High-throughput chemical screens for GPCR functional selectivity

项目摘要 G蛋白偶联受体(GPCRs)是FDA批准的三分之一以上药物的靶标，并且经常进入 对相似类别的分子有反应的家族。任何给定的GPCR都可以通过多个细胞内信号 信号通路，其中一些导致预期的治疗效果，而另一些则是多余的或 对毒品活动有害的。出于这些原因，我们正在构建一个平台，以实现高通量筛查 功能选择性激动剂-那些与正确的受体结合并触发正确的细胞内信号传递的药物 小路。在这里，我们使用这个系统来开发功能选择性和偏向的人类激动剂。 黑素皮质素受体4，抗肥胖药物开发的长期靶点。 我们在两个目标上实现了这一点：第一，通过设计多组细胞系用于多路、基于测序的分析 通过MC4R和相关受体的信号活动；第二，通过构建一个高通量的平台 小分子的微型化学合成。这些工具结合在一起，将支持直接评估功能 高通量形式的选择性和配基偏向，并创建丰富的多维结构-活性关系 以前所未有的规模，加速开发用于控制的口服可用临床前铅分子 肥胖症。 目标1：GPCR功能选择性的高通量筛选平台：在这里，我们将寻求应用 Ocant的经过验证的多路转录报告技术与黑素皮质素受体家族的结合。具体来说， 我们将把这项技术集中在MC1R、MC3R、MC4R和MC5R受体上，创建一个系统来测量 每个受体在多条细胞内信号通路上的反应。为了做到这一点，我们设计、合成和 鉴定新的信号通路特异性启动子元件并使用下一代RNA测序来测量 这些生物传感器。 目标2：构建高通量化学合成平台：我们将使用声学液体处理 机器人技术为一步化学合成建立自动化系统。有了这个系统，我们将创建 通过一步合成(每次反应约1nmol)合成微尺度的小分子。我们将专注于化学 对自动化的特性具有很强的抵抗力。这个平台将使人们能够探索结构偏向和结构- 在大片化学空间中的选择性关系。 意义和创新：控制GPCRs的信号偏向通常具有重要的药理学意义 只有特定的细胞内信号通路与治疗相关，而其他通路可能会导致副作用。偏见是 通常在药物开发的后期阶段确定，在此阶段，先导化合物的改变可能会证明 困难的或不可能的。这也使得事后很难理解为什么特定的配体在某些情况下是偏向的 方式。大多数新化学物质的初级筛选仍然依赖于单目标、单信号系统。我们的工作将 能够使用配体偏差作为主要筛选指标，并将能够收集跨GPCR的配体偏差 非常大的一组化学相关的小分子。

Project Summary G protein-coupled receptors (GPCRs) are the target of more than one-third of FDA-approved drugs, and often come in families that respond to similar classes of molecules. Any given GPCR can signal through multiple intracellular signalling pathways, some of which lead to desired therapeutic effects, and others of which are superfluous or deleterious to drug activity. For these reasons, we are constructing a platform to enable high-throughput screening for functionally selective agonists — those that bind to the right receptors and trigger the right intracellular signalling pathways. Here, we use this system for the development of functionally selective and biased agonists of the human melanocortin receptor 4, a long-standing target for anti-obesity drug development. We achieve this in two aims: first, by engineering sets of cell lines for the multiplexed, sequencing-based analysis of signalling activity by MC4R and related receptors; and secondly, by constructing a high-throughput platform for microscale chemical synthesis of small molecules. Together, these tools will enable direct assessment of functional selectivity and ligand bias in a high-throughput format and create rich multidimensional structure-activity relationships on an unprecedented scale, accelerating the development of orally available pre-clinical lead molecules for the control of obesity. Aim 1: A high-throughput screening platform for GPCR functional selectivity: Here we will seek to apply Octant’s validated multiplexed transcriptional reporter technology to the melanocortin receptor family. Specifically, we will focus this technology onto MC1R, MC3R, MC4R, and MC5R receptors, creating a system to measure the response of each receptor on multiple intracellular signalling pathways. To do this, we design, synthesize, and characterize new signalling-pathway-specific promoter elements and use next generation RNA sequencing to measure these biosensors. Aim 2: Construction of a high-throughput chemical synthesis platform: We will use acoustic liquid handling robotics to build an automated system for single-step chemical synthesis. With this system, we will create libraries of small molecules in microscale formats by single-step synthesis (~1 nmol per reaction). We will focus on chemistries robust to the idiosyncracies of automation. This platform will enable exploration of structure-bias and structure- selectivity relationships across wide swaths of chemical space. Significance & Innovation: Control of the signalling bias at GPCRs is pharmacologically important, as oftentimes only certain intracellular signalling pathways are therapeutically relevant, while others may lead to side effects. Bias is often identified in later stages of drug development where alterations of lead compounds for improved bias may prove difficult or impossible. This also makes it difficult to understand post-facto why a particular ligand is biased in certain ways. Most primary screens of novel chemical matter still rely on single-target, single-signal systems. Our work will enable the use of ligand bias as a primary screening metric, and will enable the collection of GPCR ligand bias across very large sets of chemically related small molecules.