Design, synthesis and systematic evaluation of all possible cyclic dinucleotides (CDNs) that activate human stimulator of interferon genes (STING) variants

Article abstract of DOI:10.1007/s11426-019-9662-5

Cyclic dinucleotides (CDNs) are known to activate stimulator of interferon genes (STING) and induce type I interferon responses, therefor possess great potentials to be of immunotherapeutic value for cancers and infectious diseases. However, the existence

Full text of DOI:10.1007/s11426-019-9662-5

SCIENCE CHINA
Chemistry
•ARTICLES•
https://doi.org/10.1007/s11426-019-9662-5
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Design, synthesis and systematic evaluation of all possible cyclic
dinucleotides (CDNs) that activate human stimulator of interferon
genes (STING) variants
Zheng-Hua Wang1†, Can-Can Zhao2†, Qiang-Zhe Zhang2, Chuan-Lin Wang1, Hang Zhang1,
De-Jun Ma1, Da-Wei Wang1, Xin Wen1, Lu-Yuan Li2* & Zhen Xi1,3,4*
1State Key Laboratory of Elemento-organic Chemistry, Department of Chemical Biology, College of Chemistry, Nankai University,
Tianjin 300071, China;
2State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Tianjin Key Laboratory of Molecular Drug Research,
Nankai University, Tianjin 300071, China;
3Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300071, China;
4National Pesticide Engineering Research Center (Tianjin), Tianjin 300071, China
Received September 5, 2019; accepted December 13, 2019; published online March 13, 2020
Cyclic dinucleotides (CDNs) are known to activate stimulator of interferon genes (STING) and induce type I interferon
responses, therefor possess great potentials to be of immunotherapeutic value for cancers and infectious diseases. However, the
existence of different single nucleotide polymorphism (SNP) of human STING (hSTING) gene poses an obstacle to achieve
broad-spectrum activation by CDNs. We reported here the design and synthesis of a total of 36 CDNs, representing all structural
variations, that contain four bases (A, G, C, U) and two linkage directions (2′-5′-linked and 3′-5′-linked phosphodiester).
Through systematic evaluation of IFN-β induction with a dual-luciferase reporter assay, we discovered that wild type hSTING
and two isoforms (HAQ and AQ) showed strong response while hSTING-R232H and R293Q exhibited the relatively weak
response to CDNs stimulation. For the first time, we found that the c[G(2′,5′)U(2′,5′)] showed excellent activity against all five
hSTING variants even equivalent to the endogenous ligand c[G(2′,5′)A(3′,5′)]. Furthermore, we have also demonstrated that 3′-3′
CDNs with two 3′-5′ phosphodiesters showed higher serum and hydrolase stability than 2′-2′ CDNs with two 2′-5′ phospho-
diesters and 2′-3′ CDNs with one 2′-5′ and one 3′-5′ phosphodiester. It is very interesting to note that 2′-2′ CDNs has been found
for the first time to show strong activity. These findings will stimulate our exploration for the new functional role of CDNs, and
provide guidelines to design CDNs based hSTING targeted drugs.
cyclic dinucleotides (CDNs), stimulator of interferon genes (STING), pyrimidine CDNs, interferon β, ecto-nucleotide
pyrophosphatase/phosphodiesterase 1 (ENPP1)
Citation:
Wang ZH, Zhao CC, Zhang QZ, Wang CL, Zhang H, Ma DJ, Wang DW, Wen X, Li LY, Xi Z. Design, synthesis and systematic evaluation of all
possible cyclic dinucleotides (CDNs) that activate human stimulator of interferon genes (STING) variants. Sci China Chem, 2020, 63, https://doi.org/
10.1007/s11426-019-9662-5
1 Introduction
portant newly discovered second messengers both in pro-
karyotes and eukaryotes [1,2]. While double-stranded DNA
(dsDNA) as the main stimulator activates cyclic GMP-AMP
synthase (cGAS) in the cytoplasm to produce second mes-
senger c[G(2′,5′)A(3′,5′)] in metazoan [3–7]. After the
synthesis of c[G(2′,5′)A(3′,5′)] by cGAS, c[G(2′,5′)A(3′,5′)]
Cyclic dinucleotides (CDNs) have been recognized as im-
†These authors contributed equally to this work.
*Corresponding authors (liluyuan@nankai.edu.cn; zhenxi@nankai.edu.cn)
© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020
chem.scichina.com link.springer.com

