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

细胞信号是所有生命中至关重要和不可或缺的一部分，它控制着生物体的内部工作，使它们能够做出反应、适应和生存。这个具有挑战性的项目最终将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转导中的作用，而P2Y受体属于G蛋白偶联受体超家族。到目前为止，P2Y家族由已经克隆的8个人类亚型组成，在许多脊椎动物中都发现了这些亚型。P2Y2受体(P2Y2R)属于核苷酸激活的G蛋白偶联受体(GPCRs)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)的阐明和开发一直落后于A组GPCRs的许多成员，因此，缺乏有效和选择性的化学P2Y2R探针，作为阐明受体的生理作用所必需的药理工具。这项研究建议的引人注目的目标在于应用一种新兴的基于GPCR荧光的合成化学平台技术，结合对一系列未优化的P2Y2R拮抗剂中单个氮原子引发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，以验证这种未被研究的生物受体的拮抗作用的临床益处。非类药物但高亲和力的P2Y2R拮抗剂将作为化学支架，在计算化学的指导下，通过化学修饰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拮抗作用的结构要求，并用于研究受体所在的细胞群体。拟议的结合合成化学、计算化学和药理学的研究将在医疗保健、科学创造就业和培训领域具有广泛的重要性。新的证据表明，对P2Y2R的拮抗作用在未得到满足的医疗需求的关键领域具有潜在的意义，例如，最近的披露表明，P2Y2R在某些癌细胞中表达，并可能在肿瘤细胞增殖中发挥关键作用。癌症是一大类疾病的总称，这些疾病可能会影响到身体的任何部位；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。癌症是全球主要的死亡原因，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迅速产生异常细胞，这些细胞超出它们通常的边界生长，然后可以侵袭身体的邻近部分，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，一种高亲和力的荧光标记的P2Y2R拮抗剂可能会为在这一领域尚未满足临床需求的新的小分子化学先导和生物工具的发现打开令人兴奋的机会。然而，缺乏高亲和力和稳定的选择性P2Y2R配体阻碍了对这种重要生物受体的全面药理学评估。总之，这一应用的目的将是确保合成高亲和力和稳定的化学荧光探针，使未来基于片段的药物发现拨款应用成为可能。本计划中发现的高亲和力P2Y2R拮抗剂配体的合成将用于探索未检测疾病状态下的P2Y2R拮抗剂的生物潜力，而荧光高亲和力的P2Y2R拮抗剂探针将用于建立新的高通量生物检测方法，以便将来对这一重要受体进行筛选。

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.