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，一种在骨髓谱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。异常的胞质dsDNA导致炎性疾病， 这表明cGAS的抑制可能在治疗上是有益的。我们最近开发了一种质量- 使用小鼠cGAS和鉴定的小分子抑制剂进行基于光谱的高通量筛选（HTS） 这在小鼠细胞测定中产生了cGAS的第一个活性和特异性抑制剂，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，更经济的HTS用于识别人类 cGAS抑制剂使用化学发光和筛选的300，000种化合物。后续药物 化学优化鉴定了人cGAS的有效和特异性抑制剂，其在主要干扰素- 产生包括原代巨噬细胞和PBMC的细胞类型。我们还解决了共晶结构的 抑制剂与人类cGAS，建议结构和计算指导的优化路径的铅 抑制剂的我们建议确定新的命中化合物，以获得额外的药物支架，通过扩大 药物库筛选，以继续支持我们的药物化学计划。使用最有效的， 特异性抑制剂，我们希望以更多样化的方式表征其抑制作用和途径特异性。 细胞的范围。我们的长期目标是开发cGAS失调相关疾病的治疗方法。 本研究的主要目的有三：（1）利用高温超导技术识别新的化学支架，并应用于 化学信息学分析和药物化学命中优化。我们将利用我们新成立的， 用于鉴定人cGAS抑制剂的经验证的HTS方法，以筛选100，000个新可用文库 化合物.新的命中化合物将优先用于进一步衍生化，以优化效力和药物样活性。 特色(2)优化新支架和导线的结构/计算引导设计 人cGAS抑制剂。我们将使用从Corneor-cGAS共晶结构中获得的见解来设计， 合成和测试衍生物，以探测最有效和有前途的药物样cGAS抑制剂。（三） 开发新的细胞验证试验，用于鉴定强效和高选择性cGAS 抑制剂的我们建议通过评估新的和最近发现的抑制剂， 使用已建立的和新的细胞测定法（包括非髓系细胞测定法）， 具有染色体不稳定性的癌细胞系、原代骨髓细胞和患者来源的骨髓细胞。 虽然cGAS最初被发现是抗病毒活性的中心dsDNA传感器，但它也正在成为抗病毒活性的参与者。 肥胖、神经变性疾病、肿瘤发生和癌症转移。特征良好，经过验证， 在研究cGAS-STING生物学的社区中以及在研究cGAS-STING生物学的社区中， 治疗cGAS相关的炎症性疾病！

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!