Nonlinear Imaging Methods for High-Throughput Protein and Drug Screening

描述（由申请人提供）：对鉴定和定量低分子量药物化合物与生物膜相互作用的分析方法的需求日益增长。脂质双层阵列的使用具有提供能够解决这些问题的廉价且有效的高通量分析方法的潜力。如果脂质微阵列要达到DNA微阵列测定的成功水平，需要解决几个关键问题。其中最主要的是需要一种非侵入性的方法来检测药物协会的脂质微阵列表面和随后的扰动的脂质基质由于药物相互作用。紫外-可见和频成像（UV-Vis SFG）和红外-可见和频成像（IR-Vis SFG）等非线性成像技术有望解决这一问题。非线性成像的几个属性使其成为一种有前途的方法，用于检测微阵列上的药物-膜相互作用，包括定量检测信号的能力，高光学分辨率和固有的表面特异性。关键的第一步，实施紫外-可见光SFG和红外-可见光SFG成像的脂质微阵列上的药物缔合的调查已经实现。具体目标#1将侧重于开发用于检测平面支撑脂质双层（PSLB）中低分子量分子的UV-Vis SFG。使用紫外-维斯SFG测量蛋白质吸附膜，这是一个扩展的目标，前一个赠款期，也将继续进行。将研究来自几种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目标#1中控制其通过UV-Vis SFG检测的因素。在目标#2中探索了UV-Vis SFG用于筛选潜在离子通道抑制剂的应用。在目标#3中探索了通过IR-维斯SFG光谱和IR-维斯SFG高通量成像检查药物对脂膜的热致相变和相行为的影响。这些研究旨在证明非线性成像方法与微图案化流体脂质双层阵列结合用于高通量药物筛选应用的实用性。公共卫生相关性：膜蛋白，无论是相关的或整体的，占已知药物靶点的三分之二。药物分配到脂质膜的疏水环境中控制了这些材料的生物利用度。拟议的研究的目标是开发分析工具，用于非侵入性的，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学筛选和药物-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.