Background/Purpose: Anti-malarial drugs (AMD) such as Hydroxychloroquine (HCQ) and Quinacrine (QC) are effective in the treatment of skin rash and arthritis in systemic lupus erythematosus (SLE). AMD have multiple modes of action, but precisely which mechanism(s) are responsible for their beneficial action is uncertain. Type I interferon, (IFN-I) is strongly implicated in the pathogenesis of SLE as well as rare monogenic ‘interferonopathies’ such as Aicardi-Goutieres Syndrome (AGS). Recently, a new DNA activated IFN-I pathway, cyclic GMP-AMP (cGAMP) synthase (cGAS), was discovered and linked to AGS. To identify potential inhibitors of the DNA-cGAS interaction, we performed in silico screening of chemical and drug libraries.

Methods: In silico structure-based drug screening were provided by the CANDO docking algorithm. Predictions made by CANDO were confirmed by Autodock Vina and analyzed via PyMOL. cGAS activity/cGAMP production was analyzed by Thin Layer Chromatography (TLC). DNA-binding to cGAS in the presence or absence of AMD was determined by Electrophoretic Mobility Shift Assay (EMSA). Following DNA cell transfections, cytokines were quantified by qPCR, ELISA or an ISRE-luciferase reporter assay.

Results: In silico screening of chemical and drug libraries identified several antimalarial drugs (AMD) including HCQ, QC, Chloroquine (CQ), Primaquine (PQ) and 9-amino-6-chloro-2-methoxyacridine (ACMA), which could potentially inhibit cGAS activity by interacting with the cGAS/DNA dimer complex. These AMD inhibited cGAS activity/cGAMP production in a dose dependent manner. Interestingly, the in silico predicted binding affinities of these AMD correlated well with their potency (QC>ACMA>HCQ>CQ>PQ) to inhibit cGAS activity, validating the prediction of our computational analysis. EMSA revealed that AMD disrupted the double stranded DNA-cGAS complex in a dose dependent manner, indicating that AMD blocked dsDNA/cGAS binding. These AMD also inhibited IFN-I expression in THP1 cells transfected with dsDNA and in 293T cells transfected with cGAS/STING plasmids validating that cGAS is a target of AMD. Based on these results and in silico modeling, we synthesized several new AMD. One of these compounds, X6, had excellent water solubility and cell penetration. Fluorescence microscopy revealed that X6 localized to the cytosol and had a lower toxicity profile compared to QC. In vitro and cell based studies, revealed that X6 was a more potent inhibitor of IFN-I production following dsDNA transfection into reporter cells than HCQ. X6 was also more potent than HCQ in the inhibition of IFN-a production following CpGA stimulation of PBMC.

Conclusion: Our studies identify the cytosolic DNA sensor, cGAS, as a target of AMD activity, which provide a novel mechanism of action of these AMD. This observation together with decades of experience of AMD in human diseases, suggest that this widely used family of drugs with a strong safety profile could be repurposed to target interferonopathies and possibly other autoimmune disorders related to cGAS over-activity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-mechanism-of-action-of-anti-malarial-drugs-in-the-inhibition-of-type-i-interferon-production/