Nitroalkene-based inhibitors to target sting-dependent inflammation

Abstract

A method that includes administering to a subject having a stimulators of interferon genes (STING)-dependent inflammatory or autoimmune condition a therapeutically effective amount of a compound that includes a nitrovinyl moiety conjugated to an aromatic moiety.

Description

[0001]8123-112123-02 05 / 16 / 25 06666NITROALKENE-BASED INHIBITORS TO TARGET STING-DEPENDENT INFLAMMATION CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No.63 / 649,203, filed May 17, 2024, which is incorporated by reference herein in its entirety. ACKNOWLEDGMENT OF GOVERNMENT SUPPORT This invention was made with government support under grant number GM125944 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND The Stimulator of Interferon Genes (STING) is a key player in the inflammatory response initiated by cytosolic DNA. Dysregulated STING activation is implicated in the pathogenesis of numerous inflammatory diseases. Despite the urgent need for effective STING inhibitors, the development of such compounds has been hindered, with no viable candidates beyond the preclinical stage. SUMMARY Disclosed herein is a method comprising administering to a subject having a stimulators of interferon genes (STING)-dependent inflammatory or autoimmune condition a therapeutically effective amount of a compound that includes a nitrovinyl moiety conjugated to an aromatic moiety. In certain embodiments, the nitrovinyl moiety conjugated to an aromatic moiety is a β-nitrostyrene moiety. Other illustrative nitrovinyl moieties conjugated to an aromatic moieties include 2- vinylpyridine and vinylindoles. Also disclosed herein is a compound, or a pharmaceutically acceptable salt thereof, having a structure of: Z-(CH2)x-C(NO2)=CH-A-(CH2)y-(CH=CH)z-C(=O)O-R1wherein x and y are each independently 0 to 10; z is 0 or 1; Z is CH3, cycloalkyl, substituted cycloalkyl, aryl, or substituted aryl; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.8123-112123-02 05 / 16 / 25 06666Further disclosed herein is a compound, or a pharmaceutically acceptable salt thereof, having a structure of: Y-A-CH=C(NO2)-(CH2)x-(CH=CH)z-C(=O)O-R1wherein x is 1 to 10; z is 0 or 1; Y is CH3, halogen, alkyl, substituted alkyl, or hydroxyl; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl. The foregoing and other objects, features, and advantages will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS FIG.1: Nitroalkene compounds inhibit STING based on the length of the fatty acyl chain and the relative position of the nitroalkene group. The cells were pre-treated with specified concentrations of testing compounds for 2 h, followed by cGAMP stimulation (4 µg / mL) for 24 h (unless otherwise stated below) (FIG.1A) Levels of NF-κB and IRF activation in THP-1-Dual cells treated with indicated nitroalkene compounds (10 µM) or H151 (1 µM) and cGAMP stimulation. (FIG.1B) CXCL-10 release in THP-1s wt cells treated with indicated nitroalkene compounds (10 µM). (FIG. 1C) CXCL-10 release in THP-1 wt cells treated with indicated nitroalkene compounds (2, 5 and 10 µM), CKA (15, 20 and 30 µM), and nitroalkene dicarboxylates (di-COOH-C3, 2, 5, 10 and 15 µM) or H151 (0.5 and 1 µM). (FIG.1D) Immunoblot analysis of whole-cell lysate of THP-1 wt cells to evaluate levels of STING, pSTING, TBK1, pTBK1, IRF3, and pIRF3 after treatment with indicated nitroalkene compounds (5 and 10 µM), OA (10 µM), CKA (20 and 30 µM), nitroalkene dicarboxylates (di-COOH-C3, 5 and 10 µM) or H151 (0.5 and 1 µM), followed by 3 h cGAMP stimulation. (FIG.1E) Immunoblot analysis of whole-cell lysate of THP-1 wt cells to evaluate levels of IFIT1 and ISG15 after treatment with indicated nitroalkene compounds (5 and 10 µM), 9-NO2OA and OA (10 µM), CKA (20 and 30 µM), nitroalkene dicarboxylates (di-COOH-C3, 5 and 10 µM) or H151 (1 µM). (FIG.1F) Type I IFN release in SAVI fibroblasts treated with indicated