High-throughput chemical screens for GPCR functional selectivity

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 andamp; 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.Project Narrative We will develop a platform for the rapid, microscale chemical synthesis and screening of small molecules for the potential treatment of obesity. We target an entire family of human GPCR proteins. Our platform can detect ligand bias and functionality selectivity within closely related GPCR families in a single assay at the level of a primary screen.