New chemical biology approaches to study P2Y2 receptors in health and disease

The process in which cells communicate is a vital and integral part of all life and controls the inner workings of organisms allowing them to respond, adapt and survive. G protein-coupled receptors (GPCRs) are a large group of related proteins that are located on the surface of cells. The receptors can detect molecules outside the cell and activate cellular responses within the cells through a process called cell signalling. This project will address fundamental questions as we will be able to visualise and finely tune a biological receptor's response through the synthesis of molecules that contain a fluorescent group allowing us to visualise the key processes in this critical cell signalling process. We will make molecules that can turn on (agonise) or turn of (antagonise) the ability of the receptor to signal. Membrane-bound P2-receptors mediate the actions of small molecules (nucleotides) in cell-to-cell signalling and P2Y-receptors belong to the family of GPCRs. The P2Y2 receptor (P2Y2R) is notable that elucidation and exploitation of the both blockers (antagonists) and activators (agonists) of this receptor has lagged behind that of many other GPCRs. As such, there is a lack of potent and selective chemical P2Y2R probes, required as pharmacological tools to elucidate the physiological roles of the receptor in areas of un-met medical need. The compelling goal of this research proposal lies in the application of a fluorescence-based synthetic chemistry platform technology in combination with a serendipitous discovery of the discovery of new molecules that inhibit the action of this key receptor. We will synthesise new high-affinity P2Y2R antagonist and agonist probes for use in future drug discovery research programmes in order to prove the clinical benefit of this understudied biological receptor. We will establish new ways to study the receptor function, through the establishment of novel biochemical assays and visualisation techniques enabling us to understand better the structural requirements for activation of the P2Y2R and for studying cell populations where the receptor is located. The research spanning synthetic and computational chemistry with pharmacology, has widespread importance in the areas of healthcare, scientific job creation and training. Emerging evidence suggests antagonism of the P2Y2R has roles in key areas of unmet medical need. Recent disclosures have demonstrated that P2Y2Rs are expressed in certain cancer cells and could play a vital role in tumour cell proliferation. Cancer is a leading cause of death worldwide and is a generic term for a large group of diseases that can affect any part of the body such as; lung, stomach, liver, colon and breast. One defining feature of cancer is the rapid creation of abnormal cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs. This process known as metastasis is the major cause of death from cancer. Interfering with G protein-coupled P2Y receptor signalling is a promising therapeutic alternative to treat aggressive and difficult-to-manage cancers. In addition, the role of the P2Y2R as a key mediator in fibrotic lung diseases was demonstrated. Idiopathic pulmonary fibrosis (IPF) is a condition in which the lungs become scarred and breathing becomes increasingly difficult. It is a devastating disease with few available treatment options. In conclusion, this application will secure the synthesis of the high affinity and stable chemical fluorescent probes to enable future drug discovery grant applications. The synthesis of high affinity P2Y2R antagonist and agonist ligands discovered in this programme will be used to probe the biological potential of P2Y2R in unmet disease states and the fluorescent high affinity P2Y2R chemical probes will be used to establish novel high-throughput biological assays to enable future screening of this important receptor.

Activation of purinergic P2Y2 receptors by ATP and UTP released from damaged cells plays a significant role in inflammatory conditions, and the receptor is an important therapeutic target in many areas of clinical need. The elucidation of the receptor signalling and subsequent drug discovery has lagged behind that of other GPCRs due to the lack of commercially available P2Y2R radio-labelled ligands and stable high affinity P2Y2R ligands. We will expand on our previously reported structure activity relationship (SAR) in a series of stable P2Y2R antagonists, increasing the affinity of these compounds alongside improving physicochemical properties, such as solubility. We will synthesise P2Y2R antagonist chemical scaffolds with attachment points to carry out high speed synthetic chemistry to explore linking groups from the chemical scaffold to a fluorescent probe. We have previously reported on the synthesis of such ligands, however our present plan is to increase the affinity of the fluorescently labelled ligands to develop novel fluorescence based ligand binding assays for future lead generation programmes. We will design and synthesise novel, non-nucleotide P2Y2R agonists and P2Y2R agonist chemical scaffolds to explore linking groups from the chemical scaffold to fluorescent probes. The high affinity selective P2Y2R agonist probes will be used to gain valuable insight into cell signalling processes in both healthy and diseased cells. The chemical biology tools will study the interaction between the P2Y2R and other key mediators, such as VEGFR2, responsible in life threatening diseases such as cancer and IPF. In combination with the chemistry-driven design programmes, structural work will be explored through a combination of the emerging P2Y2R homology model and the information generated within this research project. Valuable insights could be applied to other purinergic receptors, offering the design of new ligands for therapeutic benefit.