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1.
J Neurooncol ; 168(1): 125-138, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38563850

RESUMO

PURPOSE: Triple-negative breast cancer (TNBC) often metastasizes to the central nervous system (CNS) and has the highest propensity among breast cancer subtypes to develop leptomeningeal disease (LMD). LMD is a spread of cancer into leptomeningeal space that speeds up the disease progression and severely aggravates the prognosis. LMD has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD. METHODS: A small-molecule screen involving TNBC cell lines identified benzimidazoles as potential therapeutic agents for further study. In vitro migration assays were used to evaluate cell migration capacity and the effect of MBZ. For in vivo testing, CNS metastasis was introduced into BALB/c athymic nude mice through internal carotid artery injections of brain-tropic MDA-MB-231-BR or MCF7-BR cells. Tumor growth and spread was monitored by bioluminescence imaging and immunohistochemistry. MBZ was given orally at 50 and 100 mg/kg doses. MBZ bioavailability was assayed by mass spectrometry. RESULTS: Bioinformatic analysis and migration assays revealed higher migratory capacity of TNBC compared to other breast cancer subtypes. MBZ effectively slowed down migration of TNBC cell line MDA-MB-231 and its brain tropic derivative MDA-MB-231-BR. In animal studies, MBZ reduced leptomeningeal spread, and extended survival in brain metastasis model produced by MDA-MB-231-BR cells. MBZ did not have an effect in the non-migratory MCF7-BR model. CONCLUSIONS: We demonstrated that MBZ is a safe and effective oral agent in an animal model of TNBC CNS metastasis. Our findings are concordant with previous efforts involving MBZ and CNS pathology and support the drug's potential utility to slow down leptomeningeal spread.


Assuntos
Movimento Celular , Reposicionamento de Medicamentos , Mebendazol , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias de Mama Triplo Negativas , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/patologia , Animais , Humanos , Feminino , Mebendazol/farmacologia , Mebendazol/uso terapêutico , Camundongos , Movimento Celular/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Linhagem Celular Tumoral , Neoplasias do Sistema Nervoso Central/secundário , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos
2.
Res Sq ; 2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38405839

RESUMO

Purpose: Triple-negative breast cancer (TNBC) is an aggressive subtype that often metastasizes to the brain. Leptomeningeal disease (LMD), a devastating brain metastasis common in TNBC, has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD. Methods: A small-molecule screen involving TNBC cell lines identified benzimidazoles as potential therapeutic agents for further study. In vitro migration assays were used to evaluate cell migration capacity and the effect of MBZ. For in vivo testing, LMD was introduced into BALB/c athymic nude mice through internal carotid artery injections of brain-tropic MDA-MB-231-BR or MCF7-BR cells. Tumor growth and spread was monitored by bioluminescence imaging. MBZ was given orally at 50 and 100 mg/kg doses. MBZ bioavailability was assayed by mass spectrometry. Results: Bioinformatic analysis and migration assays revealed higher migratory capacity of TNBC compared to other breast cancer subtypes. MBZ effectively slowed down migration of TNBC cell line MDA-MB-231 and its brain tropic derivative MDA-MB-231-BR. In animal studies, MBZ reduced tumor growth and extended survival in the LMD model produced by MDA-MB-231-BR cells. MBZ did not have an effect in the non-migratory MCF7-BR model. Conclusions: We demonstrated that MBZ is a safe and effective oral agent in an animal model of TNBC LMD. Our findings are concordant with previous efforts involving MBZ and central nervous system pathology and further support the drug's potential utility as an alternative therapeutic for TNBC LMD.

3.
J Clin Invest ; 133(19)2023 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-37581931

RESUMO

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family of receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, ErbB2, and ErbB4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, proinflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production, and disruption of blood-brain barrier integrity in microfluidics-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof of principle for a repurposed, ErbB-targeted approach to combat emerging viruses.


