Antitumor Effects Of Novel NEK2 Inhibitors MBM-17 And MBM-55 And Study On The Molecular Mechanisms Of NEK2 Regulating KDM5B/H3K4me3

Background:NEK2,one of serine/threonine kinase family members,is a cell cycle regulatory protein.NEK2 can participate in the process of mitosisincluding assembling centrosome,regulating the centrosome separation,promoting chromosome condensation and separation.Dysfunction of NEK2 can lead to oncogenesis.NEK2 is frequently overexpressed in a wide variety of human cancers.Up-regulated NEK2 can promote the degree of tumor malignant transformation and may induce tumor resist to drug therapies at the same time.Furthermore,NEK2 is also associated with the poor prognosis of tumor.Therefore,NEK2,a proto-oncogene,is expected to be a new therapeutic target for variety human cancers.Purpose:So far,the reported NEK2 inhibitors have lots of disadvantages,such as low biological activity,poor selectivity and weak antitumor effect in vivo.Although many novel interaction partners of NEK2 have been found,the detailed signaling pathways are still not completely clear.Therefore,in view of the problems mentioned above,we designed a series of compounds which blocking-up the binding site between ATP and NEK2.This subject aimed to screen out compounds with high activity and selectivity in inhibiting NEK2 kinase,and evaluated the anti-tumor activity in vitro and in vivo at the same time.Furthermore,the molecular mechanisms of NEK2 will be deeply explored.Methods:(1)Using Mobility shift assay for evaluating the enzyme spectrum selectivity of MBM-55;(2)Using MTT assay to evaluate the anti-proliferation activity against several kinds of cancer cells;(3)Using Western blot to detect the change of NEK2 expression influenced by MBM-17 and MBM-55;(4)Using PI test to verify the influence on cell cycle made by MBM-17 and MBM-55;(5)Using AnnexinV-FITC method test the influence on cell apoptosis made by MBM-17 and MBM-55;(6)The pharmacokinetic parameters of MBM-17S and MBM-55S were determined by intravenous injection of SD rats;(7)Evaluating the antitumor effect of MBM-17S and MBM-55S in HCT-116 xenografts in nude mice;(8)Silent NEK2 in MDA-MB-468 breast cell line for detecting the proteins level and mRNA level that may be changed;Results:(1)Compounds MBM-17 and MBM-55 have good activity in inhibiting NEK2 kinase,meanwhile,MBM-55 have good global kinase selectivity for Nek2;(2)Compound MBM-17 and MBM-55 can inhibit proliferation in a variety of tumor cell lines,especially for human gastric cancer cell line MGC-803 and human colon cancer cell line HCT-116;(3)Compounds MBM-17 and MBM-55 can decrease the NEK2 protein level in MGC-803 cell;(4)Compounds MBM-17 and MBM-55 can arrest cells in G2/M phase in a dose dependent manner,inducing polyploidy at the same time;(5)Compounds MBM-17 and MBM-55 have the ability to induce apoptosis and necrosis;(6)Compounds MBM-17S and MBM-55S exhibit bad pharmacokinetic parameters in vivo;(7)Compounds MBM-17 and MBM-55 exhibit good antitumor activity with no obvious toxicity in vivo;(8)Compounds MBM-17 and MBM-55 increased the protein level of H3K4me3 in MGC-803 cell line;(9)Silence NEK2 in MDA-MB-468 can significantly decreased the protein level of KDM5B but have no influence on the mRNA level.Conclusions:Compounds MBM-17 and MBM-55 can effectively inhibit NEK2 kinase activity and decrease the NEK2 protein level,exhibiting potent antitumor activity in vitro.Both of them can arrest tumour cells in G2/M phase and induce polyploidy in vitro at the same time.MBM-17 and MBM-55 also have good antitumor activity in vivo without obvious toxicity.It is possible that KDM5B is a potential downstream target.Therefore,the study provides the experimental basis and research ideas for the development of NEK2 inhibitors,and provides the theoretical basis for the study of NEK2 mechanism.