Discovery of Clonixeril as a Sub-Femtomolar Modulator of the Human STING Receptor.

Stimulator of interferon genes (STING) is a transmembrane endoplasmic reticulum (ER) resident protein involved in innate immunity. STING activation occurs by binding of cyclic guanosine-(2'→5')-monophosphate-adenosine-(3'→5')-monophosphate (2',3'-cGAMP) to STING, which leads to downstream production of type 1 interferons (IFN-1). We generated molecular dynamics (MD) equilibrated agonist and antagonist models of human STING (hSTING) for computer-based screening and now report the discovery of clonixeril (CXL) as the most potent non-nucleotide hSTING modulator discovered to date. We demonstrate in vitro and in cellulo that CXL has two modes of interaction with hSTING, one with an EC₅₀ above 1 nM and the other with an EC₅₀ in the 1 fM-100 aM range (10^-15-10^-16 M). In cell-based experiments, when CXL is titrated below 1 nM, it displays inverse dose-dependent antagonistic behavior toward hSTING. We have substantiated that CXL displays this exceptionally strong inhibitory effect on hSTING mediated IFN-1 production using a THP-1 cell luciferase reporter for interferon regulatory factor 3 (IRF3). Further characterization of CXL was performed in HEK293 cells by using biophysical and biochemical techniques.