HTS Assays for Targeting the cGAS-STING Pathway in Autoimmune Diseases and Cancer - Project Summary
Cyclic GMP-AMP synthase (cGAS) is a recently discovered enzyme that acts as a foreign DNA sensor to elicit
an immune response to pathogens via activation of the STING (stimulator of interferon genes) receptor. Though
there are other DNA sensors (e.g., TLR9 ), the cGAS-cGAMP-STING pathway appears to be essential for DNA-
mediated immune response irrespective of cell type or DNA sequence. DNA binds to a specific site on cGAS in
a non-sequence-dependent manner and activates its catalytic activity, resulting in the production of a unique
cyclic nucleotide G(2'-5')pA(3'-5')p (cGAMP) from ATP and GTP precursors. cGAMP binds to the STING
receptor with nanomolar affinity and induces expression of type I interferons. Thus, cGAMP plays a fundamental
role in human immunity, acting upstream of both T and B cells to trigger an innate immune response.
Shortly after its discovery in 2013, aberrant
activation of cGAS by self-DNA was shown to
contribute to debilitating and sometimes fatal
autoimmune diseases, such as systemic lupus
erythematosus (SLE), and knocking out cGAS
abrogates disease in animal models. The
cGAS-STING pathway has also been shown to
play a key role in the innate immune response
to tumors, and intratumoral injection of cGAMP
analogs is an emerging strategy for cancer
immunotherapy. Modulating cGAS activity is
autoimmunity and cancer.
Tumor immunityATP
GTP
cGAS cGAMP
Autoimmune diseases
IRF
Antimicrobial Immunity 3 STING
NF¿B
Type I IFNs
Activation of cGAS by cytoplasmic DNA initiates activation of the innate immune
response via induction of Type I IFNs which induce tumor cell specific T cell
responses in cancer but induce autoantibodies and cause extensive tissue
damage in autoimmune diseases such as SLE.
therefore a very compelling therapeutic strategy, both for
Though basic research on the cGAS/STING pathway and efforts to target it therapeutically have expanded
rapidly, the difficulty in detecting cGAMP has seriously hindered progress on both fronts. It functions at
nanomolar concentrations, whereas other nucleotides, including its precursors ATP and GTP, are present at
levels as much as 1000-fold higher, making specific detection of cGAMP in cell or tissue extracts extremely
challenging. Currently, the only method used is methanol extraction followed by HPLC purification and LC/MS.
Similarly, methods for detecting cGAS enzymatic activity involve chromatographic separation of radioactive
cGAMP from ATP and GTP. Development of drugs that modulate cGAS will require sensitive, homogenous
assay methods for detecting cGAMP with exquisite specificity that can be used for biochemical and cellular HTS
assays, and eventually for biomarker assays to support translational research.
In Phase I, we produced monoclonal antibodies that recognize cGAMP with more than 1000-fold selectivity vs.
other nucleotides and used one of them to develop homogenous detection assays with fluorescent readouts (FP
and TR-FRET). We validated the assays as a robust biochemical HTS platform using purified human cGAS;
these assays will allow us to begin screening for cGAS modulators under separate grant applications. In Phase
II, we will develop more sensitive cGAMP detection reagents and methods that can be used for cellular HTS and
biomarker assays. Taking inspiration from recent examples of sandwich-based assays for small molecules, we
will use cGAMP antigen design approaches and in vitro evolution to generate a pair of single chain Fv's that bind
cGAMP simultaneously with picomolar affinity to allow direct detection of cGAMP in cell and tissue lysates with
TR-FRET and/or ELISA assays. The effort will include contributions from two outstanding academic scientists
to buttress BellBrook's assay development expertise. Dr. Karl Wittrup, Director of the Koch Institute at MIT and
inventor of the yeast display system that we will use for scFv affinity maturation, will act as a consultant for the
critical in vitro evolution step. Dr. Keith Elkon, Head of the Division of Rheumatology at University of Washington,
one of the world's leading experts on the molecular and genetic basis for autoimmune diseases, who is
pioneering efforts to elucidate the role of cGAS/STING in SLE, will collaborate on validation of the biomarker
assay with samples from animal models and patients.
The development of simple, quantitative cGAMP assays would have very significant scientific and medical
impact. The biochemical and cellular cGAMP assays will enable BellBrook and collaborators to pursue HTS-
driven efforts to develop first-in-class lead molecules targeting the cGAS/STING pathway for autoimmunity and
cancer immunotherapy. More broadly, commercialization of the assays as kits will fill critical gaps in the tools
needed for basic cellular and biochemical studies of the cGAS/STING pathway.
