RESUMO
Ferroptosis is a form of nonapoptotic cell death for which key regulators remain unknown. We sought a common mediator for the lethality of 12 ferroptosis-inducing small molecules. We used targeted metabolomic profiling to discover that depletion of glutathione causes inactivation of glutathione peroxidases (GPXs) in response to one class of compounds and a chemoproteomics strategy to discover that GPX4 is directly inhibited by a second class of compounds. GPX4 overexpression and knockdown modulated the lethality of 12 ferroptosis inducers, but not of 11 compounds with other lethal mechanisms. In addition, two representative ferroptosis inducers prevented tumor growth in xenograft mouse tumor models. Sensitivity profiling in 177 cancer cell lines revealed that diffuse large B cell lymphomas and renal cell carcinomas are particularly susceptible to GPX4-regulated ferroptosis. Thus, GPX4 is an essential regulator of ferroptotic cancer cell death.
Assuntos
Carbolinas/farmacologia , Morte Celular/efeitos dos fármacos , Glutationa Peroxidase/antagonistas & inibidores , Piperazinas/farmacologia , Animais , Carcinoma de Células Renais/tratamento farmacológico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Xenoenxertos , Humanos , Linfoma de Células B/tratamento farmacológico , Camundongos , Transplante de Neoplasias , Neoplasias/tratamento farmacológico , Fosfolipídeo Hidroperóxido Glutationa PeroxidaseRESUMO
The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: (1) associate with specific cancer-genomic alterations and (2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (http://www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene ß-catenin with sensitivity to the Bcl-2 family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and to accelerate discovery of drugs matched to patients by their cancer genotype and lineage.
Assuntos
Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas , Neoplasias/tratamento farmacológico , Antineoplásicos/química , Linhagem Celular Tumoral , Humanos , Neoplasias/genéticaRESUMO
Pills are a cornerstone of medicine but can be challenging to swallow. While liquid formulations are easier to ingest, they lack the capacity to localize therapeutics with excipients nor act as controlled release devices. Here we describe drug formulations based on liquid in situ-forming tough (LIFT) hydrogels that bridge the advantages of solid and liquid dosage forms. LIFT hydrogels form directly in the stomach through sequential ingestion of a crosslinker solution of calcium and dithiol crosslinkers, followed by a drug-containing polymer solution of alginate and four-arm poly(ethylene glycol)-maleimide. We show that LIFT hydrogels robustly form in the stomachs of live rats and pigs, and are mechanically tough, biocompatible and safely cleared after 24 h. LIFT hydrogels deliver a total drug dose comparable to unencapsulated drug in a controlled manner, and protect encapsulated therapeutic enzymes and bacteria from gastric acid-mediated deactivation. Overall, LIFT hydrogels may expand access to advanced therapeutics for patients with difficulty swallowing.
RESUMO
Chordoma is a rare malignant tumor demonstrating notochordal differentiation. It is dependent on brachyury (TBXT), a hallmark notochordal gene and transcription factor, and shares histologic features and the same anatomic location as the notochord. This study involved a molecular comparison of chordoma and notochord to identify dysregulated cellular pathways. The lack of a molecular reference from appropriate control tissue limits our understanding of chordoma and its relationship to notochord. Therefore, an unbiased comparison of chordoma, human notochord, and an atlas of normal and cancerous tissue was conducted using gene expression profiling to clarify the chordoma/notochord relationship and potentially identify novel drug targets. The study found striking consistency in gene expression profiles between chordoma and notochord, supporting the hypothesis that chordoma develops from notochordal remnants. A 12-gene diagnostic chordoma signature was identified and the TBXT/transforming growth factor beta (TGF-ß)/SOX6/SOX9 pathway was hyperactivated in the tumor, suggesting that pathways associated with chondrogenesis were a central driver of chordoma development. Experimental validation in chordoma cells confirmed these findings and emphasized the dependence of chordoma proliferation and survival on TGF-ß. The computational and experimental evidence provided the first molecular connection between notochord and chordoma and identified core members of a chordoma regulatory pathway involving TBXT. This pathway provides new therapeutic targets for this unique malignant neoplasm and highlights TGF-ß as a prime druggable candidate.
