A Novel Time-Dependent CENP-E Inhibitor with Potent Antitumor Activity

<div><p>Centromere-associated protein E (CENP-E) regulates both chromosome congression and the spindle assembly checkpoint (SAC) during mitosis. The loss of CENP-E function causes chromosome misalignment, leading to SAC activation and apoptosis during prolonged mitotic arrest. Here, we describe the biological and antiproliferative activities of a novel small-molecule inhibitor of CENP-E, Compound-A (Cmpd-A). Cmpd-A inhibits the ATPase activity of the CENP-E motor domain, acting as a time-dependent inhibitor with an ATP-competitive-like behavior. Cmpd-A causes chromosome misalignment on the metaphase plate, leading to prolonged mitotic arrest. Treatment with Cmpd-A induces antiproliferation in multiple cancer cell lines. Furthermore, Cmpd-A exhibits antitumor activity in a nude mouse xenograft model, and this antitumor activity is accompanied by the elevation of phosphohistone H3 levels in tumors. These findings demonstrate the potency of the CENP-E inhibitor Cmpd-A and its potential as an anticancer therapeutic agent.</p></div

Cmpd-A induces prolonged mitotic arrest accompanied by SAC activation.

<p>(A) Cell cycle histogram of synchronous HeLa cells treated with Cmpd-A (200 nM) or DMSO. Cmpd-A was added at the G2 phase (7 h after dT block release), and the cells were collected at the indicated time points for FACS analysis. (B) pHH3 in synchronous HeLa cells treated with Cmpd-A (200 nM) or DMSO. Cmpd-A was added at the G2 phase (7 h after dT block release), and the cells were collected 12 h after dT block release. Representative results are shown. (C) pHH3 elevation in synchronous HeLa cells treated with Cmpd-A (200 nM) or DMSO. Cmpd-A was added at the G2 phase (7 h after dT block release), and the cells were collected at the indicated time points for FACS analysis of pHH3 staining. The graph indicates quantified pHH3-positive cells (mean ± standard deviation; n = 3). Red and blue lines indicate Cmpd-A- and DMSO-treated HeLa cells, respectively. (D) Immunoblotting of mitosis markers in synchronous HeLa cells treated with Cmpd-A or DMSO. The cells were treated with Cmpd-A or DMSO as described in Fig 2A and collected 12 h after dT block release for immunoblotting.</p

Time-dependent antiproliferative activity of Cmpd-A in HeLa cells.

<p>(A) Experimental schemes to asses time-dependent antiproliferative activity of Cmpd-A. HeLa cells were treated with Cmpd-A at the indicated concentrations for 4, 8, 24, 48, and 72 h (red arrows) and then the cells were cultured in Cmpd-A-free medium for 72 h (black arrows). Cells were collected 72 h after treatment for cell viability analysis. (B) Time-dependent antiproliferative activity of Cmpd-A in HeLa cells. The relative ATP concentration was calculated based on chemiluminescence compared with the 0 nM chemiluminescence value (control). Data are presented as mean ± standard deviation (n = 8). (C) Quantitative RT-PCR analysis of CENP-E in cancer cell lines and human skin fibroblasts (MRC5). CENP-E expression ratios were quantified using GAPDH expression in each cell line as a control. Data are presented as mean ± standard deviation (n = 3).</p

Cmpd-A exhibits potent antiproliferative activity in multiple cancer cell lines.

<p>(A) Antiproliferative activity of Cmpd-A in multiple cancer cell lines. DU145, COLO205, NIH-OVCAR3, RKO, ES2, SK-OV3, PC-3, SW620, and CAPAN-2 cell lines were treated with Cmpd-A for 3 days at the indicated concentrations. The relative ATP concentration was calculated based on the chemiluminescence compared with the 0 nM chemiluminescence value (control). Data are presented as mean ± standard deviation (n = 3). (B) Correlation between the antiproliferative activity of Cmpd-A and CENP-E mRNA expression in cancer cell lines. The X and Y axes indicate the relative ATP level at 300 nM Cmpd-A treatment and CENP-E mRNA levels in the 14 indicated cancer cell lines, respectively. The relative ATP concentration was calculated based on chemiluminescence compared with the 0 nM chemiluminescence value (control) in each cell line. The raw data of CENP-E mRNA expression was downloaded from the Cancer Cell Line Encyclopedia (<a href="http://www.broadinstitute.org/ccle/data/browseData" target="_blank">http://www.broadinstitute.org/ccle/data/browseData</a>) and processed with MAS 5.0 algorithm.</p

CENP-E inhibitor Cmpd-A induces chromosome misalignment during mitosis.

<p>(A) Cmpd-A is a time-dependent inhibitor with an ATP competitive-like behavior. Red and black lines indicate the dose-dependent activity of Cmpd-A in the presence of low (1.25 μM) and high (500 μM) concentrations of ATP, respectively. The blue line indicates the activity of Cmpd-A with a high concentration of ATP, following 1 h of preincubation with CENP-E. The X-axis and Y-axis indicate the concentration of Cmpd-A and % inhibition of CENP-E ATPase activity, respectively. (B) Representative mitotic HeLa cells treated with Cmpd-A (200 nM) or DMSO. Arrows indicate misaligned chromosomes. Blue, green, and red signals indicate 4′,6-diamidino-2-phenylindole (DAPI)-stained DNA, α-tubulin, and CENP-B (kinetochores), respectively. (C) Quantitative analysis of mitotic morphology in the DMSO- or Cmpd-A-treated HeLa cells. The cells were treated for 3 h with 200 nM Cmpd-A or DMSO after dT block release. The DMSO- and Cmpd-A-treated mitotic cells (105 and 106 cells, respectively) were then counted. (D) Inter-kinetochore distance of aligned and misaligned chromosomes in HeLa cells treated with Cmpd-A or DMSO. Prometaphase (left) and metaphase (middle) cells were used as controls for misaligned and aligned chromosomes, respectively. The inter-kinetochore distance was measured between the outer kinetochore markers (HEC1) of individual chromosomes. Statistical analysis was performed using Student’s t-test. Differences were considered significant at P ≤ 0.05 (*) and P ≤ 0.01 (**).</p

PK/PD and antitumor efficacy of Cmpd-A in a COLO205 xenograft nude mouse model.

