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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：
10355152
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/10355152
关键词：
ADORA2A gene Adenosine Adverse event Affinity Agonist Agreement Antiinflammatory Effect 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 GTP-Binding Protein alpha Subunits, Gs 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 base 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