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

摘要 胞质核酸提醒免疫系统注意入侵的病原体，并触发强大的I型干扰素 (干扰素)通过激活刺激物(干扰素基因刺激物)受体而产生的反应。用于测量的传感器 最近发现胞质核酸是一种环状GMP-AMP合成酶，它产生一种 独一无二的环二核苷酸第二信使，cGAMP，作为刺痛受体的激动剂。 CGAS-sting通路的异常激活正在迅速成为一种重要的潜在原因 包括系统性红斑狼疮(SLE)在内的衰弱甚至有时致命的自身免疫性疾病， 硬皮病和Aicardi-Goutieres综合征(AGS)；cGAS是治疗干预的明显靶点。 此外，最近的研究表明，刺激刺痛通路可能是一种有效的策略 用于癌症免疫治疗。CGAS小分子抑制剂和激活剂的研究进展 需要探索的治疗策略。然而，同质化的发展；即混合阅读 对于小分子的高通量筛选(HTS)，cGAS分析是相当具有挑战性的，因为它需要 在底物、ATP和GTP存在的情况下，对cGAMP产物进行特异性检测。电流化验 方法涉及从放射性标记底物中产生的放射性cGAMP的层析分离。从… 在细胞和组织样本中检测cGAMP的更广泛、简单、均一的方法将是 针对cGAS的基础研究、药物发现和转化研究的极其强大的工具- 刺痛路径。我们的长期目标(第一阶段-第二阶段)是开发强大的、与HTS兼容的cGAS酶 检测和细胞cGAMP检测加速化合物的发现和临床翻译 调节刺痛介导的免疫反应。 在第一阶段，我们将建立cGAMP的荧光偏振均相免疫检测方法 (FP)和时间分辨Förster共振能量转移(TR-FRET)信号，并将它们合并到 生化HTS法检测cGAS。Bellbrook开创了HTS检测的先河，其基础是 检测核苷酸，我们的初步研究表明，我们将成功地生产一种 CGAMP的单抗，具有cGAS酶分析所需的选择性。我们会 完成cGAMP单抗的研制和鉴定，合成与cGAMP结合的荧光示踪剂 并开发基于FP-FRET和TRRET的竞争免疫分析方法。然后我们将生产 重组人cGAS在大肠杆菌中的高效表达及亲和纯化方法的优化 CGAS酶分析。最后，我们将使用Lopac药理学文库进行初步筛选 活性化合物和20K多样性集，以评估分析干扰的水平并展示健壮性 HTS性能。建议的化验将满足高温超导的关键要求，包括同质 可检测性、健壮性(良好的动态范围、低信号变化性)、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分析的开发，包括全面， 用于商业分析试剂盒的稳定试剂的可重复性生产。我们还将开发、优化和 验证试剂以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，在动物的表型HTS分析和翻译研究中作为终点。发展中的 生物样品中cGAMP的简单HTS均质方法与cGAS酶分析相结合将 为发现和表征调节刺痛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。

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.