High-throughput chemical screens for GPCR functional selectivity

项目摘要 G蛋白偶联受体（GPCR）是超过三分之一的FDA批准的药物的靶点， 对类似分子有反应的家族。任何给定的GPCR可以通过多个细胞内信号传导 信号传导途径，其中一些导致期望的治疗效果，而另一些则是多余的， 对药物活性有害。基于这些原因，我们正在构建一个平台，以实现高通量筛选， 功能选择性激动剂--与正确受体结合并触发正确细胞内信号传导的激动剂 途径。在这里，我们使用这个系统的发展功能选择性和偏见的激动剂的人类 黑皮质素受体4，抗肥胖药物开发的长期目标。 我们通过两个目标实现了这一点：首先，通过工程化细胞系的多路复用，基于测序的分析， 通过MC 4 R和相关受体的信号传导活性;其次，通过构建高通量平台， 小分子的微尺度化学合成。总之，这些工具将能够直接评估功能 选择性和配体偏好，并创建丰富的多维结构-活性关系 以前所未有的规模，加速开发口服可用的临床前先导分子， 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 Octant的验证多重转录报告技术的黑皮质素受体家族。具体地说， 我们将把这项技术集中在MC 1 R、MC 3R、MC 4 R和MC 5 R受体上，建立一个测量 每个受体对多种细胞内信号传导途径的反应。为了做到这一点，我们设计，合成， 表征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 机器人技术来建立一个单步化学合成的自动化系统。有了这个系统，我们将创建 通过单步合成以微尺度形式的小分子（每个反应约1 nmol）。我们将专注于化学 对自动化的特性来说是健壮的。这个平台将使结构偏见和结构的探索， 广泛的化学空间的选择性关系。 意义和创新：控制GPCR的信号偏差非常重要，因为通常情况下， 只有某些细胞内信号传导途径是治疗相关的，而其他的可能导致副作用。偏压 通常在药物开发的后期阶段被发现，其中改变先导化合物以改善偏倚可能证明 困难或不可能。这也使得事后难以理解为什么特定配体在某些情况下是有偏见的。 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 能够使用配体偏倚作为主要筛选指标，并将能够收集GPCR配体偏倚， 非常大的化学相关的小分子的集合。

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.