Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents—real hits or promiscuous artifacts?

doi:10.1016/j.cbpa.2010.10.022

Current Opinion in Chemical Biology

Volume 15, Issue 1, February 2011, Pages 174-182

https://doi.org/10.1016/j.cbpa.2010.10.022 Get rights and content

Redox cycling compounds (RCCs) generate μM concentrations of hydrogen peroxide (H₂O₂) in the presence of strong reducing agents, common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays. H₂O₂ generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine, or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H₂O₂-mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H₂O₂ in cells are the Nox enzyme/SOD systems, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling at both the microsomal and mitochondrial sites of electron transport. Given the role of H₂O₂ as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds that can generate intracellular H₂O₂ by enzyme mediated redox cycling would have pleiotropic effects. RCCs can therefore have serious negative consequences for the probe and/or lead generation process: primary HTS assay hit rates may be inflated by RCC false positives; crucial resources will be diverted to develop and implement follow up assays to distinguish RCCs from real hits; and screening databases will become annotated with the promiscuous activity of RCCs. In an attempt to mitigate the serious impact of RCCs on probe and lead generation, two groups have independently developed assays to indentify RCCs.

Introduction

In the pursuit of small molecules that modulate the activity of isolated protein targets or cellular phenotypes the selection of the screening assay format is arguably one of the most crucial factors that influences whether a chemical biology endeavor will be successful [1, 2]. The pairing of a particular target or cellular phenotype with an assay technology not only requires the development, optimization, and validation of an assay that is biologically appropriate, but also one that provides a robust and reproducible signal window with sufficient throughput and capacity to screen the compound diversity of interest [1, 2, 3, 4]. The potential for compound interference with an assay format must also be considered, especially since it typically requires the integration of orthogonal counter screens into the follow up paradigm to identify and eliminate false positives [1, 2, 5••, 6••]. Assay interference can be attributed to both chemical and physical compound properties [1, 5••, 6••, 7, 8, 9, 10••]. Compounds that are colored, fluorescent, or that form aggregates have the potential to promiscuously interfere with a wide variety of biochemical and cell based assay formats [1, 2, 5••, 6••, 11]. Alternatively, some inhibitors of the firefly luciferase (FLuc) enzyme that stabilize intracellular FLuc can produce a gain in signal in cell based luciferase reporter assays that may be misconstrued as transcriptional activation, and represent a more restricted example of assay format interference [6••, 7, 12]. Redox cycling compounds (RCCs) generate hydrogen peroxide (H₂O₂) in the presence of strong reducing agents like dithiothreitol (DTT) (Figure 1a) or tris(2-carboxyethyl)phosphine (TCEP), common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays [8, 9, 10••, 13, 14]. H₂O₂ generated by RCCs in HTS assay buffers containing DTT/TCEP can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine, or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H₂O₂-mediated inactivation; protein tyrosine phosphatases (PTPs), cysteine proteases (cathepsins and caspases), and metalloenzymes (Figure 1a) [8, 9, 10••, 13, 14]. The current review focuses on RCCs as nuisance/pan assay interference compounds with the potential to annotate screening databases with promiscuous bioactivity profiles (Figure 1b) [6••, 8, 9, 10••].

Section snippets

False positive hits due to redox cycling compounds

A HTS of >700,000 compounds to identify inhibitors of caspase-8 produced a high hit rate (∼1%), and it was found that 85% of the inhibitors from the initial set of 20,000 compounds were RCCs that inhibited the enzyme through the generation of H₂O₂ in the presence of DTT [14]. 97% of the actives in a HTS campaign to identify activators of glucokinase (GK) activity were found to be nuisance RCCs that interfered with the resazurin coupled assay format independently of the GK enzyme [9]. A

Functional characteristics of redox cycling compounds

Several hallmark characteristics have been utilized to distinguish the behavior of RCCs from hit compounds that modulate target activities directly [8, 13, 14, 16•, 17, 18]. The degree of inhibition of the target protein activity by the RCC increases over time, consistent with the time dependent redox cycling generation of H₂O₂. The inhibition of the target protein activity by the RCC can be abolished by the addition of catalase (CAT) to the assay to degrade any H₂O₂ produced. RCCs can inhibit

