Identification Of Monobenzone As A Nevel Potential Anti-leukemic Agent By RRM2-targeted Inhibition

Leukemia is a kind of malignant proliferative disease originated from hematopoietic stem cells.At present,the anti-leukemic clinical therapies still exist several problems such as toxic and side effects,drug-resistance,and so on.It's still an significant task to develop novel anti-leukemic agents with low toxicity,high selectivity and bio-activity.Ribonucleotide reductase(RR)is responsible for reducing ribonucleotides to their corresponding deoxyribonucleoties,which are the building blocks required for DNA replication and DNA damage repair.Human ribonucleotide reductase is composed of large subunits(RRM1)with catalytic functions and small subunits(RRM2 and RRM2B)performing regulating effects.In recent years,more and more studies indicate that RR represents an important node in tumorigenesis and development.The use of RR inhibitors,such as gemcitabine and hydroxyurea,either as a single agent or combined with other therapies,has been proven to be a promising approach for treating malignancies.Based on the 3D structure of RRM2,we virtual screened a database of old drug compounds and obtained monobenzone,which might bind to RRM2.It used to be approved by FDA to be added into a cream as an external drug for vitiligo patients.However,its mechanisms are not clear.In silico docking results suggested that monobenzone formed hydrogen bonds with Cys270 and Tyr323 residues of RRM2 and had other non-bonded interacions with certain amino acid residues of RRM2.The estimated free binding energy of the ligand to the target was about-4.84kcal/mol to-4.76kcal/mol.Monobenzone is a tyrosinase inhibitor,which can inhibit the synthesis of melanin.In this study,we found that monobenzone potently inhibited the recombinant RR enzyme activity in vitro with IC50 at 480nM.Also,it could inhibit the growth of hematological malignancies including leukemia,lymphoma and multiple myeloma.Moreover,monobenzone showed better anti-proliferation effect with IC50 around 5uM to 20uM in leukemia cells.Softagar experiments showed that monobenzone could effectively inhibit the formation of AML colony.Flow cytometry assays demonstrated that monobenzone induced cell-cycle arrest at S phase with a dose-dependent manner in AML and lymphoma cells.Monobenzone upregulated y-H2AX protein level.Furthermore,monobenzone displayed synergistic anti-proliferation and pro-apoptosis efficacy on AML cells in combination with the Bcl-2 inhibitor ABT-737.In AML mouse xenograft model,the administration of monobenzone by intraperitoneal injection for 12 days produced a significant anti-leukemic effect in a dose-dependent manner with a weak toxicity at the dosage of 150mg/kg.Talking together,we demonstrate that RR is the novel target of monobenzone,which possesses a potent activity in vitro and in vivo for potential treatment of leukemia.We also propose that the simultaneous use of the anti-proliferatory RR inhibitors with pro-apoptosis agents like Bcl-2 inhibitors can enhance the inhibitory effect on hematological neoplasms,which provides theoretical basis for clinical therapies.