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Utility of Resazurin, Horseradish Peroxidase, and NMR Assays to Identify Redox-Related False-Positive Behavior in High-Throughput Screens

Tarnowski, Matthew; Barozet, Amélie; Johansson, Carina; Eriksson, Per Olof; Engkvist, Ola; Walsh, Jarrod and Nissink, J. Willem M. (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
publishing date
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
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
2019-09-17 04:31:17
@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},
  keyword      = {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},
  volume       = {16},
  year         = {2018},
}