RESUMEN
The importance of protein kinases as a major class of drug targets across multiple diseases has generated a critical need for technologies that enable the identification of potent and selective kinase inhibitors. Bruton's tyrosine kinase (Btk) is a compelling drug target in multiple therapeutic areas, including systemic lupus erythematosus, asthma, rheumatoid arthritis, and B cell malignancies. We have combined potent, selective kinase inhibition through chemical genetics with gene expression profiling to identify a "fingerprint" of transcriptional changes associated with selective Btk kinase inhibition. The Btk transcriptional fingerprint shows remarkable relevance for Btk's biological roles and was used for functional selectivity profiling of two kinase inhibitor compounds. The fingerprint was able to rank the compounds by relative selectivity for Btk, and revealed broader off-target effects than observed in a broad panel of biochemical kinase cross screens. In addition to being useful for functional selectivity profiling, the fingerprint genes are themselves potential preclinical and clinical biomarkers for developing Btk-directed therapies.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Riñón/metabolismo , Mapeo Peptídico/métodos , Inhibidores de Proteínas Quinasas/administración & dosificación , Inhibidores de Proteínas Quinasas/análisis , Proteínas Quinasas/metabolismo , Factores de Transcripción/metabolismo , Bioensayo/métodos , Línea Celular , Humanos , Riñón/efectos de los fármacos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas Quinasas/genética , Factores de Transcripción/genéticaRESUMEN
While standard transgenic and knockout mouse technologies have provided a wealth of information for target selection and validation, there have been great advances in using more sophisticated modeling techniques to achieve temporal and spatial regulation of individual genes in adult animals. Recent developments in RNA interference (RNAi) technology in in vivo models promise to further improve upon the static and irreversible features of gene knockouts. Chemical genetic approaches create novel functional alleles of targets and allow fine modulation of protein function in vivo by small molecules, providing the most pharmacologically relevant target validation. Using these advanced models, one can not only ask whether the function of the target is critical for the initiation and maintenance of the disease, but also whether therapies designed to alter the function of the target would be safe and efficacious. In this review, we describe various in vivo tools for target validation in mouse models, discuss advantages and disadvantages of each approach, and give examples of their impact on drug discovery.
Asunto(s)
Ingeniería Genética , Ratones Noqueados/genética , Ratones Transgénicos/genética , Animales , Ratones , Mutágenos/toxicidad , Mutación/genética , Mutación/fisiología , ARN/genética , Interferencia de ARNRESUMEN
Fluorescent-sandwich immunoassays on microarrays hold appeal for proteomics studies, because equipment and antibodies are readily available, and assays are simple, scalable, and reproducible. The achievement of adequate sensitivity and specificity, however, requires a general method of immunoassay amplification. We describe coupling of isothermal rolling-circle amplification (RCA) to universal antibodies for this purpose. A total of 75 cytokines were measured simultaneously on glass arrays with signal amplification by RCA with high specificity, femtomolar sensitivity, 3 log quantitative range, and economy of sample consumption. A 51-feature RCA cytokine glass array was used to measure secretion from human dendritic cells (DCs) induced by lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha). As expected, LPS induced rapid secretion of inflammatory cytokines such as macrophage inflammatory protein (MIP)-1beta, interleukin (IL)-8, and interferon-inducible protein (IP)-10. We found that eotaxin-2 and I-309 were induced by LPS; in addition, macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC), soluble interleukin 6 receptor (sIL-6R), and soluble tumor necrosis factor receptor I (sTNF-RI) were induced by TNF-alpha treatment. Because microarrays can accommodate approximately 1,000 sandwich immunoassays of this type, a relatively small number of RCA microarrays seem to offer a tractable approach for proteomic surveys.
Asunto(s)
Citocinas/biosíntesis , Citocinas/inmunología , Replicación del ADN , Perfilación de la Expresión Génica/métodos , Inmunoensayo/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Anticuerpos/inmunología , Línea Celular , Citocinas/genética , ADN Circular/biosíntesis , ADN Circular/genética , Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Humanos , Lipopolisacáridos/farmacología , Reacción en Cadena de la Polimerasa , Proteoma/biosíntesis , Proteoma/genética , Proteoma/inmunología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sensibilidad y Especificidad , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Concurrent advances in both high-throughput chemistry and genomics have given rise to the field of chemical genetics as a discipline for elucidating and validating drug targets, and generating novel therapeutics. Indeed, chemical genetic approaches to drug discovery have now been applied to several important drug target classes, especially those involved in signal transduction. Chemical genetics is distinct from the broader term "chemogenomics" which is defined as the description of all possible drugs against all possible targets (reviewed in [1]). This review covers several "orthogonal" chemical genetic approaches and focuses on a unique analog sensitive kinase technology and its applications to kinase drug discovery.