Background/Purpose: The cGAS enzyme is a sensor of cytosolic double-strand (ds) DNA. It serves to detect viruses and elicits an acute, transient cGAMP-STING mediated type I interferon (IFN) response as part of anti-viral immunity. Recently, evidence has accumulated pointing to chronic stimulation of cGAS through endogenous (self) cytosolic dsDNA in the context of a wide variety diseases associated with a type I IFN signature. Clear examples of such auto-inflammatory diseases include Aicardi-Goutières Syndrome (AGS) and Systemic Lupus Erythematosus (SLE), which can harbour loss-of-function mutations in the gene encoding a 3′ repair exonuclease, TREX1, which functions to prevent accumulation of cytosolic dsDNA. Indeed, mice deficient for Trex1 exhibit severe chronic systemic inflammation, which can be completely reversed by concomitant deletion of cGAS or STING. Other diseases associated with strong activation of cGAS include, among others, dermatomyositis, age-related macular degeneration, and Parkinson’s disease (PD).

Methods: We report on the discovery and optimization of a novel series of small-molecule inhibitors of cGAS. The molecular binding mode is supported by crystallography, direct binding assays and multiple enzymatic assay formats. Compounds are assessed across different cellular contexts (including monocytes, microglia and retinal epithelial cells) using distinct sources of dsDNA as a stimulus. Lead compounds are prioritized based on DMPK (Drug Metabolism and Pharmacokinetics) properties, including distribution to the brain in mice. Mouse models have been used to prioritize compounds based on the ability to suppress acute HSV-mediated cGAS activation and chronic systemic autoimmunity (in Trex1-/- mice).

Results: Best compounds in the series have low nM potency in enzymatic assays using either mouse or human cGAS. No cross-reactivity with other enzymes that also use ATP or GTP as substrates were detected. Unlike many previously reported cGAS inhibitors, our compounds exhibit potent inhibition of cGAMP production and IFN response in cells, either stimulated with an exogenous source of dsDNA or mimicking a pathologic chronic stimulation of the pathway (e.g. using TREX1 deficient monocytes). Likewise, examples of the series display potent inhibition of microglia activation using alpha synuclein fibrils. The orally bioavailable lead compound, which displayed robust brain distribution, showed dose-dependent inhibition of autoimmunity across tissue types (PBMCs, kidney, lungs, heart and brain) in Trex1-/- mice and was significantly superior to the previously reported STING inhibitor, H151. In most cases, inflammation markers were restored to levels of WT mice within 2 weeks of dosing our cGAS inhibitor to Trex1-/- mice.

Conclusion: We discovered novel cGAS inhibitors and demonstrated their potential in preclinical models as therapeutics to treat peripheral auto-immune diseases such as AGS and SLE, and neurodegenerative diseases associated with a strong neuro-inflammation component such as PD.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/selective-and-potent-inhibition-of-cyclic-gmp-amp-synthase-cgas-fully-normalizes-autoinflammation-across-tissues-in-a-trex1-mouse-model-for-type-i-interferonopathies/