RESUMO
We previously demonstrated that natural product-inspired 3,4-dihydropyrrolo[1,2-a]pyrazin-1(2H)-ones derivatives delivered potent and selective PIM kinases inhibitors however with non-optimal ADME/PK properties and modest oral bioavailability. Herein, we describe a structure-based scaffold decoration and a stereoselective approach to this chemical class. The synthesis, structure-activity relationship studies, chiral analysis, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Compound 20c demonstrated excellent potency on PIM1 and PIM2 with exquisite kinases selectivity and PK properties that efficiently and dose-dependently promoted c-Myc degradation and appear to be promising lead compounds for further development.
Assuntos
Alcaloides , Antineoplásicos , Alcaloides/farmacologia , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
In this article we describe the identification of unprecedented ATP-competitive ChoKα inhibitors starting from initial hit NMS-P830 that binds to ChoKα in an ATP concentration-dependent manner. This result is confirmed by the co-crystal structure of NMS-P830 in complex with Δ75-ChoKα. NMS-P830 is able to inhibit ChoKα in cells resulting in the reduction of intracellular phosphocholine formation. A structure-based medicinal chemistry program resulted in the identification of selective compounds that have good biochemical activity, solubility and metabolic stability and are suitable for further optimization. The ChoKα inhibitors disclosed in this article demonstrate for the first time the possibility to inhibit ChoKα with ATP-competitive compounds.
Assuntos
Trifosfato de Adenosina/antagonistas & inibidores , Colina Quinase/antagonistas & inibidores , Cicloexanos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Trifosfato de Adenosina/metabolismo , Colina Quinase/metabolismo , Cicloexanos/síntese química , Cicloexanos/química , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-AtividadeRESUMO
Background Pharmacological inhibition of polo-like kinase 1 (PLK1) represents a new approach for the treatment of solid tumors. This study was aimed at determining the first cycle dose-limiting toxicities (DLTs) and related maximum tolerated dose (MTD) of NMS-1286937, a selective ATP-competitive PLK1-specific inhibitor. Secondary objectives included evaluation of its safety and pharmacokinetic (PK) profile in plasma, its antitumor activity, and its ability to modulate intracellular targets in biopsied tissue. Methods This was a Phase I, open-label, dose-escalation trial in patients with advanced/metastatic solid tumors. A treatment cycle comprised 5 days of oral administration followed by 16 days of rest, for a total of 21 days (3-week cycle). Results Nineteen of 21 enrolled patients with confirmed metastatic disease received study medication. No DLTs occurred at the first 3 dose levels (6, 12, and 24 mg/m2/day). At the subsequent dose level (48 mg/m2/day), 2 of 3 patients developed DLTs. An intermediate level of 36 mg/m2/day was therefore investigated. Four patients were treated and two DLTs were observed. After further cohort expansion, the MTD and recommended phase II dose (RP2D) were determined to be 24 mg/m2/day. Disease stabilization, observed in several patients, was the best treatment response observed. Hematological toxicity (mostly thrombocytopenia and neutropenia) was the major DLT. Systemic exposure to NMS-1286937 increased with dose and was comparable between two cycles of treatment following oral administration of the drug. Conclusions This study successfully identified the MTD and DLTs for NMS-1286937 and characterized its safety profile.
