High-Throughput Small Molecule Screening Using Photonic Crystal Technology: Appli

描述(由申请人提供)：现代药物发现行业在过去的120年里得到了发展，主要受Emil Fischer在1890年提出的酶和抑制剂的“锁和钥匙”假说和Paul Ehrich关于选择性药物的“神奇子弹”假说的影响。今天，酶的小分子抑制和受体激动剂/拮抗剂仍然是药物开发策略的主要内容。然而，有一种新的认识是，人们需要超越酶和受体来开发药物，特别是对于无法治愈的疾病的治疗。所有的生物途径都依赖于蛋白质-DNA和蛋白质-蛋白质的相互作用，从而使它们成为疾病治疗中小分子干扰的主要目标。不幸的是，虽然酶抑制剂可以很容易地通过使用显色/荧光读数的体外测试来发现，但蛋白质-DNA和蛋白质-蛋白质的相互作用更难检测。缺乏通用和方便的分析是没有为这些目标开发更多化合物的一个主要原因。在前三年的资助期(在撰写本文时，我们刚刚完成第二年)，我们开发了一种基于光子晶体(PC)生物传感器的新技术来检测蛋白质-DNA相互作用。在这次更新中，我们将在这一成功的基础上扩展这项技术，将其应用于蛋白质-蛋白质相互作用，并利用它来发现和验证抑制AIF-DNA相互作用和细胞色素c-APAF-1相互作用的化合物。拟议项目的具体目标旨在进一步发展PC生物传感器技术，将其作为一种通用的药物筛选工具，从而扩大其能够解决的具有生物学意义的应用范围。为此，我们的目标是利用PC生物传感器格式的能力，提供高分辨率的生化和细胞结合到其表面的空间图像，并开发一种新的“自参考”微板格式，使高通量地直接测量小分子-蛋白质相互作用成为可能。我们的目标是通过继续关注与细胞凋亡途径相关的分析来展示这些能力。具体地说，我们正在使用PC生物传感器来识别破坏凋亡诱导因子(AIF)-DNA相互作用的小分子，以及细胞色素c-APAF-1相互作用，这些生物结合事件具有很高的药物相关性，但不适用于标准的高通量筛选(HTS)方法。通过这些PC生物传感器筛选获得的化合物将在一系列分层的体外和基于细胞的分析中进行评估。通过计划目标的执行，拟议的工作将为小分子药物发现中使用的广泛分析(即抑制分析、直接结合分析、初步筛选、二次筛选和基于细胞的验证)开发、演示和验证PC技术，同时专注于与人类健康高度相关的应用。我们的目标是使用这些化合物来验证AIF-DNA和细胞色素c-APAF-1的相互作用作为治疗过早细胞死亡疾病(如帕金森氏病)的易处理靶点。这项工作的长期影响将是开发和广泛传播可广泛应用于药物发现的高度敏感、高通量的无标签分析方法，开发化合物并验证治疗帕金森氏病的治疗目标。帕金森氏症是一种毁灭性的疾病，目前困扰着65岁及以上人口的1%。 与公众健康相关：拟议项目的具体目标是进一步发展光子晶体(PC)生物传感器技术，作为一种通用的药物筛选工具，从而扩大它们可以解决的具有生物学意义的应用范围。其中一个主要目标是发展PC生物传感器的能力，通过在1536孔生物传感器微板内采用三重参考方法，并使用介电纳米棒表面来增强小分子结合信号，从而使小分子结合到固定的蛋白质靶标上具有很强的筛选性。我们的目标也是扩展基于PC生物传感器的筛查能力，包括蛋白质-DNA和蛋白质-蛋白质相互作用的抑制剂和增强剂，继续专注于与细胞凋亡途径相关的检测。具体地说，我们正在使用PC生物传感器来识别破坏凋亡诱导因子(AIF)-DNA相互作用的小分子，这种生物相互作用具有很高的药用价值，但不适用于标准的HTS方法。通过这种PC生物传感器筛选获得的化合物将在一系列分层的体外和基于细胞的分析中进行验证和评估。

DESCRIPTION (provided by applicant): The modern drug discovery industry has evolved over the last ~120 years, largely influenced by Emil Fischer's enunciation of the "lock-and-key" hypothesis for enzymes and inhibitors in 1890 and Paul Ehrich's "magic bullet" hypothesis for selective pharmaceutical agents. Today, small molecule inhibition of enzymes and receptor agonism/antagonism remain mainstays of drug development strategies. However, there is an emerging recognition that one needs to move beyond enzymes and receptors for the development of drugs, especially for the treatment of diseases for which there are no cures. All biological pathways rely on protein- DNA and protein-protein interactions, thus making them prime targets for disruption with small molecules in disease treatment. Unfortunately, while enzyme inhibitors can be readily discovered through in vitro assays using chromogenic/fluorescent readouts, protein-DNA and protein-protein interactions are much more difficult to detect. This lack of general and convenient assays has been a major reason that more compounds have not been developed for these targets. In the previous 3-year funding period, (at the time of this writing, we have just completed Year 2) we developed a novel technology based on Photonic Crystal (PC) biosensors to detect protein-DNA interactions. In this renewal, we will build off this success and extend this technology, apply it to protein-protein interactions, and utilize it to discover and validate compounds that inhibit the AIF- DNA interaction and the cytochrome c-Apaf-1 interaction. The specific aims of the proposed project are designed to further the development of PC biosensor technology as a