<p>(A) Expression of pHH3 in COLO205 xenografts at the indicated time points after the intraperitoneal administration of Cmpd-A at a dose of 100 mg/kg. (B) Time-dependent PK and PD of Cmpd-A. The green, blue, and red lines indicate plasma concentration, tumor concentration, and tumor pHH3 intensity, respectively. The pHH3 intensity was quantified using the results shown in (A). (C) Immunohistochemistry of pHH3 in the tumor sections from COLO205 xenograft nude mice 24 h after the intraperitoneal administration of Cmpd-A at 100 mg/kg. Black arrows indicate misaligned chromosomes in sections from COLO205 xenografts treated with Cmpd-A. (D) Antitumor efficacy of Cmpd-A in the COLO205 xenograft nude mouse model. COLO205 xenografted nude mice intraperitoneally injected with Cmpd-A at 100 mg/kg or vehicle three times (at 0, 8, and 24 h) on the first day of the study. Representative tumors 8 days after the administration of vehicle or Cmpd-A are shown. (E) Efficacy data plotted as the mean tumor volume (mm³ ± standard error of the mean; n = 5) in COLO205 xenograft nude mice treated with Cmpd-A (red) or vehicle (black). Statistical analysis was performed using Student’s t-test. Differences were considered significant at P ≤ 0.05 (*) and P ≤ 0.01 (**). (F) Bodyweight comparison of COLO205 xenograft nude mice 8 days after the administration of Cmpd-A (red) or vehicle (black). Data are presented as mean ± standard deviation (n = 5). Statistical analysis was performed using Student’s t-test. Differences were considered significant at P ≤ 0.05 (*) and P ≤ 0.01 (**).</p

Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors

Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor <b>53a</b> exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of <b>53a</b> and its analog <b>52a</b> to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds <b>52a</b> and <b>53a</b> represent novel molecular probes for further study of eIF4A3, the EJC, and NMD

Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors

Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor <b>53a</b> exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of <b>53a</b> and its analog <b>52a</b> to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds <b>52a</b> and <b>53a</b> represent novel molecular probes for further study of eIF4A3, the EJC, and NMD

Discovery of Novel 1,4-Diacylpiperazines as Selective and Cell-Active eIF4A3 Inhibitors

Eukaryotic initiation factor 4A3 (eIF4A3), a member of the DEAD-box RNA helicase family, is one of the core components of the exon junction complex (EJC). The EJC is known to be involved in a variety of RNA metabolic processes typified by nonsense-mediated RNA decay (NMD). In order to identify molecular probes to investigate the functions and therapeutic relevance of eIF4A3, a search for selective eIF4A3 inhibitors was conducted. Through the chemical optimization of 1,4-diacylpiperazine derivatives identified via high-throughput screening (HTS), we discovered the first reported selective eIF4A3 inhibitor <b>53a</b> exhibiting cellular NMD inhibitory activity. A surface plasmon resonance (SPR) biosensing assay ascertained the direct binding of <b>53a</b> and its analog <b>52a</b> to eIF4A3 and revealed that the binding occurs at a non-ATP binding site. Compounds <b>52a</b> and <b>53a</b> represent novel molecular probes for further study of eIF4A3, the EJC, and NMD

Synthetic Studies on Centromere-Associated Protein‑E (CENP-E) Inhibitors: 2. Application of Electrostatic Potential Map (EPM) and Structure-Based Modeling to Imidazo[1,2‑<i>a</i>]pyridine Derivatives as Anti-Tumor Agents

To develop centromere-associated protein-E (CENP-E) inhibitors for use as anticancer therapeutics, we designed novel imidazo­[1,2-<i>a</i>]­pyridines, utilizing previously discovered 5-bromo derivative <b>1a</b>. By site-directed mutagenesis analysis, we confirmed the ligand binding site. A docking model revealed the structurally important molecular features for effective interaction with CENP-E and could explain the superiority of the inhibitor (<i>S</i>)-isomer in CENP-E inhibition vs the (<i>R</i>)-isomer based on the ligand conformation in the L5 loop region. Additionally, electrostatic potential map (EPM) analysis was employed as a ligand-based approach to optimize functional groups on the imidazo­[1,2-<i>a</i>]­pyridine scaffold. These efforts led to the identification of the 5-methoxy imidazo­[1,2-<i>a</i>]­pyridine derivative (+)-(<i>S</i>)-<b>12</b>, which showed potent CENP-E inhibition (IC₅₀: 3.6 nM), cellular phosphorylated histone H3 (p-HH3) elevation (EC₅₀: 180 nM), and growth inhibition (GI₅₀: 130 nM) in HeLa cells. Furthermore, (+)-(<i>S</i>)-<b>12</b> demonstrated antitumor activity (<i>T</i>/<i>C</i>: 40%, at 75 mg/kg) in a human colorectal cancer Colo205 xenograft model in mice