Gozal D, Sachleben LR Jr, Kausar H, Trent JO, Gozal E, Rane MJ. Akt-mediated valosin-containing protein 97 phosphorylation regulates its association with ubiquitinated proteins. J Biol Chem 280(36):31870-81, 2005.

Kozlowski PM, Nazarenko VV, Jarzecki AA. DFT analysis of interligand vibrations in a hydroperoxo complex of cobalt bleomycin. Inorg Chem. 45(4):1424-6, 2006.

Telang S, Yalcin A, Clem AL, Bucala R, Lane AN, Eaton JW, Chesney J. Ras transformation requires metabolic control by 6-phosphofructo-2-kinase. Oncogene 2006 May 22; [Epub ahead of print]

Wattenberg BW, Pitson SM, Raben DM. The sphingosine and diacylglycerol kinase superfamily of signaling kinases: localization as a key to signaling function. J Lipid Res 47(6):1128-39, 2006.