RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive type of pancreatic cancer, which rapidly develops resistance to the current standard of care. Several oncolytic Human AdenoViruses (HAdVs) have been reported to re-sensitize drug-resistant cancer cells and in combination with chemotherapeutics attenuate solid tumour growth. Obstacles preventing greater clinical success are rapid hepatic elimination and limited viral replication and spread within the tumour microenvironment. We hypothesised that higher intratumoural levels of the virus could be achieved by altering cellular epigenetic regulation. Here we report on the screening of an enriched epigenetics small molecule library and validation of six compounds that increased viral gene expression and replication. The greatest effects were observed with three epigenetic inhibitors targeting bromodomain (BRD)-containing proteins. Specifically, BRD4 inhibitors enhanced the efficacy of Ad5 wild type, Ad∆∆, and Ad-3∆-A20T in 3-dimensional co-culture models of PDAC and in vivo xenografts. RNAseq analysis demonstrated that the inhibitors increased viral E1A expression, altered expression of cell cycle regulators and inflammatory factors, and attenuated expression levels of tumour cell oncogenes such as c-Myc and Myb. The data suggest that the tumour-selective Ad∆∆ and Ad-3∆-A20T combined with epigenetic inhibitors is a novel strategy for the treatment of PDAC by eliminating both cancer and associated stromal cells to pave the way for immune cell access even after systemic delivery of the virus.
Assuntos
Carcinoma Ductal Pancreático , Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias Pancreáticas , Humanos , Proteínas Nucleares/genética , Epigênese Genética , Vírus Oncolíticos/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patologia , Terapia Viral Oncolítica/métodos , Adenoviridae/genética , Microambiente Tumoral , Proteínas que Contêm Bromodomínio , Proteínas de Ciclo Celular/metabolismoRESUMO
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.
Assuntos
Cinesinas , Tiazóis , Humanos , Linhagem Celular Tumoral , Centrossomo/metabolismo , Cinesinas/antagonistas & inibidores , Cinesinas/genética , Cinesinas/metabolismo , Mitose , Fuso Acromático/metabolismo , Tiazóis/química , Tiazóis/farmacologiaRESUMO
BACKGROUND: Activating mutations in the Fms-like tyrosine kinase 3 (FLT3) are among the most prevalent oncogenic mutations in acute myeloid leukaemia. Inhibitors selectively targeting FLT3 kinase have shown promising clinical activity; their success in the clinic, however, has been limited due to the emergence of acquired resistance. METHODS: CCT245718 was identified and characterised as a dual Aurora A/FLT3 inhibitor through cell-based and biochemical assays. The ability of CCT245718 to overcome TKD-mediated resistance was evaluated in a cell line-based model of drug resistance to FLT3 inhibitors. RESULTS: CCT245718 exhibits potent antiproliferative activity towards FLT3-ITD + AML cell lines and strongly binds to FLT3-ITD and TKD (D835Y) mutants in vitro. Activities of both FLT3-ITD and Aurora A are also inhibited in cells. Inhibition of FLT3 results in reduced phosphorylation of STAT5, downregulation of survivin and induction of apoptotic cell death. Moreover, CCT245718 overcomes TKD-mediated resistance in a MOLM-13-derived cell line containing FLT3 with both ITD and D835Y mutations. It also inhibits FLT3 signalling in both parental and resistant cell lines compared to FLT3-specific inhibitor MLN518, which is only active in the parental cell line. CONCLUSIONS: Our results demonstrate that CCT245718 is a potent dual FLT3/Aurora A inhibitor that can overcome TKD-mediated acquired resistance.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Imidazóis/farmacologia , Leucemia Mieloide Aguda/enzimologia , Tirosina Quinase 3 Semelhante a fms/genética , Aurora Quinase A/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/química , Leucemia Mieloide Aguda/tratamento farmacológico , Mutação , Fosforilação , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT5/metabolismo , Survivina/metabolismo , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Tirosina Quinase 3 Semelhante a fms/químicaRESUMO
Internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML); it causes constitutive activation of FLT3 kinase and is associated with high relapse rates and poor survival. Small-molecule inhibition of FLT3 represents an attractive therapeutic strategy for this subtype of AML, although resistance from secondary FLT3 tyrosine kinase domain (FLT3-TKD) mutations is an emerging clinical problem. CCT241736 is an orally bioavailable, selective, and potent dual inhibitor of FLT3 and Aurora kinases. FLT3-ITD+ cells with secondary FLT3-TKD mutations have high in vitro relative resistance to the FLT3 inhibitors quizartinib and sorafenib, but not to CCT241736. The mechanism of action of CCT241736 results in significant in vivo efficacy, with inhibition of tumor growth observed in efficacy studies in FLT3-ITD and FLT3-ITD-TKD human tumor xenograft models. The efficacy of CCT241736 was also confirmed in primary samples from AML patients, including those with quizartinib-resistant disease, which induces apoptosis through inhibition of both FLT3 and Aurora kinases. The unique combination of CCT241736 properties based on robust potency, dual selectivity, and significant in vivo activity indicate that CCT241736 is a bona fide clinical drug candidate for FLT3-ITD and TKD AML patients with resistance to current drugs.
