Design, Synthesis, and Biological Evaluation of Novel Cyclic Adenosine-Inosine Monophosphate (cAIMP) Analogs That Activate Stimulator of Interferon Genes (STING).

We describe novel STING-activating cyclic dinucleotides whose constituent nucleosides are adenosine and inosine and that vary by ribose substitution, internucleotide linkage position, and phosphate modification. In mammalian cells in vitro, some of these cAIMP analogs induce greater STING-dependent IRF and NF-κB pathway signaling than do the reference agonists for murine (DMXAA) or human (2',3'-cGAMP) STING. In human blood ex vivo, they induce type I interferons (IFNs) and proinflammatory cytokines: for the former, 3',3'-cAIMP (9; EC50 of 6.4 µM) and analogs 52-56 (EC50 of 0.4-4.7 µM), which contain one or two 2'-fluoro-2'-deoxyriboses and/or bis-phosphorothioate linkages, are more potent than 2',3'-cGAMP (EC50 of 19.6 µM). Interestingly, 9 induces type I IFNs more strongly than do its linkage isomers 2',3'-cAIMP (10), 3',2'-cAIMP (23), and 2',2'-cAIMP (27). Lastly, some of the cAIMP analogs are more resistant than 2',3'-cGAMP to enzymatic cleavage in vitro. We hope to exploit our findings to develop STING-targeted immunotherapies.

Structural determinants for N1/N7 cyclization of nicotinamide hypoxanthine 5'-dinucleotide (NHD+) derivatives by ADP-ribosyl cyclase from aplysia californica: Ca2+-mobilizing activity of 8-substituted cyclic inosine 5'-diphosphoribose analogues in T-lymphocytes.

A series of nicotinamide hypoxanthine 5'-dinucleotide (NHD+) analogues modified at C-8 (2-5) and 7-deaza-NHD+ were synthesized, and cyclization in the presence of Aplysia ADP-ribosyl cyclase was studied. All 8-substituted NHD+ analogues were converted into their N1-cyclic forms by the enzyme, while in contrast, 7-deaza-NHD+ 17 was hydrolyzed into 7-deazainosine 5'-diphosphoribose (7-deaza-IDPR) 25. Correlations are made showing that the conformation of the NHD+ substrate is the key to successful cyclization. The pharmacological activities of these novel cIDPR derivatives were evaluated in both permeabilized and intact Jurkat T-lymphocytes. The results show that in permeabilized cells both 8-iodo 1g and 8-N3-N1-cIDPR 1d have an activity comparable to that of cADPR, while 8-iodo 1g and 8-phenyl-N1-cIDPR 1c have a small but significant effect in intact cells and can therefore be regarded as membrane-permeant; thus, cIDPR derivatives are emerging as important novel biological tools to study cADPR-mediated Ca2+ release in T-cells.