Application of alantolactone and derivative thereof as STING inhibitor and preparation of medicine for treating STING dependent diseases

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of alantolactone and derivatives thereof as STING inhibitors and in preparation of medicines for treating STING dependent diseases. Cys309 is found as a targeting site for selective STING inhibition of a pathway, alantolactone is found as a treatment candidate molecule with dual functions, pathological signal transduction is inhibited through covalent modification while steady-state transport is maintained, and a basis is provided for development of drugs for treating STING dependent diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of costunolide and its derivatives as STING inhibitors and in the preparation of drugs for treating STING-dependent diseases. Background Technology

[0002] The cyclic GMP-AMP synthase (cGAS)-interferon gene-stimulating protein (STING) pathway is a key surveillance mechanism for detecting physiological and pathological abnormalities, including microbial invasion and cell damage, and plays a central role in innate immunity. cGAS, acting as a cytoplasmic DNA sensor, recognizes misplaced double-stranded DNA (dsDNA) and catalyzes the synthesis of the second messenger 2′,3′-cyclic GMP-AMP (cGAMP). cGAMP binding induces conformational rearrangement of the transmembrane domain of STING, leading to its oligomerization into side-by-side tetramers, which are subsequently transported from the endoplasmic reticulum (ER) to the ER-Golgi intercompartment (ERGIC) and the Golgi apparatus. Activated STING recruits TANK-binding kinase 1 (TBK1), which phosphorylates STING and interferon regulatory factor 3 (IRF3), ultimately driving the expression of type I interferons (IFNs) and interferon-stimulated genes (ISGs).

[0003] While optimal STING activation is crucial for pathogen defense and immune homeostasis, dysregulation of STING activity is associated with a variety of diseases, including autoimmune diseases, autoinflammatory syndromes, fibrotic diseases, and neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases, and Parkinson's disease. Overactivation of the cGAS-STING axis can induce neuronal apoptosis and neuroinflammation, highlighting the therapeutic potential of pharmacological STING inhibitors.

[0004] The development of STING inhibitors follows two distinct pharmacological strategies: non-covalent competitive inhibitors and covalent modifiers. Non-covalent drugs such as Compound1, Astin C, SN-011, and ATS exert their effects by reversibly occupying the cGAMP binding pocket of STING, thereby blocking the production of downstream interferons and cytokines. Although these reversible inhibitors dominate current pharmacological strategies, their therapeutic efficacy remains limited because their effectiveness largely depends on their binding affinity to the target protein. To overcome the limitations of non-covalent inhibitors, the covalent STING inhibitor H-151 exerts its effects by inhibiting activation-induced palmitoylation of STING. It establishes a strong, irreversible interaction with the Cys91 site of STING, thereby improving binding affinity and potency. Due to its potent STING inhibitory effect, it is currently widely used in basic research. Using H-151 as a lead compound, compound 42 was developed, which showed superior inhibitory potency and improved oral pharmacokinetic (PK) properties and bioavailability in a mouse model. In addition, endogenous nitro fatty acid metabolites—nitro fatty acids (NO2-FA) and 10-nitrooleic acid (CXA-10)—have been shown to covalently bind to the Cys88 and Cys91 sites of STING, inhibiting its palmitoylation and thereby reducing the production of pro-inflammatory cytokines and interferons.

[0005] However, the clinical translation of covalent inhibitors targeting Cys88 / 91 (such as H-151 and C-176) is limited by species-specific ineffectiveness and non-specific alkylation across the proteome. Nitrofuran-based compounds (C-176 / C-178) exhibit mixed reactivity, leading to off-target effects and poor inhibitory activity against human STING. Similarly, CXA-10 can inhibit STING, but it also suffers from poor specificity. Therefore, screening for novel STING inhibitors that can selectively inhibit pathological STING overactivation while preserving physiological immune surveillance functions, thereby advancing clinically viable therapies for STING-related diseases and cancer immunotherapy, remains a key technical challenge. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0007] As one aspect of the present invention, the present invention provides the application of calceolone as a STING inhibitor.

[0008] In this context, the calomelactone is a covalent inhibitor that selectively targets Cys309, which is a target of STING.

[0009] This invention also provides the application of calomelactin in the preparation of drugs for treating STING-dependent diseases.

[0010] The STING-dependent diseases include STING-dependent autoimmune diseases, STING-dependent neurodegenerative diseases, autoinflammatory syndromes, or fibrotic diseases.

[0011] The STING-dependent neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis, lysosomal storage diseases, and Parkinson's disease.

[0012] The present invention also provides the application of arugula lactone-related derivatives as STING inhibitors, wherein the arugula lactone-related derivatives include isoarugula lactone.

[0013] The aforementioned derivatives of gentian lactone include one or more of the following: gentian lactone, deoxygentianin, and isodeoxygentianin.

[0014] This invention also provides the application of inosinol-related derivatives in the preparation of drugs for treating STING-dependent diseases.