Non-enzymatic redox cycling generation of H₂O₂

A reaction scheme for the non-enzymatic generation of H₂O₂ by redox cycling between a quinone RCC and DTT in aqueous solutions containing oxygen has been proposed (Figure 2) [8, 10••, 13]. (1) DTT reacts with the quinone RCC to form dihydroxydithiane (ox-DTT) and a hydroquinone. (2) When the hydroquinone and quinone RCC are present together they undergo a synproportionation to form a transient semiquinone radical anion species (RCC radical dot ⁻) and 2H⁺. (3) The semiquinone radical anion reacts with O₂ to

H₂O₂ and signal transduction

Of all the reactive oxygen species produced by cells, H₂O₂ best meets the criteria of an intracellular second messenger involved in the transduction of external signals [20••, 21••, 22, 23]. H₂O₂ is a small molecule that diffuses rapidly, can cross membranes, and is rapidly synthesized and destroyed in cells in response to external stimuli [20••, 21••, 22, 23, 24•]. Many cell types generate low levels of H₂O₂ in response to a variety of extracellular stimuli including; cytokines (TNF-α, IL-1,

HTS assays to identify redox cycling compounds

Until recently, the process to identify and eliminate RCCs from HTS hit lists involved the development and implementation of multiple counter screens and secondary assays: (i) performing a detailed enzyme kinetic analysis to verify the time-dependent and concentration-dependent inhibition of target activity by RCCs in the presence of DTT or TCEP; (ii) testing whether CAT abolishes the target inhibition by RCCs in DTT or TCEP; (iii) investigating RCC inhibition in the presence of weaker reducing

Activities of redox cycling compounds in cells

The main sources of H₂O₂ in cells are the Nox enzyme/SOD systems described above, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling [20••, 21••, 23, 38, 39, 40]. Within the cellular environment and in the presence of molecular oxygen and metal ions, phenols, catechols, and hydoquinones are converted to quinones by intracellular monoxygenase and peroxidase enzymes [38]. In cells, quinones undergo enzymatic redox cycling at both the microsomal and

Conclusions

To illustrate the complexities and challenges that RCCs represent to the chemical biology approach, a recent case history is discussed [10••, 41, 42, 43]. Seven pyrimidotriazinedione hits, structurally related to the RCCs presented here (Figure 1, Figure 4) or that have been characterized previously [8, 9, 10••, 14], were identified in an HTS campaign to identify small molecules that disrupt the interaction between the C-terminal peptide of heat shock protein 90 (Hsp90) and the TPR2A domain of

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest
•• of outstanding interest

References (43)

P.A. Johnston et al.
Cellular platforms for HTS: three case studies
Drug Discov Today
(2002)
C. Brideau et al.
Improved statistical methods for hit selection in high-throughput screening
J Biomol Screen
(2003)
D.S. Auld et al.
Characterization of chemical libraries for luciferase inhibitory activity
J Med Chem
(2008)
A. Simeonov et al.
Fluorescence spectroscopic profiling of compound libraries
J Med Chem
(2008)
P. Johnston et al.
Development and implementation of a 384-well homogeneous fluorescence intensity high-throughput screening assay to identify mitogen-activated protein kinase phosphatase-1 dual-specificity protein phosphatase inhibitors
Assay Drug Dev Technol
(2007)
P.A.F.C. Johnston et al.
Characterization of the Cdc25B dual specificity phosphatase inhibitor hits identified in a high throughput screen of the NIH compound library
Assay Drug Dev Technol
(2009)
M. Brisson et al.
Redox regulation of Cdc25B by cell-active quinolinediones
Mol Pharmacol
(2005)
E. Miller et al.
Aquaporin-3 mediates hydrogen peroxide uptake to regulate downstream intracellular signaling
Proc Natl Acad Sci USA
(2010)
S.R.Y.K. Lee et al.
Reversible inactivation of the tumor suppressor PTEN by H2O2
J Biol Chem
(2002)
M. Bembenek et al.
A fluorescence-based coupling reaction for monitoring the activity of recombinant human NAD synthetase
Assay Drug Dev Technol
(2005)

