Design, Synthesis and Biological Evaluation of (2′,5′ and 3′5′-Linked) cGAMP Analogs that Activate Stimulator of Interferon Genes (STING)

Abstract

Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor transmembrane protein that plays a pivotal role in innate immune system. STING agonists, such as endogenous cyclic dinucleotide (CDN) cyclic GMP-AMP (cGAMP), have been used in diverse clinical research for immunogenic tumor clearance, antiviral treatments and vaccine adjuvants. CDNs containing noncanonical mixed 3′-5′ and 2′-5′ phosphodiester linkages show higher potency in the activation of the STING pathway. In this study, a series of 2′3′-CDNs were designed and synthesized through a modified one-pot strategy. We then established a surface plasmon resonance (SPR)-based binding assay to quantify the binding affinities of synthesized CDNs for human STING, which requested a minuscule amount of sample without any pretreatment. Using this assay, we identified compound 8d (KD = 0.038 μM), a novel CDN that showed higher binding affinity with hSTING than cGAMP (KD = 0.543 μM). Cellular assays confirmed that 8d could trigger the expression of type I IFNs and other proinflammatory cytokines more robust than cGAMP. 8d also exhibited more resistant than cGAMP to enzymatic cleavage in vitro, indicating the successful improvement in drug availability. These findings provide guidelines for the design and structural optimization of CDNs as STING agonists.

2′,3′‐Cyclic GMP‐AMP Dinucleotides for STING‐Mediated Immune Modulation: Principles, Immunotherapeutic Potential, and Synthesis

“…[30,148] As mentioned above, phosphorothioate nucleotides are typically prepared by oxidative sulfurization of phosphite triesters synthesized via a P(III)-based approach, such as the Hphosphonate or phosphoramidite method. [141,146] The synthesis…”

“…Recently, Wang et al developed a phosphoramidite cyclization method to synthesize 2',3'-cGAMP analogues that contain one purine and one pyridine nucleoside. [146] The synthesis starts with 2'-O-TBS-pyridine nucleoside 92 (Scheme 6), which is coupled with protected guanosine phosphoramidite 61 followed by phosphite oxidation to afford linear dinucleotide 93. Then, the key cyclization step is efficiently achieved using bifunctional phosphoramide 94, which reacts with the free 3'-OH and 5'-OH of 93.…”

CDK inhibitor Palbociclib targets STING to alleviate autoinflammation

“…After identifying Palbociclib as a STING-binding compound, we went on to confirm the interaction of Palbociclib with STING using a real-time oblique-incidence reflectivity difference (OI-RD) assay. Palbociclib had a strong binding affinity to recombinant STING protein with a K D of 6.01 nM (Fig 4A), which was much stronger than the affinity of cGAMP to STING (K D = 543 nM) (Xie et al, 2020). We further generated biotin-conjugated Palbociclib by linking biotin to the amidogen residue of Palbociclib (Fig 4B).…”

Mn-phenolic networks as synergistic carrier for STING agonists in tumor immunotherapy

“…Presently, ADU-S100 is undergoing phase I/II clinical trials for treating advanced metastatic solid tumors and lymphomas [ [26] , [27] , [28] ]. Nonetheless, ADU-S100 confronts several challenges, including issues related to stability, negative charge-associated impediments to effective cellular targeting, inefficient cytoplasmic transport, and heightened hydrophilicity [ 18 , 23 , [29] , [30] , [31] ]. Consequently, the predominant mode of ADU-S100 administration remains intratumoral delivery.…”