[0015] The beneficial effects of this invention: This invention discovers that human Cys309 (NCBI ID: 340061) or mouse Cys308 (NCBI ID: 72512) is a key allosteric hub regulating STING activation. The C309A mutation, while preserving classical STING transport, eliminates TBK1 recruitment by disrupting the stability of the active tetramer. Based on this discovery, this invention identifies Alantolactone (ALA), a sesquiterpene lactone compound derived from the Asteraceae family, as a covalent STING inhibitor targeting Cys309, achieving selective binding via Michael addition. ALA inhibits STING signaling assembly without impairing its transport to the Golgi apparatus, thus effectively decoupling structural dynamics from signal transduction function. Crucially, ALA retains its inhibitory efficacy against STING gain-of-function (GOF) mutants associated with autoinflammatory diseases. In vivo, ALA treatment reduces Trex1 levels. - / - The invention reduced mouse mortality and prolonged the survival of ALS model mice, while alleviating neuroinflammation and multi-organ damage. It identified Cys309 as a target site for selective STING inhibition in one pathway and ALA as a therapeutic candidate molecule with a dual role—maintaining homeostatic transport while covalently inhibiting pathological signal transduction—providing a basis for the development of drugs to treat STING-dependent diseases. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein: Figure 1 This verifies the necessity of cysteine ​​residues for STING activation. Figure 1 In the table, A shows the immunoblotting of diABZI-mediated STING activation using NEM as a cysteine ​​blocking agent; B shows the statistical analysis of changes in p-STING and p-TBK1 after diABZI-mediated STING activation using NEM as a cysteine ​​blocking agent; C shows the immunofluorescence detection of diABZI-mediated STING translocation changes and p-STING fluorescence intensity using NEM as a cysteine ​​blocking agent; D shows the statistical analysis of diABZI-mediated changes in p-STING fluorescence intensity fold change using NEM as a cysteine ​​blocking agent; E shows the statistical analysis of diABZI-mediated changes in STING aggregation area using NEM as a cysteine ​​blocking agent; F shows the immunoblotting of diABZI-mediated STING activation after transfection with empty vector, STING-WT, and STING-C10A, respectively; G shows the statistical analysis of diABZI-mediated changes in p-STING and p-TBK1 after transfection with empty vector, STING-WT, and STING-C10A, respectively. Changes in p-STING and p-TBK1 after iABZI-mediated STING activation; H represents the immunofluorescence detection of diABZI-mediated STING translocation changes and p-STING fluorescence intensity after transfection with empty vector, STING-WT, and STING-C10A, respectively; I represents the statistical analysis of diABZI-mediated STING aggregation area changes after transfection with empty vector, STING-WT, and STING-C10A, respectively; J represents the statistical analysis of diABZI-mediated p-STING fluorescence intensity changes after transfection with empty vector, STING-WT, and STING-C10A, respectively; K represents the detection of diABZI-mediated IFNβ fluorescein fold change after transfection with empty vector, STING-WT, and STING-C10A, respectively; L represents the detection of diABZI-mediated ISRE fluorescein fold change after transfection with empty vector, STING-WT, and STING-C10A, respectively.

[0017] Figure 2 This is a verification result of Cys309 mutation inhibiting STING signaling. Figure 2In the table, A represents the changes in diABZI-mediated relative mNRA levels of IFNB after transfection with empty vector, STING-WT, and different STING cysteine ​​mutations, respectively; B represents the diABZI-mediated STING activation after transfection with empty vector GFP, STING-WT-GFP, and STING-C309A-GFP, respectively; C represents the changes in diABZI-mediated relative mNRA levels after transfection with empty vector GFP, STING-WT-GFP, and STING-C309A-GFP, respectively; STING-WT-GFP, STING-C309A-GFP: Changes in p-STING and p-TBK1 after diABZI-mediated STING activation were statistically analyzed; D: Immunofluorescence detection of diABZI-mediated STING translocation changes and p-STING fluorescence intensity after transfection with empty vector STING-WT-GFP, STING-C88A / C91A-GFP, and STING-C309-GFP, respectively; E: DiABZI-mediated changes in p-STING fluorescence intensity after transfection with empty vector, STING-WT-GFP, STING-C88A / C91A-GFP, and STING-C309-GFP, respectively; F: DiABZI-mediated changes in STING aggregation area after transfection with empty vector, STING-C88A / C91A-GFP, and STING-C309-GFP, respectively; G: Transfection with empty vector, STING-WT-GFP, STING-C88A / C91A-GFP, and STING-C309-GFP, respectively. STING-WT-Flg and STING-C309A-Flag were used to detect changes in the interaction between STING-WT and STING-C309A and TBK1 after diABZI-mediated STING activation by co-immunoprecipitation and Western blotting; H represents transfection of empty vectors, respectively. STING-WT-Flg and STING-C309A-Flag were used to statistically analyze the changes in the interaction between STING-WT and STING-C309A and TBK1 after diABZI-mediated STING activation. I represents the changes in the STING-WT and STING-C309A tetramer model in molecular dynamics simulations. J represents the changes in the minimum distance of the STING-WT and STING-C309A tetramers in molecular dynamics simulations. K represents the changes in the root mean square deviation of the STING-WT and STING-C309A tetramers in molecular dynamics simulations. L represents the transfection of HEK293T cells with empty vector, STING-WT, STING-C309A, STING-N154S, STING-N154S / C309A, STING-R281Q, and STING-R281Q / C309A, respectively, and the diABZI-mediated STING activation was examined by immunoblotting.M represents the transfection of empty vectors, STING-WT, STING-C309A, STING-N154S, STING-N154S / C309A, STING-R281Q, and STING-R281Q / C309A, respectively. The changes in p-STING and p-TBK1 after diABZI-mediated STING activation were statistically analyzed.

