Microcantilever Biosensors Based on Ligand Gated Ion Channel Receptors

描述(由申请人提供)：该项目的主要目标是开发一种用于药物开发的传感器，模拟人脑受体。该传感器将能够在短时间内评估大量候选药物的结合情况，从而大大提高识别潜在治疗药物的速度。该项目的主要目标是利用基于最近发现的乙酰胆碱结合蛋白(AChBP)的受体蛋白来开发改进的微悬臂梁生物传感器。AChBP是一种可溶性蛋白质，其结构和药理与中枢和外周神经系统中存在的烟碱型乙酰胆碱受体(NAChR)惊人地相似。由于nAChR是配体门控离子通道(LGIC)超家族的成员，该项目最终将开发用于这类神经元受体的高通量药物筛选和药物发现的传感器。具体来说，我们的目标是：1)利用AChBP及其衍生物作为生物传感器蛋白，研制一种微悬臂梁生物传感器。2)研究基本表面共轭化学反应，建立微悬臂梁传感的修饰方案。该项目在很大程度上利用了我们在LGIC结构和功能(Schulte博士)和微悬臂设计(纪万昌博士)方面的综合专业知识。目标1将评估AChBP本身作为一种潜在的有用的生物传感器分子。我们将通过探索该传感器与nAChR、GABAR、GlycineR和5-HT3R配体的相互作用，初步展示该传感器在高通量药物筛选中的应用。由于AChBP可以通过突变来改变其配基特异性，因此通过定点突变和/或嵌合构建获得的衍生物也将被开发出来。由于AChBP通常被用作模拟配基门控离子通道受体的模板，这项工作的另一个好处是扩大了对这些重要受体的配基特异性的基础的了解。Aim 2将利用AChBP和其他受体蛋白，通过改进的连接化学结合改进的微悬臂阵列技术来改善传感器的响应

DESCRIPTION (provided by applicant): The primary goal of this project is to develop a sensor for use in drug development that mimics human brain receptors. This sensor will be capable of evaluating the binding of large numbers of drug candidates in a short period of time; thus greatly enhancing the rate at which potential therapeutic agents can be identified. The primary goal of this project is to develop improved microcantilever-based biosensors using receptor proteins based on a recently discovered acetylcholine binding protein (AChBP). The AChBP is a soluble protein that displays structure and pharmacology that is strikingly similar to the nicotinic acetylcholine receptor (nAChR) present in the central and peripheral nervous systems. Since the nAChR is a member of a large super-family of ligand gated ion channels (LGICs), this project will ultimately develop sensors that will be useful in high throughput drug screening and drug discovery for this class of neuronal receptors. Specifically, we aim to: 1) Develop a microcantilever biosensor utilizing the AChBP and its derivatives as biological sensor proteins. 2) Investigate fundamental surface conjugation chemistries and establish modification protocols for microcantilever sensing. The project draws heavily on our combined expertise in LGIC structure and function (Dr. Schulte) and microcantilever design (Dr. Ji). Aim 1 will evaluate the AChBP itself as a potentially useful biosensor molecule. We will initially demonstrate the use of this sensor in high-throughput drug screening by exploring its interaction with nAChR, GABAR, glycineR and 5-HT3R ligands. Since the AChBP can be mutated to alter its ligand specificity, derivatives obtained via site directed mutagenesis and/or chimeric construction will also be developed. Since the AChBP is commonly used as a template for modeling of ligand gated ion channel receptors, an additional benefit of this work will be an expanded knowledge of the basis of ligand specificity at these important receptors. Aim 2 will utilize the AChBP and other receptor proteins to improve sensor response through improved conjugation chemistries in combination with improved microcantilever array technology developed in Dr. Ji's laboratory