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Wang et al. Sci China Chem
binds and activates the human stimulator of interferon genes
(hSTING) proteins to trigger the innate immune signal
pathway through the TBK1-IRF3 signal transduction, finally
inducing the production of type I interferon (IFN-β) and
other pro-inflammatory factors. Besides c[G(2′,5′)A(3′,5′)]
in mammals, bacterial CDNs including c[G(3′,5′)G(3′,5′)],
c[A(3′,5′)A(3′,5′)] and c[G(3′,5′)A(3′,5′)] can also bind and
activate STING for the downstream signal transduction [8–
11]. There is a growing body of literature that recognizes the
importance of CDNs as the new general small molecule drug
in immune-therapy. The combination of CDNs with PD-1
antibody [12,13], PD-L1 antibody [14], CAR-T [15], radia-
tion therapeutics [16], and small-molecule drug 5-FU [17],
respectively, showed better anti-tumor activity than mono-
therapy. Recently, two CDN drugs ADU-S100
phosphotriester-approach. The name of CDNs followed the
principle as shown in Figure 1. The activity of IFN-β in-
duction on wild type hSTING and isoforms was obtained
through dual-luciferase assays. Furthermore, both of the
serum and ecto-nucleotide pyrophosphatase/phosphodies-
terase 1 (ENPP1) hydrolase stability of CDNs were also
studied.
2 Experimental
2.1 Chemical materials, general procedures, and
methods
All solvents and reagents were purchased from commercial
sources and used without further purification. The reaction
was monitored by thin layer chromatography (TLC) on silica
gel GF254 with detection under UV light. Nuclear magnetic
resonance spectroscopy (NMR) spectra were recorded on
Bruker AVANCE 400M (Germany) instrument at 298 K.
Chemical shifts were relative to tetramethylsilane (TMS)
(0.00). The following abbreviations were used to explain the
multiplicities: s (singlet), d (doublet), t (triplet), q (quartet),
m (multiplet). The number of protons (n) for a given re-
sonance was indicated as nH. High resolution mass spectrum
(HRMS) (MALTI-TOF) was obtained from Varian 7.0T
FTMS (USA). UPLC-HRMS (ESI) spectra were recorded on
Waters Xevo G2-XS Q-TOF mass spectrometry (USA).
HPLC was achieved using Agilent 1260 (USA). Pre-
parative column with an ASB C18 column (10 μm, 21.2 mm
(diameter)×250 mm (height)) (Agela Technologies) and the
preparative LC with Agela OCTOPUS purification System
were used. 1 M triethylammonium bicarbonate (TEAB) buffer
was prepared by bubbling CO2 through a 1 M Et3N solution
in H2O until pH~8.5. 1 M triethylamine acetate (TEAA)
buffer was prepared by adding CH3COOH to Et3N in water
until pH 7.0 and then filtering through a 0.45 μm membrane
filter. Both TEAA and TEAB buffer were kept in 4 °C.
(NCT02675439,
NCT03172936)
and
MK1454
(NCT03010176) have been approved for the clinical trials
for cancer therapy.
Although CDNs act as good candidate of small molecule
immune stimulator, the interaction mode of hSTING and
CDNs in the structural binding and activation was less un-
derstood. Firstly, Kao et al. [18] have identified that 57.9%
hSTING-WT, 20.4% hSTING-R71H-G230A-R293Q (HAQ),
13.7% hSTING-R232H, 5.2% hSTING-G230A-R293Q
(AQ) and 1.5% hSTING-R293Q were distributed in the
human populations. These single nucleotide polymorphisms
(SNPs) in hSTING have been shown different responses to
bacterial CDNs [18]. The categories of CDNs targeting all
the hSTING variants were relatively rare, and it would be
very interesting to explore the structural varieties of CDNs to
gain the wide spectrum activators with high potency. Re-
cently, Kranzusch et al. [19] found that bacterial cGAS/
DncV like nucleotidyltransferases (CD-NTases) could syn-
thesize pyrimidine CDNs. But these CDNs could not activate
the mouse STING (mSTING) dependent type I interferon
signaling in mammalian cells. However, all of these reported
CDNs with pyrimidine bases were linked by two 3′-5′
phosphodiester bonds, which was different from CDNs
linked with a non-canonical 2′-5′ phosphodiester bond in
mammal cells [11,19]. The linkage mode might regulate the
different signal pathway for diverse functions in cells.
Hence, a systematic evaluation of all possible CDNs in-
cluding four bases (A, G, C, U bases) and two linkage di-
rections (2′-5′-linked phosphodiester and 3′-5′-linked
phosphodiester) against all the reported hSTING variants
was necessary to establish the relationship of the structure
and activity. These understanding on the interaction mode of
CDNs and hSTING variants will help us to find efficient
hSTING activators with good serum stability.
2.2 Biological materials and general procedures
HEK293T and THP-1-lucia cells were maintained in culture
In this article, we reported the synthesis and biological
activity of CDNs with all the possible combination of A, U,
C, G bases and 2′-5′ and 3′-5′ phosphodiester linkages
(Figure 1). The CDNs and its analogs were obtained through
Figure 1 General structure of CDNs with different bases, phosphodiester
linkage and the name principle of CDNs (color online).

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Wang et al. Sci China Chem
medium (DMEM and RPMI 1640, separately) supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin/
streptomycin under the condition of 37 °C and 5% CO2. The
biological materials were purchased from the corresponding
commercial sources and used as received: Anti-TMEM173
(hSTING) antibody [EPR13130] (Abcam, ab181125), Anti-
GAPDH mouse monoclonal antibody (CWBIO, CW0100M),
Goat-anti-mouse IgG HRP-conjugated (CWBIO, CW0102S),
Goat-anti-Rabbit IgG HRP-conjugated (CWBIO, CW0103S),
Recombinant Human ENPP1 protein (R&D, 6136-EN-010),
Digitonin (Abcam, ab141501), DMXAA (MCE, HY-10964),
Polyjet (Signagen, SL100688), FBS-Australian Originecl
(Life, 10099-141), ECL reagent (GE Healthcare), Dual-Lu-
ciferase® Reporter Assay System (Promega, E1960), TRIzon
Reagent (CWBIO, CW0580S).
2.5 In vitro stability assay in serum and ENPP1 solu-
tions
Each CDNs (0.1 μg/mL) was incubated in either a ENPP1
solutions (30 μL) containing 2 nM ENPP1, 2 mM Ca2+,
0.2 mM Zn2+, 20 mM Tris-HCl pH 9.0, 0.2% NP-40; or 20%
FBS, 20 mM PBS pH 7.4, 5 mM Mg2+ or in water (as the
negative control) at 37 °C. At various time, aliquots of the
reaction mixture was collected and stopped by adding 5 μL
EDTA (0.5 M, pH 8.0) and diluted with 80 μL water. The
reaction mixture was then centrifuged at 10,000 r/min for
10 min, leaving the 100 μL supernatant for the high perfor-
mance liquid chromatography (HPLC) assay. 20 μL of each
aliquot was injected directly into HPLC (Agilent 1260 In-
finity HPLC equipped with a UV detector; column Agilent
ZORABX SB-C18 5 μm (4.6×150 mm); detection at
254 nm; column temperature: 25 °C) for analysis. 10 mM
TEAA buffer (solvent A) and MeCN (solvent B) were used
as the mobile phase with a flow rate of 1 mL/min. The gra-
dient was set as following: 0–2 min: 100% A; 2–12 min:
100% A to 80% A/0% B to 20% B; 12–13.5 min: 80% A to
0% A/20% B to 100% B; 13.5–15 min: 100% B; 15–17 min:
0% A to 100% A/100% B to 0% B.
2.3 Plasmid constructions
The pGL3-IFN-β plasmid was a gift from prof. Nicolas
Mannel (Addgene 102597) [20]. The pGL4.74-Rluc-TK
plasmid was purchased from Promega (E6921). The full-
length hSTING-WT gene sequence was synthesized by
AuGCT DNA-SYN Biotechnology Company (Beijing,
China) and cloned into pcDNA3.1 plasmids vector.
pcDNA3.1-hSTING-R232H,
pcDNA-hSTING-HAQ,
2.6 Molecular dynamics simulations
pcDNA3.1-hSTING-AQ, and pcDNA3.1-hSTING-R293Q
plasmids were constructed by site-directed mutagenesis
method using the polymerase chain reaction (PCR). The
primers were shown in Table S1 (Supporting Information
online). The gene sequence encoding the C-terminal domain
(CTD) of hSTING (aa 139-379) was inserted into the
pET28b(+) vector between NdeI and XhoI sites, resulting N-
terminal and C-terminal hexahistidine tag. Mutants were
generated by site-directed mutation through PCR using the
pET28b(+)-hSTING-WT vector as the template and con-
firmed by sequencing.
We carried out a investigation on the equilibrium con-
formations of hSTING in the presence and absence of li-
gands by molecular dynamics simulations using Amber 14
molecular modelling package [22,23]. The X-ray structure of
hSTING was retrieved from the Protein Data Bank (PDB
code: 4F5D). Ala230 in the crystal structure of hSTING
cytoplasmic domain was mutated back to the wild type re-
sidues Gly230. Amber ff14SB force field [24] was used to
parameterize amino acids residues of hSTING. The force
field parameters of CDNs come from previous work [25].
Each system was immersed in a truncated octahedral box of
explicit TIP3P water molecules. Particle Mesh Ewald (PME)
[26] and periodic boundary condition was employed to deal
with the long-range electrostatic interactions. SHAKE
method [27] was used to constrain bonds involving hydrogen
atoms in order to tolerate a time step of 2 fs. The system was
gradually heated from 0 to 300 K over 50 ps. The equili-
brating calculation was executed at 1 atm and at 300 K for
50 ps. Then, 15 ns MD simulations were performed under
300 K and 1 atm. The snapshot of the system was taken
every 1 ps.
2.4 Dual-luciferase reporter assay
5×104 HEK293T cells were seeded in 24-well plates.
pcDNA3.1-hSTING (50 ng), pGL3-IFNβ (firefly) (400 ng),
and pGL4.74-Rluc-TK (renilla) (50 ng) were co-transfected.
After 12 h, cells were stimulated with CDNs (5 μM) using
digitonin
permeabilization
(50 mM
N-2-hydro-
xyethylpiperazine-N-2-ethane sulfonic acid (HEPES),
100 mM KCl, 3 mM MgCl2, 0.1 mM dithiothreitol (DTT),
85 mM sucrose, 0.2% bovine serum albumin (BSA), 1 mM
adenosine triphosphate (ATP), 0.1 mM GTP, and 10 μg/mL
digitonin) [21]. After 30 min, the stimulation mixture was
removed and the normal media was added. After 12 h, cell
lysates were prepared and the relative luciferase activity
between the Firefly luciferase and the Renilla luciferase was
measured using the dual-luciferase reporter assay system.
2.7 Docking calculations of c[G(2′,5′)U(2′,5′)] and
hSTING-R232H
The structure of hSTING-R232H (aa 139-379) (PDB ID:
4LOH) was download from PDB, the water molecules and

