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

项目摘要 免疫检查点对于维持免疫稳态至关重要。它们可以防止 免疫反应。然而，在某些情况下，免疫检查点途径的异常激活可能是一个重要的因素。 癌症降低抗肿瘤免疫力，使肿瘤增殖。免疫靶向治疗 检查点CTLA 4、PD-1和PDL-1已成功治疗各种难治性 癌的然而，它们的疗效仅限于一小部分患者，因此需要 靶向额外的免疫检查点途径。在这方面，腺苷-腺苷的阻断 使用高亲和力拮抗剂的A2 A受体（A2 AR）免疫检查点激活已显示出前景， 临床前研究。由于拮抗剂全面阻断腺苷-A2 AR免疫检查点， 都在预料之中。相反，负变构调节剂（NAM）可以想象地阻断 腺苷-A2 AR免疫检查点仅在腺苷水平升高的肿瘤部位。我们 设想用NAM阻断腺苷-A2 AR免疫检查点激活是疾病部位的 特异性，因此是增强抗肿瘤免疫力的更直接的方法。我们已经证明 先前，腺苷-A2 AR免疫检查点服从正变构调节， 通过合成的小分子。然而，腺苷-A2 AR的负变构调节 免疫检查点尚未报道。为了测试我们的想法，我们设计并合成了一个 超过300种可能含有腺苷-A2 AR NAMs的化合物的文库。在本提案中，我们将 利用我们实验室建立的体外筛选模式鉴定A2 AR NAM。目标1：确定 腺苷-A2 AR免疫检查点的NAM。(a)化合物库将在细胞中筛选- 使用稳定表达A2 AR的CHO细胞的基于cAMP的测定。NAM将根据 腺苷介导的cAMP生成抑制。(b)将在放射性标记的 使用CHO-A2 AR膜的激动剂结合测定。A2 AR的结合亲和力降低 选择性激动剂CGS 21680指示NAM。目的2：确定具有最佳免疫增强作用的NAM 活性和A2 AR选择性。(a)将筛选NAM逆转腺苷-A2 AR的能力。 通过α-⑶ 3/⑶ 28刺激的⑶ 8 + T细胞介导的IFN-γ产生的抑制。(b)NAMs with the 将评价基于细胞的cAMP中A1 R、A2 BR和A3 R活性的最佳免疫增强活性 利用稳定表达受体的细胞系的测定。如果成功，这将是第一次鉴定 腺苷-A2 AR免疫检查点的NAM，并证明了一种新的靶向方法 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.