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

doi:10.1007/978-1-0716-1130-2_9

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 proceeding › Chapter

14 Scopus citations

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)
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press Inc.
Pages	121-137
Number of pages	17
DOIs	https://doi.org/10.1007/978-1-0716-1130-2_9
State	Published - 2021

Publication series

Name	Methods in Molecular Biology
Volume	2248
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
Humans
Molecular Conformation
Structure-Activity Relationship
Computational Biology/methods
Receptors, Tumor Necrosis Factor, Type I/chemistry
Fluorescence Resonance Energy Transfer
Genes, Reporter
Cell Line
Drug Discovery/methods
Gene Expression
Molecular Dynamics Simulation
Small Molecule Libraries
Biosensing Techniques
Fluorescent Antibody Technique
Protein Binding
Ligands
High-Throughput Nucleotide Sequencing
Molecular Docking Simulation
Software
Microscopy, Fluorescence

PubMed: MeSH publication types

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

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Publisher link

10.1007/978-1-0716-1130-2_9

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Cite this

Fluorescence-Based TNFR1 Biosensor for Monitoring Receptor Structural and Conformational Dynamics and Discovery of Small Molecule Modulators. / Lo, Chih Hung; Schaaf, Tory M.; Thomas, David D. et al.
Methods in Molecular Biology. Humana Press Inc., 2021. p. 121-137 (Methods in Molecular Biology; Vol. 2248).

Research output: Chapter in Book/Report/Conference proceeding › Chapter

@inbook{d40b7171507a435ea7afdd930a222bcb,

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

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.",

keywords = "NF-κB inhibition, Receptor conformational dynamics, Receptor–receptor interaction, Time-resolved FRET, Tumor necrosis factor receptor 1, Humans, Molecular Conformation, Structure-Activity Relationship, Computational Biology/methods, Receptors, Tumor Necrosis Factor, Type I/chemistry, Fluorescence Resonance Energy Transfer, Genes, Reporter, Cell Line, Drug Discovery/methods, Gene Expression, Molecular Dynamics Simulation, Small Molecule Libraries, Biosensing Techniques, Fluorescent Antibody Technique, Protein Binding, Ligands, High-Throughput Nucleotide Sequencing, Molecular Docking Simulation, Software, Microscopy, Fluorescence",

author = "Lo, {Chih Hung} and Schaaf, {Tory M.} and Thomas, {David D.} and Sachs, {Jonathan N.}",

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.",

year = "2021",

doi = "10.1007/978-1-0716-1130-2_9",

language = "English (US)",

series = "Methods in Molecular Biology",

publisher = "Humana Press Inc.",

pages = "121--137",

booktitle = "Methods in Molecular Biology",

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TY - CHAP

T1 - Fluorescence-Based TNFR1 Biosensor for Monitoring Receptor Structural and Conformational Dynamics and Discovery of Small Molecule Modulators

AU - Lo, Chih Hung

AU - Schaaf, Tory M.

AU - Thomas, David D.

AU - Sachs, Jonathan N.

N1 - 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.

PY - 2021

Y1 - 2021

N2 - 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.

AB - 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.

KW - NF-κB inhibition

KW - Receptor conformational dynamics

KW - Receptor–receptor interaction

KW - Time-resolved FRET

KW - Tumor necrosis factor receptor 1

KW - Humans

KW - Molecular Conformation

KW - Structure-Activity Relationship

KW - Computational Biology/methods

KW - Receptors, Tumor Necrosis Factor, Type I/chemistry

KW - Fluorescence Resonance Energy Transfer

KW - Genes, Reporter

KW - Cell Line

KW - Drug Discovery/methods

KW - Gene Expression

KW - Molecular Dynamics Simulation

KW - Small Molecule Libraries

KW - Biosensing Techniques

KW - Fluorescent Antibody Technique

KW - Protein Binding

KW - Ligands

KW - High-Throughput Nucleotide Sequencing

KW - Molecular Docking Simulation

KW - Software

KW - Microscopy, Fluorescence

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UR - http://www.scopus.com/inward/citedby.url?scp=85096202865&partnerID=8YFLogxK

U2 - 10.1007/978-1-0716-1130-2_9

DO - 10.1007/978-1-0716-1130-2_9

M3 - Chapter

C2 - 33185872

AN - SCOPUS:85096202865

T3 - Methods in Molecular Biology

SP - 121

EP - 137

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

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

Abstract

Publication series

Bibliographical note

Keywords

PubMed: MeSH publication types

UN SDGs

Publisher link

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Olympus Fluoview FV1000 IX2 Inverted Confocal with FLIM Detector

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

Abstract

Publication series

Bibliographical note

Keywords

PubMed: MeSH publication types

UN SDGs

Publisher link

Find It

Other files and links

Fingerprint

University Assets

Light Microscopy

Olympus Fluoview FV1000 IX2 Inverted Confocal with FLIM Detector

University Imaging Centers

Cite this