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Wang et al. Sci China Chem
the ligand in the crystal structure were removed by PyMol.
c[G(2′,5′)U(2′,5′)] was constructed by SYBYL-X 2.0 (Tri-
pos, Inc., USA) and subsequently optimized with the con-
jugate-gradient and steepest-descent algorithm to a conver-
gence criterion of 0.005 kcal/mol. The parameters were
prepared by standard methods using Autodock Tools Pack-
age before docking. Docking calculations of receptor and
ligands were performed on AutoDock 4.2. For each ligand,
the docking runs were set to 200. After calculation, the re-
sults were clustered, and the best binding modes were se-
lected by the docking energy as well as by comparison with
the reference co-crystal ligand.
protecting group of phosphate was then introduced with the
3-hydroxypropionitrile. The two different protecting groups
could be removed under mild conditions. After the 5′-OH
protecting group DMTr was removed in a solution of 3%
dichloroacetic acid (DCA) in dichloromethane, the second
building blocks 4a–4d and 6a–6d were obtained. As shown
in Scheme 2, the coupling reactions of 5′-O-DMTr-2′-O-
TBDMS-3′-phosphodiester nucleotides 3a–3d with 5′-OH-
2′-O-TBDMS-3′-phosphotriester nucleotides 4a–4d were
achieved in the presence of 1-(2-mesitylenesulfonyl)-3-ni-
tro-1H-1,2,4-triazole (MSNT) in anhydrous pyridine to form
linear dimeric nucleotide 7 following the elimination of 5′-
DMTr under the acidic condition. When the linear inter-
mediates were obtained, phosphate protecting groups were
removed under tBuNH2-MeCN mixture and the cyclization
was achieved with the help of the coupling reagent MSNT in
anhydrous pyridine to obtain the fully protected cyclic di-
nucleotides 8a–8j, which were subsequently treated with N1,
N1,N3,N3-tetramethylguanidinium (TMG) and syn-pyr-
idine-2-carboxaldoximate (PBO) complex in water and 1,4-
dioxane solution to remove the 2-chlorophenyl groups. In the
following step, the base protection with benzoyl and acetyl
groups were removed in 33 wt% MeNH2-EtOH solution and
tert-butyldimethylsilyl (TBDMS) groups were removed
using Et3N•3HF mixture. The 3′-3′ CDNs with two 3′-5′
phosphodiester linkages 9a–9j were achieved with pre-
parative RP-HPLC. As shown in Scheme 3, the 2′-2′ CDNs
with two 2′-5′ phosphodiester linkages 12a–12j were obtained
through the coupling reaction of the 2′-O-TBDMS-3′-phos-
phorylate building blocks 5a–5d with 6a–6d followed by the
same cyclization and deprotection steps. The combination of
3′-O-TBDMS-2′-phosphorylated intermediates 3a–3d with
2′-O-TBDMS-3′-phosphorylated intermediates 6a–6d or in-
termediates 4a–4d with 5a–5d resulted in the formation of
2′-3′ CDNs with one 3′-5′ phosphodiester linkage and one 2′-
5′ phosphodiester linkage 16a–16p (Scheme 4). In summary,
2.8 Protein expression and purification
All proteins were expressed in E. coli BL21(DE3) and in-
duced with 0.5 mM isopropyl-β-d-1-thiogalactopyranoside
(IPTG) when OD600 reached 0.5–0.6 and grew overnight at
20 °C. Cells were spun down and lysed in lysis buffer
(20 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM imidazole).
After centrifugation and removal of cell debris, supernatant
was incubated with Ni-NTA beads. Ni-beads were washed
extensively and protein was eluted in wash buffer (20 mM
Tris-HCl pH 7.5, 300 mM NaCl, 300 mM imidazole). Eluted
protein was further cleaned by GE Disposable PD-10 col-
umns in assay buffer (20 mM Tris-HCl pH7.5, 150 mM
NaCl). Proteins were flash frozen in liquid nitrogen and
stored at −80 °C.
2.9 Isothermal titration calorimetry
The thermodynamic parameters of binding reactions of
hSTING-CTD with CDNs were measured by isothermal ti-
tration calorimetry using a PEAQ-ITC (Malvern) at 25 °C.
2.10 IFN-β induction in THP-1-lucia cells
5×105 THP-1-lucia cells were seeded in 24-well plate. 10 μM
CDNs were dissolved in the culture media directly without
any transfection reagent. After stimulation with 24 h, the
luciferase were measured for the IFN-β induction.
3 Results and discussion
3.1 Synthesis of CDNs
All possible CDNs including two phosphodiester linkage
bonds (2′-5′ and 3′-5′) and four bases (A, G, U, C) were
synthesized through the phosphotriester approach. Firstly, 2-
chlorophenyl phosphorodichloridate was chosen as phos-
phorylation reagent and was introduced onto the 3′-OH or 2′-
OH of the nucleosides 1a–1d and 2a–2d to get the first
building blocks 3a–3d and 5a–5d (Scheme 1). The second
Scheme 1 Synthesis of intermediates 3a–3d, 4a–4d, 5a–5d, 6a–6d. Re-
agents and conditions: (i) 1) 1,2,4-triazole, Et3N, CH2Cl2; 2) TEAB buffer;
(ii) 1) 3-hydroxypropionitrile, MSNT, pyridine; 2) 3% DCA, CH2Cl2 (color
online).

