Function- and interaction-based discovery of negative allosteric modulators of the A2A Receptor

项目摘要 免疫检查点是维持免疫动态平衡的关键。它们可以防止过度激活 免疫反应。然而，在某些情况下，免疫检查点通路的异常激活 癌症降低了抗肿瘤的免疫力，使肿瘤得以扩散。针对免疫的治疗方法 CTLA4、PD-1和PDL-1检查站已经成功地治疗了各种难治性 癌症。然而，它们的疗效仅限于一小部分患者，强调了 瞄准更多的免疫检查点路径。在这方面，腺苷-腺苷的阻断 高亲和力拮抗剂激活A2a受体(A2AR)免疫检查点在 临床前研究。当拮抗剂在全球范围内阻断腺苷-A2AR免疫检查点时，不良事件 都是意料之中的。相反，负变构调节剂(NAM)可想而知地阻止激活 腺苷-A2AR免疫检查点仅在腺苷水平升高的肿瘤部位。我们 设想用NAMS阻断腺苷-A2AR免疫检查点激活是疾病的部位 因此，这是一种增强抗肿瘤免疫的更有针对性的方法。我们已经证明了 在此之前，腺苷-A2AR免疫检查点对正变构调节是适用的 通过合成的小分子。然而，腺苷-A2AR的负变构调节 免疫检查点尚未报告。为了测试我们的想法，我们设计并合成了一个 包含300多种可能含有腺苷-A2AR NAMS的化合物的文库。在这项提案中，我们将 利用本实验室建立的体外筛选范式鉴定A2ARNAMS。目标1：确定 腺苷-A2AR免疫检查点的NAMS。(A)化合物文库将在一个单元格中进行筛选- 以稳定表达A2AR的CHO细胞为基础的cAMP检测。NAM将在以下基础上确定 腺苷对cAMP产生的抑制作用。(B)NAMS将在无线电标记中得到确认 利用CHO-A2AR膜进行激动剂结合分析。A2AR结合亲和力的降低 选择性激动剂CGS 21680是NAMS的征象。目标2：确定免疫增强效果最好的NAMS 活性和A2AR选择性。(A)将对NAM进行筛查，以确定其逆转腺苷-A2AR-的能力 α-CD3/CD28刺激的CD8 T细胞产生干扰素的抑制作用(B)NAMS与 以细胞为基础的cAMP中的A1R、A2BR和A3R活性将被评为最佳免疫增强活性 利用稳定表达受体的细胞系进行分析。如果成功，这将是第一次识别 腺苷-A2AR免疫检查点NAMS的构建及靶向新方法的验证 一样的。

Project Summary Immune checkpoints are critical for maintaining immune homeostasis. They prevent overactivation of the immune response. However, aberrant activation of immune checkpoints pathways in certain cancers reduces anti-tumor immunity allowing tumors to proliferate. Therapies targeting immune checkpoints CTLA4, PD-1 and PDL-1 have been successful in treating a wide variety of refractory cancers. However, their efficacy is limited to a small percentage of patients highlighting the need for targeting additional immune checkpoint pathways. In this regard, the blockade of adenosine-adenosine A2A receptor (A2AR) immune checkpoint activation with high-affinity antagonists has shown promise in preclinical studies. As antagonists block adenosine-A2AR immune checkpoint globally, adverse events are anticipated. In contrast, negative allosteric modulators (NAM) conceivably block the activation of adenosine-A2AR immune checkpoint only at tumor sites where adenosine levels are elevated. We envision that blocking the adenosine-A2AR immune checkpoint activation with NAMs is disease-site specific and thus a more directed approach to enhance anti-tumor immunity. We have demonstrated previously that adenosine-A2AR immune checkpoint is amenable to positive allosteric modulation through synthetic small molecules. However, negative allosteric modulation of the adenosine-A2AR immune checkpoint has not been reported. To test our notion, we have designed and synthesized a library of over 300 compounds potentially containing adensoine-A2AR NAMs. In this proposal, we will identify A2AR NAMs utilizing in vitro screening paradigms established in our laboratory. Aim 1: Identify NAMs of the adenosine-A2AR immune checkpoint. (a) The compound library will be screened in a cell- based cAMP assay utilizing CHO cells stably expressing the A2AR. NAMs will be identified on the basis of adenosine-mediated inhibition of cAMP production. (b) NAMs will be confirmed in a radiolabeled agonist binding assay utilizing CHO-A2AR membranes. A reduction in the binding affinity of the A2AR selective agonist CGS 21680 is indicative of NAMs. Aim 2: Identify NAMs with best immune-enhancing activity and A2AR selectivity. (a) NAMs will be screened for their ability to reverse the adenosine-A2AR- mediated inhibition of IFN-g production by a-CD3/CD28-stimulated CD8+ T cells. (b) NAMs with the best immune enhancing activity will be evaluated for A1R, A2BR and A3R activity in cell-based cAMP assays utilizing cell lines stably expressing the receptors. If successful, this will be the first identification of NAMs of the adenosine-A2AR immune checkpoint and demonstration of a novel approach targeting the same.