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

描述(由申请人提供)：快速和选择性地拮抗特定蛋白质和/或分子复合体的能力是区分健康和疾病组织中活跃的潜在细胞过程的关键。对于90%以上的已知基因组来说，这种类型的控制仍然难以捉摸。这在一定程度上源于这样一个事实，即大多数大分子相互作用分布在大界面上，没有提供开发高亲和力、小分子拮抗剂所需的足够空穴。另一方面，将多个弱相互作用分布在一个大的界面上可以产生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新的方法，使用基于二价肽的试剂，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体中剥离单个组分。类似于血管内皮生长因子陷阱，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马达复合体的变构调节器。这些试剂还提供了对动力蛋白中介过程(例如细胞器运输)的时间尺度的第一次洞察。通过这种新方法实现的这种信息目前对于其他技术(例如，siRNA和微注射)是不可用的。基于强大的初步数据，我们建议通过以下方式扩展这一方法：1)开发一种可光裂解的类似物，以允许分子陷阱的快速解离；2)开发随机可诱导二聚体文库，用于针对指定目标的正向遗传筛选。这些试剂的成功开发和它们的用途的展示将导致在生物学和神经及其他疾病的治疗开发中的多种应用。此外，将这些试剂与特定的启动子相结合，将允许以组织特定的方式进行可逆捕获，并提供整个有机体内的分子信息。 与公共卫生相关：尽管人类基因组的完成、快速的SNP分析以及新的成像和计算技术已经为神经系统和其他致病过程带来了新的和重要的见解，但在我们选择性地针对感兴趣的分子并询问它们在复杂和纠缠的过程中的作用的能力方面，仍然存在着显著的差距。如果没有这种控制，很难辨别这些分子的点突变是如何导致疾病的。在这里，我们提出了一种新的方法来选择和产生可诱导的、具有小分子性质的二价抑制剂，以及一种新的试剂来提供对所选靶标活性的空间和时间控制。这一提议产生的试剂的成功开发将为研究人员提供新的工具，最终将导致对正在研究的疾病机制的新见解。

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.