[0018] Figure 3 This provides the results of verification regarding the inhibitory effect and mechanism of ALA on the STING pathway. Figure 3 In the diagram, A represents the changes in p-STING after diABZI-mediated STING activation in human HMC3 cells treated with different small molecules, as shown in the heatmap; B represents the changes in p-STING after DMXAA-mediated STING activation in mouse BV2 cells treated with different small molecules, as shown in the heatmap; C represents the molecular structure of alanine lactone, abbreviated as ALA; and D represents the diABZI-mediated STING activation detected by Western blotting after treatment with different concentrations of alanine lactone in human HMC3 cells. E shows the immunoblotting analysis of DMXAA-mediated STING activation in mouse BV2 cells treated with different concentrations of inosinol; F shows the comparison of the half-maximal inhibitory rates (ICP-50) of H-151 and inosinol using the IFNβ fluorescein reporter system; G shows the immunofluorescence detection of diABZI-mediated STING translocation changes and p-STING fluorescence intensity after treatment with DMSO, H-151, and inosinol, respectively; H shows the statistical analysis of diABZI-mediated STING activation after treatment with DMSO, H-151, and inosinol, respectively. Changes in p-STING fluorescence intensity; I represents the statistical changes in diABZI-mediated STING aggregation area after treatment with DMSO, H-151, and calceolone, respectively; J represents the changes in diABZI-mediated STING aggregates in non-denaturing gels, DSS-SDS gels, and SDS gels after treatment with DMSO and calceolone, respectively; K represents the changes in diABZI-mediated STING activation after treatment with DMSO, H-151, and calceolone, respectively, by co-precipitation and immunoblotting. The interaction between STING and TBK1 was observed; L was treated with DMSO, H-151 and calceolone respectively, and the changes in the interaction between STING and TBK1 after diABZI-mediated STING activation were statistically analyzed; M consisted of HEK293T cells transfected with empty vector, STING-WT, STING-N154S and STING-R281Q respectively, treated with calceolone, and the diABZI-mediated STING activation was examined by immunoblotting; N consisted of HEK293T cells transfected with empty vector, STING-WT, STING-N154S and STING-R281Q respectively, treated with calceolone, and the changes in p-STING and p-TBK1 after diABZI-mediated STING activation were statistically analyzed.

[0019] Figure 4 The figure shows the validation results of ALA-specific targeting of STING Cys309. Figure 4 In the table, A shows the changes in STING protein stability detected by Western blotting after treatment with inosinol in human HMC3 cells followed by different temperature treatments; B shows the changes in relative gray intensity of STING after treatment with inosinol in human HMC3 cells followed by different temperature treatments; C shows the molecular weight of STING covalently bound at position 309 by inosinol and STING using mass spectrometry; D shows the synthesized biotinylated inosinol (Bio-ALA); E shows the STING protein level detected by immunoprecipitation and Western blotting after treatment with different concentrations of biotinylated inosinol in human HMC3 cells; F shows the STING protein level detected by immunoprecipitation and Western blotting after pretreatment with NEM and subsequent treatment with biotinylated inosinol in human HMC3 cells; G shows the STING protein level detected by immunoprecipitation and Western blotting after pretreatment with inosinol and subsequent treatment with biotinylated inosinol in human HMC3 cells; H shows the STING protein level detected by immunoprecipitation and Western blotting after treatment with human HeLa... STING knockout cells were transfected with STING-WT, STING-C309A, STING-C10A, and STING-C9A (A309C), respectively, and treated with biotinylated costunolide. STING protein levels were assessed by co-precipitation and Western blotting. Image I shows purified STING-WT and STING-C309A proteins, treated with biotinylated costunolide, and STING protein levels were assessed by Coomassie Brilliant Blue and Western blotting. Image J shows human HeLa STING knockout cells transfected with STING-WT and STING-C309A, treated with costunolide, and then subjected to different temperature treatments; STING protein stability was detected by Western blotting. Image K shows human HeLa... In STING knockout cells, STING-WT and STING-C309A were transfected, treated with aucubin, and then subjected to different temperature treatments. The changes in STING-WT protein stability were statistically analyzed. L represents the changes in STING-C309A protein stability in human HeLaSTING knockout cells, transfected with STING-WT and STING-C309A, treated with aucubin, and then subjected to different temperature treatments.

[0020] Figure 5 This is a verification diagram of ALA binding to STING. Figure 5In the diagram, A shows the STING protein level in mouse BV2 cells after treatment with different concentrations of biotinylated costunolide, followed by immunoprecipitation and Western blotting; B shows the STING protein level in mouse BV2 cells after pretreatment with NEM followed by treatment with biotinylated costunolide; C shows the STING protein level in mouse BV2 cells after pretreatment with costunolide followed by treatment with biotinylated costunolide, followed by immunoprecipitation and Western blotting; D shows the docking diagram of STING-WT and costunolide; and E shows the amino acid interaction site diagram of the docking between STING-WT and costunolide.

[0021] Figure 6 To suppress Trex1 in ALA - / - Verification diagrams showing inflammation and mortality in mice. Figure 6 In the diagram, A represents wild-type and Trex1. - / - Schematic diagram of mouse treatment with inosinol; B represents wild-type and Trex1. - / - Plot showing survival percentages of mice treated with inosinol; C represents wild-type and Trex1. - / - Mice treated with inosinolone showed changes in LDH levels; D was wild-type and Trex1. - / - Mice treated with inosinol showed changes in spleen size; E represents wild-type and Trex1. - / - Changes in spleen / body weight in mice treated with inosinol; F represents wild-type and Trex1. - / - Mice treated with inosinol showed histopathological changes in heart and kidney tissues; G was wild-type and Trex1. - / - Changes in cardiac histopathological scores in mice treated with inosinol; H represents wild-type and Trex1. - / - Changes in kidney histopathological scores in mice treated with inosinol; I represents wild-type and Trex1. - / - Mice treated with inosinol showed changes in immunoblotting of spleen, heart, and kidney tissues; J represents wild-type and Trex1. - / - Mice treated with Trexone lactone had their spleen tissue altered by real-time quantitative PCR, detecting changes in If1b, Irf7, Isg15, and Cxcl10 levels. K represented wild-type and Trex1... - / - Mice treated with Trexone lactone had their levels of If1b, Irf7, Isg15, and Cxcl10 in cardiac tissue detected by real-time quantitative PCR; L represented wild-type and Trex1... - / - Mice were treated with inosinol, and changes in If1b, Irf7, Isg15, and Cxcl10 in kidney tissue were detected by real-time quantitative PCR.

