RESUMEN

The existence of multiple centrosomes in some cancer cells can lead to cell death through the formation of multipolar mitotic spindles and consequent aberrant cell division. Many cancer cells rely on HSET (KIFC1) to cluster the extra centrosomes into two groups to mimic the bipolar spindle formation of non-centrosome-amplified cells and ensure their survival. Here, we report the discovery of a novel 2-(3-benzamidopropanamido)thiazole-5-carboxylate with micromolar in vitro inhibition of HSET (KIFC1) through high-throughput screening and its progression to ATP-competitive compounds with nanomolar biochemical potency and high selectivity against the opposing mitotic kinesin Eg5. Induction of the multipolar phenotype was shown in centrosome-amplified human cancer cells treated with these inhibitors. In addition, a suitable linker position was identified to allow the synthesis of both fluorescent- and trans-cyclooctene (TCO)-tagged probes, which demonstrated direct compound binding to the HSET protein and confirmed target engagement in cells, through a click-chemistry approach.

Asunto(s)

Cinesinas , Tiazoles , Humanos , Línea Celular Tumoral , Centrosoma/metabolismo , Cinesinas/antagonistas & inhibidores , Cinesinas/genética , Cinesinas/metabolismo , Mitosis , Huso Acromático/metabolismo , Tiazoles/química , Tiazoles/farmacología

RESUMEN

BOS172722 (CCT289346) is a highly potent, selective, and orally bioavailable inhibitor of spindle assembly checkpoint kinase MPS1. BOS172722 treatment alone induces significant sensitization to death, particularly in highly proliferative triple-negative breast cancer (TNBC) cell lines with compromised spindle assembly checkpoint activity. BOS172722 synergizes with paclitaxel to induce gross chromosomal segregation defects caused by MPS1 inhibitor-mediated abrogation of the mitotic delay induced by paclitaxel treatment. In in vivo pharmacodynamic experiments, BOS172722 potently inhibits the spindle assembly checkpoint induced by paclitaxel in human tumor xenograft models of TNBC, as measured by inhibition of the phosphorylation of histone H3 and the phosphorylation of the MPS1 substrate, KNL1. This mechanistic synergy results in significant in vivo efficacy, with robust tumor regressions observed for the combination of BOS172722 and paclitaxel versus either agent alone in long-term efficacy studies in multiple human tumor xenograft TNBC models, including a patient-derived xenograft and a systemic metastasis model. The current target indication for BOS172722 is TNBC, based on their high sensitivity to MPS1 inhibition, the well-defined clinical patient population with high unmet need, and the synergy observed with paclitaxel.

Asunto(s)

Puntos de Control del Ciclo Celular , Pirimidinas/farmacología , Huso Acromático/metabolismo , Triazoles/farmacología , Neoplasias de la Mama Triple Negativas/patología , Animales , Disponibilidad Biológica , Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Segregación Cromosómica/efectos de los fármacos , Cromosomas Humanos/genética , Sinergismo Farmacológico , Humanos , Ratones , Fosfohidrolasa PTEN/metabolismo , Paclitaxel/farmacología , Paclitaxel/uso terapéutico , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/química , Huso Acromático/efectos de los fármacos , Triazoles/química , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico

RESUMEN

Monopolar spindle 1 (MPS1) occupies a central role in mitosis and is one of the main components of the spindle assembly checkpoint. The MPS1 kinase is an attractive cancer target, and herein, we report the discovery of the clinical candidate BOS172722. The starting point for our work was a series of pyrido[3,4- d]pyrimidine inhibitors that demonstrated excellent potency and kinase selectivity but suffered from rapid turnover in human liver microsomes (HLM). Optimizing HLM stability proved challenging since it was not possible to identify a consistent site of metabolism and lowering lipophilicity proved unsuccessful. Key to overcoming this problem was the finding that introduction of a methyl group at the 6-position of the pyrido[3,4- d]pyrimidine core significantly improved HLM stability. Met ID studies suggested that the methyl group suppressed metabolism at the distant aniline portion of the molecule, likely by blocking the preferred pharmacophore through which P450 recognized the compound. This work ultimately led to the discovery of BOS172722 as a Phase 1 clinical candidate.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Descubrimiento de Drogas , Microsomas Hepáticos/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/química , Pirimidinas/farmacología , Triazoles/química , Triazoles/farmacología , Animales , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Ensayos Clínicos Fase I como Asunto , Femenino , Humanos , Masculino , Metilación , Ratones , Microsomas Hepáticos/efectos de los fármacos , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Inhibidores de Proteínas Quinasas/farmacocinética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacocinética , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Distribución Tisular , Triazoles/farmacocinética

RESUMEN

Monopolar spindle 1 (MPS1) plays a central role in the transition of cells from metaphase to anaphase and is one of the main components of the spindle assembly checkpoint. Chromosomally unstable cancer cells rely heavily on MPS1 to cope with the stress arising from abnormal numbers of chromosomes and centrosomes and are thus more sensitive to MPS1 inhibition than normal cells. We report the discovery and optimization of a series of new pyrido[3,4-d]pyrimidine based inhibitors via a structure-based hybridization approach from our previously reported inhibitor CCT251455 and a modestly potent screening hit. Compounds in this novel series display excellent potency and selectivity for MPS1, which translates into biomarker modulation in an in vivo human tumor xenograft model.