Assuntos
Cordoma , Humanos , Cordoma/genética , Cordoma/patologia , Notocorda/metabolismo , Notocorda/patologia , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
We describe our design, synthesis, and chemical study of a set of functional epidithiodiketopiperazines (ETPs) and evaluation of their activity against five human cancer cell lines. Our structure-activity relationship-guided substitution of ETP alkaloids offers versatile derivatization while maintaining potent anticancer activity, offering exciting opportunity for their use as there are no examples of complex and potently anticancer (nM) ETPs being directly used as conjugatable probes or warheads. Our synthetic solutions to strategically designed ETPs with functional linkers required advances in stereoselective late-stage oxidation and thiolation chemistry in complex settings, including the application of novel reagents for dihydroxylation and cis-sulfidation of diketopiperazines. We demonstrate that complex ETPs equipped with a strategically substituted azide functional group are readily derivatized to the corresponding ETP-triazoles without compromising anticancer activity. Our chemical stability studies of ETPs along with cytotoxic evaluation of our designed ETPs against A549, DU 145, HeLa, HCT 116, and MCF7 human cancer cell lines provide insights into the impact of structural features on potency and chemical stability, informing future utility of ETPs in chemical and biological studies.
Assuntos
Antineoplásicos , Piperazinas , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Dicetopiperazinas/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Piperazinas/farmacologia , Relação Estrutura-AtividadeRESUMO
A unified enantioselective total synthesis and anticancer evaluation of all known epoxide-containing communesin alkaloids and related derivatives is described. Our synthesis is predicated on the convergent and modular diazene-directed assembly of two complex fragments to secure the critical C3a-C3a' linkage followed by a guided biomimetic aminal reorganization to deliver the heptacyclic core of these alkaloids. Concise enantioselective syntheses of the fragments were devised, with highlights including the application of a rationally designed sulfinamide chiral auxiliary, an efficient calcium trifluoromethanesulfonate promoted intramolecular amination, and a diastereoselective epoxidation that simultaneously converts the new chiral auxiliary to a versatile amine protective group. The modularity of our convergent approach enabled the rapid synthesis of all epoxide-containing members of the communesin family from a single heterodimeric intermediate, including the first total synthesis of communesins C-E, and G-I, and facilitated our stereochemical revision of (-)-communesin I, the most recently isolated communesin alkaloid. Furthermore, the generality of our biogenetically inspired heterodimer rearrangement was demonstrated in a guided synthesis of a communesin derivative with an unnatural topology. Finally, we report the first comparative analysis of the anticancer activities of all naturally occurring communesin alkaloids A-I and eight complex derivatives against five human cancer cell lines. From these data, we have identified (-)-communesin B as the most potent natural communesin and discovered that derivatives with N8'-sulfonamide substitution exhibit up to a 10-fold increase in potency over the natural alkaloids.
Assuntos
Alcaloides/farmacologia , Antineoplásicos/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Alcaloides/síntese química , Alcaloides/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Humanos , Conformação Molecular , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Collagen overproduction is a feature of fibrosis and cancer, while insufficient deposition of functional collagen molecules and/or the secretion of malformed collagen is common in genetic disorders like osteogenesis imperfecta. Collagen secretion is an appealing therapeutic target in these and other diseases, as secretion directly connects intracellular biosynthesis to collagen deposition and biological function in the extracellular matrix. However, small molecule and biological methods to tune collagen secretion are severely lacking. Their discovery could prove useful not only in the treatment of disease, but also in providing tools for better elucidating mechanisms of collagen biosynthesis. We developed a cell-based, high-throughput luminescent assay of collagen type I secretion and used it to screen for small molecules that selectively enhance or inhibit that process. Among several validated hits, the Hsp90 inhibitor 17-allylaminogeldanamycin (17-AAG) robustly decreases the secretion of collagen-I by our model cell line and by human primary cells. In these systems, 17-AAG and other pan-isoform Hsp90 inhibitors reduce collagen-I secretion post-translationally and are not global inhibitors of protein secretion. Surprisingly, the consequences of Hsp90 inhibitors cannot be attributed to inhibition of the endoplasmic reticulum's Hsp90 isoform, Grp94. Instead, collagen-I secretion likely depends on the activity of cytosolic Hsp90 chaperones, even though such chaperones cannot directly engage nascent collagen molecules. Our results highlight the value of a cell-based high-throughput screen for selective modulators of collagen secretion and suggest an unanticipated role for cytosolic Hsp90 in collagen secretion.
Assuntos
Colágeno Tipo I/química , Proteínas de Choque Térmico HSP90/química , Ensaios de Triagem em Larga Escala , Glicoproteínas de Membrana/química , Benzoquinonas/farmacologia , Linhagem Celular , Colágeno Tipo I/biossíntese , Colágeno Tipo I/metabolismo , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Lactamas Macrocíclicas/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Isoformas de Proteínas/químicaRESUMO
Changes in cellular gene expression in response to small-molecule or genetic perturbations have yielded signatures that can connect unknown mechanisms of action (MoA) to ones previously established. We hypothesized that differential basal gene expression could be correlated with patterns of small-molecule sensitivity across many cell lines to illuminate the actions of compounds whose MoA are unknown. To test this idea, we correlated the sensitivity patterns of 481 compounds with â¼19,000 basal transcript levels across 823 different human cancer cell lines and identified selective outlier transcripts. This process yielded many novel mechanistic insights, including the identification of activation mechanisms, cellular transporters and direct protein targets. We found that ML239, originally identified in a phenotypic screen for selective cytotoxicity in breast cancer stem-like cells, most likely acts through activation of fatty acid desaturase 2 (FADS2). These data and analytical tools are available to the research community through the Cancer Therapeutics Response Portal.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Aflatoxinas/química , Aflatoxinas/farmacologia , Western Blotting , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Simulação por Computador , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Estrutura Molecular , Análise de Componente Principal , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Lysosomes perform a critical cellular function as a site of degradation for diverse cargoes including proteins, organelles, and pathogens delivered through distinct pathways, and defects in lysosomal function have been implicated in a number of diseases. Recent studies have elucidated roles for the lysosome in the regulation of protein synthesis, metabolism, membrane integrity, and other processes involved in homeostasis. Complex small-molecule natural products have greatly contributed to the investigation of lysosomal function in cellular physiology. Here we report the discovery of a novel, small-molecule modulator of lysosomal acidification derived from diversity-oriented synthesis through high-content screening.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Lisossomos/efeitos dos fármacos , Lisossomos/enzimologia , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lisossomos/metabolismo , Macrolídeos/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidoresRESUMO
Dexras1, a small G-protein localized predominantly to the brain, is transcriptionally upregulated by the synthetic glucocorticoid dexamethasone. It has close homology to the Ras subfamily but differs in that Dexras1 contains an extended 7 kDa C-terminal tail. Previous studies in our laboratory showed that NMDA receptor activation, via NO and Dexras1, physiologically stimulates DMT1, the major iron importer. A membrane-permeable iron chelator substantially reduces NMDA excitotoxicity, suggesting that Dexras1-mediated iron influx plays a crucial role in NMDA/NO-mediated cell death. We here report that iron influx is elicited by nitric oxide but not by other proapoptotic stimuli, such as H2O2 or staurosporine. Deletion of Dexras1 in mice attenuates NO-mediated cell death in dissociated primary cortical neurons and retinal ganglion cells in vivo. Thus, Dexras1 appears to mediate NMDA-elicited neurotoxicity via NO and iron influx.
Assuntos
Córtex Cerebral/fisiologia , Ácido Glutâmico/toxicidade , N-Metilaspartato/toxicidade , Células Ganglionares da Retina/fisiologia , Proteínas ras/fisiologia , Animais , Córtex Cerebral/efeitos dos fármacos , Ácido Glutâmico/fisiologia , Células HEK293 , Humanos , Ferro/metabolismo , Ferro/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/fisiologia , N-Metilaspartato/fisiologia , Óxido Nítrico/fisiologia , Óxido Nítrico/toxicidade , Células PC12 , Ratos , Proteínas ras/deficiênciaRESUMO
High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.
RESUMO
A challenge for screening new candidate drugs to treat cancer is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels to propagate cells. Which nutrients are available can influence how cancer cells use metabolism to proliferate and impact sensitivity to some drugs, but a general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To enable screening of compounds to determine how the nutrient environment impacts drug efficacy, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We used this system to screen several small molecule libraries and found that compounds targeting metabolic enzymes were enriched as having differential efficacy in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.
RESUMO
A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.
Assuntos
Técnicas de Cultura de Células , Ensaios de Triagem em Larga Escala , Humanos , Linhagem Celular , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) influences cytotoxicity, translocating to the nucleus during apoptosis. Here we report a signalling pathway in which nitric oxide (NO) generation that follows apoptotic stimulation elicits S-nitrosylation of GAPDH, which triggers binding to Siah1 (an E3 ubiquitin ligase), nuclear translocation and apoptosis. S-nitrosylation of GAPDH augments its binding to Siah1, whose nuclear localization signal mediates translocation of GAPDH. GAPDH stabilizes Siah1, facilitating its degradation of nuclear proteins. Activation of macrophages by endotoxin and of neurons by glutamate elicits GAPDH-Siah1 binding, nuclear translocation and apoptosis, which are prevented by NO deletion. The NO-S-nitrosylation-GAPDH-Siah1 cascade may represent an important molecular mechanism of cytotoxicity.
Assuntos
Apoptose/fisiologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/fisiologia , Proteínas Nucleares/metabolismo , S-Nitrosotióis/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Cisteína/metabolismo , Citoplasma/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Modelos Biológicos , Mutação , N-Metilaspartato/farmacologia , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase Tipo I , Óxido Nítrico Sintase Tipo II , Proteínas Nucleares/genética , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , S-Nitrosoglutationa/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismo , Ubiquitina-Proteína LigasesRESUMO
Hepatokines, secretory proteins from the liver, mediate inter-organ communication to maintain a metabolic balance between food intake and energy expenditure. However, molecular mechanisms by which hepatokine levels are rapidly adjusted following stimuli are largely unknown. Here, we unravel how CNOT6L deadenylase switches off hepatokine expression after responding to stimuli (e.g., exercise and food) to orchestrate energy intake and expenditure. Mechanistically, CNOT6L inhibition stabilizes hepatic Gdf15 and Fgf21 mRNAs, increasing corresponding serum protein levels. The resulting upregulation of GDF15 stimulates the hindbrain to suppress appetite, while increased FGF21 affects the liver and adipose tissues to induce energy expenditure and lipid consumption. Despite the potential of hepatokines to treat metabolic disorders, their administration therapies have been challenging. Using small-molecule screening, we identified a CNOT6L inhibitor enhancing GDF15 and FGF21 hepatokine levels, which dramatically improves diet-induced metabolic syndrome. Our discovery, therefore, lays the foundation for an unprecedented strategy to treat metabolic syndrome.
Assuntos
Síndrome Metabólica , Estabilidade de RNA , Animais , Ingestão de Alimentos , Metabolismo Energético/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 15 de Diferenciação de Crescimento/genética , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Fígado/metabolismo , Síndrome Metabólica/metabolismo , Camundongos , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , Ribonucleases/metabolismoRESUMO
To accelerate the translation of cancer nanomedicine, we used an integrated genomic approach to improve our understanding of the cellular processes that govern nanoparticle trafficking. We developed a massively parallel screen that leverages barcoded, pooled cancer cell lines annotated with multiomic data to investigate cell association patterns across a nanoparticle library spanning a range of formulations with clinical potential. We identified both materials properties and cell-intrinsic features that mediate nanoparticle-cell association. Using machine learning algorithms, we constructed genomic nanoparticle trafficking networks and identified nanoparticle-specific biomarkers. We validated one such biomarker: gene expression of SLC46A3, which inversely predicts lipid-based nanoparticle uptake in vitro and in vivo. Our work establishes the power of integrated screens for nanoparticle delivery and enables the identification and utilization of biomarkers to rationally design nanoformulations.
Assuntos
Antineoplásicos , Biomarcadores Farmacológicos , Proteínas de Transporte de Cobre , Composição de Medicamentos , Sistemas de Liberação de Fármacos por Nanopartículas , Nanopartículas , Neoplasias , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Proteínas de Transporte de Cobre/genética , Expressão Gênica , Genômica , Humanos , Lipossomos , Camundongos , Nanomedicina , Nanopartículas/administração & dosagem , Nanopartículas/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismoRESUMO
Nanoformulations of therapeutic drugs are transforming our ability to effectively deliver and treat a myriad of conditions. Often, however, they are complex to produce and exhibit low drug loading, except for nanoparticles formed via co-assembly of drugs and small molecular dyes, which display drug-loading capacities of up to 95%. There is currently no understanding of which of the millions of small-molecule combinations can result in the formation of these nanoparticles. Here we report the integration of machine learning with high-throughput experimentation to enable the rapid and large-scale identification of such nanoformulations. We identified 100 self-assembling drug nanoparticles from 2.1 million pairings, each including one of 788 candidate drugs and one of 2,686 approved excipients. We further characterized two nanoparticles, sorafenib-glycyrrhizin and terbinafine-taurocholic acid both ex vivo and in vivo. We anticipate that our platform can accelerate the development of safer and more efficacious nanoformulations with high drug-loading capacities for a wide range of therapeutics.
