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患病组织中活性的潜在细胞过程至关重要。这种类型的控制对于超过90%的已知基因组来说仍然是难以捉摸的。部分原因是大多数大分子相互作用分布在大的界面上，并且不存在开发高亲和力小分子拮抗剂所需的足够的空腔。另一方面，在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的操作特征（例如，抗原-抗体相互作用）。认识到这一原则，我们假设并开发了一种新的方法，使用二价肽基试剂，可以通过添加小分子来调节，以阻断大分子界面或从指定的复合物中剥离单个组分。与VEGF陷阱类似，我们创造了这些诱导型分子陷阱。使用这些诱导型分子陷阱，我们已经表明，动力蛋白轻链作为动力蛋白马达复合物的变构调节剂。这些试剂还提供了对动力蛋白介导过程的时间尺度的第一次洞察（例如，细胞器运输）。通过这种新颖的方法实现的这种信息目前不能通过其他技术获得（例如，siRNA和显微注射）。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）开发可光裂解的类似物以允许分子陷阱的快速解离，以及2）开发随机诱导的二聚体文库用于针对指定靶标的正向遗传筛选。这些试剂的成功开发及其实用性的证明将导致在神经系统和其他疾病的生物学和治疗开发中的多种应用。此外，将这些试剂与特异性启动子组合将允许以组织特异性方式进行可逆捕获并提供整个生物体内的分子信息。 公共卫生相关性：虽然人类基因组的完成，快速SNP分析以及新的成像和计算技术已经导致对神经学和其他致病过程的新颖和重要的见解，但我们选择性靶向感兴趣的分子并询问其在复杂和纠缠过程中的作用的能力仍然存在显着差距。如果没有这种控制，就很难辨别这些分子中的点突变如何导致疾病。在此，我们提出了一种新的方法来选择和产生诱导型，二价抑制剂，提供小分子样的特性，以及一种新的试剂，以提供空间和时间的控制选定的目标的活性。该提案所产生的试剂的成功开发将为研究人员提供新的工具，最终将导致对所研究疾病机制的新见解。

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.