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1.
Br J Cancer ; 119(9): 1118-1128, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30377337

RESUMO

BACKGROUND: AKT is commonly overexpressed in tumours and plays an important role in the metabolic reprogramming of cancer. We have used magnetic resonance spectroscopy (MRS) to assess whether inhibition of AKT signalling would result in metabolic changes that could potentially be used as biomarkers to monitor response to AKT inhibition. METHODS: Cellular and metabolic effects of the allosteric AKT inhibitor MK-2206 were investigated in HT29 colon and PC3 prostate cancer cells and xenografts using flow cytometry, immunoblotting, immunohistology and MRS. RESULTS: In vitro treatment with MK-2206 inhibited AKT signalling and resulted in time-dependent alterations in glucose, glutamine and phospholipid metabolism. In vivo, MK-2206 resulted in inhibition of AKT signalling and tumour growth compared with vehicle-treated controls. In vivo MRS analysis of HT29 subcutaneous xenografts showed similar metabolic changes to those seen in vitro including decreases in the tCho/water ratio, tumour bioenergetic metabolites and changes in glutamine and glutathione metabolism. Similar phosphocholine changes compared to in vitro were confirmed in the clinically relevant orthotopic PC3 model. CONCLUSION: This MRS study suggests that choline metabolites detected in response to AKT inhibition are time and microenvironment-dependent, and may have potential as non-invasive biomarkers for monitoring response to AKT inhibitors in selected cancer types.


Assuntos
Antineoplásicos/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Inibidores Enzimáticos/farmacologia , Compostos Heterocíclicos com 3 Anéis/farmacologia , Neoplasias da Próstata/patologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Espectroscopia de Ressonância Magnética/métodos , Masculino
2.
Nat Chem Biol ; 11(12): 973-980, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26502155

RESUMO

There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small-molecule inhibitor of the WNT pathway discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a type 1 binding mode involving insertion of the CDK8 C terminus into the ligand binding site. In contrast to type II inhibitors of CDK8 and CDK19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogs alter WNT pathway-regulated gene expression and other on-target effects of modulating CDK8 and CDK19, including expression of genes regulated by STAT1. Consistent with this, we find that phosphorylation of STAT1(SER727) is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors.


Assuntos
Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinase 8 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Sondas Moleculares/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Compostos de Espiro/farmacologia , Linhagem Celular Tumoral , Quinase 8 Dependente de Ciclina/genética , Quinases Ciclina-Dependentes/genética , Humanos , Modelos Moleculares , Sondas Moleculares/química , Estrutura Molecular , Inibidores de Proteínas Quinases/química , Piridinas/química , Compostos de Espiro/química
3.
BMC Biol ; 10: 29, 2012 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-22439642

RESUMO

BACKGROUND: There is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed. RESULTS: To address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease. CONCLUSIONS: We have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.


Assuntos
Antineoplásicos/farmacologia , Avaliação de Medicamentos/métodos , Células-Tronco Embrionárias/efeitos dos fármacos , Modelos Biológicos , Animais , Benzoquinonas/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Furanos/farmacologia , Proteínas de Choque Térmico HSP90/farmacologia , Lactamas Macrocíclicas/farmacologia , Camundongos , Piridinas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Microambiente Tumoral/efeitos dos fármacos
4.
J Med Chem ; 66(8): 5907-5936, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37017629

RESUMO

CCT251236 1, a potent chemical probe, was previously developed from a cell-based phenotypic high-throughput screen (HTS) to discover inhibitors of transcription mediated by HSF1, a transcription factor that supports malignancy. Owing to its activity against models of refractory human ovarian cancer, 1 was progressed into lead optimization. The reduction of P-glycoprotein efflux became a focus of early compound optimization; central ring halogen substitution was demonstrated by matched molecular pair analysis to be an effective strategy to mitigate this liability. Further multiparameter optimization led to the design of the clinical candidate, CCT361814/NXP800 22, a potent and orally bioavailable fluorobisamide, which caused tumor regression in a human ovarian adenocarcinoma xenograft model with on-pathway biomarker modulation and a clean in vitro safety profile. Following its favorable dose prediction to human, 22 has now progressed to phase 1 clinical trial as a potential future treatment for refractory ovarian cancer and other malignancies.