Assuntos
COVID-19 , Hepatite C Crônica , Animais , Humanos , Camundongos , Antivirais/farmacologia , Citocinas , Inflamação/tratamento farmacológico , Lapatinib/farmacologia , SARS-CoV-2
4.
bioRxiv ; 2023 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34159337

RESUMO

Targeting host factors exploited by multiple viruses could offer broad-spectrum solutions for pandemic preparedness. Seventeen candidates targeting diverse functions emerged in a screen of 4,413 compounds for SARS-CoV-2 inhibitors. We demonstrated that lapatinib and other approved inhibitors of the ErbB family receptor tyrosine kinases suppress replication of SARS-CoV-2, Venezuelan equine encephalitis virus (VEEV), and other emerging viruses with a high barrier to resistance. Lapatinib suppressed SARS-CoV-2 entry and later stages of the viral life cycle and showed synergistic effect with the direct-acting antiviral nirmatrelvir. We discovered that ErbB1, 2 and 4 bind SARS-CoV-2 S1 protein and regulate viral and ACE2 internalization, and they are required for VEEV infection. In human lung organoids, lapatinib protected from SARS-CoV-2-induced activation of ErbB-regulated pathways implicated in non-infectious lung injury, pro-inflammatory cytokine production, and epithelial barrier injury. Lapatinib suppressed VEEV replication, cytokine production and disruption of the blood-brain barrier integrity in microfluidic-based human neurovascular units, and reduced mortality in a lethal infection murine model. We validated lapatinib-mediated inhibition of ErbB activity as an important mechanism of antiviral action. These findings reveal regulation of viral replication, inflammation, and tissue injury via ErbBs and establish a proof-of-principle for a repurposed, ErbB-targeted approach to combat emerging viruses.

5.
Nat Commun ; 13(1): 6796, 2022 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-36357388

RESUMO

When the protein or calcium homeostasis of the endoplasmic reticulum (ER) is adversely altered, cells experience ER stress that leads to various diseases including neurodegeneration. Genetic deletion of an ER stress downstream effector, CHOP, significantly protects neuron somata and axons. Here we report that three tricyclic compounds identified through a small-scale high throughput screening using a CHOP promoter-driven luciferase cell-based assay, effectively inhibit ER stress by antagonizing their common target, histamine receptor H1 (HRH1). We further demonstrated that systemic administration of one of these compounds, maprotiline, or CRISPR-mediated retinal ganglion cell (RGC)-specific HRH1 inhibition, delivers considerable neuroprotection of both RGC somata and axons and preservation of visual function in two mouse optic neuropathy models. Finally, we determine that maprotiline restores ER homeostasis by inhibiting HRH1-mediated Ca2+ release from ER. In this work we establish maprotiline as a candidate neuroprotectant and HRH1 as a potential therapeutic target for glaucoma.


Assuntos
Maprotilina , Células Ganglionares da Retina , Camundongos , Animais , Células Ganglionares da Retina/metabolismo , Maprotilina/metabolismo , Maprotilina/farmacologia , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Modelos Animais de Doenças , Homeostase , Receptores Histamínicos/metabolismo
6.
Cell Rep ; 41(4): 111505, 2022 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-36288715

RESUMO

Gene-based therapeutic strategies to lower ataxin-2 levels are emerging for the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). Additional strategies to lower levels of ataxin-2 could be beneficial. Here, we perform a genome-wide arrayed small interfering RNA (siRNA) screen in human cells and identify RTN4R, the gene encoding the RTN4/NoGo-Receptor, as a potent modifier of ataxin-2 levels. RTN4R knockdown, or treatment with a peptide inhibitor, is sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro, and Rtn4r knockout mice have reduced ataxin-2 levels in vivo. We provide evidence that ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy. These data define the RTN4/NoGo-Receptor as a novel therapeutic target for ALS and SCA2 and implicate the targeting of ataxin-2 as a potential treatment following nerve injury.


