Diaphanous-related formins, DRF, cytoskeleton, Diaphanous Autoregulatory Domain, Diaphanous Inhibitory Domain, actin nucleation, fluorescence anisotropy, mDia2
Benson, Brittany E. and Wallar, Bradley J., "Studying Potential Drug Interactions Involved in the Regulation of the Diaphanous-related Formins" (2008). Student Summer Scholars Manuscripts. 10.
Diaphanous-related formins (DRFs) are a conserved family of proteins that are involved in the regulation of cellular shape, motility, and cell division by regulating the structure of the cellular “skeleton” (cytoskeleton); any disruption of this regulation can result in cell death. Normally, the DRFs are kept in an inactive state by the intramolecular binding of two regions of the protein: the Diaphanous Autoregulatory Domain (DAD) and Diaphanous Inhibitory Domain (DID). This binding can be alleviated by various naturally-occurring mechanisms and proper regulation of the activity of these proteins is vital to cell survival because prolonged activation of DRFs can result in cell death. In a recent scan of 10,000 chemical compounds, two compounds (I and II) were identified to bind to the DID region and activate DRFs indefinitely resulting in the killing of breast and colon cancer cells. In this study, we intend to prove that these compounds are actually alleviating DID-DAD binding (the essential step in DRF activation) by directly binding to DID. In addition, the specific location on DID that binds these compounds must be elucidated. We have hypothesized three amino acid residues that contribute to the binding of these compounds, have generated them using site-directed mutagenesis, and have evaluated their ability to bind the compounds using fluorescence anisotropy. Further experiments will test how these DID mutations affect the ability for these compounds to directly bind to DID using isothermal titration calorimetry. By characterizing the DID-compound bound structure, these studies will allow for the design of new compounds that could bind more tightly to DID, thereby resulting in a drug that could efficiently kill cancer cells.