Compound C Reducing Interferon Expression by Inhibiting cGAMP Accumulation

Abstract

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a major DNA sensor responsible for cytosolic DNA-mediated innate immune response. Inhibition of cGAS may be an effective strategy for treating autoimmune diseases such as Aicardi-Goutieres syndrome and systemic lupus erythematosus. Compound C (also known as Dorsomorphin) has been annotated as a potent and reversible inhibitor for AMPKs as well as ALK protein kinases. Here, we report a new function of Compound C which can suppress dsDNA-dependent type I interferon induction. These effects were not dependent on the activities of AMPK proteins. In vitro assays and liquid chromatograph-mass spectrometry data show that Compound C has the capability of reducing cGAMP accumulation, suggesting that Compound C may function as a modulator involved in the cGAS-STING-mediated DNA sensing pathway. Furthermore, Compound C is able to rescue the autoimmune phenotypes in a mouse model carrying the Trex1 gene deficiency. These data demonstrate a new and inverse correlation between Compound C and type I interferon production in response to dsDNA signaling.

Keywords: DNA sensing; cGAMP; cGAS; compound C; dorsomorphin; type I interferon.

Figure 1

Compound C inhibited type I IFN production in dsDNA signaling. (A) L929 cells were treated with the different doses of Compound C for 24 h, and subjected to the CCK-8 assay. (B) Compound C inhibited the AMP-activated protein kinase (AMPK) activity as tested by Western blotting. (C) Enzyme-Linked Immunosorbent Assay (ELISA) analyses of IFNβ expression in L929 cells. The cells were transfected with HT-DNA after treated with the different doses of Compound C for 1 h. (D, E) ELISA analyses of IFNβ expression in L929 and BJ cells. The cells were transfected with HT-DNA and poly (I:C) after treated with indicate doses of Compound C. The experiments were performed at least three times. The statistical analyses were performed by Student’s-t test and the data are presented as mean ± SD (n = 3, ^* P < 0.05, **P < 0.01,***P < 0.001).

Figure 2

Compound C suppressed the expression of IFNβ by transfected dsDNA, but not cGAMP. (A, C, E) RT-qPCR analyses of IFNβ expression in L929 (A), BJ (C), THP1 (E) cells. The cells were transfected with HT-DNA after treated with indicated doses of Compound C. (B, D, F) RT-qPCR analyses of IFNβ expression in L929 (B), BJ (D), THP1 (F) cells. The cells were transfected with poly (I:C) after treated with indicated doses of Compound C for 1 h. (G) Western blot analyses of STING, P-STING, TBK1, P-TBK1, and GAPDH expression in BJ cells. The cell was transfected with HT-DNA, cGAMP and poly (I:C) after treated with indicate doses of Compound C. The experiments were performed at least three times. Data in bar graphs are presented as mean ± SD (n = 3−5) with asterisks indicating significant changes (NS Nonsignificance, *P < 0.05, **P < 0.01).

Figure 3

Compound C inhibited the dsDNA-dependent pathway which was not dependent on the activity of AMP-activated protein kinases (AMPKs). (A) Western blot analyses of phorsphorylated AMPK in L929 wild-type and L929-AMPK^-/- cells. The cells were transfected with HT-DNA and poly (I:C). (B, C) RT-qPCR analyses of IFNβ expression in L929 and L929-AMPK^-/- cells. The cells were transfected with HT-DNA (B) or poly (I:C) (C) after treated with indicated dose of Compound C. (D–F) RT-qPCR analyses of IFNβ and CXCL10 expression in L929 wild-type and L929-AMPK^-/- cells. The cells were transfected HT-DNA (D), cGAMP (E); Analyses of IFNβ expression in L929 wild-type and L929-AMPK^-/- cells. The cells were transfected with poly (I:C) (F). The experiments were performed at least three times. Data in bar graphs are presented as mean ± SD (n = 5) with asterisks indicating significant changes between the indicated bars (NS, Nonsignificance, ^* P < 0.05, **P < 0.01,***P < 0.001).

Figure 4

The target of Compound C was in the upstream of TBK1. (A, B) RT-qPCR analyses of IFNβ expression levels in THP1 wild-type (A) and THP1-STING^-/- (B) cells. The cells were transfected with HT-DNA and pcDNA 3.1-TBK1-Flag after treated with indicated dose of Compound C. (C) Western blot analyses of TBK1, p-TBK1, IRF3, and p-IRF3 expression in 293T cells. The 293T cells were treated with 10 μM Compound C followed by transfection with pcDNA3.1-TBK1-Flag and pcDNA3.1-IRF3-Flag. (D, E) RT-qPCR analyses of IFNβ (D) and ISG56 (E) expression in 293T cells. The 293T cells were treated with 10 μM Compound C followed by transfection with pcDNA3.1-TBK1-Flag for 12 h and 24 h, respectively. The experiments were performed at least three times. The statistical analyses were performed by Student’s-t test and the data are presented as mean ± SD (n = 3) with asterisks indicating significant changes (^* P < 0.05, **P < 0.01).

Figure 5

Compound C lowered the cGAMP level. (A) RT-qPCR analyses of IFNβ induction in THP1-ISG-liciferase cells. The cells were treated with the increasing amounts of cGAMP. (B) RT-qPCR analyses of IFNβ induction in THP1-ISG-liciferase cells. The cells were treated with the S100 fractions which were extracted from the BJ cells transfected with HT-DNA after treated with indicated doses of Compound C for 1h. (C, D) RT-qPCR analyses of IFNβ (C), CXCL10 (D) expression in THP1-ISG-liciferase cells treated with the S100 fractions which were extracted from the BJ cells transfected with HT-DNA after treated with indicated doses of Compound C for 1h. (E, F) LC-MS analyses of the content of cGAMP in the S100 fractions which were extracted from the BJ cells transfected with HT-DNA after treated with 10 μM Compound C. The experiments were performed at least three times. Data in bar graphs are presented as mean ± SD (n = 3−5) with asterisks indicating significant changes (NS, Nonsignnificant, ^* P < 0.05, **P < 0.01,***P < 0.001).

Figure 6

The inhibitory effect of Compound C on cGAS-STING signaling was not dependent on the cGAS enzymatic activity. (A) Western blot analyses of p-AMPK, AMPK, and cGAS expression in L929 wild-type and L929-AMPK^-/- cells. The cells were transfected with HT-DNA and poly (I:C) after treated with indicated doses of Compound C. (B) Enzyme-Linked Immunosorbent Assay (ELISA) analyses of IFNβ expression in THP1-Luci cells following stimulation by cGAMP at the different concentrations for 16 h with or without 5 μM Compound C treatments. (C, D) The cGAS enzyme activity was evaluated by the (ATP consumption. The experiments were performed at least three times. Data in bar graphs are presented as mean ± SD (n = 3−5) with asterisks indicating significant changes (**P < 0.01).

Figure 7

Compound C is active in cells loss of Trex1 gene. (A–D) RT-qPCR analyses of CXCL10 (A), ISG15 (B), ISG56(C), and IFIT3 (D) expression in mouse embryonic fibroblast (MEF) wild-type and MEF-Trex1^-/- cells. The cells were treated with indicated doses of Compound C for 3 h. The experiments were performed at least three times. Data in bar graphs are presented as mean ± SD (n = 3) with asterisks indicating significant changes (^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001).