Cytochrome P450 bio-affinity detection coupled to gradient HPLC: On-line screening of affinities to cytochrome P4501A2 and 2D6

Kool, Jeroen; Liempd, Sebastiaan van; Harmsen, Stefan; Beckman, J.; Elswijk, Danny van; Commandeur, Jan N. M.; Irth, H.; Vermeulen, Nico

doi:10.1016/j.jchromb.2007.08.005

Cited by 15 publications

(16 citation statements)

References 37 publications

(55 reference statements)

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“…ligands temporarily inhibit fluorescent product formation, which is monitored by a fluorescence (FlD) detector. estrone (e1) 23 , hexestrol 24 , 17β-estradiol (e2) 25 , estriol (e3) 26 , benzophenone-3 (bP3) 27 , aldrin 28 , testosterone 29 , tramadol 30 , imidazole 31 , and ketoconazole 32 ) was measured in the standard eaD setup. compounds (3 nmol, with the exception of ketoconazole [300 pmol]) were introduced into the system by flow injection into a potassium phosphate (100 mm; ph 7.4) carrier solution at a flow rate of 100 µl/min.…”

Section: Optimization Of the Bm3 Ead In Fia Modementioning

confidence: 99%

“…It has already been shown by Kool et al [29][30][31] that flow injection analysis (FIa) can be used to determine affinities of ligands toward P450s in an enzyme affinity detection (eaD) setup. the aim of this study was to use the same strategy to develop an alternative approach to screen bm3 mutants for diversity, and for this purpose, 3 different eaD setups were used.…”

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confidence: 99%

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Application of a Fluorescence-Based Continuous-Flow Bioassay to Screen for Diversity of Cytochrome P450 BM3 Mutant Libraries

et al. 2011

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A fluorescence-based continuous-flow enzyme affinity detection (EAD) setup was used to screen cytochrome P450 BM3 mutants on-line for diversity. The flow-injection screening assay is based on the BM3-mediated O-dealkylation of alkoxyresorufins forming the highly fluorescent product resorufin, and can be used in different configurations, namely injection of ligands, enzymes and substrates. Screening conditions were optimized and the activity of a library of 32 BM3 mutants towards the recently synthesized new probe substrate allyloxyresorufin was measured in flow-injection analysis (FIA) mode and it was shown that large activity differences between the mutants existed. Next, six BM3 mutants containing mutations at different positions in the active site were selected for which on-line enzyme kinetics were determined. Subsequently, for these six BM3 mutants affinity towards a set of 30 xenobiotics was determined in FIA EAD mode. It was demonstrated that significant differences existed for the affinity profiles of the mutants tested and that these differences correlated to alterations in the BM3 mutant-generated metabolic profiles of the drug buspirone. In conclusion, the developed FIA EAD approach is suitable to screen for diversity within BM3 mutants and this alternative screening technology offers new perspectives for rapid and sensitive screening of compound libraries towards BM3 mutants.

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Section: Optimization Of the Bm3 Ead In Fia Modementioning

confidence: 99%

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confidence: 99%

Application of a Fluorescence-Based Continuous-Flow Bioassay to Screen for Diversity of Cytochrome P450 BM3 Mutant Libraries

