RESUMO
All coronaviruses (CoVs) encode for a conserved macrodomain (Mac1) located in nonstructural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that binds and hydrolyzes mono-ADP-ribose from target proteins. Previous work has shown that Mac1 is important for virus replication and pathogenesis. Within Mac1, there are several regions that are highly conserved across CoVs, including the GIF (glycine-isoleucine-phenylalanine) motif. To determine how the biochemical activities of these residues impact CoV replication, the isoleucine and the phenylalanine residues were mutated to alanine (I-A/F-A) in both recombinant Mac1 proteins and recombinant CoVs, including murine hepatitis virus (MHV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The F-A mutant proteins had ADP-ribose binding and/or hydrolysis defects that led to attenuated replication and pathogenesis in cell culture and mice. In contrast, the I-A mutations had normal enzyme activity and enhanced ADP-ribose binding. Despite increased ADP-ribose binding, I-A mutant MERS-CoV and SARS-CoV-2 were highly attenuated in both cell culture and mice, indicating that this isoleucine residue acts as a gate that controls ADP-ribose binding for efficient virus replication. These results highlight the function of this highly conserved residue and provide unique insight into how macrodomains control ADP-ribose binding and hydrolysis to promote viral replication.
RESUMO
Herpes simplex virus 1 (HSV-1) is a ubiquitous virus that results in lifelong infections due to its ability to cycle between lytic replication and latency. As an obligate intracellular pathogen, HSV-1 exploits host cellular factors to replicate and aid in its life cycle. HSV-1 expresses infected cell protein 0 (ICP0), an immediate-early regulator, to stimulate the transcription of all classes of viral genes via its E3 ubiquitin ligase activity. Here we report an automated, inexpensive, and rapid high-throughput approach to examine the effects of small molecule compounds on ICP0 transactivator function in cells. Two HSV-1 reporter viruses, KOS6ß (wt) and dlx3.1-6ß (ICP0-null mutant), were used to monitor ICP0 transactivation activity through the HSV-1 ICP6 promoter:lacz expression cassette. A ≥10-fold difference in ß-galactosidase activity was observed in cells infected with KOS6ß compared to dlx3.1-6ß, demonstrating that ICP0 potently transactivates the ICP6 promoter. We established the robustness and reproducibility with a Z'-factor score of ≥0.69, an important criterium for high-throughput analyses. Approximately 19,000 structurally diverse compounds were screened and 76 potential inhibitors of the HSV-1 transactivator ICP0 were identified. We expect this assay will aid in the discovery of novel inhibitors and tools against HSV-1 ICP0. Using well-annotated compounds could identify potential novel factors and pathways that interact with ICP0 to promote HSV-1 gene expression.
Assuntos
Herpesvirus Humano 1/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/genética , Ativação Transcricional/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Coleta de Dados , Expressão Gênica , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Bibliotecas de Moléculas Pequenas/farmacologia , Ativação Transcricional/genéticaRESUMO
BACKGROUND: Pancreatic cancer has an enrichment of stem-like cancer cells (CSCs) that contribute to chemoresistant tumors prone to metastasis and recurrence. Drug screening assays based on cytotoxicity cannot identify specific CSC inhibitors, because CSCs comprise only a small portion of cancer cell population, and it is difficult to propagate stable CSC populations in vitro for high-throughput screening (HTS) assays. Based on the important role of cancer cell epithelial-to-mesenchymal transition (EMT) in promoting CSCs, we hypothesized that inhibition of EMT can be a useful strategy for inhibiting CSCs, and therefore a feasible approach for HTS can be built for identification of CSC inhibitors, based on assays detecting EMT inhibition. METHODS: An immunofluorescent assay was established and optimized for HTS to identify compounds that enhance E-cadherin expression, as a hallmark of inhibition of EMT. Four chemical libraries containing 41,472 compounds were screened in PANC-1 pancreatic cancer cell line. Positive hits were validated for EMT and CSC inhibition in vitro using sphere formation assay, western blotting, immune fluorescence, and scratch assay. RESULTS: Initial hits were refined to 73 compounds with a secondary screening, among which 17 exhibited concentration dependent induction of E-cadherin expression. Six compounds were selected for further study which belonged to 2 different chemical structural clusters. A novel compound 1-(benzylsulfonyl) indoline (BSI, Compound #38) significantly inhibited pancreatic cancer cell migration and invasion. BSI inhibited histone deacetylase, increased histone 4 acetylation preferably, resulting in E-cadherin up-regulation. BSI effectively inhibited tumor spheres formation. Six more analogues of BSI were tested for anti-migration and anti-CSC activities. CONCLUSION: This study demonstrated a feasible approach for discovery of agents targeting EMT and CSCs using HTS, and identified a class of novel chemicals that could be developed as anti-EMT and anti-CSC drug leads.