Fluorescence-Based TNFR1 Biosensor for Monitoring Receptor Structural and Conformational Dynamics and Discovery of Small Molecule Modulators

Chih Hung Lo, Tory M. Schaaf, David D. Thomas, Jonathan N. Sachs

Research output: Chapter in Book/Report/Conference proceedingChapter

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 languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages121-137
Number of pages17
DOIs
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2248
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Bibliographical note

Funding Information:
We thank Samantha Yuen and Prachi Bawaskar from the Thomas group and Benjamin Grant from Fluorescence Innovations for technical discussions. The pRH132 plasmid was a gift from the Reuben Harris lab at UMN. Flow cytometry and FACS were performed at the UMN Lillehei Heart Institute, confocal fluorescence microscopy was conducted at the UMN Imaging Center, compound dispensing at the UMN Institute of Therapeutics Discovery and Development, and spectroscopy measurements at the UMN Biophysical Technology Center. This study was supported by U.S. NIH grants to J.N.S. (R01 GM107175 and R35 GM131814) and D.D.T. (R01 GM27906, R37 AG26260, R42 DA03762). C.H.L. was supported by a Doctoral Dissertation Fellowship from the UMN.

Keywords

  • NF-κB inhibition
  • Receptor conformational dynamics
  • Receptor–receptor interaction
  • Time-resolved FRET
  • Tumor necrosis factor receptor 1

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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