Study On The Antitumor Activity Of Novel Nitroheterocyclic Compounds Based On LSD1 And LSD1 Neddylation

Lysine specific demethylase 1A(LSD1)is a flavin-dependent demethylase.It could remove mono-and di-methyl groups from the fourth and ninth lysine residue on histone 3.LSD1 is highly expressed in a variety of tumor tissues and plays an important role in tumorigenesis,growth,metastasis,invasion and differentiation.As a new target for the development of anti-tumor drugs,LSD1 has attracted wide attention.Therefore,it is a new approach for cancer treatment to develop LSD1-targeting inhibitors or induce endogenous degradation of LSD1According to the previous synthesis experience of LSD 1 in our research group and literature research,it is shown that heterocyclic compounds with N-heterocyclic ring as the skeleton are an important class of LSD 1 inhibitor.Therefore,it is of great significance to explore the N-heterocyclic compounds targeting LSD1 and their anti-tumor activities.Firstly,LSD1 was expressed in Ecoli.BL21(DE3)and purified by affinity chromatography,gel chromatography and ion exchange chromatography.Then,three kinds of N-heterocyclic compounds with different structure types were screened by fluorescence-based method.The mechanism of the potent compound against LSD1 was discussed.And their anti-tumor activity was further evaluated.In addition,the effect of neddylation on the stability of LSD 1 and the modification sites were also studied.Research contents were listed as below:(1)Identification of aromatic hydrazine LSD1 inhibitors and evaluation of antitumor activitiesBy introducing different groups into the heterocyclic skeleton[1,2,4]triazolo[1,5-a]pyrimidine,31 compounds were synthesized.After LSD1 activity screening,it was found that compound D8(IC50=882.3 ±1.06 nM)containing hydrazine had the strongest inhibitory activity on LSD1.It indicated that compound D8 is a reversible,selective and substrate competitive LSD1 inhibitor.In A549 cell line,it indicated that compound D8 could bind to LSD1 and inhibit LSD1 activity.In addition,D8 could inhibit the epithelial-mesenchymal transition process and metastasis of A549 cells.In vivo,it demonstrated that compound D8 could inhibit lung cancer cell metastasis,which wa consistent with the results of cell experiment.It indicated that aromatic hydrazine structure was a novel LSD1 inhibitor skeleton,which provides guidance for obtaining more effective LSD1-targeting inhibitors.(2)Identification of osimertinib as a dual inhibitor of EGFR/LSD1 and evaluation of antitumor activityBy screening partial EGFR-TKI,we found that osimertinib containing pyrimidine skeleton could inhibit LSD1 activity with IC50 value of 3.98±0.30 μM.Osimertinib could inhibit LSD 1 reversibly,selectively and competitively with FAD.In addition,osimertinib could suppress the proliferation of NCI-H1975 cell line and inhibit the activity of LSD 1 at cellular level.At the same time,osimertinib could significantly inhibit the metastasis of NCI-H1975 cells.Molecular docking results showed that the protonated N,N-dimethyl group formed ionic contact with Asp556,the carbonyl oxygen atom of the amide group had a hydrogen bond interaction with Thr335.As the first dual inhibitor,osimertinib provides a new molecular skeleton for the development of potential LSD1 and EGFR inhibitors.(3)Identification of isoquinoline alkaloid LSD1 inhibitors and evaluation of antitumor activitiesBy screening some alkaloids,it was found that isoquinoline alkaloid had LSD1 inhibitory activity by fluorescence method.Epiberberine with IC50 value of 0.14±0.01 μM,the most potent compound,was further evaluated the antitumor effect in vitro and in vivo.The mechanism study indicated that epiberberine selectly inactivated LSD1 in a reversible manner.The specific binding between epiberberine and LSD1 was in a manner of fast association and slow dissociation.,epiberberine could inhibit proliferation of acute myelocytic leukemia cell lines,inhibit the activity of LSD 1 at cellular level and promote cell differentiation.Finally,animal experiments revealed that epiberberine inhibited tumor growth and prolong survival NOD/SCID mice bearing THP-1 cells,suggesting that epiberberine had anti-tumor activity in vivo.The discovery of isoquinoline alkaloid based LSD1 inhibitor provides a novel skeleton for obtaining more effective LSD1-targeting inhibitors.(4)Study on LSD1 neddylationnedd8,involved in the protein post-translation,is a ubiquitin-like protein.The process,binding to target protein,is called neddylation.In this study,the overexpression of nedd8 and the inhibitor MLN4924 were used to verify the positive and negative stimulation,and the preliminary conclusion was drawn that necddylation reduced the stability of LSD 1.Subsequently,four neddylation sites,K5/6/63/117,were found in the first 165 aa of LSD1 by truncation,deletion and site-specific mutation.These results laid a foundation for further study on the biological function of LSD 1 neddylation.In conclusion,three kinds of LSD1 inhibitor containing N-heterocyclic was identified and the antitumor activities of D8,osimertinib and epiberberine were evaluated.In addition,reducing stability of LSD1 by neddylation and general modification sites(K5/6/63/117)were revealed.These findings provide experimental and theoretical basis for further biological study of LSD 1,as well as for the development of anti-tumor drugs based on LSD1 inhibitory.