Abstract
Inhibition of tumor necrosis factor receptor 1 (TNFR1) is a billion-dollar industry for treatment of autoimmune and inflammatory diseases. As current therapeutics of anti-TNF leads to dangerous side effects due to global inhibition of the ligand, receptor-specific inhibition of TNFR1 signaling is an intensely pursued strategy. To monitor directly the structural changes of the receptor in living cells, we engineered a fluorescence resonance energy transfer (FRET) biosensor by fusing green and red fluorescent proteins to TNFR1. Expression of the FRET biosensor in living cells allows for detection of receptor–receptor interactions and receptor structural dynamics. Using the TNFR1 FRET biosensor, in conjunction with a high-precision and high-throughput fluorescence lifetime detection technology, we developed a time-resolved FRET-based high-throughput screening platform to discover small molecules that directly target and modulate TNFR1 functions. Using this method in screening multiple pharmaceutical libraries, we have discovered a competitive inhibitor that disrupts receptor–receptor interactions, and allosteric modulators that alter the structural states of the receptor. This enables scientists to conduct high-throughput screening through a biophysical approach, with relevance to compound perturbation of receptor structure, for the discovery of novel lead compounds with high specificity for modulation of TNFR1 signaling.
Original language | English (US) |
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Title of host publication | Methods in Molecular Biology |
Publisher | Humana Press Inc. |
Pages | 121-137 |
Number of pages | 17 |
DOIs | |
State | Published - 2021 |
Publication series
Name | Methods in Molecular Biology |
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Volume | 2248 |
ISSN (Print) | 1064-3745 |
ISSN (Electronic) | 1940-6029 |
Bibliographical note
Publisher Copyright:© 2020, Springer Science+Business Media, LLC, part of Springer Nature.
Keywords
- NF-κB inhibition
- Receptor conformational dynamics
- Receptor–receptor interaction
- Time-resolved FRET
- Tumor necrosis factor receptor 1