Discovery And Anti-inflammatory Activity Evaluation Of A Novel CDK8 Inhibitor Through Upregulation Of Il-10 For The Treatment Of Inflammatory Bowel Disease

Inflammatory bowel disease（IBD）is a chronic noncommunicable disease mediated by the immune system characterized by uncontrolled chronic inflammation in the gastrointestinal tract,damage to the epithelial barrier,and microbial dysbiosis.It is generally accepted that the progression of IBD is related to the production of inflammatory cytokines in the immune system.Genetic studies have found that deletion mutations in IL-10 or IL-10 receptor gene function can lead to spontaneous,severe enterocolitis.Therefore,increasing the body’s IL-10 level is a promising strategy for treating IBD.CDK8 is a regulator of IL-10 production,its inhibition increases the transcriptional activity of activator protein 1（AP-1）and thus upregulates IL-10 production.Therefore,we propose to develop CDK8 inhibitors to upregulate IL-10 levels as a development strategy for anti-IBD drug.In this study,we describe the complete discovery process of a safe and effective CDK8 inhibitor,including structure-based design,biological activity evaluation,the structure-activity relationship study of the compounds,target verification,mechanism study and in vivo safety and efficacy evaluation of target compounds,which provides experience and ideas for CDK8 inhibitors as anti-IBD drugs.Wogonin has the chemical structure of a classical kinase inhibitor and has recently been confirmed as a weak CDK8 inhibitor with an IC₅₀of 5.9μM.Based on the crystal structure of CDK8 protein and CCT-251545（PDB ID:5BNJ）,we simulated the binding pattern of CDK8 protein to Wogonin and identified it as a hit compound.In order to increase the CDK8 inhibitory activity of the compounds,improve the IL-10 production capacity and AP-1 transcriptional activity in BMDCs,we designed and synthesized a total of 95 compounds after a series of structural optimizations under the strategy of receptor-based drug design and scaffold jumping.Finally,lead compound 85 was found,which has a strong inhibitory effect on CDK8 activity with an IC₅₀value of 55.9 n M,increases the level of IL-10 in BMDCs by 88%and the transcriptional activity of AP-1by a fold of 3.1,and has good selectivity in the CDK family.To verify the targeting of compound 85,we performed target validation.We selected HCT-116,a colon cancer cell line with high expression of CDK8 protein,for a cell heat transfer analysis experiment to demonstrate the direct interaction between compound 85 and CDK8.The results showed that compound 85 could improve the thermal stability of CDK8 protein and reduce the degradation of CDK8 protein in a concentration-dependent manner.Not only that,but we also explored the effects of compound 85 on the biological function of the CDK8 protein.Dephosphorylation of STAT1 Ser727 and C-JUN Ser243 is a specific biological indicator of CDK8 inhibition.The study showed that compound 85 inhibited IFN-γand R848 induced phosphorylation of STAT1 Ser727 and C-JUN Ser243 in a concentration-dependent manner,respectively.The ELISA results showed that the target compound significantly increased the level of anti-inflammatory cytokine IL-10 and reduced the level of pro-inflammatory cytokine IL-6,and the activity was significantly better than that of the positive drug BRD6989.In addition,we investigated the in vitro anti-inflammatory mechanism of action of compound 85.It was found that compound 85 significantly inhibited the activation of TLR7 and NF-κB/MAPK signaling pathways mediated by TLR7.To further evaluate the druggable properties of compound 85,we tested the metabolic stability of compound 85 in human liver microsomes,Caco-2 cell permeability,CYP450 enzyme induction capacity,PK characteristics in rats and the safety in mice.The data showed that compound 85 had good stability in human liver microsomes.Compound 85 showed moderate permeability in Caco-2 cells and there is no obvious efflux phenomenon,and compound 85 had no obvious inhibitory effect on CYP450 enzyme.The pharmacokinetic study showed that compound 85 had good oral bioavailability in SD rats（F=29.3%）,which provided theoretical support for the determination of administration route and frequency in subsequent pharmacodynamic experiments.The results of acute toxicity study in vivo showed that after oral administration of 1000 mg/kg compound 85,the mice showed normal physiological status compared with mice in normal group,body weight showed a normal growth trend,and no obvious inflammatory damage in tissue was observed.In a mouse model of acute colitis induced by dextran sodium sulfate（DSS）,compound 85 exhibited a significant therapeutic effect in a dose-dependent manner.Compared with the model group（DSS group）,the colons of mice in compound 85 group were longer,and the disease activity index（DAI）score and MPO activity in colon tissues were also significantly lower than those in the model group.The results of HE staining showed that the inflammatory damage in the colon tissue of the mice in compound 85 group was obviously alleviated.Not only that,compound 85 also significantly weakened the inflammatory response in mice,leading to the changes in expression of inflammatory cytokines in colon tissues and serum.To further investigate the mechanism of compound 85 in vivo,we performed western blotting to analyze the protein expression in colon tissue.The results show that compound 85 exerts anti-inflammatory effects in vivo by inhibiting the activation of TLR7 signaling and TLR7 mediated NF-κB and MAPK signaling pathways,and also inhibits JAK1/STAT3 signaling pathway,which is a key signaling pathway in colitis.The above results show that compound 85 is a promising CDK8 inhibitor with anti-IBD activity.Overall,in this study,through analyzing the binding mode between Wogonin and CDK8 protein,we designed and synthesized 94 compounds,multi-angle screening methods were used to evaluate the biological activity of compounds.Among them,compound 85 was identified as a novel CDK8 inhibitor,which has good CDK8inhibitory activity,anti-inflammatory activity in vitro and anti-IBD activity in vivo,which has important implications for enriching development strategies to inhibit CDK8 for the treatment of IBD.