Quantitative Whole-Cell Cytochrome P450 Measurement Suitable for High-Throughput Application

The recombinant expression of cytochrome P450 enzymes involved in drug metabolism is of interest to the pharmaceutical and biotechnological industries due to the versatile catalytic properties of these enzymes. Accurate quantification of cytochrome P450 enzymes expressed in bacterial culture generally depends on disruption and fractionation of cells to prepare membranes for spectral analysis. Although whole-cell methods for spectral determination have been reported, problems with poor reproducibility and low signal-to-noise ratio confound the use of such techniques where P450 hemoprotein expression levels are relatively low, such as in cultures of certain mammalian forms. In particular, interference from bacterial hemoproteins often obscures the P450 peak. In the current study, the combination of culture concentration, incubation under microaerobic conditions, and a modified method of baseline correction enabled reproducible quantification of cytochrome P450s in whole cells. This whole-cell method is well suited to high-throughput application, as large sets or libraries of enzymes can be expressed in parallel and relative expression levels measured without downstream cell processing. ( Journal of Biomolecular Screening 2008:135-141)

Download Full-text

Evaluation of the Effects of Mitragyna speciosa Alkaloid Extract on Cytochrome P450 Enzymes Using a High Throughput Assay

Molecules ◽

10.3390/molecules16097344 ◽

2011 ◽

Vol 16 (9) ◽

pp. 7344-7356 ◽

Cited By ~ 45

Author(s):

Wai Mun Kong ◽

Zamri Chik ◽

Murali Ramachandra ◽

Umarani Subramaniam ◽

Raja Elina Raja Aziddin ◽

...

Keyword(s):

Cytochrome P450 ◽

High Throughput ◽

Cytochrome P450 Enzymes ◽

Mitragyna Speciosa ◽

High Throughput Assay

Download Full-text

Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4

Biochemical Pharmacology ◽

10.1016/j.bcp.2018.10.006 ◽

2018 ◽

Vol 158 ◽

pp. 134-140 ◽

Cited By ~ 4

Author(s):

Francisco Esteves ◽

Diana Campelo ◽

Philippe Urban ◽

Sophie Bozonnet ◽

Thomas Lautier ◽

...

Keyword(s):

Cytochrome P450 ◽

High Throughput ◽

Activity Assay ◽

Whole Cell ◽

Human Cytochrome ◽

P450 Expression

Download Full-text

Union is strength: target-based and whole-cell high throughput screens in antibacterial discovery

Journal of Bacteriology ◽

10.1128/jb.00477-21 ◽

2021 ◽

Author(s):

Cristina Landeta ◽

Adrian Mejia-Santana

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Enzymatic Reactions ◽

Whole Cell ◽

Bacterial Physiology ◽

Whole Cells ◽

Discovery Research ◽

High Throughput Screens

Antimicrobial resistance is one of the greatest global health challenges today. For over three decades antibacterial discovery research and development has been focused on cell-based and target-based high throughput assays. Target-based screens use diagnostic enzymatic reactions to look for molecules that can bind directly and inhibit the target. Target-based screens are only applied to proteins that can be successfully expressed, purified and the activity of which can be effectively measured using a biochemical assay. Often times the molecules found in these in vitro screens are not active in cells due to poor permeability or efflux. On the other hand, cell-based screens use whole cells and look for growth inhibition. These screens give higher number of hits than target-based assays and can simultaneously test many targets of one process or pathway in their physiological context. Both strategies have pros and cons when used separately. In the past decade and a half our increasing knowledge of bacterial physiology has led to the development of innovative and sophisticated technologies to perform high throughput screening combining these two strategies and thus minimizing their disadvantages. In this review we discuss recent examples of high throughput approaches that used both target-based and whole-cell screening to find new antibacterials, the new insights they have provided and how this knowledge can be applied to other in vivo validated targets to develop new antimicrobials.

Download Full-text

Evaluation of Luminogenic Substrates as Probe Substrates for Bacterial Cytochrome P450 Enzymes: Application to Mycobacterium tuberculosis

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555219853220 ◽

2019 ◽

Vol 24 (7) ◽

pp. 745-754

Author(s):

Sandra Ortega Ugalde ◽

Dongping Ma ◽

James J. Cali ◽

Jan N. M. Commandeur

Keyword(s):

