A non-nucleotide agonist that binds covalently to cysteine residues of STING.

Stimulator of interferon genes (STING) is an ER-localized transmembrane protein and the receptor for 2',3'-cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which is a second messenger produced by cGAMP synthase (cGAS), a cytosolic double-stranded DNA sensor. The cGAS-STING pathway plays a critical role in the innate immune response to infection of a variety of DNA pathogens through the induction of the type I interferons. Pharmacological activation of STING is a promising therapeutic strategy for cancer, thus the development of potent and selective STING agonists has been pursued. Here we report that mouse STING can be activated by phenylarsine oxide (PAO), a membrane permeable trivalent arsenic compound that preferentially reacts with thiol group of cysteine residue (Cys). The activation of STING with PAO does not require cGAS or cGAMP. Mass spectrometric analysis of the peptides generated by trypsin and chymotrypsin digestion of STING identifies several PAO adducts, suggesting that PAO covalently binds to STING. Screening of STING variants with single Cys to serine residues (Ser) reveals that Cys88 and Cys291 are critical to the response to PAO. STING activation with PAO, as with cGAMP, requires the ER-to-Golgi traffic and palmitoylation of STING. Our results identify a non-nucleotide STING agonist that does not target the cGAMP-binding pocket, and demonstrate that Cys of STING can be a novel target for the development of STING agonist.Key words: STING agonist, cysteine modification, innate immunity, phenylarsine oxide.

Supercritical fluid chromatography-mass spectrometry enables simultaneous measurement of all phosphoinositide regioisomers.

Phosphoinositide species, differing in phosphorylation at hydroxyls of the inositol head group, play roles in various cellular events. Despite the importance of phosphoinositides, simultaneous quantification of individual phosphoinositide species is difficult using conventional methods. Here we developed a supercritical fluid chromatography-mass spectrometry method that can quantify the molecular species of all seven phosphoinositide regioisomers. We used this method to analyze (1) profiles of phosphoinositide species in mouse tissues, (2) the effect of lysophosphatidylinositol acyltransferase 1-depletion on phosphoinositide acyl-chain composition in cultured cells, and (3) the molecular species of phosphatidylinositol-3-phosphate produced during the induction of autophagy. Although further improvement is needed for the absolute quantification of minor phosphoinositide regioisomers in biological samples, our method should clarify the physiological and pathological roles of phosphoinositide regioisomers at the molecular species level.