Biophysical Methods for Attomolar Detection of Compounds Acting on Protein Oligomer States

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 drug candidates. 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 endogenous ligand and/or analyte. We demonstrate the utilization of these methods through measurements of stimulator of interferon genes (STING) exposure to 2’,3’-cGAMP and the drug candidate clonixeril along with several analog compounds that were created for lead optimization.