67 research outputs found
Reversed-phase liquid chromatography coupled on-line to estrogen receptor bioaffinity detection based on fluorescence polarization
We describe the development and validation of a high-resolution screening (HRS) platform which couples gradient reversed-phase high-performance liquid chromatography (RP-HPLC) on-line to estrogen receptor Ī± (ERĪ±) affinity detection using fluorescence polarization (FP). FP, which allows detection at high wavelengths, limits the occurrence of interference from the autofluorescence of test compounds in the bioassay. A fluorescein-labeled estradiol derivative (E2-F) was synthesized and a binding assay was optimized in platereader format. After subsequent optimization in flow-injection analysis (FIA) mode, the optimized parameters were translated to the on-line HRS bioassay. Proof of principle was demonstrated by separating a mixture of five compounds known to be estrogenic (17Ī²-estradiol, 17Ī±-ethinylestradiol and the phytoestrogens coumestrol, coumarol and zearalenone), followed by post-column bioaffinity screening of the individual affinities for ERĪ±. Using the HRS-based FP setup, we were able to screen affinities of off-line-generated metabolites of zearalenone for ERĪ±. It is concluded that the on-line FP-based bioassay can be used to screen for the affinity of compounds without the disturbing occurrence of autofluorescence
Determination of the Proteolytic Cleavage Sites of the Amyloid Precursor-Like Protein 2 by the Proteases ADAM10, BACE1 and Ī³-Secretase
Regulated intramembrane proteolysis of the amyloid precursor protein (APP) by the protease activities Ī±-, Ī²- and Ī³-secretase controls the generation of the neurotoxic amyloid Ī² peptide. APLP2, the amyloid precursor-like protein 2, is a homolog of APP, which shows functional overlap with APP, but lacks an amyloid Ī² domain. Compared to APP, less is known about the proteolytic processing of APLP2, in particular in neurons, and the cleavage sites have not yet been determined. APLP2 is cleaved by the Ī²-secretase BACE1 and additionally by an Ī±-secretase activity. The two metalloproteases ADAM10 and ADAM17 have been suggested as candidate APLP2 Ī±-secretases in cell lines. Here, we used RNA interference and found that ADAM10, but not ADAM17, is required for the constitutive Ī±-secretase cleavage of APLP2 in HEK293 and SH-SY5Y cells. Likewise, in primary murine neurons knock-down of ADAM10 suppressed APLP2 Ī±-secretase cleavage. Using mass spectrometry we determined the proteolytic cleavage sites in the APLP2 sequence. ADAM10 was found to cleave APLP2 after arginine 670, whereas BACE1 cleaves after leucine 659. Both cleavage sites are located in close proximity to the membrane. Ī³-secretase cleavage was found to occur at different peptide bonds between alanine 694 and valine 700, which is close to the N-terminus of the predicted APLP2 transmembrane domain. Determination of the APLP2 cleavage sites enables functional studies of the different APLP2 ectodomain fragments and the production of cleavage-site specific antibodies for APLP2, which may be used for biomarker development
Advances in mass spectrometry-based post-column bioaffinity profiling of mixtures
In the screening of complex mixtures, for example combinatorial libraries, natural extracts, and metabolic incubations, different approaches are used for integrated bioaffinity screening. Four major strategies can be used for screening of bioactive mixtures for protein targetsāpre-column and post-column off-line, at-line, and on-line strategies. The focus of this review is on recent developments in post-column on-line screening, and the role of mass spectrometry (MS) in these systems. On-line screening systems integrate separation sciences, mass spectrometry, and biochemical methodology, enabling screening for active compounds in complex mixtures. There are three main variants of on-line MS based bioassays: the mass spectrometer is used for ligand identification only; the mass spectrometer is used for both ligand identification and bioassay readout; or MS detection is conducted in parallel with at-line microfractionation with off-line bioaffinity analysis. On the basis of the different fields of application of on-line screening, the principles are explained and their usefulness in the different fields of drug research is critically evaluated. Furthermore, off-line screening is discussed briefly with the on-line and at-line approaches
Fluorescent Probes for Cytochrome P450 Structural Characterization and Inhibitor Screening
We have synthesized two luminescent probes (D-4-Ad and D-8-Ad) that target cytochrome P450cam. D-4-Ad luminescence is quenched by Fƶrster energy transfer upon binding (K_d = 0.83 Ī¼M) but is restored when the probe is displaced from the active site by camphor. In contrast, D-8-Ad (K_d ā 0.02 Ī¼M) is not displaced from the enzyme, even in the presence of a large excess of camphor. The 2.2 Ć
resolution crystal structure of the D-8-Ad:P450cam complex reveals extensive hydrophobic contacts between the probe and the enzyme, which result from the conformational flexibility of the Bā, F, and G helices. Probes with properties similar to those of D-4-Ad potentially could be useful for screening P450 inhibitors
Fluorescent Probes for Cytochrome P450 Structural Characterization and Inhibitor Screening
ContinuousāFlow Systems for Ligand Binding and Enzyme Inhibition Assays Based on Mass Spectrometry
Rapid dereplication of estrogenic compounds in pomegranate (Punica granatum) using on-line biochemical detection coupled to mass spectrometry
