Human cGAS inhibitor

Specific inhibitor of human cGAS

Inhibition of human cGAS signaling by G140

G140 is a small-molecule inhibitor of human cGAS (cyclic GMP-AMP synthase; cGAMP synthase). cGAS is the primary sensor of cytosolic double-stranded DNA (dsDNA) originating either from pathogenic infections, mitochondrial dsDNA misprocessing, or micronuclei rupture.  Direct DNA binding to cGAS catalyzes the production of 2', 3'-cGAMP, a cyclic dinucleotide, which in turn activates the STING pathway, resulting in IRF (interferon regulatory factor) and NF‑κB-mediated expression of pro-inflammatory cytokine and type-I interferons [2-4].
G140 is a promising probe for the development of drugs targeting human cGAS for preventing auto-inflammation and treating interferonopathies [5]. G140 was co-developed along with another chemotype, G150, and both molecules display similar potency at inhibiting cGAS [1].  
 More details

Mode of action:

Co-crystallization studies with another lead compound, G150, revealed that the small molecule targets the human cGAS catalytic pocket, competing with cGAS substrates ATP/GTP, thereby inhibiting the synthesis of  2', 3'-cGAMP [1, 4].

Key features:

• Potent and selective inhibitor of human cGAS
• No substantial cellular toxicity [1]
• Inhibition of IRF and NF-κB signaling validated in cellular assays
• InvitroFit™ grade: each lot is highly pure (≥95%) and functionally tested

Note: InvivoGen also offers RU.521, a specific inhibitor of murine cGAS.

References:

1. Lama, L. et al., 2019. Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression. Nat Commun 10, 2261.
2. Gao, D. et al., 2015. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases. PNAS 112, E5699-5705.
3. Abe, T. et al., 2014. Cytosolic-DNA-mediated, STINGdependent proinflammatory gene induction necessitates canonical NF-kappaB activation through TBK1. J Virol 88, 5328-5341.
4. Hertzog, J. & Rehwinkel J., 2020. Regulation and inhibition of the DNA sensor cGAS. EMBO Reports 21(12):e51345.
5. Tan J. et al., 2021. Synthesis and pharmacological evaluation of Tetrahydro-γ-carboline derivatives as potent anti-inflammatory agents targeting cyclic GMP-AMP synthase. J. Med Chem. 64(11):7667-7690.

Figures

G140 inhibits human cGAS in a dose-dependent response. THP1-Dual™ cells were incubated with increasing concentrations (0 - 25 μM) of G140 for 3 hours. Following this, G3-YSD (1 μg/ml), a specific cGAS agonist, was complexed with LyoVec™ and transfected into the cells. After overnight incubation, activation of cGAS was assessed by measuring IRF-dependent Lucia activity using QUANTI‑Luc™, a detection reagent. Data are shown as a percentage (%) of cGAS activity ± std error.

G140 inhibits human cGAS in a dose-dependent response. THP1-Dual™ cells were incubated with increasing concentrations (0 - 25μM) of G140 for 3 hours. Following this, G3-YSD (1 μg/ml), a specific cGAS agonist, was complexed with LyoVec™ and transfected into the cells. After overnight incubation, activation of cGAS was assessed by measuring NF‑κB -dependent SEAP activity using QUANTI-Blue™ Solution, a detection reagent. Data are shown as a percentage (%) of cGAS activity ± std error.

Specific inhibition of human cGAS by G140. THP1-Dual™ cells were incubated in the presence or absence of 6 μM G140 for 3 hours. Following this, G3-YSD (1 μg/ml), a specific cGAS agonist, or 3p-hpRNA (1 μg/ml), a specific RLR agonist were complexed with LyoVec™ and transfected into the cells. After overnight incubation, the IRF response was assessed using QUANTI‑Luc™, a detection reagent. Data are shown as a percentage (%) of IRF-induced Lucia luciferase activity.

Specific inhibition of human cGAS by G140. THP1‑Dual™ cells (WT) and THP-Dual™ KO-cGAS (KO-cGAS) cells were incubated in the presence or absence of 6 μM G140 for 3 hours. Following this, 2’3’-cGAMP (10 μg/ml), a STING agonist was added to the cells. After overnight incubation, the IRF response was assessed using QUANTI‑Luc™. Data are shown as a percentage (%) of STING-induced Lucia luciferase activity.

Specifications

Formula: C₁₇H₁₆Cl₂N₄O₂

Molecular weight: 379.24 g/mol

Solubility: 5 mg/ml (13.18 mM) in DMSO

Working concentration: 1 - 20 µM for cell culture assays.

Quality control:

• Purity ≥ 95% (UHPLC).
• Inhibition of cGAS has been confirmed using cellular assays
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK‑Blue™ TLR4 cells.

Contents

• 2 mg G140 (provided as a dried powder)

G140 is shipped at room temperature.

Store at -20°C.

Resuspended product is stable for at least 6 months when properly stored.

Avoid repeated freeze-thaw cycles.

Details

G140 and G150 are two small molecules that have been chemically optimized from the same parent compound to potently and specifically inhibit human cGAS [1].  G140 and G150 feature a methyl-pyrazole and a 2-aminopyridine moiety, respectively.

• They both show no substantial cellular toxicity and similar cellular IC₅₀ when measuring the expression of ISGs (interferon-stimulated genes) in human THP-1 monocytes or primary human macrophages [1].
• A 50 to 100 fold concentration difference between LD₅₀ and IC₅₀ provides a significant window between efficacy and toxicity for drug testing [1].
• G150 with human cGAS co-crystallization reveals that the inhibitor (and at least one related chemotype) binds to the enzyme catalytic pocket [1].
• G140 efficiently inhibits IRF signaling induced by dsDNA of different lengths in a dose-dependent manner [1].
• G140 has been used as a reference compound in developing highly potent small-molecule inhibitors of cGAS to treat inflammatory diseases [2]. 

1. Lama, L. et al. 2019. Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression. Nat Commun 10, 2261.
2. Tan J. et al., 2021. Synthesis and pharmacological evaluation of Tetrahydro-γ-carboline derivatives as potent anti-inflammatory agents targeting cyclic GMP-AMP synthase. J. Med Chem. 64(11):7667-7690.