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/toxicidade , Antígeno CD24/metabolismo , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Molécula de Adesão da Célula Epitelial/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Receptores de Hialuronatos/metabolismo , Microscopia de Fluorescência , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Bibliotecas de Moléculas Pequenas/química , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Fatores de Transcrição da Família Snail/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Biomolecular screening research frequently searches for the chemical compounds that are most likely to make a biochemical or cell-based assay system produce a strong continuous response. Several doses are tested with each compound and it is assumed that, if there is a dose-response relationship, the relationship follows a monotonic curve, usually a version of the median-effect equation. However, the null hypothesis of no relationship cannot be statistically tested using this equation. We used a linearized version of this equation to define a measure of pharmacological effect size, and use this measure to rank the investigated compounds in order of their overall capability to produce strong responses. The null hypothesis that none of the examined doses of a particular compound produced a strong response can be tested with this approach. The proposed approach is based on a new statistical model of the important concept of response detection limit, a concept that is usually neglected in the analysis of dose-response data with continuous responses. The methodology is illustrated with data from a study searching for compounds that neutralize the infection by a human immunodeficiency virus of brain glioblastoma cells.
Assuntos
Fármacos Anti-HIV/administração & dosagem , Limite de Detecção , Modelos Estatísticos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Infecções por HIV/tratamento farmacológico , Humanos , Projetos de Pesquisa/estatística & dados numéricos , Resultado do TratamentoRESUMO
Nuclear factor erythroid 2-related factor 2 is a master regulator that promotes transcription of cytoprotective genes in response to oxidative/electrophilic stress. A large number of natural dietary compounds are thought to protect against oxidative stress, and a few have been reported to induce genes involved in antioxidant defense through activating nuclear factor erythroid 2-related factor 2. Therefore, a library of 54 natural compounds were collected to determine whether they are nuclear factor erythroid 2-related factor 2 activators and to compare their efficacy and potency to activate nuclear factor erythroid 2-related factor 2. The assay utilized AREc32 cells that contain a luciferase gene under the control of antioxidant response element promoters. Each natural compound was tested at 13 concentrations between 0.02 and 30 µM. Known nuclear factor erythroid 2-related factor 2 activators tert-butylhydroquinone and 2-cyano-3,12-dioxooleana-1,9-diene-28-imidazolide were used as positive controls in parallel with the natural compounds. Among the 54 tested natural compounds, andrographolide had the highest efficacy, followed by trans-chalcone, sulforaphane, curcumin, flavone, kahweol, and carnosol, all of which had better efficacy than tert-butylhydroquinone. Among the compounds tested, 2-cyano-3,12-dioxooleana-1,9-diene-28-imidazolide was the most potent, having an EC50 of 0.41 µM. Seven of the natural compounds, namely andrographolide, trans-chalcone, sulforaphane, curcumin, flavone, kahweol, and cafestol had lower EC50 values than tert-butylhydroquinone but higher than 2-cyano-3,12-dioxooleana-1,9-diene-28-imidazolide. The present study provides insights into which natural compounds activate the Keap1-nuclear factor erythroid 2-related factor 2 pathway and thus might be useful for detoxifying oxidative/electrophilic stress.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Elementos de Resposta Antioxidante , Antioxidantes/farmacologia , Linhagem Celular/efeitos dos fármacos , Diterpenos/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Hidroquinonas/farmacologia , Proteína 1 Associada a ECH Semelhante a Kelch , Redes e Vias Metabólicas/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacosRESUMO
The half maximal inhibitory concentration (IC50) has several limitations that make it unsuitable for examining a large number of compounds in cytotoxicity studies, particularly when multiple exposure periods are tested. This article proposes a new approach to measure drug effectiveness, which allows ranking compounds according to their toxic effects on live cells. This effectiveness measure, which combines all exposure times tested, compares the growth rates of a particular cell line in the presence of the compound with its growth rate in the presence of DMSO alone. Our approach allows measuring a wider spectrum of toxicity than the IC50 approach, and allows automatic analyses of a large number of compounds. It can be easily implemented in linear regression software, provides a comparable measure of effectiveness for each investigated compound (both toxic and non-toxic), and allows statistically testing the null hypothesis that a compound is non-toxic versus the alternative that it is toxic. Importantly, our approach allows defining an automated decision rule for deciding whether a compound is significantly toxic. As an illustration, we describe the results of a cellbased study of the cytotoxicity of 24 analogs of novobiocin, a C-terminal inhibitor of heat shock protein 90 (Hsp90); the compounds were ranked in order of cytotoxicity to a panel of 18 cancer cell lines and 1 normal cell line. Our approach may also be a good alternative to computing the half maximal effective concentration (EC50) in studies searching for compounds that promote cell growth.
Assuntos
Antineoplásicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Ensaios de Triagem em Larga Escala/métodos , Novobiocina/farmacologia , Antineoplásicos/química , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Humanos , Concentração Inibidora 50 , Modelos Biológicos , Modelos Estatísticos , Neoplasias/tratamento farmacológico , Novobiocina/análogos & derivadosRESUMO
To identify genes that contribute to chemotherapy resistance in glioblastoma, we conducted a synthetic lethal screen in a chemotherapy-resistant glioblastoma-derived cell line with the clinical alkylator temozolomide (TMZ) and an siRNA library tailored toward "druggable" targets. Select DNA repair genes in the screen were validated independently, confirming the DNA glycosylases uracil-DNA glycosylase (UNG) and A/G-specific adenine DNA glycosylase (MYH) as well as methylpurine-DNA glycosylase (MPG) to be involved in the response to high dose TMZ. The involvement of UNG and MYH is likely the result of a TMZ-induced burst of reactive oxygen species. We then compared the human TMZ sensitizing genes identified in our screen with those previously identified from alkylator screens conducted in Escherichia coli and Saccharomyces cerevisiae. The conserved biologic processes across all three species compose an alkylation functionome that includes many novel proteins not previously thought to impact alkylator resistance. This high-throughput screen, validation and cross-species analysis was then followed by a mechanistic analysis of two essential nodes: base excision repair (BER) DNA glycosylases (UNG, human and mag1, S. cerevisiae) and protein modification systems, including UBE3B and ICMT in human cells or pby1, lip22, stp22 and aim22 in S. cerevisiae. The conserved processes of BER and protein modification were dual targeted and yielded additive sensitization to alkylators in S. cerevisiae. In contrast, dual targeting of BER and protein modification genes in human cells did not increase sensitivity, suggesting an epistatic relationship. Importantly, these studies provide potential new targets to overcome alkylating agent resistance.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Dacarbazina/análogos & derivados , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Alquilação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Reparo do DNA , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Escherichia coli/genética , Escherichia coli/metabolismo , Glioblastoma/metabolismo , Humanos , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Temozolomida , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Uracila-DNA Glicosidase/genética , Uracila-DNA Glicosidase/metabolismoRESUMO
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor that induces a battery of cytoprotective genes involved in antioxidant defense through binding to Antioxidant Response Elements (ARE) located in the promoter regions of these genes. To identify Nrf2 activators for the treatment of oxidative/electrophilic stress-induced diseases, the present study developed a high-throughput assay to evaluate Nrf2 activation using AREc32 cells that contain a luciferase gene under the control of ARE promoters. Of the 47,000 compounds screened, 238 (top 0.5% hits) of the chemicals increased the luminescent signal more than 14.4-fold and were re-tested at eleven concentrations in a range of 0.01-30 µM. Of these 238 compounds, 231 (96%) increased the luminescence signal in a concentration-dependent manner. Chemical structure relationship analysis of these 231 compounds indicated enrichment of four chemical scaffolds (diaryl amides and diaryl ureas, oxazoles and thiazoles, pyranones and thiapyranones, and pyridinones and pyridazinones). In addition, 30 of these 231 compounds were highly effective and/or potent in activating Nrf2, with a greater than 80-fold increase in luminescence, or an EC50 lower than 1.6 µM. These top 30 compounds were also screened in Hepa1c1c7 cells for an increase in Nqo1 mRNA, the prototypical Nrf2-target gene. Of these 30 compounds, 17 increased Nqo1 mRNA in a concentration-dependent manner. In conclusion, the present study documents the development, implementation, and validation of a high-throughput screen to identify activators of the Keap1-Nrf2-ARE pathway. Results from this screening identified Nrf2 activators, and provide novel insights into chemical scaffolds that might prevent oxidative/electrophilic stress-induced toxicity and carcinogenesis.
Assuntos
Elementos de Resposta Antioxidante/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Compostos Orgânicos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes/métodos , Camundongos , Estrutura Molecular , NAD(P)H Desidrogenase (Quinona)/genética , Compostos Orgânicos/química , Ligação Proteica/efeitos dos fármacos , Piridonas/química , Piridonas/farmacologia , Pirimidinonas/química , Pirimidinonas/farmacologia , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Bibliotecas de Moléculas PequenasRESUMO
Discovering chemosensitivity pathways or nodes is an attractive strategy for formulating new drug combinations for cancer. Microtubules are among the most successful anticancer drug targets. Therefore, we implemented a small interfering RNA (siRNA) synthetic lethal screen targeting 5520 unique druggable genes to identify novel chemosensitivity nodes for vinblastine, a microtubule-destabilizing agent used clinically. We transiently transfected human glioblastoma cells with siRNAs for 48 h and then treated cells with a sublethal concentration of vinblastine. Forty-eight hours later, we analyzed cell viability and, using a series of statistical methods, identified 65 gene products that, when suppressed, sensitized glioblastoma cells to vinblastine. After completion of the secondary assays, we focused on one siRNA, B-cell lymphoma extra large (BCL-xL), because of its role in the intrinsic apoptosis signaling pathway as well as the availability of pharmacological inhibitors. We found that nontoxic concentrations of 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide (ABT-263), an inhibitor of the BCL-2 family members (BCL-2, BCL-xL, and BCL-w), sensitized glioblastoma and non-small-cell lung cancer cells to vinblastine and induced apoptosis through the intrinsic cell death pathway. These results illustrate the usefulness of unbiased siRNA screens as a method for identifying potential novel anticancer therapeutic combinations.
Assuntos
Compostos de Anilina/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , RNA Interferente Pequeno , Sulfonamidas/farmacologia , Vimblastina/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Relação Dose-Resposta a Droga , Genes bcl-2 , Glioblastoma/genética , Ensaios de Triagem em Larga Escala , Humanos , Mitocôndrias/efeitos dos fármacos , Transfecção , Moduladores de Tubulina/farmacologiaRESUMO
The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core was established in 2002 at the University of Kansas with support from an NIH grant and the state of Kansas. It collaborates with investigators from national and international academic, nonprofit and pharmaceutical organizations in executing HTS-ready assay development and screening of chemical libraries for target validation, probe selection, hit identification and lead optimization. This is part two of a contribution from the KU HTS laboratory.
Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Tecnologia Farmacêutica/métodos , Comportamento Cooperativo , Humanos , Kansas , Patentes como Assunto , Bibliotecas de Moléculas Pequenas/metabolismo , UniversidadesRESUMO
The University of Kansas High-Throughput Screening (KU HTS) core is a state-of-the-art drug-discovery facility with an entrepreneurial open-service policy, which provides centralized resources supporting public- and private-sector research initiatives. The KU HTS core applies pharmaceutical industry project-management principles in an academic setting by bringing together multidisciplinary teams to fill critical scientific and technology gaps, using an experienced team of industry-trained researchers and project managers. The KU HTS proactively engages in supporting grant applications for extramural funding, intellectual-property management and technology transfer. The KU HTS staff further provides educational opportunities for the KU faculty and students to learn cutting-edge technologies in drug-discovery platforms through seminars, workshops, internships and course teaching. This is the first instalment of a two-part contribution from the KU HTS laboratory.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Avaliação Pré-Clínica de Medicamentos , Kansas , Laboratórios , Preparações Farmacêuticas/química , UniversidadesRESUMO
High throughput screening (HTS) facilitates screening large numbers of compounds against a biochemical target of interest using validated biological or biophysical assays. In recent years, a significant number of drugs in clinical trails originated from HTS campaigns, validating HTS as a bona fide mechanism for hit finding. In the current drug discovery landscape, the pharmaceutical industry is embracing open innovation strategies with academia to maximize their research capabilities and to feed their drug discovery pipeline. The goals of academic research have therefore expanded from target identification and validation to probe discovery, chemical genomics, and compound library screening. This trend is reflected in the emergence of HTS centers in the public domain over the past decade, ranging in size from modestly equipped academic screening centers to well endowed Molecular Libraries Probe Centers Network (MLPCN) centers funded by the NIH Roadmap initiative. These centers facilitate a comprehensive approach to probe discovery in academia and utilize both classical and cutting-edge assay technologies for executing primary and secondary screening campaigns. The various facets of academic HTS centers as well as their implications on technology transfer and drug discovery are discussed, and a roadmap for successful drug discovery in the public domain is presented. New lead discovery against therapeutic targets, especially those involving the rare and neglected diseases, is indeed a Mount Everestonian size task, and requires diligent implementation of pharmaceutical industry's best practices for a successful outcome.
Assuntos
Acesso à Informação , Descoberta de Drogas/métodos , Indústria Farmacêutica/métodos , Ensaios de Triagem em Larga Escala/métodos , Setor Público , Acesso à Informação/legislação & jurisprudência , Animais , Descoberta de Drogas/legislação & jurisprudência , Descoberta de Drogas/tendências , Avaliação Pré-Clínica de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/tendências , Indústria Farmacêutica/legislação & jurisprudência , Indústria Farmacêutica/tendências , Ensaios de Triagem em Larga Escala/tendências , Humanos , Preparações Farmacêuticas/química , Setor Público/legislação & jurisprudência , Setor Público/tendências , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Universidades/legislação & jurisprudência , Universidades/tendênciasRESUMO
Glioblastoma multiforme (GBM) is a high-grade brain malignancy arising from astrocytes. Despite aggressive surgical approaches, optimized radiation therapy regimens, and the application of cytotoxic chemotherapies, the median survival of patients with GBM from time of diagnosis remains less than 15 months, having changed little in decades. Approaches that target genes and biological pathways responsible for tumorigenesis or potentiate the activity of current therapeutic modalities could improve treatment efficacy. In this regard, several genomic and proteomic strategies promise to impact significantly on the drug discovery process. High-throughput genome-wide screening with short interfering RNA (siRNA) is one strategy for systematically exploring possible therapeutically relevant targets in GBM. Statistical methods and protein-protein interaction network databases can also be applied to the screening data to explore the genes and pathways that underlie the pathological basis and development of GBM. In this study, we highlight several genome-wide siRNA screens and implement these experimental concepts in the T98G GBM cell line to uncover the genes and pathways that regulate GBM cell death and survival. These studies will ultimately influence the development of a new avenue of neurosurgical therapy by placing the drug discovery process in the context of the entire biological system.
Assuntos
Neoplasias Encefálicas/genética , Descoberta de Drogas/métodos , Genômica/métodos , Glioblastoma/genética , Neoplasias Encefálicas/tratamento farmacológico , Morte Celular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Regulação Neoplásica da Expressão Gênica , Testes Genéticos/métodos , Estudo de Associação Genômica Ampla/métodos , Glioblastoma/tratamento farmacológico , Humanos , Modelos Genéticos , Farmacogenética/métodos , Mapeamento de Interação de Proteínas , Proteômica/métodos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêuticoRESUMO
A major challenge for the treatment of cancers, such as glioblastoma multiforme (GBM), has been resistance to radiation and cancer chemotherapeutics. Short interfering RNA (siRNA) based screening may facilitate the identification of genes and pathways essential for cancer cell survival and could enable a more targeted therapeutic approach for the treatment of GBM. Although the commercial availability of siRNA libraries has expanded greatly, detailed methods for the implementation and analysis of genome-scale screens are largely lacking. To annotate the essential genes and pathways for glioma cell survival, we designed, optimized, and implemented a high-throughput siRNA screen in the highly drug and radiation resistant T98G glioma cell line. We developed a rapid, readily available, and simple strategy to optimize siRNA transfection assays in a 384-well plate format based on immunofluorescence studies and inhibition of the non-essential, endogenous gene lamin A/C. We used these transfection conditions to successfully screen a library of 1056 siRNAs targeting 352 unique human genes in a cell-based one gene per well format to identify the genes essential for glioma cell survival and assess the quality of the screening conditions prior to large-scale screening. After developing and applying a median-based outlier detection algorithm for post-screen analysis, we identified the Ras oncogene family member RAN as an essential gene for glioma cell survival. Successful implementation and analysis of this siRNA screen validates our transfection optimization approach and provides guidance for the rapid development of high-throughput siRNA screens in human glioma cells.