Assuntos
Leucemia Mieloide Aguda , Compostos de Fenilureia , Aurora Quinases , Benzotiazóis , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Tirosina Quinase 3 Semelhante a fms/genéticaRESUMO
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.
Assuntos
Pontos de Checagem do Ciclo Celular , Pirimidinas/farmacologia , Fuso Acromático/metabolismo , Triazóis/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Animais , Disponibilidade Biológica , Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Segregação de Cromossomos/efeitos dos fármacos , Cromossomos Humanos/genética , Sinergismo Farmacológico , Humanos , Camundongos , PTEN Fosfo-Hidrolase/metabolismo , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/química , Fuso Acromático/efeitos dos fármacos , Triazóis/química , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
CCT241736 is a dual fms-like tyrosine kinase 3 (FLT3)/Aurora kinase inhibitor in development for the treatment of acute myeloid leukaemia. The successful development of any new drug relies on adequate safety testing including preclinical toxicology studies. Selection of an appropriate preclinical species requires a thorough understanding of the compound's metabolic clearance and pathways, as well as other pharmacokinetic and pharmacodynamic considerations. In addition, elucidation of the metabolising enzymes in human facilitates improved clinical prediction based on population pharmacokinetics and can inform drug-drug interaction studies. Intrinsic clearance (CLint) determination and metabolite profiling of CCT241736 in human and four preclinical species (dog, minipig, rat and mouse) was undertaken in cryopreserved hepatocytes and liver microsomes. Recombinant human cytochrome P450 bactosomes (rCYP) were utilised to provide reaction phenotyping data and support prediction of metabolic pathways. CCT241736 exhibited low CLint in both hepatocytes and liver microsomes of human, dog, minipig and rat, but considerably higher CLint in mouse. CYP3A4 and CYP3A5 were identified as the major enzymes responsible for biotransformation of CCT241736 in human, exclusively forming five out of seven metabolites. Minipig showed greatest similarity to human with regard to both overall metabolic profile and abundance of specific metabolites relative to parent compound, and is therefore proposed as the most appropriate toxicological species. The greatest disparity was observed between human and dog. Based on metabolic profile, either mouse or rat is a suitable rodent species for toxicology studies.
Assuntos
Aurora Quinases/antagonistas & inibidores , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidores , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Avaliação Pré-Clínica de Medicamentos , Feminino , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos Endogâmicos ICR , Microssomos Hepáticos/metabolismo , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos Sprague-Dawley , Especificidade da Espécie , Suínos , Porco Miniatura , Testes de ToxicidadeRESUMO
Cellular models for siRNA and small molecule high-throughput screening have been widely used in the last decade to identify targets for drug discovery. As an example, we present a twofold readout approach based on cell viability and multipolar phenotype. To maximize the discovery of potential targets and at the same time reduce the number of false positives in our dataset, we have combined focused and rationally designed custom siRNA libraries with small molecule inhibitor libraries. Here we describe a cellular model for centrosome amplification as an example of how to design and perform a multiple readout/multiple screening strategy.
Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , RNA Interferente Pequeno/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Biblioteca Gênica , Ensaios de Triagem em Larga Escala/métodos , Humanos , Interferência de RNA/efeitos dos fármacosRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Descoberta de Drogas , Microssomos Hepáticos/metabolismo , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirimidinas/química , Pirimidinas/farmacologia , Triazóis/química , Triazóis/farmacologia , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Ensaios Clínicos Fase I como Assunto , Feminino , Humanos , Masculino , Metilação , Camundongos , Microssomos Hepáticos/efeitos dos fármacos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Distribuição Tecidual , Triazóis/farmacocinéticaRESUMO
Accurate chromosome segregation is dependent on the spindle assembly checkpoint (SAC). In current models, the key direct role of Aurora B in the SAC has been suggested to be to promote rapid kinetochore localisation of MPS1, allowing MPS1 to generate the checkpoint signal. However, Aurora B is also thought to play an indirect role in the SAC through the destabilisation of kinetochore-microtubule (KT-MT) attachments. Here, we demonstrate that Aurora B activity is not required for the kinetochore recruitment of the majority of SAC proteins. More importantly, we show that the primary role of Aurora B in the SAC is to prevent the premature removal of SAC proteins from the kinetochore, which is strictly dependent on KT-MT interactions. Moreover, in the presence of KT-MT interactions, Aurora B inhibition silences a persistent SAC induced by tethering MPS1 to the kinetochore. This explains the highly synergistic interaction between Aurora B and MPS1 inhibitors to override the SAC, which is lost when cells are pre-arrested in nocodazole. Furthermore, we show that Aurora B and MPS1 inhibitors synergistically kill a panel of breast and colon cancer cell lines, including cells that are otherwise insensitive to Aurora B inhibitors alone. These data demonstrate that the major role of Aurora B in SAC is to prevent the removal of SAC proteins from tensionless kinetochores, thus inhibiting premature SAC silencing, and highlights a therapeutic strategy through combination of Aurora B and MPS1 inhibitors.
RESUMO
The cell adhesion glycoprotein E-cadherin (CDH1) is commonly inactivated in breast tumors. Precision medicine approaches that exploit this characteristic are not available. Using perturbation screens in breast tumor cells with CRISPR/Cas9-engineered CDH1 mutations, we identified synthetic lethality between E-cadherin deficiency and inhibition of the tyrosine kinase ROS1. Data from large-scale genetic screens in molecularly diverse breast tumor cell lines established that the E-cadherin/ROS1 synthetic lethality was not only robust in the face of considerable molecular heterogeneity but was also elicited with clinical ROS1 inhibitors, including foretinib and crizotinib. ROS1 inhibitors induced mitotic abnormalities and multinucleation in E-cadherin-defective cells, phenotypes associated with a defect in cytokinesis and aberrant p120 catenin phosphorylation and localization. In vivo, ROS1 inhibitors produced profound antitumor effects in multiple models of E-cadherin-defective breast cancer. These data therefore provide the preclinical rationale for assessing ROS1 inhibitors, such as the licensed drug crizotinib, in appropriately stratified patients.Significance: E-cadherin defects are common in breast cancer but are currently not targeted with a precision medicine approach. Our preclinical data indicate that licensed ROS1 inhibitors, including crizotinib, should be repurposed to target E-cadherin-defective breast cancers, thus providing the rationale for the assessment of these agents in molecularly stratified phase II clinical trials. Cancer Discov; 8(4); 498-515. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Caderinas/deficiência , Crizotinibe/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Anilidas/farmacologia , Anilidas/uso terapêutico , Animais , Antígenos CD/genética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias da Mama/metabolismo , Caderinas/genética , Linhagem Celular Tumoral , Crizotinibe/uso terapêutico , Feminino , Humanos , Camundongos , Mutação , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Quinolinas/farmacologia , Quinolinas/uso terapêuticoRESUMO
Triple negative breast cancers (TNBCs) lack recurrent targetable driver mutations but demonstrate frequent copy number aberrations (CNAs). Here, we describe an integrative genomic and RNAi-based approach that identifies and validates gene addictions in TNBCs. CNAs and gene expression alterations are integrated and genes scored for pre-specified target features revealing 130 candidate genes. We test functional dependence on each of these genes using RNAi in breast cancer and non-malignant cells, validating malignant cell selective dependence upon 37 of 130 genes. Further analysis reveals a cluster of 13 TNBC addiction genes frequently co-upregulated that includes genes regulating cell cycle checkpoints, DNA damage response, and malignant cell selective mitotic genes. We validate the mechanism of addiction to a potential drug target: the mitotic kinesin family member C1 (KIFC1/HSET), essential for successful bipolar division of centrosome-amplified malignant cells and develop a potential selection biomarker to identify patients with tumors exhibiting centrosome amplification.
