cGAS Inhibitors for ALS Therapeutics: Targeting Upstream mtDNA Recognition

The cGAS-STING pathway drives neuroinflammation in ALS through aberrant recognition of cytoplasmic mitochondrial DNA released following TDP-43 pathology. Rather than targeting the downstream effector STING, therapeutic intervention at the upstream sensor cGAS (MB21D1) offers a more proximal approach to pathway inhibition. cGAS contains a distinct N-terminal DNA-binding domain and C-terminal nucleotidyltransferase catalytic domain connected by a flexible linker region. Upon mtDNA binding, cGAS undergoes liquid-liquid phase separation, forming membraneless condensates that concentrate the enzyme and its DNA substrate to amplify cGAMP production. This phase separation is mediated by intrinsically disordered regions in the linker domain and requires specific electrostatic interactions between positively charged lysine residues and the phosphate backbone of mtDNA. Small molecule inhibitors targeting the cGAS active site, such as RU.521 and G140, demonstrate selective inhibition of cGAMP synthesis without affecting other nucleotidyltransferases. These compounds bind to the ATP/GTP binding pocket and prevent the conformational changes required for catalytic activity.

The cGAS-STING pathway drives neuroinflammation in ALS through aberrant recognition of cytoplasmic mitochondrial DNA released following TDP-43 pathology. Rather than targeting the downstream effector STING, therapeutic intervention at the upstream sensor cGAS (MB21D1) offers a more proximal approach to pathway inhibition. cGAS contains a distinct N-terminal DNA-binding domain and C-terminal nucleotidyltransferase catalytic domain connected by a flexible linker region. Upon mtDNA binding, cGAS undergoes liquid-liquid phase separation, forming membraneless condensates that concentrate the enzyme and its DNA substrate to amplify cGAMP production. This phase separation is mediated by intrinsically disordered regions in the linker domain and requires specific electrostatic interactions between positively charged lysine residues and the phosphate backbone of mtDNA. Small molecule inhibitors targeting the cGAS active site, such as RU.521 and G140, demonstrate selective inhibition of cGAMP synthesis without affecting other nucleotidyltransferases. These compounds bind to the ATP/GTP binding pocket and prevent the conformational changes required for catalytic activity. Additionally, cGAS activity is regulated by post-translational modifications including SUMOylation at lysine 335 and 372, which reduces DNA binding affinity and enzymatic activity. Drug repurposing screens have identified FDA-approved compounds like suramin and quinacrine that can inhibit cGAS through allosteric mechanisms. In ALS mouse models, cGAS knockout or pharmacological inhibition reduces microglial activation, decreases pro-inflammatory cytokine production, and preserves motor neuron survival. Targeting cGAS offers advantages over STING inhibition by preventing the initial inflammatory trigger while maintaining STING's potential beneficial roles in cellular homeostasis and antimicrobial defense, providing a more selective therapeutic approach for ALS-associated neuroinflammation.