Assuntos
Esclerose Lateral Amiotrófica , Ataxias Espinocerebelares , Animais , Camundongos , Humanos , Ataxina-2/genética , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , RNA Interferente Pequeno , Receptores Nogo/metabolismo , Ataxias Espinocerebelares/genética , Camundongos Knockout , Peptídeos/metabolismo , Proteínas Nogo/genética , Proteínas Nogo/metabolismo
7.
Oncogene ; 39(9): 2029, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31659253

RESUMO

The original version of this Article contained an error in the spelling of the author David Solow-Cordero, which was incorrectly given as David Solow-Codero. This has now been corrected in both the PDF and HTML versions of the Article.

8.
Proc Natl Acad Sci U S A ; 116(36): 18009-18014, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31427509

RESUMO

Citrus greening disease, also known as huanglongbing (HLB), is the most devastating disease of Citrus worldwide. This incurable disease is caused primarily by the bacterium Candidatus Liberibacter asiaticus and spread by feeding of the Asian Citrus Psyllid, Diaphorina citriCa L. asiaticus cannot be cultured; its growth is restricted to citrus phloem and the psyllid insect. Management of infected trees includes use of broad-spectrum antibiotics, which have disadvantages. Recent work has sought to identify small molecules that inhibit Ca L. asiaticus transcription regulators, based on a premise that at least some regulators control expression of genes necessary for virulence. We describe a synthetic, high-throughput screening system to identify compounds that inhibit activity of Ca L. asiaticus transcription activators LdtR, RpoH, and VisNR. Our system uses the closely related model bacterium, Sinorhizobium meliloti, as a heterologous host for expression of a Ca L. asiaticus transcription activator, the activity of which is detected through expression of an enhanced green fluorescent protein (EGFP) gene fused to a target promoter. We used this system to screen more than 120,000 compounds for compounds that inhibited regulator activity, but not growth. Our screen identified several dozen compounds that inhibit regulator activity in our assay. This work shows that, in addition to providing a means of characterizing Ca L. asiaticus regulators, an S. meliloti host can be used for preliminary identification of candidate inhibitory molecules.


Assuntos
Antibacterianos , Proteínas de Bactérias/antagonistas & inibidores , Rhizobiaceae/metabolismo , Transativadores/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Citrus/microbiologia , Avaliação Pré-Clínica de Medicamentos , Doenças das Plantas/microbiologia , Rhizobiaceae/genética , Transativadores/genética , Transativadores/metabolismo
9.
J Clin Invest ; 128(12): 5307-5321, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30371505

RESUMO

After the initial responsiveness of triple-negative breast cancers (TNBCs) to chemotherapy, they often recur as chemotherapy-resistant tumors, and this has been associated with upregulated homology-directed repair (HDR). Thus, inhibitors of HDR could be a useful adjunct to chemotherapy treatment of these cancers. We performed a high-throughput chemical screen for inhibitors of HDR from which we obtained a number of hits that disrupted microtubule dynamics. We postulated that high levels of the target molecules of our screen in tumors would correlate with poor chemotherapy response. We found that inhibition or knockdown of dynamin 2 (DNM2), known for its role in endocytic cell trafficking and microtubule dynamics, impaired HDR and improved response to chemotherapy of cells and of tumors in mice. In a retrospective analysis, levels of DNM2 at the time of treatment strongly predicted chemotherapy outcome for estrogen receptor-negative and especially for TNBC patients. We propose that DNM2-associated DNA repair enzyme trafficking is important for HDR efficiency and is a powerful predictor of sensitivity to breast cancer chemotherapy and an important target for therapy.


Assuntos
Antineoplásicos/farmacologia , Dinaminas/metabolismo , Reparo de DNA por Recombinação , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/enzimologia , Animais , Células CHO , Cricetulus , Dinamina II , Dinaminas/genética , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Mol Cancer Res ; 16(5): 745-753, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29440447

