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.

Fig 1. Characterization of cGAS enzyme activity.

Measurement of cGAMP production was conducted by LC-MS as described in Methods. (A) Time course of cGAS (15 nM) activity; (B) titration of dsDNA activation of cGAS (1nM) activity; (C) cGAS enzyme titration; (D) inhibition of cGAS (1 nM) activity by CuBr.

Fig 2. Characterization of cGAMP FP assay.

(A) mAb titration with Cy5-cGAMP (2 nM); (B) competition of Cy5-cGAMP (2 nM) binding to mAb 80–2 with: cGAMP, cAMP, cGMP, ATP or GTP; (C) Z’ results of FP assay in subset screen; (D) Distribution of compound activity from subset screen.

Fig 3. Binding affinities and in vitro activities of cGAS inhibitors.

Fig 4. Characterization of compound 15 binding to cGAS.

(A) 1D ¹H spectra of 2´,3´-cGAMP (top) and ¹H STD of 2´,3´-cGAMP interacting with cGAS (bottom). (B) 1D ¹H spectra of 2´,3´-cGAMP (orange) and compound 15 (green) (top) and ¹H STD of a mixture of 2´,3´-cGAMP and compound 15 showing compound 15 has out competed 2´,3´-cGAMP for interacting with cGAS (bottom). (C) SPR sensorgram of compound 15 with binding fit inset. (D) Compound 15 in either its hydroxyl (15a) or keto (15b) tautomeric forms. (E) cGAS active site showing residues that interact with compound 15; Fo-Fc electron density omit map (green) for compound 15 (brown) is contoured at 3 Sigma and shows all density within 4 Å of compound 15.

Fig 5. PF-06928215 is a high affinity cGAS inhibitor.

(A) Concentration-dependent inhibition in cGAMP FP assay. (B) SPR sensorgram of PF-06928215 binding to cGAS, 2-fold variations in the concentration of PF-06928215 are shown in rainbow registry with kinetic association and dissociation fits in black. (C) cGAS active site showing residues that interact with PF-06928215, Fo-Fc electron density omit map (green) for PF-06928215 (brown) is contoured at 3 Sigma and shows all density within 4 Å of PF-06928215. Figure was generated using Pymol.