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URL: https://dx.doi.org/DOI:10.17504/protocols.io.jfxcjpn

Authors: Les Dakin

Summary: Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay 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 ʹ, 3 ʹ -cGAMP (cGAMP), a cyclic dinucleotide second messenger with mixed 2ʹ-5ʹ and 3ʹ-5ʹ 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.

Associated Publications: Hall J, Brault A, Vincent F, Weng S, Wang H, Dumlao D, Aulabaugh A, Aivazian D, Castro D, Chen M, Culp J, Dower K, Gardner J, Hawrylik S, Golenbock D, Hepworth D, Horn M, Jones L, Jones P, Latz E, Li J, Lin L, Lin W, Lin D, Lovering F, Niljanskul N, Nistler R, Pierce B, Plotnikova O, Schmitt D, Shanker S, Smith J, Snyder W, Subashi T, Trujillo J, Tyminski E, Wang G, Wong J, Lefker B, Dakin L, Leach K (2017) Discovery of PF-06928215 as a high affinity inhibitor of cGAS enabled by a novel fluorescence polarization assay. PLoS ONE 12(9): e0184843. doi: 10.1371/journal.pone.0184843

Affiliations: Pfizer

External URL: https://doi.org/10.1371/journal.pone.0184843

Version: 1

Publication Date: 2017

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