[0022] Figure 7 A diagram used to validate ALA in reducing ALS in a mouse model. Figure 7 In the table, A shows the changes in STING pathway protein activation in the brain of TDP-43 rNLS8 mice induced and uninduced by tetracycline DOX, as detected by Western blotting; B shows the survival percentage of TDP-43 rNLS8 mice treated with inosinol; C shows the treatment pattern of TDP-43 rNLS8 mice with inosinol; D shows the leg-hugging changes in TDP-43 rNLS8 mice treated with inosinol; E shows the body weight changes in male TDP-43 rNLS8 mice treated with inosinol; F shows the body weight changes in female TDP-43 rNLS8 mice treated with inosinol; G shows the grip strength score changes in TDP-43 rNLS8 mice treated with inosinol; H shows the rotarod drop time changes in TDP-43 rNLS8 mice treated with inosinol; I shows the TDP-43 rNLS8 mice treated with inosinol. Changes in total movement distance in rNLS8 mice treated with inosinol; J represents TDP-43 rNLS8 mice induced and uninduced by tetracycline DOX, treated with inosinol, and changes in STING pathway protein activation in the brain were detected by immunoblotting; K represents TDP-43 rNLS8 mice treated with inosinol, and changes in If1b, Irf7, Isg15, and Cxcl10 in kidney tissue were detected by real-time quantitative PCR.

[0023] Figure 8 Validation of STING inhibitors for screening human HMC3 cells. Figure 8 In the table, A represents the changes in the diABZI-mediated STING activation pathway in human HMC3 cells treated with different small molecules CI-1 to CI-9, detected by Western blotting; B represents the changes in the diABZI-mediated STING activation pathway in human HMC3 cells treated with different small molecules CI-10 to CI-18, detected by Western blotting; C represents the changes in diABZI-mediated p-STING levels in human HMC3 cells treated with different small molecules CI-1 to CI-18, with a p-STING level below 1.5 defined as a threshold, and values ​​below 1.5 indicating an effective inhibitory molecule; D represents the structure of the effective inhibitory molecule for STING, where CI-2 is costunolide, CI-5 isocostunolide, CI-7 isogentenolide, CI-9 is deoxygentenin, and CI-11 isodeoxygentenin. Detailed Implementation

[0024] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0025] Materials and Methods: Cell culture: HeLa, HEK293T, HMC3, and BV2 cells were cultured in DMEM medium (containing L-glutamine and 4.5 g / L glucose); all media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Doxycycline-induced stable cell lines were constructed as follows: HP138-STING-WT-GFP (or HP138-STING-C309A-GFP) and HP216 plasmids were co-transfected into HeLa STING knockout (KO) cells using Lipofectamine 2000 (Invitrogen), and selection was performed with 10 μg / ml puromycin. All cells were cultured at 37°C and 5% CO2.

[0026] Doxycycline (#A603456) and puromycin (#A606719) were purchased from Sangon® Biotech.

[0027] N-ethylmaleimide (NEM, #BD144276), EDCI (#BD19757), DMAP (#BD17199) and N-biotinyl-6-aminohexanoic acid (#BD43051) were purchased from Bidepharm.

[0028] DMXAA (#S1537), diABZI (#S8796), and H-151 (#S6652) were purchased from SelleckChem.

[0029] 2'3'-cGAMP purchased from Invivogen.

[0030] Alantolactone (#PU0800-0025) was purchased from Chengdu Push Bio-technology Co., Ltd.

[0031] 1β-Hydroxyalantolactone (#CFN92600) was purchased from ChemFaces.

[0032] Anti-β-tubulin (#M1305-2) and anti-GFP tag (#ET1607-31) were purchased from HUABIO.

[0033] The anti-Flag-tagged HRP direct antibody (#M185-7) and the anti-HA-tagged HRP direct antibody (#M180-7) were purchased from MBL.

[0034] Anti-STING (#19851-1-AP) purchased from Proteintech.

[0035] Antiphosphorylated STING (Ser365, #72971), antiphosphorylated STING (Ser366, #19781), anti-TBK1 (#380066), antiphosphorylated TBK1 (Ser172, #5843), anti-IRF3 (#4302), and antiphosphorylated IRF3 (Ser396, #29047) were purchased from Cell Signaling Technology.

[0036] Anti-streptavidin-HRP direct labeling antibody (#A0305-1) was purchased from Beyotime.

[0037] Anti-Flag (DYKDDDDK) affinity gel (#B23102) and anti-HA magnetic beads (#B26202) were purchased from Bimake.

[0038] The fluorescent secondary antibodies used for immunofluorescence, namely goat anti-rabbit Alexa Fluor 546 (#A-11010) and goat anti-mouse Alexa Fluor 405 (#A-31553), were purchased from Thermo Fisher Scientific.

[0039] C57BL / 6J mice and Trex1-deficient mice (strain number T013987) were purchased from GemPharmatech, Nanjing, China. To evaluate the efficacy of ALA in Trex1- / - mice, the following procedures were performed: wild-type (WT) or Trex1-deficient mice (male, 4 weeks old, n = 12 per group) were intraperitoneally injected once daily with ALA (20 mg / kg) or a carrier dissolved in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% PBS for 56 consecutive days. Mice were sacrificed by inducing anesthesia in a CO2 chamber followed by cervical dislocation.

[0040] B6;C3-Tg(NEFH-tTA)8Vle / J (JAX strain number 025397) and B6;C3-Tg(tetO-TARDBP*)4Vle / J (JAX strain number 014650) mice were purchased from The Jackson Laboratory. The experimental double transgenic rNLS8 model (genotype: B6;C3-Tg(NEFH-tTA)8Vle Tg(tetO-TARDBP*)4Vle / J) was obtained by hybridization of these strains. Both the double transgenic animals and their breeding parents were given doxycycline (Dox, 2 mg / mL) in their drinking water to inhibit TDP-43 protein expression. To evaluate the efficacy of ALA in this model, 8-week-old rNLS8 mice (equal number of males and females, n = 4 per group) were given intraperitoneal injections daily: ALA (20 mg / kg) or DMSO solvent control group (5% DMSO + 40% PEG300 + 5% Tween80 + 50% PBS), with weekly weight monitoring and behavioral phenotype assessment.

