Development of chemical induced molecular traps for time resolved, in vivo studie

描述（由申请人提供）：快速和选择性拮抗特定蛋白质和/或分子复合物的能力对于区分健康和病变组织中活跃的潜在细胞过程至关重要。这种类型的控制对于已知基因组的90%以上仍然是难以捉摸的。在某种程度上，这源于这样一个事实，即大多数大分子相互作用分布在大的界面上，并且没有足够的空腔来发展高亲和力的小分子拮抗剂。另一方面，在一个大的界面上分布多个弱相互作用可以导致高的表观亲和力和精致的特异性，并且似乎是生物系统中普遍存在的操作特征（例如抗原-抗体相互作用）。认识到这一原理，我们假设并开发了一种使用二价肽基试剂的新方法，该试剂可以通过添加小分子来调节，以阻断大分子界面或从指定复合物中剥离单个组分。与VEGF陷阱类似，我们创造了这些可诱导的分子陷阱。利用这些可诱导的分子陷阱，我们首次证明了动力蛋白轻链作为动力蛋白运动复合物的变构调节因子。这些试剂也提供了动力蛋白介导过程（例如，细胞器运输）的时间尺度的第一个见解。这种新方法所提供的信息是目前其他技术（例如siRNA和显微注射）所无法获得的。基于强有力的初步数据，我们建议通过以下方式扩展该方法：1)开发可光切割的类似物以允许分子陷阱的快速解离；2)开发随机诱导二聚体文库，用于针对指定目标的正向遗传筛选。这些试剂的成功开发及其实用性的证明将导致生物学和神经系统疾病和其他疾病的治疗发展的多种应用。此外，将这些试剂与特定启动子结合，将允许以组织特定的方式进行可逆捕获，并提供整个生物体内的分子信息。

DESCRIPTION (provided by applicant): The ability to rapidly and selectively antagonize specific proteins and/or molecular complexes is essential to differentiate the underlying cellular processes active in healthy and diseased tissues. This type of control remains elusive for over 90% of the known genome. In part, this stems from the fact that most macromolecular interactions are spread over large interfaces and do not present sufficient cavities necessary to develop a high affinity, small molecular antagonist. On the other hand, distributing multiple weak interactions over a large interface can lead to a high apparent affinity and exquisite specificity, and appears to be a prevalent operational feature in biological systems (e.g., antigen-antibody interactions). In recognition of this principle, we hypothesized and developed a novel approach using bivalent peptide-based reagents that can be regulated by the addition of a small molecule to either block a macromolecular interface or strip an individual component from a designated complex. In analogy to the VEGF trap, we have coined these inducible molecular traps. Using these inducible molecular traps, we have shown for the first time that the dynein light chains act as allosteric regulators of the dynein motor complex. These reagents also provide the first insight to the timescales of dynein mediate processes (e.g., organelle transport). Such information enabled by this novel methodology is not currently available with other techniques (e.g., siRNA and microinjection). Based on strong preliminary data, we propose to extend this methodology by 1) developing a photocleavable analog to permit rapid dissociation of the molecular traps and 2) developing random inducible dimeric libraries for forward genetic screens against a designated target. The successful development of these reagents and the demonstration of their utility will lead to multiple applications in biology and therapeutic development of neurological and other disorders. Moreover, combining these reagents with specific promoters will permit reversible trapping in a tissue specific manner and provide molecular information within a whole organism. PUBLIC HEALTH RELEVANCE: While the completion of the human genome, rapid SNP analyses, and new imaging and computational techniques has lead to novel and significant insight to neurological and other pathogenic processes, there remains a significant gap in our ability to selectively target molecules of interest and interrogate their action in complex and entangled processes. Without this control, it is difficult to discern how point mutations in these molecules lead to disease. Herein, we propose a novel method to select and generate inducible, bivalent inhibitors that afford small molecule-like properties as well as a new reagent to afford spatial and temporal control of the activity of selected targets. The successful development of the reagents emanating from this proposal will provide researchers new tools that will ultimately lead to novel insight to the mechanism of disease under study.