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

基于功能和相互作用的 A2A 受体负变构调节剂的发现

基本信息

批准号：
10625971
负责人：
EDWARD P AMENTO
金额：
$ 9.75万
依托单位：
MOLECULAR MEDICINE RESEARCH INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-23 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10625971
关键词：
ADORA2A gene Adenosine Adverse event Affinity Agonist Agreement Anti-Inflammatory Agents Antitumor Response Autoimmunity Binding Biological Assay CD28 gene CD3 Antigens CD8-Positive T-Lymphocytes CTLA4 gene Cells Chinese Hamster Ovary Cell Clinic Clinical Cyclic AMP Disease Feedback Future Homeostasis Human Immune Immune response Immune system Immunosuppression Immunotherapeutic agent In Vitro Inflammation Inflammatory Interferon Type II Laboratories Libraries Malignant Neoplasms Mediating Membrane Mus Natural Immunity Natural Killer Cells Pathway interactions Patients Pre-Clinical Model Production Proliferating Radiolabeled Receptor Activation Receptor Inhibition Reporting Site Skin Structure-Activity Relationship Testing Tissues Tumor Escape Tumor Immunity adaptive immunity antagonist anti-tumor immune response cancer cell checkpoint receptors chemotherapeutic agent cytokine design experience immune activation immune checkpoint immune checkpoint blockade in vitro Assay in vivo joint inflammation mouse model novel strategies pharmacokinetics and pharmacodynamics positive allosteric modulator preclinical study prevent programmed cell death ligand 1 programmed cell death protein 1 receptor receptor function refractory cancer screening sensor side effect small molecule stable cell line targeted treatment tumor tumor growth tumor microenvironment

项目摘要

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.

项目概要免疫检查点对于维持免疫稳态至关重要。它们可以防止过度激活免疫反应。然而，某些免疫检查点通路的异常激活癌症会降低抗肿瘤免疫力，导致肿瘤增殖。针对免疫的治疗检查点 CTLA4、PD-1 和 PDL-1 已成功治疗多种难治性癌症。然而，它们的功效仅限于一小部分患者，这突出表明需要针对其他免疫检查点途径。在这方面，腺苷-腺苷的阻断使用高亲和力拮抗剂激活 A2A 受体 (A2AR) 免疫检查点已在以下方面显示出前景：临床前研究。由于拮抗剂在全球范围内阻断腺苷-A2AR 免疫检查点，不良事件是预期的。相反，负变构调节剂（NAM）可能会阻止腺苷-A2AR 免疫检查点仅位于腺苷水平升高的肿瘤部位。我们设想用 NAM 阻断腺苷-A2AR 免疫检查点激活是疾病部位增强抗肿瘤免疫力的具体且更有针对性的方法。我们已经证明了以前认为腺苷-A2AR 免疫检查点适合正向变构调节通过合成小分子。然而，腺苷-A2AR 的负变构调节免疫检查点尚未见报道。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 NAM 的化合物的库。在本提案中，我们将利用我们实验室建立的体外筛选范例来识别 A2AR NAM。目标 1：识别腺苷-A2AR 免疫检查点的 NAM。 (a) 化合物库将在细胞中进行筛选- 基于 cAMP 测定，利用稳定表达 A2AR 的 CHO 细胞。 NAM 将根据腺苷介导的 cAMP 产生抑制。 (b) NAM 将在放射性标记中得到确认利用 CHO-A2AR 膜进行激动剂结合测定。 A2AR 的结合亲和力降低选择性激动剂 CGS 21680 代表 NAM。目标 2：识别具有最佳免疫增强作用的 NAM 活性和 A2AR 选择性。 (a) 将筛选 NAM 逆转腺苷-A2AR-的能力介导α-CD3/CD28刺激的CD8+T细胞产生IFN-g的抑制。 (b) 不结盟运动最佳免疫增强活性将根据细胞 cAMP 中的 A1R、A2BR 和 A3R 活性进行评估利用稳定表达受体的细胞系进行测定。如果成功，这将是第一次识别腺苷-A2AR 免疫检查点的 NAM 的研究以及一种新的靶向方法的演示相同。