RESUMEN
We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Benzamidas , Extractos Celulares , Cromatografía de Afinidad , Receptor con Dominio Discoidina 1 , Enzimas Inmovilizadas/antagonistas & inhibidores , Células HeLa , Humanos , Mesilato de Imatinib , Concentración 50 Inhibidora , Células K562 , Preparaciones Farmacéuticas , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Pirimidinas/farmacología , Quinona Reductasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
In recent years mass spectrometry-based proteomics has moved beyond a mere quantitative description of protein expression levels and their possible correlation with disease or drug action. Impressive progress in LC-MS instrumentation together with the availability of new enabling tools and methods for quantitative proteome analysis and for identification of posttranslational modifications has triggered a surge of chemical and functional proteomics studies dissecting mechanisms of action of cancer drugs and molecular mechanisms that modulate signal transduction pathways. Despite the tremendous progress that has been made in the field, major challenges, relating to sensitivity, dynamic range, and throughput of the described methods, remain. In this review we summarize recent advances in LC-MS-based approaches and their application to cancer drug discovery and to studies of cancer-related pathways in cell culture models with particular emphasis on mechanistic studies of drug action in these systems. Moreover we highlight the emerging utility of pathway and chemical proteomics techniques for translational research in patient tissue.
Asunto(s)
Antineoplásicos/uso terapéutico , Atención a la Salud , Descubrimiento de Drogas , Oncología Médica/métodos , Neoplasias/tratamiento farmacológico , Proteómica/métodos , Animales , Ensayos Clínicos como Asunto , HumanosRESUMEN
Phenotypic screens for bactericidal compounds against drug-resistant tuberculosis are beginning to yield novel inhibitors. However, reliable target identification remains challenging. Here, we show that tetrahydropyrazo[1,5-a]pyrimidine-3-carboxamide (THPP) selectively pulls down EchA6 in a stereospecific manner, instead of the previously assigned target Mycobacterium tuberculosis MmpL3. While homologous to mammalian enoyl-coenzyme A (CoA) hydratases, EchA6 is non-catalytic yet essential and binds long-chain acyl-CoAs. THPP inhibitors compete with CoA-binding, suppress mycolic acid synthesis, and are bactericidal in a mouse model of chronic tuberculosis infection. A point mutation, W133A, abrogated THPP-binding and increased both the in vitro minimum inhibitory concentration and the in vivo effective dose 99 in mice. Surprisingly, EchA6 interacts with selected enzymes of fatty acid synthase II (FAS-II) in bacterial two-hybrid assays, suggesting essentiality may be linked to feeding long-chain fatty acids to FAS-II. Finally, our data show that spontaneous resistance-conferring mutations can potentially obscure the actual target or alternative targets of small molecule inhibitors.
Asunto(s)
Antituberculosos/farmacología , Proteínas Bacterianas/metabolismo , Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos Esenciales/metabolismo , Genes Esenciales , Mycobacterium tuberculosis/metabolismo , Pirazoles/farmacología , Pirimidinas/farmacología , Animales , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Proteínas de Unión a Ácidos Grasos/genética , Ratones , Pruebas de Sensibilidad Microbiana , Mutación Missense , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Mutación Puntual , Unión Proteica , Mapeo de Interacción de Proteínas , Tuberculosis/microbiología , Tuberculosis/patología , Técnicas del Sistema de Dos HíbridosRESUMEN
Jun dimerization protein 2 (JDP2) was identified as a bZIP protein that forms dimers with Jun proteins. JDP2 represses transcriptional activation of reporter constructs containing 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements (TRE) or cyclic AMP responsive elements (CRE). JDP2, overexpressed by the avian retroviral vector RCAS, induces partial oncogenic transformation of chicken embryo fibroblasts. JDP2-expressing cells form multilayered foci in monolayer cultures but do not show anchorage-independent growth. Both the carboxyl and the amino terminus of JDP2 are required for the transforming activity. Chimeric constructs of JDP2 carrying the leucine zipper domain of Fos, GCN4 or EB1 fail to transform CEF. The leucine zipper of Fos mediates only heterodimerization; it cannot homodimerize. In contrast, the leucine zippers of GCN4 and of EB1 exclusively homodimerize and do not form dimers with other bZip proteins. The results with the JDP2 chimeras suggest that the JDP2 homodimer and the JDP2/Jun heterodimer (or other bZip heterodimers formed with the Fos leucine zipper) are nontransforming, leaving as possible transforming combination the JDP2/Fos heterodimer. The unexpected transforming activity of a negative regulator of TRE- and CRE-dependent transcription raises an important question concerning the mechanisms of transformation by the related bZIP proteins Jun and Fos that address the same target sequences.
Asunto(s)
Transformación Celular Neoplásica , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación Neoplásica de la Expresión Génica , Proteínas Represoras/fisiología , Factores de Transcripción/genética , Factor de Transcripción Activador 2 , Animales , Embrión de Pollo , Fibroblastos , Elementos de Respuesta/genética , Acetato de Tetradecanoilforbol/metabolismo , TransfecciónRESUMEN
Transcription factor Nuclear Factor One (NFI) proteins are derived from a small family of four vertebrate genes (NFIA, B, C and X), all of which produce a fair number of protein variants by alternative splicing. In order to ultimately locate RNA signal sequences around exon/intron borders for the production of regulated splice variants, we have determined the exon structure of the chicken NFIB gene as the last of the four vertebrate genes for which the gene structure was not yet elucidated. This made it possible to compile nine newly isolated and sequenced mouse NFI cDNA sequences together with all previously available ones and to deduce corresponding splicing patterns for the orthologous vertebrate genes of all four paralogous gene types. Results from the analysis of alternative splicing and of NFI gene mapping in the genome of human and mouse argue for a phylogenetic route in which the four vertebrate NFI genes result from a single duplication of a genomic segment containing two NFI intermediate genes rather than from two independent duplications of two separated single ancestor genes.
Asunto(s)
Empalme Alternativo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas de Unión al ADN , Factores de Transcripción/genética , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , ADN Complementario/química , ADN Complementario/genética , Exones , Femenino , Genes/genética , Hibridación Fluorescente in Situ , Intrones , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Factores de Transcripción NFI , Proteínas Nucleares , Isoformas de Proteínas/genética , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Proteína 1 de Unión a la Caja YRESUMEN
The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.