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

  • PLoS One. 2017 Sep 21;12(9):e0184843. doi: 10.1371/journal.pone.0184843.
Justin Hall  1 ,  Amy Brault  1 ,  Fabien Vincent  1 ,  Shawn Weng  2 ,  Hong Wang  1 ,  Darren Dumlao  1 ,  Ann Aulabaugh  1 ,  Dikran Aivazian  3 ,  Dana Castro  3 ,  Ming Chen  1 ,  Jeffrey Culp  1 ,  Ken Dower  4 ,  Joseph Gardner  5 ,  Steven Hawrylik  1 ,  Douglas Golenbock  6 ,  David Hepworth  7 ,  Mark Horn  3 ,  Lyn Jones  7 ,  Peter Jones  7 ,  Eicke Latz  6  8 ,  Jing Li  7 ,  Lih-Ling Lin  4 ,  Wen Lin  1 ,  David Lin  1 ,  Frank Lovering  7 ,  Nootaree Niljanskul  1 ,  Ryan Nistler  2 ,  Betsy Pierce  1 ,  Olga Plotnikova  1 ,  Daniel Schmitt  1 ,  Suman Shanker  1 ,  James Smith  1 ,  William Snyder  3 ,  Timothy Subashi  1 ,  John Trujillo  1 ,  Edyta Tyminski  2 ,  Guoxing Wang  2 ,  Jimson Wong  1 ,  Bruce Lefker  7 ,  Leslie Dakin  7 ,  Karen Leach  2
Affiliations
  • 1. Medicine Design, Pfizer, Groton, Connecticut, United States of America.
  • 2. Pfizer Centers for Therapeutic Innovation (CTI), Boston, Massachusetts, United States of America.
  • 3. Pfizer Centers for Therapeutic Innovation (CTI), San Diego, California, United States of America.
  • 4. Inflammation and Immunology, Pfizer, Cambridge, Massachusetts, United States of America.
  • 5. External Research Solutions, Pfizer, Groton, Connecticut, United States of America.
  • 6. University of Massachusetts Medical School, Worcester, Massachusetts, United States of America.
  • 7. Medicine Design, Pfizer, Cambridge, Massachusetts, United States of America.
  • 8. Institute of Innate Immunity, University Hospitals Bonn, Bonn, Germany.
PMID: 28934246 DOI: 10.1371/journal.pone.0184843
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.

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