M. Goetz et al.

Reactive species: a cell damaging rout assisting to chemical carcinogens

Cancer Lett

(2008)

D. Auld et al.

Literature search and review

Assay Drug Dev Technol

(2008)

J. Inglese et al.

High-throughput screening assays for the identification of chemical probes

Nat Chem Biol

(2007)

N. Malo et al.

Statistical practice in high-throughput screening data analysis

Nat Biotechnol

(2006)

J. Baell et al.

New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays

J Med Chem

(2010)

N. Thorne et al.

Apparent activity in high-throughput screening: origins of compound-dependent assay interference

Curr Opin Chem Biol

(2010)

P. Johnston et al.

Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents

Assay Drug Dev Technol

(2008)

L. Lor et al.

A simple assay for detection of small-molecule redox activity

J Biomol Screen

(2007)

K. Soares et al.

Profiling the NIH small molecule repository for compounds that generate H(2)O(2) by redox cycling in reducing environments

Assay Drug Dev Technol

(2010)

N. Thorne et al.

Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology

Chem Biol

(2010)

M.P. Bova et al.

The oxidative mechanism of action of ortho-quinone inhibitors of protein-tyrosine phosphatase [alpha] is mediated by hydrogen peroxide

Arch Biochem Biophys

(2004)

Cited by (68)

