C4′-Me-modified 2′,3′-cGAMP elicits a robust innate immune response in cells harboring the loss-of-function hSTING-R232H variant

The 2',3'-cyclic GMP-AMP dinucleotide (2',3'-cGAMP) is a potent endogenous agonist of the STING (stimulator of interferon genes), playing a critical role in innate immune activation and representing a promising therapeutic candidate for the treatment of infections, cancer, and autoimmune diseases. However, its clinical utility is hindered by poor cellular permeability, limited nuclease stability, and reduced efficacy in individuals carrying loss-of-function STING variants, such as R232H. In this study, we report the design, synthesis, and characterization of a novel C4'-methyl-guanosine-modified analogue, 2',3'-cG^4'-MeAMP. Structural investigations using NMR spectroscopy and replica exchange molecular dynamics simulations reveal that the C4'-Me modification induces only minor conformational changes. Notably, this modification substantially improves resistance to 3' exonucleases and enhances serum stability. Functional assays demonstrate that 2',3'-cG^4'-MeAMP elicits a stronger innate immune response in human cells expressing the STING-R232H variant compared to unmodified 2',3'-cGAMP. These results position 2',3'-cG^4'-MeAMP as a compelling lead candidate for STING-targeted immunotherapy, especially for patients with the STING-R232H variant that fails to respond to native 2',3'-cGAMP.