RESUMO

Activation of the unfolded protein response (UPR) signaling pathways is linked to multiple human diseases, including cancer. The inositol-requiring kinase 1α (IRE1α)-X-box binding protein 1 (XBP1) pathway is the most evolutionarily conserved of the three major signaling branches of the UPR. Here, we performed a genome-wide siRNA screen to obtain a systematic assessment of genes integrated in the IRE1α-XBP1 axis. We monitored the expression of an XBP1-luciferase chimeric protein in which luciferase was fused in-frame with the spliced (active) form of XBP1. Using cells expressing this reporter construct, we identified 162 genes for which siRNA inhibition resulted in alteration in XBP1 splicing. These genes express diverse types of proteins modulating a wide range of cellular processes. Pathway analysis identified a set of genes implicated in the pathogenesis of breast cancer. Several genes, including BCL10, GCLM, and IGF1R, correlated with worse relapse-free survival (RFS) in an analysis of patients with triple-negative breast cancer (TNBC). However, in this cohort of 1,908 patients, only high GCLM expression correlated with worse RFS in both TNBC and non-TNBC patients. Altogether, our study revealed unidentified roles of novel pathways regulating the UPR, and these findings may serve as a paradigm for exploring novel therapeutic opportunities based on modulating the UPR.Implications: Genome-wide RNAi screen identifies novel genes/pathways that modulate IRE1α-XBP1 signaling in human tumor cells and leads to the development of improved therapeutic approaches targeting the UPR.Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/5/745/F1.large.jpg Mol Cancer Res; 16(5); 745-53. ©2018 AACR.


Assuntos
Genoma Humano/genética , Proteína 1 de Ligação a X-Box/genética , Humanos , Interferência de RNA , Transfecção
11.
Mol Cancer Ther ; 15(9): 2055-65, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27307600

RESUMO

Using a luciferase reporter-based high-throughput chemical library screen and topological data analysis, we identified N-acridine-9-yl-N',N'-dimethylpropane-1,3-diamine (DAPA) as an inhibitor of the inositol requiring kinase 1α (IRE1α)-X-box binding protein-1 (XBP1) pathway of the unfolded protein response. We designed a collection of analogues based on the structure of DAPA to explore structure-activity relationships and identified N(9)-(3-(dimethylamino)propyl)-N(3),N(3),N(6),N(6)-tetramethylacridine-3,6,9-triamine (3,6-DMAD), with 3,6-dimethylamino substitution on the chromophore, as a potent inhibitor. 3,6-DMAD inhibited both IRE1α oligomerization and in vitro endoribonuclease (RNase) activity, whereas the other analogues only blocked IRE1α oligomerization. Consistent with the inhibition of IRE1α-mediated XBP1 splicing, which is critical for multiple myeloma cell survival, these analogues were cytotoxic to multiple myeloma cell lines. Furthermore, 3,6-DMAD inhibited XBP1 splicing in vivo and the growth of multiple myeloma tumor xenografts. Our study not only confirmed the utilization of topological data analysis in drug discovery but also identified a class of compounds with a unique mechanism of action as potent IRE1α-XBP1 inhibitors in the treatment of multiple myeloma. Mol Cancer Ther; 15(9); 2055-65. ©2016 AACR.


Assuntos
Acridinas/farmacologia , Antineoplásicos/farmacologia , Endorribonucleases/metabolismo , Mieloma Múltiplo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 1 de Ligação a X-Box/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Modelos Animais de Doenças , Descoberta de Drogas , Ensaios de Seleção de Medicamentos Antitumorais , Endorribonucleases/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Camundongos , Mieloma Múltiplo/genética , Proteínas Serina-Treonina Quinases/genética , Proteína 1 de Ligação a X-Box/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Oncotarget ; 7(8): 8653-62, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26840025

RESUMO

The transcription factor CREB (cAMP Response Element Binding Protein) is an important determinant in the growth of Acute Myeloid Leukemia (AML) cells. CREB overexpression increases AML cell growth by driving the expression of key regulators of apoptosis and the cell cycle. Conversely, CREB knockdown inhibits proliferation and survival of AML cells but not normal hematopoietic cells. Thus, CREB represents a promising drug target for the treatment of AML, which carries a poor prognosis. In this study, we performed a high-throughput small molecule screen to identify compounds that disrupt CREB function in AML cells. We screened ~114,000 candidate compounds from Stanford University's small molecule library, and identified 5 molecules that inhibit CREB function at micromolar concentrations, but are non-toxic to normal hematopoietic cells. This study suggests that targeting CREB function using small molecules could provide alternative approaches to treat AML.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/patologia , Regiões Promotoras Genéticas/genética , Elementos de Resposta/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Luciferases/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Sci Transl Med ; 7(306): 306ra148, 2015 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-26400909

RESUMO

Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.


