Characterization of cGAS enzyme activity.

<p>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.</p

Binding affinities and <i>in vitro</i> activities of cGAS inhibitors.

<p>Binding affinities and <i>in vitro</i> activities of cGAS inhibitors.</p

Characterization of cGAMP FP assay.

<p>(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.</p

Characterization of compound 15 binding to cGAS.

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