Assuntos
Genômica/métodos , Neoplasias de Mama Triplo Negativas/genética , Pontos de Checagem do Ciclo Celular/genética , Variações do Número de Cópias de DNA/genética , Dano ao DNA/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Inativação Gênica/fisiologia , Humanos , Cinesinas/genética , Interferência de RNARESUMO
Thousand-and-one amino acid kinases (TAOK) 1 and 2 are activated catalytically during mitosis and can contribute to mitotic cell rounding and spindle positioning. Here, we characterize a compound that inhibits TAOK1 and TAOK2 activity with IC50 values of 11 to 15 nmol/L, is ATP-competitive, and targets these kinases selectively. TAOK inhibition or depletion in centrosome-amplified SKBR3 or BT549 breast cancer cell models increases the mitotic population, the percentages of mitotic cells displaying amplified centrosomes and multipolar spindles, induces cell death, and inhibits cell growth. In contrast, nontumorigenic and dividing bipolar MCF-10A breast cells appear less dependent on TAOK activity and can complete mitosis and proliferate in the presence of the TAOK inhibitor. We demonstrate that TAOK1 and TAOK2 localize to the cytoplasm and centrosomes respectively during mitosis. Live cell imaging shows that the TAOK inhibitor prolongs the duration of mitosis in SKBR3 cells, increases mitotic cell death, and reduces the percentages of cells exiting mitosis, whereas MCF-10A cells continue to divide and proliferate. Over 80% of breast cancer tissues display supernumerary centrosomes, and tumor cells frequently cluster extra centrosomes to avoid multipolar mitoses and associated cell death. Consequently, drugs that stimulate centrosome declustering and induce multipolarity are likely to target dividing centrosome-amplified cancer cells preferentially, while sparing normal bipolar cells. Our results demonstrate that TAOK inhibition can enhance centrosome declustering and mitotic catastrophe in cancer cells, and these proteins may therefore offer novel therapeutic targets suitable for drug inhibition and the potential treatment of breast cancers, where supernumerary centrosomes occur. Mol Cancer Ther; 16(11); 2410-21. ©2017 AACR.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Centrossomo/efeitos dos fármacos , Feminino , Humanos , Mitose/efeitos dos fármacos , Inibidores de Proteínas Quinases/síntese química , Proteínas Quinases/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/efeitos dos fármacosRESUMO
BACKGROUND: F-box and WD40 repeat domain-containing 7 (FBXW7) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of multiple oncogenic substrates. The tumour suppressor function is frequently lost in multiple cancers through genetic deletion and mutations in a broad range of tumours. Loss of FBXW7 functionality results in the stabilisation of multiple major oncoproteins, culminating in increased cellular proliferation and pro-survival pathways, cell cycle deregulation, chromosomal instability and altered metabolism. Currently, there is no therapy to specifically target FBXW7-deficient tumours. METHODS: We performed a siRNA kinome screen to identify synthetically lethal hits to FBXW7 deficiency. RESULTS: We identified and validated cyclin G-associated kinase (GAK) as a potential new therapeutic target. Combined loss of FBXW7 and GAK caused cell cycle defects, formation of multipolar mitoses and the induction of apoptosis. The synthetic lethal mechanism appears to be independent of clathrin-mediated receptor endocytosis function of GAK. CONCLUSIONS: These data suggest a putative therapeutic strategy for a large number of different types of human cancers with FBXW7 loss, many of which have a paucity of molecular abnormalities and treatment options.