Assuntos
Portadores de Fármacos/química , Ensaios de Triagem em Larga Escala/métodos , Nanopartículas/química , Sorafenibe/farmacologia , Terbinafina/farmacologia , Animais , Candida albicans/efeitos dos fármacos , Simulação por Computador , Portadores de Fármacos/síntese química , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Difusão Dinâmica da Luz , Excipientes/química , Feminino , Ácido Glicirrízico/química , Humanos , Aprendizado de Máquina , Camundongos Endogâmicos , Absorção Cutânea , Sorafenibe/química , Sorafenibe/farmacocinética , Ácido Taurocólico/química , Terbinafina/química , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Dexras1 is a 30 kDa G protein in the Ras subfamily whose discovery was based on its pronounced inducibility by the glucocorticoid dexamethasone. It binds to neuronal nitric oxide synthase (nNOS) via the adaptor protein CAPON, eliciting S-nitrosylation and activation of Dexras1. We report that Dexras1 binds to the peripheral benzodiazepine receptor-associated protein (PAP7), a protein of unknown function that binds to cyclic AMP-dependent protein kinase and the peripheral benzodiazepine receptor. PAP7 in turn binds to the divalent metal transporter (DMT1), an iron import channel. We have identified a signaling cascade in neurons whereby stimulation of NMDA receptors activates nNOS, leading to S-nitrosylation and activation of Dexras1, which, via PAP7 and DMT1, physiologically induces iron uptake. As selective iron chelation prevents NMDA neurotoxicity in cortical cultures, the NMDA-NO-Dexras1-PAP7-DMT1-iron uptake signaling cascade also appears to mediate NMDA neurotoxicity.
Assuntos
Homeostase/fisiologia , Ferro/metabolismo , Óxido Nítrico/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/fisiologia , Proteínas ras/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Aldeídos/farmacologia , Animais , Western Blotting/métodos , Proteínas de Transporte de Cátions/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Embrião de Mamíferos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Imunofluorescência/métodos , Homeostase/efeitos dos fármacos , Humanos , Hidrazonas/farmacologia , Imunoprecipitação/métodos , Quelantes de Ferro/farmacologia , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Knockout , Modelos Biológicos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo I/deficiência , Óxido Nítrico Sintase Tipo I/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptores de GABA-A/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transfecção/métodosRESUMO
Kidney stones and ureteral stents can cause ureteral colic and pain. By decreasing contractions in the ureter, clinically prescribed oral vasodilators may improve spontaneous stone passage rates and reduce the pain caused by ureteral stenting. We hypothesized that ureteral relaxation can be improved via the local administration of vasodilators and other smooth muscle relaxants. Here, by examining 18 candidate small molecules in an automated screening assay to determine the extent of ureteral relaxation, we show that the calcium channel blocker nifedipine and the Rho-kinase inhibitor ROCKi significantly relax human ureteral smooth muscle cells. We also show, by using ex vivo porcine ureter segments and sedated pigs that, with respect to the administration of a placebo, the local delivery of a clinically deployable formulation of the two drugs reduced ureteral contraction amplitude and frequency by 90% and 50%, respectively. Finally, we show that standard oral vasodilator therapy reduced contraction amplitude by only 50% and had a minimal effect on contraction frequency. Locally delivered ureteral relaxants therefore may improve ureter-related conditions.
Assuntos
Contração Muscular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Ureter/efeitos dos fármacos , Vasodilatadores/administração & dosagem , Animais , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Nifedipino/administração & dosagem , Inibidores de Proteínas Quinases/administração & dosagem , Sus scrofaRESUMO
Cancer is often seen as a disease of mutations and chromosomal abnormalities. However, some cancers, including pediatric rhabdoid tumors (RTs), lack recurrent alterations targetable by current drugs and need alternative, informed therapeutic options. To nominate potential targets, we performed a high-throughput small-molecule screen complemented by a genome-scale CRISPR-Cas9 gene-knockout screen in a large number of RT and control cell lines. These approaches converged to reveal several receptor tyrosine kinases (RTKs) as therapeutic targets, with RTK inhibition effective in suppressing RT cell growth in vitro and against a xenograft model in vivo. RT cell lines highly express and activate (phosphorylate) different RTKs, creating dependency without mutation or amplification. Downstream of RTK signaling, we identified PTPN11, encoding the pro-growth signaling protein SHP2, as a shared dependency across all RT cell lines. This study demonstrates that large-scale perturbational screening can uncover vulnerabilities in cancers with "quiet" genomes.