Enzymatic Preparation of 2′–5′,3′–5′-Cyclic Dinucleotides, Their Binding Properties to Stimulator of Interferon Genes Adaptor Protein, and Structure/Activity Correlations

Cyclic dinucleotides are second messengers in the cyclic
GMP–AMP
synthase (cGAS)–stimulator of interferon genes (STING) pathway,
which plays an important role in recognizing tumor cells and viral
or bacterial infections. They bind to the STING adaptor protein and
trigger expression of cytokines via TANK binding kinase 1 (TBK1)/interferon
regulatory factor 3 (IRF3) and inhibitor of nuclear factor-κB
(IκB) kinase (IKK)/nuclear factor-κB (NFκB) signaling
cascades. In this work, we describe an enzymatic preparation of 2′–5′,3′–5′-cyclic
dinucleotides (2′3′CDNs) with use of cyclic GMP–AMP
synthases (cGAS) from human, mouse, and chicken. We profile substrate
specificity of these enzymes by employing a small library of nucleotide-5′-triphosphate
(NTP) analogues and use them to prepare 33 2′3′CDNs.
We also determine affinity of these CDNs to five different STING haplotypes
in cell-based and biochemical assays and describe properties needed
for their optimal activity toward all STING haplotypes. Next, we study
their effect on cytokine and chemokine induction by human peripheral
blood mononuclear cells (PBMCs) and evaluate their cytotoxic effect
on monocytes. Additionally, we report X-ray crystal structures of
two new CDNs bound to STING protein and discuss structure–activity
relationship by using quantum and molecular mechanical (QM/MM) computational
modeling.