Assuntos
Antineoplásicos , Neoplasias Ovarianas , Humanos , Feminino , Fatores de Transcrição/metabolismo , Neoplasias Ovarianas/patologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia
5.
J Med Chem ; 66(8): 5892-5906, 2023 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-37026591

RESUMO

B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.


Assuntos
Linfoma Difuso de Grandes Células B , Quinolonas , Animais , Humanos , Camundongos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Linfoma Difuso de Grandes Células B/patologia , Proteínas Proto-Oncogênicas c-bcl-6/química , Fatores de Transcrição
6.
Cells ; 11(20)2022 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-36291153

RESUMO

Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.


Assuntos
Proteínas Associadas à Resistência a Múltiplos Medicamentos , Timidilato Sintase , Humanos , Células CACO-2 , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Timidilato Sintase/metabolismo , Metilenotetra-Hidrofolato Redutase (NADPH2) , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína 2 Associada à Farmacorresistência Múltipla , Proteínas de Membrana Transportadoras , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Permeabilidade , Ácido Fólico , Metionina , Carbono/metabolismo
7.
J Med Chem ; 65(12): 8191-8207, 2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35653645

RESUMO

The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.


Assuntos
Carcinogênese , Regulação Neoplásica da Expressão Gênica , Animais , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo
8.
Autophagy ; 16(6): 1044-1060, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31517566

RESUMO

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.


Assuntos
Antineoplásicos/farmacologia , Autofagossomos/enzimologia , Autofagossomos/metabolismo , Colina-Fosfato Citidililtransferase/metabolismo , Furanos/farmacologia , Macroautofagia , Fosfatidilcolinas/biossíntese , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Células CHO , Linhagem Celular Tumoral , Colina/metabolismo , Colina-Fosfato Citidililtransferase/genética , Cricetulus , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Macroautofagia/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Metabolômica , Camundongos , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
9.
J Med Chem ; 63(8): 4047-4068, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32275432

RESUMO

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.


Assuntos
Benzimidazóis/administração & dosagem , Benzimidazóis/química , Sistemas de Liberação de Medicamentos/métodos , Descoberta de Drogas/métodos , Proteínas Proto-Oncogênicas c-bcl-6/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
10.
Cancer Res ; 67(12): 5840-50, 2007 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-17575152

RESUMO

Extensive evidence implicates activation of the lipid phosphatidylinositide 3-kinase (PI3K) pathway in the genesis and progression of various human cancers. PI3K inhibitors thus have considerable potential as molecular cancer therapeutics. Here, we detail the pharmacologic properties of a prototype of a new series of inhibitors of class I PI3K. PI103 is a potent inhibitor with low IC50 values against recombinant PI3K isoforms p110alpha (2 nmol/L), p110beta (3 nmol/L), p110delta (3 nmol/L), and p110gamma (15 nmol/L). PI103 also inhibited TORC1 by 83.9% at 0.5 micromol/L and exhibited an IC50 of 14 nmol/L against DNA-PK. A high degree of selectivity for the PI3K family was shown by the lack of activity of PI103 in a panel of 70 protein kinases. PI103 potently inhibited proliferation and invasion of a wide variety of human cancer cells in vitro and showed biomarker modulation consistent with inhibition of PI3K signaling. PI103 was extensively metabolized, but distributed rapidly to tissues and tumors. This resulted in tumor growth delay in eight different human cancer xenograft models with various PI3K pathway abnormalities. Decreased phosphorylation of AKT was observed in U87MG gliomas, consistent with drug levels achieved. We also showed inhibition of invasion in orthotopic breast and ovarian cancer xenograft models and obtained evidence that PI103 has antiangiogenic potential. Despite its rapid in vivo metabolism, PI103 is a valuable tool compound for exploring the biological function of class I PI3K and importantly represents a lead for further optimization of this novel class of targeted molecular cancer therapeutic.


