Development of small molecule cGAS inhibitors for repression of dsDNA-triggered interferon expression

环 GMP-AMP 合酶 (cGAS) 是胞质 dsDNA 的主要传感器，产生环二核苷酸 cGAMP，在髓系细胞类型亚群中启动细胞因子产生的第二信使， 负责提供先天免疫。异常的胞浆 dsDNA 会导致炎症性疾病， 表明抑制 cGAS 可能具有治疗益处。我们最近开发了一种大规模 使用小鼠 cGAS 进行基于光谱分析的高通量筛选 (HTS) 并鉴定出小分子抑制剂 在小鼠细胞试验中产生了第一个活性和特异性的 cGAS 抑制剂，尽管没有抑制作用 人体细胞中的活性。我们现在开发了一种更快、更具成本效益的 HTS，用于识别人类 使用化学发光法筛选 cGAS 抑制剂并筛选了 300,000 种化合物。后续药用 化学优化确定了对主要干扰素活性的人类 cGAS 有效且特异性的抑制剂 产生细胞类型，包括初级巨噬细胞和 PBMC。我们还解决了共晶结构 人类 cGAS 抑制剂，提出了先导化合物的结构和计算引导的优化路径 抑制剂。我们建议通过扩展来识别新的热门化合物以获取更多的药物支架 药物库筛选以继续支持我们的药物化学项目。使用最有效且 特异性抑制剂，我们希望以更多样化的方式表征它们的抑制作用和途径特异性 细胞范围。我们的长期目标是开发治疗 cGAS 失调相关疾病的疗法。 该提案有 3 个目标：(1) 通过 HTS 识别新的化学支架并应用 化学信息分析和药物化学命中优化。我们将利用我们新成立的和 经过验证的 HTS 方法可用于鉴定人类 cGAS 抑制剂，以筛选包含 100,000 个新可用的库 化合物。新的热门化合物将优先进行进一步衍生化，以优化效力和类药性 特点。 (2) 结构/计算引导设计，优化新支架和引线 人类 cGAS 抑制剂。我们将利用从抑制剂-cGAS 共晶结构中获得的见解来设计， 合成并测试衍生物，以探索最有效、最有前途的类药 cGAS 抑制剂。 (3) 开发新的细胞验证方法来鉴定有效且高选择性的 cGAS 抑制剂。我们建议通过评估新的和最近发现的抑制剂来评估和验证它们 使用已建立的和新的细胞检测（包括非骨髓细胞检测）的功效、选择性和生物相容性 具有染色体不稳定的癌细胞系、原代骨髓细胞和患者来源的骨髓细胞。 尽管最初被发现是作为抗病毒活性的中心 dsDNA 传感器，但 cGAS 也正在成为以下领域的重要参与者： 肥胖、神经退行性疾病、肿瘤发生和癌症转移。特征明确、经过验证、 研究 cGAS-STING 生物学以及 治疗cGAS相关炎症性疾病！

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for cytosolic dsDNA, producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid-lineage cell types and responsible for providing innate immunity. Aberrant cytosolic dsDNA contributes to inflammatory diseases, suggesting that inhibition of cGAS may be therapeutically beneficial. We recently developed a mass- spectrometry-based high throughput screen (HTS) using mouse cGAS and identified small-molecule inhibitors that yielded the first active and specific inhibitors of cGAS in mouse cellular assays, albeit with no inhibitory activity in human cells. We now developed a faster and more cost effective HTS for identification of human cGAS inhibitors using chemiluminescence and screened 300,000 compounds. Subsequent medicinal chemistry optimization identified potent and specific inhibitors for human cGAS active in major interferon- producing cell types including primary macrophages and PBMCs. We have also solved co-crystal structures of inhibitors with human cGAS, suggesting structure- and computational-guided optimization path for the lead inhibitors. We propose to identify new hit compounds to access additional drug scaffolds through extended drug library screening to continue supporting our medicinal chemistry program. Using the most potent and specific inhibitors, we wish to characterize their inhibitory effect and pathway specificity in a more diverse range of cells. Our long-term goal is to develop therapeutics for cGAS-dysregulation associated diseases. This proposal is organized in 3 aims: (1) Identify new chemical scaffolds by HTS and apply cheminformatic analysis and medicinal chemistry hit optimization. We will use our newly established and validated HTS method for identification of human cGAS inhibitors to screen a newly available library of 100,000 compounds. New hit compounds will be prioritized for further derivatization to optimize potency and drug-like characteristics. (2) Structure/computation-guided design for optimization of new scaffolds and lead human cGAS inhibitors. We will use the insights gained from inhibitor-cGAS co-crystal structures to design, synthesize, and test derivatives to probe for the most potent, and promising drug-like cGAS inhibitor(s). (3) Develop new cellular validation assays for the identification of potent and highly selective cGAS inhibitors. We propose to evaluate and validate new and recently identified inhibitors by assessing their efficacy, selectivity, and biocompatibility using established and new cellular assays including non-myeloid cancer cell lines with chromosomal instability, primary myeloid cells, and patient-derived myeloid cells. Although initially discovered as central dsDNA sensor for antiviral activity, cGAS is also emerging as a player in obesity, neurodegenerative diseases, tumorigenesis, and cancer metastasis. Well-characterized, validated, potent, and specific cGAS inhibitors are needed in the community studying cGAS-STING biology as well as for treating cGAS-related inflammatory diseases!