Rapid Screening of Allosteric Effectors Using Two-Dimensional Infrared Spectroscopy

摘要 对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 用于支持药物发现的抑制剂。变构作用涉及将远程位置的效应器结合到酶上 这会导致活动站点的属性发生变化。发现起作用的候选药物分子 然而，变构是困难的，因为筛选应该识别与酶结合的分子 但只有在活动站点以外的站点上明确执行此操作并导致 催化性能。虽然筛选方法可以很容易地识别结合事件，但确定 约束的方式或对活动的影响需要额外评估。这样做的中心目标是 建议评估二维红外(2D IR)的适宜性和性能 光谱学作为筛选酶的变构效应的工具。2D IR测量 振动发色团周围环境的构象动力学和非均质性 飞秒时间分辨率，以及以前对蛋白质的应用表明，它对细微的变化很敏感 改变。以下两个目标将实现本项目的目标：目标1.加速和自动化 用于快速筛选的2D IR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光束方法来实现 2D IR中的主动背景减除，引入拟合方法提取动态观测值 从少量的测量，并开发多井样品保持器和自动化 在快速屏幕上连续测量大量样本的2D IR数据的过程。在第二个 AIM，它完全独立于第一个，三个具有已知变构抑制剂的模型系统将 以确定变构效应器的结合对测量的动力学的影响 用二维红外光谱测定活性中心。这些模型是一组研究得很好的各种酶，HIV-1逆转 转录酶、β-内酰胺酶和p38丝裂原活化蛋白激酶。探头是氰基苯丙氨酸， 将突变为每一个取代2D IR中现有的活性中心苯丙氨酸残基 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。如果成功，2D IR将是一项新的使能技术 用于药物研发。拟议的研究具有重要意义，因为它们将提供关键的证据-- 提出了2D IR的技术途径，使其适用于快速筛选。 最终，2D IR有可能成为药物发现的有力工具。

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.