SHG based screening assay for detecting effectors of brain localized GPCRs that can be used to probe mental illness related phenotypes

摘要 Biodesy开发了Biodesy Delta设备，这是一个基于二次谐波产生（SHG）的平台， 允许在配体结合时实时和高分辨率地测量生物分子结构的变化， 通量方式（使用多孔板）。Biodesy Delta将用于开发一种新的，高- 高通量、基于SHG的商业筛选平台，用于鉴定新的结合剂和研究已知的 直接调节G蛋白偶联受体（GPCR）的结构和功能的配体。SHG是一个 非线性光学过程对SH活性370 Da染料探针的取向变化高度敏感， 共价连接到感兴趣的蛋白质上，并且需要非常少量的蛋白质来获得阅读。使用 SHG，Biodesy已经测量了靶蛋白和小分子之间的一系列分子相互作用， 片段、肽和抗体。为了能够研究GPCR，我们成功地开发了一种 原型表面，允许束缚蛋白质，并耐用于溶解和稳定的洗涤剂 溶液中的膜蛋白样品。新的平板表面表现出最小的非特异性结合， 纯化的、标记的、热稳定的神经降压素受体1（NTR 1）GPCR。我们还证明， SHG染料标记的NTR 1拴系到表面保持功能，通过检查两个对照的结合， 配体的蛋白质。在这个项目中，我们将开发一种SHG检测方法，可用于GPCR， 尚未通过诱变稳定，并且是药物发现工作的相关靶点，或者需要更多的 深入了解其功能。腺苷A2 A受体（A2 AR）将是模型GPCR，因为 大量的结构、生物物理和生物化学数据、功能测定和非稳定的 构建，我们可以用于我们的SHG测定验证。首先，我们将使用 Biodesy Delta多个A2 AR构建体的C末端尾的长度不同，其非常灵活， 可能会干扰与表面的结合。这将使我们能够选择保留完整活动的最佳A2 AR 在标记SHG并附着到井表面之后。然后，我们将创建一个版本的检测， 利用1536孔板更有效地筛选化学文库。最后，我们将在一个 包含20，000个小分子的文库的概念验证筛选。

Abstract Biodesy has developed the Biodesy Delta device, a platform based on second harmonic generation (SHG) that allows one to measure changes in biomolecule structure upon ligand binding in real-time and in a high- throughput manner (using multi well plates). The Biodesy Delta will be used to develop a novel, high- throughput, SHG-based, commercial screening platform for identifying new binders and studying known ligands that directly modulate the structure and function of G-protein coupled receptors (GPCRs). SHG is a non-linear optical process that is highly sensitive to orientational changes of a SH-active, 370Da dye probe, covalently attached to a protein of interest, and requires very small amount of protein to get a reading. Using SHG, Biodesy has measured a range of molecular interactions between target proteins and small molecules, fragments, peptides and antibodies. To enable the study of GPCRs, we have successfully developed a prototype surface that allows tethering proteins and is resistant to detergents used to solubilize and stabilize membrane protein samples in solution. The new plate surface demonstrated minimal non-specific binding of the purified, labeled, thermostabilized, neurotensin receptor 1 (NTR1) GPCR. We have also demonstrated that SHG dye labeled NTR1 tethered to the surface remains functional by examining the binding of two control ligands to the tethered protein. In this project, we will develop a SHG assay that can be utilized for GPCRs that have not been stabilized by mutagenesis, and are relevant targets for drug discovery efforts or require a more in-depth understanding of their function. The Adenosine A2A Receptor (A2AR) will be the model GPCR, due to the large amount of structural, biophysical and biochemical data, functional assays and a non-stabilized construct that we can use for our SHG assay validation. First, we will design and test for activity with the Biodesy Delta multiple A2AR constructs that differ in the length of the C-terminal tail, which is very flexible and can interfere with binding to the surface. This will allow us to choose the optimal A2AR that retains full activity after labeling for SHG and attachment to the well surface. We will then create a version of our assay that can utilize 1536-well plates for more efficient screening of chemical libraries. Finally, we will test our assay in a proof of concept screen of a library that contains 20,000 small molecules.