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Wang et al. Sci China Chem
Scheme 2 Synthesis of 3′-3′ CDNs 9a–9j. Reagents and conditions: (i) 1) MSNT, pyridine; 2) DCA, CH2Cl2; (ii) 1) tBuNH2, MeCN; 2) MSNT, pyridine;
(iii) 1) TMG, PBO, 1,4-dioxane, H2O; 2) MeNH2, EtOH; 3) pyridine, Et3N, Et3N•3HF.
Scheme 3 Synthesis of 2′-2′ CDNs 12a–12j. Reagents and conditions: (i) 1) MSNT, pyridine; 2) DCA, CH2Cl2; (ii) 1) tBuNH2, MeCN; 2) MSNT, pyridine;
(iii) 1) TMG, PBO, 1,4-dioxane, H2O; 2) MeNH2, EtOH; 3) pyridine, Et3N, Et3N•3HF.
36 analogues of CDNs (9a–9j, 12a–12j, and 16a–16p) with
different bases and different phosphodiester linkage combi-
nations were successfully synthesized and characterized.
the presence of hSTING-expressing plasmids, reporter
plasmids pGL3-IFNβ (firefly) and pGL4.74-Rluc-TK (re-
nilla) were also co-transfected into HEK293T cells and in-
cubated for 12 h. The transfection of CDNs needs the help of
digitonin permeabilization [21,32]. After the cell lysis, all
five hSTING proteins could be detected by immune blotting
but no endogenous hSTING proteins were observed, in-
dicating the over-expression of exogenous hSTING proteins
in cells (Figure S1). As shown in Figure 2, in the absence of
CDNs, the transfection of different hSTING-expressing
plasmids (50 ng/well) exhibited different activity to activate
IFN-β pathway. The relative activity was ranked as follows:
R232H (12.2 fold)>WT (8.3 fold)>HAQ (3.8 fold)>R293Q
(2.3 fold)>AQ (1.0 fold). When the endogenous ligand 16e
(c[G(2′,5′)A(3′,5′)]) was added, all five hSTING proteins
responded to 16e differently with the following ranking ac-
tivity: AQ (66.9 fold)>R293Q (49.6 fold)>HAQ (18.4 fold)
>WT (14.9 fold)>R232H (8.3 fold) although the overall
activity was upregulated for all hSTING proteins compared
with the cells only transfected hSTING expression plasmids.
Among five hSTING proteins, 16e exerted the greatest ac-
tivation to hSTING-AQ, indicating the possible strong in-
teraction between 16e and hSTING-AQ. The results
3.2 IFN-β luciferase induction through activating wild
type hSTING and its SNPs elicited by CDNs
After the stimulation of CDNs, STING recruits TBK1 kinase
to induce the phosphorylation of the transcription factor
IRF3, which subsequently dimerizes and translocates into the
nucleus and binds IFN-β promoter to induce the production
of type I interferons [28–30]. Based on this principle, IRF3-
responsive dual-luciferase reporter assay was established to
determine the activity in IFN-β activation [5,20,31]. Herein,
we determined the activity of 36 CDNs through the IRF3-
binding induced fluorescence increase when dual-luciferase
reporter vectors and hSTING expressing vectors were co-
transfected into HEK293T cell.
To compare the sensitivity of hSTING variants to all these
CDNs, we firstly introduced five hSTING variants contain-
ing different SNPs (WT, HAQ, AQ, R232H, R293Q) in the
expressing vectors [18], which was confirmed by Sanger
sequencing (Figure S1, Supporting Information online). In

