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.
Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we evaluate the activity of 3′,3′-c-di(2′F,2′dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2′ position of 3′,3′-c-di(2′F,2′dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.
The cGAS-STING (cyclic GMP-AMP synthase−stimulator of interferon genes) pathway plays a crucial role in inducing an antiviral and antitumor immune response. We studied the effects of synthetic STING agonists on several immune populations and related cytokine production. In comparison with the toll-like receptor 7 (TLR7) agonist, STING agonists induced secretion of a broader proinflammatory cytokine spectrum. Unlike the TLR7 agonist, the structurally diverse STING agonists partially depleted B and NK cells and completely depleted CD14+ monocytes via induction of apoptosis. The TANKbinding kinase 1 inhibitor efficiently prevented interferon alpha (IFNα) secretion and cell depletion, suggesting their possible dependence on the cGAS-STING pathway activation. Finally, IFNα, tumor necrosis factor alpha, interleukin 6, and interleukin 1 beta secretion and CD14+ monocyte apoptosis were primary responses to STING agonists, whereas IFNγ was secreted secondarily. These findings bring new insights into the cGAS-STING pathway immunomodulation that is of future therapeutic importance.
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