Assuntos
Antineoplásicos , Proteínas de Ciclo Celular/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Administração Oral , Idoso , Antineoplásicos/administração & dosagem , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Relação Dose-Resposta a Droga , Feminino , Humanos , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/metabolismo , Neoplasias/patologia , Neutropenia/induzido quimicamente , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/farmacocinética , Trombocitopenia/induzido quimicamente , Resultado do Tratamento , Quinase 1 Polo-LikeRESUMO
Cancer cell survival is frequently dependent on the elevated levels of members of the Bcl-2 family of prosurvival proteins that bind to and inactivate BH3-domain pro-apoptotic cellular proteins. Small molecules that inhibit the protein-protein interactions between prosurvival and proapoptotic Bcl-2 family members (so-called "BH3 mimetics") have a potential therapeutic value, as indicated by clinical findings obtained with ABT-263 (navitoclax), a Bcl-2/Bcl-xL antagonist, and more recently with GDC-0199/ABT-199, a more selective antagonist of Bcl-2. Here, we report study results of the functional role of the prosurvival protein Mcl-1 against a panel of solid cancer cell lines representative of different tumor types. We observed silencing of Mcl-1 expression by small interfering RNAs (siRNAs) significantly reduced viability and induced apoptosis in almost 30% of cell lines tested, including lung and breast adenocarcinoma, as well as glioblastoma derived lines. Most importantly, we provide a mechanistic basis for this sensitivity by showing antagonism of Mcl-1 function with specific BH3 peptides against isolated mitochondria induces Bak oligomerization and cytochrome c release, therefore demonstrating that mitochondria from Mcl-1-sensitive cells depend on Mcl-1 for their integrity and that antagonizing Mcl-1 function is sufficient to induce apoptosis. Thus, our results lend further support for considering Mcl-1 as a therapeutic target in a number of solid cancers and support the rationale for development of small molecule BH3-mimetics antagonists of this protein.
Assuntos
Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Apoptose , Linhagem Celular Tumoral , Sobrevivência Celular , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
BACKGROUND: Activated anaplastic lymphoma kinase (ALK) gene fusions are recurrent events in a small fraction of colorectal cancers (CRCs), although these events have not yet been exploited as in other malignancies. METHODS: We detected ALK protein expression by immunohistochemistry and gene rearrangements by fluorescence in situ hybridisation in the ALKA-372-001 phase I study of the pan-Trk, ROS1, and ALK inhibitor entrectinib. One out of 487 CRCs showed ALK positivity with a peculiar pattern that prompted further characterisation by targeted sequencing using anchored multiplex PCR. RESULTS: A novel ALK fusion with the carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene (CAD-ALK fusion gene) was identified. It resulted from inversion within chromosome 2 and the fusion of exons 1-35 of CAD with exons 20-29 of ALK. After failure of previous standard therapies, treatment of this patient with the ALK inhibitor entrectinib resulted in a durable objective tumour response. CONCLUSIONS: We describe the novel CAD-ALK rearrangement as an oncogene and provide the first evidence of its drugability as a new molecular target in CRC.
Assuntos
Antineoplásicos/uso terapêutico , Aspartato Carbamoiltransferase/genética , Benzamidas/uso terapêutico , Carbamoil Fosfato Sintase (Glutamina-Hidrolizante)/genética , Neoplasias Colorretais/tratamento farmacológico , Di-Hidro-Orotase/genética , Rearranjo Gênico , Indazóis/uso terapêutico , Receptores Proteína Tirosina Quinases/genética , Quinase do Linfoma Anaplásico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
VCP (also known as p97 or Cdc48p in yeast) is an AAA(+) ATPase regulating endoplasmic reticulum-associated degradation. After high-throughput screening, we developed compounds that inhibit VCP via different mechanisms, including covalent modification of an active site cysteine and a new allosteric mechanism. Using photoaffinity labeling, structural analysis and mutagenesis, we mapped the binding site of allosteric inhibitors to a region spanning the D1 and D2 domains of adjacent protomers encompassing elements important for nucleotide-state sensing and ATP hydrolysis. These compounds induced an increased affinity for nucleotides. Interference with nucleotide turnover in individual subunits and distortion of interprotomer communication cooperated to impair VCP enzymatic activity. Chemical expansion of this allosteric class identified NMS-873, the most potent and specific VCP inhibitor described to date, which activated the unfolded protein response, interfered with autophagy and induced cancer cell death. The consistent pattern of cancer cell killing by covalent and allosteric inhibitors provided critical validation of VCP as a cancer target.
