Design,Synthesis,and Biological Activity Evaluations Of Novel STING Receptor Agonists

Innate immunity is the first-line natural barrier of the host for the elimination of pathogenic infections that can be triggered by pattern recognition receptors?PRRs?,such as cytosolic DNA sensors.STING is both a pattern-recognition receptor,which directly binds cyclic dinucleotides?CDNs?via its C-terminal domain,and an adaptor molecule,which can be activated by multiple cytoplasmic receptors of dsDNA.STING plays a vital role in the activation of innate immune responses.Accumulated evidence has demonstrated that the cyclic GMP-AMP synthase?cGAS?is the major dsDNA cytosolic sensor allowing STING to function.The essential mechanism for innate immune sensing of various pathogens and tumours exists in the cGAS–STING pathway in which cGAS is activated by cytoplasmic DNA to subsequently catalyse the production of cyclic GMP-AMP?cGAMP?.2'3'-cGAMP is a non-canonical CDN that binds to STING and induces it to oligomerize,leading to a downstream signalling cascade including recruiting the serine/threonine-protein kinase1?TBK1?,phosphorylating the interferon regulatory transcription factor3?IRF3?and nuclear factor-?B?NF-?B?,and producing cytokines and proteins such as the type I interferons?IFNs?,interleukin 6?IL-6?,and tumour necrosis factor??TNF??.Therefore,STING is regarded as a promising target in various fields,including immunization,autoinflammation,and cancer therapeutics.The main contents of this research work include:1.Design and Synthesis of novel STING agonists based on STING crystal complexesPharmacologic activation of STING-dependent signaling has shown promise in diverse clinically impactful applications including broad-acting antiviral treatments,vaccine adjuvants and immunogenic tumor clearance.This has led to academic and commercial efforts to formulate CDNs for pharmaceutical use including their advancement to an ongoing clinical trial.Unfortunately,CDNs may be chemically undesirable for research and clinical work since:1)They violate Lipinski rules for druglikeness and are not amenable to large structural changes;2)They are susceptible to phosphodiesterase-mediated degradation;3)Their size and hydrophilicity render them impermeable to cell membranes.Small molecular STING activators can mitigate these factors,as well-exemplified by the mouse-specific compound 5,6-dimethylxanthenone-4-acetic acid?DMXAA?.Identification of novel small molecule STING agonists that are efficacious across species are thus highly sought since they may develop valuable research tools to understand STING-mediated processes.Furthermore,their use in animals enables broad assessment of safety and biological mechanisms.The seminal contributions of Patel and co-workers,which includes subtle mutations in protein sequences of STING now able to endow human STING with DMXAA sensitivity,inspire us to begin our study.Hence,our innovations of molecule design originate from comprehensively analysing the similarities of DMXAA and 10-carboxymethyl-9-acridanone?CMA?,especially the relationship between molecules and proteins.The following information has been revealed:1)Maintaining the structure of three rings guarantees the interaction between two small molecules;2)The 5,6-dimethyl substitutes of DMXAA are highly significant since it can produce a strong hydrophobic effect with the surrounding amino acids;3)The mutant substitutions?S162A and Q266I?of hSTING at the region of binding-pocket correspond to the positions of DMXAA at C1/C2 and C7;4)Referencing the structure of DMXAA,the carboxyl group on the N atom of CMA molecule can be transferred to C4 position.Previous work synthesized C7-position DMXAA derivatives that contain a hydrogen bonding donor and acceptor or a halide,but none of the compounds can bind to hSTING.Thus,we designed and synthesized four types of small molecules of a relevance skeleton whose changes concentrated on assorted groups at C1/C2 and C7 positions of compounds.At present,the synthetic routes of various small molecule compounds have been opened up,and more than 30 novel XAA or acridone derivatives have been synthesized.Next,the work of biological activity screening will be introduced,and the screening results will help us to further optimize small molecules.2.Screening and identification of acridone derivatives as broad-spectrum STING agonists at the cellular levelTo thoroughly evaluate the four kinds of analogues,we elaborately selected three cell lines in which the STING gene has been transfected or knocked out:293T-hSTING-R232 cells,293T-mSTING cells,and THP1-KO-STING cells.STING is either deactivated,undetectable,or not expressed in certain cell lines,such as human embryonic kidney cells?HEK293?.Therefore,human or murine specific STING agonist stimulation can be respectively assessed in 293T-hSTING-R232 cells or293T-mSTING cells by monitoring IFN-stimulated response elements and?ISRE?