RESUMEN
Accumulating evidence has indicated that pre-mRNA splicing plays critical roles in a variety of physiological processes, including development of multiple diseases. In particular, alternative splicing is profoundly involved in cancer progression through abnormal expression or mutation of splicing factors. Small-molecule splicing modulators have recently attracted considerable attention as a novel class of cancer therapeutics, and several splicing modulators are currently being developed for the treatment of patients with various cancers and are in the clinical trial stage. Novel molecular mechanisms modulating alternative splicing have proven to be effective for treating cancer cells resistant to conventional anticancer drugs. Furthermore, molecular mechanism-based combination strategies and patient stratification strategies for cancer treatment targeting pre-mRNA splicing must be considered for cancer therapy in the future. This review summarizes recent progress in the relationship between druggable splicing-related molecules and cancer, highlights small-molecule splicing modulators, and discusses future perspectives of splicing modulation for personalized and combination therapies in cancer treatment.
RESUMEN
BACKGROUND: Endothelial cell senescence is the state of permanent cell cycle arrest and plays a critical role in the pathogenesis of age-related diseases. However, a comprehensive understanding of the gene regulatory network, including genome-wide alternative splicing machinery, involved in endothelial cell senescence is lacking. RESULTS: We thoroughly described the transcriptome landscape of replicative senescent human umbilical vein endothelial cells. Genes with high connectivity showing a monotonic expression increase or decrease with the culture period were defined as hub genes in the co-expression network. Computational network analysis of these genes led to the identification of canonical and non-canonical senescence pathways, such as E2F and SIRT2 signaling, which were down-regulated in lipid metabolism, and chromosome organization processes pathways. Additionally, we showed that endothelial cell senescence involves alternative splicing. Importantly, the first and last exon types of splicing, as observed in FLT1 and ACACA, were preferentially altered among the alternatively spliced genes during endothelial senescence. We further identified novel microexons in PRUNE2 and PSAP, each containing 9 nt, which were altered within the specific domain during endothelial senescence. CONCLUSIONS: These findings unveil the comprehensive transcriptome pathway and novel signaling regulated by RNA processing, including gene expression and splicing, in replicative endothelial senescence.
Asunto(s)
Empalme Alternativo , Redes Reguladoras de Genes , Senescencia Celular/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , TranscriptomaRESUMEN
Cholesterol 24-hydroxylase (CH24H) is a brain-specific enzyme that converts cholesterol into 24S-hydroxycholesterol, the primary mechanism of cholesterol catabolism in the brain. The therapeutic potential of CH24H activation has been extensively investigated, whereas the effects of CH24H inhibition remain poorly characterized. In this study, the therapeutic potential of CH24H inhibition was investigated using a newly identified small molecule, soticlestat (TAK-935/OV935). The biodistribution and target engagement of soticlestat was assessed in mice. CH24H-knockout mice showed a substantially lower level of soticlestat distribution in the brain than wild-type controls. Furthermore, brain-slice autoradiography studies demonstrated the absence of [3H]soticlestat staining in CH24H-knockout mice compared with wild-type mice, indicating a specificity of soticlestat binding to CH24H. The pharmacodynamic effects of soticlestat were characterized in a transgenic mouse model carrying mutated human amyloid precursor protein and presenilin 1 (APP/PS1-Tg). These mice, with excitatory/inhibitory imbalance and short life-span, yielded a remarkable survival benefit when bred with CH24H-knockout animals. Soticlestat lowered brain 24S-hydroxycholesterol in a dose-dependent manner and substantially reduced premature deaths of APP/PS1-Tg mice at a dose lowering brain 24S-hydroxycholesterol by approximately 50%. Furthermore, microdialysis experiments showed that soticlestat can suppress potassium-evoked extracellular glutamate elevations in the hippocampus. Taken together, these data suggest that soticlestat-mediated inhibition of CH24H may have therapeutic potential for diseases associated with neural hyperexcitation.
