Many of the body’s cell-surface receptors—the G protein-coupled receptors (GPCRs) that control everything from heart rate to mood—can fire off signals even when no activating molecule is present. This spontaneous, or “basal,” activity is linked to conditions such as hyperthyroidism and retinitis pigmentosa, and drugs called inverse agonists exist to suppress it. But no one knows exactly what these drugs look like when they bind to a receptor and a G protein together, because the relevant molecular structures have never been solved. Static techniques like cryo-electron microscopy fail on the floppy, sparsely populated states of the ligand-free receptor. This project will use solution NMR—a method that captures proteins in motion—to map the conformations of the β1-adrenoceptor as its basal activity is tuned by point mutations and then quenched by inverse agonists. The result will be the first detailed molecular model of inverse agonism for any GPCR. Because the β1AR is a stand-in for the entire GPCR superfamily (over 800 members), the framework should guide rational design of inverse agonists for many diseases. This is fundamental science: it explains a core rule of receptor signalling, with no immediate clinical product, but past work on GPCR structure has directly spawned blockbuster drugs.
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Many receptors display spontaneous (constitutive) basal activity as they generate a signalling response without agonist stimulation. Elevated spontaneous basal activity has been reported for numerous members of the G protein-coupled receptor (GPCR) superfamily and has physiological, pathological, as well as therapeutic implications (e.g. hyperthyroidism and retinitis pigmentosa). Such is the extent of elevated spontaneous activity for some receptors that molecules known as inverse agonists have been developed that suppress their basal activity. While this has opened promising therapeutic windows for disease treatment, rational design of inverse agonists has been hindered as the underlying molecular/structural details of inverse agonism and basal activation remain opaque. Major questions persist about the fundamental molecular details of basal activation, including what receptor conformations are involved and how basal activity is reduced by inverse agonists. The issues are compounded since, to date, there are no published structures of GPCRs bound to inverse agonists in a (ternary) complex with a G protein. This is despite reports that some inverse agonists may distinguish between different signalling outcomes (e.g. ghrelin receptor) and the notion that G protein pre-coupling to a ligand-free GPCR is also discussed as an on-path intermediate of agonist-stimulation. Our proposal aims to address these questions regarding basal activation. Typically, for structural information the GPCR field relies primarily on immobilised proteins (as has been extensively used for agonist activation). In the context of ligand-free receptors and their G protein complexes, however, static methods such as cryo-EM and X-ray crystallography have shown little success as low population of relevant states and unfavourable conformational dynamics of the apo (ligand-free) receptor pose challenges. Hence, insight is required from complementary methods such as NMR that can inform on the conformational sampling of GPCRs. Building on our recent agonist-focused studies with ß1-adrenoceptor (ß1AR) we will use solution NMR to provide a molecular explanation of basal activation and inverse agonism, resolving the questions above. We have developed a platform where through point mutations we can tune basal receptor activity. We will use this to explore the receptor conformational landscape. Combining NMR with complementary biophysical methods (that assess the kinetics of G protein interaction) and a wide range of cellular assays (which reveal the signalling specifics in a cellular context), we propose a comprehensive multidisciplinary approach to establish the molecular framework driving basal activation and inverse agonist action. Our findings will foster inverse agonist development in the future. Our proposal pursues three fundamental objectives: Establish how inverse agonists stabilise the apo-state of GPCRs. Obtain a comprehensive molecular description of apo-receptor-G protein complexes and explore the role of inverse agonists in signal abrogation. Assess the role of allosteric modulators in controlling spontaneous GPCR activity. Successful completion of these objectives will provide the first detailed model of inverse agonism for GPCRs. While this project focusses on the ß1AR its true cost-effectiveness and impact will be realised through its general applicability to the entire GPCR superfamily (>800 members). As such, the research has the potential to unlock future production of novel therapies promoting increased long-term life expectancy and improved quality of life; key national priorities for the UK (BBSRC’s ‘Healthy ageing across the Life course’ and Innovate UK’s ‘Securing better health, ageing and wellbeing’ areas of support). The project focuses on essential aspects of GPCR functioning, which fits the BBSRC's ‘Understanding the Rules of Life’.
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