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

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.

Activation of purinergic P2Y2 receptors by ATP released from damaged cells plays a significant role in inflammatory conditions and as such, the receptor is an important therapeutic target in many areas of clinical need. However, the elucidation of the receptor signalling and drug discovery has lagged behind that of other GPCRs due in part to the lack of commercially available P2Y2R radio labelled ligands and stable high affinity P2Y2R ligands. We will initially evaluate and expand on the known structure activity relationship (SAR) in a series of stable P2Y2R antagonists, where the antagonist activity has been recovered through the serendipitous discovery of the molecular recognition invoked by a single nitrogen atom, thereby giving a series of stable non acidic, non-phosphate containing P2Y2R antagonists. We will aim to synthesize a set of P2Y2R antagonist chemical scaffolds with suitable attachment points and groups suitable to carry out high speed synthetic chemistry where we will explore linking groups from the P2Y2R antagonist chemical scaffold to a BODIPY fluorescent probe. The resulting high affinity selective P2Y2R antagonist probes will be used to visualize, through for example confocal microscopy, cell populations where the P2Y2R is expressed, gaining valuable insight into cell signalling processes in both healthy and diseased cells. The resulting high affinity selective P2Y2R antagonist probes will be used to develop a novel fluorescence based ligand binding assay for future fragment-based lead generation programs to discover novel P2Y2R ligands. In combination with the chemistry-driven design programs, structural work will be explored through a combination of the emerging P2Y2R homology model and the SAR to be generated within this research project. Finally, valuable insights gleaned from this research could be applied to other purinergic receptors, offering insights into cell signalling and ultimately the design of new ligands for therapeutic benefit.

The primary impact of the proposed work will be directly to extend knowledge of the molecular basis of antagonist action and purinergic P2Y2 receptors. The work has direct relevance to the Academic Community, Pharmaceutical Industry and ultimately the Healthcare sector as there has been extensive therapeutic interest in P2Y2 receptor drugs. For example P2Y2 receptor agonism has direct relevance in the treatment of dry eye syndrome where the P2Y2 receptor agonist diquafosol tetrasodium is an available treatment and for numerous other potential therapeutic diseases, such as pain control, cystic fibrosis and CNS disorders that cannot yet be clinically validated due to the lack of high-quality drug-like P2Y2 receptor agonists and antagonists. The project will provide robust models and pharmacological tools that can be used to develop novel and more selective antagonists to assist in the future identification and development of new treatments for these debilitating diseases. Ultimately, the development of new P2Y2 receptor drugs through the sharing of our work/collaborating with the Pharmaceutical Industry could have impact on the UK-based pharmaceutical companies. Our planned research therefore has the potential to provide a significant long term impact in meeting clinical need, delivering and improving therapies for a range of debilitating conditions and contributing to human health and wellbeing.

This project will train research staff in multidisciplinary skills essential in both the pharmaceutical and academic sector working on the structure-function and pharmacological evaluation of the distinct P2 receptor family. The postdoctoral researcher will benefit from continued scientific training and will receive training in the interpretation of molecular modelling, drug discovery, synthetic chemistry and pharmacology. In addition there will be continuing on-going professional development of presentation skills (oral, written and numerical analysis). The project will also provide training opportunities for MPharm students doing 4th year research projects. This will give them direct practical experience as well as training in presentation skills and time management.
The post-doctoral research scientist will take a leading role in the design and synthesis of the P2Y2 receptor antagonist ligands and will evolve in collaboration with the PIs and CIs forward thinking techniques to facilitate scientists' abilities' to analyse both current and future G protein-coupled receptors for the structural requirements that elicit a biological response. It is proposed that key findings from this research would offer huge potential benefit in exploration of other areas of GPCR-based receptor signalling including other purinergic P2 receptor sub-types.

Due to the novel approaches involved, it is believed that through a combination of publications in peer-reviewed journals and presentations at leading international conferences, the quality of the work will lead to increasing and highlighting the expertise of the PIs research group within the area and will assist in securing future grant funding, leading to further support and training for the key scientists of tomorrow. It is essential that the work is also communicated to a wider, potentially a younger lay audience to enhance uptake of training and education in science.

The impact of our work therefore will be initially through the publication of our research findings, according to the research council's and the University of Nottingham's open access policy, that will be of interest to the academic community and pharmaceutical industry. In the medium term this may translate into the development of new P2Y receptor drugs that have the long term potential to impact on health and wellbeing as well as have economic returns.