The synthesis and evaluation of new fluorescent-labelled P2Y2 receptor chemical probes.

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Cell signalling is a vital and integral part of all life and controls the inner workings of organisms allowing them to respond, adapt and survive. This challenging project will ultimately address fundamental questions within cell signalling research of how to visualise and finely tune a biological receptor's response. Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling and P2Y-receptors belong to the superfamily of G-protein-coupled receptors (GPCRs). So far, the P2Y family is composed out of 8 human subtypes that have been cloned and are found in many vertebrates. The P2Y2 receptor (P2Y2R) belongs to the family of nucleotide-activated G protein coupled receptors (GPCRs) and it is notable that elucidation and exploitation of the of both blockers (antagonists) and activators (agonists) of the purinergic P2Y2R has lagged behind that of many members of group A GPCRs and, as such, there is a lack of potent and selective chemical P2Y2R probes, which are required as pharmacological tools to elucidate the physiological roles of the receptor. The compelling goal of this research proposal lies in the application of an emerging GPCR fluorescence-based synthetic chemistry platform technology in combination with a unique and serendipitous discovery of the molecular recognition invoked by a single nitrogen atom in a series of unoptimised P2Y2R antagonists to synthesise novel high-affinity stable P2Y2R antagonist probes for use in future biomedical and drug discovery research programmes in order to validate the clinical benefit for antagonism of this understudied biological receptor. The non-drug like, but high affinity P2Y2R antagonists will act as chemical scaffolds which, guided by computational chemistry, will be chemically elaborated to generate new high-affinity fluorescently labelled P2Y2R antagonist chemical probes to understand the structural requirements for antagonism for the P2Y2R and for studying cell populations where the receptor is located.The proposed research combining synthetic chemistry, computational chemistry and pharmacology, would have widespread importance in the areas of healthcare, scientific job creation and training. Emerging evidence suggests antagonism of the P2Y2R has implication in key areas of unmet medical need, for example, 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 generic term for a large group of diseases that can affect any part of the body; lung, stomach, liver, colon and breast cancer cause the most cancer deaths each year. Cancer is a leading cause of death worldwide, accounting for 7.6 million deaths (around 13% of all deaths) in 2008. 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 is referred to as metastasis and metastases are the major cause of death from cancer. Therefore, a high-affinity fluorescent labelled P2Y2R antagonist might open exciting opportunities for the discovery of new small molecule chemical leads and biological tools in this area of unmet clinical need. However a lack of high affinity and stable selective P2Y2R ligands has held back full pharmacological evaluation of this important biological receptor.In conclusion, the aim of this application will be to secure the synthesis of the high affinity and stable chemical fluorescent probes to enable future fragment-based drug discovery grant applications. The synthesis of high affinity P2Y2R antagonist ligands discovered in this programme will be used to probe the biological potential of P2Y2R antagonists in unmet disease states and the fluorescent high affinity P2Y2R antagonist probes will be used to establish novel high-throughput biological assays to enable future screening of this important receptor.