nitroalkene compounds (1, 2, and 5 µM) or H151 (2 and 5 µM). (FIG.1G) Immunoblot analysis of whole-cell lysate of SAVI fibroblasts to evaluate levels of STING, pSTING, TBK1, pTBK1, IRF3, and pIRF3 after treatment with indicated nitroalkene compounds (5 and 10 µM) or H151 (1 and 2 µM), followed by 3 h cGAMP stimulation. Bars indicate the mean ± SEM of two to three replicates and are normalized to levels reached with cGAMP activation. The dotted lines denote 75 % inhibition of NF-8123-112123-02 05 / 16 / 25 06666kB activation, CXCL-10 response, and type I IFN response, or 50 % inhibition of IRF activation induced by cGAMP. FIG.2: Determining the in vitro therapeutic index of second-generation nitroalkene-based compounds. In vitro therapeutic index based on inhibition of CXCL-10 release plotted against level of cytotoxicity. For CXCL-10 release, THP-1 wt cells were pre-treated with indicated nitroalkene compounds in different concentrations (2, 5, 10, and 15 µM) for 2 h, followed by cGAMP stimulation (4 µg / mL) for 24 hrs. The cytotoxicity was measured by MTT assay, and THP-1s wt cells were treated with indicated nitroalkene compounds in different concentrations (2, 5, 10, 15, 20, 30, 50, 70 and 100 µM) for 24 h. Data of the therapeutic index represent the mean ± SEM of three replicates. The CXCL-10 release is normalized to the cGAMP condition, expressed as a percentage. The dotted line denotes 75 % inhibition of the CXCL-10 response induced by cGAMP. The cytotoxicity is normalized to untreated cells, expressed as percentage viability. The EC50 value is determined by fitting the curve with nonlinear regression, and compounds with EC50 values above 25 are considered to have low cytotoxicity (bold numbers). The asterisk and bold axes indicate the lead compounds with a high in vitro therapeutical index, due to high potency to inhibit CXCL-10 production with minimal cytotoxicity. FIG.3: Inhibition of STING by second-generation nitroalkene-based compounds and assessment of physicochemical characterization. (FIG.3A) In vitro therapeutic index based on inhibition of type I IFN release plotted against level of cytotoxicity. For type I IFN release, SAVI fibroblasts were pre-treated with indicated nitroalkene compounds (2, 5, 10 and 15 µM) for 2 h, followed by cGAMP stimulation (4 µg / mL) for 24 h. The cytotoxicity was measured by MTT assay, SAVI fibroblasts were treated with indicated nitroalkene compounds in different concentrations (2, 5, 10, 15, 20, 30, 50, 70 and 100 µM) for 24 h. Data of the therapeutic index represent the mean ± SEM of two (IFN) and three (MTT) replicates. The type I IFN release is normalized to levels reached with cGAMP treatment, expressed as percentage. The cytotoxicity is normalized to untreated cells, expressed as percentage viability. The dotted line denotes 75 % inhibition of the type I IFN response induced by cGAMP. The EC50value is determined by fitting the curve with nonlinear regression. The asterisk and bold axes indicate the lead compounds with a high in vitro therapeutical index, due to high potency to inhibit type I IFN production with minimal cytotoxicity. (FIG.3B) Level of NF-κB and IRF activation in THP-1-Dual cells after pre-treatment with indicated nitroalkene compounds (10, 20, and 30 µM) or 9-NO2OA (10 µM), CP-8b (20 µM) and OA (30 µM) for 2 h, followed by cGAMP stimulation (4 µg / mL) for 24 h. Bars indicate the mean ± SEM of three replicates and are normalized to cGAMP treatment levels, expressed as the percentage. The dotted lines denote 50 % or 75 % inhibition of the IRF and NF-kB activation induced by cGAMP, respectively. (FIG.3C) Immunoblot analysis of whole-cell lysate of SAVI fibroblasts to evaluate levels of pSTING, STING, pTBK1, TBK1, pIRF3, and IRF3 after treated with indicated nitroalkene compounds (2, 5, and 10 µM) or CP-8123-112123-02 05 / 16 / 25 066668b (5 and 10 µM) for 2 h, followed by cGAMP stimulation (4 µg / mL) for 