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Colesterol 24-Hidroxilasa/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Piperidinas/farmacología , Piridinas/farmacología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encefalopatías/tratamiento farmacológico , Encefalopatías/metabolismo , Encefalopatías/fisiopatología , Colesterol 24-Hidroxilasa/deficiencia , Colesterol 24-Hidroxilasa/genética , Inhibidores Enzimáticos del Citocromo P-450/química , Inhibidores Enzimáticos del Citocromo P-450/farmacocinética , Modelos Animales de Enfermedad , Desarrollo de Medicamentos , Femenino , Humanos , Hidroxicolesteroles/metabolismo , Longevidad/efectos de los fármacos , Longevidad/genética , Longevidad/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Piperidinas/química , Piperidinas/farmacocinética , Presenilina-1/genética , Presenilina-1/metabolismo , Piridinas/química , Piridinas/farmacocinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Drug-induced liver injury (DILI) is a leading cause of termination in drug development programs and removal of drugs from the market; this is partially due to the inability to identify patients who are at risk1. In this study, we developed a polygenic risk score (PRS) for DILI by aggregating effects of numerous genome-wide loci identified from previous large-scale genome-wide association studies2. The PRS predicted the susceptibility to DILI in patients treated with fasiglifam, amoxicillin-clavulanate or flucloxacillin and in primary hepatocytes and stem cell-derived organoids from multiple donors treated with over ten different drugs. Pathway analysis highlighted processes previously implicated in DILI, including unfolded protein responses and oxidative stress. In silico screening identified compounds that elicit transcriptomic signatures present in hepatocytes from individuals with elevated PRS, supporting mechanistic links and suggesting a novel screen for safety of new drug candidates. This genetic-, cellular-, organoid- and human-scale evidence underscored the polygenic architecture underlying DILI vulnerability at the level of hepatocytes, thus facilitating future mechanistic studies. Moreover, the proposed 'polygenicity-in-a-dish' strategy might potentially inform designs of safer, more efficient and robust clinical trials.
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Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Herencia Multifactorial , Polimorfismo de Nucleótido Simple , Alelos , Benzofuranos/uso terapéutico , Estudios de Casos y Controles , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/tratamiento farmacológico , Enfermedad Hepática Inducida por Sustancias y Drogas/epidemiología , Estudios de Cohortes , Conjuntos de Datos como Asunto/estadística & datos numéricos , Perfilación de la Expresión Génica , Frecuencia de los Genes , Predisposición Genética a la Enfermedad/genética , Estudio de Asociación del Genoma Completo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Análisis por Micromatrices , Herencia Multifactorial/genética , Sulfonas/uso terapéuticoRESUMEN
In our pursuit of developing a novel, potent, and selective cell division cycle 7 (Cdc7) inhibitor, we optimized the previously reported thieno[3,2-d]pyrimidinone analogue I showing time-dependent Cdc7 kinase inhibition and slow dissociation kinetics. These medicinal chemistry efforts led to the identification of compound 3d, which exhibited potent cellular activity, excellent kinase selectivity, and antitumor efficacy in a COLO205 xenograft mouse model. However, the issue of formaldehyde adduct formation emerged during a detailed study of 3d, which was deemed an obstacle to further development. A structure-based approach to circumvent the adduct formation culminated in the discovery of compound 11b (TAK-931) possessing a quinuclidine moiety as a preclinical candidate. In this paper, the design, synthesis, and biological evaluation of this series of compounds will be presented.
