Development Of P2 Receptor Ligands

对药物化学家来说，克隆至少15种亚型的P2核苷酸受体是一个独特的挑战：在几乎没有现有线索的情况下，针对这种多样性的受体设计选择性激动剂和拮抗剂。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肉的功能。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，既可用作探测受体功能的药理工具，也可用作潜在的治疗剂。P2X受体是一种配体门控离子通道。P2Y受体是G蛋白偶联受体，作为第二信使与磷脂酰肌醇途径相连。作为代谢性嘌呤和嘧啶核苷酸受体的代表的人P2Y1受体可以基于视紫红质模板进行建模，所得到的模型与药理学和诱变结果高度一致。跨膜区和胞外区的带电残基以及受体激活所必需的两个二硫键已被鉴定。选择性的P2Y1受体拮抗剂，如腺嘌呤核苷酸MRS 2179(N-methyl-2‘-deoxyadenosine-3’，5‘-bisphosphate)和它的碳环类似物正在开发中。我们还合成了含有构象受限核糖环的核苷酸，以冻结在P2受体上提供良好亲和力和/或选择性的构象。因此，我们已经确定了P2Y1受体对核糖的Northern环构象的构象偏好。这一结论既适用于激动剂，也适用于拮抗剂。通过冻结受体优先构象中的核糖替代品，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受体的作用发生了质的变化：核糖类似物没有活性，而构象受限类似物(北方甲基卡巴)是中等强度的激动剂。 MRS 2500是已知的最有效的P2Y1受体拮抗剂，它是一种2-碘腺嘌呤-甲基卡巴二磷酸核苷，其核糖环被锁定在北构象中。这种拮抗剂已被证明能有效地抑制ADP诱导的人血小板聚集。MRS 2365是2-甲硫基-ADP的(N)甲基卡巴类似物，是第一个选择性的P2Y1激动剂，用于诱导血小板变形而不产生聚集。这支持了P2Y1和P2Y12受体的激活都是ADP诱导的聚集所必需的观点。这些药理学工具将有助于验证P2Y1拮抗剂作为抗血小板药物的可能性。 构象受限核苷酸的使用也已扩展到P2Y2、P2Y4、P2Y6和P2Y11亚型。除P2Y6外，北方甲基卡巴环系统在上述所有亚型的激动剂中都能保持高效力。P2Y6受体的构象要求目前正在我们的部分进行探索。此外，该亚型与细胞凋亡，即程序性细胞死亡之间的关系也已被发现。表达P2Y6受体的星形细胞瘤细胞，当被UDP激活时，可以保护对照细胞免受肿瘤坏死因子-肿瘤坏死因子诱导的细胞凋亡。这种保护包括蛋白激酶C的激活，以及随后被称为ERK的信号激酶的激活。这可能与涉及肿瘤坏死因子的退行性和炎症性疾病有关。使用类似的构象策略(MRS 2279，它还包含代替核糖的Northern甲烷卡巴环系统)开发了一种高度有效和选择性的P2Y1亚型拮抗剂。该化合物已被证明能抑制ADP诱导的大鼠和人血小板聚集。它的氚版本是第一个报道的具有普遍适用性的P2Y1受体的高亲和力放射性配体。我们正在继续探索这一系列有效和选择性的P2Y1受体拮抗剂的构效关系。由于不存在细胞外核苷酸结合区的模板，因此还没有实现对P2X受体的建模。然而，已经鉴定出该亚型的选择性拮抗剂MRS 2220和增强剂MRS 2219。两者在结构上都基于吡哆醛-5‘-磷酸拮抗剂(如PPADS)，其SAR正在P2受体的所有亚型中进行检测。

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 such as the adenine nucleotide MRS 2179 (N-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and it carbocyclic analogue 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. 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. Modeling of P2X receptors has not been achieved, since no template for the extracellular nucleotide binding region exists. Nevertheless, a selective antagonist, MRS 2220, and a potentiator, MRS 2219, of this subtype have been identified. Both are based structurally on pyridoxal-5'-phosphate antagonists (such as PPADS), for which the SAR is being examined at all of the P2 receptor subtypes.