Rapid Screening of Allosteric Effectors Using Two-Dimensional Infrared Spectroscopy

使用二维红外光谱快速筛选变构效应器

基本信息

批准号：
10457468
负责人：
CHRISTOPHER M CHEATUM
金额：
$ 19.17万
依托单位：
UNIVERSITY OF IOWA
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10457468
关键词：
Active Sites Allosteric Regulation Amyloid Fibrils Area Binding Binding Sites Biological Biological Models Communication Data Data Collection Development Distal Drug Interactions Eligibility Determination Environment Enzymes Event Funding Mechanisms Goals HIV-1 Heterogeneity Measurement Measures Methodology Methods Modeling Molecular Conformation Mutate Noise Performance Pharmaceutical Preparations Phenylalanine Process Property Proteins Public Health RNA-Directed DNA Polymerase Rapid screening Research Research Project Grants Resolution Sampling Screening procedure Sensitivity and Specificity Series Signal Transduction Site Spectrum Analysis Structure System Technology Technology Development Study Time Work amyloid formation beta-Lactamase chromophore data acquisition drug candidate drug discovery experimental study high risk human disease improved infrared spectroscopy inhibitor innovation molecular recognition novel therapeutics p38 Mitogen Activated Protein Kinase protein structure screening small molecule tool two-dimensional vibration

项目摘要

Summary There is an unmet need for an experimental approach to screen small molecules as potential allosteric inhibitors to support drug discovery. Allostery involves binding an effector at a remote site on an enzyme that causes changes in the properties of the active site. Discovering drug candidate molecules that act allosterically is difficult, however, because the screen should identify molecules that bind to the enzyme but only those that do so specifically, at sites other than the active site, and result in a change in the catalytic properties. Although screening methods can readily identify binding events, determining the mode of binding or the effect on activity requires additional assessment. The central objective of this proposal is to evaluate the appropriateness and performance of two-dimensional infrared (2D IR) spectroscopy as a tool for screening for allosteric effectors of enzymes. 2D IR measures the conformational dynamics and heterogeneity of the environment around a vibrational chromophore with femtosecond time resolution, and previous applications to proteins show that it is sensitive to subtle changes. The following two aims will achieve the goals of this project: Aim 1. Accelerate and automate 2D IR data collection for rapid screening; and Aim 2. Assess the sensitivity and specificity of 2D IR as a probe of allosteric binding. The first aim will be accomplished by implementing a dual-beam approach for active background subtraction in 2D IR, introducing fitting methods to to extract the dynamic observables from a small number of measurements, and developing a multi-well sample holder and automating the process of serially measuring the 2D IR data for a large set of samples in a rapid screen. In the second aim, which is entirely independent of the first, three model systems with known allosteric inhibitors will be studied to determine the effects of the binding of the allosteric effector on the dynamics measured at the active site by 2D IR. These models are a diverse group of well-studied enzymes, HIV-1 reverse transcriptase, b-lactamase, and p38 mitogen-activated protein kinase. The probe, a cyanophenylalanine, will be mutated into each in place of an existing active-site phenylalanine residue for the 2D IR experiments. The approach is innovative because it develops a new and substantively different way of discovering allosteric effectors of enzymes. If successful, 2D IR would be a new and enabling technology for drug discovery. The proposed studies are significant because they will provide the critical proof-of- concept for the idea and advance the technical approach to 2D IR to make it suitable for rapid screening. Ultimately, 2D IR has the potential to be a powerful tool for drug discovery.

概括对于筛选小分子作为潜在变构的实验方法的需求尚未得到满足支持药物发现的抑制剂。变构涉及在酶的远程位点结合效应子导致活动站点的属性发生变化。发现有效的候选药物分子然而，变构很困难，因为筛选应该识别与酶结合的分子但仅限于那些在活动站点以外的站点上专门这样做的站点，并导致了催化特性。尽管筛选方法可以很容易地识别结合事件，但确定结合方式或对活性的影响需要额外评估。本次活动的中心目标提案是评估二维红外（2D IR）的适用性和性能光谱学作为筛选酶变构效应子的工具。 2D IR 测量振动发色团周围环境的构象动力学和异质性飞秒时间分辨率以及之前对蛋白质的应用表明它对微妙的敏感变化。以下两个目标将实现该项目的目标：目标 1. 加速和自动化 2D IR 数据采集，用于快速筛查；目标 2. 评估 2D IR 作为一种方法的敏感性和特异性变构结合探针。第一个目标将通过实施双光束方法来实现二维红外中的主动背景扣除，引入拟合方法来提取动态可观测值通过少量测量，开发多孔样品架并实现自动化在快速屏幕中连续测量大量样品的 2D IR 数据的过程。在第二个目标，完全独立于第一个，具有已知变构抑制剂的三个模型系统将进行研究以确定变构效应物的结合对在通过 2D IR 检测活性位点。这些模型是一组经过充分研究的酶，HIV-1 逆转转录酶、b-内酰胺酶和 p38 丝裂原激活蛋白激酶。探针是氰基苯丙氨酸，将突变为每个取代 2D IR 现有活性位点苯丙氨酸残基实验。这种方法是创新的，因为它开发了一种新的、本质上不同的方式发现酶的变构效应子。如果成功，2D IR 将成为一项全新的支持技术用于药物发现。拟议的研究意义重大，因为它们将提供关键的证据- 该想法的概念并推进了 2D IR 的技术方法，使其适合快速筛选。最终，二维红外有可能成为药物发现的强大工具。