The Anti-tumor Effect And Mechanisms Study Of Toosendanin And New Berberine Derivates Through Downregulation Of IDO1 Expression

Indoleamine 2,3-dioxygenase 1(IDO 1)is an enzyme that catalyze the first-and rate-limiting step associated with the catabolic conversion of tryptophan(Trp)into kynurenine(Kyn).Coincidently,the depletion of Trp and accumulation of Kyn has been demonstrated to induce effector T-cell and NK-cell apoptosis/dysfunction and immunosuppressive regulatory T-cell induction,respectively.Similar to other immune checkpoints,IDO1 is suggested to be an important target for immunotherapeutic intervention.Meanwhile,as promising immunotherapy candidates,Indoleamine 2,3-dioxygenase 1(IDO1)inhibitors including Epacadostat and Indoximod have already entered clinic trial.Especially,combination of small-molecule IDO inhibitors with PD-1 antibodies could significantly improve the objective response rate(ORR)in specific tumor treatment.Therefore,it is a promising therapeutic strategy to develop small-molecule IDO1 inhibitors or modulators and provide new components for cancer immunotherapy combination.In the first part of the experiment,fragments of the human IDO1(indoleamine 2,3-dioxygenase 1)gene 5'-UTR(untranslated 1245 bp region)promoters were amplified by PCR and cloned into pGL4.20 vector and named as pGL4-IDO1-luc.A549 cells were transfected with the constructed plasmid and IDO1 inhibitor screening model was established with dual-luciferase reporter assay.Based on the model,we screened of natural small moleculeswhich could down-regulate the expression of IDO1 on tumor cells.The anti-tumor role and regulation of IDO1 expression of the positive candidate were examined by MTT,Western blotting and LDH release assays.Our results demonstrated that NS-180(toosendanin,TSN)down-regulated the IDO1 expressionin many kinds of cancer cells and it did not inhibit the activity of IDO1 directly.Toosendanin can inhibit STAT1 phosphorylation and nuclear translocation induced by IFN-? and it can inhibit IDO1 mRNA level in A549 cells.Moreover,toosendanin significantly increased the cytotoxity of co-cultured NK cells onA549 cells as demonstrated by LDH release assays.In summary,toosendanin is a novel and potent IDO1 inhibitor,which has an antitumor activity and is expected to be acancer immunotherapies candidate compound.In the second part of the experiment,to discover small-molecule cancer immunotherapy candidates through targeting Indoleamine 2,3-dioxygenase 1(IDO1),twenty-five new berberine(BBR)derivatives defined with substituents on position 3 or 9 were synthesized and examined for repression of IFN-y-induced IDO1 promoter activities.Structure-activity relationship(SAR)indicated that large volume groups at the 9-position might be beneficial for potency.Among them,compounds 2f,2i,2n,2o and 8b exhibited increased activities,with inhibition rate of 71-90%compared with BBR.Their effects on IDO1 expression were further confirmed by protein level as well.Furthermore,compounds 2i and 2n exhibited anticancer activity by enhancing the specific lysis of NK cells to A549 through IDO1,but not cytotoxicity.Preliminary mechanism revealed that both of them inhibited IFN-y-induced IDO1 expression through activating AMPK and subsequent inhibition of STAT1 phosphorylation.Therefore,compounds 2i and 2n have been selected as IDO1 modulators for small-molecule cancer immunotherapy for next investigation.