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

• 批准号: 10283983
• 负责人: CHRISTOPHER M CHEATUM
• 金额: $ 21.95万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 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/10283983
• 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/antagonist; innovation; molecular recognition; novel therapeutics; p38 Mitogen Activated Protein Kinase; protein structure; screening; small molecule; tool; two-dimensional; vibration

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.

总结 对于筛选小分子作为潜在的变构剂的实验方法存在未满足的需求。 抑制剂来支持药物发现。异源性包括在酶的远端结合效应物 导致活性位点的性质发生变化。发现药物候选分子， 然而，从变构的角度来看是困难的，因为筛选应该识别与酶结合的分子 但只有那些在活性位点以外的位点特异性地这样做，并导致 催化性能尽管筛选方法可以容易地鉴定结合事件，但是确定结合事件的分子量是非常困难的。 结合方式或对活动的影响需要进一步评估。其核心目标是 建议是评估二维红外（2D IR）的适当性和性能 光谱学作为筛选酶的变构效应物的工具。二维红外测量 构象动力学和环境的异质性周围的振动发色团与 飞秒时间分辨率，和以前的应用蛋白质表明，它是敏感的微妙 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 用于快速筛选的二维红外数据收集;以及目标2。评估2D IR作为一种 变构结合探针。第一个目标将通过实施双波束方法来实现， 在二维红外图像中采用主动背景扣除法，引入拟合方法提取动态观测量 从少量的测量，并开发一个多井样品保持器和自动化， 在快速屏幕上连续测量大量样品的2D IR数据的过程。在第二 目的，这是完全独立的第一，三个模型系统与已知的变构抑制剂将 研究以确定变构效应物的结合对在以下条件下测量的动力学的影响： 这些模型是一组不同的经过充分研究的酶，HIV-1逆转录酶， 转录酶、β-内酰胺酶和p38丝裂原活化蛋白激酶。探针，氰基苯丙氨酸， 将突变成每一个取代现有的活性位点苯丙氨酸残基的2D IR 实验这种方法是创新的，因为它开发了一种新的和实质上不同的方式， 发现酶的变构效应物。如果成功，2D IR将成为一种新的技术， 用于药物发现。拟议的研究是重要的，因为它们将提供关键的证据- 概念的想法和先进的技术方法，二维红外，使其适用于快速筛选。 最终，2D IR有可能成为药物发现的强大工具。