Asunto(s)
Antineoplásicos/uso terapéutico , Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazolonas/uso terapéutico , Pirimidinas/uso terapéutico , Pirimidinonas/uso terapéutico , Quinuclidinas/uso terapéutico , Tiofenos/uso terapéutico , Animales , Antineoplásicos/síntesis química , Antineoplásicos/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Diseño de Fármacos , Descubrimiento de Drogas , Formaldehído/química , Humanos , Ratones , Simulación del Acoplamiento Molecular , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Pirazolonas/farmacología , Pirimidinas/farmacología , Pirimidinonas/síntesis química , Pirimidinonas/metabolismo , Quinuclidinas/síntesis química , Quinuclidinas/metabolismo , Relación Estructura-Actividad , Tiofenos/síntesis química , Tiofenos/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The DEAD-box family of RNA helicases plays essential roles in both transcriptional and translational mRNA degradation; they unwind short double-stranded RNA by breaking the RNA-RNA interactions. Two DEAD-box RNA helicases, eukaryotic translation initiation factor 4A3 (eIF4A3) and DEAD-box helicase 3 (DDX3X), show high homology in the ATP-binding region and are considered key molecules for cancer progression. Several small molecules that target eIF4A3 and DDX3X have been reported to inhibit cancer cell growth; however, more potent compounds are required for cancer therapeutics, and there is a critical need for high-throughput assays to screen for RNA helicase inhibitors. In this study, we developed novel fluorescence resonance energy transfer-based high-throughput RNA helicase assays for eIF4A3 and DDX3X. Using these assays, we identified several eIF4A3 allosteric inhibitors whose inhibitory effect on eIF4A3 ATPase showed a strong correlation with inhibitory effect on helicase activity. From 102 compounds that exhibited eIF4A3 ATPase inhibition, we identified a selective DDX3X inhibitor, C1, which showed stronger inhibition of DDX3X than of eIF4A3. Small-molecule helicase inhibitors can be valuable for clarifying the molecular machinery of DEAD-box RNA helicases. The high-throughput quantitative assays established here should facilitate the evaluation of the helicase inhibitory activity of compounds.
Asunto(s)
ARN Helicasas DEAD-box/antagonistas & inhibidores , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , ARN Helicasas DEAD-box/metabolismo , Descubrimiento de Drogas/métodos , Evaluación Preclínica de Medicamentos/métodos , Pruebas de Enzimas/métodos , Factor 4A Eucariótico de Iniciación/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
The RNA helicase EIF4A3 regulates the exon junction complex and nonsense-mediated mRNA decay functions in RNA transcript processing. However, a transcriptome-wide network definition of these functions has been lacking, in part due to the lack of suitable pharmacological inhibitors. Here we employ short-duration graded EIF4A3 inhibition using small molecule allosteric inhibitors to define the transcriptome-wide dependencies of EIF4A3. We thus define conserved cellular functions, such as cell cycle control, that are EIF4A3 dependent. We show that EIF4A3-dependent splicing reactions have a distinct genome-wide pattern of associated RNA-binding protein motifs. We also uncover an unanticipated role of EIF4A3 in the biology of RNA stress granules, which sequester and silence the translation of most mRNAs under stress conditions and are implicated in cell survival and tumour progression. We show that stress granule induction and maintenance is suppressed on the inhibition of EIF4A3, in part through EIF4A3-associated regulation of G3BP1 and TIA1 scaffold protein expression.
Asunto(s)
Ciclo Celular/genética , Gránulos Citoplasmáticos/metabolismo , ARN Helicasas DEAD-box/genética , Factor 4A Eucariótico de Iniciación/genética , Estrés Fisiológico/genética , Transcriptoma , Regulación Alostérica/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Biología Computacional/métodos , Gránulos Citoplasmáticos/efectos de los fármacos , ARN Helicasas DEAD-box/antagonistas & inhibidores , ARN Helicasas DEAD-box/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , Inhibidores Enzimáticos/farmacología , Factor 4A Eucariótico de Iniciación/antagonistas & inhibidores , Factor 4A Eucariótico de Iniciación/metabolismo , Regulación de la Expresión Génica , Células HCT116 , Células HeLa , Humanos , Proteínas de Unión a Poli-ADP-Ribosa/genética , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas con Motivos de Reconocimiento de ARN/genética , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Estrés Fisiológico/efectos de los fármacos , Antígeno Intracelular 1 de las Células T/genética , Antígeno Intracelular 1 de las Células T/metabolismoRESUMEN
Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931-induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS-wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.
