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

Abstract

Redox cycling compounds (RCCs) generate µM concentrations of hydrogen peroxide (H 2 O 2 ) 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. H 2 O 2 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 H 2 O 2 -mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H 2 O 2 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 H 2 O 2 as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds which can generate intracellular H 2 O 2 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; critical 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.

Reconfiguring the AR-TIF2 Protein–Protein Interaction HCS Assay in Prostate Cancer Cells and Characterizing the Hits from a LOPAC Screen

“…[39][40][41]57,58 We will perform chemical classification and clustering of the remaining hits and apply computational filters (PAINS/REOS) to identify and eliminate nuisance compounds and to evaluate their drug-like properties. [39][40][41]57,58 We will evaluate the ADME/Tox properties of the hits, their chemical tractability; perform database, patent, and literature searches to evaluate the intellectual property position; and devise a preliminary synthetic strategy for development. Finally, we will perform similarity and sub-structure searches of the core compound libraries to identify and select structurally related analogs to purchase for potency determinations and to assess the preliminary structure-activity relationship.…”

A Sensitive Luminescent Assay for the Histone Methyltransferase NSD1 and Other SAM-Dependent Enzymes

“…Inspection of the structures in Figure 2 reveals that many of the confirmed hits comprise structural features known to generate hydrogen peroxide in the presence of DTT, thereby irreversibly inactivating cysteine-containing enzymes. [21][22][23] The pilot HTS assay buffer contained DTT, and NSD1 possesses a cysteine-rich region adjacent to the catalytic site that activates the enzyme on binding to DNA. 8 Therefore, it seemed likely that many of the hits were artifacts which interacted with the assay buffer to inactivate NSD1.…”

“…[21][22][23] Three observations suggested that many of the confirmed hits in Table 2 were DTT-reactive ''nuisance compounds'': DTT was present in the pilot HTS assay buffer, several of the structures in Figure 2 include functionality previously found to be problematic, and NSD1 contains a cysteine-rich AWS site that accommodates DNA binding essential for activity. It has been previously shown [21][22][23] that substitution of DTT with less potent reducing agents such as cysteine or BME prevents oxidative enzyme inactivation. Therefore, we tested NSD1 activity in the presence of cysteine or BME in place of DTT.…”

“…This is consistent with the role of catalase as a peroxide scavenger that protects cysteine-containing enzymes from oxidative inactivation. 21 A further 2 compounds, 8 and 9, showed some loss of activity when DTT was replaced with BME or supplemented with catalase, but not to a conclusive degree. Nevertheless, it is likely that these 2 compounds are oxidative inactivators of NSD1, because they have previously been reported to undergo redox cycling in the presence of DTT.…”

Development and Implementation of a High-Throughput High-Content Screening Assay to Identify Inhibitors of Androgen Receptor Nuclear Localization in Castration-Resistant Prostate Cancer Cells

“…Small molecules with promiscuous biological activity identified in a cross target query of the PubChem database often interfere with the HTS assay format 36 or with multiple signaling pathways and therefore were not considered further. Any compound with <95% purity and IC 50 > 10 mM was also eliminated.…”