RESUMEN
Developing molecularly targeted therapeutics with minimal off-target effects is facilitated by an understanding of compound selectivity. However, for HDAC inhibitors, a clear understanding of specificity has been challenging. In particular, it has been suggested that use of nonspecific substrates and the presence of multiple HDAC activities in enzyme preparations may complicate interpretation of inhibitor experiments. To overcome these and other potential limitations of activity-based HDAC assays, the authors have developed an assay format based on measurement of the binding affinity of inhibitors rather than measurement of enzyme activity. One advantage of this format is that it does not require use of a substrate and thus ameliorates concerns about lack of specificity of existing substrates. This assay is based on an Alexa Fluor® 647-labeled HDAC inhibitor or "tracer," which binds with a high affinity to Class I and Class IIb HDACs. Unlike activity assays, which can be affected by the presence of residual untagged endogenous HDACs from the host expression system, the signal in this format is dependent on the presence of an epitope tag on the specific HDAC of interest. The authors demonstrate the utility of this method by determining the potencies of commonly used inhibitors for six human HDACs.
Asunto(s)
Pruebas de Enzimas/métodos , Transferencia Resonante de Energía de Fluorescencia/métodos , Inhibidores de Histona Desacetilasas/farmacología , Histona Desacetilasas/metabolismo , Humanos , Cinética , Unión Proteica/efectos de los fármacosRESUMEN
The human pregnane X nuclear receptor (PXR) is a xenobiotic-regulated receptor that is activated by a range of diverse chemicals, including antibiotics, antifungals, glucocorticoids, and herbal extracts. PXR has been characterized as an important receptor in the metabolism of xenobiotics due to induction of cytochrome P450 isozymes and activation by a large number of prescribed medications. Developing methodologies that can efficiently detect PXR ligands will be clinically beneficial to avoid potential drug-drug interactions. To facilitate the identification of PXR ligands, a time-resolved fluorescence resonance energy transfer (TR-FRET) assay was miniaturized to a 1,536-well microtiter plate format to employ quantitative high-throughput screening (qHTS). The optimized 1,536-well TR-FRET assay showed Z'-factors of >or=0.5. Seven- to 15-point concentration-response curves (CRCs) were generated for 8,280 compounds using both terbium and fluorescein emission data, resulting in the generation of 241,664 data points. The qHTS method allowed us to retrospectively examine single concentration screening datasets to assess the sensitivity and selectivity of the PXR assay at different compound screening concentrations. Furthermore, nonspecific assay artifacts such as concentration-based quenching of the terbium signal and compound fluorescence were identified through the examination of CRCs for specific emission channels. The CRC information was also used to define chemotypes associated with PXR ligands. This study demonstrates the feasibility of profiling thousands of compounds against PXR using the TR-FRET assay in a high-throughput format.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Receptores de Esteroides/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Ligandos , Receptor X de Pregnano , Receptores de Esteroides/análisis , Relación Estructura-ActividadRESUMEN
Caspase-3 is an intracellular cysteine protease, activated as part of the apoptotic response to cell injury. Its interest as a therapeutic target has led many to pursue the development of inhibitors. To date, only one series of nonpeptidic inhibitors have been described, and these have limited selectivity within the caspase family. Here we report the properties of a series of anilinoquinazolines (AQZs) as potent small molecule inhibitors of caspase-3. The AQZs inhibit human caspase-3 with Ki values in the 90 to 800 nM range. A subset of AQZs are equipotent against caspase-6, although most lack activity against this isoform and caspase-1, -2, -7, and -8. The AQZs inhibit endogenous caspase-3 activity toward a cell permeable, exogenously added substrate in staurosporine-treated SH-SY5Y cells. The AQZs reduce biochemical and cellular features of apoptosis that are thought to be a consequence of caspase-3 activation including DNA fragmentation, TUNEL staining, and the various morphological features that define the terminal stages of apoptotic cell death. Moreover, the AQZs also inhibit apoptosis induced by nerve growth factor withdrawal from differentiated PC12 cells. Thus, the AQZs represent a new and structurally novel class of inhibitors, some of which selectively inhibit caspase-3 and will thereby allow evaluation of the role of caspase-3 activity in various cellular models of apoptosis.