Assuntos
Acetanilidas/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Antineoplásicos/farmacologia , Benzotiazóis/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Acetanilidas/química , Adenosina Trifosfatases/metabolismo , Regulação Alostérica/efeitos dos fármacos , Antineoplásicos/química , Benzotiazóis/química , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Neoplasias/metabolismo , Relação Estrutura-Atividade , Proteína com ValosinaRESUMO
Compound 1, a hit from the screening of our chemical collection displaying activity against JAK2, was deconstructed for SAR analysis into three regions, which were explored. A series of compounds was synthesized leading to the identification of the potent and orally bioavailable JAK2 inhibitor 16 (NMS-P830), which showed an encouraging tumour growth inhibition in SET-2 xenograft tumour model, with evidence for JAK2 pathway suppression demonstrated by in vivo pharmacodynamic effects.
Assuntos
Amidas/síntese química , Antineoplásicos/síntese química , Janus Quinase 2/antagonistas & inibidores , Leucemia Megacarioblástica Aguda/tratamento farmacológico , Inibidores de Proteínas Quinases/síntese química , Pirróis/síntese química , Amidas/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Janus Quinase 2/química , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Leucemia Megacarioblástica Aguda/enzimologia , Leucemia Megacarioblástica Aguda/genética , Leucemia Megacarioblástica Aguda/patologia , Células Progenitoras de Megacariócitos/efeitos dos fármacos , Células Progenitoras de Megacariócitos/enzimologia , Células Progenitoras de Megacariócitos/patologia , Camundongos , Camundongos Nus , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteínas Quinases/farmacologia , Pirróis/farmacologia , Relação Estrutura-Atividade , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.
Assuntos
Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Quinazolinas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Estrutura Terciária de Proteína , Quinazolinas/síntese química , Quinazolinas/farmacologia , Relação Estrutura-AtividadeRESUMO
We report herein the discovery, structure guided design, synthesis and biological evaluation of a novel class of JAK2 inhibitors. Optimization of the series led to the identification of the potent and orally bioavailable JAK2 inhibitor 28 (NMS-P953). Compound 28 displayed significant tumour growth inhibition in SET-2 xenograft tumour model, with a mechanism of action confirmed in vivo by typical modulation of known biomarkers, and with a favourable pharmacokinetic and safety profile.
Assuntos
Antineoplásicos/farmacologia , Janus Quinase 2/antagonistas & inibidores , Neoplasias Experimentais/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Pirróis/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Janus Quinase 2/metabolismo , Camundongos , Camundongos SCID , Modelos Moleculares , Estrutura Molecular , Neoplasias Experimentais/patologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Pirimidinas/síntese química , Pirimidinas/química , Pirróis/síntese química , Pirróis/química , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
A novel series of PIM inhibitors was derived from a combined effort in natural product-inspired library generation and screening. The novel pyrrolo[1,2-a]pyrazinones initial hits are inhibitors of PIM isoforms with IC50 values in the low micromolar range. The application of a rational optimization strategy, guided by the determination of the crystal structure of the complex in the kinase domain of PIM1 with compound 1, led to the discovery of compound 15a, which is a potent PIM kinases inhibitor exhibiting excellent selectivity against a large panel of kinases, representative of each family. The synthesis, structure-activity relationship studies, and pharmacokinetic data of compounds from this inhibitor class are presented herein. Furthermore, the cellular activities including inhibition of cell growth and modulation of downstream targets are also described.
Assuntos
Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-pim-1/antagonistas & inibidores , Pirazinas/química , Pirazinas/farmacologia , Descoberta de Drogas , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/síntese química , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-pim-1/química , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Pirazinas/síntese químicaRESUMO
5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92, I95, and F99 pockets normally occupied by pro-apoptotic protein Bim. An X-ray crystal structure confirmed the proposed binding mode. Observation of cytochrome c release from isolated mitochondria in MV-411 cells provides further evidence of target inhibition. Compounds demonstrated submicromolar antiproliferative activity in Bcl-2/Bcl-xL dependent cell lines.
