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

J Biomol Screen. 2014 Jun;19(5):715-26. doi: 10.1177/1087057113516861. Epub 2013 Dec 26.


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 (Ni(2+)-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.

Keywords: AlphaScreen; assay development; drug discovery; frequent hitter; high-throughput screening; protein-protein interaction.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Automation
  • Biological Assay
  • Drug Discovery / methods*
  • Escherichia coli / metabolism
  • Fluorescence Resonance Energy Transfer
  • High-Throughput Screening Assays / methods*
  • Kinetics
  • Nitrilotriacetic Acid / analogs & derivatives
  • Nitrilotriacetic Acid / chemistry
  • Organometallic Compounds / chemistry
  • Protein Binding
  • Protein Interaction Mapping
  • Recombinant Proteins / chemistry
  • Small Molecule Libraries / chemistry*


  • Organometallic Compounds
  • Recombinant Proteins
  • Small Molecule Libraries
  • nickel nitrilotriacetic acid
  • Nitrilotriacetic Acid