-induced secreted embryonic alkaline phosphatase?SEAP?production.Then,we employed THP1-KO-STING cells to confirm whether the compound exhibits the function of STING-dependent cytokine induction.The screening process was divided into two steps.Together,six potent compounds were discovered from the various synthetic precursors and their derivatives by two-step selection.Three were non-species-specific agonists?compound 11,compound 28 and compound 34?,and none of these compounds exhibited any IRF or NF-?B activities in the knockout hSTING cells,while the other compounds?compound 2,compound 3 and compound 33?just activated the murine STING pathway.Fortunately,some of the acridone analogues we discovered were powerful and helpful,especially compound 34,which displayed approximately similar activities to 2'3'-cGAMP at activating both pathways(human:EC₅₀=7.45?M,murine:EC₅₀=10.23?M).Additionally,some information provided by comprehensive and detailed SARs implied that removing any substituents of compound 34 could weaken its activity.Therefore,in our next study,compound 34can be used as the lead compound,mainly modifying the positions of N,C-4 and C-7,so as to enhance the activating activity of acridone analogues to the STING pathway.3.Mechanism study of acridone STING agonistsWe mainly studied and explored the mechanisms of protein levels and cytokine induction levels.According to whether STING agonists directly bind to STING protein to exert immune effect,STING agonists can be divided into direct and indirect types.CDNs and DMXAA are both direct STING agonists,which have strong activation and clear binding mechanism,and can be used as tool molecules to study the new mechanism of STING pathway.However,the activations of indirect STING agonists were generally inferior.They may activate upstream or downstream proteins of the STING pathway to induce the production of immune factors,and may even indirectly activate the STING pathway by activating other pathways.Therefore,it is very important to verify whether the active compounds directly bind to STING protein at the protein level for the activity evaluation of STING agonists.To verify whether acridones agonists directly activate the STING-TBK1-IRF3 pathway through STING,we first used human STING binding kits provided by Cisbio.We successfully demonstrated that acridone STING agonists activate their pathway for induction of immune factors by directly binding to STING protein.The competitive binding data identified compound 34(IC₅₀=1.92?M)as exhibiting the strongest ligand-human STING interaction among all our synthetic agonists.To further cross-validate the direct binding of compound 34 with hSTING CTD,we conducted a universal and authoritative method called isothermal titration calorimetry?ITC?to measure the Kd value of compound 34.The results of ITC were consistent with that of the binding kits,and compound 34 is a direct STING agonist.At the same time,the binding mechanism of acridone molecules with STING was also discussed.Finally,we investigated the mechanism of cytokine induction levels of compound 34.STING pathway mainly produced the cytokines,including IFN?,TNF?,IL-6 and IP-10.To confirm the functional consequence of binding,we employed Real-time Quantitative Polymerase Chain Reaction?qPCR?to examine the effects of the best compound 34 on proinflammatory-cytokine gene expression in a native system?THP-1 cells?,using well-known STING binders 2'3'-c GAMP as a control.At the same time,two gradients of concentration and time were set for STING agonists in the experiments.By comprehensive analysis,we observed that compound 34 and2'3'-cGAMP generally exhibited potent action in excreting cytokines.Although the former gave a more fast,powerful,and durable induction,the results can be explained by the different mechanisms between compound 34 and 2'3'-cGAMP.We boldly presumed that compound 34 can induce both STING pathways,and the activation of NF-?B-pathway is more active than 2'3'-cGAMP.The innovations of the paper are as follows:1)Based on the STING protein crystal complexes,in order to obtain broad-spectrum STING agonists,four kinds of compounds were reasonably designed and more than 30 small molecular compounds with novel structures were successfully synthesized.2)After activity evaluation of the target compounds synthesized by different cell lines,acridone compounds were identified as novel small molecular STING agonists;these acridone STING agonists successfully broke through species-specificity and showed excellent activation activity in both human and mouse STING cells.3)After collating and summarizing the experimental data,the basic structure-activity relationships of the compounds were analyzed and summarized;For acridone STING agonists,the methoxy group at C2 is the key group,and the dimethyl substitution at position 5,6 is the key to obtain humanization.4)The mechanism of acridone compounds activating STING pathway was also studied;The experimental results displayed that acridone STING agonists directly bind to STING to activate the pathway,and can also activate STING-IRF3and STING-NF-?B pathways.In this paper,2'3'-cGAMP was selected as the positive control for the following reasons:1)it is a natural STING agonist and the representative of CDNs compounds;2)it is the gold standard for the study of STING agonists and pathways,and it is the most convincing compared with it.It is worth noting that the activation effect of most non-CDNs STING agonists is much lower than that of 2'3'-cGAMP,but by many aspects of evaluation,we found that the activation activity of acridone compounds is slightly stronger than that of 2'3'-cGAMP.In summary,acridone STING agonists have the advantages of strong activation effect,small molecular weight,large modification space and clear action mechanism,which not only lay a foundation for the discovery of new mechanisms of STING pathway,but also provide new ideas for anti-tumor,antibacterial and antiviral immunotherapy.