Assuntos
Antineoplásicos/síntese química , Pirazóis/síntese química , Pirimidinas/síntese química , Sulfonamidas/síntese química , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína bcl-X/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Citocromos c/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Moleculares , Ligação Proteica , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Pirazóis/farmacologia , Pirimidinas/farmacologia , Sulfonamidas/farmacologia , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/química , Proteína bcl-X/metabolismoRESUMO
A series of phenylacylsulfonamides has been prepared as antagonists of Bcl-2/Bcl-xL. In addition to potent binding affinities for both Bcl-2 and Bcl-xL, these compounds were shown to induce classical markers of apoptosis in isolated mitochondria. Overall weak cellular potency was improved by the incorporation of polar functionality resulting in compounds with moderate antiproliferative activity.
Assuntos
Antineoplásicos/síntese química , Mitocôndrias/efeitos dos fármacos , Sulfonamidas/síntese química , Proteína X Associada a bcl-2/antagonistas & inibidores , Proteína bcl-X/antagonistas & inibidores , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Citocromos c/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Mitocôndrias/metabolismo , Modelos Moleculares , Sulfonamidas/farmacologia , Proteína X Associada a bcl-2/química , Proteína bcl-X/químicaRESUMO
Design, synthesis, and SAR development led to the identification of the potent, novel, and selective pyrazole based inhibitor (7f) of Coactivator Associated Arginine Methyltransferase (CARM1).
Assuntos
Inibidores Enzimáticos/química , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Pirazóis/química , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Humanos , Proteína-Arginina N-Metiltransferases/metabolismo , Pirazóis/síntese química , Pirazóis/farmacologia , Relação Estrutura-AtividadeRESUMO
Poly(ADP-ribose) polymerase-1 (PARP-1) is an enzyme involved in signaling and repair of DNA single strand breaks. PARP-1 employs NAD+ to modify substrate proteins via the attachment of poly(ADP-ribose) chains. PARP-1 is a well established target in oncology, as testified by the number of marketed drugs (e.g., Lynparza, Rubraca, Zejula, and Talzenna) used for the treatment of ovarian, breast, and prostate tumors. Efforts in investigating an uncharted region of the previously identified isoindolinone carboxamide series delivered (S)-13 (NMS-P515), a potent inhibitor of PARP-1 both in biochemical (K d: 0.016 µM) and cellular (IC50: 0.027 µM) assays. Cocrystal structure allowed explaining NMS-P515 stereospecific inhibition of the target. After having ruled out potential loss of enantiopurity in vitro and in vivo, NMS-P515 was synthesized in an asymmetric fashion. NMS-P515 ADME profile and its antitumor activity in a mouse xenograft cancer model render the compound eligible for further optimization.
RESUMO
High-content analysis (HCA) is a term used to describe techniques involving multiplexed analysis of fluorescent markers to measure multiple cellular responses to biological stimuli or drug treatment. HCA is usually based on automated microscopy or related technologies, and its value lies in providing multiparametric information on single cells within a population. During the last decade, several HCA approaches have been developed and applied to assess cellular mechanism of action of pharmacologically relevant compounds identified through biochemical screening or similar in vitro methods. With automation and instrument development, these approaches have evolved to the extent that the technique is now routinely used in screening applications, including primary HTS on compound collections. Here, we review the field and discuss in particular the application of HCA to the discovery of small molecule inhibitors targeting kinases which are implicated in Oncology.
