Description
A fast and easy way to identify new binders to mouse STING WT.
Stimulator of interferon genes (STING), also known as transmembrane protein 173 (TMEM173), is a protein playing a major role in innate immunity. Upon intracellular cytosolic DNA release from pathogens such as viruses and bacteria, 2’-3’cGAMP binds to STING protein and triggers the secretion of type 1 interferon. Identifying new STING ligands is therefore a way to control immunity and fight autoinflammatory diseases.
HTRF assays offer many advantages over other technologies:
Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
Assay principle
The HTRF STING binding assay is a competitive assay format which uses d2-labeled STING ligand, a 6His tagged mouse STING protein, and an anti 6His Cryptate-labeled antibody. Your compound competes with the STING ligand-d2 and thereby prevents FRET from occurring.
Assay protocol
The Mouse STING binding assay can be run in a 96- or 384-well low volume white plate (20 µL final). As described here, samples or standards are dispensed directly into the assay plate, the mouse His-tagged STING protein is then added followed by the dispensing of the HTRF reagents: the anti 6His antibody labeled with Terbium cryptate and the STING ligand labeled with d2. The reagents labelled with HTRF fluorophores may be pre-mixed and added in a single dispensing step. No washing steps are needed. The protocol can be further miniaturized or upscaled by simply resizing each addition volume proportionally.
Compound screening
Various compounds known to be STING ligands were added to the assay.
2'3'cGAMP displays a high affinity for the mouse protein in the nM range. The other bacterial compounds, cyclic di-AMP and cyclic di-GMP are in the tens nM range.
DMXAA and CMA, two well known compounds specific for mouse STING compete the labelled ligand, with a Ki in good correlation with the litterature.
STING simplified pathway
STING, for STimulator of INterferon Genes, is a cytoplasmic homodimeric protein localized in the endoplasmic reticulum, and which plays an essential role in innate immunity. Upon pathogen infection or mitochrondrial shrinking during apoptosis, floating dsDNAs bind and activate a DNA sensor, cyclic GMP-AMP synthase (cGAS). Activated cGAS leads to the production of 2’-3’cGAMP, a cyclic dinucleotide, which then binds to STING proteins. In turn, phosphorylated STING interacts with TANK-binding-kinase-I (TBK1), leading to the recruitment and activation of the active interferon regulatory factor (IRF3) dimer. Nuclear translocation of the IRF3 dimer leads to the transcription of genes encoding IFN-α/β. In addition, the STING pathway controls NF-κB dependent inflammatory cytokine expression. As a negative feedback loop, the DNA-stimulated cGAS-STING-TBK1 pathway also triggers STING protein degradation through p62 SQSTM1 associated autophagy, to switch off IFNb production.
