A Phosphotriester-Masked Dideoxy-cGAMP Derivative as a Cell-Permeable STING Agonist

Abstract

2',3'-Cyclic GMP-AMP (cGAMP) is a cyclic dinucleotide second messenger in which guanosine and adenosine are connected by one 3'-5' and one 2'-5' phosphodiester linkage. It is formed in the cytosol upon detection of pathogenic DNA by the enzyme guanosine-monophosphate-adenosine monophosphate synthase (cGAS). cGAMP subsequently binds to the adaptor protein stimulator of interferon genes (STING) to elicit an innate immune response leading to the production of type I interferons and cytokines. STING agonists are a highly promising avenue for an immuno-oncological anticancer therapy. A particular challenge with cyclic dinucleotide STING agonists are the two negative charges of the phosphodiester linkages, which strongly reduce the ability of such compounds to penetrate cell membranes. The development of cell-permeable STING agonists that can stimulate the immune system enhancing their anticancer potency is currently of utmost importance in the field. Herein, we report the development of a dideoxy derivative of cGAMP as a phosphotriester prodrug, where the negative charge of the phosphate backbone has been masked with a thioester. We found that this thioester-protected compound features a dramatic increase in its cellular potency that rises from EC₅₀=5 μM to 25 nM. The new compound is envisioned to enable an efficient STING-agonist-based anticancer therapy.

Keywords: STING agonists; cyclic dinucleotides; immuno-oncology; prodrugs; thioesters.

Figure 1

A) cGAMP 1, dd‐cGAMP 2 and the thioester‐protected cyclic dinucleotide 3 prepared for this study. B) Mechanism of cleavage of the thioester protecting group.

Scheme 1

A) Synthesis of 3’‐deoxy guanosine 12. B) Synthesis of bis‐HTE‐dd‐cGAMP 3. C) Synthesis of the photolabile protecting group 6. C) Synthesis of the HTE‐protecting reagents 9 and 15. Conditions: a) 6, 18‐crown‐6, NaH, THF, 0 °C—rt, 14 h, 61 % yield; b) TBAF, THF, rt, 14 h, 84 % yield; c) DMTrCl, DMAP, pyridine, rt, 2 d, 77 % yield; d) 2‐Cyanoethyl N,N,N’,N’‐tetraisopropylphosphorodiamidite, DIPAT, CH₂Cl₂, rt, overnight; e) 1. BTT, CH₃CN, rt, 2 h. 2. TBHP, rt, 30 min; f) 3 % v/v DCA, CH₂Cl₂, rt, 10 min, 58 % yield; g) 7, 18‐crown‐6, NaH, THF, 0 °C–rt, 24 h, 85 % yield; h) TBAF, THF, rt, overnight, 62 % yield; i) DMTrCl, DMAP, pyridine rt, 2 d, 82 % yield; j) 16, DIPAT, CH₂Cl₂, rt, overnight; k) 1. 12, 16, BTT, CH₃CN, rt, 2 h. 2. TBHP, rt, 30 min; l) 3 % v/v DCA, CH₂Cl₂, rt, 10 min, 75 % yield; m) tBuNH₂, CH₃CN rt, 30 min, 71 % yield; n) 2,4,6‐triisopropylbenzenesulfonyl chloride, NMI, THF, rt, 2 d, 58 % yield; q) hν (365 nm), CH₃CN, rt, 12 min, 63 % yield p) CDI, THF, 0 °C, 1 h, rt, 30 min, 71 % yield; q) DCC, CH₃CN, 0 °C–rt, overnight, 62 % yield; r) bis(diisopropylamino)chlorophosphine, Et₃N, Et₂O, rt, 18 h, 92 % yield. PPG: photolabile protecting group.

Figure 2

A) HPLC chromatograms of the in vitro cleavage of bis‐HTE‐dd‐cGAMP 3 with enzyme CES1 after 0 h (top) and 24 h (bottom). The amount of 3 decreases (orange dotted‐line box) with ongoing incubation time in the presence of CES1. Simultaneously, the amount of unprotected dd‐cGAMP 2 increases (blue dotted‐line box) with incubation time (see Figures S1 for more time‐points). During the cleavage, an intermediate is observed that carries one HTE protecting group (black dotted‐line box). B) Extracted ion chromatograms representing the intracellular cleavage in THP1 cells after 30 min (top) and 4 h (bottom) of 3 (left) to 2 (right). After 30 min, we can see a peak for 3 and a small peak of the cleaved dd‐cGAMP 2. After 4 h, a small peak is seen for 3 but a larger peak of 2 is observed.

Figure 3

Dose‐dependent response of THP1‐Dual^TM cells to dd‐cGAMP 2 (orange) and bis‐HTE‐dd‐cGAMP 3 (green) in comparison to the natural STING ligand cGAMP 1 (blue). Dots represent the mean of at least three biologically independent experiments, the shade represents the 95 % confidence interval (CI).

Figure 4

A volcano plot illustrating differentially expressed proteins in THP1 cells treated with bis‐HTE‐dd‐cGAMP 3 for 18 h compared to untreated cells (n=4). Proteins with significant upregulation (cut‐off p‐value 0.05 and fold‐change >2) are highlighted in red. DAVID analysis identified 3 highly enriched clusters within this group. Cluster‐specific proteins are depicted as follows: proteins in cluster 1 are represented by blue dots, those in both cluster 1 and 2 by blue rectangles, and proteins present in all 3 clusters are labeled and shown as green rectangles with blue border.