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

描述(由申请人提供)：这项提案的目标是开发一种高通量分析方法，以确定Forin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方法。第二个目标是开发用于表征mDia1抑制剂的特异性、作用机制和在活细胞中的有效性的方法。Forin蛋白已经成为肌动蛋白细丝成核的关键调节因子，是细胞质分裂、细胞极性和发育所必需的。尽管它们很重要，但关于哺乳动物的15种异构体中的任何一种的细胞功能的详细信息都很少。除了Forins，另外两个主要的肌动蛋白成核因子Arp2/3复合体和Spir已经被确定，特定的核因子对细胞中特定的肌动蛋白结构的相对贡献是一个主要的悬而未决的问题。在这里，我们关注mDia1中普遍表达的Form。目前在哺乳动物细胞中进行mDia1功能丧失研究的策略受到低效(RNAi)和其他mdia1亚型代偿性上调(小鼠敲除)的限制。快速灭活mDia1的细胞渗透性抑制剂将缓解这些挑战，极大地促进其独特功能的阐明，并可能对癌症具有潜在的治疗作用。MDia1受自身抑制的调节，我们已经提出，自我抑制的蛋白质可能对变构抑制剂敏感，变构抑制剂可以稳定非活性构象。这类抑制剂应该比那些直接针对高度保守的催化FH2结构域的抑制剂表现出更大的靶向选择性，这一假说将在目标2中直接进行验证。我们建议使用纯化的重组蛋白进行两次平行筛选，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类别的假阳性，这些假阳性是由直接针对肌动蛋白或非特异性干扰蛋白的化合物引起的。未来的工作将把这一筛选应用于更大的化合物集合，我们设想应用类似的策略来开发其他福尔马林的抑制剂。

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.