Development of a high-throughput TR-FRET screening assay for LAG-3/FGL1 interaction

SLAS Discov. 2023 Jun;28(4):188-192. doi: 10.1016/j.slasd.2023.04.003. Epub 2023 Apr 28.

Abstract

Lymphocyte activation gene 3 (LAG-3) is a negative immune checkpoint and a key regulator of immune homeostasis with multiple biological activities related to T-cell functions. Fibrinogen-like protein 1 (FGL1) is a major LAG-3 functional ligand that is upregulated in various human cancers. LAG-3 positive T cells bind FGL1 expressed by cancer cells, which inhibits T-cell activation and cytokine secretion via indirect blocking of T cell receptor (TCR) signaling. High expression of LAG-3 and FGL1 in patients with solid tumors is associated with drug resistance and decreased survival in response to FDA-approved immune checkpoint inhibitors. Therefore, targeting the LAG-3/FGL1 pathway represents a promising therapeutic strategy to maximize the number of patients benefiting from checkpoint blockade therapy. However, there are no small molecules in existence that target LAG-3/FGL1 interaction. Herein, we report a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to evaluate the ability of small molecules to inhibit LAG-3/FGL1 interaction. We further demonstrate the implementation of the developed assay in screening chemical libraries of small molecules from the NCI Diversity Set VII, FDA-approved drugs, and a focused library of NF-κB modulators. This work will pave the way for drug discovery efforts focused on therapeutic targeting of LAG-3/FGL1 interaction using small molecules.

Keywords: Assay development; Cancer immunotherapy; High-throughput screening; Immune checkpoints; TR-FRET.

MeSH terms

  • Drug Discovery
  • Fibrinogen
  • Fluorescence Resonance Energy Transfer*
  • High-Throughput Screening Assays*
  • Humans
  • Lymphocyte Activation
  • Small Molecule Libraries / pharmacology

Substances

  • Small Molecule Libraries
  • FGL1 protein, human
  • Fibrinogen