[0041] Rotating bar test: Motor function was assessed using a rotating bar instrument (XR-6C, Shanghai Xinruan Information Technology Co., Ltd.). Training phase: Mice received 5 minutes of accelerated rotating bar training daily for 3 consecutive days—initially at a low speed for adaptation, then accelerated to 20 rpm until the mouse fell. Formal test after training: Three trials were conducted at a constant speed of 20 rpm, with 15-minute intervals between each trial. The fall latency for each trial was recorded, and the average of the three trials was used for statistical analysis.

[0042] Hind limb grasp assessment: Grasp the mouse by the base of its tail and suspend it in the air, ensuring it is free from surrounding objects. Observe the hind limb posture for 10 seconds and score according to the following criteria: 0 points: Continuous abduction of hind limbs; 1 point: The duration of unilateral hind limb contraction towards the abdomen is greater than 50% of the suspension time; 2 points: The duration of bilateral hind limb contraction towards the abdomen is greater than 50% of the suspension time; 3 points: The duration of complete contraction of both hind limbs against the abdomen is greater than 50% of the suspension time.

[0043] Immediately after the test, the mice were returned to their cages, and their hind limb grasp scores were recorded.

[0044] Virtual screening: STING_HUMAN (UniProt ID: Q86WV6) was retrieved from the UniProt database, and the dimer structure of the core target protein STING was predicted using AlphaFold3. A 3D model of the protein structure was downloaded. Covalent docking of the STING dimer structure with small molecules was performed using Maestro v13.5. Protein preparation and small molecule conformation optimization were first performed in Maestro. C309 was selected as the covalent reaction residue for Michael addition-type covalent docking. The C=C double bond of the small molecule formed a covalent bond with the sulfur atom of the cysteine ​​(Cys) residue. The final binding score for the inhibitor PSX028 was -4.939, and the binding score for Alantolactone was -4.241. Hydrogen bond interactions between the STING dimer and small molecules were analyzed using PyMOL v2.5.4, and a 3D interaction map was generated.

[0045] PSX028 forms hydrogen bonds with residues L299 and N307 of STING, and also covalently interacts with residue C309 of STING. Inosinol forms hydrogen bonds with residue L311 of STING, and also covalently interacts with residue C309 of STING. Protein structures are shown using a cartoon model, while smaller molecules and interacting amino acid residues are shown using stick models. Red dashed lines represent hydrogen bonds, and the numbers above the hydrogen bonds indicate bond lengths.

[0046] Preparation of biotinylated ALA (Bio-ALA): 1β-hydroxycaryophyllene (10 mg, 40 μmol) and EDCI (16 mg, 80 μmol) were added to a solution of dichloromethane (DCM, 5 ml) containing N-biotinyl-6-aminohexanoic acid (57 mg, 160 μmol) and DMAP (10 mg, 80 μmol). The mixture was heated overnight at 60°C. LC-MS showed the reaction was complete. The reaction solution was cooled to 20°C and washed three times with water (3 ml). The organic phase was concentrated under vacuum to obtain a residue, which was diluted with methanol (MeOH, 1 ml), purified by preparative HPLC (prep-HPLC), and filtered to obtain the product biotinylated ALA (Bio-ALA, 15 mg, 64% yield) as a white powder.

[0047] Mass spectrometry analysis: Purified STING protein and ALA were incubated overnight at 4°C in buffer (20 mM Tris pH 8.0, 150 mM NaCl, 0.03% DDM, 0.006% CHS, 0.5 mM TECP) to perform a covalent binding reaction between ALA and STING protein. The reaction was terminated by adding SDS loading buffer containing DTT / TCEP, heating at 95°C, and separating on a 10% SDS-PAGE gel. The gel was stained with Coomassie Brilliant Blue, destained, and then the STING band was excised for downstream analysis.

[0048] The protein was reduced with 5 mM DTT and alkylated with 11 mM iodoacetamide, followed by intra-solution enzymatic digestion with sequencing-grade trypsin overnight at 37°C. The next day, the reaction was terminated by adjusting the pH to below 2 with 10% TFA. The peptides were extracted twice with a 50% acetonitrile (ACN) aqueous solution containing 0.1% trifluoroacetic acid (TFA) for 1 hour each time, and then dried in a SpeedVac. The peptides were reconstituted with 25 μl of 0.1% TFA, and 5 μl of the extracted peptides were analyzed using an Orbitrapexploris 480 mass spectrometer.

[0049] For LC-MS / MS analysis, peptides were separated using a Thermo-Dionex Ultimate 3000 HPLC system with a 120-minute gradient elution at a flow rate of 0.30 µl / min. This system was directly coupled to an Orbitrap exploris 480 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The analytical column was a self-made fused silica capillary column (75 µm inner diameter, 150 mm length; Upchurch, Oak Harbor, WA) packed with C-18 resin (300 Å pore size, 5 µm particle size, Varian, Lexington, MA). Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 100% acetonitrile containing 0.1% formic acid. The Orbitrap mass spectrometer was operated in data-dependent acquisition (DDA) mode and controlled using Xcalibur 4.5 software. First, a full-scan mass spectrum acquisition was performed in Orbitrap (scan range 300-1500 m / z, resolution 120,000), followed by a data-dependent MS / MS scan of the precursor ions in the Ion Routing Multipole (collision energy HCD 30%, acquisition time 2 seconds).

[0050] The database search criteria are as follows: Complete trypsin digestion specificity is required; carbamoyl methylation is defined as a fixed modification; methionine oxidation (M) and C... 15 H 20 O2 (modified C) is a variable modification; the precursor ion mass tolerance for all MS1 spectra acquired in the Orbitrap mass analyzer is 20 ppm; the fragment ion mass tolerance for all MS2 spectra acquired in the Orbitrap is 0.02 Da. "Hit" peptides were screened using a high confidence score filter (FDR < 1%), and their corresponding MS / MS spectra were manually reviewed.