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Wang et al. Sci China Chem
Scheme 4 Synthesis of 2′-3′ CDNs 16a–16p. Reagents and conditions: (i) 1) MSNT, pyridine; 2) DCA, CH2Cl2; (ii) 1) tBuNH2, MeCN; 2) MSNT, pyridine;
(iii) 1) TMG, PBO, 1,4-dioxane, H2O; 2) MeNH2, EtOH; 3) pyridine, Et3N, Et3N•3HF.
imidine ring recovered their activity like 12d c[U(2′,5′)U
(2′,5′)], 12g (c[G(2′,5′)U(2′,5′)]), and 12i (c[G(2′,5′)C
(2′,5′)]) targeting hSTING-WT and hSTING-AQ. 12d failed
to activate hSTING-HAQ. Among all the 2′-2′ CDNs (12a–
12j), interestingly, only 12g was observed to activate all the
five hSTING variants. What is more, the 12g (106.2 fold)
had higher ability to activate hSTING-AQ than 16e (87.1
fold).
Finally, the activity of 2′-3′ CDNs (16a–16p) was also
Figure 2 IFN-β luciferase induction activity of WT hSTING and SNPs
evaluated (Figure 5). We found that CDNs with two purine
bases containing one 2′-5′ phosphodiester and one 3′-5′
phosphodiester bond could induce robust activation to
hSTING-WT, hSTING-HAQ, and hSTING-AQ proteins. 16f
(c[G(3′,5′)A(2′,5′)]) decreased the capacity of activating
hSTING-R232H and hSTING-R293Q. 16a failed to activate
hSTING-R293Q. Similarly, 2′-3′ CDNs with mixed pyr-
imidines like 16g (c[U(3′,5′)C(2′,5′)]), 16i (c[G(2′,5′)U
(3′,5′)]), 16j (c[G(3′,5′)U(2′,5′)]), 16l (c[A(3′,5′)U(2′,5′)])
and 16p (c[A(3′,5′)C(2′,5′)]) also activated hSTING-WT and
hSTING-AQ but failed to activate hSTING-R232H and
hSTING-R293Q. Only 16i and 16j could partly activate
hSTING-HAQ. Among all the 2′-3′ CDNs (16a–16j), only
16e could effectively activate five hSTING variants.
Previous studies have confirmed that the purine rings G
and A could easily be able to stack with Tyr167 and Arg238
of hSTING, which made the CDNs with GG, AA, and GA
showing excellent activity to activate hSTING immune
pathway [32]. Comparing the activity of 3′-3′ CDNs, 2′-2′
CDNs, and 2′-3′ CDNs, we found that all the CDNs with
purine bases (A and G) showed good activity to induce IFN-β
luciferase production as expected. The CDNs constructed
with G and A bases, like 9e, 12e, 16e, and 16f, exhibited
relative strong activities to all five hSTING variants. And
what is more, the metazoan ligand 16e was the strongest
hSTING activator. It was taken for granted that hSTING
should not sense CDNs with mixed pyrimidine bases be-
with or without stimulated with 16e (5 μM) for 12 h in HEK293T cells.
Data were presented as mean±s.e.m; n=5.
demonstrated the feasibility of using IRF3 responsive dual-
luciferase reporter assay to evaluate the potency of CDNs.
We further evaluated the activity of 3′-3′ CDNs with two
3′-5′ phosphodiester linkage (9a–9j). As shown in Figure 3,
only bacterial CDNs such as 9a (c[G(3′,5′)G(3′,5′)]), 9b (c[A
(3′,5′)A(3′,5′)]) and 9e (c[G(3′,5′)A(3′,5′)]) could activate
hSTING-WT and hSTING-AQ. The introduction of U and
(or) C base did not trigger hSTING-dependent IRF pathway.
In contrast with hSTING-WT, hSTING-HAQ was less sen-
sitive to 9a (Figure 3(c)) while hSTING-R232H showed
much weaker response to 9a and 9e. Interestingly, the mu-
tation of R232H nearly abolished its response to 9b, which
revealed the important role of Arg232 in binding 9b (Figure
3(d)). Among all the 3′-3′CDNs, only 9e could effectively
activate five hSTING variants, which might be a broad-
spectrum hSTING activator.
Subsequently, we evaluated the activity of 2′-2′ CDNs with
two 2′-5′ phosphodiester linkage (12a–12j) (Figure 4). The
results showed that 2′-2′ CDNs with two purine rings could
activate hSTING-WT, hSTING-HAQ and hSTING-AQ
variants while showing low activation to hSTING-R232H
and R293Q variants. 12e (c[G(2′,5′)A(2′,5′)]) also showed
good activities to all five mutants, except much lower to
R232H. Unlike 3′-3′ CDNs, 2′-2′ CDNs with mixed pyr-

7
Wang et al. Sci China Chem
Figure 3 The activity of 3′-3′ CDNs against five hSTING variants. (a) The general structure of 3′-3′ CDNs; (b–f) HEK293T cells were cotransfected with
IFN-β-luciferase reporter plasmids and the test plasmids expressing (b) hSTING-WT, (c) hSTING-HAQ, (d) hSTING-R232H, (e) hSTING-AQ, and (f)
hSTING-R293Q. After 24 h, cells were further stimulated for 12 h in the presence of CDNs (5 μM) and then used for the luciferase assay. Data were
presented as mean±s.e.m; n=5.
Figure 4 The activity of 2′-2′ CDNs against five hSTING variants. (a) The general structure of 2′-2′ CDNs; (b–d) HEK293T cells were cotransfected with
IFN-β-luciferase reporter plasmids and the test plasmids expressing (b) hSTING-WT, (c) hSTING-HAQ, (d) hSTING-R232H, (e) hSTING-AQ, and (f)
hSTING-R293Q. After 24 h, cells were further stimulated for 12 h in the presence of CDNs (5 μM) and then used for the luciferase assay. Data were
presented as mean±s.e.m; n=5.
cause the pyrimidine ring would not be able to reach Tyr167
for the favorable staking interaction [32]. Surprisingly, we
have found here that CDNs with mixed pyrimidine bases
12d, 12g, 12i, 16g, 16i, 16j, 16l, and 16p could activate
hSTING, in which 12g showed as same if not better activity
as CDNs with purine bases, and 12g exhibited excellent
activity to all the five hSTING variants. As we found, all the
CDNs with mixed pyrimidines containing at least one 2′-5′
linkage bond stimulated the IFN-β induction. The 2′-5′
linked phosphate group have a higher propensity than 3′-5′
linked phosphate to form hydrogen bonding with Arg232
[33], which might also provide the explanation that hSTING-
R232H responded poorly to the majority of CDNs.
When hSTING expression plasmids were transfected into
HEK293T cells, the IFN-β Luc reporter was also induced
without stimulation of CDNs. We hypothesized this phe-
nomenon may be caused by the hSTING self-activation.
hSTING self-activation was more obviously in some gain-

