JHU/CCR Fellowship in Molecular Targets and Drug Discovery Technologies Project Details

Project Sponsor/Mentor:	Allan Weissman
Title:	Lab/Branch Chief
Address:	Bldg. 560 Rm. 22-103 NCI-Frederick
Telephone:	301-846-7540
Fax:	301-846-1666
Email:	amw@nih.gov
Sponsoring Laboratory/Branch:	LPDS

Project Title:	Small Molecule Inhibitors of ERAD
Target(s) of Interest:	gp78, ERAD

Project Synopsis:
The Frederick campus of the NCI-Center for Cancer Research provides a unique collaborative atmosphere for drug discovery and development in an environment that includes biologists with a wide variety of expertise including biophysics, biochemistry, molecular and cell biology, and chemists who specialize in either synthetic medicinal or natural products chemistry. A successful candidate for this position will have the opportunity to play a central role in a drug discovery effort involving such a team of scientists who are developing means to inhibit a cellular process closely-linked to cancer and metastasis. As part of this research team the trainee will obtain hands-on experience in a wide variety of techniques in high-throughput screening, cell biology, biochemistry and state of the art optical microscopy and molecular modeling. Our group within the Laboratory of Protein Dynamics and Signaling has a primary interest in the ubiquitin-proteasome system. This system of protein regulation plays essential roles in many cellular processes through targeted and exquisitely timed protein degradation in the proteasome. Moreover, many of the substrates and enzymes of this system are implicated in disorders ranging from neurodegenerative diseases to cancer. A particular area of focus, and an area where we have played a significant role in advancing the field, is in endoplasmic reticulum (ER)-associated degradation (ERAD). This is a critical process in which the ubiquitin-proteasome system maintains protein homeostasis in the crowded molecular milieu of the ER. The ER is a large organelle where, among other things, proteins are processed for transport to other organelles, to the cell membrane or for secretion into the bloodstream. When proteins fail to fold or assemble properly into oligomeric complexes, the ERAD pathway prevents proteotoxicity by targeting these proteins to the proteasome for degradation. ERAD also plays essential roles in regulating proteins that are critical to a number of cellular processes. Relevant to the two projects described below, is the recent finding by our group that a specific component of the ERAD ubiquitination machinery, known as gp78, promotes cancer metastasis by targeting the metastasis suppressor KAI1 (aka CD82) for destruction. Our data suggest that there is a link between ERAD and the spread of cancer cells throughout the body (metastasis), which is the cause of 90% of cancer deaths. We also have preliminary evidence that blocking ERAD in general, which promotes a condition known as ER stress, will help block tumor progression and spread and can be particularly effective in treatment of Multiple Myeloma. This is a tumor of the blood and bone marrow that is recalcitrant to most therapeutic modalities. Our group, together with our collaborators, are carrying out two interrelated projects. The first involves screening for potential leads for cancer treatment that block the multi-step process of ERAD. The second is oriented towards identifying compounds that specifically block the function of gp78. A successful candidate would have the opportunity to work on both of these projects. The first project employs a cell-based and phenotypic-based screen to identify natural product extracts that contain novel molecules that cross the cell membrane and block ERAD. This project employs an initial screen using well-characterized fluorescent substrates that accumulate when ERAD is disrupted and can be followed by confocal microscopy or measured quantitatively with a fluorescence plate reader. Confirmed hits from the primary screen will be followed up by a combination of chemical purification from extracts and secondary assays to interrogate the molecular effects of the molecules on ERAD and cancer cell death in tissue culture. Compounds will eventually be assessed in animal models of cancer as a prelude to development of therapeutics for human disease. Concurrently, studies will be carried out to identify the specific molecular targets of these molecules. The second project is oriented towards evaluating small molecules for their capacity to disrupt the interaction of gp78 with cellular targets and with interacting components of the ubiquitin-proteasome system. This project, which is at an earlier stage of development than the first, will provide the trainee with the opportunity to develop reagents and assays to monitor the disruption of interactions between gp78 and its ERAD substrates, including KAI1, in living cells. The types of reagents to be developed would include a variety of chimeric fluorescent proteins. These constructs would permit the use of advanced microscopy techniques such as fluorescence resonance energy transfer (FRET), to examine protein-protein interactions and their disruption by small molecules. Additionally, photobleaching techniques will be used to examine the kinetics of molecular processes within both normal and cancer cells. As with the first project, a variety of secondary assays will be employed with the goal being preventing cancer cell growth and spread in animal models with the ultimate goal being development of therapeutics for human disease. The trainee will have the opportunity to be mentored by scientists with expertise that reflects the multi-disciplinary nature of these projects. In particular, this research involves a close collaboration between our group in the Laboratory of Protein Dynamics and Signaling, the Optical Microscopy and Analysis Laboratory and the Molecular Targets Laboratory. This work will also take advantage of recent discoveries related to the structure of gp78 made by our group in collaboration with the Structural Biophysics Laboratory and the Macromolecular Crystallography Laboratory. The successful candidate will therefore also gain exposure to world class technology for determining protein structure.
Fellow Research Plan:
The fellow will function as an integral member of my group within the Laboratory of Protein Dynamics and Signaling. The trainee will participate in our group meetings and in the Cancer Genetics and Signaling Seminar Series and will meet regularly with me. The trainee will have the opportunity to become expert in mammalian cell culture by deriving clones expressing proteins of interest as well as by adapting various cellular endpoint assays to a variety of cancer cell lines. The fellow will also play critical roles in development of plasmids expressing a variety of chimeric proteins for expression in cells. The fellow will also use and develop a variety of techniques in cell biology and advanced optical microscopy. The overarching goal of this experience will be to provide the successful candidate with both the necessary skill set and portfolio of scientific approaches to embark on a successful career in drug discovery research.