The Use Of Novel Polymer-Lipid Nanoparticles To Study G-Protein-Coupled Receptor Activation And Dynamics.

Most chemical messengers in the body, work through proteins on the cell surface known as receptors. The biggest class of receptors is the G-protein-coupled receptor (GPCRs) family, which are important for cell signalling and as drug targets. GPCRs are thus of enormous interest, both in terms of basic biology and also for commercial exploitation by the pharmaceutical industry. Understanding how GPCRs work at the molecular level is fundamentally important and is one of the 'big questions' in biology today. For a few receptors we have a crystal structure showing their 3-dimensional shape when bound to a 'blocking drug'. This has deepened our understanding of receptor architecture which should aid future drug discovery. However, it is clear that GPCRs are very flexible proteins that change their shape on binding hormones and drugs, whereas the crystal structures provide snap-shots of a single conformation, usually of the non-signalling receptor. To fully understand GPCRs, we need to understand how they change their shape during signalling. This is also important for 'allosteric' agents that bind to a different part of the receptor to the natural hormone to modify receptor activity. This opens the way to a whole new class of drugs. In this application we describe a new approach to understanding GPCRs. We have found a novel way to make large quantities of natural GPCRs 'solubilised' within very small particles of their membrane environment without the need for detergent. Previously, there has been an absolute requirement for detergent to solubilise receptors. This formed a major barrier to progress in this field as its presence disrupted the normal properties and shape of the receptor, de-stabilising the protein. For the first time anywhere, we can now circumvent the detergent barrier to progress. This important advance opens up the prospect of applying powerful biophysical techniques to provide detailed information on how GPCRs dynamically change their shape in response to drugs of different classes. The project brings together four scientists from the Universities of Birmingham and Aston, with complementary skills to exploit our new technologies. We will study the adenosine A2a receptor (A2aR) as an example of the GPCR family. This is a well-characterised receptor with a large literature, defined drugs, useful tools and importantly it is one of the few receptors for which there is an atomic level crystal structure. In addition, our industrial collaborators Heptares Therapeutics will provide A2aRs constrained into functionally-defined shapes (conformations) which will be a great help to us. Finally we have a formal collaboration with an experienced computational chemist at Essex University who will construct molecular models and dynamic simulations of different functional states of the receptor based on our experimental data. We are ahead of the field and our pilot data show that we have the capability at this very moment to produce very large amounts of A2aR by growing them in a yeast and to rapidly 'solubilise' active receptors in their natural state in a styrene maleic acid lipid particle (SMALP). We will engineer A2aR so that 'reporter groups', including fluorescent groups, can be introduced at defined locations in the receptor architecture. We can then study A2aR-SMALP by a range of techniques. For example, fluorescence can tell us about the changes in the environment around important parts of the receptor when drugs bind. A related technique can also be used to provide a molecular ruler that allows us to calculate the distance between two parts of the receptor to determine how it changes on binding drugs of different signalling capability or in conformationally-constrained A2aRs provided by Heptares Therapeutics. Overall, this project will provide insight into the changes of shape that underlie GPCR activation and may aid rational drug design in the future.

G-protein-coupled receptors (GPCRs) are the largest class of receptors and the largest target for prescription drugs. The processes underlying GPCR activation at the molecular level remain poorly understood. Recent crystal structures have provided static snap-shots of a few of these highly flexible proteins. A full understanding of GPCR signalling requires knowledge of the various states adopted by the receptor in response to ligands of differing efficacy (full agonist, inverse agonist) and the dynamics of the inter-conversions of these states. A formidable barrier to progress in this field is that the detergents required to solubilise GPCRs de-stabilise the receptors and disrupt conformational states. We have developed novel technologies to 'solubilise' GPCRs in a styrene maleic acid lipid particle (SMALP), without detergent, preserving native conformations and enabling powerful biophysical analyses - a world first. We aim to exploit this advantage to study the adenosine A2a receptor (A2aR) a GPCR. Conformationally stabilised receptors (StaRs) provided by Heptares Therapeutics will be an asset and the published antagonist-A2aR crystal structure provides a defined structure for modelling A2aR conformational changes. We will: 1) Enable A2aR for site-specific incorporation of reporters to allow downstream biophysical analyses. 2) Characterise constructs in HEK293T cells, which will then be over-expressed in P. pastoris, solubilised by spontaneous embedding into SMALPs and purified. 3) Use a battery of biophysical techniques to: i) Define the changes in A2aR conformation underlying activation and characterise the dynamics of inter-conversions. ii) Identify differences in conformation when the receptor is occupied by ligands of different efficacy (full agonist, partial agonist, inverse agonist). iii) Define A2aR conformation modifications induced by allosteric ligands. iv) Use constraints from biophysical data to build unified structural models of GPCR activation.

The research described in this proposal will increase understanding of the molecular processes underlying GPCR activation and how these processes are initiated or blocked by the receptor binding different classes of ligand (agonist/antagonist/inverse agonist/allosteric modulator). This will have a significant impact in the field of cell signalling in academia and also within the Pharmaceutical Industry. The insights provided may lead to rational drug design in the future producing drugs which are better able to activate or block discrete aspects of receptor signalling. This would lead to improved medicines which would increase the quality of life. Our research is directly relevant to the Pharmaceutical Industry in the UK as is evident from the 'Letter of Support and Collaboration' from our industrial collaborator Heptares Therapeutics Ltd. (attached). Any advance we make would be exploited by Pharma companies. Our technologies may be applicable to studying other membrane proteins thereby extending the impact of our research beyond cell signalling. Two PDRAs will be trained in a wide range of multidisiplinary skills which will be an asset to their career development and to subsequent employers.