Assuntos
Antibacterianos/uso terapêutico , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/tratamento farmacológico , Virulência/efeitos dos fármacos , Animais , Azóis/uso terapêutico , Isoindóis , Camundongos , Compostos Organosselênicos/uso terapêutico
14.
Chem Biol ; 20(11): 1352-63, 2013 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-24183972

RESUMO

Phenotypic high-throughput chemical screens allow for discovery of small molecules that modulate complex phenotypes and provide lead compounds for novel therapies; however, identification of the mechanistically relevant targets remains a major experimental challenge. We report the application of sequential unbiased high-throughput chemical and ultracomplex small hairpin RNA (shRNA) screens to identify a distinctive class of inhibitors that target nicotinamide phosphoribosyl transferase (NAMPT), a rate-limiting enzyme in the biosynthesis of nicotinamide adenine dinucleotide, a crucial cofactor in many biochemical processes. The lead compound STF-118804 is a highly specific NAMPT inhibitor, improves survival in an orthotopic xenotransplant model of high-risk acute lymphoblastic leukemia, and targets leukemia stem cells. Tandem high-throughput screening using chemical and ultracomplex shRNA libraries, therefore, provides a rapid chemical genetics approach for seamless progression from small-molecule lead identification to target discovery and validation.


Assuntos
Antineoplásicos/farmacologia , Benzamidas/farmacologia , Citocinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Nicotinamida Fosforribosiltransferase/antagonistas & inibidores , Picolinas/farmacologia , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Benzamidas/química , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/genética , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Estrutura Molecular , Nicotinamida Fosforribosiltransferase/genética , Nicotinamida Fosforribosiltransferase/metabolismo , Fenótipo , Picolinas/química , Relação Estrutura-Atividade , Células Tumorais Cultivadas
15.
J Clin Invest ; 123(8): 3600-13, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23867624

RESUMO

Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Células Endoteliais/fisiologia , Hipertensão Pulmonar/tratamento farmacológico , Tacrolimo/farmacologia , Animais , Apoptose , Proteína Morfogenética Óssea 4/fisiologia , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Hipóxia Celular , Linhagem Celular Tumoral , Proliferação de Células , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Ensaios de Triagem em Larga Escala , Humanos , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/patologia , Proteína 1 Inibidora de Diferenciação/genética , Proteína 1 Inibidora de Diferenciação/metabolismo , Masculino , Camundongos , Camundongos Knockout , Microvasos/patologia , Neointima/tratamento farmacológico , Neointima/metabolismo , Neointima/patologia , Artéria Pulmonar/patologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Proteínas Smad/metabolismo , Proteína 1A de Ligação a Tacrolimo/metabolismo
16.
Proc Natl Acad Sci U S A ; 109(37): E2476-85, 2012 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-22895790

RESUMO

Up-regulation of the folding machinery of the heat-shock protein 90 (Hsp90) chaperone protein is crucial for cancer progression. The two Hsp90 isoforms (α and ß) play different roles in response to chemotherapy. To identify isoform-selective inhibitors of Hsp90(α/ß)/cochaperone p23 interactions, we developed a dual-luciferase (Renilla and Firefly) reporter system for high-throughput screening (HTS) and monitoring the efficacy of Hsp90 inhibitors in cell culture and live mice. HTS of a 30,176 small-molecule chemical library in cell culture identified a compound, N-(5-methylisoxazol-3-yl)-2-[4-(thiophen-2-yl)-6-(trifluoromethyl)pyrimidin-2-ylthio]acetamide (CP9), that binds to Hsp90(α/ß) and displays characteristics of Hsp90 inhibitors, i.e., degradation of Hsp90 client proteins and inhibition of cell proliferation, glucose metabolism, and thymidine kinase activity, in multiple cancer cell lines. The efficacy of CP9 in disrupting Hsp90(α/ß)/p23 interactions and cell proliferation in tumor xenografts was evaluated by non-invasive, repetitive Renilla luciferase and Firefly luciferase imaging, respectively. At 38 h posttreatment (80 mg/kg × 3, i.p.), CP9 led to selective disruption of Hsp90α/p23 as compared with Hsp90ß/p23 interactions. Small-animal PET/CT in the same cohort of mice showed that CP9 treatment (43 h) led to a 40% decrease in (18)F-fluorodeoxyglucose uptake in tumors relative to carrier control-treated mice. However, CP9 did not lead to significant degradation of Hsp90 client proteins in tumors. We performed a structural activity relationship study with 62 analogs of CP9 and identified A17 as the lead compound that outperformed CP9 in inhibiting Hsp90(α/ß)/p23 interactions in cell culture. Our efforts demonstrated the power of coupling of HTS with multimodality molecular imaging and led to identification of Hsp90 inhibitors.


