RESUMO
Abundant evidence suggests that a unifying principle governing the molecular pathology of cancer is the co-dependent aberrant regulation of core machinery driving proliferation and suppressing apoptosis. Anomalous proteins engaged in support of this tumorigenic regulatory environment most probably represent optimal intervention targets in a heterogeneous population of cancer cells. The advent of RNA-mediated interference (RNAi)-based functional genomics provides the opportunity to derive unbiased comprehensive collections of validated gene targets supporting critical biological systems outside the framework of preconceived notions of mechanistic relationships. We have combined a high-throughput cell-based one-well/one-gene screening platform with a genome-wide synthetic library of chemically synthesized small interfering RNAs for systematic interrogation of the molecular underpinnings of cancer cell chemoresponsiveness. NCI-H1155, a human non-small-cell lung cancer line, was employed in a paclitaxel-dependent synthetic lethal screen designed to identify gene targets that specifically reduce cell viability in the presence of otherwise sublethal concentrations of paclitaxel. Using a stringent objective statistical algorithm to reduce false discovery rates below 5%, we isolated a panel of 87 genes that represent major focal points of the autonomous response of cancer cells to the abrogation of microtubule dynamics. Here we show that several of these targets sensitize lung cancer cells to paclitaxel concentrations 1,000-fold lower than otherwise required for a significant response, and we identify mechanistic relationships between cancer-associated aberrant gene expression programmes and the basic cellular machinery required for robust mitotic progression.
Assuntos
Genes Letais/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Paclitaxel/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Análise Mutacional de DNA , Relação Dose-Resposta a Droga , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Neoplasias Pulmonares/tratamento farmacológico , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/metabolismo , Fuso Acromático/patologiaRESUMO
The Wnt family of secreted proteins coordinate cell fate decision-making in a broad range of developmental and homeostatic contexts. Corruption of Wnt signal transduction pathways frequently results in degenerative diseases and cancer. We have used an iterative genome-wide screening strategy that employs multiple nonredundant RNAi reagents to identify mammalian genes that participate in Wnt/beta-catenin pathway response. Among the genes that were assigned high confidence scores are two members of the TCF/LEF family of DNA-binding proteins that control the transcriptional output of the pathway. Surprisingly, we found that the presumed cancer-promoting gene TCF7L2 functions instead as a transcriptional repressor that restricts colorectal cancer (CRC) cell growth. Mutations in TCF7L2 identified from cancer genome sequencing efforts abolish its ability to function as a transcriptional regulator and result in increased CRC cell growth. We describe a growth-promoting transcriptional program that is likely activated in CRC tumors with compromised TCF7L2 function. Taken together, the results from our screen and studies focused on members of the TCF/LEF gene family refine our understanding of how aberrant Wnt pathway activation sustains CRC growth.
Assuntos
Neoplasias Colorretais/genética , RNA Interferente Pequeno/farmacologia , Transdução de Sinais , Fatores de Transcrição TCF/fisiologia , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proliferação de Células , Neoplasias Colorretais/patologia , Genes Neoplásicos , Genoma Humano , Humanos , Interferência de RNA , Fatores de Transcrição TCF/genética , Proteína 2 Semelhante ao Fator 7 de TranscriçãoRESUMO
Several hundred PDZ (postsynaptic density-95, Drosophila disks-large, ZO-1) domain-containing proteins have been identified in the human genome. PDZ domains play a critical role in organization and function of cellular signaling pathways. Thus, small molecule inhibitors of PDZ domain association with their targets have wide potential applications as research and therapeutic agents. PDZ domains typically bind to a carboxyl-terminal tail of the target protein. Here we describe a high-throughput screening (HTS) assay for small molecule inhibitors of association between Mint1-PDZ domains and N-type Ca2+ channel carboxyl-terminal peptide (NC peptide). The performance of a homogeneous time-resolved fluorescence resonance energy transfer (HTRF) and an amplified luminescent proximity homogeneous assay (ALPHA) were systematically compared in parallel pilot HTS experiments with glutathione S-transferase-Mint1-PDZ1/2 protein and biotinylated NC peptide. Both of the two assays showed similar sensitivities in our target protein assay. Using HTRF-based assay we screened a library of 100,000 small molecule compounds and identified a number of potential "hits." The activity of isolated "hits" was confirmed by ALPHA assay. However, further evaluation revealed that isolated "hits" most likely act as "promiscuous binders," not as specific Mint-PDZ inhibitors, and that additional screening will be required to identify the true Mint-PDZ inhibitors. The assays described provided an example of HTS for a small molecule inhibitor of Mint-PDZ domain that can be easily adapted to other PDZ domain-mediated interactions.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Glutationa Transferase/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , Domínios PDZ/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Transferência Ressonante de Energia de Fluorescência , Indicadores e Reagentes , Luz , Luminescência , Oxigênio/química , Oxigênio/efeitos da radiação , Ratos , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/efeitos da radiação , Proteínas Recombinantes/química , Estreptavidina/química , Especificidade por SubstratoRESUMO
Plasmodium falciparum is the causative agent of the most serious and fatal malarial infections, and it has developed resistance to commonly employed chemotherapeutics. The de novo pyrimidine biosynthesis enzymes offer potential as targets for drug design, because, unlike the host, the parasite does not have pyrimidine salvage pathways. Dihydroorotate dehydrogenase (DHODH) is a flavin-dependent mitochondrial enzyme that catalyzes the fourth reaction in this essential pathway. Coenzyme Q (CoQ) is utilized as the oxidant. Potent and species-selective inhibitors of malarial DHODH were identified by high-throughput screening of a chemical library, which contained 220,000 drug-like molecules. These novel inhibitors represent a diverse range of chemical scaffolds, including a series of halogenated phenyl benzamide/naphthamides and urea-based compounds containing napthyl or quinolinyl substituents. Inhibitors in these classes with IC(50) values below 600 nm were purified by high pressure liquid chromatography, characterized by mass spectroscopy, and subjected to kinetic analysis against the parasite and human enzymes. The most active compound is a competitive inhibitor of CoQ with an IC(50) against malarial DHODH of 16 nm, and it is 12,500-fold less active against the human enzyme. Site-directed mutagenesis of residues in the CoQ-binding site significantly reduced inhibitor potency. The structural basis for the species selective enzyme inhibition is explained by the variable amino acid sequence in this binding site, making DHODH a particularly strong candidate for the development of new anti-malarial compounds.