Identification of Small-Molecule Frequent Hitters of Glutathione S-Transferase–Glutathione Interaction

Abstract

In high-throughput screening (HTS) campaigns, the binding of glutathione S-transferase (GST) to glutathione (GSH) is used for detection of GST-tagged proteins in protein-protein interactions or enzyme assays. However, many false-positives, so-called frequent hitters (FH), arise that either prevent GST/GSH interaction or interfere with assay signal generation or detection. To identify GST-FH compounds, we analyzed the data of five independent AlphaScreen-based screening campaigns to classify compounds that inhibit the GST/GSH interaction. We identified 53 compounds affecting GST/GSH binding but not influencing His-tag/Ni 2+ -NTA interaction and general AlphaScreen signals. The structures of these 53 experimentally identified GST-FHs were analyzed in chemoinformatic studies to categorize substructural features that promote interference with GST/GSH binding. Here, we confirmed several existing chemoinformatic filters and more importantly extended them as well as added novel filters that specify compounds with anti-GST/GSH activity. Selected compounds were also tested using different antibody-based GST detection technologies and exhibited no interference clearly demonstrating specificity toward their GST/GSH interaction. Thus, these newly described GST-FH will further contribute to the identification of FH compounds containing promiscuous substructures. The developed filters were uploaded to the OCHEM website (http://ochem.eu) and are publicly accessible for analysis of future HTS results.

Targeting TRAF6 E3 ligase activity with a small-molecule inhibitor combats autoimmunity

“…S1B). After removing AlphaScreen frequent hitters using our own developed chemoinformatics filters (23,24) and by employing strict selection criteria (dose-response and cell-based NF-B activation assays), we were able to select the small molecule C25 as the most promising scaffold for further evaluation (Fig. S1, C-E).…”

Epigenetic assays for chemical biology and drug discovery

“…It is prudent to develop a panel of assays with different readout modes, as these are suitable for the hit validation stage thus allowing confirmation as to whether the activities of compounds translate to more than one assay format thereby adding confidence that they are not assay artefacts [33–36]. This is important as it is now known that assays that make use of specific tagged proteins in the AlphaScreen™ assay format often yield specific interfering compounds as false positive hits [37, 38]. Assay formats that have enhanced the capabilities relative to phenotypic assays include label-free impedance-based [39, 40] dynamic mass redistribution [41, 42] and multiplex assays [43, 44], and these have been successfully applied in screening against small-molecule libraries.…”

“…Colorimetric assays are also particularly prone to optical interference due to coloured compounds which are commonly found in small-molecule libraries. These optically interfering compounds are likely to result in many of these being identified as apparent hits in a small-molecule screening campaign but subsequently shown not to be genuine modulators of the activity of the target protein [34, 37, 38, 67–69]. These false positives need to be identified and removed prior to the progression of compounds for drug discovery purposes.…”

A High-Throughput Screening Strategy for Development of RNF8-Ubc13 Protein–Protein Interaction Inhibitors

“…Distinct AlphaScreen assays with different target proteins but identical protein tags and concentrations allow the identification of molecules that (1) interfere with the AlphaScreen technology, (2) bind to protein tags (GST/ His), or (3) bind to the corresponding affinity matrices (GSH/Ni-NTA). 17,18 We established an OTUB1-Ubc13 PPI assay to uncover unspecific Ubc13 binders. As already shown before, OTUB1 competes for binding of RNF8 to Ubc13 in the AlphaScreen system (Fig.…”