The cyclic GMP-AMP synthase - Stimulator of Interferon Genes (cGAS-STING) immune pathway is pivotal in detecting pathogen-associated (PAMPs) and damage-associated molecular patterns (DAMPs) [1]. Cyclic dinucleotides (CDNs) serve as second messengers transducing signals between cGAS and STING, if dsDNA is detected in the cytosol by cGAS [2]. Activation of STING by CDNS leads to downstream signal transduction resulting in induction of expression of proinflammatory cytokines (TNF-α, IL-1β) and the type I interferons (IFN-α, IFN-β) [3]. The cGAS-STING pathway thus has a crucial role in defence against pathogen infection, immune surveillance of tumour cells, and maintaining the normal immune functions of the body [4].
In this work, we describe the enzymatic and/or chemical synthesis of CDNs with 7-deazapurine modifications, with which we were able to introduce bulky substitutions while preserving the activity of modified CDNs when compared to unmodified. For the preparation of modified CDNs, we utilized mouse cyclic GMP-AMP synthase (cGAS) and bacterial dinucleotide synthases from Vibrio cholerae (DncV) and Bacillus thuringiensis (DisA). Moreover, the Suzuki-Myiaura cross-coupling reaction of 7-iodinated 7-deazapurine CDNs with aryl and heteroaryl boronic acids was used to introduce substituents too bulky for enzymatic synthesis. In total, we have prepared 24 new CDNs, which were characterised by biochemical, cell-based assays and for effect on human peripheral blood mononuclear cells (PBMCs). To explain the effect of these aromatic substituents we have solved four X-ray structures of complexes of 7-aryl substituted 7-deazapurine containing CDNs with human STING. These structures explain the reshaping of STING's binding site enabling the formation of a complex of STING with CDNs carrying such bulky substitutions. Moreover, potentially enables further modifications with even larger substitutions escaping the binding site, while still preserving further signal transduction of signal, which hasn't been possible until now.