[0051] Example 1: Cysteine ​​residues are crucial for STING activation: To assess the functional role of cysteine ​​residues in STING activation, we irreversibly modified free cysteine ​​residues using the thiol alkylating agent N-ethylmaleimide (NEM)—a well-established strategy for blocking thiol groups in the functional assessment of cysteine-dependent signaling pathways. In HeLa cells stably expressing STING-HA, NEM pretreatment reduced diABZI-induced STING phosphorylation and TBK1 activation. Figure 1 (A, B in the text). Confocal microscopy showed that NEM blocked diABZI-driven transport and phosphorylation of STING to the perinuclear compartment. Figure 1 To eliminate off-target effects of NEM, we systematically mutagenized all ten cysteine ​​residues of human STING by alanine substitution. Transfection of wild-type (WT) or C10A (all 10 Cys mutated to Ala) STING into STING-deficient HeLa cells showed that the C10A mutation attenuated STING activation (CE). Figure 1 In F and G). Compared to WT, the C10A mutation also impaired the transport of STING to the Golgi apparatus (essential for activation) and reduced the level of phosphorylated STING (pSTING). Figure 1 (HJ in the text). Parallel experiments in HEK293T cells showed that, compared with STING WT, STING C10A inhibited IFN-β and ISRE promoter activity, confirming the necessity of cysteine ​​residues for STING activation. Figure 1 (K, L in the residues). In summary, these findings indicate that cysteine ​​residues are essential for STING activation, and that pharmacological blocking or gene knockout of these residues disrupts phosphorylation and downstream signal transduction.

[0052] Cys309 Mutation Eliminates STING Signaling: Emerging evidence suggests that specific cysteine ​​residues play a crucial role in regulating STING post-translational modifications and oligomeric states. Groundbreaking research identified Cys88, Cys91, and Cys148 as molecular determinants of STING palmitoylation, supramolecular assembly, and redox sensing. Building on this, systematic cysteine ​​mutagenesis unexpectedly revealed that, in HEK293T cells, C309A substitution uniquely eliminated diABZI-induced IFNb mRNA expression, a phenotype distinct from other cysteine ​​mutants. Figure 2 Similarly, this mutation significantly reduced diABZI-induced phosphorylation of TBK1 and STING, and decreased polymer formation (A). Figure 2 B, C and Figure 8(A in the text). Notably, although C309A attenuated agonist-induced TBK1 phosphorylation, classical STING transport kinetics remained intact, and the C309A and C88A / C91A double mutants retained transport efficiency from the ER to the Golgi apparatus compared to the WT control group. Figure 2 DF and Figure 8 Phylogenetic analysis further highlighted the cross-species conservation of this residue (Cys309 in human samples and Cys308 in mouse samples). DSS crosslinking experiments showed that tetramer formation was impaired in both human C309A and mouse C308A mutants compared to WT. Figure 8 The C in the figure. These biochemical observations were confirmed mechanistically by microsecond-level all-atom molecular dynamics simulations, which showed that the C309A tetramer exhibits significant quaternary destabilization, manifested as increased inter-proton spacing and elevated RMSF values ​​(C). Figure 2 The IK in the C309A mutant was confirmed by Co-IP and immunofluorescence assays, which showed a reduction in STING-TBK1 interaction. Figure 2 G, H and Figure 8 (D in the original text). To further characterize its inhibitory effect, we tested the ability of C309A to inhibit the self-activation of gain-of-function (GOF) mutants (N154S at the linker loop and R281Q at the multimer interface) in human STING, mutations associated with infancy-onset STING-associated vascular disease (SAVI)—an autoinflammatory disease caused by the TMEM173GOF mutation. Notably, the introduction of C309A partially attenuated the self-activation of these GOF mutants (D in the original text). Figure 2 (L, M in). In summary, these findings establish Cys309 as a novel allosteric hub that stabilizes the active STING tetramer to facilitate TBK1 recruitment and downstream signaling while maintaining classical transport kinetics, thus revealing a previously unknown cysteine-dependent regulatory axis in STING activation.

[0053] Identification of ALA as a covalent STING inhibitor: This invention, based on the reported human STING dimer structure (PDBID: 6NT5), used Maestro software to perform virtual screening of STING dimer proteins and covalent small molecules. First, protein preparation and small molecule conformation optimization were performed in Maestro. Cys309 was selected as the covalently reacting amino acid based on Michael addition-based covalent docking to identify potential covalent inhibitors. This computational strategy screened α-methylene-γ-lactone derivatives from a library containing 842 covalent inhibitors as high-affinity ligands for the Cys309 binding pocket. Subsequently, 18 natural products containing this electrophilic scaffold were preferentially selected, and functional validation of STING pathway inhibition was performed using human HMC3 and mouse BV2 microglia models. Figure 3 A, B and Figure 8 AC in the middle.