8
Wang et al. Sci China Chem
Figure 5 The activity of 2′-3′ CDNs against five hSTING variants. (a) The general structure of 2′-3′ CDNs; (b–f) HEK293T cells were cotransfected with
IFN-β-luciferase reporter plasmids and the test plasmids expressing (b) hSTING-WT, (c) hSTING-HAQ, (d) hSTING-R232H, (e) hSTING-AQ, and (f)
hSTING-R293Q. After 24 h, cells were further stimulated for 12 h in the presence of CDNs (5 μM) and then used for the luciferase assay. Data were
presented as mean±s.e.m; n=5.
Table 1 Dynamic data distributed in the range of 33.77±1 Å
of-function mutations like R248S, R281Q, R284G, V155M,
N154S, etc, which were found in autoimmune disease pa-
tients called STING-associated vasculopathy with onset in
infancy (SAVI) [34–37]. Without the stimulation of CDNs,
these patients showed higher level of IFN-β than healthy
people. Depending on the general understanding of hSTING-
activation mode, when hSTING bound to CDNs, the two
protomers of hSTING will shift closely. As a result, the
hSTING was changed from an “open” conformation to a
“closed” conformation [5,6]. Thus, the degree of “opening”
or “closing” of the two arms of the V-shaped hSTING dimer
was evaluated here by the distance between two Thr181-Cα
from each hSTING protomer. Depending on the reported
structures of apo-hSTING and hSTING-CDNs complexes,
the distances were distributed in a range of 33.77–48.33 Å
(Figure S2) [5,32,38–42]. We defined the distance dis-
tributed in a range of 33.77±1 Å as the active conformation
of hSTING. Molecular dynamics simulations for hSTING in
the presence and absence of CDNs were carried out to
generate the equilibrium conformations with Amber14
software. The probability of active conformation was cal-
culated. The results were shown in Table 1 and Figure S3.
The probability distribution of apo-hSTING, 16e-hSTING,
and 9a-hSTING were 0.13, 0.56, and 0.58, respectively. It
indicated that without any ligands, the apo-hSTING could
also form the active conformation by itself with a certain
probability. This means that the hSTING protein could be
activated and induce downstream signaling transduction
pathway without the stimulation of ligands. The Western
Blot results indicated that the expression level of each
hSTING variants were similar (Figure S1) [18,31], but the
ability of self-activation or CDNs-mediated activation were
much different. And the sensitively of each hSTING iso-
Apo-hSTING
1,728
16e-hSTING
9a-hSTING
Amounts of active
conformation
7,281
7,537
Possibility of
active conformation
0.13
0.56
0.58
forms response to 16e were also much different. The
hSTING-AQ had the lowest activity without the stimulation
of 16e, but it was most sensitive to 16e.
All five hSTING variants showed different response to 36
CDNs, indicating the interaction complexity between CDNs
and hSTING proteins. The hSTING is ER-membrane located
protein, which makes it difficult to achieve the full-length
crystal structure for the study of hSTING-CDNs binding
mode before. Fortunately, the cryo-EM structure of full-
length hSTING (PDB: 6NT5) has been dissolved [41]. Ac-
cording to the apo-hSTING full-length structure, Arg71 site
was located between the transmembrane helix α2 and helix
α3 of the N-terminus and the Arg293 site was located on the
helix α3 of CDN binding domain (CBD) of hSTING con-
tacting with the transmembrane domains and forming
charge-charge interaction with Glu149 and Arg76 (Figure 6
(a, b)) [41]. Both of two sites had no direct contact with
ligands but might help stabilize the dimerization of hSTING.
Resulting in the mutations of R293Q or R71H interfered with
the translocation or aggregation of hSTING to initiate the
IFN-β pathway. It might be more important for R293Q
mutation to affect the rotation of each hSTING protomer
induced by CDN binding [41].
Furthermore, there were two sites affecting the CDNs
binding like Gly230 and Arg232 sites located in the highly
flexible loop region between β2 and β3 strands of hSTING

