Homogeneous and bioluminescent biochemical and cellular assay for monitoring cGAMP and enzymes that generate and degrade cGAMP

The cyclic GMP-AMP synthase-stimulator of the interferon gene (cGAS-STING) signaling pathway is considered an essential pattern recognition and effector pathway in the natural immune system and is mainly responsible for recognizing DNA molecules present in the cytoplasm and activating downstream signaling pathways to generate type I interferons (IFN-I) and other inflammatory factors. STING, a crucial junction protein in the innate immune system, exerts an essential role in host resistance to external pathogen invasion. The DNA introduced by pathogens or tumors is recognized by the cytoplasmic nucleic acid receptor cGAS, and a second messenger, cGAMP, is generated using intracellular guanosine triphosphate (GTP) and adenosine triphosphate (ATP). Furthermore, cellular and extracellular cGAMP concentrations are also controlled by ENPP1, an enzyme that breaks down cGAMP to AMP and GMP. Therefore, the role of the cGAS-STING signaling pathway has generated great interest in inflammatory and cancer research. To advance our understanding of innate immune system and in particular the STING pathway, we have developed a homogeneous, bioluminescent cGAMP detection assay that is very sensitive and highly selective against other nucleotides, cyclic nucleotides, and dicyclic nucleotides. The assay can be also used to monitor the activity of cGAS and ENPP1 to enable the development of inhibitors of both enzymes which might be used for therapeutic applications.