[0054] ALA (code CI-2) is a sesquiterpene lactone derived from *Inula helenium* L., possessing anti-inflammatory, antioxidant, and neuroprotective properties. Its α-methylene-γ-lactone group can covalently modify cysteine ​​residues via Michael addition, suggesting its potential as a covalent STING inhibitor. This potential is supported by experimental evidence, as ALA exhibits potent cross-species inhibitory activity, demonstrating micromolar potency against both human and mouse STING. Figure 3 (CE in the text). Comparative dose-response analysis showed that ALA was significantly superior to the reference covalent inhibitor H-151, with an IC50 of 80 nM for inhibiting IFN-β in diABZI-stimulated cells, compared to 279 nM for H-151. Figure 3 The F in the text is missing. Immunofluorescence analysis further confirmed that neither ALA nor H-151 interfered with the transport of STING to the Golgi apparatus, but reduced the level of phosphorylated STING (pSTING). Figure 3 (GI in the middle). Consistent with in vitro and in vivo observations, DSS crosslinking experiments showed that ALA disrupted STING tetramers and oligomers ( Figure 3 The oligomer destabilization pharmacologically mimics the C309A mutation phenotype, confirming the common mechanistic convergence point of disrupting the STING quaternary structure by targeting Cys309. Furthermore, Co-IP experiments showed that ALA treatment attenuated diABZI-induced STING-TBK1 interactions (J). Figure 3 To assess the therapeutic potential of ALA, we examined its ability to inhibit the spontaneous activation of human STING GOF mutants N154S and R281Q. The results showed that ALA significantly inhibited the spontaneous activation of these SAVI-related STING mutants (K, L). Figure 3(M, N in). Notably, this drug intervention reproduced the disruptive effects observed with the C309A mutation, manifesting as similar damage to signal body assembly. In summary, these experimental results establish ALA as a novel covalent STING inhibitor. ALA selectively targets Cys309, disrupting stimulus-dependent tetramerization and downstream signal transduction while preserving classical STING transport kinetics, thereby decoupling the structural kinetics of STING from its signal transduction function and providing a new strategy for pathway-selective intervention.

[0055] Example 2: ALA specifically targets STING Cys309: After confirming the antagonistic effect of ALA on the STING pathway, this invention investigated whether ALA directly targets STING to mediate this inhibition. Cellular thermal displacement analysis (CETSA) showed that the thermostability of STING increased after ALA treatment, indicating the existence of direct ligand-protein interactions. Figure 4 (A, B in the original text). To further verify the covalent binding of ALA and STING, the purified STING protein was co-incubated with ALA overnight in vitro. LC-MS / MS analysis confirmed that ALA covalently modified STING at the Cys309 site. Figure 4 (C in the text). We then synthesized biotin-conjugated ALA (Bio-ALA) and verified its dose-dependent interaction with endogenous STING in human and mouse cell lysates using pull-down assays. Figure 4 D, E and Figure 5 In A), this interaction can be competitively bound by excess free ALA or NEM pretreatment (A). Figure 4 F, G and Figure 5 (B, C). Immunoprecipitation experiments using cell lysates showed that Bio-ALA binding was eliminated in the STING C309A mutant, but was restored in the STING C9(A309C) variant (i.e., Cys309 reintroduced into the C9A mutant background). Figure 4 (H in the text). Further in vitro experiments confirmed the covalent binding of Bio-ALA to STING WT, but this interaction was weakened when Bio-ALA was incubated with the C309A mutant. Figure 4 (I) As a summary of target binding validation, CETSA showed that ALA treatment selectively stabilized wild-type STING, but had no thermostabilizing effect on the C309A mutant. Figure 4 JL in the middle). Molecular docking analysis of the full-length STING dimer showed that ALA covalently binds to Cys309 of STING through hydrogen bonding between its reactive group and the Leu311 residue (JL in the middle). Figure 5 (D, E in the text). This thermal shift characteristic ultimately identified Cys309 as a core residue regulating two interdependent processes: covalent binding of ALA via Michael addition, and stabilizing the active tetrameric conformation of STING—this dual mechanism couples target occupancy with quaternary reinforcement. These findings ultimately establish ALA as a selective covalent inhibitor of STING Cys309, outlining a novel therapeutic pathway to regulate STING-dependent pathology by targeting and disrupting specific cysteine ​​residues that activate it.

[0056] Example 3: ALA inhibits Trex1 - / - Inflammation and death in mice Normal function of the triphosphate repair exonuclease 1 (Trex1) is crucial for maintaining homeostasis of cytoplasmic autoDNA levels. Accumulated waste autoDNA can directly activate the cGAS-STING pathway, leading to excessive IFN production in Trex1-deficient mice, thereby triggering severe autoimmune inflammation and autoimmune diseases. In this model, STING knockout completely rescued fatal autoinflammatory reactions, further highlighting the key role of STING in the pathogenesis of Trex1 deficiency diseases. Therefore, we hypothesize that ALA can be used to therapeutically alleviate STING-mediated autoinflammatory diseases. To evaluate the role of ALA in Trex1... - / - Protective efficacy in mice, compared to age-matched WT and Trex1 mice. - / - Litter mice (4 weeks old) were intraperitoneally injected daily with ALA (20 mg / kg) or a vector control for two months. Figure 6 (A) In the treatment process, 12 untreated Trex1 mice - / - Three mice died, while none of the 12 mice treated with ALA died. Figure 6 (B in the text). ALA treatment normalized pathological splenomegaly and reduced the spleen / body weight ratio relative to untreated animals. Figure 6 In addition, serum biochemical analysis showed that ALA treatment significantly reduced lactate dehydrogenase (LDH) levels compared with the untreated control group. Figure 6 (C) These findings suggest that ALA intervention reduced multi-organ damage after treatment administration. Histopathological examination of heart and kidney tissues by hematoxylin and eosin (H&E) staining revealed untreated Trex1 - / - Mice exhibited severe inflammatory infiltration. In contrast, the ALA treatment group showed a significant reduction in inflammation, with preservation of tissue structure. Figure 6 (in FH). Consistently, Trex1 - / -The elevated levels of Il1b, Irf7, Isg15, and Cxcl10 mRNA in the heart, spleen, and kidneys of mice were largely suppressed by ALA treatment. Figure 6 (IL in the middle). In summary, in vivo data indicate that ALA effectively reduces Trex1. - / - The systemic inflammation in mice suggests its potential to improve STING-dependent autoimmune diseases.