9
Wang et al. Sci China Chem
mutant. But there was no much different while 9a bound to
hSTING-WT and R232H CTD (2.82 and 2.84 μM). As
previous reported, while 9a bound to hSTING-WT-CTD, the
Kd value was two-fold compared to the hSTING-G230A-
CTD [18]. When 9a bound to hSTING-AQ-CTD, the Kd
value was also half (1.26 μM) compared to hSTING-WT-
CTD. It indicated that the R293Q mutation has low influence
to the binding affinity of CDNs to hSTING-CTD. So de-
pending on the structure and bioactivity data analysis, we
forecasted that the R71H, R232H, and R293Q were loss-of-
function mutations, but the G230A was a kind of gain-of-
function mutation. But all of these mutations did not affect
the responses to the metazoan ligand 16e and the synthetic
ligand 12g.
Figure 6 Comparison of the structure of hSTING SNP variants. (a) Full
length of apo-hSTING-WT (PDB: 6NT5). (b) Close view of Arg71 and
Arg293 sites located in apo-hSTING-WT (PDB: 6NT5). (c) Comparison of
the structure changes when 16e bound to hSTING-WT (PDB:4KSY, ma-
genta) and hSTING-H232 (PDB:4LOH). (d) Comparison of the lip region
change when 9a bound to hSTING-WT (PDB:4F5Y, green) and hSTING-
G230A (PDB:4F5D, hotpink). (e) Comparison of the structure changes
when 9b bound to hSTING-WT (PDB:6CFF, cyan) and hSTING-G230A
(PDB:6CYT, orange) (color online).
The previous study explored that the purine rings would
have more potency to form stacking interactions with
Arg238 and Tyr167 of hSTING than pyrimidine rings [6]. As
shown in Figure S5, when 9a bind to hSTING (PDB: 4F5D),
the G1 base would have potency to form stacking with
A_Tyr167 (Tyr167 residue located in hSTING protomer A)
and B_Arg238 (Arg238 residue located in hSTING protomer
B), and the interactions we labeled as Π1 and Π2, separately.
The G2 ring could form stacking with B_Tyr167 and
A_Arg238, and the interactions were also labeled as Π3 and
Π4, separately (Figure S5). Here, we calculated the stacking
interactions between the bases of CDNs like 9a, 9b, 9c, 9d,
9e, and 16e with hSTING Tyr167 and Arg238 residues. We
got all of the data after 2 ns (Figures S6–S11). We counted
the data distributed in the range of 0–20° dihedral anger and
3.4-4 Å distance. As shown in Table 2, the purine rings have
more potency to form stacking interactions than pyrimidine
rings, especially the interaction with Arg238 residues. These
results further supported the idea that CDNs with purines
would have a higher binding affinity with hSTING. The two
U rings of 9d also showed favored stacking interaction with
Arg238 and Tyr167. But when the 9c bind to hSTING, only
one C ring could form stacking with Arg238 and Tyr167. The
U ring was more favored to stack with Tyr167 and Arg238
than C ring. It indicated that CDNs with pyrimidines, espe-
cially the U rings could also bind to hSTING with a certain
binding affinity. However, the 9d only showed limited ac-
tivity to activated hSTING-AQ (12.8 fold) (Figure 3(e)).
While for pyrimidine CDNs contained 2′-5′ phosphodiester
linkage, their ability to activate hSTING was increased
(Figures 4, 5 and S4). Except for the 9g, CDNs with G and U
bases combination (12g, 16i, and 16j) could activate
hSTING-WT, hSTING-AQ, and hSTING-HAQ with higher
activity than other pyrimidine CDNs (Figure S4). Further-
more, 12g also showed good activity to induce IFN-β Lu-
ciferase production mediated by hSTING-R232H and
R293Q isoforms.
protein. When CDNs bound to hSTING, this flexible loop
region would transform into four β-sheets covering on top of
the binding-pocket [6], which would increase the binding
affinity of hSTING protein to CDNs. It could be well vali-
dated that the loop region in hSTING-WT did not form the
ordered β-sheet [39] but the loop region in hSTING-G230A
formed the ordered sheet when bound by 9a (Figure 6(d))
[38]. However, there was no obvious difference between
hSTING-WT and G230A when bound by 9b (Figure 6(e))
[43]. Since G230A mutated hSTING favored the closed
conformation in the presence of some CDNs [18], hSTING-
HAQ and hSTING-AQ were largely sensitive in response to
different CDNs (Figure 6(c)). For Arg232 mutation, we took
the crystal complex of 16e and hSTING-WT (PDB:4KSY)
as the example [6], and found that the two phosphate groups
could form hydrogen bonding with Arg232, which was ab-
sent in the crystal complex of 16e and hSTING-R232H
(PDB: 4LOH) [5]. It revealed that the R232H mutation de-
creased the activation of hSTING and provided an ex-
planation of the weak activation to hSTING-R232H by the
majority of CDNs in our hands. We also recorded the iso-
thermal titration calorimetric (ITC) binding curves for the
complex formation of hSTING-WT, R232H and AQ variants
C-terminal domain (CTD) (aa 139–379) with 9a, 9e and 16e
as shown in Figure S13 and the ITC-based thermodynamic
parameters as shown in Table S2. Kd value of the complexes
formation of 16e and 9e to hSTING-WT-CTD were 5.91 nM
and 1.59 μM, separately. But while 16e and 9e bound to
hSTING-R232H-CTD, the Kd value were dramatically in-
creased (3.33 and 5.44 μM, separately). It indicated the
binding affinity was decreased while CDNs bind to R232H
Since we found several CDNs had excellent ability to
activate all five hSTING isoforms like 12g and 16e, we

10
Wang et al. Sci China Chem
further investigated the mechanism of the broad-spectrum
activity based on the binding mode of 12g and hSTING-
R232H (PDB:4LOH) [5] through the docking calculations
which was performed on AutoDock 4.2. When 12g bound to
hSTING-R232H protein, 12g showed a similar conformation
to 16e in that the uracil group forms the same stacking with
Arg238 and Tyr167 (Figure 7(a, c)). Meanwhile, the O2 and
O4 part of the uracil group had the high propensity to form
hydrogen bonding with Thr263 and Arg238, respectively
(Figure 7(d)). Both of the 3′-OH of the ribose also had high
potency to form favorable hydrogen bonding interaction with
Ser162 (3.8 and 2.8 Å, respectively) (Figure 7(b)). The
phosphate groups could interact with Arg238 through hy-
drogen bonding and charge-charge interactions (Figure 7(d)).
Hence, it might reveal the general interaction mode for five
hSTING variants binding to 12g. The docking data indicated
that 12g may interact with hSTING protein with high binding
affinity. The ITC binding curves and ITC-based thermo-
dynamic parameters for the complex formation of 12g bound
to hSTING-CTD protein were also recorded (Figure S13 and
Table S2). The dissociation constants of 12g bound to
hSTING-WT-CTD and hSTING-R232H-CTD were Kd=3.68
and 6.75 μM, separately. Both of the docking data and ITC
test indicated that 12g may interact with hSTING protein
with high binding affinity.
The activity of CDNs inducing IFN-β production in THP-1
cells was also studied. THP-1-lucia cells were incubated in a
culture media containing 10 μM of CDNs without any
transfection reagents for 24 h. The IFN-β production activity
was measured through luciferase reporter activity (Figure
S14). CDNs constructed by G and A bases (9e, 12e, 16e and
16f) could activate IFN-β induction dramatically. And the
endogenous 16e showed the highest activation activity. Cells
did not give responses of IFN-β induction to CDNs with
mixed pyrimidine except 12g. It indicated that 12g could
activate IFN-β induction both in HEK293T (pre-transduced
with hSTING) and THP-1 cell lines.
3.3 Stability of CDNs in serum and hydrolase
To achieve the long-lasting efficiency of CDNs for clinical
applications, the serum stability and hydrolase stability of
CDNs were indispensable. CDNs were constructed by two
internucleotide phosphodiester bonds, which might be ra-
pidly hydrolyzed by specific enzymes in serum. The in-
stability in serum could restrict their in vivo potency. As
reported, the only hydrolase called ENPP1 was proven to
degrade 16e in cultured cells while it was still unknown
whether CDNs with other structures could be also degraded
by ENPP1 [44,45]. Hence, we here intended to determine the
stability of CDNs both in serum and ENPP1 solutions by
HPLC.
Table 2 Dynamic calculation of the stacking interactions between the
bases of CDNs with hSTING Y167 and R238 residues. Data counted a
range of dihedral anger in 0–20° and distance in 3.4–4 Å
Π1
10,627
1,009
5
Π2
1,244
1,922
1
Π3
10,649
14
Π4
23
After time-scale determination of the changes of CDNs in
10 mM phosphate buffer saline (PBS) buffer (pH 7.4) con-
taining 20% FBS and 1 mM Mg2+, we found that 3′-3′ CDNs
with two 3′-5′ linkages (T1/2>50 h) were more stable than 2′-2′
CDNs with two 2′-5′ linkages (T1/2=6–20 h) and 2′-3′ CDNs
with one 3′-5′ linkage and one 2′-5′ linkage (T1/2=20–40 h)
(Figure 8(a)). The results of the incubation of CDNs in
ENPP1 were shown in Figure 8(b). CDNs with two 3′-5′
linkages were more stable than CDNs with 2′-5′ linkage
while no significant difference between 2′-3′ CDNs with one
2′-5′ and one 3′-5′ phosphodiester bond and 2′-2′ CDNs with
two 2′-5′ phosphodiester bonds was observed. When CDNs
were treated with ENPP1 (2 nM) for 48 h, 80% of 3′-3′
CDNs were not degraded but only less than 20% of 2′-2′
CDNs and 2′-3′ CDNs were left, which was consistent with
the stability assay in serum. Overall, the 3′-3′ CDNs with two
3′-5′ linkages provided a more hydrolase-resistant con-
formation. Contrary to expectations, 16a and 12b did not
show much degradation in 72 h. This may be caused by the
substrate selectivity of the catalytically pocket of ENPP1.
To further confirm that the degradation of CDNs was
caused by hydrolase, we also identified that the CDNs in
water could be stable for more than a month at room tem-
perature. Through the calculation and NMR tests, we pro-
posed that the 2′-5′ phosphodiester linked ribose preferred
9a
9b
9c
1,883
576
4,134
9d
9e
8,339
124 (A)
9,871(G)
142
9,912
3,028
308(G)
6,527(A)
1,150(A)
700(G)
10,494(G)
920(A)
16e
Figure 7 Docking calculation of 12g and hSTING-R232H (aa 139–379).
12g marked in green and 16e marked in magenta (color online).

