Assy principle

The HTRF H232 STING binding assay is a competitive assay format which uses d2-labeled H232 STING ligand, a 6His tagged human H232 STING protein, and an anti 6His Cryptate-labeled antibody. Your compound competes with the H232 STING ligand-d2 and thereby prevents FRET from occurring.

Assay protocol

The Human H232 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 human His-tagged H232 STING protein is then added, followed by the dispensing of the HTRF reagents: the anti 6His antibody labeled with Terbium cryptate and  the H232 STING ligand labeled with d2. The reagents labeled 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 low affinity for the H232 variant in the µM range, compared its high affinity against WT Sting (Ki=2.5nM). Similarly, the other bacterial compounds such as 3'3' cGAMP, cyclic di-AMP, and cyclic d-GMP, or the reference compound ADU-S100, show Ki constants in the µM.

The assay standard corresponding to a derivative of the labeled ligand is more potent, with a Ki at 13.4nM.

DMXAA, a non-specific compound and also a well-known mouse STING binder, has no effect on the assay, confirming the human specificity of the kit.

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.