Utility of Resazurin, Horseradish Peroxidase, and NMR Assays to Identify Redox-Related False-Positive Behavior in High-Throughput Screens
(2018) In Assay and Drug Development Technologies 16(3). p.177-191- Abstract
Discerning false positives from true actives in high-throughput screening (HTS) output is fraught with difficulty as the reason of anomalous activity seen for compounds is often not clear-cut. In this study, we introduce a novel medium-throughput NMR assay for the identification of redox-cycling compounds (RCCs), which is based on detection of oxidation of a reducing agent. We compare its outcomes to those from horseradish peroxidase (HRP)/phenol red and resazurin (RZ)-based assays that are more commonly used for triaging HTS outputs. Data from NMR, RZ, and HRP redox assay are shown to correlate, with the NMR assay showing the greatest accuracy. In addition, historical data analysis was used to identify compounds frequently active in... (More)
Discerning false positives from true actives in high-throughput screening (HTS) output is fraught with difficulty as the reason of anomalous activity seen for compounds is often not clear-cut. In this study, we introduce a novel medium-throughput NMR assay for the identification of redox-cycling compounds (RCCs), which is based on detection of oxidation of a reducing agent. We compare its outcomes to those from horseradish peroxidase (HRP)/phenol red and resazurin (RZ)-based assays that are more commonly used for triaging HTS outputs. Data from NMR, RZ, and HRP redox assay are shown to correlate, with the NMR assay showing the greatest accuracy. In addition, historical data analysis was used to identify compounds frequently active in assays for redox-susceptible targets. We provide examples of compound classes found and conclude that the NMR redox assay offers a novel and reliable way of identifying RCCs at a medium throughput. The HRP and RZ assays are reasonable higher-throughput alternatives, with both showing similar sensitivity to redox-cycling and false-positive compounds. The RZ assay has a higher hit rate, reflecting its ability to pick up multiple modes of action.
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- author
- Tarnowski, Matthew ; Barozet, Amélie ; Johansson, Carina ; Eriksson, Per Olof ; Engkvist, Ola ; Walsh, Jarrod and Nissink, J. Willem M.
- publishing date
- 2018-04-01
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- horse-radish peroxidase/phenol red assay, HTS output deconvolution, NMR, redox false positives, resazurin-based redox assay
- in
- Assay and Drug Development Technologies
- volume
- 16
- issue
- 3
- pages
- 15 pages
- publisher
- Mary Ann Liebert, Inc.
- external identifiers
-
- scopus:85045545758
- pmid:29608094
- ISSN
- 1540-658X
- DOI
- 10.1089/adt.2017.838
- language
- English
- LU publication?
- no
- id
- fd5d1436-2ae4-4fe8-9178-380de3903e3c
- date added to LUP
- 2018-04-27 11:53:17
- date last changed
- 2024-04-01 05:07:55
@article{fd5d1436-2ae4-4fe8-9178-380de3903e3c,
abstract = {{<p>Discerning false positives from true actives in high-throughput screening (HTS) output is fraught with difficulty as the reason of anomalous activity seen for compounds is often not clear-cut. In this study, we introduce a novel medium-throughput NMR assay for the identification of redox-cycling compounds (RCCs), which is based on detection of oxidation of a reducing agent. We compare its outcomes to those from horseradish peroxidase (HRP)/phenol red and resazurin (RZ)-based assays that are more commonly used for triaging HTS outputs. Data from NMR, RZ, and HRP redox assay are shown to correlate, with the NMR assay showing the greatest accuracy. In addition, historical data analysis was used to identify compounds frequently active in assays for redox-susceptible targets. We provide examples of compound classes found and conclude that the NMR redox assay offers a novel and reliable way of identifying RCCs at a medium throughput. The HRP and RZ assays are reasonable higher-throughput alternatives, with both showing similar sensitivity to redox-cycling and false-positive compounds. The RZ assay has a higher hit rate, reflecting its ability to pick up multiple modes of action.</p>}},
author = {{Tarnowski, Matthew and Barozet, Amélie and Johansson, Carina and Eriksson, Per Olof and Engkvist, Ola and Walsh, Jarrod and Nissink, J. Willem M.}},
issn = {{1540-658X}},
keywords = {{horse-radish peroxidase/phenol red assay; HTS output deconvolution; NMR; redox false positives; resazurin-based redox assay}},
language = {{eng}},
month = {{04}},
number = {{3}},
pages = {{177--191}},
publisher = {{Mary Ann Liebert, Inc.}},
series = {{Assay and Drug Development Technologies}},
title = {{Utility of Resazurin, Horseradish Peroxidase, and NMR Assays to Identify Redox-Related False-Positive Behavior in High-Throughput Screens}},
url = {{http://dx.doi.org/10.1089/adt.2017.838}},
doi = {{10.1089/adt.2017.838}},
volume = {{16}},
year = {{2018}},
}