scholarly journals Development of a Miniaturized 384-Well High Throughput Screen for the Detection of Substrates of Cytochrome P450 2D6 and 3A4 Metabolism

2001 ◽  
Vol 6 (2) ◽  
pp. 91-99 ◽  
Author(s):  
Ilona Kariv ◽  
Mark P. Fereshteh ◽  
Kevin R. Oldenburg
Keyword(s):  
Drug Metabolism ◽  
High Throughput ◽  
High Throughput Screening ◽  
Biologically Active ◽  
In Vitro Assays ◽  
Main Concern ◽  
Assay Sensitivity ◽  
Cyp Enzymes ◽  
Assay Optimization

The identification of a large number of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with druglike properties early during the lead prioritization and development process. One of the major steps in lead prioritization is the assessment of drug metabolism mediated by the cytochrome P450 (CYP) enzymes to evaluate the potential drug-drug interactions. CYP2D6 and CYP3A4 comprise the main human CYP enzymes involved in drug metabolism. The recent availability of specific CYP cDNA expression systems and the development of specific fluorescent probes have accelerated the ability to develop robust in vitro assays in HTS format. The aim of this study was to optimize conditions for the CYP2D6 and CYP3A4 HTS assays and subsequently adapt those assays to a miniaturized 384-well format. Assay conversion to a miniaturized format presents certain difficulties, such as robustness of the signal and of compound delivery. Thus the assay optimization involved the comparison of different substrates to identify those most suitable for use in a miniaturized format. Because of current technical limitations in liquid dispensing of nanoliter volumes, assay sensitivity to organic solvents also provides a main concern during assay miniaturization. Therefore, compound activity from redissolved dry films and from DMSO stocks directly delivered into assay buffer was compared. The data indicate that compound activity was comparable in both formats. The data support the conclusion that CYP2D6 and CYP3A4 in vitro metabolism assays can be successfully performed in 384-well plate format and the substrate potencies, as evaluated by the IC50 values, determined.

scholarly journals Investigating Molecular Mechanisms of Immunotoxicity and the Utility of ToxCast for Immunotoxicity Screening of Chemicals Added to Food

2021 ◽  
Vol 18 (7) ◽  
pp. 3332
Author(s):  
Olga V. Naidenko ◽  
David Q. Andrews ◽  
Alexis M. Temkin ◽  
Tasha Stoiber ◽  
Uloma Igara Uche ◽  
...  
Keyword(s):  
Immune System ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Molecular Mechanisms ◽  
Specific Activity ◽  
Laboratory Animals ◽  
In Vitro Assays ◽  
Toxicological Studies

The development of high-throughput screening methodologies may decrease the need for laboratory animals for toxicity testing. Here, we investigate the potential of assessing immunotoxicity with high-throughput screening data from the U.S. Environmental Protection Agency ToxCast program. As case studies, we analyzed the most common chemicals added to food as well as per- and polyfluoroalkyl substances (PFAS) shown to migrate to food from packaging materials or processing equipment. The antioxidant preservative tert-butylhydroquinone (TBHQ) showed activity both in ToxCast assays and in classical immunological assays, suggesting that it may affect the immune response in people. From the PFAS group, we identified eight substances that can migrate from food contact materials and have ToxCast data. In epidemiological and toxicological studies, PFAS suppress the immune system and decrease the response to vaccination. However, most PFAS show weak or no activity in immune-related ToxCast assays. This lack of concordance between toxicological and high-throughput data for common PFAS indicates the current limitations of in vitro screening for analyzing immunotoxicity. High-throughput in vitro assays show promise for providing mechanistic data relevant for immune risk assessment. In contrast, the lack of immune-specific activity in the existing high-throughput assays cannot validate the safety of a chemical for the immune system.

scholarly journals Optimization of a High-Throughput 384-Well Plate-Based Screening Platform with Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 15442 Biofilms

2020 ◽  
Vol 21 (9) ◽  
pp. 3034 ◽  
Author(s):  
Shella Gilbert-Girard ◽  
Kirsi Savijoki ◽  
Jari Yli-Kauhaluoma ◽  
Adyary Fallarero
Keyword(s):  
Staphylococcus Aureus ◽  
Pseudomonas Aeruginosa ◽  
High Throughput ◽  
High Throughput Screening ◽  
Bacterial Infections ◽  
Rapid Screening ◽  
Bacterial Biofilms ◽  
Main Concern ◽  
Assay Optimization ◽  
Screening Platform

In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.

scholarly journals High Throughput Screening of Pharmacokinetics and Metabolism in Drug Discovery (II) —Investigation on In vitro and In vivo Correlation in Drug Metabolism Screening—

2005 ◽  
Vol 125 (1) ◽  
pp. 131-139 ◽  
Author(s):  
Hiroshi KOMURA ◽  
Kenichi MATSUDA ◽  
Yukie SHIGEMOTO ◽  
Iichiro KAWAHARA ◽  
Rieko ANO ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Drug Metabolism ◽  
High Throughput ◽  
High Throughput Screening

