Structural insight into the cGAS active site explains differences between therapeutically relevant species

Abstract

Cyclic GMP-AMP synthase (cGAS) is an intracellular sensor of double-stranded DNA that triggers a pro-inflammatory response upon binding. The interest in cGAS as a drug discovery target has increased substantially over the past decade due to growing evidence linking its activation to numerous peripheral and neurological diseases. Here, we report the binding mode of previously described cGAS inhibitors while also uncovering the structural basis for the interspecies potency shifts within this chemotype. A single threonine to isoleucine substitution between human and mouse cGAS drives compound activity, as demonstrated by biochemical, cellular, and in vivo studies. Finally, we utilize a structurally enabled design approach to engineer a novel chemical inhibitor with excellent potency for both human and mouse enzymes by targeting key interactions within the enzyme active site. Overall, this work provides the framework for rational optimization of cGAS inhibitors and potential preclinical translational strategies.

Fig. 1. G-chemotype cGAS inhibitors have an ATP-uncompetitive and GTP-competitive mechanism of action.

A Chemical structures of G150, G140, and PF-06928215. B, C G150 h-cGAS biochemical potency as determined by the inhibition of cGAMP production measured by LC-MS. GTP concentrations were varied while ATP concentrations were fixed at 50 μM (B) or ATP concentrations were varied while GTP concentrations were fixed at 50 μM (C) cGAS was activated by stimulation with 25 nM of 100 bp dsDNA. Graphs depict one of three independent experiments. Error bars represent standard deviations. D IC₅₀ values from (B) and (C) representing the mean ± SD of three independent experiments. E, F Thermal shift experiment of h-cGAS melting temperature in complex with 25 bp dsDNA and cGAS inhibitors (E) or in complex with dsDNA, ATP, and cGAS inhibitors (F). The graphs depict the mean ± SD of one of two independent experiments. G SPR relative response measurements depicting G140 binding to h-cGAS, h-cGAS + 45 bp dsDNA, h-cGAS + ATP, and h-cGAS + ATP + dsDNA. The K_D value represents the mean ± SD of two independent experiments.

Fig. 2. G150 binds within the GTP pocket of human cGAS in the presence of ATP.

A Cartoon representation of the h-cGAS catalytic domain (cyan) in the dsDNA bound active conformation in complex with AMP-PNP (blue) and G150 (green). 2F_O-F_C electron map of ligands contoured at 1.0 sigma. Ligands are represented as sticks and Mg metals are represented as spheres. B Key residues are identified while the interactions between AMP-PNP, Mg²⁺, G150, and h-cGAS are depicted by dotted lines. C Superimposed m-cGAS structure (PDB: 4K97, magenta) with G150 bound h-cGAS structure (cyan). Key residues involved in AMP-PNP and G150 binding are presented as sticks. D T321 is not conserved between human and mouse. Mouse I309 spatially clashes with the pyridine-ring of G150 bound to the GTP pocket.

Fig. 3. Identification of A-ring methyl compound 2 with high potency for both human and mouse cGAS.

A Reported h-cGAS and m-cGAS biochemical potencies for G150 and G015 show that removal of the D-ring improves mouse potency but erodes human activity. Previously published cGAMP LC-MS IC₅₀ values from Ref. are shown. B Design strategy to expand from the A-ring leading to the identification of compound 2 and mean IC₅₀ values from at least 2 independent experiments in indicated human and mouse assays. n.d. not determined. C Co-crystal structure of compound 2 (pink) bound to h-cGAS and AMP-PNP superimposed with the G150 (green) co-crystal structure.

Fig. 4. GTP pocket threonine to isoleucine substitution is responsible for the potency shifts of G-chemotype inhibitors in human and mouse cGAS.

A Structures and potencies of representative G-chemotype cGAS inhibitors in the cGAMP LC-MS biochemical assay. IC₅₀ values represent the mean of two independent experiments. The fold shift between WT and mutant enzymes is in green. B Representative dose response curves of G150 and compound 2 in WT and mutant enzyme cGAMP LC-MS biochemical assays. Error bars represent standard deviations. C–E cGAS inhibitor potency in (C) WT h-cGAS vs WT m-cGAS and (D) WT h-cGAS vs I309T m-cGAS. IC₅₀ values were determined using cGAS Kinase-glo biochemical assays and represent the mean of at least 2 independent experiments. Grey circles represent GTP pocket binding inhibitors while specific compounds or chemotypes are identified by color. Indicated G-chemotype (green), Hall et al., (purple), RU.521 (pink), WO 2021/233854 (blue), WO 2024/035622 (orange). E Structures and potencies of representative inhibitors from differentiated chemotypes in cGAS Kinase-Glo biochemical assay. The fold shift between WT h-cGAS vs WT m-cGAS or WT- h-cGAS vs I309T m-cGAS is depicted in green. IC₅₀ values represent the mean of at least 2 independent experiments.

Fig. 5. Threonine to isoleucine substitution correlates with cGAS inhibitor potency in multiple preclinical species.

A Sequence alignment of cGAS enzymatic pocket amino acids from indicated mammalian species. Threonine or isoleucine residues at human position 321 are highlighted. B Chemical structures and potencies of representative G-chemotype cGAS inhibitors in the cGAS Kinase-Glo biochemical assay of indicated mammalian species stimulated with 100 bp dsDNA. Values represent the mean of at least 3 independent experiments. C Representative dose response curves of indicated inhibitors in cGAS Kinase-Glo biochemical assay. Error bars represent standard deviations.

Fig. 6. G140 inhibits in vivo cGAS activity in cGAS^I309T, but not WT mice.

A, B Dose response curves of (A) G140 and (B) compound 2 inhibition of 100 bp dsDNA transfected WT or cGAS^I309T BMDM secreted CXCL10. The IC₅₀ value represents the mean ± SD of three independent experiments. C, D Dose response curves of (C) G140 and (D) compound 2 inhibition of 100 bp dsDNA transfected WT or cGAS^I309T mouse whole blood plasma CXCL10. The IC₅₀ value represents the mean ± SD of two independent experiments. E Blood concentration of G140 after 100 mg/kg PO dosing in WT and cGAS^I309T mice at indicated timepoints. Mouse whole blood IC₅₀ values from (C) are indicated. F Splenic cGAMP levels 16 h post ConA treatment of WT and cGAS^I309T mice pretreated with G140 dosed 100 mg/kg PO. The results from one of two independent experiments is depicted. G Terminal blood concentrations of G140 in ConA treated mice. Statistical analysis represents 1-way ANOVA with Dunnett’s multiple comparisons test. *** p < 0.0001.