Design, synthesis, and cell-based in vitro assay of deoxyinosine-mixed SATE-dCDN prodrugs that activate all common STING variants

Developing therapeutic strategies to modulate the activity of all prevalent variants (wild-type, HAQ, R232H, AQ, and R293Q) of the stimulator of interferon genes (STING) is still of great interest to treating immune-related diseases. Herein, we synthesized six novel deoxyinosine-mixed deoxyribose cyclic dinucleotide prodrugs (SATE-dCDN) including a combination of hypoxanthine and other bases (A, U, C, T, and G) for a cell-based in vitro assay. The HPLC assay indicated that deoxyinosine-mixed SATE (S-acylthioalkyl ester)-dCDN prodrugs retained high serum stability. The IRF3-responsive luciferase assay in THP1-Lucia cells showed that the activity of the prodrugs with purine bases (SATE-3′,3′-c-di-dIMP, SATE-3′,3′-c-di-dIdAMP, and SATE-3′,3′-c-di-dIdGMP) was higher than that of the prodrugs with pyrimidine bases (SATE-3′,3′-c-di-dIdUMP, SATE-3′,3′-c-di-dIdTMP, and SATE-3′,3′-c-di-dIdCMP), among which prodrug 14a (SATE-3′,3′-c-di-dIdAMP) with hypoxanthine and adenine bases exhibited the highest activity with an EC50 value of 0.046 μM. The IRF3 responsive dual-luciferase reporter assay in HEK293T cells transfected with plasmids expressing different STING variants further showed that prodrug 14a could activate all five most common hSTING variants, including the refractory hSTINGR232H and hSTINGQ variants. Furthermore, prodrug 14a also induced the production of the highest levels of mRNA of IFN-β, CXCL10, IL-6 and TNF-α through STING-dependent IRF and NF-κB signaling pathways in THP-1 cells. These results suggested that the combination of deoxyinosine with a SATE-dCDN prodrug could modulate the broad-spectrum activity of all common STING variants.