Rationale. Taxanes, epitomized by the drug Taxol, and their derivatives represent a state-of-the-art anti-cancer therapy. They bind to tubulin and disrupt the dynamic remodeling of the microtubule cytoskeleton, leading to inhibition of cell motility and proliferation; cells respond by initiating programmed cell death. These drugs are limited clinically, however, by their side effects and tumor cell resistance. Thus, there is a need to further identify anti-cancer compounds that disrupt cytoskeletal function. Guiding Hypothesis. Rho small GTPases act as critical regulators of cellular shape during growth factor responses and cell division; their activity is also required for oncogenic transformation. Diaphanous related formins (Drf), members of the formin family of cytoskeletal regulators, are activated by Rho proteins upon binding to the Drf GTPase binding domains (GBDs). The interaction disrupts an intramolecular association between the GBD and the Dia-autoregulatory domain (DAD). Introduction of a DAD-derived peptide into cells is sufficient to deregulate Drf proteins; DAD mimics Rho binding, thereby disrupting intramolecular autoinhibition. The result is the inappropriate stabilization of the cytoskeleton. Like taxanes, DAD peptide causes cells to eventually undergo apoptosis. We hypothesize that DAD peptide represents a novel class of anti-cancer drug that targets and deregulates Drfs in cancer cells. The proposed experiments will test our guiding hypothesis.
Specific Aims. (1) Identify the Diaphanous-related formin(s) that is/are targeted by DAD peptide (beginning with DAD peptide derived from the Drf3 protein) in Taxol-sensitive and -resistant breast and prostate cancer cell lines, (2) determine the structure of DAD peptide bound to its target, the GTPase binding domain, and (3) develop a screening method for identifying novel molecules that disrupt Drf autoregulation that is amenable to high-throughput screening (HTS) of drug libraries. If our hypothesis is correct, then DAD peptide will be sufficient to kill even Taxol-resistant cancer cells. DAD variants, that fail to interact with the GBD of cellular Drfs, will be inactive, and the disrupted sites will correspond to molecular determinants that define the structure of DAD bound to the GBD. Finally, DAD peptides will disrupt the GBD-DAD interaction in vitro; the assays will then be optimized, using DAD peptides and their derivatives, for screening strategies that will allow us to identify additional molecules that harbor Drf-deregulating activity.
