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，很大程度上受到埃米尔·费舍尔（Emil Fischer）在1890年对酶和抑制剂提出的“锁与钥匙”假说和保罗·埃里奇（Paul Ehrich）对选择性药物制剂提出的“魔弹”假说的影响。今天，酶的小分子抑制和受体激动/拮抗仍然是药物开发策略的主要内容。然而，人们逐渐认识到，开发药物，特别是治疗无法治愈的疾病，需要超越酶和受体。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，因此它们在疾病治疗中被小分子破坏的主要目标。不幸的是，虽然酶抑制剂可以很容易地通过体外分析发现使用显色/荧光读数，蛋白质- dna和蛋白质-蛋白质相互作用更难检测。缺乏通用和方便的检测方法是没有针对这些靶标开发更多化合物的主要原因。在之前的3年资助期内（在撰写本文时，我们刚刚完成了第2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，我们将建立这一成功并扩展这项技术，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生物传感器技术作为通用药物筛选工具，从而扩大其可以解决的生物学重要应用范围。为此，我们的目标是利用PC生物传感器格式的能力，提供生物化学和细胞结合到其表面的高分辨率空间图像，并开发一种新的“自我参考”微孔板格式，该格式将能够以高通量的方式直接测量小分子-蛋白质相互作用。我们的目标是通过继续关注与细胞凋亡途径相关的检测来证明这些能力。具体来说，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相互作用和细胞色素c-Apaf-1相互作用的小分子，这些生物结合事件具有高度的医学相关性，但不适合标准的高通量筛选（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测定中进行评估。通过项目目标的执行，计划的工作将开发、演示和验证PC技术在小分子药物发现中广泛使用的分析（即抑制分析、直接结合分析、初级筛选、次级筛选和基于细胞的验证），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相互作用作为治疗过早细胞死亡疾病（如帕金森病）的可处理靶点。这项工作的长期影响将是开发和广泛传播高灵敏度、高通量无标签分析方法，这些方法可以广泛应用于药物发现，以及开发用于治疗帕金森病的化合物和治疗靶点的验证。帕金森病是一种毁灭性的疾病，目前折磨着65岁及以上人口的1%。

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.