The catalytic mechanism of cyclic GMP‐AMP synthase (cGAS) and implications for innate immunity and inhibition

Abstract

Cyclic GMP‐AMP synthase (cGAS) is activated by ds‐DNA binding to produce the secondary messenger 2′,3′‐cGAMP. cGAS is an important control point in the innate immune response; dysregulation of the cGAS pathway is linked to autoimmune diseases while targeted stimulation may be of benefit in immunoncology. We report here the structure of cGAS with dinucleotides and small molecule inhibitors, and kinetic studies of the cGAS mechanism. Our structural work supports the understanding of how ds‐DNA activates cGAS, suggesting a site for small molecule binders that may cause cGAS activation at physiological ATP concentrations, and an apparent hotspot for inhibitor binding. Mechanistic studies of cGAS provide the first kinetic constants for 2′,3′‐cGAMP formation, and interestingly, describe a catalytic mechanism where 2′,3′‐cGAMP may be a minor product of cGAS compared with linear nucleotides.

STING pathway agonism as a cancer therapeutic

“…cGAS becomes fully active when dsDNA stabilizes cGAS residues Gly 207 ‐Asn 210 . A small molecule capable of stabilizing cGAS at these residues could activate cGAS in the absence of DNA and might have therapeutic potential …”

“…A small molecule capable of stabilizing cGAS at these residues could activate cGAS in the absence of DNA and might have therapeutic potential. 32 cGAS binding to DNA does not depend on the DNA sequence but does have a minimum length requirement for optimal activation. This ranges from 45 to 70 base pairs at 1 mg/mL to 800-2000 bp at lower, more physiological DNA concentrations.…”

A simple model for determining affinity from irreversible thermal shifts

“…(b) v plot for irreversible unfolding. Proteins include the published values for BACE1 (diamonds), cGAS (squares), the NBD1 domain of CFTR (triangles), or other proteins available at Pfizer (circles). Fit and trendline for all proteins is shown; K D span ca 10 to 0.05 μM.…”

“…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.…”