Design, synthesis and systematic evaluation of all possible cyclic dinucleotides (CDNs) that activate human stimulator of interferon genes (STING) variants

Abstract

Cyclic dinucleotides (CDNs) are known to activate stimulator of interferon genes (STING) and induce type I interferon responses, therefor possess great potentials to be of immunotherapeutic value for cancers and infectious diseases. However, the existence of different single nucleotide polymorphism (SNP) of human STING (hSTING) gene poses an obstacle to achieve broad-spectrum activation by CDNs. We reported here the design and synthesis of a total of 36 CDNs, representing all structural variations, that contain four bases (A, G, C, U) and two linkage directions (2′-5′-linked and 3′-5′-linked phosphodiester). Through systematic evaluation of IFN-β induction with a dual-luciferase reporter assay, we discovered that wild type hSTING and two isoforms (HAQ and AQ) showed strong response while hSTING-R232H and R293Q exhibited the relatively weak response to CDNs stimulation. For the first time, we found that the c[G(2′,5′)U(2′,5′)] showed excellent activity against all five hSTING variants even equivalent to the endogenous ligand c[G(2′,5′)A(3′,5′)]. Furthermore, we have also demonstrated that 3′-3′ CDNs with two 3′-5′ phosphodiesters showed higher serum and hydrolase stability than 2′-2′ CDNs with two 2′-5′ phosphodiesters and 2′-3′ CDNs with one 2′-5′ and one 3′-5′ phosphodiester. It is very interesting to note that 2′-2′ CDNs has been found for the first time to show strong activity. These findings will stimulate our exploration for the new functional role of CDNs, and provide guidelines to design CDNs based hSTING targeted drugs.