Assuntos
Acetamidas/farmacologia , Benzoquinonas/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Oxirredutases Intramoleculares/metabolismo , Lactamas Macrocíclicas/farmacologia , Neoplasias/metabolismo , Tioacetamida/análogos & derivados , Tiofenos/farmacologia , Animais , Western Blotting , Linhagem Celular Tumoral , Descoberta de Drogas , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Ensaios de Triagem em Larga Escala , Humanos , Imidazóis , Imunoprecipitação , Chumbo/farmacologia , Luciferases de Vaga-Lume , Luciferases de Renilla , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Tomografia por Emissão de Pósitrons , Prostaglandina-E Sintases , Dobramento de Proteína , Isoformas de Proteínas/metabolismo , Pirazinas , Bibliotecas de Moléculas Pequenas , Tioacetamida/farmacologia , Tomografia Computadorizada por Raios X , Trítio
17.
Sci Transl Med ; 3(94): 94ra70, 2011 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-21813754

RESUMO

Identifying new targeted therapies that kill tumor cells while sparing normal tissue is a major challenge of cancer research. Using a high-throughput chemical synthetic lethal screen, we sought to identify compounds that exploit the loss of the von Hippel-Lindau (VHL) tumor suppressor gene, which occurs in about 80% of renal cell carcinomas (RCCs). RCCs, like many other cancers, are dependent on aerobic glycolysis for ATP production, a phenomenon known as the Warburg effect. The dependence of RCCs on glycolysis is in part a result of induction of glucose transporter 1 (GLUT1). Here, we report the identification of a class of compounds, the 3-series, exemplified by STF-31, which selectively kills RCCs by specifically targeting glucose uptake through GLUT1 and exploiting the unique dependence of these cells on GLUT1 for survival. Treatment with these agents inhibits the growth of RCCs by binding GLUT1 directly and impeding glucose uptake in vivo without toxicity to normal tissue. Activity of STF-31 in these experimental renal tumors can be monitored by [(18)F]fluorodeoxyglucose uptake by micro-positron emission tomography imaging, and therefore, these agents may be readily tested clinically in human tumors. Our results show that the Warburg effect confers distinct characteristics on tumor cells that can be selectively targeted for therapy.


Assuntos
Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/tratamento farmacológico , Transportador de Glucose Tipo 1/metabolismo , Neoplasias Renais/tratamento farmacológico , Trifosfato de Adenosina/biossíntese , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose , Carcinoma de Células Renais/metabolismo , Glucose/metabolismo , Glicólise , Humanos , Neoplasias Renais/metabolismo
18.
Blood ; 117(4): 1311-4, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21081713

RESUMO

Activation of the adaptive Ire1-XBP1 pathway has been identified in many solid tumors and hematologic malignancies, including multiple myeloma (MM). Here, we report the identification of STF-083010, a novel small-molecule inhibitor of Ire1. STF-083010 inhibited Ire1 endonuclease activity, without affecting its kinase activity, after endoplasmic reticulum stress both in vitro and in vivo. Treatment with STF-083010 showed significant antimyeloma activity in model human MM xenografts. Similarly, STF-083010 was preferentially toxic to freshly isolated human CD138(+) MM cells compared with other similarly isolated cell populations. The identification of this novel Ire1 inhibitor supports the hypothesis that the Ire1-XBP1 axis is a promising target for anticancer therapy, especially in the context of MM.