Assuntos
Bioensaio/métodos , Células/enzimologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fosfotransferases/antagonistas & inibidores , Fosfotransferases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Corantes FluorescentesRESUMO
PURPOSE: Purpose of this study has been the assessment of nuclear factor-kappaB (NF-kappaB) as a survival factor in human mesothelial cells (HMC), transformed HMC and malignant mesothelioma (MMe) cells. We aimed at verifying whether the proteasome inhibitor Bortezomib could abrogate NF-kappaB activity in MMe cells, leading to tumor cell death and may be established as a novel treatment for this aggressive neoplasm. EXPERIMENTAL DESIGN: In HMC and MMe cells, NF-kappaB nuclear translocation and DNA binding were studied by electrophoretic mobility shift assay, following treatment with tumor necrosis factor-alpha (TNF-alpha). The IKK inhibitor Bay11-7082 was also tested to evaluate its effects on HMC, transformed HMC, and MMe cell viability upon exposure to asbestos fibers. Following Bortezomib treatment, cytotoxicity of MMe cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, whereas apoptosis and cell-cycle blockade were investigated by high-content analysis. Bortezomib was also given to mice bearing i.p. xenografts of MMe cells, and its effects on tumor growth were evaluated. RESULTS: Here, we show that NF-kappaB activity is a constitutive survival factor in transformed HMC, MMe cells, and acts as a survival factor in HMC exposed to asbestos fibers. Bortezomib inhibits NF-kappaB activity in MMe cells and induces cell cycle blockade and apoptosis in vitro as well as tumor growth inhibition in vivo. CONCLUSIONS: Inhibition of NF-kappaB constitutive activation in MMe cells by Bortezomib resulted in in vitro cytotoxicity along with apoptosis and in vivo tumor regression. Our results support the use of Bortezomib in the treatment of MMe and has led to a phase II clinical trial currently enrolling in Europe.
Assuntos
Ácidos Borônicos/farmacologia , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , NF-kappa B/metabolismo , Pirazinas/farmacologia , Animais , Antineoplásicos/farmacologia , Bortezomib , Linhagem Celular Tumoral , Sobrevivência Celular , Transformação Celular Neoplásica , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Nitrilas/farmacologia , Inibidores de Proteassoma , Sulfonas/farmacologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Chordomas are rare bone tumors with no approved therapy. These tumors express several activated tyrosine kinase receptors, which prompted attempts to treat patients with tyrosine kinase inhibitors. Although clinical benefit was observed in phase II clinical trials with imatinib and sorafenib, and sporadically also with EGFR inhibitors, therapies evaluated to date have shown modest activity. With the goal of identifying new drugs with immediate therapeutic potential for chordoma patients, we collected clinically approved drugs and other advanced inhibitors of MET, PDGFRß, and EGFR tyrosine kinases, and assessed their antiproliferative activity against a panel of chordoma cell lines. Chordoma cell lines were not responsive to MET and PDGFRß inhibitors. U-CH1 and UM-Chor1 were sensitive to all EGFR inhibitors, whereas the remaining cell lines were generally insensitive to these drugs. Afatinib was the only EGFR inhibitor with activity across the chordoma panel. We then investigated the molecular mechanisms behind the responses observed and found that the antiproliferative IC50s correlate with the unique ability of afatinib to promote degradation of EGFR and brachyury, an embryonic transcription factor considered a key driver of chordoma. Afatinib displayed potent antitumor efficacy in U-CH1, SF8894, CF322, and CF365 chordoma tumor models in vivo In the panel analyzed, high EGFR phosphorylation and low AXL and STK33 expression correlated with higher sensitivity to afatinib and deserve further investigation as potential biomarkers of response. These data support the use of afatinib in clinical trials and provide the rationale for the upcoming European phase II study on afatinib in advanced chordoma. Mol Cancer Ther; 17(3); 603-13. ©2017 AACR.
