HTS Assays for Targeting the cGAS-STING Pathway in Autoimmune Diseases and Cancer

针对自身免疫性疾病和癌症中的 cGAS-STING 通路的 HTS 检测

• 批准号: 9347049
• 负责人: Robert G Lowery
• 金额: $ 28.87万
• 依托单位: BELLBROOK LABS, LLC
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2018-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9347049
• 关键词: Accelerometer; Adoption; Affinity Chromatography; Agonist; Animal Model; Animals; Antibodies; Autoimmune Diseases; Basic Science; Binding; Biochemical; Biological; Biological Assay; Cells; Characteristics; Coupled; Cyclic GMP; DNA; Detection; Development; Dinucleoside Phosphates; Diversity Library; Drug Targeting; Energy Transfer; Enzymes; Escherichia coli; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Genes; Goals; Guanosine Triphosphate; Human; Immune response; Immune system; Immunoassay; Innate Immune Response; Interferon Type I; Interferons; Invaded; Knock-out; Laboratories; Lanthanoid Series Elements; Libraries; Lupus; Malignant Neoplasms; Measures; Mediating; Methods; Monitor; Monoclonal Antibodies; Nucleic Acids; Nucleotides; Pathway interactions; Performance; Periodicity; Pharmaceutical Preparations; Pharmacology; Phase; Phenotype; Play; Production; Radioactive; Radiolabeled; Radiometry; Reagent; Receptor Gene; Recombinants; Reproducibility; Research Personnel; Role; Sampling; Scientist; Scleroderma; Second Messenger Systems; Signal Transduction; Signaling Molecule; System; Systemic Lupus Erythematosus; Therapeutic; Therapeutic Intervention; Thin Layer Chromatography; Time; Tissue Sample; Tracer; Translating; Variant; base; cancer immunotherapy; clinical candidate; clinical translation; commercialization; drug discovery; enzyme activity; high throughput screening; immune activation; inhibitor/antagonist; pathogen; prevent; pseudotoxoplasmosis syndrome; receptor; residence; response; screening; sensor; small molecule; small molecule inhibitor; stability testing; targeted treatment; tool; translational study; tumor; weapons

SUMMARY Cytoplasmic nucleic acids alert the immune system to invading pathogens and trigger a robust type I interferon (IFN) response via activation of the STING (stimulator of interferon genes) receptor. The sensor for cytoplasmic nucleic acids was recently discovered to be a cyclic GMP-AMP synthase, which produces a unique cyclic dinucleotide second messenger, cGAMP, that serves as an agonist for the STING receptor. Aberrant activation of the cGAS-STING pathway is rapidly emerging as an important underlying cause of debilitating and sometimes fatal autoimmune disorders including systemic lupus erythematosus (SLE), scleroderma, and Aicardi–Goutieres Syndrome (AGS); cGAS is an obvious target for therapeutic intervention. In addition, very recent studies have indicated that stimulating the STING pathway may be an effective strategy for cancer immunotherapy. Development of small molecule inhibitors and activators of cGAS are clearly therapeutic strategies that need to be explored. However, development of homogenous; i.e., mix-and-read cGAS assays for high throughput screening (HTS) of small molecules is quite challenging, as it requires specific detection of the cGAMP product in the presence of the substrates, ATP and GTP. Current assay methods involve chromatographic isolation of radioactive cGAMP produced from radiolabeled substrates. From a broader perspective, simple, homogenous methods for detecting cGAMP in cell and tissue samples would be an extremely powerful tool for basic research, drug discovery and translational studies targeting the cGAS- STING pathway. Our long term (Phase I-Phase II) goal is to develop robust, HTS compatible cGAS enzymatic assays and cellular cGAMP assays to accelerate discovery and clinical translation of compounds that modulate STING mediated immune responses. In Phase I we will develop homogenous immunodetection methods for cGAMP with fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals and incorporate them into biochemical HTS assays for cGAS. BellBrook has pioneered the development of HTS assays based on detection of nucleotides, and our preliminary studies indicate that we will be successful in producing a monoclonal antibody (mAb) for cGAMP with the selectivity required for a cGAS enzymatic assay. We will complete development and characterization of cGAMP mAbs, synthesize fluorescent tracers that bind to the mAbs, and develop the FP- and TR-FRET- based competitive immunoassays. We will then produce recombinant human cGAS using well defined E. coli expression and affinity purification methods and optimize the cGAS enzymatic assays. Lastly, we will perform pilot screens with a LOPAC library of pharmacologically active compounds and a 20K diversity set to assess the level of assay interference and to demonstrate robust HTS performance. The proposed assays will fulfill the key requirements for HTS, including homogenous detection, robustness (good dynamic range, low signal variability), low levels of interference, and outstanding reagent and signal stability. In Phase II, we will complete development of the biochemical cGAS HTS assay, including full scale, reproducible production of stable reagents for commercial assay kits. We will also develop, optimize, and validate the reagents for the more challenging requirements of detecting cGAMP in cell lysates and tissue samples; i.e., as an endpoint in phenotypic HTS assays and translational studies in animals. Development of simple, HTS homogenous methods for cGAMP in biological samples combined with cGAS enzyme assays will provide a powerful platform for discovering and characterizing compounds that modulate STING mediated immune responses and translating them into clinical candidates.

