Label-Free Screening Using Photonic Crystal Sensors for the Apoptotic Pathway

描述（由申请人提供）：在本提案中，我们描述了一种新型无标记测定法，可用于快速筛选任何其他方法都不容易筛选的蛋白质-小分子相互作用。无标记检测是基于一种称为“光子晶体”结构的光学生物传感器技术，该结构由连续的塑料薄膜片廉价制造，并结合到标准的96孔和384孔板中。通过消除对标记的需要，该测定对由活性结合表位的构象变化或阻断引起的误差和伪影不太敏感。一旦完全开发，设想该技术将用于化学文库的初级筛选和/或作为用于测量蛋白质-小分子组合的剂量响应特征的次级筛选。该项目开发了一种新型的无标记检测方法，称为“Spot Protocol”，其中所需的靶蛋白体积<0.1 μ l/孔，并且自动消除了由于体积折射率变化和非特异性结合引起的误差源。该新测定利用基于图像的方法来实现小分子结合信号的高灵敏度检测，但结合了新的图像处理算法来提供简单的数值输出，该数值输出是蛋白质和分析物之间的结合相互作用的直接测量。所提出的工作的成功完成将代表光子晶体分析技术的高通量筛选（HTS）应用的第一个应用程序。通过将最初的演示集中在不起酶作用并且因此难以通过更标准的HTS过程靶向的蛋白质上，该技术对靶向传统上被视为“不可药物化”的蛋白质的益处将是显而易见的。因此，我们的目标是将这种测定技术发展到任何研究人员都可以使用这种无标记检测方法来鉴定感兴趣蛋白质的小分子配体的程度，即使该蛋白质不是酶，因此没有基于活性的间接监测读数。为了使社区意识到这种方法的潜力，从而促进其转移到其他实验室，我们靶向了凋亡途径中的蛋白质。通过关注调节细胞凋亡的生化途径，该项目旨在对理解程序性细胞死亡中涉及的基本过程以及这些过程的失调如何导致癌症和神经退行性疾病产生影响。选择和鉴定具有选择性诱导癌细胞凋亡或防止健康细胞凋亡的能力的化合物是开发用于许多疾病的新药物疗法的关键之一。

DESCRIPTION (provided by applicant): In this proposal we describe a novel label-free assay that can be used to rapidly screen protein-small molecule interactions that are not easily screened by any other method. The label-free assay is based upon an optical biosensor technology called a "photonic crystal" structure that is inexpensively manufactured from continuous sheets of plastic film and incorporated into standard 96- and 384-well plates. By eliminating the need for a label, the assay is less susceptible to errors and artifacts caused by conformational change or blocking of active binding epitopes. Once fully developed, it is envisioned that the technology will be used in the context of a primary screen of a chemical library and/or as a secondary screen for measuring dose- response characteristics of a protein-small molecule combination. The proposed project develops a new type of label-free assay, called the "Spot Protocol," in which the volume of target protein required is <0.1 mu l/well, and error sources due to bulk refractive index variability and nonspecific binding are automatically eliminated. The new assay utilizes an image-based method to enable high sensitivity detection of small- molecule binding signals, but incorporates a novel image-processing algorithm to provide a simple numerical output that is a direct measurement of the binding interaction between protein and analyte. The successful completion of the proposed work would represent the first application of the photonic crystal assay technology to a high-throughput screening (HTS) application. By focusing the initial demonstrations on proteins that do not function as enzymes and are thus difficult to target through more standard HTS processes, the benefits of this technology towards the targeting of proteins traditionally viewed as "nondrugable" will be obvious. Thus, our goal is to develop this assay technology to the point where any researcher could use this label-free detection method to identify a small molecule ligand to a protein of interest, even if that protein in not an enzyme and thus has no conveniently-monitored activity-based readout. To make the community aware of the potential of such a method and thus facilitate its transfer into other laboratories, we have targeted proteins in the apoptotic pathway. By focusing on biochemical pathways that regulate apoptosis, the proposed project aims to make an impact on understanding the fundamental processes involved in programmed cell death, and on how misregulation of such processes are result in cancer and neurodegenerative disorders. The selection and identification of chemical compounds with the ability to selectively induce apoptosis of cancer cells, or to prevent apoptosis of healthy cells is one key to the development of new drug therapies for many diseases.