Assuntos
Antineoplásicos/farmacologia , Furanos/farmacologia , Neoplasias/tratamento farmacológico , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Inibidores de Fosfoinositídeo-3 Quinase , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Citometria de Fluxo , Humanos , Immunoblotting , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Clin Cancer Res ; 25(21): 6487-6500, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31345839

RESUMO

PURPOSE: Radiotherapy is important in managing pelvic cancers. However, radiation enteropathy may occur and can be dose limiting. The gut microbiota may contribute to the pathogenesis of radiation enteropathy. We hypothesized that the microbiome differs between patients with and without radiation enteropathy.Experimental Design: Three cohorts of patients (n = 134) were recruited. The early cohort (n = 32) was followed sequentially up to 12 months post-radiotherapy to assess early radiation enteropathy. Linear mixed models were used to assess microbiota dynamics. The late cohort (n = 87) was assessed cross-sectionally to assess late radiation enteropathy. The colonoscopy cohort compared the intestinal mucosa microenvironment in patients with radiation enteropathy (cases, n = 9) with healthy controls (controls, n = 6). Fecal samples were obtained from all cohorts. In the colonoscopy cohort, intestinal mucosa samples were taken. Metataxonomics (16S rRNA gene) and imputed metataxonomics (Piphillin) were used to characterize the microbiome. Clinician- and patient-reported outcomes were used for clinical characterization. RESULTS: In the acute cohort, we observed a trend for higher preradiotherapy diversity in patients with no self-reported symptoms (P = 0.09). Dynamically, diversity decreased less over time in patients with rising radiation enteropathy (P = 0.05). A consistent association between low bacterial diversity and late radiation enteropathy was also observed, albeit nonsignificantly. Higher counts of Clostridium IV, Roseburia, and Phascolarctobacterium significantly associated with radiation enteropathy. Homeostatic intestinal mucosa cytokines related to microbiota regulation and intestinal wall maintenance were significantly reduced in radiation enteropathy [IL7 (P = 0.05), IL12/IL23p40 (P = 0.03), IL15 (P = 0.05), and IL16 (P = 0.009)]. IL15 inversely correlated with counts of Roseburia and Propionibacterium. CONCLUSIONS: The microbiota presents opportunities to predict, prevent, or treat radiation enteropathy. We report the largest clinical study to date into associations of the microbiota with acute and late radiation enteropathy. An altered microbiota associates with early and late radiation enteropathy, with clinical implications for risk assessment, prevention, and treatment of radiation-induced side-effects.See related commentary by Lam et al., p. 6280.


Assuntos
Bactérias/genética , Trato Gastrointestinal/microbiologia , Neoplasias Pélvicas/radioterapia , Lesões por Radiação/genética , Idoso , Bactérias/classificação , Bactérias/efeitos da radiação , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/efeitos da radiação , Trato Gastrointestinal/patologia , Trato Gastrointestinal/efeitos da radiação , Humanos , Mucosa Intestinal/microbiologia , Mucosa Intestinal/patologia , Mucosa Intestinal/efeitos da radiação , Masculino , Pessoa de Meia-Idade , Neoplasias Pélvicas/complicações , Neoplasias Pélvicas/microbiologia , Neoplasias Pélvicas/patologia , RNA Ribossômico 16S/genética , Exposição à Radiação/efeitos adversos , Lesões por Radiação/microbiologia , Lesões por Radiação/patologia
12.
Assay Drug Dev Technol ; 5(3): 391-401, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17638539

RESUMO

In this age of molecularly targeted drug discovery, robust techniques are required to measure pharmacodynamic (PD) responses in tumors so that drug exposures can be associated with their effects on molecular biomarkers and efficacy. Our aim was to develop a rapid screen to monitor PD responses within xenografted human tumors as an important step towards a clinically applicable technology. Currently there are various methods available to measure PD end points, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction, gene expression profiling, and western blotting. These may require relatively large samples of tumor, surrogate tissue, or peripheral blood lymphocytes with subsequent analyses taking several days. The phosphoinositide 3-kinase (PI3-kinase) pathway is frequently deregulated in cancer and is also important in diabetes and autoimmune conditions. In this paper, optimization of the Meso Scale Discovery (MSD) (Gaithersburg, MD) platform to quantify changes in phospho-AKT and phospho-glycogen synthase kinase-3beta in response to a PI3-kinase inhibitor, LY294002, is described, initially in vitro and then within xenografted solid tumors. This method is highly practical with high throughput since large number of samples can be run simultaneously in 96-well format. The assays are robust (coefficient of variation for phospho-AKT 13.4%) and offer significant advantages (in terms of speed and quantitation) over western blots. This optimized procedure can be used for both in vitro and in vivo analysis, unlike an established fixed-cell ELISA with a time-resolved fluorescent end point.


Assuntos
Cromonas/uso terapêutico , Quinase 3 da Glicogênio Sintase/metabolismo , Morfolinas/uso terapêutico , Neoplasias Experimentais/química , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/análise , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Transplante de Neoplasias , Neoplasias Experimentais/tratamento farmacológico , Reprodutibilidade dos Testes , Manejo de Espécimes , Transplante Heterólogo
13.
Mol Cancer Ther ; 5(3): 522-32, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16546966

RESUMO

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in maintaining the correct conformation and stability of its client proteins. This study investigated the effects of Hsp90 inhibitors on client protein expression and key cellular functions required for tumor angiogenesis. The benzoquinone ansamycin Hsp90 inhibitors geldanamycin and/or its derivatives 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin inhibited production of vascular endothelial growth factor (VEGF)-A by tumor cells and blocked proliferative responses of human endothelial cells at nanomolar concentrations. 17-AAG also significantly reduced endothelial cell migration, tubular differentiation, invasion through Matrigel, and secretion of urokinase-type plasminogen activator at concentrations at or below those that inhibited proliferation. 17-AAG significantly reduced expression of VEGF receptor (VEGFR)-2 and established Hsp90 client proteins in human endothelial cells in vitro as well as in mouse vena cava, mesenteric vessels, and blood vessels within human tumor xenografts in vivo; this was associated with decreased tumor microvessel density. Finally, we showed for the first time that Hsp90 inhibitors also reduce expression of VEGFR-1 on human vascular endothelial cells, VEGFR-3 on lymphatic endothelial cells in vitro, and all three VEGFRs on mouse vasculature in vivo. Thus, we identify Hsp90 inhibitors as important regulators of many aspects of tumor angiogenesis (and potentially lymphangiogenesis) and suggest that they may provide therapeutic benefit not only via direct effects on tumor cells but also indirectly by inhibiting the production of angiogenic cytokines and responses of activated endothelial cells that contribute to tumor progression and metastasis.


Assuntos
Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Neoplasias/irrigação sanguínea , Neovascularização Patológica/metabolismo , Rifabutina/análogos & derivados , Benzoquinonas , Capilares/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Regulação para Baixo , Endotélio Vascular/efeitos dos fármacos , Quinase 1 de Adesão Focal/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas , Quinonas/farmacologia , Receptores de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Rifabutina/farmacologia , Cordão Umbilical/citologia , Cordão Umbilical/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
Mol Oncol ; 11(8): 996-1006, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28432815

RESUMO

Targeted inhibition of anaplastic lymphoma kinase (ALK) is a successful approach for the treatment of many ALK-aberrant malignancies; however, the presence of resistant mutations necessitates both the development of more potent compounds and pharmacodynamic methods with which to determine their efficacy. We describe immunoassays designed to quantitate phosphorylation of ALK, and their use in preclinical models of neuroblastoma, a pediatric malignancy in which gain-of-function ALK mutations predict a poor overall outcome to conventional treatment. Validation of the immunoassays is presented using a panel of neuroblastoma cell lines and evidence of on-target ALK inhibition provided by treatment of a genetically engineered murine model of neuroblastoma with two clinical ALK inhibitors, crizotinib and ceritinib, highlighting the superior efficacy of ceritinib.


Assuntos
Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/metabolismo , Neuroblastoma/enzimologia , Pirazóis/farmacologia , Piridinas/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Sulfonas/farmacologia , Quinase do Linfoma Anaplásico , Crizotinibe , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Imunoensaio , Fosforilação/efeitos dos fármacos
15.
Elife ; 52016 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-27935476

RESUMO

Mediator-associated kinases CDK8/19 are context-dependent drivers or suppressors of tumorigenesis. Their inhibition is predicted to have pleiotropic effects, but it is unclear whether this will impact on the clinical utility of CDK8/19 inhibitors. We discovered two series of potent chemical probes with high selectivity for CDK8/19. Despite pharmacodynamic evidence for robust on-target activity, the compounds exhibited modest, though significant, efficacy against human tumor lines and patient-derived xenografts. Altered gene expression was consistent with CDK8/19 inhibition, including profiles associated with super-enhancers, immune and inflammatory responses and stem cell function. In a mouse model expressing oncogenic beta-catenin, treatment shifted cells within hyperplastic intestinal crypts from a stem cell to a transit amplifying phenotype. In two species, neither probe was tolerated at therapeutically-relevant exposures. The complex nature of the toxicity observed with two structurally-differentiated chemical series is consistent with on-target effects posing significant challenges to the clinical development of CDK8/19 inhibitors.


Assuntos
Anti-Inflamatórios/administração & dosagem , Antineoplásicos/administração & dosagem , Quinase 8 Dependente de Ciclina/antagonistas & inibidores , Quinases Ciclina-Dependentes/antagonistas & inibidores , Complexo Mediador/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Animais , Anti-Inflamatórios/efeitos adversos , Anti-Inflamatórios/toxicidade , Antineoplásicos/efeitos adversos , Antineoplásicos/toxicidade , Modelos Animais de Doenças , Xenoenxertos , Humanos , Hiperplasia/tratamento farmacológico , Camundongos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/toxicidade , Resultado do Tratamento
16.
J Med Chem ; 45(3): 590-7, 2002 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-11806711

RESUMO

Two short routes to novel methylated pentacyclic quinoacridinium salts have been devised. New compounds display telomerase-inhibitory potency (<1 microM) in the TRAP assay. 3,11-Difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (12d, RHPS4, NSC 714187) has a higher selectivity for triplex and quadruplex DNA structures than the 3,6,8,11,13-pentamethyl analogue (12c, RHPS3, NSC 714186) and a low overall growth-inhibitory activity in the NCI 60 cell panel (mean GI(50) 13.18 microM); in addition, the activity profile of 12d does not COMPARE with agents of the topoisomerase II class. Compound 12d is soluble in water, stable in the pH range of 5-9, efficiently transported into tumor cells, and is currently the lead structure for further elaboration in this new class of telomerase inhibitor.


Assuntos
Acridinas/síntese química , Inibidores Enzimáticos/síntese química , Telomerase/antagonistas & inibidores , Acridinas/química , Acridinas/metabolismo , Acridinas/farmacologia , Núcleo Celular/metabolismo , Cristalografia por Raios X , DNA/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Confocal , Modelos Moleculares , Solubilidade , Relação Estrutura-Atividade , Telomerase/química , Células Tumorais Cultivadas
17.
J Med Chem ; 46(21): 4463-76, 2003 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-14521409

RESUMO

The synthesis and evaluation for telomerase-inhibitory and quadruplex DNA binding properties of three related series of rationally designed trisubstituted acridine derivatives are described. These are substituted on the acridine ring at the 2,6,9; 2,7,9; and 3,6,9 positions. The ability of several of the most potent compounds to interact with and stabilize an intramolecular G-quadruplex DNA was evaluated by surface plasmon resonance methods, and affinities were found to correlate with potency in a telomerase assay. The interactions of a number of compounds with a parallel quadruplex DNA structure were simulated by molecular modeling methods. The calculated interaction energies were compared with telomerase activity and showed generally consistent correlations between quadruplex affinity and telomerase inhibition. These data support a model for the action of these compounds that involves the stabilization of intermediate quadruplex structures that inhibit the elongation of telomeric DNA by telomerase in tumor cells.


Assuntos
Acridinas/síntese química , Acridinas/farmacologia , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Telomerase/antagonistas & inibidores , Cristalografia por Raios X , DNA/efeitos dos fármacos , DNA/metabolismo , DNA de Neoplasias/biossíntese , DNA de Neoplasias/metabolismo , Humanos , Cinética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rodaminas/química , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Células Tumorais Cultivadas
18.
Phytomedicine ; 21(12): 1717-24, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25442282

RESUMO

BACKGROUND: The lichen compound (+)-protolichesterinic acid (+)-PA, isolated from Iceland moss, has anti-proliferative effects on several cancer cell lines. The chemical structure of (+)-PA is similar to a known fatty acid synthase (FASN) inhibitor C75. AIMS: To test whether the anti-proliferative activity of (+)-PA is associated with effects on FASN and HER2 (human epidermal growth factor receptor 2) and major signalling pathways. Synergism between (+)-PA and lapatinib, a HER2 active drug, was also evaluated. MATERIALS AND METHODS: Pure compound was isolated by preparative high-performance liquid chromatography (HPLC) and purity of (+)-PA analyzed by analytical HPLC. Cell viability was assessed using Crystal violet staining. FASN and HER2 expression was estimated by immunofluorescence. The Meso Scale Discovery (MSD)(®) assay was used to measure activation of ERK1/2 and AKT. Synergism was estimated by the CalcuSyn software. RESULTS: Treatment with (+)-PA increased FASN expression in SK-BR-3 cells, which overexpress FASN and HER2, implying a compensatory response to inhibition of FASN activity. HER2 expression was decreased suggesting secondary downregulation. ERK1/2 and AKT signalling pathways were inhibited, probably due to reduced levels of HER2. No effects were observed in T-47D cells. Synergism between (+)-PA and lapatinib was observed in the SK-BR-3 cells. CONCLUSION: Results suggest that the primary effect of (+)-PA is inhibition of FASN activity. Synergistic effects with lapatinib were seen only in SK-BR-3 cells, and not T-47D cells, further supporting the notion that (+)-PA acts by inhibiting FASN with secondary effects on HER2 expression and signalling. (+)-PA could therefore be a suitable agent for further testing, alone or in combination treatment against HER2-overexpressing breast cancer.


Assuntos
4-Butirolactona/análogos & derivados , Neoplasias da Mama/metabolismo , Ácido Graxo Sintases/metabolismo , Líquens/química , Transdução de Sinais/efeitos dos fármacos , 4-Butirolactona/farmacologia , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Humanos , Lapatinib , Estrutura Molecular , Quinazolinas/farmacologia , Receptor ErbB-2/metabolismo
19.
Eur J Cancer ; 49(11): 2512-21, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23582742

RESUMO

BACKGROUND: Acquired resistance to tyrosine kinase inhibitors (TKIs) is becoming a major challenge in the treatment of many cancers. Epidermal growth factor receptor (EGFR) is overexpressed in squamous carcinomas, notably those of the head and neck (HNSCC), and can be targeted with several TKIs. We aimed to identify soluble proteins suitable for development as markers of EGFR TKI resistance in cancer patients to aid in early and minimally invasive assessment of therapeutic responses. METHODS: Resistant HNSCC cell lines were generated by exposure to an EGFR TKI, gefitinib, in vitro. Cell lines were characterised for their biological behaviour in vitro (using growth inhibition assays, flow cytometry, western blots, antibody arrays and/or immunoassays) and in vivo (using subcutaneous tumour xenografts). Sera from EGFR-treated and -untreated HNSCC patients were analysed by immunoassay. RESULTS: Two independent sublines of CAL 27 and a PJ34 subline with acquired resistance to EGFR TKIs (gefitinib, erlotinib and afatinib) were developed. Resistant cells grew as highly aggressive xenografts leading to reduced host survival rates compared with EGFR-TKI sensitive cells. This suggested a link between resistance in vitro and poor prognosis in vivo. A significant upregulation of proteins linked to tumour angiogenesis and invasion was identified in resistant cells. This 'resistance-associated protein signature' (RAPS) was detected in the sera of a small cohort of HNSCC patients and was associated with reduced survival. CONCLUSION: We have identified a protein signature associated with EGFR-TKI resistance that may also be linked to poor prognosis and warrants further investigation as a potential clinical biomarker.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma de Células Escamosas/sangue , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/sangue , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas de Neoplasias/sangue , Inibidores de Proteínas Quinases/farmacologia , Animais , Carcinoma de Células Escamosas/enzimologia , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Biologia Computacional , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Feminino , Fluoruracila/administração & dosagem , Gefitinibe , Neoplasias de Cabeça e Pescoço/enzimologia , Humanos , Camundongos , Camundongos Nus , Quinazolinas/administração & dosagem , Carcinoma de Células Escamosas de Cabeça e Pescoço , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Clin Cancer Res ; 19(21): 5940-51, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-23918606

RESUMO

PURPOSE: To provide rationale for using phosphoinositide 3-kinase (PI3K) and/or mitogen-activated protein kinase (MAPK) pathway inhibitors to treat rhabdomyosarcomas, a major cause of pediatric and adolescent cancer deaths. EXPERIMENTAL DESIGN: The prevalence of PI3K/MAPK pathway activation in rhabdomyosarcoma clinical samples was assessed using immunohistochemistry. Compensatory signaling and cross-talk between PI3K/MAPK pathways was determined in rhabdomyosarcoma cell lines following p110α short hairpin RNA-mediated depletion. Pharmacologic inhibition of reprogrammed signaling in stable p110α knockdown lines was used to determine the target-inhibition profile inducing maximal growth inhibition. The in vitro and in vivo efficacy of inhibitors of TORC1/2 (AZD8055), MEK (AZD6244), and P13K/mTOR (NVP-BEZ235) was evaluated alone and in pairwise combinations. RESULTS: PI3K pathway activation was seen in 82.5% rhabdomyosarcomas with coactivated MAPK in 36% and 46% of alveolar and embryonal subtypes, respectively. p110α knockdown in cell lines over the short and long term was associated with compensatory expression of other p110 isoforms, activation of the MAPK pathway, and cross-talk to reactivate the PI3K pathway. Combinations of PI3K pathway and MAP-ERK kinase (MEK) inhibitors synergistically inhibited cell growth in vitro. Treatment of RD cells with AZD8055 plus AZD6244 blocked reciprocal pathway activation, as evidenced by reduced AKT/ERK/S6 phosphorylation. In vivo, the synergistic effect on growth and changes in pharmacodynamic biomarkers was recapitulated using the AZD8055/AZD6244 combination but not NVP-BEZ235/AZD6244. Pharmacokinetic analysis provided evidence of drug-drug interaction with both combinations. CONCLUSIONS: Dual PI3K/MAPK pathway activation and compensatory signaling in both rhabdomyosarcoma subtypes predict a lack of clinical efficacy for single agents targeting either pathway, supporting a therapeutic strategy combining a TORC1/2 with a MEK inhibitor.


Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Benzimidazóis/administração & dosagem , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases , Modelos Animais de Doenças , Sinergismo Farmacológico , Ativação Enzimática/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Humanos , Morfolinas/administração & dosagem , Morfolinas/farmacologia , Fosfatidilinositol 3-Quinases/genética , Rabdomiossarcoma/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
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