11
Wang et al. Sci China Chem
Figure 8 Time-scale HPLC analysis of CDNs in (a) 20% FBS and (b) 2 nM ENPP1 (color online).
the C2′-endolike (south-type) conformation and the 3′-5′
phosphodiester linked ribose preferred C3′-endolike (north-
type) conformation (Figure S12(a)) [25,33,46]. The special
structure might endow CDNs the non-specific nuclease re-
sistance. Based on the crystal-structure of ENPP1 in complex
with 9e obtained by Nureki et al. [44], they proposed that the
ENPP1 favors to cleave the 2′-5′ phosphodiester of 16e in a
conformation suitable for catalysis reaction instead of 3′-5′
phosphodiester (Figure S12(b)), which was in accordance
with our findings. The 16e and 12g showed the same stability
in ENPP1. But 16e (T1/2~30 h) showed more stable than 12g
(T1/2 less than 12 h) in serum. 16e contains one 3′-5′ phos-
phodiester bond and one 2′-5′ phosphodiester bond and 12g
contains two 2′-5′ phosphodiester bonds. According to the
cell-based activity, the two CDNs showed same IFN-β in-
duction level in HEK293T cell with transduction of hSTING
R232H and AQ. However, HEK293T cells expressing
hSTING-WT, HAQ and R293Q, 16e showed higher activity
than 12g. When 16e and 12g were transfected into THP-1 cells
through co-incubation in serum-containing culture media,
16e also showed much higher activity than 12g. The higher
stability of 16e may also contribute the higher activity.
In summary, CDNs with two 3′-5′ phosphodiester linkage
were more stable than CDNs with one or two 2′-5′ phos-
phodiester linkages in serum and ENPP1, and the base type
in CDNs contributed little to the stability of CDNs. However,
depending on the IFN-β-induction results, CDNs with 2′-5′
phosphodiester linkage had more potency to activate
hSTING, especially for the mixed pyrimidine CDNs. Taken
together, in order to balance the activity and stability of
CDNs, these results suggested that a more stable 2′-5′
phosphodiester substitution would be better suited to the
clinical application.
36) CDNs that containing four natural bases (A, G, C, U) and
two linkage directions (2′-5′-linked and 3′-5′-linked phos-
phodiester) have herein been synthesized and evaluated
against all five hSTING SNPs reported so far. Surprisingly,
we have found for the first time that c[G(2′,5′)U(2′,5′)] has
expressed excellent activation activity toward IFN-β induc-
tion on all five hSTING variants, comparable to c[G(2′,5′)A
(3′,5′)]. The serum stability screen by serum/ENPP1 hydro-
lase has shown that 3′-3′ analogues showed more stable than
2′-3′ and 2′-2′ analogues. Our results showed that different
CDNs activate all five hSTING protein variants differently,
normally with lower activity against hSTING-R232H and
hSTING-R293Q. It is very interesting to note that three
distinctive phosphodiester linkage types (3′-3′, 2′-3′, 2′-2′) all
yield purine-purine CDNs with good activity, but only c[G
(2′,5′)U(2′,5′)] showing excellent activation activity against
all five hSTING mutants. Our docking study showed that the
Tyr167 and Arg238 of hSTING may provide the stacking
interaction with Uracil to enhance the CDN activation. Until
now,
3′-3′
CDNs
such
as
c[G(3′,5′)G(3′,5′)],
c[A(3′,5′)A(3′,5′)], c[G(3′,5′)A(3′,5′)] and c[A(3′,5′)U(3′,5′)]
were discovered in bacteria and 2′-3′ CDNs such as c[G
(2′,5′)A(3′,5′)] regulates immune system in mammalian.
However, 2′-2′ CDNs are not found yet in natural sources.
What is more, no CDNs are found in the plant yet. It causes
our great interests in founding whether CDNs, especially the
2′-2′ CDNs, could also play as second messengers in the
plant to regulate part of physiological bioactivities. Further
study is ongoing to gain our understanding on the mechan-
ism.
Acknowledgements This work was supported by the National Key Re-
search and Development Program of China (2017YFD0200500) and the
National Natural Science Foundation of China (21740002, 21837001). We
also thanks Prof. Junmin Quan for the support of THP-1-Lucia cells and
Prof. Dongsheng Guo for the help of ITC assay.
4 Conclusions
Conflict of interest The authors declare that they have no conflict of
interest.
Cyclic dinucleotides (CDNs) have been recognized as im-
portant secondary messengers both in prokaryotes and eu-
karyotes recently. For the first time, all possible (a total of
Supporting information The supporting information is available online at
http://chem.scichina.com and http://link.springer.com/journal/11426. The

12
Wang et al. Sci China Chem
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