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方法，以鉴定双胍蛋白mDia1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方法。第二个目标是开发用于表征mDia1抑制剂在活细胞中的特异性、作用机制和功效的检测方法。双胍蛋白已成为细胞分裂、细胞极性和发育所需的肌动蛋白丝成核的关键调节因子。尽管它们很重要，但关于15种哺乳动物双胍异构体中任何一种的细胞功能的详细信息很少。除了形成蛋白，另外两个主要的肌动蛋白成核因子，Arp2/3复合物和Spir，已经被确定，特定成核因子对细胞中特定肌动蛋白结构的相对贡献是一个主要的开放性问题。在这里，我们关注的是mDia1中无处不在的表达。目前在哺乳动物细胞中对mDia1进行功能丧失研究的策略受到疗效差（RNAi）和其他mDia1亚型代偿上调（小鼠敲除）的限制。快速灭活mDia1的细胞渗透性抑制剂将减轻这些挑战，并极大地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。mDia1受自抑制调节，我们提出自抑制蛋白可能易受变构抑制剂的影响，这些变构抑制剂可以稳定非活性构象。这些抑制剂应该比直接靶向高度保守的催化FH2结构域的抑制剂表现出更大的靶向选择性，这一假设将在Aim 2中直接验证。我们建议使用纯化的重组蛋白同时进行两种筛选，这两种蛋白将共同识别mDia1的变构小分子抑制剂。这种双重筛选还将消除主要预期的假阳性类别，这些假阳性是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的。未来的工作将把这种筛选应用到更大的化合物集合中，我们设想应用类似的策略来开发其他形式的抑制剂。

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.