Cytoplasmic nucleic acids alert the immune system to invading pathogens and trigger a robust type I interferon (IFN) response via activation of the STING (stimulator of interferon genes) receptor. The sensor for cytoplasmic nucleic acids was recently discovered to be a cyclic GMP-AMP synthase, which produces a unique cyclic dinucleotide second messenger, cGAMP, that serves as an agonist for the STING receptor. Aberrant activation of the cGAS-STING pathway is rapidly emerging as an important underlying cause of debilitating and sometimes fatal autoimmune disorders including systemic lupus erythematosus (SLE), scleroderma, and Aicardi?Goutieres Syndrome (AGS); cGAS is an obvious target for therapeutic intervention. In addition, very recent studies have indicated that stimulating the STING pathway may be an effective strategy for cancer immunotherapy. Development of small molecule inhibitors and activators of cGAS are clearly therapeutic strategies that need to be explored. However, development of homogenous; i.e., mix-and-read cGAS assays for high throughput screening (HTS) of small molecules is quite challenging, as it requires specific detection of the cGAMP product in the presence of the substrates, ATP and GTP. Current assay methods involve chromatographic isolation of radioactive cGAMP produced from radiolabeled substrates. From a broader perspective, simple, homogenous methods for detecting cGAMP in cell and tissue samples would be an extremely powerful tool for basic research, drug discovery and translational studies targeting the cGAS- STING pathway. Our long term (Phase I-Phase II) goal is to develop robust, HTS compatible cGAS enzymatic assays and cellular cGAMP assays to accelerate discovery and clinical translation of compounds that modulate STING mediated immune responses. In Phase I we will develop homogenous immunodetection methods for cGAMP with fluorescence polarization (FP) and time resolved Frster resonance energy transfer (TR-FRET) signals and incorporate them into biochemical HTS assays for cGAS. BellBrook has pioneered the development of HTS assays based on detection of nucleotides, and our preliminary studies indicate that we will be successful in producing a monoclonal antibody (mAb) for cGAMP with the selectivity required for a cGAS enzymatic assay. We will complete development and characterization of cGAMP mAbs, synthesize fluorescent tracers that bind to the mAbs, and develop the FP- and TR-FRET- based competitive immunoassays. We will then produce recombinant human cGAS using well defined E. coli expression and affinity purification methods and optimize the cGAS enzymatic assays. Lastly, we will perform pilot screens with a LOPAC library of pharmacologically active compounds and a 20K diversity set to assess the level of assay interference and to demonstrate robust HTS performance. The proposed assays will fulfill the key requirements for HTS, including homogenous detection, robustness (good dynamic range, low signal variability), low levels of interference, and outstanding reagent and signal stability. In Phase II, we will complete development of the biochemical cGAS HTS assay, including full scale, reproducible production of stable reagents for commercial assay kits. We will also develop, optimize, and validate the reagents for the more challenging requirements of detecting cGAMP in cell lysates and tissue samples; i.e., as an endpoint in phenotypic HTS assays and translational studies in animals. Development of simple, HTS homogenous methods for cGAMP in biological samples combined with cGAS enzyme assays will provide a powerful platform for discovering and characterizing compounds that modulate STING mediated immune responses and translating them into clinical candidates.
The ability to sense foreign nucleic acids and mount an immune response is an important weapon in the body's armory against invading pathogens, however when this system becomes aberrantly activated by self-DNA, it causes debilitating autoimmune disorders such as lupus and scleroderma. We plan to develop simple, rapid assays to detect a recently discovered signaling molecule that plays a central role in the immune response to nucleic acids; the assays will enable scientists to search for drugs that prevent aberrant immune activation in autoimmune diseases.
