Study On Activity Of Melanoma Targeted Prodrugs Induced By H₂O₂ And Tyrosinase

Melanoma is one of the deadliest and most threatening skin cancers.It has prominent characteristics such as high recurrence,easy metastasis,and low survival rate.In recent years,the morbidity and mortality rate have been increasing.The chemotherapy is one of the main treatment for melanoma.However,the side effects are the main obstacle of chemotherapy drugs for clinical application.To circumvent this issue,we proposed a dual biomarker-cascade activated prodrug strategy based on the high levels of reactive oxygen species（ROS）and high expression of tyrosinase（TYR）in melanoma and carried out the following researchs:1.Firstly,we synthesized two representative prodrugs,coumarin-quinazolin one-phenylboronic acid（CQBA）and coumarin-quinazolinone-phenylboronic acid pinacol ester（CQB）.The nuclear magnetic resonance was used to characterize the structure of compounds.2.The cascaded activation of prodrug CQBA/CQB were verified.High performance liquid chromatography analysis showed that the prodrug CQBA/CQB were converted to coumarin-quinazolinone phenol（CQP）after H₂O₂treatment.The results of mass spectrometry analysis showed that CQP was specifically oxidized to coumarin-quinazolinone-o-quinone conjugate（CQQ）by tyrosinase.3.The cytotoxicity experiments of CQBA/CQB were used to explore their high selectivity to melanoma cells.The results showed that the CQBA only induced the death of melanoma cells(IC₅₀=17.01μM)and they had no obvious toxicity to other cells(IC₅₀>100μM).4.Cell imaging showed that the prodrug CQBA located in the mitochondria with a colocalization coefficient of 84%.In addition,the level of reactive oxygen species in melanoma cells was increased in the presence of CQBA/CQB（5 folds as control for CQBA,3 folds as control for CQB）.The glutathione level was also decreased by half after CQBA/CQB treatment,which indicated the redox balance disruption in melanoma cells.CQBA/CQB also induced G0/G1-Phase arrest in melanoma cells,which disrupted the cell cycle and replication.The results of Western Blot experiments showed that the apoptosis executive protein caspase 3 was cleaved and the expression of crucial protein GPX4 of ferroptosis decreased in the presence of CQBA/CQB,indicating the occurrence of apoptosis and ferroptosis.5.Finally,a melanoma xenograft mouse model was established to further study the anti-tumor activity of CQB in vivo.The results showed that the tumor volume of mice treated with the prodrug CQB were significantly reduced,but there were no obvious lesions in main organs.All these results indicated that the prodrug CQB had good anti-tumor activity and good drug safety in vivo.In summary,this cascade-activated prodrug strategy could precisely target melanoma cells to induce programmed death and greatly reduce toxic side effects.It enriched the prodrug strategy of cascade induction and provided new ideas for the precise treatment of melanoma in the future.