Discovery And In Vivo Anti-inflammatory Activity Evaluation Of A Novel CDK8 Inhibitor For The Treatment Of IBD

Inflammatory bowel disease(（IBD）is a noninfectious disease mediated by the chronic immune system,characterized by a high morbidity and a high recurrence rate.The IBD includes two clinical manifestations:Crohn’s disease and ulcerative colitis,characterized by uncontrolled chronic inflammation in the gastrointestinal tract,epithelial barrier disruption,and a dysregulated microbial population.Interleukin-10（IL-10）is a multicellular-derived multifunctional cytokine that promotes immune homeostasis by regulating cell growth and differentiation through multiple mechanisms,including inhibition of proinflammatory cytokines produced by innate immune cells and promotion of regulatory T cell function.Genetic studies have found that loss-of-function mutations in the IL-10 or IL-10 receptor genes cause severe,frequent enterocolitis in children.Therefore,raising the level of the anti-inflammatory cytokine interleukin-10（IL-10）is a promising strategy to inhibit the progression of IBD.It has been shown that inhibition of cell cycle-dependent kinase 8（CDK8）selectively upregulates IL-10 production by activated myeloid cells,thereby avoiding the toxicity associated with elevated systemic IL-10 levels,implying that CDK8 It is an effective target for the treatment of IBD.In this study,55 compounds were designed and synthesized based on the active site of CDK8.Through kinase activity test and IL-10 abundance detection,compound R53was identified as the target compound,which showed a strong CDK8 inhibitory activity,with an IC₅₀value of 75.5 n M,and increased the IL-10 level in activated bone marrow-derived dendritic cells（BMDCs）by 71.6%.In vitro studies indicated that R53reduced the phosphorylation of STAT1 at Ser727 caused by interferon-γ（IFN-γ）and the phosphorylation of c-Jun at Ser243 mediated by R848 in BMDCs,indicating that R53affected CDK8 related biological functions.To further explore the mechanism of R53regulated the expression of IL-10,TLR7/NF-κB/MAPK pathway was investigated by western blotting.R848 activates TLR7 signaling in BMDCs and causes an increase in MYD88 expression,and activation of TLR7 simultaneously causes activation of downstream inflammatory signals.However,after R53 treatment,the phosphorylation of related inflammatory signals was inhibited,indicating that R53 can play an anti-inflammatory effect by regulating the TLR7/NF-κB/MAPKs signaling pathway.We used dextran sulfate sodium（DSS）-induced IBD mouse model to evaluate the in vivo anti-inflammatory effect of R53.The results showed that R53 could significantly relieve the symptoms of IBD,as the DAI decreased and the colon length increased compared with the model group.HE staining showed that the colonic tissue of the R53treatment group was relatively complete,the goblet cells were significantly increased,and the inflammatory phenomena were significantly relieved.Analysis of cytokines in serum and colon tissue of experimental mice showed that R53 could significantly increase the levels of anti-inflammatory cytokines IL-10 and decrease the levels of pro-inflammatory cytokines IL-6 and IL-17.Autophagy is a cellular stress response that plays a key role in the disease progression of IBD.R53 may modulate the autophagic activity during DSS-induced colitis through the AMPK-m TOR signaling pathway,thereby alleviating the inflammatory response.In conclusion,compound R53 can be used as a leading compound in the treatment of IBD,which is of great significance for the treatment of IBD and the discovery of CDK8 inhibitors.