Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay

Abstract

Cyclic GMP-AMP synthase (cGAS) initiates the innate immune system in response to cytosolic dsDNA. After binding and activation from dsDNA, cGAS uses ATP and GTP to synthesize 2′, 3′ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2′-5′ and 3′-5′ phosphodiester bonds. Inappropriate stimulation of cGAS has been implicated in autoimmune disease such as systemic lupus erythematosus, thus inhibition of cGAS may be of therapeutic benefit in some diseases; however, the size and polarity of the cGAS active site makes it a challenging target for the development of conventional substrate-competitive inhibitors. We report here the development of a high affinity (KD = 200 nM) inhibitor from a low affinity fragment hit with supporting biochemical and structural data showing these molecules bind to the cGAS active site. We also report a new high throughput cGAS fluorescence polarization (FP)-based assay to enable the rapid identification and optimization of cGAS inhibitors. This FP assay uses Cy5-labelled cGAMP in combination with a novel high affinity monoclonal antibody that specifically recognizes cGAMP with no cross reactivity to cAMP, cGMP, ATP, or GTP. Given its role in the innate immune response, cGAS is a promising therapeutic target for autoinflammatory disease. Our results demonstrate its druggability, provide a high affinity tool compound, and establish a high throughput assay for the identification of next generation cGAS inhibitors.

Human DNA-PK activates a STING-independent DNA sensing pathway

“…The cGAS-STING antiviral response has become the subject of intense development in the pharmaceutical industry, including efforts to develop inhibitors of cGAS and STING to treat human autoimmune diseases (35)(36)(37), as well as agonists of STING to improve immune responses to tumors (38)(39)(40). Our discovery of a second DNA-activated antiviral response in human cells has important implications for these efforts.…”

A simple model for determining affinity from irreversible thermal shifts

“…Proteins and ligands are not identified if they are part of an active therapeutic research program. Nucleotide‐binding domain 1 (NBD1) of Cystic Fibrosis Transmembrane Conductance Regulator, and the cyclic GMP‐AMP Synthase (cGAS) were expressed and purified as described . Other proteins were purified at Pfizer using standard growth and purification techniques.…”

“…Nucleotide-binding domain 1 (NBD1) of Cystic Fibrosis Transmembrane Conductance Regulator, and the cyclic GMP-AMP Synthase (cGAS) were expressed and purified as described. 15,16 Other proteins were purified at Pfizer using standard growth and purification techniques. All the proteins used in these experiments were shown to be greater than 95% pure by gel.…”

“…Ligand affinities for BASE1 are from the work reported by Lo et al, 7 SPR affinities for cGAS and NBD1 were determined as described. [15][16][17] Other affinities shown here were determined at 25 C using SPR or estimated from KI. Activity assays were performed using commercial kits following the manufacturer's protocol.…”

The Role of Nucleic Acid Sensing in Controlling Microbial and Autoimmune Disorders

“…Given these advances in developing small molecules and methods to activate innate immunity to reduce microbial burden, methods to inhibit hyperactive innate immune signaling may aid in the development of therapies for autoimmune disorders. Already, small molecules have been developed that target cGAS and reduce its activity (An et al, 2015; Hall et al, 2017; Vincent et al, 2017). Given the evolutionary arms race between pathogens and host, especially with respect to the co-evolution of DNA-and retro-viruses with humans (Elde and Malik, 2009), might it be possible to develop virally based strategies or molecules to inhibit hyperactive innate immunity?…”