[0057] Example 4: ALA alleviates disease in ALS mouse models The accumulation of 43 kDa TAR DNA-binding protein (TDP-43) in the cytoplasm is a pathological marker of ALS. Evidence of this accumulation suggests that nuclear depletion coupled with cytoplasmic mislocalization drives a toxic gain-of-function mechanism in ALS pathogenesis. Emerging mechanistic studies have established STING activation as a key pathogenic driver of ALS progression, and its dysregulation is associated with the neuroinflammatory cascade and motor neuron degeneration. These findings collectively establish pharmacological inhibition of the cGAS-STING axis as a viable therapeutic strategy to mitigate neuroinflammatory damage in ALS. Therapeutic relevance is that several STING-targeting compounds currently in preclinical development could be rapidly repurposed for ALS treatment once their safety and efficacy are validated in clinical trials.

[0058] The rNLS8 mouse model expresses tetracycline transactivator (tTA) under the human NEFH promoter and human TDP-43 (hTDP-43) with a mutant nuclear localization signal (ΔNLS) under the tetO promoter. This system enables doxycycline (DOX)-repressible cytoplasmic hTDP-43 expression in neurons, creating a reversible ALS model for preclinical testing. Comparative analysis of DOX-treated and untreated mice showed upregulation of STING levels and activation of STING-related inflammatory pathways. Figure 7 The A in this study is consistent with previous reports. To further evaluate the therapeutic effect of ALA in this ALS model, we administered 50 mg / kg ALA via daily intraperitoneal injection. Compared with the DMSO-treated control group, ALA-treated rNLS8 mice showed a modest lifespan extension, demonstrating the blood-brain barrier penetration and neuroprotective potential of this compound. Figure 7 (B in the text). We further observed that, at week 3 after DOX withdrawal, ALA-treated mice exhibited a significantly reduced incidence of clinging behavior, indicating improved neuromuscular function. Figure 7 (C, D in the original text). Interestingly, at week 4 post-treatment, ALA-treated female mice showed a significant difference in body weight compared to vector-treated female mice. Figure 7(F in the text), while there was no significant difference in weight trajectory between the treatment groups in the male group (F in the text). Figure 7 The sex-specific metabolic response may suggest that ALA has sex-specific pharmacokinetic or pharmacodynamic properties, warranting further investigation. To assess motor function, we performed hindlimb hold and rotarod tests. Two weeks after DOX withdrawal, DMSO-treated mice showed significantly increased hindlimb hold scores, while ALA-treated mice maintained lower scores, indicating delayed disease progression in this mouse model. Notably, the carrier-treated control group began showing progressive functional decline at week 2 post-treatment, with significantly reduced walking distance and rotarod dwell time. In contrast, ALA intervention significantly alleviated these deficiencies, achieving statistical significance in motor function preservation by week 3. Figure 7 (G, H, I). After a one-month treatment regimen, surviving animals were euthanized for a comprehensive pathological evaluation. Subsequent analysis of rNLS8 mouse brain tissue showed that ALA treatment reduced activation of the STING pathway, as evidenced by decreased phosphorylation levels of key signaling molecules: phosphorylated STING (p-STING), phosphorylated TBK1 (p-TBK1), and phosphorylated p65 (p-p65). Figure 7 Consistent with these molecular changes, ALA administration significantly suppressed the upregulation of disease-associated pro-inflammatory transcripts, including Il1b, Irf7, Isg15, and Cxcl10, compared to the control group. Figure 7 In summary, these findings demonstrate that pharmacological inhibition of the STING pathway through ALA treatment has neuroprotective and disease-modifying effects in the rNLS8 ALS model, manifested as prolonged survival, improved neuromuscular function, preservation of motor function, and reduced neuroinflammation, thus validating STING as a therapeutic target for ALS intervention.

[0059] In summary, this invention identified Cys309 as a previously unrecognized cysteine ​​residue crucial for STING activation. Through integrated virtual screening and experimental validation, ALA was characterized as a selective covalent inhibitor targeting the STING Cys309 residue. In both murine and human cell models, ALA exhibited potent inhibition of STING phosphorylation and its subsequent downstream signaling cascade. Notably, ALA administration ameliorated autoimmune pathology and completely prevented death in Trex1-deficient mice. Furthermore, in a rNLS8-associated ALS murine model, ALA treatment improved survival outcomes while alleviating neuroinflammatory responses. These findings not only establish Cys309 as a novel targetable site on the STING protein but also lay the mechanistic foundation for developing covalent STING inhibitors targeting STING-related pathologies.

[0060] Example 5: ALA-related derivatives target STING Cys309: This invention screened α-methylene-γ-lactone derivatives from 842 covalent inhibitors as high-affinity ligands for the Cys309 binding pocket. Subsequently, 18 natural products containing this electrophilic scaffold were selected, and the function of STING pathway inhibition was verified using a human HMC3 cell model. Figure 8 The results showed that CI-2 (auslanolide), CI-5 (isoauslanolide), CI-7 (iso-gentiolide), CI-9 (deoxygentiolide), and CI-11 (isodeoxygentiolide) were screened out as covalent inhibitors that could effectively inhibit STING activation.

[0061] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. Application of inosinol as a STING inhibitor.

2. The application according to claim 1, characterized in that: The calomel lactone is a covalent inhibitor that selectively targets Cys309, a component of STING.

3. Application of inosinol in the preparation of drugs for treating STING-dependent diseases.

4. The application according to claim 3, characterized in that: The STING-dependent diseases include STING-dependent autoimmune diseases, STING-dependent neurodegenerative diseases, autoinflammatory syndromes, or fibrotic diseases.

5. The application according to claim 3, characterized in that: The STING-dependent neurodegenerative diseases include Alzheimer's disease, amyotrophic lateral sclerosis (ALS), lysosomal storage diseases, and Parkinson's disease.

6. The application of inosinol-related derivatives as STING inhibitors, characterized by: The related derivatives of arugula lactone include isoarugula lactone.

7. The application according to claim 6, characterized in that: The related derivatives of the gentian lactone include one or more of the following: gentian lactone, deoxygentianin, and isodeoxygentianin.

8. Application of inosinol-related derivatives in the preparation of drugs for treating STING-dependent diseases.

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