Design,Synthesis And In Vitro Evaluation Of Terminal Functionalized Thiourea-containing Dipeptides Targeting Proteasome

The ubiquitin-proteasome pathway and autophagy-lysosome pathway are two of the most important mechanisms of protein degradation in all eukaryotes.Ubiquitin-proteasome pathway is specifically involved in many intracellular physiologicaland biochemical processes.It is often act as an important mechanism of neurological disease,inflammation development and diabetes,and also closely related to malignancy development.Proteasome which associated with the ubiquitin-proteasome system is the cellular target for chemotherapy.Proteasome inhibitors are a new class of effective anticancer drugs,by inhibiting the proteasome activity,disturbing original function of tumor cells to causing growth inhibition and apoptosis of cells.An increasing number of tumor-associated proteasome inhibitors have been found to development,and the mechanism of pharmacological activities have been continuously explored in recent years.However,the clinical application is limited by its side effects and drug resistance,so looking for efficiency and low toxicity,overcoming the resistance of the new proteasome inhibitors in recent years become the primary objective.Proteasome inhibitors Bortezomib was chosen as the lead compound,twenty nine novel dipeptide derivatives containing thiourea moiety were designed and synthesized with high target-selectivity and an optimal inhibition profile in our study.All compounds were characterized by means of 1H NMR,13C NMR and HR-MS.The anti-proliferative activities were determined by the standard method(MTT assay)against six human tumor cell lines(T24,Spca-2,SKOV-3,NCl-H460,HepG2 and MGC-803).The activities of derivatives against NCl-H460/DOX and HepG2/DOX cancer resistance strain were examined to test the reverse multidrug resistance.The activities of derivatives against HL-7702 cells were also examined to test the toxicity of these compounds against human normal cells.The resulted showed that most of derivatives had significant antitumor activities and the IC50 of most target compounds was lower than that of the commercial anticancer drug doxorubicin.Especially,compound 6k exhibited the highest cytotoxicity against NCI-H460 cancer cells with IC50 values of 1.70±1.16?M.Structure-activity relationship shows that,for most compounds,electron-withdrawing groups at the same time as terminal substituent group is favorable to the cytotoxicity.Most compounds were first detected for their inhibitory potency against the ChT-L activity of the human 20S proteasome.These compounds exhibited significant selectivity toward the ?5-subunit of the human 20S proteasome with IC50 values at submicromolar concentrations.Docking study shows that the key of the hydrogen bonding interaction between proteasome and dipeptide thiourea derivatives in which the thiourea moiety and a nitro group are quite important for improving activity.Compounds 6k were selected as a representative to investigate their mechanism of antitumor against NCI-H460 cell line.Flow cytometric analysis(Annexin V-FITC/propidium iodide test)and fluorescence staining assays(Hoechst 33258 staining and JC-1 staining)showed that compounds 6k induced apoptosis.In order to further investigate their mechanism of antitumor,further analysis indicated that these derivatives increased the intracellular level of ROS and Ca2+production.And flow cytometry test results also show that compounds 6k involved S phase cell cycle arrest on NCI-H460 cell line.The levels of caspase-9,caspase-3,caspase-12,calpain,caspase-4,CHOP,GRP78,p-PERK and p-eIF2a of compound 6k were measured by Western blot.Our results suggest that 6k increased the expression levels of caspase-9,caspase-3,caspase-12,calpain,caspase-4,CHOP and GRP78,and induced p-PERK and p-eIF2a phosphorylation,which was an important signal of apoptosis.From the result,it can be suggested compound 6k could induce NCI-H460 cells apoptosis.In summary,the structural change introduced by transposing the thiourea and nitrophenyl to dipeptide residues could significantly improve the anticancer activities of proteasome inhibitor.Thus,we conclude that dipeptide derivatives containing thiourea moiety are the dual inhibitors of proteasome with the ability to reverse multidrug resistance.