Assuntos
Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/genética , Proteínas F-Box/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mitose/genética , Neoplasias/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Clatrina/antagonistas & inibidores , Proteína 7 com Repetições F-Box-WD , Humanos , Interferência de RNA , RNA Interferente Pequeno , Sulfonamidas/farmacologia , Mutações Sintéticas Letais , Tiazolidinas/farmacologiaRESUMO
BACKGROUND: The main role of the cell cycle is to enable error-free DNA replication, chromosome segregation and cytokinesis. One of the best characterised checkpoint pathways is the spindle assembly checkpoint, which prevents anaphase onset until the appropriate attachment and tension across kinetochores is achieved. MPS1 kinase activity is essential for the activation of the spindle assembly checkpoint and has been shown to be deregulated in human tumours with chromosomal instability and aneuploidy. Therefore, MPS1 inhibition represents an attractive strategy to target cancers. METHODS: To evaluate CCT271850 cellular potency, two specific antibodies that recognise the activation sites of MPS1 were used and its antiproliferative activity was determined in 91 human cancer cell lines. DLD1 cells with induced GFP-MPS1 and HCT116 cells were used in in vivo studies to directly measure MPS1 inhibition and efficacy of CCT271850 treatment. RESULTS: CCT271850 selectively and potently inhibits MPS1 kinase activity in biochemical and cellular assays and in in vivo models. Mechanistically, tumour cells treated with CCT271850 acquire aberrant numbers of chromosomes and the majority of cells divide their chromosomes without proper alignment because of abrogation of the mitotic checkpoint, leading to cell death. We demonstrated a moderate level of efficacy of CCT271850 as a single agent in a human colorectal carcinoma xenograft model. CONCLUSIONS: CCT271850 is a potent, selective and orally bioavailable MPS1 kinase inhibitor. On the basis of in vivo pharmacodynamic vs efficacy relationships, we predict that more than 80% inhibition of MPS1 activity for at least 24 h is required to achieve tumour stasis or regression by CCT271850.
Assuntos
Proteínas de Ciclo Celular/genética , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Células HCT116 , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Descoberta de Drogas , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/químicaRESUMO
Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided 7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights provide options for the design of compounds interacting with the DFG motif or with Thr217.
Assuntos
Aurora Quinases/antagonistas & inibidores , Aurora Quinases/química , Imidazóis/síntese química , Imidazóis/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/síntese química , Pirazóis/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalização , Relação Dose-Resposta a Droga , Humanos , Imidazóis/química , Camundongos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Modelos Moleculares , Estrutura Molecular , Pirazóis/química , Piridinas/química , Relação Estrutura-AtividadeRESUMO
Acquired resistance to therapy is perhaps the greatest challenge to effective clinical management of cancer. With several inhibitors of the mitotic checkpoint kinase MPS1 in preclinical development, we sought to investigate how resistance against these inhibitors may arise so that mitigation or bypass strategies could be addressed as early as possible. Toward this end, we modeled acquired resistance to the MPS1 inhibitors AZ3146, NMS-P715, and CCT251455, identifying five point mutations in the kinase domain of MPS1 that confer resistance against multiple inhibitors. Structural studies showed how the MPS1 mutants conferred resistance by causing steric hindrance to inhibitor binding. Notably, we show that these mutations occur in nontreated cancer cell lines and primary tumor specimens, and that they also preexist in normal lymphoblast and breast tissues. In a parallel piece of work, we also show that the EGFR p.T790M mutation, the most common mutation conferring resistance to the EGFR inhibitor gefitinib, also preexists in cancer cells and normal tissue. Our results therefore suggest that mutations conferring resistance to targeted therapy occur naturally in normal and malignant cells and these mutations do not arise as a result of the increased mutagenic plasticity of cancer cells.
Assuntos
Proteínas de Ciclo Celular/genética , Resistencia a Medicamentos Antineoplásicos/genética , Mutação Puntual , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Compostos de Anilina/química , Compostos de Anilina/metabolismo , Compostos de Anilina/farmacologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Células HCT116 , Células HEK293 , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/metabolismo , Compostos Heterocíclicos com 2 Anéis/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Pirazóis/química , Pirazóis/metabolismo , Pirazóis/farmacologia , Quinazolinas/química , Quinazolinas/metabolismo , Quinazolinas/farmacologiaRESUMO
The quest for potent and selective small molecule inhibitors of the Aurora kinases has been long and resource intensive with multiple agents progressed to the clinic. To definitively explore the potential for clinical efficacy at well-tolerated dosing schedules requires a well-characterized, selective inhibitor with pharmacokinetic properties, flexible dosing regimen, and suite of target engagement biomarkers suitable for clinical use. AMG900 is a promising opportunity to definitively test the clinical benefit of dual Aurora kinase A and B inhibition.
Assuntos
Aurora Quinases/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ftalazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Humanos , Neoplasias/enzimologiaRESUMO
Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC.
Assuntos
Antineoplásicos/farmacologia , Proteínas do Citoesqueleto/deficiência , Neoplasias Pulmonares/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Cisplatino/farmacologia , Proteínas do Citoesqueleto/genética , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Pulmonares/genética , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Ftalazinas , Piperazinas , Rad51 Recombinase/genéticaRESUMO
The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions.