RESUMEN
This review highlights the concepts, recent applications and limitations of High Throughput Screening (HTS) flow cytometry-based efflux inhibitory assays. This platform has been employed in mammalian and yeast efflux systems leading to the identification of small molecules with transporter inhibitory capabilities. This technology offers the possibility of substrate multiplexing and may promote novel strategies targeting microbial efflux systems. This platform can generate a comprehensive dataset that may support efforts to map the interface between chemistry and transporter biology in a variety of pathogenic systems.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Transporte Biológico , Humanos , Bibliotecas de Moléculas Pequeñas/química , Especificidad por SustratoRESUMEN
ATP binding cassette (ABC) transmembrane efflux pumps such as P-glycoprotein (ABCB1), multidrug resistance protein 1 (ABCC1), and breast cancer resistance protein (ABCG2) play an important role in anticancer drug resistance. A large number of structurally and functionally diverse compounds act as substrates or modulators of these pumps. In vitro assessment of the affinity of drug candidates for multidrug resistance proteins is central to predict in vivo pharmacokinetics and drug-drug interactions. The objective of this study was to identify and characterize new substrates for these transporters. As part of a collaborative project with Life Technologies, 102 fluorescent probes were investigated in a flow cytometric screen of ABC transporters. The primary screen compared substrate efflux activity in parental cell lines with their corresponding highly expressing resistant counterparts. The fluorescent compound library included a range of excitation/emission profiles and required dual laser excitation as well as multiple fluorescence detection channels. A total of 31 substrates with active efflux in one or more pumps and practical fluorescence response ranges were identified and tested for interaction with eight known inhibitors. This screening approach provides an efficient tool for identification and characterization of new fluorescent substrates for ABCB1, ABCC1, and ABCG2.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Citometría de Flujo/métodos , Colorantes Fluorescentes/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Línea Celular , Humanos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/antagonistas & inhibidores , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Unión ProteicaRESUMEN
Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)-driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Pirazoles/farmacología , Pirimidinas/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular , Resistencia a Antineoplásicos , Citometría de Flujo , Ensayos Analíticos de Alto Rendimiento , Humanos , Concentración 50 Inhibidora , Ratones , Ratones SCID , Proteínas de Neoplasias/metabolismo , Bibliotecas de Moléculas Pequeñas , Relación Estructura-Actividad , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
By comparing the patterns of evolution in the coding and upstream noncoding regions of yeast ribosomal protein (RP) genes duplicated in a genome duplication, we find that although nonsynonymous sites in the coding sequences show strong evidence for the fixation of recent gene conversion events, similar patterns are less evident among the synonymous positions and noncoding regulatory elements. This result suggests a potential explanation for the somewhat puzzling fact that duplicated RP genes are not functionally redundant despite their very high protein sequence identity. An analysis of the patterns of regulatory network evolution after genome duplication also indicates that the duplicated proteins have diverged considerably in expression despite their similar protein sequences.
Asunto(s)
Conversión Génica , Duplicación de Gen , Proteínas Ribosómicas/genética , Saccharomyces/genética , Expresión Génica , Perfilación de la Expresión Génica , Genoma Fúngico , Sistemas de Lectura AbiertaRESUMEN
We analyze the structure of the yeast transcriptional regulation network, as revealed by chromatin immunoprecipitation experiments, and characterize the molecular evolution of both its transcriptional regulators and their target (regulated) genes. We test the hypothesis that highly connected genes are more important to the function of gene networks. Three lines of evidence-the rate of molecular evolution of network genes, the rate at which network genes undergo gene duplication, and the effects of synthetic null mutation in network genes-provide no strong support for this hypothesis. In addition, we ask how network genes diverge in their transcriptional regulation after duplication. Both loss (subfunctionalization) and gain (neofunctionalization) of transcription factor binding play a role in this divergence, which is often rapid. On the one hand, gene duplicates experience a net loss in the number of transcription factors binding to them, indicating the importance of losing transcription factor binding sites after gene duplication. On the other hand, the number of transcription factors that bind to highly diverged duplicates is significantly greater than would be expected if loss of binding played the only role in the divergence of duplicate genes.