Study Of Anti-cancer Effects And Mechanisms Of Novel NEK2Kinase Inhibitor MBM-5

NEK2, a member of the serine-threonine kinase family, plays an important role in mitosis. Uncontrolled NEK2 activity can lead to chromosomal instability as well as abnormal chromosome content and the formation of multinucleate cell. NEK2 is overexpression in a variety of cancer cells. One CIN gene in particular, NEK2, was highly correlated with drug resistance, rapid relapse, and poor outcome in multiple cancers.Therefore, pharmacologic modulation of NEK2 with a single agent may effect several mechanisms important for tumor growth, survival, progression, and metastasis.Recently, the characteristics of the NEK2 inhibitors are poor selectivity, no cell activity and weak antitumor effect in vivo. Development of specificity NEK2 inhibitors has a broad prospect. It is reported that change the ATP binding site of NEK2 can further complicate NEK2 inhibitor design. In our previous study, we synthesize a series of compounds with potential NEK2 inhibitory activity by interrupting the connection between NEK2 kinase and ATP. Here we evaluated the antitumor activity of these compounds and studied their mechanism through molecules, cells and animal levels.In our study, kinase assay (EZ Reader and HTRF(?) KinEASETM-STK) indicated that MBM-5 has better inhibition than other compunds on the target kinase NEK2 with IC50=0.494μM. And cell viability assay proved the growth of cancer cell with IC50 of micromole range especially on GC and CC. So we choosed human gastric carcinoma cell line MGC-803 and human colcnic carcinoma cell line HCT-116 to finish the next experiment. The in vivo anti-tumor efficacy of MBM-5 was evaluated on HCT-116 and MGC-803 xenografts in a parallel comparison with another antineoplastic agent irinotecan. MBM-5 group demonstrated superior antitumor efficacy than control. In two xenografts, the TGI was 29.35% and 42.35%, respectively, but compared with positive drug group are still gap. Pharmacokinetic experiment showed MBM-5 metabolized fast in vivo, resulting the poor effect of tumor inhibition. We herein optimize the structure of compound to obtain better results.Meanwhile, MBM-5 demonstrated anti-tumor effect in MGC-803 and HCT-116 cells in vitro by inducing cell cycle arrest and apoptosis. After treating with MBM-5, the cell cycle of more than 60% cells were blocked in the G2/M phases. At the same time, we noticed some polyploidy cells. As inactivation Nek2 was reported to trigger mitotic abnormalities, especially spindle configuration changes and chromosome misalignment, we then tested whether MBM-5 treatment would elicit a similar cellular phenotype. In contrast to control DMSO (dimethyl sulfoxide) treatment, cells displayed increased chromosomal misalignment after 12 h of treatment with MBM-5. Consistently, multinucleate cells and chromosomal misalignment in the mitotic population were aggravated in a dose and time-dependent manner after MBM-5 treatment.Over time, the accumulation of chromosomal and spindle abnormalities led to cell death. To determine whether mitotic abnormalities induced by MBM-5 leads to apoptosis and necrosis, we performed flow cytometry with Annexin-V/propidium iodide staining. The percentages of apoptotic and necrotic cells were higher in the MBM-5-treated MGC-803 and HCT-116 cells (72.84% and 45.43% respectively), compared with 8.57,7.60% in DMSO treated cells. Next, we examined the apoptosis related proteins expression level. With the increasing of dose and time, the expression of c-Capase 3 and c-PARP-1 became higher in MGC-803 and HCT-116. Together, these results suggested that MBM-5 cause mitotic catastrophe leading to cell death.To delineate the cellular consequences upon MBM-5 treatment, we first examined NEK2 protein levels in MGC-803 cells. In a time course study, NEK2 level was remarkably reduced by>95% after 24 h of treatment with 1 μM MBM-5, the same situation was observed in Hec1 expression. However, MBM-5 treatment did not affect the stability of other mitotic molecules, including cyclin B and two mitotic kinases, Aurora A and Aurora B. Consistent with our previous finding, we found MBM-5 arrested cell cycle by inhibiting the expression of NEK2 and its related protein Hec1 without influencing other cyclin kinase such as Aurora A and Aurora B. As NEK2 is degraded through the proteasome pathway at prometaphase, we then co-treated cells with MBM-5 and the proteasome inhibitor MG132 and found MG132 treatment prevented the MBM-5-induced degradation of NEK2.Therefore, for the purpose of changing the ATP binding site of NEK2 to design the compound MBM-5 has excellent effect of blocking cell cycle progression and inducing cell apoptosis. MBM-5 also suppressed the activity of NEK2 kinase and indicated obvious antitumor effect. Theses evidences indicate that MBM-5 is a potent and potential kinase inhibitor with a unique antiproliferative profile, and provide strong confidence for further development of NEK2 inhibitor for cancer therapy.