ALARM NMR for HTS triage and chemical probe validation

Jayme L. Dahlin, Matthew Cuellar, Gurpreet Singh, Kathryn M. Nelson, Jessica Strasser, Todd Rappe, Youlin Xia, Gianluigi Veglia, Michael A. Walters

Research output: Contribution to journalArticle

4 Scopus citations

Abstract

Nonspecific target engagement by test compounds and purported chemical probes is a significant source of assay interference and promiscuous bioactivity in high-throughput screening (HTS) and chemical biology. Most counter-screens for thiol-reactive compounds utilize mass spectrometry or fluorescence detection, and non-proteinaceous reporters like glutathione that may not always approximate the reactivity of protein side-chains. By contrast, a La assay to detect reactive molecules by nuclear magnetic resonance (ALARM NMR) is an industry-developed protein-based [1H-13C]-heteronuclear multiple quantum coherence (HMQC) NMR counter-screen to identify nonspecific protein interactions by test compounds by reporting their tendencies to modulate the human La antigen conformation. This Current Protocol is a users-guide to the production of the 13C-labeled La antigen reporter protein, the reaction of test compounds with this reporter protein, as well as the collection and analysis of characteristic NMR spectra. Combined with other assay interference counter-screens, this assay will enhance chemical biology by helping researchers better prioritize chemical matter and which will increase the number of tractable HTS screening actives and aid in the development of better chemical probes.

Original languageEnglish (US)
Pages (from-to)91-117
Number of pages27
JournalCurrent protocols in chemical biology
Volume10
Issue number1
DOIs
StatePublished - Mar 1 2018

Keywords

  • NMR
  • assay interference
  • bioassay promiscuity
  • chemical biology
  • chemical probes
  • drug discovery
  • high-throughput screening
  • thiol reactivity

Fingerprint Dive into the research topics of 'ALARM NMR for HTS triage and chemical probe validation'. Together they form a unique fingerprint.

  • Cite this