The Preliminary Investigation In The Regulation Of The CGAS-STING Pathway By Potential Active Compounds

The cGAS-STING pathway not only can mediate immune defense against various external pathogens,but also detect DNA from tumor to generate internal anti-tumor immunity.However,abnormal activation of the cGAS pathway can cause autoimmune diseases.Thus,it would be invaluable to regulate the cGAS-STING pathway in the clinical therapy.To screen the modulators for this pathway,we chose two different series of compounds from different sources in the first attempt: One is de novo syntheses of peptide nucleic acids,which are supposed as cGAMP analogues.Their potential activity as cGAMP antagonists was evaluated by the level of the downstream factor IFNb with the stimulation after the establishment of the screening system.The other one is the extracts from Quasipaa spinosa powder(containing a large number of active short peptides with broad-spectrum antibacterial,antiviral and anti-tumor activities)and their activity also were estimated by the similar protocol after the purification to figure out the potential modulator for the cGAS-STING pathway.As the important second messenger in the cGAS-STING pathway,cGAMP can also be used to regulate the immune response by disturbing the cascade physiological arousal in the living system.Thus,the syntheses of cGAMP and its antagonists are worth to investigate.In this chapter,we designed and synthesized a kind of cGAMP analogues of peptide nucleic acids(c PNA)after the extensive investigation of the related references,which was considered as the analogue of cGAMP.Because c PNA itself has no charge,it can resist the degradation of nuclease and protease and provide good biological stability.Hence it was speculated that it might provoke similar biological activity as cGAMP and work as an antagonist by the competitive integration of the downstream protein receptors.To address this hypothesis,six analogues of cGAMP were synthesized and preliminarily investigated to estimate their possibility in the THP1-lucia-ISG cell line.Among them,c PNA-U might inhibit this DNA pathway,while c PNA-A might promote the RNA pathway after the immunological trials.The further experiments about m RNA levels will be investigated.Nomally,frogs are considered as an important reservoir of bioactive peptides,including antimicrobial peptides from skin secretion.Antimicrobial peptides may provide some anti-tumor activity,such as chemotaxis on immune cells,enhancing the uptake and presentation of tumor antigens in the antigen-presenting cells,inducing the secretion of IL12,IFNγ and other immune factors,then provoke the immune response of the body.Accordingly,we chose Quasipaa spinosa powder as the research object to optimize the extraction process of the bioactive extracts and explored the effect on innate immune pathways.We applied the previously established THP1-lucia-ISG cell screening system to evaluate the activity of Quasipaa spinosa powder extract solution and its concentrate with ultrasound-microwave synergistic mtheod.Positive results showed that the extract solution can enhance the expression of IFN beta induced by ds DNA,then irritate the cellular immune.Compared with the concentrated product,treatment with the original extract solution showed the better effect in the expression of IFN beta with better correlation with drug concentrations.In the optimization of extraction,we found that the pure water extract solution provided the best progressive effect in the expression of IFN beta.In the subsequent study about the optimal concentration of extract solution,the acivity of the extract solutions(WF-1)with different extraction time were evaluated and found that solution with extraction time for 45 min(WF-1-45 min),working concentration of 4 A/m L,showed the best effect in the expression of IFN beta induced by ds DNA,up to 2.4 times of the control group.Finally,after three years of postgraduate study,the following achievements have been acquired:(1)Six peptide nucleic acid monomers and six peptide nucleic acid cGAMP analogues were successfully prepared and evaluated their immunological activity in the THP1-Lucia-ISG screening system.Among them,c PNA-U might inhibit the DNA pathway,while c PNA-A might promote the RNA pathway after the immunological trials.(2)An efficient extraction of active ingredients from Quasipaa spinosa powder was established after optimization,then their immunologic activity were estimated in the same way as aboved.The identified extract solution which using pure water as extraction solvent and extraction time is 45 min(WF-1-45 min),showed the best effect in the expression of IFN beta induced by ds DNA,up to 2.4 times of the control group.