During recent years, phytoestrogens have been receiving an increasing amount of interest, as several lines of evidence suggest a possible role in preventing a range of diseases, including the hormonally dependent cancers. In this context, various parts of the pomegranate fruit (Punica granatum; Punicaceae), e.g. seed oil, juice, fermented juice and peel extract, have been shown to exert suppressive effects on human breast cancer cells in vitro. On-line biochemical detection coupled to mass spectrometry (LC-BCD-MS) was applied to rapidly profile the estrogenic activity in the pomegranate peel extract. The crude mixture was separated by HPLC, after which the presence of biologically active compounds, known or unknown, was detected by means of an on-line Ī²-estrogen receptor (ER) bioassay. Chemical information, such as molecular weight and MS/MS fingerprint, was obtained in real time by directing part of the HPLC effluent towards a mass spectrometer. Using this approach in total three estrogenic compounds, i.e. luteolin, quercetin and kaempferol, were detected and identified by comparing the obtained molecular weights and negative ion APCI MS/MS spectra with the data of an estrogenic compound library. Although well known in literature and widely distributed in nature, the presence of these phytoestrogenic compounds in pomegranate peel extract was not reported previously. Compared to traditional screening approaches of complex mixtures, often characterized by a repeating cycle of HPLC fractionation and biological screening, LC-BCD-MS was shown to profoundly accelerate the time required for compound description and identification. Ā© 2003 Elsevier Ltd. All rights reserved
High resolution screening of plant natural product extracts for estrogen receptor alpha and beta binding activity using an online HPLC-MS biochemical detection system
Laserinduzierte Fluoreszenz-Detektion auf mikrostrukturierten Probentraegern fuer die Analytik im Umweltbereich (LINDAU). Teilvorhaben: Etablierung des immunanalytischen Testsystems Abschlussbericht
In the field of environmental analysis, immunological methods have become more and more popular. These procedures have proven to be fast and simple, particularly because no or little sample preparation is required. Typical test volumes employed are between 50-250 #mu#l. The costs of the assays can drastically be reduced by decreasing the volumes below 1 #mu#l. Thus the screening of large numbers of samples will become more feasible. We investigated the feasibility of immunoassays on microstructured sample carriers (nanotiterplates). Well sizes of 0.55 to 0.75 mm edge length and volumes of 50-70 nl were realised (BIAS). The sample handling was accomplished by using nanoliterpumps with piezoelectric actuators. Available droplet volumes were between 50 pl and 500 pl and could be delivered very accurately (#+-#2%). In order to avoid the evaporation of the dispensed liquid, the nanotiterplate was handled in a humid chamber and cooled immediately up to the point of condensation by means of a peltier element. After finishing the dispensing actions, the nanotiterplates were sealed with a transparent tape. This allowed the storage of the filled plates for several hours. Fluorescence read-out was performed using a laser scanning device especially developed for the nanowell array (Perkin-Elmer, Laser-Laboratorium Goettingen LLG). We have developed a competitive homogeneous phase immunoassay based on resonant energy transfer. As donor acceptor pair two red (long wavelength excitable) fluorescent dyes were chosen (Cy5 as donor and Cy5.5 as acceptor / Amersham). In this way, background fluorescence arising from assay compounds could be reduced. The assays for the analytes under investigation (simazine, atrazine, 2,4-D and isoproturon) were firstly set up in microtiterplates (volume of 200 #mu#l). The limit of determination were for three of these analytes (simazine, atrazine, and isoproturon) below the limit of the drinking water regulations of various European countries (0.1 #mu#g/l). The limit of determination of the 2,4-D assay was at 0.65 #mu#g/l. The transfer of the established immunoassays into the nanotireplates (volume of 50-70 nl) was performed with the analyte simazine. In order to avoid adsorption of the assay compounds to the cavity walls a special surface chemistry was developed. The results obtained in the nanotireplates coincide with the results from the reference assays (microtitreplate). The sample volume has successfully been reduced by a factor of 5000. Fields of application: analysis of high sample numbers in the field of environmental monitoring, clinical diagnostics and, more recently, pharmaceutical high throughput screening. (orig.)Available from TIB Hannover: F99B1401 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Bildung und Forschung (BMBF), Bonn (Germany)DEGerman
Development of a novel cytochrome P450 bioaffinity detection system coupled online to gradient reversed-phase high-performance liquid chromatography
A high-resolution screening platform, coupling online affinity detection for mammalian cytochrome P450s (Cyt P450s) to gradient reversed-phase high-performance liquid chromatography (HPLC), is described. To this end, the online Cyt P450 enzyme affinity detection (EAD) system was optimized for enzyme (Ī²-NF-induced rat liver microsomes), probe substrate (ethoxyresorufine), and organic modifier (methanol or acetonitrile). The optimized Cyt P450 EAD system has first been evaluated in a flow injection analysis (FIA) mode with 7 known ligands of Cyt P450 1A1/1A2 (Ī±-naphthoflavone, Ī²- naphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, I
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