Compound C Affects Interferon Immune Response By Regulating SLC10A3

Cyclic guanosine-adenylate synthase(cyclic GMP-AMP,c GAS)is a nucleotide transferase and known as a DNA recognition receptor.It can recognize and bind to double-stranded DNA(ds DNA)in the cytoplasm and use ATP and GTP to synthesize the second messengers 2’,3’-c GAMP,activating the membrane protein stimulator of interferon genes(STING)to activate the type I interferon(IFN-β)immune response.In recent years,an increasing number of studies have found that inhibition of interferon and interferonstimulating genes(ISGs)are effective in alleviating the phenotypes caused by autoimmune diseases,such as Aicardi-Goutières syndrome(AGS)and systemic lupus erythematosus(SLE)and present promising therapeutic potential.Therefore,it is important to find key target genes and related small molecule compounds that can effectively inhibit interferon expression.Our research group previously found that Compound C can inhibit the expression of IFN-βby regulating c GAS-STING pathway,but the mechanism remains to be further studied,especially the relevant gene targets remain unclear.In this dessertation,the structure of Compound C was optimized,five Compound C analogueswere synthesized,and their functional mechanisms further studied.We tested the effects of Compound C and its analogs on the c GAS-STING pathway and interferon expression by Western blotting and q RTPCR.The results showed that:(1)Compound C significantly lower the expression induced by Herring-Testis DNA(HT-DNA)but not Poly(I:C)stimulation;(2)Five Compound C analogs s,Compound 1,Compound 2,Compound 4,Compound 5 and Compound 6,were developed and synthesized based on the Compound C structure backbone,and their effectiveness was verified at the same time.Compound 4,Compound 5 and Compound 6 could effectively inhibit IFN-β expression induced by HT-DNA,while Compound 1and Compound 2 had no effect;(3)To investigate the potential of Compound C and its analogs in the treatment of autoimmune diseases,we demonstrated that Compound C and its analogs can inhibit the abnomal expression of type I interferon and interferon stimulator genes induced by TREX1 deletion in the cell models.Then we studied the relevant mechanism.The second-generation sequencing results of RNA-seq showed that Compound C affected the interferon immune response by regulating solute carrier family 10(sodium/bile acid cotransporter family)member 3-SLC10A3.SLC protein family belongs to a class of common solute carrier proteins,which are mainly involved in regulating the transport of intracellular or intercellular organic ions,short peptides and cholesterol derivatives and other signaling molecules.SLC19A1,one of the family members,has been reported to be involved in the transport of c GAMP in cells,which in turn affects the expression of IFN-β.Therefore,we examined the relationship between Compound C and its analogs and SLC10A3 gene.The results showed:(1)The m RNA and protein expression of SLC10A3 were significantly decreased in Compound C-treated cells,and the inhibition effect was concentration dependent;(2)Compound C,Compound 4,Compound 5,and Compound 6 could inhibit SLC10A3 and the expression of ds DNA-induced IFN-β through c GAS-STING signaling pathway.However,Compound 1 or Compound 2,had no effect;(3)SLC10A3 gene deletion can significantly inhibit HT-DNA-induced IFN-β expression,and this inhibition depends on the activation of c GAS-STING signaling pathway.At the same time,SLC10A3 gene deletion did not significantly inhibit ds RNA-induced RNA immune signaling.In conclusion,we suggest that there is a certain connection between SLC10A3 gene,Compound C as well as its analogs and c GAS-STING signaling pathway,and Compound C can affect interferon immune response by regulating SLC10A3 gene.Further understanding of the function of SLC10A3 gene and the regulation mechanism between Compound C and SLC10A3 gene will contribute to the better understanding of the biological role about c GASSTING signaling pathway and provide significant support for the development of new small molecule drugs based upon the above Compound C analogs for treating autoimmune diseases in the future.