Screening of natural products extracts for the presence of phosphodiesterase inhibitors using liquid chromatography coupled online to parallel biochemical detection and chemical characterization

The ability to rapidly identify active compounds in a complex mixture (e.g., natural products extract) is still one of the major problems in natural products screening programs. An elegant way to overcome this problem is to separate the complex mixture by gradient liquid chromatography followed by online biochemical detection parallel with chemical characterization, referred to as high-resolution screening (HRS). To find and identify phosphodiesterase (PDE) inhibitors in natural products extracts using the HRS technology, the authors developed a continuous-flow PDE enzymatic assay. The suitability of the continuous-flow PDE enzymatic assay for natural products screening was demonstrated. After optimization of the continuous-flow PDE assay, the limit of detection for 3-isobutyl-1-methyl-xanthine (IBMX) was 1 muM, with a dynamic range from 1 to 100 muM IBMX. The applicability of the HRS technology for the detection of PDE inhibitors in natural products extracts was demonstrated by the analysis of a plant extract spiked with 2 naturally occurring PDE inhibitors. The plant extract was analyzed with 2 assay lines in parallel, enabling background fluorescence correction of the sample. The simultaneous quantification of the active compounds using evaporative light-scattering detection allowed the estimation of the IC(50) value of the active compounds directly in the crude extract.     

Development of surface plasmon resonance biosensor assays for primary and secondary screening of acetylcholine binding protein ligands

Surface plasmon resonance (SPR) biosensors recently gained an important place in drug discovery. Here we present a primary and secondary SPR biosensor screening methodology. The primary screening method is based on a direct binding assay with covalent immobilized drug target proteins. For the secondary screening method, a sequential competition assay has been developed where the captured protein is first exposed to an unknown test compound, followed directly by an exposure to a high-molecular-weight reporter ligand. Using the high-molecular-weight reporter ligand to probe the remaining free binding site on the sensor, a significant signal enhancement is obtained. Furthermore, this assay format allows the validation of the primary direct binding assay format, efficiently revealing false positive data. As a model system, acetylcholine binding protein (AChBP), which is a soluble model protein for neuronal nicotinic acetylcholine receptors, has been used. The secondary assay is lower in throughput than the primary assay; however, the signal-to-noise ratio is two times higher compared with the direct assay, and it has a z′ factor of 0.96. Using both assays, we identified the compound tacrine as a ligand for AChBP.     

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.     

A flow-through fluorescence polarization detection system for measuring GPCR-mediated modulation of cAMP production

A flow-through fluorescence polarization (FP) detection system that makes use of a novel high-performance liquid chromatography (HPLC) fluorescence detector modified with polarization filters was developed. This flow-through FP detection system was evaluated by using a novel and very cost-effective bioassay for cyclic adenosine monophosphate (cAMP). The bioassay was first evaluated and optimized in an FP plate reader format and subsequently in a flow-through bioassay setup. The principle of the bioassay is based on the competition of cAMP and a fluorescent cAMP derivative for the cAMP binding domain of protein kinase A. cAMP could accurately be determined over a range of 0.8 to 30 pmol/well in the plate reader FP assay and over a range of 0.3 to 50 pmol/well in the flow-through FP assay setup. High Z' factors (i.e., 0.89 for the plate reader and 0.93 for the flow-through FP cAMP assay, respectively) indicated robust assays. Finally, functional cAMP signaling of the human histamine H(3) G-protein-coupled receptor (GPCR) in cell cultures was measured with both assay formats with good sensitivities and assay windows. The pEC(50) values obtained in both assay formats were in accordance with those obtained with standard methods. The flow-through FP detection system could thus be used as a cost-effective alternative to FP plate reader assays. Moreover, the novel flow-through FP detection system for cAMP constitutes a good analytical tool to be used in the GPCR research field as an alternative to the use of FP plate readers or radioactive laboratories nowadays used for cAMP measurements.     

HPLC coupled on-line to ESI-MS and a DPPH-based assay for the rapid identification of anti-oxidants in Butea superba

A reversed-phase HPLC coupled on-line to a radical scavenging detection system and MS/MS was developed in order to combine separation, activity determination and structural identification of anti-oxidants in complex mixtures in one run. The sample was separated by HPLC and the eluate split into two flows. The major portion was fed into an electrospray ionisation MS/MS system, while the minor part was mixed with a free radical, 2,2'-diphenyl-1-picrylhydrazyl (DPPH), and the reaction determined spectrophotometrically. The negative peaks, which indicated the presence of anti-oxidant activity, were monitored by measuring the decrease in absorbance at 517 nm. The developed method was successfully applied to the identification of anti-oxidant compounds in a fraction, obtained by solid-phase extraction, of an extract of a Thai medicinal plant, Butea superba Roxb. The anti-oxidant compounds were separated and identified as procyanidin B2, (-)-epicatechin and procyanidin B5.     

