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. 2023 Nov 16;30(11):1366-1376.e7.
doi: 10.1016/j.chembiol.2023.07.002. Epub 2023 Aug 2.

Arabinose- and xylose-modified analogs of 2',3'-cGAMP act as STING agonists

Wei Xie  1 , Lodoe Lama  2 , Xuejing Yang  3 , Vitaly Kuryavyi  4 , Shibani Bhattacharya  5 , Ilona Nudelman  6 , Guangli Yang  7 , Ouathek Ouerfelli  7 , J Fraser Glickman  6 , Roger A Jones  8 , Thomas Tuschl  9 , Dinshaw J Patel  10
Affiliations

Arabinose- and xylose-modified analogs of 2',3'-cGAMP act as STING agonists

Wei Xie et al. Cell Chem Biol. .

Abstract

Stimulator of interferon genes (STING) agonists are promising candidates for vaccine adjuvants and antitumor immune stimulants. The most potent natural agonist of STING, 2',3'-cyclic GMP-AMP (2',3'-cGAMP), is subject to nuclease-mediated inherent metabolic instability, thereby placing limits on its clinical efficacy. Here, we report on a new class of chemically synthesized sugar-modified analogs of 2',3'-cGAMP containing arabinose and xylose sugar derivatives that bind mouse and human STING alleles with high affinity. The co-crystal structures demonstrate that such analogs act as 2',3'-cGAMP mimetics that induce the "closed" conformation of human STING. These analogs show significant resistance to hydrolysis mediated by ENPP1 and increased stability in human serum, while retaining similar potency as 2',3'-cGAMP at inducing IFN-β secretion from human THP1 cells. The arabinose- and xylose-modified 2',3'-cGAMP analogs open a new strategy for overcoming the inherent nuclease-mediated vulnerability of natural ribose cyclic nucleotides, with the additional benefit of high translational potential as cancer therapeutics and vaccine adjuvants.

Keywords: 2′,3′-cGAMP; analogs targeting STING; arabinose and xylose analogs; cancer therapeutics; resistance to ENPP1; stability in human serum; vaccine adjuvants.

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Conflict of interest statement

Declaration of interests T.T. is a cofounder and scientific advisor, and D.J.P. and L.L. are consultants of Ventus Therapeutics.