Assuntos
Descoberta de Drogas/métodos , Genômica/métodos , RNA Interferente Pequeno/farmacologia , Automação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Perfilação da Expressão Gênica/métodos , Biblioteca Genômica , Glioma/genética , Humanos , Transfecção/métodosRESUMO
To assist in screening existing drugs for use as potential radioprotectors, we used a human unbiased 16,560 short interfering RNA (siRNA) library targeting the druggable genome. We performed a synthetic protection screen that was designed to identify genes that, when silenced, protected human glioblastoma T98G cells from gamma-radiation-induced cell death. We identified 116 candidate protective genes, then identified 10 small molecule inhibitors of 13 of these candidate gene products and tested their radioprotective effects. Glyburide, a clinically used second-generation hypoglycemic drug, effectively decreased radiation-induced cell death in several cell lines including T98G, glioblastoma U-87 MG, and normal lung epithelial BEAS-2B and in primary cultures of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective in vivo (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, P = 0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors.
Assuntos
Glibureto/farmacologia , Hipoglicemiantes/farmacologia , RNA Interferente Pequeno , Protetores contra Radiação/farmacologia , Animais , Sequência de Bases , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ensaio de Unidades Formadoras de Colônias , Avaliação Pré-Clínica de Medicamentos , Feminino , Biblioteca Genômica , Hematopoese/efeitos dos fármacos , Hematopoese/efeitos da radiação , Humanos , Camundongos , Fenótipo , RNA Interferente Pequeno/genética , Radiação Ionizante , Irradiação Corporal TotalRESUMO
Target identification and validation remain difficult steps in the drug discovery process, and uncovering the core genes and pathways that are fundamental for cancer cell survival may facilitate this process. Glioblastoma represents a challenging form of cancer for chemotherapy. Therefore, we assayed 16,560 short interfering RNA (siRNA) aimed at identifying which of the 5520 unique therapeutically targetable gene products were important for the survival of human glioblastoma. We analyzed the viability of T98G glioma cells 96 h after siRNA transfection with two orthogonal statistical methods and identified 55 survival genes that encoded proteases, kinases, and transferases. It is noteworthy that 22% (12/55) of the survival genes were constituents of the 20S and 26S proteasome subunits. An expression survey of a panel of glioma cell lines demonstrated expression of the proteasome component PSMB4, and the validity of the proteasome complex as a target for survival inhibition was confirmed in a series of glioma and nonglioma cell lines by pharmacological inhibition and RNA interference. Biological networks were built with the other survival genes using a protein-protein interaction network, which identified clusters of cellular processes, including protein ubiquitination, purine and pyrimidine metabolism, nucleotide excision repair, and NF-kappaB signaling. The results of this study should broaden our understanding of the core genes and pathways that regulate cell survival; through either small molecule inhibition or RNA interference, we highlight the potential significance of proteasome inhibition.
Assuntos
Genes Neoplásicos/genética , Glioblastoma/genética , RNA Interferente Pequeno/genética , Antineoplásicos/uso terapêutico , Western Blotting , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Estudo de Associação Genômica Ampla , Glioblastoma/tratamento farmacológico , Glioma/tratamento farmacológico , Glioma/genética , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Mitogen-activated protein kinase phosphatase (MKP)-1 is a dual-specificity phosphatase that negatively regulates the activity of mitogen-activated kinases and that is overexpressed in human tumors. Contemporary studies suggest that induction of MKP-1 during chemotherapy may limit the efficacy of clinically used antineoplastic agents. Thus, MKP-1 is a rational target to enhance anticancer drug activity, but suitable small-molecule inhibitors of MKP-1 are currently unavailable. Here, we have used a high-content, multiparameter fluorescence-based chemical complementation assay for MKP activity in intact mammalian cells to evaluate the cellular MKP-1 and MKP-3 inhibitory activities of four previously described, quinone-based, dual-specificity phosphatase inhibitors, that is, NSC 672121, NSC 95397, DA-3003-1 (NSC 663284), and JUN-1111. All compounds induced formation of reactive oxygen species in mammalian cells, but only one (NSC 95397) inhibited cellular MKP-1 and MKP-3 with an IC(50) of 13 mumol/L. Chemical induction of MKP-1 by dexamethasone protected cells from paclitaxel-induced apoptosis but had no effect on NSC 95397. NSC 95397 phenocopied the effects of MKP-1 small inhibitory RNA by reversing the cytoprotective effects of dexamethasone in paclitaxel-treated cells. Isobologram analysis revealed synergism between paclitaxel and NSC 95397 only in the presence of dexamethasone. The data show the power of a well-defined cellular assay for identifying cell-active inhibitors of MKPs and support the hypothesis that small-molecule inhibitors of MKP-1 may be useful as antineoplastic agents under conditions of high MKP-1 expression.