Study On Antitumor Prodrugs Based On TDO Small Molecule Inhibitors

The new strategy of tumor treatment by combining chemotherapy with immunotherapy has gradually been favored by researchers and become one of the hotspots of current research,which not only overcome the drawback of monotherapy,but also effectively improve the therapeutic effect of tumor.Tryptophan2,3-dioxygenase(TDO)belong to immune checkpoint proteins,which can involve in the immunosuppressive microenvironment generation by catalyzing tryptophan metabolism to produce kynurenine as overexpression in tumor cells,finally give rise up to T cells anergy and/or apoptosis that support tumor cells survival and immune escape.Thus,inhibition of TDO expression can improve antitumor immunity by blocking the kynurenine pathway to provide more tryptophan to T cells proliferation,and at last enhance the therapeutic effect of tumors.Based on the above studies,in this paper,several series of antitumor compounds via introducing TDO inhibitors into chemotherapeutic agents with immunomodulatory function were synthesized and evaluated for their biological activities.Platinum-(II)-based drugs are the most common chemotherapy drugs in the treatment of cancer,but their clinical application is greatly limited due to the defects of toxic side effects and drug resistance.However,platinum(IV)complexes exhibit kinetic inertness and unique mechanisms compared with those of platinum(II)complexes,thus they have been widely concerned and studied.In order to overcome these drawbacks and enhance antitumor effect,two series of immunostimulatory platinum(IV)conjugates by introducing different TDO inhibitors into Pt(IV)complexes were reported.The results of cellular experimental revealed that all the resulting conjugates exhibited comparable cytotoxicity compared to that of platinum(II)-based drugs.Among them,mono-modifed Pt(IV)conjugates3-7 and 4-2 showed excellent antitumor activity and low cytotoxicity toward the normal cell lines.The mechanism study revealed that the two conjugates can improve the cellular Pt uptake in comparison with cisplatin which also can be reduced to release the TDO inhibitor in the presence of ascorbic acid(VC).Further biological assays demonstrated that they arrested Hep G2 cancer cells at the S phase and induced cell death by the mitochondrial mediated pathway,simultaneously inhibited TDO protein expression and decrease kynurenine production by blocking the kynurenine pathway,resulted in T cells activation and proliferation,finaly improved the therapeutic effect of the conjugates.In addition,in vivo tests demonstrated that 4-2could effectively inhibit tumor growth in the dose-dependent manner in Hep G2 xenograft mice without causing body weight loss,and concurrently decrease the production of kynurenine in tumor,which further indicated that 4-2 could inhibit TDO expression and enhance antitumor immune response.All of those demonstrated that combining TDO inhibitors with Pt(IV)complexes can surmount the limitations of monotherapy,and kill cancer cells directly by Platinum(II)-based drugs and simultaneously stimulate antitumor immunity by inhibiting TDO expression to enhance therapeutic efficacy.Overexpression of glutathione sulfhydryl transferase(GSTs)in tumor cells is associated with drug resistance and side effects of several drugs.Here,in view of the expression characteristics of GSTs and TDO in the liver,we demonstrated the utility of NBDHEX(GSTs inhibitor)and TDO inhibitor by the combinatorial linker design.Two novel conjugates with different linkers displayed signifcant antitumor activity against Hep G2 cancer cells.They could inhibit the expression of GSTs,increase the G1 population and induce apoptosis in cancer cells via a mitochondrial-dependent apoptosis pathway.Interestingly,5-4 displayed more effectively cytotoxicity than 5-5,with the inhibition of TDO protein expression and the decrease of Kyn production,finally improved antitumor immune response.Thus,this strategy provides new ideas for drug development of liver cancer treatment.Microtubules are filamentous cytoskeleton protein polymers consisting of ?-and?-tubulin dimers,which play an important role in various biological functions of cells and organism life activities.Thus,microtubule has become a well-known target in cancer therapy.A series of novel conjugates comprising both of tublin and TDO inhibitors were designed,synthesized and evaluated for their antiproliferative activity.Among them,6-31,composed of combretastatin A-4(CA-4)and TDO inhibitor 3-6,exhibited the most potent antitumor activity,which could hydrolyze to release a TDO inhibitor and CA-4.Mechanism studies indicated that 6-31 could inhibit tubulin polymerization and cell migration,caused G2/M phase arrest,concurrently induce apoptosis via the mitochondrial dependent apoptotic pathway and cause reactive oxidative stress generation in Hep G2 cells.Furthermore,6-31 could inhibit TDO expression and decrease kynurenine production,leading to stimulating T cells activation and proliferation to enhance antitumor immunity in vitro.In addition,6-31 could remarkably promote the antitumor activity of CA-4 in either immunocompetent H22(ICR)or immunodeficient A549(BALB/c)tumor xenograft models.More importantly,6-31 increased the level of splenic and tumor-infiltrated T cells and in turn effectively boosted the inhibition effect in H22 xenografted tumor growth.In conclusion,this combination strategy of TDO inhibitors with chemotherapy drugs for the synergistic treatment of tumors,which can improve antitumor immunomodulatory activity and concurrently improve the anticancer therapeutic effect.Therefore,this strategy is a promising way for developing new antitumor immune candidates.