HTS Assays for Targeting the cGAS STING Pathway in Autoimmune Diseases and Cancer

SUMMARY 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 F rster 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 K 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 Narrative The ability to sense foreign nucleic acids and mount an immune response is an important weapon in the bodyandapos 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