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

Abstract

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 hSTING^R232H and hSTING^Q 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.