Nonlinear Imaging Methods for High-Throughput Protein and Drug Screening

描述(由申请人提供)：越来越需要分析方法来识别和量化低分子药物化合物与生物膜的相互作用。脂类双层阵列的使用有可能提供一种能够解决这些问题的廉价和高效的高通量分析方法。如果脂质微阵列要达到DNA微阵列分析的成功水平，有几个关键问题需要解决。其中最主要的是需要一种非侵入性的方法来检测药物与脂质微阵列表面的关联以及随后由于药物相互作用而引起的脂质基质的扰动。紫外可见和频(UV-VIS SFG)和红外可见和频(IR-VIS SFG)成像的非线性技术可能解决了这个问题。非线性成像的一些特性使其成为检测微阵列上药物-膜相互作用的一种很有前途的方法，包括能够量化检测信号、高光学分辨率和固有的表面特异性。实现UV-Vis SFG和IR-Vis SFG成像用于研究脂质微阵列上的药物相互作用的关键的第一步已经实现。具体目标#1将重点开发UV-Vis SFG，用于检测平面支撑的脂质双层(PSLB)中的低分子分子。还将继续使用UV-Vis SFG测量蛋白质在膜上的吸附，这是上一个赠款期间目标的延伸。在目标1中，将研究几个模型蛋白质和药物靶标的非线性光谱响应，以试图更全面地了解控制UV-Vis SFG检测的因素。在Aim#2中，探索UV-Vis SFG在筛选潜在的离子通道抑制剂方面的应用。在Aim#3中，通过IR-Vis SFG光谱和IR-Vis SFG高通量成像来研究药物对脂膜热致相变和相行为的影响。这些研究旨在展示非线性成像方法与微图案化流体脂质双层阵列在高通量药物筛选中的应用。与公共卫生相关：膜蛋白，无论是相关的还是整体的，占已知药物靶标的三分之二。药物进入脂膜疏水环境的分配控制着这些材料的生物利用度。拟议研究的目标是开发分析工具，用于非侵入性、特定的光谱检测蛋白质和小分子(药物)与生物膜的相互作用。这些技术的实际应用将对候选药物的药理筛选和药物-膜相互作用的研究产生重大影响。

DESCRIPTION (provided by applicant): There is a growing need for analytical methods to identify and quantify the interaction of low molecular weight drug compounds with biological membranes. The use of lipid bilayer arrays has the potential to provide a cheap and efficient high- throughput analytical method capable of addressing these issues. There are several key issues which need to be resolved if lipid microarrays are to achieve the level of success obtained with DNA microarrays assays. Chief among these is the need for a noninvasive method to detect drug association to the lipid microarray surface and the subsequent perturbation of the lipid matrix due to drug interaction. The nonlinear techniques of ultraviolet-visible sum-frequency generation (UV-Vis SFG) and infrared- visible sum-frequency generation (IR-Vis SFG) imaging may hold the answer to this problem. Several attributes of nonlinear imaging make it a promising method for detecting drug-membrane interaction on microarrays, including the ability to quantify the detected signal, the high optical resolution and the inherent surface specificity. The crucial first steps to implementing UV-Vis SFG and IR-Vis SFG imaging for the investigation of drug association on lipid microarrays have already been achieved. Specific Aim #1 will focus on the development of UV-Vis SFG for the detection of low molecular weight molecules in planar supported lipid bilayers (PSLBs). The use of UV- Vis SFG for measuring protein adsorption to membranes, which is an extension of the goals of the previous grant period, will also be pursued. The nonlinear spectral response from several model protein and drug targets will be investigated in an attempt to more fully understand the factors controlling their detection by UV-Vis SFG in Aim #1. The application of UV-Vis SFG for the screening of potential ion channel inhibitors is explored in Aim #2. Examination of the influence of drugs on the thermotropic phase transition and phase behavior of lipid membranes by IR-Vis SFG spectroscopy and IR- Vis SFG high-throughput imaging is explored in Aim #3. These studies are designed to demonstrate the utility of nonlinear imaging methods in conjunction with micropatterned fluid lipid bilayer arrays for high-throughput drug screening applications. PUBLIC HEALTH RELEVANCE: Membrane proteins, either associated or integral, account for up to two thirds of known drug targets. The partitioning of drugs into the hydrophobic environment of the lipid membrane controls the bioavailability of these materials. The goals of the proposed studies are to develop analytical tools for the noninvasive, specific spectroscopic detection of proteins and small molecule (drug) interactions with biological membranes. The practical implementation of these technologies will have a significant impact on the pharmacological screening of drug candidates, and the investigation of drug-membrane interactions.