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

The 3′–5′, 3′–5′
cyclic
dinucleotides (3′3′CDNs) are bacterial second messengers
that can also bind to the stimulator of interferon genes (STING) adaptor
protein in vertebrates and activate the host innate immunity. Here,
we profiled the substrate specificity of four bacterial dinucleotide
synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia
coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5′-triphosphate analogues
and then employed these enzymes to synthesize 24 3′3′CDNs.
The STING affinity of CDNs was evaluated in cell-based and biochemical
assays, and their ability to induce cytokines was determined by employing
human peripheral blood mononuclear cells. Interestingly, the prepared
heterodimeric 3′3′CDNs bound to the STING much better
than their homodimeric counterparts and showed similar or better potency
than bacterial 3′3′CDNs. We also rationalized the experimental
findings by in-depth STING-CDN structure–activity correlations
by dissecting computed interaction free energies into a set of well-defined
and intuitive terms. To this aim, we employed state-of-the-art methods
of computational chemistry, such as quantum mechanics/molecular mechanics
(QM/MM) calculations, and complemented the computed results with the
{STING:3′3′c-di-ara-AMP} X-ray crystallographic
structure. QM/MM identified three outliers (mostly homodimers) for
which we have no clear explanation of their impaired binding with
respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG′int_rel and experimental ΔTm’s for the remaining ligands has been
very encouraging.