Allosteric, Small-Molecule Inhibitors of Actin Nucleation by the Formin mDia1

描述（由申请人提供）：本提案的目标是开发一种高通量测定法，以鉴定β-内酰胺酶蛋白mDia 1的变构小分子抑制剂，并通过筛选10，000种结构不同的化合物来验证该测定法。第二个目的是开发用于表征mDia 1抑制剂在活细胞中的特异性、作用机制和功效的测定法。形成蛋白已成为细胞质分裂、细胞极性和发育所需的肌动蛋白丝成核的关键调节因子。尽管它们的重要性，很少有详细的信息，细胞功能的任何15种哺乳动物β亚型。除了formins，其他两个主要的肌动蛋白成核因子，Arp 2/3复合物和Spir，已被确定和特定的成核剂在细胞中的特定肌动蛋白结构的相对贡献是一个主要的开放问题。在这里，我们专注于无处不在的表达mDia 1。目前在哺乳动物细胞中对mDia 1进行功能丧失研究的策略受到效力差（RNAi）和其他mDia同种型的补偿性上调（小鼠敲除）的限制。细胞可渗透的抑制剂，迅速抑制mDia 1将减轻这些挑战，大大促进其独特功能的阐明，并可能在癌症中具有潜在的治疗效用。mDia 1是由自抑制调节，我们已经提出，自抑制蛋白质可能是敏感的变构抑制剂，变构稳定的非活性构象。此类抑制剂应表现出比直接靶向高度保守的催化FH 2结构域的那些更大的靶向选择性，这一假设将在目标2中直接测试。我们建议使用纯化的重组蛋白并行进行两次筛选，这些蛋白将一起鉴定mDia 1的变构小分子抑制剂。这种双重筛选也将消除主要预期类别的假阳性，由直接靶向肌动蛋白或非特异性干扰蛋白质的化合物引起。未来的工作将应用这种屏幕更大的化合物收集，我们设想应用类似的策略来开发其他formin的抑制剂。

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.