Asunto(s)

Proteínas de Ciclo Celular/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas de Ciclo Celular/química , Descubrimiento de Drogas , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Proteínas Tirosina Quinasas/química

RESUMEN

CDK5 regulatory subunit associated protein 3 (CDK5RAP3) is a novel activator of PAK4 and processes important pro-metastatic function in hepatocarcinogenesis. However, it remains unclear if there are other mechanisms by which CDK5RAP3 promotes HCC metastasis. Here, we showed that in CDK5RAP3 stable knockdown SMMC-7721 HCC cells, p14(ARF) tumor suppressor was upregulated at protein and mRNA levels, and ectopic expression of CDK5RAP3 was found to repress the transcription of p14(ARF). Using chromatin immunoprecipitation assay, we demonstrated that CDK5RAP3 bound to p14(ARF) promoter in vivo. Furthermore, knockdown of p14(ARF) in CDK5RAP3 stable knockdown HCC cells reversed the suppression of HCC cell invasiveness mediated by knockdown of CDK5RAP3. Taken together, our findings provide the new evidence that overexpression of CDK5RAP3 promotes HCC metastasis via downregulation of p14(ARF).

Asunto(s)

Carcinoma Hepatocelular/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Neoplasias Hepáticas/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteína p14ARF Supresora de Tumor/fisiología , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Hepáticas/patología , Microscopía Confocal , Regiones Promotoras Genéticas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p14ARF Supresora de Tumor/genética , Proteínas Supresoras de Tumor

RESUMEN

Interleukin 6 (IL-6) is pleiotropic cytokine playing an important role in inflammatory response. Other than classical immune tissues, IL-6 is also produced in muscle cells under specific conditions. Four-and-a-half LIM-only protein 2 (FHL2) is preferentially expressed in skeletal and cardiac muscle cells compared to other tissues indicating it has an important role in skeletal muscle and cardiovascular system. In this report, the regulation of IL-6 by FHL2 in muscle cells was investigated. We demonstrated that FHL2 overexpression increased IL-6 mRNA level and its protein secretion in skeletal myoblasts. In contrast, the IL-6 secretion was significantly decreased after FHL2-knockdown by siRNA in response to TNFα stimulation. We further showed that FHL2-mediated induction of IL-6 was regulated by the activation of IL-6 promoter through stimulating NF-κB and p38 MAPK signaling pathway. Our results further illustrated the molecular mechanisms of IL-6 production, which provides new insights in the roles of FHL2 in post-injury inflammation or cytoprotection of muscle cells.

Asunto(s)

Interleucina-6/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Células Musculares/enzimología , Proteínas Musculares/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Western Blotting , Línea Celular , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas I-kappa B/metabolismo , Interleucina-6/genética , Luciferasas/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Células Musculares/metabolismo , Mioblastos/metabolismo , Regiones Promotoras Genéticas/genética , Proteolisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección

RESUMEN

The CDK5 kinase regulatory subunit-associated protein 3 (CDK5RAP3 or C53/LZAP) regulates apoptosis induced by genotoxic stress. Although CDK5RAP3 has been implicated in cancer progression, its exact role in carcinogenesis is not well established. In this article, we report that CDK5RAP3 has an important prometastatic function in hepatocarcinogenesis. An examination of human hepatocellular carcinoma (HCC) samples revealed at least twofold overexpression of CDK5RAP3 transcripts in 58% (39/67) of HCC specimens when compared with corresponding nontumorous livers. CDK5RAP3 overexpression was associated with more aggressive biological behavior. In HCC cell lines, stable overexpression of CDK5RAP3 promoted, and small interfering RNA-mediated knockdown inhibited, tumorigenic activity and metastatic potential. We found that overexpression of CDK5RAP3 and p21-activated protein kinase 4 (PAK4) correlated in human HCCs, and that CDK5RAP3 was a novel binding partner of PAK4, and this binding enhanced PAK4 activity. siRNA-mediated knockdown of PAK4 in CDK5RAP3-expressing HCC cells reversed the enhanced cell invasiveness mediated by CDK5RAP3 overexpression, implying that PAK4 is essential for CDK5RAP3 function. Taken together, our findings reveal that CDK5RAP3 is widely overexpressed in HCC and that overexpression of CDK5RAP3 promotes HCC metastasis through PAK4 activation.

Asunto(s)

Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas del Tejido Nervioso/biosíntesis , Quinasas p21 Activadas/metabolismo , Animales , Carcinoma Hepatocelular/enzimología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Activación Enzimática , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/enzimología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas del Tejido Nervioso/genética , Transfección , Proteínas Supresoras de Tumor

Asunto(s)

Proteínas Fluorescentes Verdes/fisiología , Interleucina-6/metabolismo , Mioblastos/metabolismo , Animales , Western Blotting , Línea Celular , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Interleucina-6/genética , Ratones , Mioblastos/citología , Plásmidos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección