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1.
J Med Chem ; 63(9): 4978-4996, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32369358

RESUMO

Diffuse intrinsic pontine glioma is an aggressive pediatric cancer for which no effective chemotherapeutic drugs exist. Analysis of the genomic landscape of this disease has led to the identification of the serine/threonine kinase ALK2 as a potential target for therapeutic intervention. In this work, we adopted an open science approach to develop a series of potent type I inhibitors of ALK2 which are orally bio-available and brain-penetrant. Initial efforts resulted in the discovery of M4K2009, an analogue of the previously reported ALK2 inhibitor LDN-214117. Although highly selective for ALK2 over the TGF-ßR1 receptor ALK5, M4K2009 is also moderately active against the hERG potassium channel. Varying the substituents of the trimethoxyphenyl moiety gave rise to an equipotent benzamide analogue M4K2149 with reduced off-target affinity for the ion channel. Additional modifications yielded 2-fluoro-6-methoxybenzamide derivatives (26a-c), which possess high inhibitory activity against ALK2, excellent selectivity, and superior pharmacokinetic profiles.

2.
Nat Commun ; 11(1): 2396, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32409666

RESUMO

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

3.
Nat Chem Biol ; 16(5): 577-586, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32094923

RESUMO

Receptor tyrosine kinases (RTKs) are transmembrane receptors of great clinical interest due to their role in disease. Historically, therapeutics targeting RTKs have been identified using in vitro kinase assays. Due to frequent development of drug resistance, however, there is a need to identify more diverse compounds that inhibit mutated but not wild-type RTKs. Here, we describe MaMTH-DS (mammalian membrane two-hybrid drug screening), a live-cell platform for high-throughput identification of small molecules targeting functional protein-protein interactions of RTKs. We applied MaMTH-DS to an oncogenic epidermal growth factor receptor (EGFR) mutant resistant to the latest generation of clinically approved tyrosine kinase inhibitors (TKIs). We identified four mutant-specific compounds, including two that would not have been detected by conventional in vitro kinase assays. One of these targets mutant EGFR via a new mechanism of action, distinct from classical TKI inhibition. Our results demonstrate how MaMTH-DS is a powerful complement to traditional drug screening approaches.

5.
Cell Stem Cell ; 24(4): 621-636.e16, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30930145

RESUMO

Tafazzin (TAZ) is a mitochondrial transacylase that remodels the mitochondrial cardiolipin into its mature form. Through a CRISPR screen, we identified TAZ as necessary for the growth and viability of acute myeloid leukemia (AML) cells. Genetic inhibition of TAZ reduced stemness and increased differentiation of AML cells both in vitro and in vivo. In contrast, knockdown of TAZ did not impair normal hematopoiesis under basal conditions. Mechanistically, inhibition of TAZ decreased levels of cardiolipin but also altered global levels of intracellular phospholipids, including phosphatidylserine, which controlled AML stemness and differentiation by modulating toll-like receptor (TLR) signaling.

6.
ACS Chem Biol ; 14(4): 751-757, 2019 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-30840432

RESUMO

Colloidal drug aggregates have been a nuisance in drug screening, yet, because they inherently comprise drug-rich particles, they may be useful in vivo if issues of stability can be addressed. As the first step toward answering this question, we optimized colloidal drug aggregate formulations using a fluorescence-based assay to study fulvestrant colloidal formation and stability in high (90%) serum conditions in vitro. We show, for the first time, that the critical aggregation concentration of fulvestrant depends on media composition and increases with serum concentration. Excipients, such as polysorbate 80, stabilize fulvestrant colloids in 90% serum in vitro for over 48 h. Using fulvestrant and an investigational pro-drug, pentyloxycarbonyl-( p-aminobenzyl) doxazolidinylcarbamate (PPD), as proof-of-concept colloidal formulations, we demonstrate that the in vivo plasma half-life for stabilized colloids is greater than their respective monomeric forms. These studies demonstrate the potential of turning the nuisance of colloidal drug aggregation into an opportunity for drug-rich formulations.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacocinética , Carbamatos/química , Carbamatos/farmacocinética , Doxorrubicina/análogos & derivados , Oxazóis/química , Oxazóis/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/farmacocinética , Animais , Antineoplásicos/sangue , Carbamatos/sangue , Coloides , Doxorrubicina/sangue , Doxorrubicina/química , Doxorrubicina/farmacocinética , Estabilidade de Medicamentos , Excipientes , Feminino , Fulvestranto/química , Humanos , Células MCF-7 , Camundongos , Transplante de Neoplasias , Oxazóis/sangue , Polissorbatos/química , Estudo de Prova de Conceito , Soro
7.
Cancer Res ; 79(9): 2426-2434, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30890619

RESUMO

Medulloblastoma (MB) is a pediatric malignant brain tumor composed of four different subgroups (WNT, SHH, Group 3, Group 4), each of which are a unique biological entity with distinct clinico-pathological, molecular, and prognostic characteristics. Although risk stratification of patients with MB based on molecular features may offer personalized therapies, conventional subgroup identification methods take too long and are unable to deliver subgroup information intraoperatively. This limitation prevents subgroup-specific adjustment of the extent or the aggressiveness of the tumor resection by the neurosurgeon. In this study, we investigated the potential of rapid tumor characterization with Picosecond infrared laser desorption mass spectrometry (PIRL-MS) for MB subgroup classification based on small molecule signatures. One hundred and thirteen ex vivo MB tumors from a local tissue bank were subjected to 10- to 15-second PIRL-MS data collection and principal component analysis with linear discriminant analysis (PCA-LDA). The MB subgroup model was established from 72 independent tumors; the remaining 41 de-identified unknown tumors were subjected to multiple, 10-second PIRL-MS samplings and real-time PCA-LDA analysis using the above model. The resultant 124 PIRL-MS spectra from each sampling event, after the application of a 95% PCA-LDA prediction probability threshold, yielded a 98.9% correct classification rate. Post-ablation histopathologic analysis suggested that intratumoral heterogeneity or sample damage prior to PIRL-MS sampling at the site of laser ablation was able to explain failed classifications. Therefore, upon translation, 10-seconds of PIRL-MS sampling is sufficient to allow personalized, subgroup-specific treatment of MB during surgery. SIGNIFICANCE: This study demonstrates that laser-extracted lipids allow immediate grading of medulloblastoma tumors into prognostically important subgroups in 10 seconds, providing medulloblastoma pathology in an actionable manner during surgery.


Assuntos
Neoplasias Cerebelares/classificação , Neoplasias Cerebelares/patologia , Cuidados Intraoperatórios , Meduloblastoma/classificação , Meduloblastoma/patologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias Cerebelares/cirurgia , Humanos , Meduloblastoma/cirurgia
8.
Cancer Cell ; 34(4): 579-595.e8, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30300580

RESUMO

MYC is an oncogenic driver that regulates transcriptional activation and repression. Surprisingly, mechanisms by which MYC promotes malignant transformation remain unclear. We demonstrate that MYC interacts with the G9a H3K9-methyltransferase complex to control transcriptional repression. Inhibiting G9a hinders MYC chromatin binding at MYC-repressed genes and de-represses gene expression. By identifying the MYC box II region as essential for MYC-G9a interaction, a long-standing missing link between MYC transformation and gene repression is unveiled. Across breast cancer cell lines, the anti-proliferative response to G9a pharmacological inhibition correlates with MYC sensitivity and gene signatures. Consistently, genetically depleting G9a in vivo suppresses MYC-dependent tumor growth. These findings unveil G9a as an epigenetic regulator of MYC transcriptional repression and a therapeutic vulnerability in MYC-driven cancers.


Assuntos
Carcinogênese/genética , Expressão Gênica/genética , Histona Metiltransferases/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Epigênese Genética/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Camundongos , Regiões Promotoras Genéticas/genética
9.
PLoS One ; 12(12): e0189670, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29253028

RESUMO

PURPOSE: The prognosis for patients diagnosed with glioblastoma multiforme (GBM) remains dismal, with current treatment prolonging survival only modestly. As such, there remains a strong need for novel therapeutic strategies. The janus kinase (JAK)2/signal transducer and activator of transcription (STAT)3 pathway regulates many cellular processes in GBM, including survival, proliferation, invasion, anti-apoptosis, and immune evasion. Here, we evaluated the preclinical efficacy of pacritinib, a novel compound targeting JAK2, using a collection of diverse patient-derived brain tumor initiating cells (BTICs). EXPERIMENTAL DESIGN: The effects of pacritinib on BTIC viability and sphere forming capacity were evaluated in vitro using the alamarBlue and neurosphere assays, respectively. On-target inhibition of JAK2/STAT3 signaling was investigated using western blotting. The efficacy of pacritinib was tested in vivo in pharmacokinetic analyses, liver microsome analyses, and Kaplan-Meier survival studies. RESULTS: In vitro, pacritinib decreased BTIC viability and sphere forming potential at low micromolar doses and demonstrated on-target inhibition of STAT3 signaling. Additionally, pacritinib was found to improve the response to temozolomide (TMZ) in TMZ-resistant BTICs. In vivo, systemic treatment with pacritinib demonstrated blood-brain barrier penetration and led to improved overall median survival in combination with TMZ, in mice orthotopically xenografted with an aggressive recurrent GBM BTIC culture. CONCLUSION: This preclinical study demonstrates the efficacy of pacritinib and supports the feasibility of testing pacritinib for the treatment of GBM, in combination with the standard of care TMZ.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Hidrocarbonetos Aromáticos com Pontes/uso terapêutico , Dacarbazina/análogos & derivados , Glioblastoma/tratamento farmacológico , Janus Quinase 2/antagonistas & inibidores , Pirimidinas/uso terapêutico , Fator de Transcrição STAT3/antagonistas & inibidores , Animais , Apoptose , Barreira Hematoencefálica/efeitos dos fármacos , Proliferação de Células , Sobrevivência Celular , Dacarbazina/uso terapêutico , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Camundongos SCID , Microssomos Hepáticos/efeitos dos fármacos , Permeabilidade , Temozolomida , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Chem Sci ; 8(9): 6508-6519, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28989676

RESUMO

Medulloblastoma (MB), the most prevalent malignant childhood brain tumour, consists of at least 4 distinct subgroups each of which possesses a unique survival rate and response to treatment. To rapidly determine MB subgroup affiliation in a manner that would be actionable during surgery, we subjected murine xenograft tumours of two MB subgroups (SHH and Group 3) to Mass Spectrometry (MS) profiling using a handheld Picosecond InfraRed Laser (PIRL) desorption probe and interface developed by our group. This platform provides real time MS profiles of tissue based on laser desorbed lipids and small molecules with only 5-10 seconds of sampling. PIRL-MS analysis of ex vivo MB tumours offered a 98% success rate in subgroup determination, observed over 194 PIRL-MS datasets collected from 19 independent tumours (∼10 repetitions each) utilizing 6 different established MB cell lines. Robustness was verified by a 5%-leave-out-and-remodel test. PIRL ablated tissue material was collected on a filter paper and subjected to high resolution LC-MS to provide ion identity assignments for the m/z values that contribute most to the statistical discrimination between SHH and Group 3 MB. Based on this analysis, rapid classification of MB with PIRL-MS utilizes a variety of fatty acid chains, glycerophosphates, glycerophosphoglycerols and glycerophosphocholines rapidly extracted from the tumours. In this work, we provide evidence that 5-10 seconds of sampling from ex vivo MB tissue with PIRL-MS can allow robust tumour subgroup classification, and have identified several biomarker ions responsible for the statistical discrimination of MB Group 3 and the SHH subgroup. The existing PIRL-MS platform used herein offers capabilities for future in vivo use.

11.
Biomaterials ; 123: 39-47, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28161682

RESUMO

We developed a novel taxane-binding peptide (TBP) modified, biodegradable polymeric micelle that overcomes limitations of drug loading and poor serum stability typically seen with particle delivery, leading to enhanced pharmacokinetics and tumor distribution of docetaxel (DTX). The use of the taxane-binding peptide to increase docetaxel loading is particularly compelling as it takes advantage of a known intracellular binding mechanism in a new way. Docetaxel is a potent chemotherapeutic with a therapeutic index often limited by the toxicity of the excipients that are necessary to enhance its solubility for intravenous delivery. Our polymeric micelle has terminal furan groups that enable facile antibody Fab conjugation by Diels-Alder chemistry for targeted delivery. Compared to the conventional ethanolic polysorbate 80 formulation (Free DTX), our nanoparticle (NP DTX) formulation exhibited a two-fold increase in exposure and tumor accumulation. Notably, the reduced toxicity of the NP DTX formulation increased the therapeutic index and allowed for higher dosing regimens, with a maximum tolerated dose (MTD) 1.6-fold higher than that of the Free DTX formulation, which is significant and similar to enhancements observed in clinical products for docetaxel and other drugs. These improved properties led to enhanced mouse survival in an orthotopic model of breast cancer; however, the targeted formulation of Fab-NP DTX did not further improve efficacy. Together, these results clearly demonstrate the benefits of the TBP-modified polymeric micelles as promising carriers for docetaxel.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Nanocápsulas/administração & dosagem , Nanocápsulas/química , Peptídeos/química , Taxoides/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Docetaxel , Avaliação Pré-Clínica de Medicamentos , Emulsões , Feminino , Camundongos , Micelas , Polímeros/química , Ligação Proteica , Taxoides/química , Resultado do Tratamento
13.
Stem Cell Reports ; 7(4): 787-801, 2016 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-27618721

RESUMO

Blood vessels are formed through vasculogenesis, followed by remodeling of the endothelial network through angiogenesis. Many events that occur during embryonic vascular development are recapitulated during adult neoangiogenesis, which is critical to tumor growth and metastasis. Current antiangiogenic tumor therapies, based largely on targeting the vascular endothelial growth factor pathway, show limited clinical benefits, thus necessitating the discovery of alternative targets. Here we report the development of a robust embryonic stem cell-based vascular differentiation assay amenable to small-molecule screens to identify novel modulators of angiogenesis. In this context, RSK and TTK were identified as angiogenic modulators. Inhibition of these pathways inhibited angiogenesis in embryoid bodies and human umbilical vein endothelial cells. Furthermore, inhibition of RSK and TTK reduced tumor growth, vascular density, and improved survival in an in vivo Lewis lung carcinoma mouse model. Our study suggests that RSK and TTK are potential targets for antiangiogenic therapy, and provides an assay system for further pathway screens.


Assuntos
Vasos Sanguíneos/embriologia , Vasos Sanguíneos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Descoberta de Drogas , Feminino , Humanos , Camundongos , Morfogênese , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Organogênese , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores
14.
Oncotarget ; 7(37): 59360-59376, 2016 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-27449082

RESUMO

Glioblastoma (GBM) is the most lethal and aggressive adult brain tumor, requiring the development of efficacious therapeutics. Towards this goal, we screened five genetically distinct patient-derived brain-tumor initiating cell lines (BTIC) with a unique collection of small molecule epigenetic modulators from the Structural Genomics Consortium (SGC). We identified multiple hits that inhibited the growth of BTICs in vitro, and further evaluated the therapeutic potential of EZH2 and HDAC inhibitors due to the high relevance of these targets for GBM. We found that the novel SAM-competitive EZH2 inhibitor UNC1999 exhibited low micromolar cytotoxicity in vitro on a diverse collection of BTIC lines, synergized with dexamethasone (DEX) and suppressed tumor growth in vivo in combination with DEX. In addition, a unique brain-penetrant class I HDAC inhibitor exhibited cytotoxicity in vitro on a panel of BTIC lines and extended survival in combination with TMZ in an orthotopic BTIC model in vivo. Finally, a combination of EZH2 and HDAC inhibitors demonstrated synergy in vitro by augmenting apoptosis and increasing DNA damage. Our findings identify key epigenetic modulators in GBM that regulate BTIC growth and survival and highlight promising combination therapies.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Proteína Potenciadora do Homólogo 2 de Zeste/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Piridonas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dexametasona/uso terapêutico , Sinergismo Farmacológico , Quimioterapia Combinada , Epigênese Genética , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Camundongos SCID , Terapia de Alvo Molecular , Piridonas/farmacologia , Bibliotecas de Moléculas Pequenas , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Clin Cancer Res ; 22(15): 3860-75, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27006494

RESUMO

PURPOSE: Glioblastoma is one of the most lethal cancers in humans, and with existing therapy, survival remains at 14.6 months. Current barriers to successful treatment include their infiltrative behavior, extensive tumor heterogeneity, and the presence of a stem-like population of cells, termed brain tumor-initiating cells (BTIC) that confer resistance to conventional therapies. EXPERIMENTAL DESIGN: To develop therapeutic strategies that target BTICs, we focused on a repurposing approach that explored already-marketed (clinically approved) drugs for therapeutic potential against patient-derived BTICs that encompass the genetic and phenotypic heterogeneity of glioblastoma observed clinically. RESULTS: Using a high-throughput in vitro drug screen, we found that montelukast, clioquinol, and disulfiram (DSF) were cytotoxic against a large panel of patient-derived BTICs. Of these compounds, disulfiram, an off-patent drug previously used to treat alcoholism, in the presence of a copper supplement, showed low nanomolar efficacy in BTICs including those resistant to temozolomide and the highly infiltrative quiescent stem-like population. Low dose DSF-Cu significantly augmented temozolomide activity in vitro, and importantly, prolonged in vivo survival in patient-derived BTIC models established from both newly diagnosed and recurrent tumors. Moreover, we found that in addition to acting as a potent proteasome inhibitor, DSF-Cu functionally impairs DNA repair pathways and enhances the effects of DNA alkylating agents and radiation. These observations suggest that DSF-Cu inhibits proteasome activity and augments the therapeutic effects of DNA-damaging agents (temozolomide and radiation). CONCLUSIONS: DSF-Cu should be considered as an adjuvant therapy for the treatment of patients with glioblastoma in both newly diagnosed and recurrent settings. Clin Cancer Res; 22(15); 3860-75. ©2016 AACR.


Assuntos
Antineoplásicos/farmacologia , Cobre/farmacologia , Dacarbazina/análogos & derivados , Dissulfiram/farmacologia , Glioblastoma/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Reparo do DNA , Dacarbazina/farmacologia , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Feminino , Perfilação da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Temozolomida , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Chemosphere ; 146: 206-15, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26735719

RESUMO

Selected aromatic azo and benzidine based dyes are priority compounds under the Government of Canada's Chemical Management Plan (CMP) for environmental risk assessments. Organic compounds undergo chemical and biological transformations when they interact with environmental matrices and biotic species; identifying the transformation products is thus a critical component of the risk assessment process. Here, we used zero valent iron (ZVI) to initiate the reduction of the diazo compound dye Disperse Yellow 7 (DY 7). Using state-of-the-art accurate mass Liquid Chromatography-Quadrupole Time of Flight-Mass Spectroscopy (LC-QToF-MS), four transformation products were conclusively identified, while a fifth product was tentatively ascertained. The conclusively established transformation products included p-phenylenediamine (p-PDA, a known genotoxin), 4-aminoazobenzene (4-AAB, a category 2 carcinogen) and 4-aminobiphenyl (4-ABP, a category 1 human carcinogen). 4-ABP is thought to form via a benzidine rearrangement; this is the first report of DY 7 undergoing a benzidine rearrangement. Given the importance of reduction processes in the metabolism of organic contaminants by aquatic species, we used LC-MS/MS to analyze sediment samples that had been generated previously upon exposure of Western clawed frogs (Silurana tropicalis) to DY 7 (at exposure levels where cellular stress was observed in S. tropicalis). We found p-PDA, 4-AAB, and 4-ABP were present in all exposures, but not in any of the sediment controls, demonstrating that upon release of DY 7 to the aquatic environment, sediment dwelling organisms will metabolize DY 7 to generate known (and suspected) human carcinogens, including through a previously unreported in vivo benzidine rearrangement to produce 4-ABP.


Assuntos
Compostos Azo/análise , Carcinógenos/análise , Corantes/análise , Monitoramento Ambiental/métodos , Sedimentos Geológicos/química , Mutagênicos/análise , Animais , Compostos Azo/química , Compostos Azo/farmacocinética , Compostos Azo/toxicidade , Biotransformação , Canadá , Carcinógenos/farmacocinética , Carcinógenos/toxicidade , Corantes/farmacocinética , Corantes/toxicidade , Humanos , Ferro/química , Mutagênicos/farmacocinética , Mutagênicos/toxicidade , Oxirredução , Espectrometria de Massas em Tandem , Xenopus/metabolismo
18.
Cancer Cell ; 27(6): 864-76, 2015 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-26058080

RESUMO

From an shRNA screen, we identified ClpP as a member of the mitochondrial proteome whose knockdown reduced the viability of K562 leukemic cells. Expression of this mitochondrial protease that has structural similarity to the cytoplasmic proteosome is increased in leukemic cells from approximately half of all patients with AML. Genetic or chemical inhibition of ClpP killed cells from both human AML cell lines and primary samples in which the cells showed elevated ClpP expression but did not affect their normal counterparts. Importantly, Clpp knockout mice were viable with normal hematopoiesis. Mechanistically, we found that ClpP interacts with mitochondrial respiratory chain proteins and metabolic enzymes, and knockdown of ClpP in leukemic cells inhibited oxidative phosphorylation and mitochondrial metabolism.


Assuntos
Endopeptidase Clp/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Animais , Endopeptidase Clp/metabolismo , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos SCID , RNA Interferente Pequeno/genética
19.
Apoptosis ; 20(7): 948-59, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25832785

RESUMO

To identify new biological vulnerabilities in acute myeloid leukemia, we screened a library of natural products for compounds cytotoxic to TEX leukemia cells. This screen identified the novel small molecule Deoxysappanone B 7,4' dimethyl ether (Deox B 7,4), which possessed nanomolar anti-leukemic activity. To determine the anti-leukemic mechanism of action of Deox B 7,4, we conducted a genome-wide screen in Saccharomyces cerevisiae and identified enrichment of genes related to mitotic cell cycle as well as vacuolar acidification, therefore pointing to microtubules and vacuolar (V)-ATPase as potential drug targets. Further investigations into the mechanisms of action of Deox B 7,4 and a related analogue revealed that these compounds were reversible microtubule inhibitors that bound near the colchicine site. In addition, Deox B 7,4 and its analogue increased lysosomal V-ATPase activity and lysosome acidity. The effects on microtubules and lysosomes were functionally important for the anti-leukemic effects of these drugs. The lysosomal effects were characteristic of select microtubule inhibitors as only the Deox compounds and nocodazole, but not colchicine, vinca alkaloids or paclitaxel, altered lysosome acidity and induced lysosomal disruption. Thus, our data highlight a new mechanism of action of select microtubule inhibitors on lysosomal function.


Assuntos
Cromonas/farmacologia , Guaiacol/análogos & derivados , Leucemia Mieloide Aguda/metabolismo , Lisossomos/efeitos dos fármacos , Moduladores de Tubulina/farmacologia , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Guaiacol/farmacologia , Humanos , Leucemia Mieloide Aguda/patologia , Lisossomos/química , Lisossomos/metabolismo , Camundongos , Saccharomyces cerevisiae , ATPases Vacuolares Próton-Translocadoras/metabolismo
20.
Proc Natl Acad Sci U S A ; 111(35): 12853-8, 2014 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-25136132

RESUMO

SET domain containing (lysine methyltransferase) 7 (SETD7) is implicated in multiple signaling and disease related pathways with a broad diversity of reported substrates. Here, we report the discovery of (R)-PFI-2-a first-in-class, potent (Ki (app) = 0.33 nM), selective, and cell-active inhibitor of the methyltransferase activity of human SETD7-and its 500-fold less active enantiomer, (S)-PFI-2. (R)-PFI-2 exhibits an unusual cofactor-dependent and substrate-competitive inhibitory mechanism by occupying the substrate peptide binding groove of SETD7, including the catalytic lysine-binding channel, and by making direct contact with the donor methyl group of the cofactor, S-adenosylmethionine. Chemoproteomics experiments using a biotinylated derivative of (R)-PFI-2 demonstrated dose-dependent competition for binding to endogenous SETD7 in MCF7 cells pretreated with (R)-PFI-2. In murine embryonic fibroblasts, (R)-PFI-2 treatment phenocopied the effects of Setd7 deficiency on Hippo pathway signaling, via modulation of the transcriptional coactivator Yes-associated protein (YAP) and regulation of YAP target genes. In confluent MCF7 cells, (R)-PFI-2 rapidly altered YAP localization, suggesting continuous and dynamic regulation of YAP by the methyltransferase activity of SETD7. These data establish (R)-PFI-2 and related compounds as a valuable tool-kit for the study of the diverse roles of SETD7 in cells and further validate protein methyltransferases as a druggable target class.


Assuntos
Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Pirrolidinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Tetra-Hidroisoquinolinas/farmacologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Fibroblastos/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Humanos , Células MCF-7 , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Mutação , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/genética , Pirrolidinas/química , Relação Estrutura-Atividade , Sulfonamidas/química , Tetra-Hidroisoquinolinas/química , Fatores de Transcrição
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