Background/Purpose: Type I interferon (IFN-I) is strongly implicated in the pathogenesis of Systemic Lupus Erythematosus (SLE) as well as rare monogenic ‘interferonopathies’ such as Aicardi-Goutieres Syndrome (AGS) caused by mutations in the DNA exonuclease, TREX1. A recently described DNA-activated IFN-I pathway, cyclic GMP-AMP (cGAMP) synthase (cGAS), was linked to subsets of AGS and Lupus. Because of these clinical associations, we performed in silico screening for cGAS inhibitors and synthesized a novel drug we named X6, that belongs to the antimalarial drug (AMD) class of aminoacridines. Our previous data showed that, X6, blocked cGAS in vitro and, in a proof of principle in vivo experiment, it significantly reduced the interferon stimulated genes (ISGs) and cGAMP production in the Trex1-/- mouse model of AGS. Here, we compared X6 to a standard care AMD for SLE, Hydroxychloroquine (HCQ), for the treatment of murine AGS and also examined the effects on ISG expression in Peripheral Blood Mononuclear Cell (PBMC) from SLE patients.

Methods: Trex1-/- mice were treated orally with either X6 or HCQ 25mg/kg/day (n=10 in each group) for 8 weeks from birth. ISGs expressions from mouse heart and spleen tissues were quantified by qPCR. Multiple Reaction Monitoring (MRM) by Ultra-Performance Liquid Chromatogram coupled with tandem Mass Spectrometer (UPLC-MS/MS) was used to quantify cGAMP. SLE PBMCs (n=7) were incubated with X6 or HCQ for 20 hours at different dose concentrations. ISG expression was quantified by qPCR.

Results: Trex1 deficiency in mice leads to an autoimmune myocarditis and lupus-like systemic autoimmunity with increased ISG expression and cGAMP production. When efficacy of X6 was compared with HCQ, X6 was significantly more effective than HCQ in attenuating cGAMP (p<0.05) production in the heart and ISGs expression in both the spleen (ISG15: p<0.01 and ISG20: p<0.01) and the heart (ISG15: p<0.05, Mx1: p<0.05, CXCL10: p<0.01, IFNb: p<0.05). Surprisingly, HCQ in contrast to X6, increased cGAMP production and certain ISGs were also increased compared to vehicle control treated mice. To explore the potential application of drug X6 in complex autoimmune diseases such as SLE and to examine whether X6 is superior to HCQ at reducing IFN signature genes in SLE PBMC, we incubated SLE PBMC with X6 or HCQ for 20 hours and quantified ISG expression by qPCR. We observed that X6 was superior to HCQ in reducing ISG expression (IFI27: p<0.05, IFI44L: p<0.01, PKR: p<0.01, MX1: p<0.05) in SLE PBMC. Interestingly HCQ increased rather than reduced ISG expression compared to untreated controls.

Conclusion: Our studies demonstrate that X6 is superior to HCQ for the treatment of a cGAS-STING (Stimulator of Interferon Genes) mediated autoimmune myocarditis in vivo. Furthermore, X6 was superior to HCQ in the suppression of ISGs expression (Interferon activity) in SLE PBMC. These results indicate that the drug X6 could potentially be beneficial for the treatment of AGS and /or Lupus.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/novel-anti-malarial-drug-derivative-inhibited-type-i-interferon-production-and-autoimmune-inflammation-in-sle-patient-pbmc-and-in-trex1-mouse-spleen-and-heart/