Mycobacterium Tuberculosis ◽

Cytochrome P450 ◽

High Throughput ◽

High Throughput Screening ◽

Drug Targets ◽

High Signal ◽

Cytochrome P450 Enzymes ◽

High Enzyme ◽

Endogenous Substrates ◽

Selection Of

Several cytochrome P450 enzymes (CYPs) encoded in the genome of Mycobacterium tuberculosis (Mtb) are considered potential new drug targets due to the essential roles they play in bacterial viability and in the establishment of chronic intracellular infection. Identification of inhibitors of Mtb CYPs at present is conducted by ultraviolet-visible (UV-vis) optical titration experiments or by metabolism studies using endogenous substrates, such as cholesterol and lanosterol. The first technique requires high enzyme concentrations and volumes, while analysis of steroid hydroxylation is dependent on low-throughput analytical methods. Luciferin-based luminogenic substrates have proven to be very sensitive substrates for the high-throughput profiling of inhibitors of human CYPs. In the present study, 17 pro-luciferins were evaluated as substrates for Mtb CYP121A1, CYP124A1, CYP125A1, CYP130A1, and CYP142A1. Luciferin-BE was identified as an excellent probe substrate for CYP130A1, resulting in a high luminescence yield after addition of luciferase and adenosine triphosphate (ATP). Its applicability for high-throughput screening was supported by a high Z’-factor and high signal-to-background ratio. Using this substrate, the inhibitory properties of a selection of known inhibitors could be characterized using significantly less protein concentration when compared to UV-vis optical titration experiments. Although several luminogenic substrates were also identified for CYP121A1, CYP124A1, CYP125A1, and CYP142A1, their relatively low yield of luminescence and low signal-to-background ratios make them less suitable for high-throughput screening since high enzyme concentrations will be needed. Further structural optimization of luminogenic substrates will be necessary to obtain more sensitive probe substrates for these Mtb CYPs.

Download Full-text

A high-throughput inhibition screening of major human cytochrome P450 enzymes using anin vitrococktail and liquid chromatography-tandem mass spectrometry

Biomedical Chromatography ◽

10.1002/bmc.3003 ◽

2013 ◽

Vol 28 (2) ◽

pp. 197-203 ◽

Cited By ~ 28

Author(s):

Chong-Zhen Qin ◽

Xian Ren ◽

Zhi-Rong Tan ◽

Yao Chen ◽

Ji-Ye Yin ◽

...

Keyword(s):

Mass Spectrometry ◽

Cytochrome P450 ◽

Liquid Chromatography ◽

Tandem Mass Spectrometry ◽

High Throughput ◽

Tandem Mass ◽

Cytochrome P450 Enzymes ◽

Chromatography Tandem Mass Spectrometry ◽

Human Cytochrome ◽

Liquid Chromatography Tandem Mass

Download Full-text

A3 - A High-throughput double cocktail approach for evaluating time-dependent inhibition of nine major human cytochrome P450 enzymes

Drug Metabolism and Pharmacokinetics ◽

10.1016/j.dmpk.2020.04.257 ◽

2020 ◽

Vol 35 (1) ◽

pp. S19

Author(s):

Helinä Kahma ◽

Laura Aurinsalo ◽

Mikko Neuvonen ◽

Jenni Viinamäki ◽

Terhi Launiainen ◽

...

Keyword(s):

Cytochrome P450 ◽

High Throughput ◽

Time Dependent ◽

Cytochrome P450 Enzymes ◽

Human Cytochrome ◽

Dependent Inhibition ◽

Time Dependent Inhibition ◽

Cocktail Approach

Download Full-text

CYP21-catalyzed production of the long-term urinary metandienone metabolite 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one: a contribution to the fight against doping

Biological Chemistry ◽

10.1515/bc.2010.002 ◽

2010 ◽

Vol 391 (1) ◽

Cited By ~ 23

Author(s):

Andy Zöllner ◽

Maria Kristina Parr ◽

Călin-Aurel Drăgan ◽

Stefan Dräs ◽

Nils Schlörer ◽

...

Keyword(s):

Cytochrome P450 ◽

Reference Material ◽

Anabolic Androgenic Steroids ◽

Reference Substance ◽

Cytochrome P450 Enzymes ◽

Whole Cell ◽

In Doping ◽

Whole Cell Biotransformation ◽

Androgenic Steroids

AbstractAnabolic-androgenic steroids are some of the most frequently misused drugs in human sports. Recently, a previously unknown urinary metabolite of metandienone, 17β-hydroxymethyl-17α-methyl-18-norandrosta-1,4,13-trien-3-one (20OH-NorMD), was discovered via LC-MS/MS and GC-MS. This metabolite was reported to be detected in urine samples up to 19 days after administration of metandienone. However, so far it was not possible to obtain purified reference material of this metabolite and to confirm its structure via NMR. Eleven recombinant strains of the fission yeastSchizosaccharomyces pombethat express different human hepatic or steroidogenic cytochrome P450 enzymes were screened for production of this metabolite in a whole-cell biotransformation reaction. 17,17-Dimethyl-18-norandrosta-1,4,13-trien-3-one, chemically derived from metandienone, was used as substrate for the bioconversion, because it could be converted to the final product in a single hydroxylation step. The obtained results demonstrate that CYP21 and to a lesser extent also CYP3A4 expressing strains can catalyze this steroid hydroxylation. Subsequent 5 l-scale fermentation resulted in the production and purification of 10 mg of metabolite and its unequivocal structure determination via NMR. The synthesis of this urinary metandienone metabolite viaS. pombe-based whole-cell biotransformation now allows its use as a reference substance in doping control assays.

Download Full-text