Asunto(s)
Antineoplásicos/farmacología , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirazolonas/farmacología , Pirimidinas/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular , Separación Celular , Supervivencia Celular , Centrosoma/efectos de los fármacos , Aberraciones Cromosómicas/efectos de los fármacos , Biología Computacional , Resistencia a Antineoplásicos/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Células HeLa , Humanos , Concentración 50 Inhibidora , Estimación de Kaplan-Meier , Ratones , Ratones Endogámicos BALB C , Mitosis/efectos de los fármacos , Modelos Animales , Mutación , Trasplante de Neoplasias , Neoplasias Pancreáticas/tratamiento farmacológico , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Proteómica , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CDC-like kinase phosphorylation of serine/arginine-rich proteins is central to RNA splicing reactions. Yet, the genomic network of CDC-like kinase-dependent RNA processing events remains poorly defined. Here, we explore the connectivity of genomic CDC-like kinase splicing functions by applying graduated, short-exposure, pharmacological CDC-like kinase inhibition using a novel small molecule (T3) with very high potency, selectivity, and cell-based stability. Using RNA-Seq, we define CDC-like kinase-responsive alternative splicing events, the large majority of which monotonically increase or decrease with increasing CDC-like kinase inhibition. We show that distinct RNA-binding motifs are associated with T3 response in skipped exons. Unexpectedly, we observe dose-dependent conjoined gene transcription, which is associated with motif enrichment in the last and second exons of upstream and downstream partners, respectively. siRNA knockdown of CLK2-associated genes significantly increases conjoined gene formation. Collectively, our results reveal an unexpected role for CDC-like kinase in conjoined gene formation, via regulation of 3'-end processing and associated splicing factors.The phosphorylation of serine/arginine-rich proteins by CDC-like kinase is a central regulatory mechanism for RNA splicing reactions. Here, the authors synthesize a novel small molecule CLK inhibitor and map CLK-responsive alternative splicing events and discover an effect on conjoined gene transcription.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Pirimidinas/farmacología , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Exones , Perfilación de la Expresión Génica , Genoma Humano , Células HCT116 , Humanos , Imidazoles/síntesis química , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/síntesis química , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/metabolismo , Relación Estructura-Actividad , Transcripción GenéticaRESUMEN
Accurate control of cytokinesis is critical for genomic stability to complete high-fidelity transmission of genetic material to the next generation. A number of proteins accumulate in the intercellular bridge (midbody) during cytokinesis, and the dynamics of these proteins are temporally and spatially orchestrated to complete the process. In this study, we demonstrated that localization of centromere-associated protein-E (CENP-E) at the midbody is involved in cytokinetic abscission. The motor activity of CENP-E and the C-terminal midbody localization domain, which includes amino acids 2659-2666 (RYFDNSSL), are involved in the anchoring of CENP-E to the center of the midbody. Furthermore, CENP-E motor activity contributes to the accumulation of protein regulator of cytokinesis 1 (PRC1) in the midbody during cytokinesis. Midbody localization of PRC1 is critical to the antiparallel microtubule structure and recruitment of other midbody-associated proteins. Therefore, CENP-E motor activity appears to play important roles in the organization of these proteins to complete cytokinetic abscission. Our findings will be helpful for understanding how each step of cytokinesis is regulated to complete cytokinetic abscission.
Asunto(s)
Centrómero/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Citocinesis/fisiología , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Transporte de Proteínas/fisiologíaRESUMEN
