RESUMO
Mammalian signal transduction occurs in the context of multiprotein complexes, yet currently available drug discovery strategies do not reflect this fact. We present a strategy for screening drugs and targets in living human cells by utilizing high content protein-fragment complementation assays. Synthetic fragments of a mutant fluorescent protein ("Venus" and/or enhanced yellow fluorescent protein) are used for protein-fragment complementation assay construction, allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. Here we describe the utility of this novel strategy for high-throughput screening of known targets, and for screening previously undrugable targets and profiling drug leads for improved selectivity and safety.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Complexos Multiproteicos/metabolismo , Sobrevivência Celular , Relação Dose-Resposta a Droga , Humanos , Ligação Proteica , RNA Interferente Pequeno , Sensibilidade e Especificidade , Frações SubcelularesRESUMO
Signal transduction pathways mediated by MAP kinases are among the most studied. Direct analysis of MAP kinase pathways has been difficult because some details of MAP kinase signaling cannot be studied in vitro. Here, we describe a strategy for directly analyzing MAP kinase signaling pathways in living cells using protein-fragment complementation assays (PCA) based on intensely fluorescent proteins. The assays allow for spatial and temporal analysis of protein complexes including those that form upstream and downstream from MAPKs as well as complexes of MAPKs with regulator and effector proteins. We describe high-content assays, high-throughput quantitative microscopic methods to follow temporal changes in complex subcellular location and quantity. Spatial and temporal changes in response to perturbations (chemical, siRNA, and hormones) allow for delineation of MAPK signaling networks and a general and high-throughput approach to identify small molecules that act directly or indirectly on MAPK pathways.
Assuntos
Técnicas de Química Combinatória , Teste de Complementação Genética/métodos , Técnicas Genéticas , Sistema de Sinalização das MAP Quinases , Fragmentos de Peptídeos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , RNA Interferente Pequeno , TransfecçãoRESUMO
We present a strategy for identifying off-target effects and hidden phenotypes of drugs by directly probing biochemical pathways that underlie therapeutic or toxic mechanisms in intact, living cells. High-content protein-fragment complementation assays (PCAs) were constructed with synthetic fragments of a mutant fluorescent protein ('Venus', EYFP or both), allowing us to measure spatial and temporal changes in protein complexes in response to drugs that activate or inhibit particular pathways. One hundred and seven different drugs from six therapeutic areas were screened against 49 different PCA reporters for ten cellular processes. This strategy reproduced known structure-function relationships and also predicted 'hidden,' potent antiproliferative activities for four drugs with novel mechanisms of action, including disruption of mitochondrial membrane potential. A simple algorithm identified a 25-assay panel that was highly predictive of antiproliferative activity, and the predictive power of this approach was confirmed with cross-validation tests. This study suggests a strategy for therapeutic discovery that identifies novel, unpredicted mechanisms of drug action and thereby enhances the productivity of drug-discovery research.