High-Content FRET-FLIM Screening in Inhibitors of Oncogenic Transcription by c-myc in Breast Cancer; Annual rept. 1 Jun 2007-31 May 2008

摘要

There is an urgent need for novel anti-breast cancer therapeutics. Our hypothesis is that by identifying small molecules that target the Myc oncogene, we will develop an effective therapeutic that will improve breast cancer patient care and contribute to the eradication of disease. Our OBJECTIVE is to identify compounds that can be used to selectively inhibit the oncogenic activity of Myc by inhibiting its interaction with one of its key binding partners TRRAP. To this end, we aim to (1) develop a novel high content screen to identify inhibitors that block Myc:TRRAP interaction; (2) determine the transcriptional signatures of Myc:TRRAP target genes; (3) screen drug and chemical libraries to identify compounds that disrupt Myc:TRRAP interaction; and, (4) validate lead compounds that disrupt Myc:TRRAP interaction and block the transformation potential of breast cancer cells. In the first year of this grant we have constructed several fluorescent fusion protein constructs of Myc and TRRAP, and evaluated their ability to bind and engage in fluorescence resonance energy transfer (FRET) in vivo. We have identified FRET pairs that are functional and established methodology using novel instrumentation that will enable the high throughput screening of chemical libraries. In the course of our work, we have shown that the cell systems we were aiming to use are unfortunately sensitive to expression of the fusion proteins. To overcome this unexpected issue, we are evaluating additional cell systems, as well as new expression constructs that will enable stable cell lines to be developed that constitutively and conditionally express the Myc and TRRAP fusion proteins, respectively. We have completed all tasks that we aimed to achieve in the first year of the grant and we are well positioned to fulfill the objectives of our proposal by the end of the second year of this grant.