Centromere-associated protein-E (CENP-E) is a mitotic kinesin which plays roles in cell division, and is regarded as a promising therapeutic target for the next generation of anti-mitotic agents. We designed novel fused bicyclic CENP-E inhibitors starting from previous reported dihydrobenzofuran derivative (S)-(+)-1. Our design concept was to adjust the electron density distribution on the benzene ring of the dihydrobenzofuran moiety to increase the positive charge for targeting the negatively charged L5 loop of CENP-E, using predictions from electrostatic potential map (EPM) analysis. For the efficient synthesis of our 2,3-dihydro-1-benzothiophene 1,1-dioxide derivatives, a new synthetic method was developed. As a result, we discovered 6-cyano-7-trifluoromethyl-2,3-dihydro-1-benzothiophene 1,1-dioxide derivative (+)-5d (Compound A) as a potent CENP-E inhibitor with promising potential for in vivo activity. In this Letter, we discuss the design and synthetic strategy used in the discovery of (+)-5d and structure-activity relationships for its analogs possessing various fused bicyclic L5 binding moieties.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Óxidos S-Cíclicos/síntesis química , Sistemas de Liberación de Medicamentos , Diseño de Fármacos , Imidazoles/síntesis química , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Óxidos S-Cíclicos/química , Óxidos S-Cíclicos/farmacología , Células HeLa , Humanos , Imidazoles/química , Imidazoles/farmacología , Concentración 50 Inhibidora , Estructura Molecular , Unión Proteica/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Antineoplásicos/farmacología , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Huso Acromático/efectos de los fármacos , Animales , Línea Celular Tumoral , Segregación Cromosómica , Xenoinjertos , Metafase , Ratones , Ratones Desnudos , Microtúbulos/metabolismo , MitosisRESUMEN
To develop centromere-associated protein-E (CENP-E) inhibitors for use as anticancer therapeutics, we designed novel imidazo[1,2-a]pyridines, utilizing previously discovered 5-bromo derivative 1a. 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 (S)-isomer in CENP-E inhibition vs the (R)-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-a]pyridine scaffold. These efforts led to the identification of the 5-methoxy imidazo[1,2-a]pyridine derivative (+)-(S)-12, 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, (+)-(S)-12 demonstrated antitumor activity (T/C: 40%, at 75 mg/kg) in a human colorectal cancer Colo205 xenograft model in mice.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proteínas Cromosómicas no Histona/antagonistas & inhibidores , Piridinas/síntesis química , Piridinas/farmacología , Animales , Sitios de Unión , Diseño de Fármacos , Células HeLa , Histonas/metabolismo , Humanos , Ligandos , Ratones , Mitosis/efectos de los fármacos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosforilación , Electricidad Estática , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The molecular mechanism responsible that determines cell fate after mitotic slippage is unclear. Here we investigate the post-mitotic effects of different mitotic aberrations--misaligned chromosomes produced by CENP-E inhibition and monopolar spindles resulting from Eg5 inhibition. Eg5 inhibition in cells with an impaired spindle assembly checkpoint (SAC) induces polyploidy through cytokinesis failure without a strong anti-proliferative effect. In contrast, CENP-E inhibition causes p53-mediated post-mitotic apoptosis triggered by chromosome missegregation. Pharmacological studies reveal that aneuploidy caused by the CENP-E inhibitor, Compound-A, in SAC-attenuated cells causes substantial proteotoxic stress and DNA damage. Polyploidy caused by the Eg5 inhibitor does not produce this effect. Furthermore, p53-mediated post-mitotic apoptosis is accompanied by aneuploidy-associated DNA damage response and unfolded protein response activation. Because Compound-A causes p53 accumulation and antitumour activity in an SAC-impaired xenograft model, CENP-E inhibitors could be potential anticancer drugs effective against SAC-impaired tumours.
Asunto(s)
Aneuploidia , Apoptosis , Daño del ADN , Puntos de Control de la Fase M del Ciclo Celular/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Caspasa 3/genética , Caspasa 3/metabolismo , Caspasa 7 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Regulación de la Expresión Génica , Células HeLa , Xenoinjertos , Humanos , Cinesinas/antagonistas & inhibidores , Ratones , Ratones Desnudos , Mitosis , Neoplasias Experimentales , Estrés FisiológicoRESUMEN
Centromere-associated protein-E (CENP-E), a mitotic kinesin that plays an important role in mitotic progression, is an attractive target for cancer therapeutic drugs. For the purpose of developing novel CENP-E inhibitors as cancer therapeutics, we investigated a fused bicyclic compound identified by high throughput screening, 4-oxo-4,5-dihydrothieno[3,4-c]pyridine-6-carboxamide 1a. Based on this scaffold, we designed inhibitors for efficient binding at the L5 site in CENP-E utilizing homology modeling as well as electrostatic potential map (EPM) analysis to enhance CENP-E inhibitory activity. This resulted in a new lead, 5-bromoimidazo[1,2-a]pyridine 7, which showed potent CENP-E enzyme inhibition (IC50: 50nM) and cellular activity with accumulation of phosphorylated histone H3 in HeLa cells. Our homology model and EPM analysis proved to be useful tools for the rational design of CENP-E inhibitors.