Reversible and irreversible inhibitors of coronavirus Nsp15 endoribonuclease
2023, Journal of Biological Chemistry
The emergence of severe acute respiratory syndrome coronavirus 2, the causative agent of coronavirus disease 2019, has resulted in the largest pandemic in recent history. Current therapeutic strategies to mitigate this disease have focused on the development of vaccines and on drugs that inhibit the viral 3CL protease or RNA-dependent RNA polymerase enzymes. A less-explored and potentially complementary drug target is Nsp15, a uracil-specific RNA endonuclease that shields coronaviruses and other nidoviruses from mammalian innate immune defenses. Here, we perform a high-throughput screen of over 100,000 small molecules to identify Nsp15 inhibitors. We characterize the potency, mechanism, selectivity, and predicted binding mode of five lead compounds. We show that one of these, IPA-3, is an irreversible inhibitor that might act via covalent modification of Cys residues within Nsp15. Moreover, we demonstrate that three of these inhibitors (hexachlorophene, IPA-3, and CID5675221) block severe acute respiratory syndrome coronavirus 2 replication in cells at subtoxic doses. This study provides a pipeline for the identification of Nsp15 inhibitors and pinpoints lead compounds for further development against coronavirus disease 2019 and related coronavirus infections.
Characterization of allosteric modulators that disrupt androgen receptor co-activator protein-protein interactions to alter transactivation–Drug leads for metastatic castration resistant prostate cancer
2023, SLAS Discovery
Three series of compounds were prioritized from a high content screening campaign that identified molecules that blocked dihydrotestosterone (DHT) induced formation of Androgen Receptor (AR) protein-protein interactions (PPIs) with the Transcriptional Intermediary Factor 2 (TIF2) coactivator and also disrupted preformed AR-TIF2 PPI complexes; the hydrobenzo-oxazepins (S1), thiadiazol-5-piperidine-carboxamides (S2), and phenyl-methyl-indoles (S3). Compounds from these series inhibited AR PPIs with TIF2 and SRC-1, another p160 coactivator, in mammalian 2-hybrid assays and blocked transcriptional activation in reporter assays driven by full length AR or AR-V7 splice variants. Compounds inhibited the growth of five prostate cancer cell lines, with many exhibiting differential cytotoxicity towards AR positive cell lines. Representative compounds from the 3 series substantially reduced both endogenous and DHT-enhanced expression and secretion of the prostate specific antigen (PSA) cancer biomarker in the C4–2 castration resistant prostate cancer (CRPC) cell line. The comparatively weak activities of series compounds in the H³-DHT and/or TIF2 box 3 LXXLL-peptide binding assays to the recombinant ligand binding domain of AR suggest that direct antagonism at the orthosteric ligand binding site or AF-2 surface respectively are unlikely mechanisms of action. Cellular enhanced thermal stability assays (CETSA) indicated that compounds engaged AR and reduced the maximum efficacy and right shifted the EC₅₀ of DHT-enhanced AR thermal stabilization consistent with the effects of negative allosteric modulators. Molecular docking of potent representative hits from each series to AR structures suggest that S1–1 and S2–6 engage a novel binding pocket (BP-1) adjacent to the orthosteric ligand binding site, while S3–11 occupies the AR binding function 3 (BF-3) allosteric pocket. Hit binding poses indicate spaces and residues adjacent to the BP-1 and BF-3 pockets that will be exploited in future medicinal chemistry optimization studies. Small molecule allosteric modulators that prevent/disrupt AR PPIs with coactivators like TIF2 to alter transcriptional activation in the presence of orthosteric agonists might evade the resistance mechanisms to existing prostate cancer drugs and provide novel starting points for medicinal chemistry lead optimization and future development into therapies for metastatic CRPC.
Monobody Inhibitor Selective to the Phosphatase Domain of SHP2 and its Use as a Probe for Quantifying SHP2 Allosteric Regulation: Monobody inhibitor selective to SHP2 PTP
2023, Journal of Molecular Biology
SHP2 is a phosphatase/adaptor protein that plays an important role in various signaling pathways. Its mutations are associated with cancers and developmental diseases. SHP2 contains a protein tyrosine phosphatase (PTP) and two SH2 domains. Selective inhibition of these domains has been challenging due to the multitude of homologous proteins in the proteome. Here, we developed a monobody, synthetic binding protein, that bound to and inhibited the SHP2 PTP domain. It was selective to SHP2 PTP over close homologs. A crystal structure of the monobody-PTP complex revealed that the monobody bound both highly conserved residues in the active site and less conserved residues in the periphery, rationalizing its high selectivity. Its epitope overlapped with the interface between the PTP and N-terminal SH2 domains that is formed in auto-inhibited SHP2. By using the monobody as a probe for the accessibility of the PTP active site, we developed a simple, nonenzymatic assay for the allosteric regulation of SHP2. The assay showed that, in the absence of an activating phospho-Tyr ligand, wild-type SHP2 and the “PTP-dead” C459E mutant were predominantly in the closed state in which the PTP active site is inaccessible, whereas the E76K and C459S mutants were in the open, active state. It also revealed that previously developed monobodies to the SH2 domains, ligands lacking a phospho-Tyr, weakly favored the open state. These results provide corroboration for a conformational equilibrium underlying allosteric regulation of SHP2, provide powerful tools for characterizing and controlling SHP2 functions, and inform drug discovery against SHP2.
Utilising acoustic mist ionisation mass spectrometry to identify redox cycling compounds in high throughput screening outputs
2022, SLAS Discovery
Citation Excerpt :
These can lead to oxidation of key residues, such as an active site cysteine, impairing protein activity and therefore manifesting as hits [4–6]. Previous HTS outputs, such as for the cysteine protease Caspase-8 [7], have demonstrated the requirement for identifying RCCs early in a screening cascade [4,7,8]. A number of high-throughput assays are currently available for detecting redox cyclers [1,4,6], including a colorimetric horse radish peroxide based assay [9] and a fluorescent resazurin based assay [10].
Rapid triage of compounds acting via undesired mechanisms is a crucial stage in a high-throughput screening (HTS) cascade to ensure time and resource is efficiently assigned to the most propitious hits. Redox cycling compounds (RCCs) produce reactive oxygen species, such as hydrogen peroxide, which can impair protein function and appear as hits against liable targets. Direct measurement of tris(2-carboxyethyl)phosphine (TCEP) oxidation has been demonstrated as a sensitive and accurate measure of redox cycling [1]. However, the current nuclear magnetic resonance (NMR) based detection method is not compatible with the throughput required for triage of a HTS campaign. Here we employ Acoustic Mist Ionisation Mass Spectrometry (AMI-MS) [2] to rapidly measure oxidation of TCEP and accurately identify redox cyclers in a high throughput manner.
Redox active or thiol reactive? Optimization of rapid screens to identify less evident nuisance compounds
2022, Drug Discovery Today
Compounds that exhibit assay interference or undesirable mechanisms of bioactivity are routinely encountered in assays at various stages of drug discovery. We observed that assays for the investigation of thiol-reactive and redox-active compounds have not been collected in a comprehensive review. Here, we review these assays and subject them to experimental optimization to improve their reliability. We demonstrate the usefulness of our assay cascade by assaying a library of bioactive compounds, chemical probes, and a set of approved drugs. These high-throughput assays should complement the array of wet-lab and in silico assays during the initial stages of hit discovery campaigns to pursue only hit compounds with tractable mechanisms of action.
Gains from no real PAINS: Where ‘Fair Trial Strategy’ stands in the development of multi-target ligands
2021, Acta Pharmaceutica Sinica B
Citation Excerpt :
Pyrimidotriazinedione is one of the active dominant scaffolds commonly used in drug design that was unfortunately identified as an RCC in the presence of 0.8 mmol/L DTT and has been promiscuously active against a number of target proteins23. More than 50% of RCCs identified in the RCC profiling screen are structurally similar to pyrimidotriazinedione and quinones; however, several other RCC pharmacophores were identified114. H2O2 is the common cellular messenger; hence, RCC interference is not limited to biochemical enzyme-based assays and can also produce promiscuous effects in cell-based analysis23.
Compounds that selectively modulate multiple targets can provide clinical benefits and are an alternative to traditional highly selective agents for unique targets. High-throughput screening (HTS) for multitarget-directed ligands (MTDLs) using approved drugs, and fragment-based drug design has become a regular strategy to achieve an ideal multitarget combination. However, the unexpected presence of pan-assay interference compounds (PAINS) suspects in the development of MTDLs frequently results in nonspecific interactions or other undesirable effects leading to artefacts or false-positive data of biological assays. Publicly available filters can help to identify PAINS suspects; however, these filters cannot comprehensively conclude whether these suspects are “bad” or innocent. Additionally, these in silico approaches may inappropriately label a ligand as PAINS. More than 80% of the initial hits can be identified as PAINS by the filters if appropriate biochemical tests are not used resulting in false positive data that are unacceptable for medicinal chemists in manuscript peer review and future studies. Therefore, extensive offline experiments should be used after online filtering to discriminate “bad” PAINS and avoid incorrect evaluation of good scaffolds. We suggest that the use of “Fair Trial Strategy” to identify interesting molecules in PAINS suspects to provide certain structure‒function insight in MTDL development.

