G protein-coupled receptors (GPCRs) are the target of more than one-third of FDA-approved drugs, and often come infamilies that respond to similar classes of molecules. Any given GPCR can signal through multiple intracellularsignalling pathways, some of which lead to desired therapeutic effects, and others of which are superfluous ordeleterious to drug activity. For these reasons, we are constructing a platform to enable high-throughput screening forfunctionally selective agonists - those that bind to the right receptors and trigger the right intracellular signallingpathways. Here, we use this system for the development of functionally selective and biased agonists of the humanmelanocortin 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 ofsignalling activity by MC4R and related receptors; and secondly, by constructing a high-throughput platform formicroscale chemical synthesis of small molecules. Together, these tools will enable direct assessment of functionalselectivity and ligand bias in a high-throughput format and create rich multidimensional structure-activity relationshipson an unprecedented scale, accelerating the development of orally available pre-clinical lead molecules for the controlof obesity.Aim 1: A high-throughput screening platform for GPCR functional selectivity: Here we will seek to applyOctant'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 theresponse of each receptor on multiple intracellular signalling pathways. To do this, we design, synthesize, andcharacterize new signalling-pathway-specific promoter elements and use next generation RNA sequencing to measurethese biosensors.Aim 2: Construction of a high-throughput chemical synthesis platform: We will use acoustic liquid handlingrobotics to build an automated system for single-step chemical synthesis. With this system, we will create libraries ofsmall molecules in microscale formats by single-step synthesis (~1 nmol per reaction). We will focus on chemistriesrobust 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 oftentimesonly certain intracellular signalling pathways are therapeutically relevant, while others may lead to side effects. Bias isoften identified in later stages of drug development where alterations of lead compounds for improved bias may provedifficult or impossible. This also makes it difficult to understand post-facto why a particular ligand is biased in certainways. Most primary screens of novel chemical matter still rely on single-target, single-signal systems. Our work willenable the use of ligand bias as a primary screening metric, and will enable the collection of GPCR ligand bias acrossvery large sets of chemically related small molecules.
Public Health Relevance Statement: 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.
Project Terms: Acoustics ; Acoustic ; Amides ; Amination ; Architecture ; Engineering / Architecture ; Automation ; Bar Codes ; barcode ; Biological Assay ; Assay ; Bioassay ; Biologic Assays ; Biology ; Cell Line ; CellLine ; Strains Cell Lines ; cultured cell line ; chemical synthesis ; Chemistry ; Couples ; Pharmaceutical Preparations ; Drugs ; Medication ; Pharmaceutic Preparations ; drug/agent ; Elements ; Engineering ; Family ; Goals ; Human ; Modern Man ; Integral Membrane Protein ; Intrinsic Membrane Protein ; Transmembrane Protein ; Transmembrane Protein Gene ; Lead ; Pb element ; heavy metal Pb ; heavy metal lead ; Libraries ; Ligands ; Methods ; Miniaturization ; Miniaturisations ; Obesity ; adiposity ; corpulence ; Pharmacology ; Robotics ; Satiation ; satiety ; Signal Pathway ; Signal Transduction ; Cell Communication and Signaling ; Cell Signaling ; Intracellular Communication and Signaling ; Signal Transduction Systems ; Signaling ; biological signal transduction ; Structure-Activity Relationship ; chemical structure function ; structure function relationship ; Technology ; Time ; Genetic Transcription ; Gene Transcription ; RNA Expression ; Transcription ; Work ; Measures ; Mediating ; promoter ; promotor ; base ; improved ; Chemicals ; Chemical Structure ; Stimulus ; Agonist ; melanocortin receptor ; Therapeutic ; fluid ; liquid ; Liquid substance ; Reporter ; tool ; Anti-Obesity Drugs ; Antiobesity Agents ; Antiobesity Drugs ; Anti-Obesity Agents ; Oral ; Reaction ; System ; experience ; Receptor Protein ; receptor ; chemical library ; small molecule libraries ; nucleophilic substitution ; biological sensor ; Biosensor ; MC4 Receptor ; Receptor, Melanocortin, Type 4 ; Melanocortin 4 Receptor ; Structure ; novel ; member ; G Protein-Complex Receptor ; G Protein-Coupled Receptor Genes ; GPCR ; G-Protein-Coupled Receptors ; response ; drug development ; High Throughput Assay ; high throughput screening ; Molecular Interaction ; Binding ; small molecule ; Mammalian Cell ; Receptor Activation ; Synthesis Chemistry ; Synthetic Chemistry ; Collection ; G-Protein Signaling Pathway ; Signaling Pathway from G-Protein Families ; Small Business Innovation Research Grant ; SBIR ; Small Business Innovation Research ; Therapeutic Effect ; Development ; developmental ; Pathway interactions ; pathway ; pre-clinical ; preclinical ; obesity treatment ; obesity intervention ; obesity therapy ; design ; designing ; next generation ; Coupled ; Coupling ; innovation ; innovate ; innovative ; FDA approved ; Systems Development ; transcriptome sequencing ; RNA Seq ; RNA sequencing ; RNAseq ; screening ; multidimensional data ; high dimensional data ; multidimensional datasets ; side effect ;
