DISCOVERING NOVEL COMPOUNDS TO STABILIZE THE E2F-RETINOBLASTOMA PROTEIN COMPLEX IN THE CELL CYCLE
Elise Brown, Cameron Pye, Tyler Liban, Seth Rubin.
University of California, Santa Cruz, Santa Cruz, CA.
The retinoblastoma protein (RB) is a key cell-cycle regulator. Mutations in the RB pathway cause rapid cell division, which is a hallmark of cancer. RB binds and inhibits the E2F transcription factor until cells are ready to divide. RB is then phosphorylated by cyclin-dependent kinases, which leads to E2F dissociation and its activation of cell-cycle genes. The structure of phosphorylated RB was previously determined, and when phosphorylated, its pocket domain and N-terminal domains associate to change the active site and release E2F. Many cancers have increased kinase levels which leads to phosphorylated RB, unbound E2F, and rapid cell division. We hypothesize that if a novel compound can be found to cause phosphorylated RB to have an affinity to E2F, a drug can be developed that stops rapid cell proliferation. Such a compound may block the site of pocket-N-terminal association or work by some other mechanism. We previously used a high-throughput fluorescence polarization (FP) anisotropy to identify lead compounds that increase E2F binding to phosphorylated RB. In current work, we have repeated the FP assay on screen hits prepared from fresh powders and followed up those results with isothermal titration calorimetry (ITC) experiments. The goal of ITC is to determine the affinity of the RB-E2F complex in the presence of the compound. To date, we have found 3 compounds that may show promise of enhancing binding of E2F to phosphorylated RB.