et al. 2011

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“…An off‐line ultrafiltration method, in which extracts were mixed and incubated with enzyme, and then compounds combined with enzyme after separation by ultrafiltration were analyzed by HPLC‐ESI‐TOF/MS, was applied to screen α ‐glucosidase inhibitors of guava leaf tea (Wang, Liu, Luo, Huang, & Wu, ). In addition, some on‐line HPLC‐BCD (biochemical detection) methods were developed to fast‐track identification of interesting and/or novel bioactive compounds, such as HPLC‐diode array detector‐1,1‐diphenyl‐2‐picrylhydrazyl/2,2'‐Azinobis‐(3‐ethylbenzthiazoline‐6‐sulphonate) (HPLC‐DAD‐DPPH/ABTS), HPLC‐DAD‐CL (chemiluminiscence) and DPPH‐CE（ (capillary electrophoresis）)‐DAD analysis of antioxidants (Shi et al, ; Ding et al, ; Chang et al, ; J. Liu, Tian, et al, ), HPLC‐DAD‐UV/FL (fluorescence) and MS analysis of acetylcholinesterase inhibitors (de Jong, Derks, Bruyneel, Niessen, & Irth, ; Marques et al, ; Peng et al, ), HPLC‐DAD‐UV analysis of α ‐glucosidase inhibitors (Li, Zhao, Xie, & Li, ; Li, Qian, & Li, ), HPLC‐DAD‐FL analysis of phosphodiesterase and P450 enzyme inhibitors (Kool et al, , ; van Liempd, Kool, Meerman, Irth, & Vermeulen, ), HPLC‐MS analysis of angiotensin‐I converting enzyme inhibitors and the receptor affinity (de Boer et al, ; Reinen, Kool, & Vermeulen, ; van Elswijk et al, ; van Elswijk, Schobel, Lansky, Irth, & van der Greef, ). These methods were effective strategies for screening bioactive compounds.…”

Section: Introductionmentioning

confidence: 99%

On‐line high‐performance liquid chromatography coupled with biochemical detection method for screening of α‐glucosidase inhibitors in green tea

Guo¹,

Shen

Fang

et al. 2018

Biomedical Chromatography

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An on-line high-performance liquid chromatography-biochemical detection (HPLC-BCD) method, in which compounds separated by HPLC were on-line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α-glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on-line HPLC-BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α-glucosidase and p-nitrophenyl α-d-glucopyranoside (PNPG). PNPG (1.25 mm), α-glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α-glucosidase inhibitors in green tea. Four components in green tea showed α-glucosidase inhibition action and three of them were identified as HHDP-galloyl glucose, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate by HPLC-fourier-transform mass spectrometry (HPLC-FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on-line HPLC-BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.

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“…[11][12][13][14] Most importantly, functional bioassays can also be implemented in these screening methodologies. 9 A functional bioassay gives the advantage of not only measuring binding affinity of a ligand, but also measuring functional activity and allowing distinction between, for example, agonism, antagonism, and allosteric modulation.…”

Section: Introductionmentioning

confidence: 99%

At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor

et al. 2016

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The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel expressed in different regions of the central nervous system (CNS). The α7-nAChR has been associated with Alzheimer's disease, epilepsy, and schizophrenia, and therefore is extensively studied as a drug target for the treatment of these diseases. Important sources for new compounds in drug discovery are natural extracts. Since natural extracts are complex mixtures, identification of the bioactives demands the use of analytical techniques to separate a bioactive from inactive compounds. This study describes screening methodology for identifying bioactive compounds in mixtures acting on the α7-nAChR. The methodology developed combines liquid chromatography (LC) coupled via a split with both an at-line calcium (Ca 2+ )-flux assay and highresolution mass spectrometry (MS). This allows evaluation of α7-nAChR responses after LC separation, while parallel MS enables compound identification. The methodology was optimized for analysis of agonists and positive allosteric modulators, and was successfully applied to screening of the hallucinogen mushroom Psilocybe Mckennaii. The crude mushroom extract was analyzed using both reversed-phase and hydrophilic interaction liquid chromatography. Matching retention times and peak shapes of bioactives found with data from the parallel MS measurements allowed rapid pinpointing of accurate masses corresponding to the bioactives.

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Cytochrome P450 bio-affinity detection coupled to gradient HPLC: On-line screening of affinities to cytochrome P4501A2 and 2D6

Cited by 15 publications

References 37 publications

Application of a Fluorescence-Based Continuous-Flow Bioassay to Screen for Diversity of Cytochrome P450 BM3 Mutant Libraries

Application of a Fluorescence-Based Continuous-Flow Bioassay to Screen for Diversity of Cytochrome P450 BM3 Mutant Libraries

On‐line high‐performance liquid chromatography coupled with biochemical detection method for screening of α‐glucosidase inhibitors in green tea

At-Line Cellular Screening Methodology for Bioactives in Mixtures Targeting the α7-Nicotinic Acetylcholine Receptor

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