Profiling Nucleotide Signalling Pathways and STING Agonist Activity with a Nucleotide Library

Out of context dsDNA is detected by cGAS, which produces the cyclic dinucleotide 2′3′-cGAMP to activate STING and trigger downstream responses including transcriptional reprogramming, cell death and autophagy. STING agonists, among them non-hydrolysable cyclic dinucleotide analogues, are in active development to enhance immune activation in cancer treatment and vaccine adjuvants. Detailed knowledge of STING’s nucleotide preferences is critical for the development of effective therapeutics, yet the full spectrum of cyclic dinucleotides capable of activating STING has not been comprehensively defined. Especially considering the recent diversity of cyclic nucleotides identified across bacteria and invertebrates, the pool of potential STING agonists to be tested has expanded considerably. Here, we systematically dissect STING nucleotide preferences and distinguish STING-dependent from STING-independent nucleotide signaling pathways by treating THP-1 monocytes and pancreatic cancer cells with a nucleotide library. We identify a set of 27 cyclic dinucleotides that induce STING activation to different extents. Our data indicate that STING’s bias toward 2′3′-linked cyclic dinucleotides underlies its ability to also be activated by nucleotides containing purine–pyrimidine hybrids. In addition, we show that STING can be activated by diverse non-hydrolysable c-di-AMP and cGAMP isomers, thereby expanding the opportunities for designing STING agonists for therapeutic applications.