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

基本信息

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
项目类别：
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

项目摘要

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.

概括细胞质核酸向免疫系统发出入侵病原体的警报，并触发强大的 I 型干扰素 (IFN) 通过激活 STING（干扰素基因刺激剂）受体做出反应。最近发现细胞质核酸是一种环状 GMP-AMP 合酶，它产生一种独特的环状二核苷酸第二信使 cGAMP，可作为 STING 受体的激动剂。 cGAS-STING 通路的异常激活正在迅速成为导致以下疾病的重要根本原因：使人衰弱，有时甚至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) 是治疗干预的明显目标。此外，最近的研究表明刺激 STING 通路可能是一种有效的策略用于癌症免疫治疗的cGAS小分子抑制剂和激活剂的开发是明确的。然而，需要探索同质的治疗策略；即混合阅读。用于小分子高通量筛选 (HTS) 的 cGAS 检测非常具有挑战性，因为它需要在底物、ATP 和 GTP 存在下对 cGAMP 产物进行特异性检测。方法涉及对放射性标记底物产生的放射性 cGAMP 进行色谱分离。用于检测细胞和组织样品中的 cGAMP 的更广阔的视角、简单、同质的方法将是一个极其强大的工具，用于针对 cGAS 的基础研究、药物发现和转化研究我们的长期（第一阶段-第二阶段）目标是开发强大的、HTS 兼容的 cGAS 酶。分析和细胞 cGAMP 分析，以加速化合物的发现和临床转化调节 STING 介导的免疫反应。在第一阶段，我们将开发具有荧光偏振的 cGAMP 均质免疫检测方法 (FP) 和时间分辨福斯特共振能量转移 (TR-FRET) 信号并将其合并到 BellBrook 率先开发了基于 cGAS 的 HTS 检测方法。检测核苷酸，我们的初步研究表明我们将成功生产 cGAMP 的单克隆抗体 (mAb) 具有 cGAS 酶测定所需的选择性。完成 cGAMP mAb 的开发和表征，合成与 mAb，并开发基于 FP 和 TR-FRET 的竞争性免疫测定法，然后我们将生产。使用明确的大肠杆菌表达和亲和纯化方法重组人 cGAS 并进行优化最后，我们将使用 LOPAC 药理学库进行试点筛选。活性化合物和 20K 多样性集，用于评估测定干扰水平并证明稳健性 HTS 性能。拟议的检测将满足 HTS 的关键要求，包括同质性。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，我们将完成生化 cGAS HTS 检测的开发，包括全尺寸、我们还将开发、优化和生产用于商业化验试剂盒的稳定试剂。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品；即作为表型 HTS 测定和动物转化研究的终点。生物样品中 cGAMP 的简单 HTS 均质方法与 cGAS 酶测定相结合，将为发现和表征调节 STING 介导的化合物提供了一个强大的平台免疫反应并将其转化为临床候选者。