general purpose pharmaceutical screening tool, and to thereby broaden the range of biologically significant applications that it can address. To do so, our goal is to utilize the ability of the PC biosensor format to provide high resolution spatial images of biochemical and cell binding to their surface, and to develop a new "self-referencing" microplate format that will enable direct measurement of small molecule- protein interactions in a high-throughput manner. Our goal is to demonstrate these capabilities by continuing to focus on assays that are relevant to the apoptosis pathway. Specifically, we are using PC biosensors to identify small molecules that disrupt the Apoptosis Inducing Factor (AIF)-DNA interaction, and the cytochrome c-Apaf-1 interaction, biological binding events of high medicinal relevance but that are not amenable to standard high-throughput screening (HTS) methods. Compounds obtained through these PC biosensor screens will be evaluated in a series of tiered in vitro and cell based assays. Through execution of the program objectives, the proposed effort will develop, demonstrate, and validate the PC technology for a broad range of the assays used in small molecule drug discovery (i.e. inhibition assays, direct binding assays, primary screening, secondary screening, and cell-based validation) while focusing on an application with a high degree of fundamental relevance to human health. Our goal is to use these compounds to validate the AIF-DNA and cytochrome c-Apaf-1 interactions as tractable targets for the treatment of diseases of premature cell death, such as Parkinson's Disease. The long-term impact of this work will be the development and wide dissemination of highly sensitive, high throughput label-free assay methods that can be broadly applied throughout drug discovery, and the development of compounds and validation of therapeutic targets for the treatment for a Parkinson's Disease, a devastating illness that currently afflicts >1% of the 65-and-older population. PUBLIC HEALTH RELEVANCE: The specific aims of the proposed project are designed to further the development of Photonic Crystal (PC) biosensor technology as a general purpose pharmaceutical screening tool, and to thereby broaden the range of biologically significant applications that they can address. One of the main goals is to develop the capability of PC biosensors as a robust screen for small molecule binding to immobilized protein targets through a "triple referencing" method within 1536-well biosensor microplates, and the use of a dielectric nanorod surface to enhance small molecule binding signals. Our goal is also to extend the capabilities of PC biosensor-based screening to include inhibitors and enhancers of protein-DNA and protein-protein interactions, by continuing to focus on assays that are relevant to the apoptosis pathway. Specifically, we are using PC biosensors to identify small molecules that disrupt the Apoptosis Inducing Factor (AIF)-DNA interaction, a biological interaction of high medicinal relevance but one that is not amenable to standard HTS methods. Compounds obtained through this PC biosensor screen will be validated and evaluated in a series of tiered in vitro and cell based assays.