NEK2 is a conserved mitotic regulator critical for cell cycle progression. inducing chromosome segregation defects and cytokinesis failure; therefore leading to accumulation of cells with 4N DNA content, which finally underwent apoptosis. More importantly, MBM-5 treatment effectively suppressed the tumor growth of human gastric and colorectal cancer cells xenografts. Taken together, we demonstrated that MBM-5 effectively inhibited the kinase activity of NEK2 and showed a potential application in anti-cancer treatment regimens. [1]. NEK2 is well recognized as a multifunctional serine/threonine kinase with key roles in cell cycle regulation, particularly with respect to the centrosome cycle. NEK2 localizes to the centrosome and triggers centrosome separation by phosphorylating centrosome cohesion proteins C-Nap1, Rootletin and Cep68 [2C4]. NEK2 also regulates microtubule organization and stabilization through phosphorylation of ninein-like protein (Nlp) [5]. Moreover, NEK2 operates a faithful DAMPA kinetochore microtubule attachments by phosphorylation of highly expressed in cancer 1 (Hec1) [6, 7]. Through direct interaction with mitotic arrest deficient-like 1 (MAD1) or phosphorylation of Hec1 and Sgo1, NEK2 also modulates chromosome alignment and the spindle assembly checkpoint (SAC) signaling, thus regulating chromosome separation [8C10]. In addition, NEK2B (a splice variant of NEK2) is observed to be required for execution of mitotic exit as NEK2B depleted cells were unable to complete cytokinesis and resulted in the formation of multinucleated cells [11]. NEK2 DAMPA has been reported to be overexpressed in a wide variety of human cancers, such as gastric cancer [12], colorectal cancer [13, 14], prostate cancer [15] and breast cancer [16, 17]. NEK2 overexpression is associated with tumorigenensis, tumor progression, drug resistance and predicts poor prognosis [18, 19]. Several preclinical studies using RNA interference targeting NEK2 have shown the efficient anti-tumor effect against different type of cancers. Suppression of the NEK2 expression with siRNA inhibited cell proliferation and induced cell death of breast cancer, cholangiocarcinoma, colorectal cancer, multiple myeloma, hepatoma and prostate cancer cells antitumor activity of any compound mentioned above has not been disclosed yet. Therefore, great efforts should be made to develop novel NEK2 inhibitors that are suitable for clinical applications. By DAMPA performing molecular docking analysis, we screened five hundred compounds from our in-house compound library to test their affinity to the ATP-site of the NEK2 kinase. Compounds with high docking score were selected to determine DAMPA their potential activities against NEK2 kinase and antitumor effects in a variety of cancer cells Because the NEK2 kinase is essential for cell proliferation, we examined the anti-proliferative effects of MBM-5 on a panel of 19 cancer cell lines, including leukemia, gastric, colorectal, prostate, breast and hepatoma cancer cells. Representative concentration-inhibition curves were drawn as shown in Figure ?Figure2A,2A, and IC50 values were calculated and listed in Figure ?Figure2B2B and Table ?Table1.1. The IC50 values ranged from 1 to 10 M and leukemia, gastric and colorectal cancer cell lines were relatively sensitive to MBM-5 than other cell lines. These data were consistent with the prediction that NEK2 kinase activity is crucial for cellular proliferation, and in line with the findings that NEK2 depletion inhibits cancer cell growth. Figure 2 MBM-5 has antitumor activities against cancer cells Table 1 IC50 values for Tlr2 inhibition of cell growth by MBM-5 measured via MTT assay MBM-5 induces chromosomal misalignment and triggers mitotic catastrophe Perturbation of NEK2 function by RNAi or overexpression of kinase-inactive NEK2 leads to mitotic abnormalities represented by spindle configuration changes and chromosome misalignment. We then detected whether MBM-5 treatment would elicit these phenotypes. In contrast to DMSO treatment, MGC-803 cells displayed increased chromosomal misalignment after treated with MBM-5 for 12 h (Figure ?(Figure3A).3A). The proportion of cells with chromosomal DAMPA misalignment increased from 0.79% in control group to 7.30% in MBM-5 treated cells (Figure ?(Figure3B).3B). Multipolar spindle configurations in the mitotic population.