Assuntos
Afatinib/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Cordoma/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Proteínas Fetais/antagonistas & inibidores , Proteínas com Domínio T/antagonistas & inibidores , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Neoplasias Ósseas/genética , Neoplasias Ósseas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cordoma/genética , Cordoma/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos Nus , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
The discovery of agents which disrupt cancer cell division by specifically targeting key components of the cell-cycle machinery represents a major focus of recent drug discovery efforts in Oncology. The drug discovery process can be greatly enhanced by multiparametric cellular analysis which can assist in confirmation, often in a few multiplexed assays, of the mechanism of action (MOA) of compounds identified through biochemical screening or similar in vitro methods. High-Content Analysis (HCA) is a technique based on automated microscopy which enables multiparametric analysis of fluorescent indicators to define cellular responses to compound treatment. Several distinct fluorescence channels can be acquired and analyzed within a single measure in the same cell population. Here we present a multiparametric HCA approach to characterize potential cell-cycle inhibitors in osteosarcoma U-2 OS adherent cell cultures. This approach allows monitoring of compound-induced cell-cycle perturbations by analyzing specific cellular markers such as nuclear morphology, DNA content or histone H3 phosphorylation. Moreover, the induction of DNA damage response or apoptosis can also be readily evaluated. By considering the profile of the investigated cellular markers at different compound concentrations, a fingerprint defines the cellular and molecular phenotype associated with each compound.
Assuntos
Ciclo Celular/efeitos dos fármacos , Microscopia de Fluorescência/métodos , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Dano ao DNA , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Histonas/metabolismo , Humanos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , FenótipoRESUMO
Chordomas are rare, slowly growing tumors with high medical need, arising in the axial skeleton from notochord remnants. The transcription factor "brachyury" represents a distinctive molecular marker and a key oncogenic driver of chordomas. Tyrosine kinase receptors are also expressed, but so far kinase inhibitors have not shown clear clinical efficacy in chordoma patients. The need for effective therapies is extremely high, but the paucity of established chordoma cell lines has limited preclinical research. Here we describe the isolation of the new Chor-IN-1 cell line from a recurrent sacral chordoma and its characterization as compared to other chordoma cell lines. Chor-IN-1 displays genomic identity to the tumor of origin and has morphological features, growth characteristics and chromosomal abnormalities typical of chordoma, with expression of brachyury and other relevant biomarkers. Chor-IN-1 gene variants, copy number alterations and kinome gene expression were analyzed in comparison to other four chordoma cell lines, generating large scale DNA and mRNA genomic data that can be exploited for the identification of novel pharmacological targets and candidate predictive biomarkers of drug sensitivity in chordoma. The establishment of this new, well characterized chordoma cell line provides a useful tool for the identification of drugs active in chordoma.
Assuntos
Cordoma/genética , Genômica , Biópsia , Linhagem Celular Tumoral , Cordoma/metabolismo , Cordoma/patologia , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Imuno-Histoquímica , Cariótipo , Masculino , Pessoa de Meia-IdadeRESUMO
Cell cycle analysis using flow cytometry (FC) to measure cellular DNA content is a common procedure in drug mechanism of action studies. Although this technique lends itself readily to cell lines that grow in suspension, adherent cell cultures must be resuspended in a cumbersome and potentially invasive procedure that normally involves trypsinization and mechanical agitation of monolayer cultures. High-content analysis (HCA), an automated microscopy-based technology, is well suited to analysis of monolayer cell cultures but provides intrinsically less accurate determination of cellular DNA content than does FC and thus is not the method of choice for cell cycle analysis. Using Cellomics's ArrayScan reader, the authors have developed a 4-color multiparametric HCA approach for cell cycle analysis of adherent cells based on detection of DNA content (4,6-diamidino-2-phenylindole [DAPI] fluorescence), together with the known cell cycle markers bromo-2-deoxyuridine (BrdU) incorporation, cyclin B1 expression, and histone H3 (Ser28) phosphorylation within a single cell population. Considering all 4 markers together, a reliable and accurate quantification of cell cycle phases was possible, as compared with flow cytometric analysis. Using this assay, specific cell cycle blocks induced by treatment with thymidine, paclitaxel, or nocodazole as test drugs were easily monitored in adherent cultures of U-2 OS osteosarcoma cells.