Language
English
Publication Date
6-13-2024
Journal
ACS Publications
DOI
10.1021/acsmedchemlett.4c00104
PMID
38894934
PMCID
PMC11181498
PubMedCentral® Posted Date
5-10-2024
PubMedCentral® Full Text Version
Post-print
Abstract
RIPK1 inhibitors have emerged as promising candidates for treating diverse diseases, including inflammatory diseases, autoimmune disorders, Alzheimer’s disease, and cancer. However, the previously reported binding assays have limited sensitivity and stability, impeding high-throughput screening and robust characterization of the RIPK1 inhibitors. To address this challenge, we introduced two probes, T2-BDP-FL and T3-BDP-FL, derived from distinct RIPK1 inhibitors with different binding modes to establish time-resolved fluorescence resonance energy transfer (TR-FRET) displacement assays. Employing our TR-FRET displacement assays, we quantified the biochemical binding affinities of a series of RIPK1 inhibitors with diverse structural and binding modes for human RIPK1. Consistent results were obtained with these two probes in the TR-FRET displacement assay. Furthermore, we developed a RIPK1 fluorescent probe, T2-BDP589, for the NanoBRET assay. This assay enabled the characterization of RIPK1 target engagement by various RIPK1 inhibitors for both human and mouse RIPK1 in live cells. Our developed fluorescent probe displacement assays offer a sensitive and high-throughput approach to identify RIPK1 inhibitors based on both biochemical and cellular activities.
Keywords
RIPK1, Fluorescent probe, TR-FRET, NanoBRET, Target engagement
Published Open-Access
yes
Recommended Citation
Yu, Xin; Lin, Hanfeng; Li, Feng; et al., "Development of Biochemical and Cellular Probes to Study RIPK1 Target Engagement" (2024). Faculty and Staff Publications. 3861.
https://digitalcommons.library.tmc.edu/baylor_docs/3861
Graphical Abstract