Research Project
7680730
[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to develop a high-throughput assay to identify allosteric, small-molecule inhibitors of the formin protein mDia1 and to validate the assay by screening a collection of 10,000 structurally diverse compounds. A second aim is to develop assays to be used to characterize mDia1 inhibitors in terms of their specificity, mechanism of action, and efficacy in live cells. Formin proteins have emerged as key regulators of actin filament nucleation required for cytokinesis, cell polarity, and development. Despite their importance, there is little detailed information on cellular functions for any of the 15 mammalian formin isoforms. In addition to the formins, two other major actin nucleation factors, Arp2/3 complex and Spir, have been identified and the relative contribution of specific nucleators to particular actin structures in cells is a major open question. Here we focus on the ubiquitously expressed formin mDia1. Current strategies to conduct loss-of-function studies on mDia1 in mammalian cells are limited by poor efficacy (RNAi) and compensatory up-regulation of other mDia isoforms (mouse knock- out). Cell-permeable inhibitors that rapidly inactivate mDia1 would mitigate these challenges and greatly facilitate the elucidation of its unique functions and could have potential therapeutic utility in cancer. mDia1 is regulated by autoinhibition and we have proposed that autoinhibited proteins may be susceptible to allosteric inhibitors that allosterically stabilize the inactive conformation. Such inhibitors should exhibit greater target selectivity than those directly targeting the highly conserved, catalytic FH2 domain, an hypothesis to be directly tested in Aim 2. We propose two screens to be conducted in parallel using purified, recombinant proteins that together will identify allosteric, small-molecule inhibitors of mDia1. This dual screen will also eliminate the major anticipated classes of false positives, caused by compounds that target actin directly or perturb proteins non-specifically. Future work will apply this screen to a much larger compound collection and we envisage applying a similar strategy to develop inhibitors for other formins. [unreadable] [unreadable] [unreadable]
