Synthesis of All Possible Canonical (3′–5′-Linked) Cyclic Dinucleotides and Evaluation of Riboswitch Interactions and Immune-Stimulatory Effects

Abstract

The cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and c-AMP-GMP are widely utilized as second messengers in bacteria, where they signal lifestyle changes such as motility and biofilm formation, cell wall and membrane homeostasis, virulence, and exo-electrogenesis. For all known bacterial CDNs, specific riboswitches have been identified that alter gene expression in response to the second messengers. In addition, bacterial CDNs trigger potent immune responses, making them attractive as adjuvants in immune therapies. Besides the three naturally occurring CDNs, seven further CDNs containing canonical 3'-5'-linkages are possible by combining the four natural ribonucleotides. Herein, we have synthesized all ten possible combinations of 3'-5'-linked CDNs. The binding affinity of novel CDNs and GEMM riboswitch variants was assessed utilizing a spinach aptamer fluorescence assay and in-line probing assays. The immune-stimulatory effect of CDNs was evaluated by induction of type I interferons (IFNs), and a novel CDN c-AMP-CMP was identified as a new immune-stimulatory agent.

The Role of the Phosphorus Atom in Drug Design

“…The GEMM term refers to the fact that they control Genes related to Environment, Membranes, and Motility . Compounds 223 – 229 can be considered as structures still not found as natural products . In fact, riboswitches have been found for all bacteria.…”

“…Compound 222 behaves as second messenger, which elicits the stimulator of interferon genes (STING) giving rise to the formation of type I interferons (IFN). In this regard, 223 was found to be a modest immune stimulator, eliciting type I IFN responses to a lesser extent than the known naturally occurring cyclic dinucleotides . The availability of these synthetic cyclic dinucleotides is a relevant area of research, potentially allowing the characterization of new signaling compounds in living organisms .…”

“…[203] Compounds 223-229 can be considered as structures still not found as natural products. [204] In fact, riboswitches have been found fora ll bacteria. It was found that sequences binding to 219 or 221 distinguish, in some way,t heir non-comparable cyclic dinucleotides.…”

Agonists and inhibitors of the STING pathway: Potential agents for immunotherapy

“…Structures of CDNs with lipid, fluorine, noncanonical base and locked nucleic acid modifications . CDNs, cyclic dinucleotides…”

Pseudo aptamer expands aptamer’s applications

“…There are also bacterial riboswitches that selectively recognize cyclic dinucleotides. It was reported that a natural riboswitch targeting 3', 3'-cGAMP is distinguished from the endogenous mammalian signal 2',3'-cGAMP binding protein by its backbone connectivity 43,44 as shown in Figure 4.…”