Virtual Screening And In Vitro Bioactivity Evaluation Of NLRP3 Inflammasome Natural Product Inhibitors Based On Molecular Docking

Innate immunity is the first barrier system of the body against external stimuli,which mainly activates downstream responses through Pattern recognition receptors(PRRs).NLRP3 inflammasome(Nod-like receptor pyrin domain containing 3 inflammasome)is the most widely studied type of inflammasome in PRRs,and it is closely related to the occurrence and development of many chronic diseases.Currently,some developed inhibitors targeting NLRP3 inflammasome have some problems,such as weak specificity,unclear target and toxic side effect.Therefore,the development of novel NLRP3 inflammasome inhibitors have very important clinical significance.In this study,the library of natural products and their derivatives was virtually screened by molecular docking,and combined with cluster analysis,ADMET properties(absorption,distribution,metabolism,excretion,and toxicity)prediction and drug-like analysis to screen out compounds that target the NLRP3 inflammasome.Finally,the biological activities of the five compounds were evaluated by vitro anti-inflammatory activity assay and antitumor cell assay.The main results are as follows:1.Virtual screening of NLRP3 inflammasome inhibitorsFirstly,using the Discovery Studio Libdock to screen the L6020 natural product library(containing 19,377 small molecules)and L6030 natural product derivative library(containing 139,752 small molecules)by molecular docking.A total of 46883 compounds with Libdock Score more than 125 were selected.Further,Ledock and Autodock Vina 1.5.7were used for secondary docking,then selecting the cross-hit molecules of the two docking software.Based on cluster analysis,ADMET prediction and Lipinski rule,9 compounds were further screened,and the interaction between compounds and proteins was observed by CDOCKER.The results showed that Ile411,Arg578,Ala227,Tyr443,Ala228,Phe575 were the key amino acid residues shared by the 9 compounds interacting with NLRP3 protein.2.The anti-inflammatory activity in vitro screening compoundsThe anti-inflammatory activity of five compounds,#1 T7926,#2 STOCK1N-87602,#3 STOCK1N-59431,#4 STOCK1N-57473 and #5 STOCK1N-97373,was verified in vitro.Mouse Bone marrow-derived macrophages(BMDMs)were used to induce inflammation model,and the anti-inflammatory activities of the 5 compounds were verified in vitro to detect the expression levels of inflammatory factors,Interleukin-1β(IL-1β)and Tumor necrosis factor-α(TNF-α).The results showed that compounds #2,#3,#4,#5 significantly inhibited the downstream inflammatory factor IL-1β of NLRP3 at 20 μmol/L,but did not affect the expression of TNF-α of the Nuclear factor-κB(NF-κB)signaling pathway.It suggested that compounds #2,#3,#4,#5 could inhibit the expression of NLRP3 inflammasome.3.Validation of antitumor activity of compounds in vitroThe anti-tumor activity of the 5 compounds screened was studied in vitro.The results showed that all the 5 compounds had certain inhibitory effects on the activity of tumor cells,among which compound #4 had significant inhibitory effects on Hep G2 and MCF-7 cells,and their IC50 were 64.59 μmol/L and 142.9 μmol/L.Compound #5 had significant inhibitory effects on A549 cells,and its IC50 was 195.8 μmol/L.In summary,this study performed virtual screening of NLRP3 inflammasome based on molecular docking,and verified the activity of compounds in vitro,which provided a new idea for the development of NLRP3 inflammasome inhibitors.