Abstracts

2008 ◽  
Vol 27 (6) ◽  
pp. 405-405
Author(s):  
David J. Dix
Keyword(s):  
Risk Assessment ◽  
High Throughput ◽  
Environmental Protection Agency ◽  
High Throughput Screening ◽  
Human Health Risk ◽  
Environmental Chemicals ◽  
In Vitro Assays ◽  
The Impact ◽  
Toxicity Pathways

The U.S. Environmental Protection Agency (EPA), National Toxicology Program (NTP), and National Institutes of Health (NIH) Chemical Genomics Center (NCGC) have complementary research programs designed to improve chemical toxicity evaluations by developing high throughput screening (HTS) methods that evaluate the impact of environmental chemicals on key toxicity pathways. These federal partners are coordinating an extension of the EPA’s ToxCast program, the NTP’s HTS initiative, and the NCGC’s Molecular Libraries Initiative into a collaborative research program focused on identifying toxicity pathways and developing in vitro assays to characterize the ability of chemicals to perturb those pathways. The goal is to develop new paradigm for high throughput toxicity testing that collects mechanistic and quantitative data from in vitro assays measuring chemical modulation of biological processes involved in the progression to toxicity. As toxicity pathways are identified, the in vitro assays can be optimized for comparison to in vivo animal studies, and for predicting effects in humans. Subsequent computational modeling of toxicity pathway responses and appropriate chemical dosimetry will need to be developed to make these predictions relevant for human health risk assessment. This work was reviewed by EPA and approved for publication but does not necessarily reflect official Agency policy. Index Terms: Toxicogenomics, High Throughput Screening/Testing, EPA ToxCast, Chemical Risk Assessment

Recombinant Microorganisms as Tools for High Throughput Screening for Nonantibiotic Compounds

1997 ◽  
Vol 2 (1) ◽  
pp. 41-49 ◽  
Author(s):  
Ronald D. Klein ◽  
Timothy G. Geary
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Heterologous Protein ◽  
Recombinant Dna ◽  
Biologically Active ◽  
Heterologous Proteins ◽  
Recombinant Dna Technology ◽  
Antibiotic Development ◽  
Recombinant Microorganisms

Microorganisms were among the first tools used for the discovery of biologically active compounds. Their utility reached a zenith during the era of antibiotic development in the 1950s and 1960s, then declined. Subsequently, a substantial role for microorganisms in the pharmaceutical industry developed with the realization that microbial fermentations were intriguing sources of nonantibiotic natural products. From recombinant DNA technology emerged another important role for microorganisms in pharmaceutical research: the expression of heterologous proteins for therapeutic products or for in vitro high throughput screens (HTSs). Recent developments in cloning, genetics, and expression systems have opened up new applications for recombinant microorganisms in screening for nonantibiotic compounds in HTSs. These screens employ microorganisms that depend upon the function of a heterologous protein for survival under defined nutritional conditions. Compounds that specifically target the heterologous protein can be identified by measuring viability of the microorganism under different nutrient selection. Advantages of this approach include a built-in selection for target selectivity, an easily measured end point that can be used for a multitude of different targets, and compatibility with automation required for HTSs. Mechanism-based HTSs using recombinant microorganisms can also address drug targets that are not readily approachable in other HTS formats, including certain enzymes; ion channels and transporters; and protein::protein, protein::DNA, and protein::RNA interactions.

17-SUBSTITUTED STEROIDAL TETRAZOLES – NOVEL LIGANDS FOR HUMAN STEROID-CONVERTING CYP ENZYMES

2021 ◽  
Author(s):  
Suzana S. Jovanović-Šanta ◽  
◽  
Aleksandar M. Oklješa ◽  
Antos B. Sachanka ◽  
Yaraslau U. Dzichenka ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Cytochromes P450 ◽  
Spectral Response ◽  
Spectrophotometric Titration ◽  
Type I ◽  
Cyp Enzymes ◽  
Corticosteroid Hormones ◽  
Compound Type

In animal and human organisms, there are many enzymes, members of the family of heme- containing proteins, cytochromes P450 (CYPs), included in the biosynthesis and metabolism of many biomolecules, as cholesterol, bile acids, sex, and corticosteroid hormones, as well as in metabolism of drugs and xenobiotics. It is also well-known that different imidazole and triazole derivatives are efficient inhibitors of CYPs activity. In this study, we present in vitro screening of binding of novel androstane derivatives with tetrazole- containing substituents in position 17 to human recombinant steroid-converting CYP enzymes: CYP7A1, CYP7B1, CYP17A1, CYP19, and CYP21. Initial screening was performed using a high throughput screening approach, while the affinity of the ligands was analyzed using spectrophotometric titration. For some among tested compounds type I spectral response (substrate-like binding) for CYP7A1 selectively, while for one compound type II spectral response (inhibitor-like binding) for CYP21 were detected, with micromolar values of Kds. Interestingly, one compound with mixed spectral response was found to bind for CYP7B1, which means that there are two optimal positions of the ligand inside the protein active site. Such results could be useful in CYP-inhibiting drug development, during a fast, high-throughput screening of pharmacological potential of novel compounds, as well as in side- effects recognizing.