Figures

Figure 1.
Figure 1.. Chemical structures of arabinose- and xylose-modified analogs of 2’,3’-cGAMP.
(A-D) Chemical formulas of 2′,3′-cGAMP (panel A), c[2’,3’-(ara-2’-G, ribo-3’-A)-MP], (panel B), c[2’,3’-(ribo-2’-G, xylo-3’-A)-MP], (panel C), and c[2’,3’-(ara-2’-G, xylo-3’-A)-MP], (panel D). The analogs contain the noncanonical 2′,5′ (GpA step) and the canonical 3′,5′ (ApG step) phosphodiester linkages, while the ribose is replaced by arabinose or xylose as indicated.
Figure 2.
Figure 2.. Syntheses of ara/xylo isomers of 2’,3’-cGAMP.
The syntheses follow a common route, starting from coupling of an adenosine H-phosphonate derivative, 2 or 5, with a guanosine phosphoramidite derivative, 7 or 10. The linear dimers produced, 11, 14, or 17, are then partially deprotected and cyclized to give the protected cyclic dimers 12, 15, or 18. Deprotection then gives the final products 13, 16, or 19.
Figure 3.
Figure 3.. The ara/ribo-13 and ribo/xylo-19 analogs of 2’,3’-cGAMP are effective binders of STING.
(A-E) Impact of 2’,3’-cGAMP (100 μM) and analogs (100 μM) on the melting temperature (Tm) of recombinant hSTING CBD (panel A) and mSTING CTD (panel B), as well as common hSTING alleles R232 (panel C), R293Q (panel D) and G230A/R293Q (panel E). d(Fluor)/dT represents the first derivative of the fluorescence melting curve. (F and G) ITC curves of 2’,3’-cGAMP and analogs to hSTING CBD (panel F) and mSTING CBD (panel G).
Figure 4.
Figure 4.. Crystal structures of hSTING CBD in complex with ara/ribo-13 and ribo/xylo-19.
(A) Crystal structure of hSTING CBD bound with ribo/xylo-19. The individual symmetry-related subunits of hSTING are color-coded in magenta and yellow in a ribbon representation, whereas the bound ribo/xylo-19 is shown in cyan in a stick representation. (B) Details of the interactions between ara/ribo-13 and the dimeric hSTING. (C) Two close-up alternate views comparing bound ara/ribo-13 (in cyan) with bound 2’,3’-cGAMP (in silver; PDB accession code 4KSY) on complex formation with hSTING. (D) Crystal structure of hSTING CTD bound with ribo/xylo-19 (in green). (E) Details of the interactions between ribo/xylo-19 and the dimeric hSTING. (F) Two close-up alternate views comparing bound ribo/xylo-19 (in green) with bound 2’,3’-cGAMP (in silver) on complex formation with hSTING.
Figure 5.
Figure 5.. The ara/ribo-13 and ribo/xylo-19 2’,3’-cGAMP analogs are resistant to ENPP1 hydrolysis.
(A) The hydrolase activity of recombinant human ENPP1 (25 nM) on ATP (100 μM) or CDNs (100 μM) was determined after 3 h incubation monitoring the consumption of each substrate using RF-MS. Data shown are mean ± S.D. for three replicates and are representation of two independent experiments. Significance was tested using unpaired two-tailed Student′s t-test (****p < 0.0001, nsp = not significant). (B) Time course hydrolysis analysis of each substrate (100 μM) by ENPP1 (10 nM) by RF-MS. AUC (area under the curve) for each sample was normalized against AUC of GTP used as an internal standard and added into each sample after the end of reaction but prior to running on RF-MS. Data shown are mean ± S.D. for three replicates and are representation of two independent experiments. Half-life measurement was obtained by one phase exponential decay fitting using Prism software. (C) Stability analysis of each substrate in human serum by RF-MS. Data shown are mean ± S.D. for two replicates and are representation of two independent experiments. Half-life measurement was obtained by one phase exponential decay fitting using Prism software.
Figure 6.
Figure 6.. The ara/ribo-13 and ribo/xylo-19 2’,3’-cGAMP analogs trigger STING signaling pathway.
(A and B) Potency of 2’,3’-cGAMP, ara/ribo-13, ribo/xylo-19, and 2’,3’-cGsAsMP was tested in human THP1-Dual cells by stimulation in the presence of a range of different ligand concentrations in media (panel A) or in complex with Lipofectamine2000 (panel B). (C and D) Potency of 2’,3’-cGAMP, ara/ribo-13, ribo/xylo-19, and 2’,3’-cGsAsMP was tested in murine RAW-Lucia cells by stimulation in the presence of a range of different ligand concentrations in media (panel C) or in complex with Lipofectamine2000 (panel D). (E and F) STING pathway activation was tested for all sugar-modified analogs and 2’,3’-cGsAsMP in THP1-Dual cells (panel E) or RAW-Lucia cells (panel F) at ligand:Lipofectamine2000 complex concentration representing 2’,3’-cGAMP EC80 value (THP1-Dual) or EC50 value (RAW-Lucia). ara/xylo-16 fails to activate STING pathway in both cell lines. (G) Dose response analysis of 2’,3’-cGAMP, ara/ribo-13, ribo/xylo-19, and 2’,3’-cGsAsMP in THP1 cells by digitonin-permeabilization based ligand treatment assay. IFNB1 mRNA was measured by qRT-PCR for each of the indicated concentration and normalized to digitonin only control. Data shown in all panels are mean ± S.D. for three replicates and are representation of two independent experiments and the cellular EC50 values were calculated using GraphPad Prism. 2’,3’-cGsAsMP data points for 10 μM and 40 μM treatments were excluded from curve-fitting analysis to determine EC50 value in (G) due to anomalous behavior of the compound at higher concentrations indicative of potential onset of cellular toxicity.
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