Journal
SLAS DISCOVERY
Volume 26, Issue 1, Pages 44-57Publisher
ELSEVIER SCIENCE INC
DOI: 10.1177/2472555220959266
Keywords
binding assay; MALDI-TOF; mass spectrometry; high-throughput screening; PTP1B
Ask authors/readers for more resources
This study introduces a ligand binding assay platform that combines automated affinity selection (AS) sample preparation and label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis, allowing for mass spectrometry-based high-throughput screening of small-molecule target interactions. The platform is capable of identifying orthosteric and allosteric ligands, enabling efficient separation of target-ligand comp
Demonstration of in vitro target engagement for small-molecule ligands by measuring binding to a molecular target is an established approach in early drug discovery and a pivotal step in high-throughput screening (HTS)-based compound triaging. We describe the setup, evaluation, and application of a ligand binding assay platform combining automated affinity selection (AS)-based sample preparation and label-free matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis. The platform enables mass spectrometry (MS)-based HTS for small-molecule target interactions from single-compound incubation mixtures and is embedded into a regular assay automation environment. Efficient separation of target-ligand complexes is achieved by in-plate size exclusion chromatography (SEC), and small-molecule ligands are subsequently identified by MALDI-TOF analysis. In contrast to alternative HTS-capable binding assay formats, MALDI-TOF AS-MS is capable of identifying orthosteric and allosteric ligands, as shown for the model system protein tyrosine phosphatase 1B (PTP1B), irrespective of protein function. Furthermore, determining relative binding affinities (RBAs) enabled ligand ranking in accordance with functional inhibition and reference data for PTP1B and a number of diverse protein targets. Finally, we present a validation screen of more than 23,000 compounds within 24 h, demonstrating the general applicability of the platform for the HTS-compatible assessment of protein-ligand interactions.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available