Abstract 1402: Monitoring the concentration of cGAMP using homogenous bioluminescent HTS-formatted assay

Abstract

Abstract Genomic instability engages the cGAS (cytosolic DNA sensor cyclic AMP-GMP synthase)-STING (stimulator of interferon genes) signaling (cGAS-cGAMP-STI NG) pathway on both sides of cancer either by being instrumental for antitumor immunity and blocking malignancy or by promoting inflammation-driven carcinogenesis and metastasis. The cGAS-STING axis also plays an essential role in initiating host immune defense against microbial invasion and STING-deficient mice are more susceptible to some viral infections. Credible evidence suggests that the cGAS-cGAMP-STI NG pathway makes fundamental contributions to at least three major cancer therapies, radiation therapy, chemotherapy, immunotherapy, and thus represents a promising pharmaceutical target. cGAS is well-positioned to act as a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP) upon binding to DNA and initiating host immune responses against pathogens and cancer. However, inappropriate activation of STING signaling causes severe and often fatal autoimmune or autoinflammatory diseases. Hence, STING is an attractive drug target for the treatment of STING-driven autoimmune and inflammatory disorders. Therefore, there is a need to identify lead compounds that effectively inhibit human STING for further drug development. An attempt to search for such molecules have been described recently with the report on a STING-specific inhibitor that showed high efficiency, specificity, and safety, paving the way for therapeutically manipulating STING-mediated clinical diseases. Towards this goal, we have developed a bioluminescent assay to monitor the concentration of cGAMP in intracellular as well as extracellular compartments. The assay relies on the use of ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) to release AMP which can be converted to ATP via a coupled enzyme reaction. The ATP generated can be quantified using luciferase/luciferin detection reagents. The assay is homogenous, HTS formatted and can be completed in less than one hour. The assay is sensitive and can detect as low as 10 nM cGAMP. We will show data on the use of agents that stimulate GAS activity such as dsDNA, and the use of reporter assays to detect cytokine release upon cGAS-STING activation. Citation Format: Said A. Goueli, kevin Hsiao. Monitoring the concentration of cGAMP using homogenous bioluminescent HTS-formatted assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1402.