View all citing articles on Scopus

View full text

Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents—real hits or promiscuous artifacts?

Introduction

Section snippets

False positive hits due to redox cycling compounds

Functional characteristics of redox cycling compounds

Non-enzymatic redox cycling generation of H2O2

H2O2 and signal transduction

HTS assays to identify redox cycling compounds

Activities of redox cycling compounds in cells

Conclusions

References and recommended reading

Drug Discov Today

J Biomol Screen

J Med Chem

J Med Chem

Assay Drug Dev Technol

Assay Drug Dev Technol

Mol Pharmacol

Proc Natl Acad Sci USA

J Biol Chem

Assay Drug Dev Technol

Cancer Lett

Assay Drug Dev Technol

High-throughput screening assays for the identification of chemical probes

Nat Chem Biol

Statistical practice in high-throughput screening data analysis

Nat Biotechnol

New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays

J Med Chem

Apparent activity in high-throughput screening: origins of compound-dependent assay interference

Curr Opin Chem Biol

Development of a 384-well colorimetric assay to quantify hydrogen peroxide generated by the redox cycling of compounds in the presence of reducing agents

Assay Drug Dev Technol

A simple assay for detection of small-molecule redox activity

J Biomol Screen

Profiling the NIH small molecule repository for compounds that generate H(2)O(2) by redox cycling in reducing environments

Assay Drug Dev Technol

Illuminating insights into firefly luciferase and other bioluminescent reporters used in chemical biology

Chem Biol

The oxidative mechanism of action of ortho-quinone inhibitors of protein-tyrosine phosphatase [alpha] is mediated by hydrogen peroxide

Arch Biochem Biophys

Redox cycling compounds generate H₂O₂ in HTS buffers containing strong reducing reagents—real hits or promiscuous artifacts?

Non-enzymatic redox cycling generation of H₂O₂

H₂O₂ and signal transduction