总结 细胞质核酸提醒免疫系统入侵病原体并触发强大的I型干扰素 (IFN)通过激活STING（干扰素基因刺激因子）受体产生免疫应答。的传感器 细胞质核酸最近被发现是一种环状GMP-AMP合酶，它产生一种 独特的环状二核苷酸第二信使，cGAMP，作为STING受体的激动剂。 cGAS-STING途径的异常激活正在迅速成为导致糖尿病的重要潜在原因。 使人衰弱且有时致命的自身免疫性疾病，包括系统性红斑狼疮（SLE）， 硬皮病和Aicardi-Goutieres综合征（AGS）; cGAS是治疗干预的明显靶标。 此外，最近的研究表明，刺激STING途径可能是一种有效的策略， 癌症免疫疗法cGAS的小分子抑制剂和激活剂的开发显然是可行的。 需要探索的治疗策略。然而，同质化的发展;即，溷读 用于小分子的高通量筛选（HTS）的CGAS测定是相当具有挑战性的，因为它需要 在底物ATP和GTP存在下特异性检测cGAMP产物。当前试验 方法包括从放射性标记的底物产生的放射性cGAMP的色谱分离。从 用于检测细胞和组织样品中的cGAMP的更广泛的视角、简单的同质方法将是 一个非常强大的工具，用于基础研究、药物发现和靶向cGAS的转化研究- STING途径。我们的长期（第一阶段-第二阶段）目标是开发稳健的、HTS兼容的cGAS酶促 用于加速化合物的发现和临床转化的细胞cGAMP测定， 调节STING介导的免疫应答。 在第一阶段，我们将开发荧光偏振的cGAMP均相免疫检测方法 (FP)和时间分辨的福斯特共振能量转移（TR-FRET）信号，并将它们纳入 用于cGAS的生物化学HTS测定。贝尔布鲁克率先开发了基于 检测核苷酸，我们的初步研究表明，我们将成功地生产一种 使用cGAMP的单克隆抗体（mAb），其具有cGAS酶促测定所需的选择性。我们将 完成cGAMP单克隆抗体的开发和表征，合成结合到 mAb，并开发基于FP和TR-FRET的竞争性免疫测定。我们将生产 重组人cGAS使用明确定义的E.大肠杆菌表达及亲和纯化方法的优化 CGAS酶测定。最后，我们将使用一个LOPAC库来执行试点屏幕， 活性化合物和20 K多样性集，以评估测定干扰水平并证明稳健性 HTS性能。拟定的检测试剂盒将满足HTS的关键要求，包括均质 检测、鲁棒性（良好的动态范围、低信号变异性）、低干扰水平和出色的 试剂和信号稳定性。 在第二阶段，我们将完成生物化学cGAS HTS检测的开发，包括全规模， 用于商业测定试剂盒的稳定试剂的可再现生产。我们还将开发、优化和 验证试剂是否满足检测细胞裂解物和组织中cGAMP的更具挑战性的要求 样本;即，作为表型HTS测定和动物转化研究的终点。发展 用于生物样品中cGAMP的简单HTS均相方法与cGAS酶测定结合， 为发现和表征调节STING介导的化合物提供了强大的平台 免疫反应并将其转化为临床候选物。