Determination and identification of estrogenic compounds generated with biosynthetic enzymes using hyphenated screening assays,high resolution mass spectrometry and off-line NMR

This paper describes the determination and identification of active and inactive estrogenic compounds produced by biosynthetic methods. A hyphenated screening assay towards the human estrogen receptor ligand binding domain (hER)α and hERβ integrating target–ligand interactions and liquid chromatography–high resolution mass spectrometry was used. With this approach, information on both biologic activity and structure identity of compounds produced by bacterial mutants of cytochrome P450s was obtained in parallel. Initial structure identification was achieved by high resolution MS/MS, while for full structure determination, P450 incubations were scaled up and the produced entities were purified using preparative liquid chromatography with automated fraction collection. NMR spectroscopy was performed on all fractions for 3D structure analysis; this included 1D-¹H, 2D-COSY, 2D-NOESY, and ¹H-¹³C-HSQC experiments. This multidimensional screening approach enabled the detection of low abundant biotransformation products which were not suitable for detection in either one of its single components. In total, the analytical scale biosynthesis produced over 85 compounds from 6 different starting templates. Inter- and intra-day variation of the biochemical signals in the dual receptor affinity detection system was less than 5%. The multi-target screening approach combined with full structure characterization based on high resolution MS(/MS) and NMR spectroscopy demonstrated in this paper can generally be applied to e.g. metabolism studies and compound-library screening.     

Derivatization of carboxylic acids with 4-APEBA for detection by positive-ion LC-ESI-MS(/MS) applied for the analysis of prostanoids and NSAID in urine

In order to develop a generic positive ionization ESI LC-MS method for a variety of interesting substance classes, a new derivatization strategy for carboxylic acids was developed. The carboxylic acid group is labeled with the bromine containing 4-APEBA reagent based on carbodiimide chemistry. The derivatization reaction can be carried out under aqueous conditions, thereby greatly simplifying sample preparation. In this paper, the derivatization of carboxylic acids is exemplified for the determination of prostanoids and non-steroidal anti-inflammatory drugs (NSAID). Optimization of the derivatization conditions was studied. In order to prove the applicability of the presented approach, we applied the described protocol to urine samples from complex regional pain syndrome (CRPS) patients and were able to detect several prostanoids not visible in the urine of healthy volunteers. Further, the determination of the non-steroidal anti-inflammatory drug ibuprofen in a urine sample was possible.     

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 (beta-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 (alpha-naphthoflavone, beta-naphthoflavone, ellipticine, 9-hydroxy-ellipticine, fluvoxamine, caffein, and phenacetin). Subsequently, IC50 values were online in FIA-mode determined and compared with those obtained with standardmicrosomal assay conditions. The IC50 values obtained with the online Cyt P450 EAD system agreed well with the IC50 values obtained in the standard assays. For high affinity ligands of Cyt P450 1A1/1A2, detection limits of 1 to 3 pmol injected (n=3; signal to noise [S/N]=3) were obtained. The individual inhibitory properties of ligands in mixtures of the ligands were subsequently investigated using an optimized Cyt P450 EAD system online coupled to gradient HPLC. Using the integrated online gradient HPLC Cyt P450 EAD platform, detection limits of 10 to 25 pmol injected (n=1; S/N=3) were obtained for high-affinity ligands. It is concluded that this novel screening technology offers new perspectives for rapid and sensitive screening of individual compounds in mixtures exhibiting affinity for liver microsomal Cyt P450s.     

Determination of denaturated proteins and biotoxins by on-line size-exclusion chromatography-digestion-liquid chromatography-electrospray mass spectrometry

A multidimensional analytical method for the rapid determination and identification of proteins has been developed. The method is based on the size-exclusion fractionation of protein-containing samples, subsequent on-line trypsin digestion and desalination, and reversed-phase high-performance liquid chromatography-electrospray mass spectrometry detection. The present system reduces digestion times to 20 min and the total analysis time to less than 100 min. Using bovine serum albumin and myoglobin as model proteins, optimization of key parameters such as digestion times and interfacing conditions between the different pretreatment steps was performed. The automated system was tested for the identification of infectious disease agents such as cholera toxin and staphylococcal enterotoxin B. This resulted typically in a positive identification by a total sequence coverage of approximately 40%.