Establishment of inhibitor screening and validation system for tryptophanyl tRNA synthetase using surface plasmon resonance.
Anal Biochem
; 623: 114183, 2021 06 15.
Article
in En
| MEDLINE
| ID: mdl-33798474
ABSTRACT
With the increase in throughput and sensitivity, biophysical technology has become a major component of the early drug discovery phase. Surface plasmon resonance technology (SPR) is one of the most widely used biophysical technologies. It has the advantages of circumventing labeling, molecular weight limitations, and neglect of low affinity interactions, etc., and provides a robust platform for hit to lead discovery and optimization. Here, we successfully established a reliable and repeatable tryptophanyl tRNA synthetase (TrpRS) SPR high-throughput screening and validation system by optimizing the TrpRS tag, TrpRS immobilization methodology, and the buffer conditions. When TrpRS was immobilized on Streptavidin (SA) sensor chip, the substrate competitive inhibitor indolmycin exhibited the best binding affinity in HBS-P (10 mM HEPES, 150 mM NaCl, 0.05% surfactant P-20, pH 7.4), 1 mM ATP and MgCl2, with a KD (dissociation equilibrium constant) value of 0.6 ± 0.1 µM. The Z-factor values determined in the screening assays were all larger than 0.9. We hope that our proposed research ideas and methods may provide a scientific basis for establishing SPR analysis of other drug targets, accelerate the discovery and optimization of target lead compounds, and assist the clinical application of next-generation drugs.
Key words
Full text:
1
Database:
MEDLINE
Main subject:
Tryptophan-tRNA Ligase
/
Surface Plasmon Resonance
/
Enzyme Inhibitors
/
High-Throughput Screening Assays
Type of study:
Diagnostic_studies
/
Screening_studies
Language:
En
Journal:
Anal Biochem
Year:
2021
Type:
Article