Development Of P2 Receptor Ligands

至少15种P2核苷酸受体亚型的克隆对药物化学家提出了一个独特的挑战：为这种多样性的受体设计选择性激动剂和拮抗剂，但现有的线索很少。这些受体调节中枢神经系统、免疫系统、心血管系统和平滑肌的功能。我们的实验室正在开发这些受体的选择性激动剂和拮抗剂，既可以作为探测受体功能的药理学工具，也可以作为潜在的治疗剂。P2X受体是配体门控离子通道。P2Y受体是作为第二信使连接到磷脂酰肌醇途径的G蛋白偶联受体。人类P2Y1受体作为代谢嘌呤和嘧啶核苷酸受体P2Y家族的代表，可以基于视紫红质模板建立模型，所得模型与药理学和诱变结果高度一致。在跨膜和细胞外区域的带电残基以及受体激活所必需的两个二硫桥已被确定。我们实验室开发的与腺嘌呤核苷酸MRS 2179 （n -甲基-2'-脱氧腺苷-3',5'-二磷酸）相关的选择性P2Y1受体拮抗剂及其碳环类似物正在开发中。我们还合成了含有构象受限的核糖样环的核苷酸，以冻结在P2受体上提供有利亲和力和/或选择性的构象。因此，我们已经确定了P2Y1受体对核糖北环构象的构象偏好。这一结论适用于激动剂和拮抗剂。通过将核糖替代物冷冻成受体优先构象，我们已经将已知的P2Y1亚型激动剂的效力提高了200-300倍。一种含有亚甲基碳连接第二和第三磷酸基团的ATP衍生物对P2Y1受体的影响在质量上发生了变化：核糖类似物是无活性的，而对位约束类似物（北方甲烷碳）是一种中等效力的激动剂。

The cloning of at least fifteen subtypes of P2 nucleotide receptors has presented a unique challenge to medicinal chemists: the design of selective agonists and antagonists for this multiplicity of receptors with few existing leads. These receptors regulate function of the central nervous system, the immune system, the cardiovascular system, and smooth muscles. Our laboratory is developing selective agonists and antagonists for these receptors, for use both as pharmacological tools for probing receptor function and as potential therapeutic agents. P2X receptors are ligand-gated ion channels. P2Y receptors are G protein coupled receptors linked to the phosphatidyl inositol pathway as second messenger. The human P2Y1 receptor as representative of the P2Y family of metabotropic purine and pyrimidine nucleotide receptors may be modeled based on a rhodopsin template, and the resulting model is highly consistent with pharmacological and mutagenesis results. Charged residues in both the transmembrane and extracellular domains and two disulfide bridges essential for receptor activation have been identified. Selective P2Y1 receptor antagonists related to the adenine nucleotide MRS 2179 (N-methyl-2'-deoxyadenosine-3',5'-bisphosphate) developed in our lab and it carbocyclic analogues are under development. We have also synthesized nucleotides containing conformationally constrained ribose-like rings, in order to freeze a conformation that provides favorable affinity and/or selectivity at P2 receptors. As a result, we have identified the conformation preference of the P2Y1 receptor for the Northern ring conformation of the ribose. This conclusion applies to both agonists and antagonists. By freezing the ribose substitute in the receptor-preferred conformation, we have enhanced the potency of known agonists at the P2Y1 subtype by 200-300 fold. One ATP derivative containing a methylene carbon joining the second and third phosphate groups was qualitatively altered in its effects on the P2Y1 receptor: The ribose analogue is inactive, and the confromationally constrained analogue (Northern methanocarba) was a moderately potent agonist. MRS 2500, a 2-iodoadenine-methanocarba bisphosphate nucelotide, in which the ribose-like ring is locked in the North conformation is the most potent known antaognist of the P2Y1 receptor. This antagonist has been shown to potently inhibit the ADP-induced aggregation of human platelets. This derivative has now been radiolabeled and shown to be useful as a receptor probe. MRS 2365, a (N)methanocarba analogue of 2-methylthio-ADP, is the first selective P2Y1 agonist and was shonwn to induce a shape change in platelets without producing aggregation. This supports the view that both P2Y1 and P2Y12 receptor activation are needed for ADP-induced aggregation. These pharmacological tools will be useful in validating the possible use of P2Y1 antagonists as antiplatelet agents. The use of conformationally constrained nucleotides has also been extended to P2Y2, P2Y4, P2Y6, and P2Y11 subtypes. The Northern methanocarba ring system results in retention of high potency in agonists at all of the above subtypes, except P2Y6. The conformational requirements of the P2Y6 receptor are currently being explored in our section. Also, a relationship between this subtype and apoptosis, programmed cell death, has been discovered. Astrocytoma cells that express the P2Y6 receptor, when activated by UDP, are protected from apoptosis induced in control cells upon exposure to TNF, tumor necrosis factor. The protection involves activation of protein kinase C and subsequently the signaling kinase known as ERK. This may have relevance for degenerative and inflammatory conditions that involve TNF. A highly potent and selective antagonist of the P2Y1 subtype has been developed using a similar conformational strategy (MRS 2279, which also contains a Northern methanocarba ring system in place of ribose). This compound has been shown to inhibit the ADP-induced aggregation of both rat and human platelets. Its tritiated version is the first high affinity radioligand for the P2Y1 receptor, of general applicability, to be reported. We are continuing the explore the structure activity relationships in this series of potent and selective P2Y1 receptor antagonists. The first antagonist of the P2Y13 receptor (MRS2211) has been developed. It is based structurally on pyridoxal-5'-phosphate antagonists (such as PPADS), for which the SAR is being examined at all of the P2 receptor subtypes.