Online biochemical detection of glutathione-S-transferase P1-specific inhibitors in complex mixtures.

A high-resolution screening (HRS) technology is described, which couples 2 parallel enzyme affinity detection (EAD) systems for substrates and inhibitors of rat cytosolic glutathione-S-transferases (cGSTs) and purified human GST P1 to gradient reversed-phase high-performance liquid chromatography (HPLC). The cGSTs and GST P1 EAD systems were optimized and validated first in flow injection analysis (FIA) mode, and optimized values were subsequently used for HPLC mode. The IC(50) values of 8 ligands thus obtained online agreed well with the IC(50) values obtained with microplate reader-based assays. For ethacrynic acid, an IC(50) value of 1.8 +/- 0.4 microM was obtained with the cGSTs EAD system in FIA mode and 0.8 +/- 0.6 microM in HPLC mode. For ethacrynic acid with the GST P1 EAD system, IC(50) values of 6.0 +/- 2.9 and 3.6 +/- 2.8 microM were obtained in FIA and HPLC modes, respectively. An HRS GST EAD system, consisting of both the cGSTs and the GST P1 EAD system in HPLC mode in parallel, was able to separate complex mixtures of compounds and to determine online their individual affinity for cGSTs and GST P1. Finally, a small library of GST inhibitors, synthesized by reaction of several electrophiles with glutathione (GSH), was successfully screened with the newly developed parallel HRS GST EAD system. It is concluded that the present online gradient HPLC-based HRS screening technology offers new perspectives for sensitive and simultaneous screening of general cGSTs and specific GST P1 inhibitors in mixtures.

Development of three parallel cytochrome P450 enzyme affinity detection systems coupled on-line to gradient high-performance liquid chromatography.

A high resolution screening (HRS) technology is described, in which gradient high-performance liquid chromatography (HPLC) is connected on-line to three parallel placed bioaffinity detection systems containing mammalian cytochromes P450 (P450s). The three so-called enzyme affinity detection (EAD) systems contained, respectively, liver microsomes from rats induced by beta-naphthoflavone (CYP1A activity), phenobarbital (CYP2B activity), and dexamethasone (CYP3A activity). Each P450-EAD system was optimized for enzyme, substrate, and organic modifier (isopropyl alcohol, methanol, and acetonitrile) in flow injection analysis mode. Characteristic P450 ligands were used to validate the P450-EAD systems. IC(50) values of the ligands were measured and found to be similar to those obtained with conventional microtiter plate reader assays. Detection limits (n = 3; signal-to-noise ratio = 3) of potent inhibitors ranged from 1 to 3 pmol for CYP1A activity, 4 to 17 pmol for CYP2B activity, and 4 to 15 pmol for CYP3A activity. The three optimized P450-EAD systems were subsequently coupled to gradient HPLC and used to screen compound mixtures for individual ligands. Finally, to increase analysis efficiency, a HRS system was constructed in which all three P450-EAD systems were coupled on-line and in parallel to gradient HPLC. The triple parallelized P450-EAD system was shown to enable rapid profiling of individual components in complex mixtures for inhibitory activity to three different P450s.

On-line coupling of high-performance liquid chromatography to a continuous-flow enzyme assay based on electrospray ionization mass spectrometry.

Liquid chromatography (LC) was coupled on-line to a continuous-flow enzymatic assay using electrospray ionization mass spectrometry (ESI-MS) as readout for the screening of enzyme inhibitors in complex samples. Inhibitors were detected by changes in the concentration of the enzymatic reaction products, indicating the inhibition of enzymatic activity. The molecular masses of the inhibitors were determined with high certainty by using retention time matching and peak shape comparison. Due to the high matching accuracy, baseline separation of coeluting analytes was not necessary in order to identify the correct masses of the bioactive compounds. The continuous-flow system was successfully applied for the screening of complex samples, such as natural extracts. For a red clover extract, detection limits of 0.3-0.8 micromol/L were obtained. System validation was performed by determining the IC(50) values of four inhibitors in the flow-injection mode. The IC(50) values were in the 0.11-5.6 micromol/L range and correspond closely to data obtained by microtiter plate assays. Detection limits were in the range of 0.018-0.35 micromol/L in the flow-injection mode, and 0.075-0.75 micromol/L in the LC mode. These values are well below the typical compound concentrations (1-10 micromol/L) used in high-throughput screening. Together with an interday precision of 12.6%, these results demonstrate the applicability of the system for bioactivity screening of complex mixtures, generating both chemical and biological information on bioactive compounds in a single run.

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 beta-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.

Rapid detection and identification of angiotensin-converting enzyme inhibitors by on-line liquid chromatography-biochemical detection, coupled to electrospray mass spectrometry.

An analytical method based on on-line liquid chromatography-biochemical detection (LC-BCD) coupled to electrospray mass spectrometry was developed for the detection and identification of angiotensin-converting enzyme (ACE) inhibitors in complex mixtures, such as hydrolyzed whey proteins. ACE inhibitory activity was detected by coupling a homogeneous, substrate conversion based bioassay on-line to high-performance liquid chromatography (HPLC). Chemical information was obtained by directing part of the HPLC effluent towards a mass spectrometer. After correlating the biochemical and chemical data, the accurate molecular masses of the bioactive peptides were used as search queries in protein databases. Combined with the recorded mass spectrometry (MS)-MS fingerprints, bioactive peptides were selected from the database search results. The results of LC-BCD-MS analyses were verified by establishing a bioactivity balance. Reference samples, containing several peptides at concentration levels similar to those observed in the hydrolyzed milk samples, were analyzed by LC-BCD-MS. High recoveries of biological activity were obtained, indicating that the correct ACE inhibitors were identified and that no co-elution of significantly bioactive molecules had occurred. Approximately, 30 ACE inhibitors were detected and identified. IC50 values of ACE inhibitors, reported in literature, ranged between 43 and 580 microM.