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

Project Sponsor/Mentor:	Michael M. Gottesman
Title:	Principal Investigator
Address:	Rm 2112, Bldg 37, 37 Convent Dr, Bethesda MD 20892
Telephone:	301-496-1921
Fax:	301-402-0450
Email:	GottesmM@mail.nih.gov
Sponsoring Laboratory/Branch:	LCB

Project Title:	Molecular target and drug discovery for cancer mul
Target(s) of Interest:	P-glycoprotein (MDR1, ABCB1), Multidrug resistant

Project Synopsis:
The fellow may chose one of two projects related to the understanding of multidrug resistance and the discovey and development of drugs to combat it. 1. High throughput drug discovery program This project would be conducted under the guidance of Dr. Matthew Hall, a postdoctoral fellow in the lab. The initial compounds discovered that preferentially kill P-gp expressing cells were identified in a bioinformatic screen of NCI DTP data derived from compounds submitted for testing. A lead compound, NSC73306, was selected from those that were predicted to demonstrate inverse activity, and is currently in pre-clinical development. A number of limitations existed in the original screen, including the poor purity of some compounds, and the narrow range of P-gp expression across cell lines in the dataset. Given the promise of P-gp inverse agents, we have developed an assay for high-throughput drug discovery that will enable us to identify potential candidates for further development (manuscript in press). The NIH Chemical Genomics Center (NCGC), located off-campus in Rockville, MD, is a drug discovery institute gearing towards adapting laboratory-based assays for high-throughput use, and the fellow would be involved in running the high-throughput screen we have developed on the NCGCs database of over 200,000 compounds, and subsequently validating any new MDR1-selective agents discovered. Furthermore, we have discovered that some MDR1-selective drugs can switch off the expression of the drug transporter. With this is mind, the fellow will work with a cell line expressing GFP-tagged transporter to develop a new high throughput screen for drugs that turn off expression of the transporter. The fellow would be involved in all aspects of assay optimization and implementation, as well as data analysis and validation of P-gp inhibitors or P-gp inverse compounds of interest that result from the screening process. The fellow would also be involved in manuscript preparation and encouraged to present their results at symposia and meetings. 2. Gene array This project would be conducted under the guidance of Dr. Jean-Pierre Gillet, a postdoctoral fellow in the lab. The development of multidrug resistance (MDR) to chemotherapy remains a major challenge in the treatment of cancer. Resistance exists against every effective anti-cancer drug and can develop by multiple mechanisms. These mechanisms can act individually or synergistically, leading to MDR, in which the cell becomes resistant to a variety of structurally and mechanistically unrelated drugs in addition to the drug initially administered. Although extensive work has been done to characterize MDR mechanisms in vitro, the translation of this knowledge to the clinic has not been successful. Therefore, identifying genes and mechanisms critical to the development of MDR in vivo could lead to progress in the translation of this knowledge to clinical practice, especially in personalized medicine. MDR resistance mechanisms are mediated by several superfamilies of genes such as ABC transporters and cytochrome p450 (CYP450). Thus, establishing a reliable method for analyzing highly homologous genes from small amounts of tissue is fundamental to achieve any significant enhancement in our understanding of multidrug resistance mechanisms. In a recent study published by our laboratory, two TaqMan qRT-PCR platforms, based on micro- and nano-fluidic systems, TLDA and BioMark 48.48 Dynamic Arrays, were singled out with the potential to be further developed for individualized cancer management. The superiority of these platforms was clearly demonstrated over established technologies in assessing ABC transporter expression profiles. Our investigations led to the refinement of a previously established database with the capability to more precisely identify compounds whose resistance is mediated by ABC transporters as well as ascertain which compounds are responsible for collateral sensitivity (Orina et al, 2009). The challenge, now, is to apply these platforms to elucidate the gene signatures for MDR in a well-designed clinical study. This project proposes to critically examine proposed mechanisms of drug resistance in cancers based on TaqMan Low Density Array (TLDA). The cancers investigated include adult acute myeloid leukemia, ovarian carcinoma, hepatocellular carcinoma, non small cell lung cancer, alveolar soft part sarcoma, melanoma and others. The goal of the study is to highlight gene signatures associated with good and poor survival that would allow clinicians to predict patient response a priori to avoid giving toxic therapies to patients unlikely to benefit from them and to devise effective treatments that circumvent common mechanisms of drug resistance. The fellow will be involved in all aspects of this project and would benefit of an automated gene expression profiling platform, of established clinical collaborations with MGH (Boston) and MDA (Houston) and of statistical support with a data mining specialist. As above-mentioned, our laboratory strongly encourages the fellow to be involved in manuscript preparation and to present his results at symposia and meetings.
Fellow Research Plan:
As part of this project, the fellow will be exposed to a wide range of techniques that will include RT-PCR, Western Blotting, MTT cytotoxicity assays, Acumen high throughput screening imagers, immunohistochemistry, gene arrays and confocal miscroscopy, bioinformatics data analysis, absorption and fluorescence spectroscopies, flow cytometry and mass spectrometry. The fellow will undertake radiation safety training and laboratory safety training, and may use radioisotope-labeled compounds as part of the investigations. The fellow will work alongside a postdoctoral fellow who will assist in the planning and execution of experiments, and results will be reviewed regularly in both formal group meeting and informal sessions. It is expected that a number of manuscripts will be yielded from either research project, along with the potential for intellectual property/patenting options. The outgoing JHU/CCR Fellow, Kyle Brimacombe, worked on drug discovery projects in the Gottesman group, and will be available to discuss questions regarding the lab and projects (brimacombek@mail.nih.gov).