Assuntos
Citotoxinas/farmacologia , Endorribonucleases/antagonistas & inibidores , Mieloma Múltiplo/patologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sulfonamidas/farmacologia , Tiofenos/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Ácidos Borônicos/administração & dosagem , Bortezomib , Células Cultivadas , Citotoxinas/uso terapêutico , Relação Dose-Resposta a Droga , Humanos , Camundongos , Modelos Biológicos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Pirazinas/administração & dosagem , Especificidade por Substrato/efeitos dos fármacos , Sulfonamidas/administração & dosagem , Sulfonamidas/uso terapêutico , Tiofenos/administração & dosagem , Tiofenos/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Proc Natl Acad Sci U S A ; 106(33): 14132-7, 2009 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-19666565

RESUMO

Inappropriate activation of the Hedgehog (Hh) signaling pathway has been implicated in a diverse spectrum of cancers, and its pharmacological blockade has emerged as an anti-tumor strategy. While nearly all known Hh pathway antagonists target the transmembrane protein Smoothened (Smo), small molecules that suppress downstream effectors could more comprehensively remediate Hh pathway-dependent tumors. We report here four Hh pathway antagonists that are epistatic to the nucleocytoplasmic regulator Suppressor of Fused [Su(fu)], including two that can inhibit Hh target gene expression induced by overexpression of the Gli transcription factors. Each inhibitor has a unique mechanism of action, and their phenotypes reveal that Gli processing, Gli activation, and primary cilia formation are pharmacologically targetable. We further establish the ability of certain compounds to block the proliferation of cerebellar granule neuron precursors expressing an oncogenic form of Smo, and we demonstrate that Hh pathway inhibitors can have tissue-specific activities. These antagonists therefore constitute a valuable set of chemical tools for interrogating downstream Hh signaling mechanisms and for developing chemotherapies against Hh pathway-related cancers.


Assuntos
Regulação Neoplásica da Expressão Gênica , Proteínas Hedgehog/antagonistas & inibidores , Proteínas Hedgehog/metabolismo , Neoplasias/metabolismo , Animais , Química Farmacêutica/métodos , Desenho de Fármacos , Epistasia Genética , Fibroblastos/metabolismo , Humanos , Camundongos , Modelos Biológicos , Células NIH 3T3 , Neurônios/metabolismo , Fenótipo , Ligação Proteica
20.
Mol Cell ; 35(2): 228-39, 2009 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-19647519

RESUMO

Signaling pathways that respond to DNA damage are essential for the maintenance of genome stability and are linked to many diseases, including cancer. Here, a genome-wide siRNA screen was employed to identify additional genes involved in genome stabilization by monitoring phosphorylation of the histone variant H2AX, an early mark of DNA damage. We identified hundreds of genes whose downregulation led to elevated levels of H2AX phosphorylation (gammaH2AX) and revealed links to cellular complexes and to genes with unclassified functions. We demonstrate a widespread role for mRNA-processing factors in preventing DNA damage, which in some cases is caused by aberrant RNA-DNA structures. Furthermore, we connect increased gammaH2AX levels to the neurological disorder Charcot-Marie-Tooth (CMT) syndrome, and we find a role for several CMT proteins in the DNA-damage response. These data indicate that preservation of genome stability is mediated by a larger network of biological processes than previously appreciated.


Assuntos
Instabilidade Genômica , RNA Interferente Pequeno/fisiologia , Transdução de Sinais , Doença de Charcot-Marie-Tooth/genética , Biologia Computacional , Dano ao DNA , Reparo do DNA/genética , Replicação do DNA/genética , Regulação para Baixo , Genes cdc , Biblioteca Genômica , Genômica , Células HeLa , Histonas/metabolismo , Humanos , Fosforilação , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo
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