Identification of Small-Molecule Frequent Hitters from AlphaScreen High-Throughput Screens

Abstract

Although small-molecule drug discovery efforts have focused largely on enzyme, receptor, and ion-channel targets, there has been an increase in such activities to search for protein-protein interaction (PPI) disruptors by applying high-throughout screening (HTS)–compatible protein-binding assays. However, a disadvantage of these assays is that many primary hits are frequent hitters regardless of the PPI being investigated. We have used the AlphaScreen technology to screen four different robust PPI assays each against 25,000 compounds. These activities led to the identification of 137 compounds that demonstrated repeated activity in all PPI assays. These compounds were subsequently evaluated in two AlphaScreen counter assays, leading to classification of compounds that either interfered with the AlphaScreen chemistry (60 compounds) or prevented the binding of the protein His-tag moiety to nickel chelate (Ni2+-NTA) beads of the AlphaScreen detection system (77 compounds). To further triage the 137 frequent hitters, we subsequently confirmed by a time-resolved fluorescence resonance energy transfer assay that most of these compounds were only frequent hitters in AlphaScreen assays. A chemoinformatics analysis of the apparent hits provided details of the compounds that can be flagged as frequent hitters of the AlphaScreen technology, and these data have broad applicability for users of these detection technologies.

A High-Throughput Screen to Identify LRRK2 Kinase Inhibitors for the Treatment of Parkinson’s Disease Using RapidFire Mass Spectrometry

“…AlphaScreen may also suffer from interference from redox reactive compounds, which could disrupt the singlet oxygen transfer on which this technology relies. 21 Overall, the issues described above are likely to result in a number of false positives or nonspecific hits, which will artificially inflate the overall hit rate of any screen for LRRK2. This is a particular issue given that it is a challenge to isolate the required quantity of high-quality recombinant LRRK2 protein, 22 and therefore it is essential that reagent not be wasted in confirming and progressing false actives.…”

Knowledge from Small-Molecule Screening and Profiling Data

“…When molecule libraries and miniaturized assays mix, there is always the potential for false positives due to an unwanted mechanism, often very specific to the assay technology in use. Schorpp et al 14 provide a case study in the identification of frequent hitters for the AlphaScreen technology.…”

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

“…Subsequently, these compounds were analyzed for AlphaScreen-FH and His-FH. 12 Hits positive for AlphaScreen-FH or His-FH were directly excluded. For identification of GST-FH compounds out of the hit list, the mean AlphaScreen signal values were calculated for (1) Hit Set 1 and Hit Set 2 as GST containing screens and for (2) Hit Set 3, Hit Set 4, and Hit Set 5 as non-GST-containing screens.…”

“…In a second criterion, the difference of the means [ (2) was conducted in 10-point titrations. 12 As an additional counter screening strategy for AlphaScreen assays, the compounds were tested in 10-point titrations and incubated with 30 nM Biotin-His peptide for 1 h and detected via streptavidin donor beads and nickel-chelate acceptor beads.…”

“…10,11 Subsequently, efforts have been made to further extend this knowledge, and chemoinformatic filters that identify FHs, which interfere with the AlphaScreen chemistry (AlphaScreen FHs) or with the His-tag/Ni 2+ -NTA interaction (His-tag FHs), have been developed and made publicly available. 12 To establish filters that would specifically detect GST-FH compounds, we analyzed data of two different AlphaScreen HTS campaigns based on the GST/GSH interaction and three independent control screenings, with each employing 25,000 small molecules. From these studies, 53 compounds were verified as being specific GST-FHs in a confirmatory AlphaScreen assay that made use of a GST-His fusion protein.…”