An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors

Chih Hung Lo, Nagamani Vunnam, Andrew K Lewis, Ting Lan Chiu, Benjamin E. Brummel, Tory M. Schaaf, Benjamin D. Grant, Prachi Bawaskar, David D Thomas, Jonathan N Sachs

Research output: Contribution to journalArticlepeer-review

15 Scopus citations

Abstract

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor–receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

Original languageEnglish (US)
Pages (from-to)950-961
Number of pages12
JournalSLAS Discovery
Volume22
Issue number8
DOIs
StatePublished - Sep 1 2017

Bibliographical note

Funding Information:
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by U.S. National Institutes of Health (NIH) grants to J.N.S. (R01 GM107175) and D.D.T. (R42 DA037622, subcontract from grant to Fluorescence Innovations, Gillispie PI).

Funding Information:
We thank Ji Li and Samantha Yuen from the Thomas group, and Gregory D. Gillispie and Kurt C. Peterson from Fluorescence Innovations, Inc., for technical discussions. Fluorescence microscopy was performed at the UMN Imaging Center, flow cytometry at the UMN Lillehei Heart Institute, compound dispensing at the UMN Institute of Therapeutic Drug Discovery and Development, and spectroscopy at the UMN Biophysical Technology Center. This work was carried out in part using computing resources at the University of Minnesota Supercomputing Institute.

Keywords

  • NF-κB inhibition
  • pre-ligand assembly domain
  • receptor–receptor interaction
  • time-resolved FRET
  • tumor necrosis factor receptor 1

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