scholarly journals Comparison of in Vitro Models for the Prediction of Compound Absorption across the Human Intestinal Mucosa

2004 ◽  
Vol 9 (7) ◽  
pp. 598-606 ◽  
Author(s):  
Silvia Miret ◽  
Leo Abrahamse ◽  
Els M. de Groene
Keyword(s):  
Cell Differentiation ◽  
High Throughput ◽  
High Throughput Screening ◽  
In Vitro Models ◽  
Assay System ◽  
In Vitro Assays ◽  
Cell Models ◽  
Artificial Membrane ◽  
Permeability Assay

Several in vitro assays have been developed to evaluate the gastrointestinal absorption of compounds. Our aim was to compare 3 of these methods: 1) the bio-mimetic artificial membrane permeability assay (BAMPA) method, which offers a high-throughput, noncellular approach to the measurement of passive transport; 2) the traditional Caco-2 cell assay, the use of which as a high-throughput tool is limited by the long cell differentiation time (21 days); and 3) The BioCoat™ high-throughput screening Caco-2 Assay System, which reduces Caco-2 cell differentiation to 3 days. The transport of known compounds (such as cephalexin, propranolol, or chlorothiazide) was studied at pH 7.4 and 6.5 in BAMPA and both Caco-2 cell models. Permeability data obtained was correlated to known values of human absorption. Best correlations ( r = 0.9) were obtained at pH 6.5 for BAMPA and at pH 7.4 for the Caco-2 cells grown for 21 days. The Caco-2 BioCoat™ HTS Caco-2 Assay System does not seem to be adequate for the prediction of absorption. The overall results indicate that BAMPA and the 21-day Caco-2 system can be complementary for an accurate prediction of human intestinal absorption.

A Fluorescence-Based High-Throughput Assay for the Identification of Anticancer Reagents Targeting Fructose-1,6-Bisphosphate Aldolase

2017 ◽  
Vol 23 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Eun Jeong Cho ◽  
Ashwini K. Devkota ◽  
Gabriel Stancu ◽  
Ramakrishna Edupunganti ◽  
Garth Powis ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Enzymatic Reaction ◽  
Current Method ◽  
High Rate ◽  
Assay Optimization ◽  
Wide Range ◽  
Probe Assay ◽  
Fructose 1,6 Bisphosphate

A high rate of glycolysis, which supplies energy and materials for anabolism, is observed in a wide range of tumor cells, making it a potential pathway to control cancer growth. ALDOA is a multifunctional enzyme in the glycolytic pathway and also promotes HIF-1α, which is of importance in hypoxic solid tumors. The current method for assaying ALDOA activity involves monitoring the consumption of NADH in vitro using absorbance or intrinsic fluorescence via a coupled enzymatic reaction. Here, we report the development of a homogeneous biochemical assay that can overcome limitations of current methods, in particular for the application of high-throughput drug screening. The assay utilizes the commercially available Elite NADH Assay Kit, which incorporates an enzymatic reaction to measure the level of NADH using a fluorescent probe. Assay optimization and validation are discussed. Its feasibility for high-throughput screening (HTS) was demonstrated by screening 65,000 compounds for the identification of small molecules that inhibit ALDOA. Through a validation screen and dose–response evaluation, four inhibitors with IC50 below 10 µM were identified. In conclusion, we demonstrate that a traditional ALDOA assay can be transformed readily into a fluorescence-based assay utilizing a commercial NADH detection kit that is rapid, sensitive, inexpensive, and HTS friendly.

scholarly journals Development of High-Throughput Screening Assays for Inhibitors of ETS Transcription Factors

2018 ◽  
Vol 24 (1) ◽  
pp. 77-85
Author(s):  
Simon L. Currie ◽  
Steven L. Warner ◽  
Hariprasad Vankayalapati ◽  
Xiaohui Liu ◽  
Sunil Sharma ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Transcription Factors ◽  
High Throughput ◽  
High Throughput Screening ◽  
In Vitro Assays ◽  
Ets Transcription Factors ◽  
Virtual Screen ◽  
Prostate Cells ◽  
Screening Assays

ETS transcription factors from the ERG and ETV1/4/5 subfamilies are overexpressed in the majority of prostate cancer patients and contribute to disease progression. Here, we have developed two in vitro assays for the interaction of ETS transcription factors with DNA that are amenable to high-throughput screening. Using ETS1 as a model, we applied these assays to screen 110 compounds derived from a high-throughput virtual screen. We found that the use of lower-affinity DNA binding sequences, similar to those that ERG and ETV1 bind to in prostate cells, allowed for higher inhibition from many of these test compounds. Further pilot experiments demonstrated that the in vitro assays are robust for ERG, ETV1, and ETV5, three of the ETS transcription factors that are overexpressed in prostate cancer.

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