Fexofenadine Inhibits TNF Signaling Through Directly Targeting To Cytosolic Phospholipase A2 And Is Therapeutic Against Rheumatoid Arthritis

Objective:Inflammatory diseases are a series of disorders and conditions characterized by chronic inflammation,which affect almost any parts of the body.It is known that TNF-a plays a central role in inflammatory diseases.Correspondingly,anti-TNF-a activity is considered the core treatment for inflammatory disease.Unfortunately,these treatments are only effective in a limited percentage of patients and bring side effects,such as potential carcinogenicity.Identification of new applications for previously approved drugs may be an expedient strategy for the development of additional anti-inflammatory therapeutics.The purpose of our study is to screen a FDA approved drug library in order to find the drugs that inhibit the TNF-a induced inflammation.Method:THP-1 and RAW264.7 cell lines were used to conduct drug library screening byβ-lactamase and luciferase report genes assay.In-vivo screening was performed with TNF-tg:NF-KB luc double mutant mice and terfenadine(TFD)and fexofenadine(FFD)were finally selected.ELISA,immunoflurence and EMSA assay were performed to detect the downstream inflammatory factor,NF-κB translocation and binding activity.TNF-tg mice and CIA model were used to test the anti-inflammation effect in vivo.Drug Affinity Responsive Target Stability(DARTS)assay and cellular thermal shift assay(CETSA)were used to find the binding target of Fexofenadine.ELISA and Western blot were used to detect the activity and phosphorylation of cPLA2.Docking assay was used to predict the binding site and domain of cPLA2 and the result was confirmed by site-mutation assay and sub-clone assay.Finally the cPLA2 dependence of the treatment was verified by RNAi and crispr-cas9 assay.Results:In vitro and in vivo drug screens of a library composed of FDA approved drugs led to the identification of TFD and its active metabolite FFD as the inhibitors of TNF signaling.Herein we present evidences demonstrating that both FFD and TFD effectively inhibited TNF/NF-κB signaling in vitro and in vivo.Additionally,these two drugs significantly ameliorated disease symptoms in various autoimmune diseases models,including TNF transgenic mice,collagen-induced arthritis,and inflammatory bowel diseases.Combined use of multiple approaches,including drug affinity responsive target stability assay,proteomics,cellular thermal shift assay,information field dynamics and molecular dynamics,led to the isolation and validation of cytosolic phospholipase A2(cPLA2)as a novel target of FFD and TFD.FFD blocked TNF-stimulated cPLA2 activity and the production of arachidonic acid(AA)through direct binding to the catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505.Further,deletion of cPLA2 abolished FFD inhibition of TNF induced AA production and downstream cytokine release.Conclusion:Collectively,these findings not only demonstrate that FFD is an antagonist of TNF-α,an inhibitor of cPLA2,but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions,particularly autoimmune diseases.