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

细胞信号传导是所有生命的重要组成部分，控制着生物体的内部运作，使它们能够做出反应、适应和生存。这个具有挑战性的项目将最终解决细胞信号研究中的基本问题，即如何可视化和微调生物受体的反应。膜结合的p2受体介导细胞外核苷酸在细胞间信号传导中的作用，而p2y受体属于g蛋白偶联受体（gpcr）超家族。到目前为止，P2Y家族由8个已克隆的人类亚型组成，并在许多脊椎动物中发现。P2Y2受体（P2Y2R）属于核苷酸激活的G蛋白偶联受体（gpcr）家族，值得注意的是，嘌呤能P2Y2R的阻滞剂（拮抗剂）和激活剂（激动剂）的阐明和开发落后于A组gpcr的许多成员，因此，缺乏有效和选择性的化学P2Y2R探针，这是阐明受体生理作用所需的药理学工具。本研究计划的主要目标在于应用新兴的基于GPCR荧光的合成化学平台技术，结合在一系列未优化的P2Y2R拮抗剂中单个氮原子引起的分子识别的独特和偶然发现，合成新的高亲和力稳定的P2Y2R拮抗剂探针，用于未来的生物医学和药物发现研究计划，以验证临床益处这种未充分研究的生物受体的拮抗作用。非药物样，但高亲和力的P2Y2R拮抗剂将作为化学支架，在计算化学的指导下，将化学阐述产生新的高亲和力荧光标记的P2Y2R拮抗剂化学探针，以了解P2Y2R拮抗剂的结构要求，并研究受体所在的细胞群。拟议的研究结合了合成化学、计算化学和药理学，将在保健、创造科学就业机会和培训领域具有广泛的重要性。新出现的证据表明，P2Y2R的拮抗作用在未满足医疗需求的关键领域具有重要意义，例如，最近披露的信息表明，P2Y2R在某些癌细胞中表达，并可能在肿瘤细胞增殖中发挥重要作用。癌症是一大类疾病的总称，可以影响身体的任何部位；肺癌、胃癌、肝癌、结肠癌和乳腺癌每年导致的癌症死亡人数最多。癌症是全世界死亡的主要原因，2008年有760万人死亡（约占所有死亡人数的13%）。癌症的一个决定性特征是异常细胞的快速产生，这些细胞的生长超出了通常的边界，然后可以侵入身体的邻近部位并扩散到其他器官。这个过程被称为转移，转移是癌症死亡的主要原因。因此，高亲和力荧光标记的P2Y2R拮抗剂可能为发现新的小分子化学先导物和生物工具开辟令人兴奋的机会，以满足这一领域未满足的临床需求。然而，缺乏高亲和力和稳定的选择性P2Y2R配体阻碍了对这一重要生物受体的全面药理学评估。总之，本申请的目的是确保高亲和力和稳定的化学荧光探针的合成，以实现未来基于片段的药物发现资助申请。在该项目中发现的高亲和力P2Y2R拮抗剂配体的合成将用于探测P2Y2R拮抗剂在未满足疾病状态下的生物学潜力，荧光高亲和力P2Y2R拮抗剂探针将用于建立新的高通量生物学测定，以便将来筛选这一重要受体。

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.