Biophysical System for the Detection and Quantification of Changes in Oligomeric States of STING at Attomolar Concentrations

Few protocols exist today that demonstrate repeatable resolution of small molecule interactions with target proteins at extremely low analyte levels, particularly at sub-femtomolar levels. We have developed two approaches for rapid screening and biophysical analysis which leverage changes in protein oligomer states to study highly potent small molecules. The first protocol employs microscale thermophoresis (MST) to measure competitive disruption of oligomerization following exposure of the target protein to its endogenous ligand. The second protocol engages dynamic light scattering (DLS) to measure the changes in physical size of oligomers after exposure to the endogenous ligand and/or analyte. STING (stimulator of interferon genes) is a protein that relies on oligomerization after ligand binding for intracellular signal transduction. Here we demonstrate the utilization of these methods through measurements of wild type STING oligomerization following exposure to 2’,3’-cGAMP and the compound clonixeril along with several analogs. Using these techniques, one can now measure small molecule inhibitor concentrations in the attomolar range when the target protein undergoes oligomerization as part of its natural biological activity.