Development and Validation of a Novel cGAS Inhibitor Screening Assay Julianna Blaylock, Yogan Khatri, and Daniel Tew Cayman Chemical Company, Ann Arbor, MI Cyclic GMP-AMP synthase (cGAS) is a mammalian dsDNA sensor found in cytosol and involved in an innate immune response. Dysregulation of this cGAS pathway can lead to chronic inflammation, autoimmune disorders, and even tumorigenesis. Activation of cGAS by pathogen-associated dsDNA and the production of 2’3’-cyclic GMP-AMP (2’3’-cGAMP) are important in host defense but may also play a role in the development of autoimmune diseases, such as systemic lupus erythematosus (SLE), which are characterized by increased expression of IFN-stimulated genes. Additionally, cGAS is activated in response to mitochondrial DNA leakage, which is associated with metastatic phenotypes and age-associated inflammation in cancer, and the accumulation of extrachromosomal telomere repeat DNA that results in IFN expression and inhibition of cell proliferation. Late stage tumors with a high level of chromosomal instability exhibit decreased protein expression of cGAS as a mechanism to evade cGAS-mediated IFN-signaling and inhibition of tumor growth. In contrast, carcinogen-induced cGAS activation and transfer of tumor cell cGAS to astrocytes through gap junctions promotes tumorigenesis and brain metastasis, respectively, in mouse models. Inhibition of cGAS activity suppresses IFN-stimulated gene expression and decreases type I IFN production in patient-derived samples and mouse models of autoimmune diseases, indicating therapeutic utility of cGAS inhibition. Proper regulation of the cGAS-STING pathway and finding modulators of cGAS is critical in maintaining immune homeostasis. To facilitate the study of cGAS modulators, we have developed a novel cGAS Inhibitor Screening Assay that directly measures the product of the cGAS reaction, 2’3’-cGAMP, formed in the presence of DNA, ATP and GTP. The cyclic dinucleotide product is quantified via enzyme-linked immunoassay (ELISA) with a 2’3’-cGAMP specific polyclonal antiserum. This report will focus on the production of an active recombinant human cGAS protein and the optimization and validation of an inhibitor screening assay using the protein.