ALARM NMR for HTS triage and chemical probe validation.
Curr Protoc Chem Biol
; 10(1): 91-117, 2018 03.
Article
in En
| MEDLINE
| ID: mdl-30034947
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Molecular Probes
/
Proteins
/
Nuclear Magnetic Resonance, Biomolecular
/
High-Throughput Screening Assays
Type of study:
Prognostic_studies
Limits:
Humans
Language:
En
Journal:
Curr Protoc Chem Biol
Year:
2018
Document type:
Article
Affiliation country:
Estados Unidos