3 h. (FIGS.3D-F) Reaction kinetics of CP-36 (top panels) and CP-45 (bottom panels) with GSH. (FIG.3D) Changes in UV-vis absorbance of CP-36 (10 µM) and CP-45 (10 µM) upon reaction with GSH (50 µM) in phosphate buffer (0.1 M, pH 7.4, 0.1 mM DTPA) at 25 °C. Spectra was recorded every 36 seconds for CP-36 and 12 seconds for CP-45, capturing approximately 10 half-lives of the reaction between GSH and the nitroalkene. The downward arrows at 323 and 333 nm indicate consumption of CP-36 and CP-45, respectively. The upward arrows at 265 / 270 nm indicate the formation of a single adduct with GSH (FIG.3E) Stopped flow kinetic traces of the reaction of CP-36 (20 µM) and CP-45 (µM) with increasing concentrations of GSH, followed at 323 and 333 nm, respectively. (FIG.3F) Exponential pseudo-first-order rate constants as a function of GSH concentration. Kobsvalues were determined from the best fit of the kinetics traces shown in (E) to a single-phase exponential with slope function and plotted against GSH concentration. FIG.4: Intraperitoneally administration of nitroalkene compound CP-36 dampens STING- dependent inflammation in vivo. (FIGS.4A-C) Mice were administrated with CP-36 i.p. (10 or 30 mg / kg) 2 h before activating STING with i.p. injection of cAIM(PS)2 Difluor (Rp / Sp) (0.2 mg / kg) for 4 h. Immunoblot analysis of homogenized and lysed (FIG.4A) lung and (FIG.4B) liver tissue to evaluate the levels of pTBK1, TBK1, pSTAT1, STAT1 and Viperin with (FIG.4C) densitometry quantification of protein levels. The plots include data from individual mice represented in immunoblots. Bars indicate mean ± SEM and are normalized to the cAIM(PS)2 Difluor (Rp / Sp) group and expressed as percentage. (FIGS.4D-F) WT or STING-KO BMDMs were pre-treated with CP-36 (5 and 10 µM) or reduced CP-36 (R-CP-36, 5 and 10 µM) for 2 h, followed by cGAMP stimulation (4 µg / mL) for either 3 h. or 6 h. (FIG.4D) Immunoblot analysis of whole-cell lysate (representative blot) to evaluate levels of pSTING, STING, pIRF3 and IRF3 and (FIG.4E) densitometry quantification of pSTING / STING protein levels after 3 h cGAMP stimulation. Three independent experiments are plotted. (FIG.4F) qPCR analysis of Ifnb1 and Cxcl-10 expression was quantified relative to 18S mRNA levels after 6 h of cGAMP stimulation. BMDM cultures obtained from 6 individual mice (3 male and 3 female mice) are plotted. Bars indicate mean ± SEM and are normalized to cGAMP treatment and expressed as percentage. Statistical analyses were performed using Welch’s t-test, yielding the following p values: For tissues: pTBK1: p = 0.0293, TBK1: p = 0.0283, Viperin: p = 0.0311, STAT1: p = 0.0159. For BMDMs: pSTING / STING: p = 0.0184, Ifnb1: p < 0.0001, Cxcl-10: *, p = 0.0367 and **, p = 0.0076. * Indicates p < 0.05, ** indicates p < 0.01, and **** indicates p < 0.0001. FIG.5: Orally administration of nitroalkene compounds CP-36 and CP-45 dampens STING- dependent inflammation in vivo. Mice were administrated with CP-36 and CP-45 orally (40 or 60 mg / kg) for 2 h before activating STING with i.p. injection of cAIM(PS)2Difluor (Rp / Sp) (0.2 mg / kg) for 4 h. (FIG.5A) Immunoblot analysis of homogenized and lyzed lung tissue to evaluate the levels of8123-112123-02 05 / 16 / 25 06666pSTING, STING, STAT1, Viperin and ISG15 tissue (representative blot) with (FIG.5B) densitometry quantification of protein levels. (FIG.5C) Cryo-preserved plasma was analyzed for levels of IFNα, IFNβ, MCP-1, MIP-1β, TNFα, and CXCL-10 using Meso Scale Discovery. DL signifies the Detection Limit for individual assays. Individual mice are plotted. Bars indicate mean ± SEM and are normalized to the cAIM(PS)2 Difluor (Rp / Sp) group and expressed as a percentage. Statistical analyses by Welch’s t-test. P values: MCP-1: p = 0.0429, TNF-α: p = 0.0464, pSTING: *, p = 0.0126 and ****, p < 0.0001, STING: p < 0.0001, STAT1: **, p = 0.0024 (CP-36, 40 mg / kg) and p = 0.0042 (CP-45, 40 mg / kg) and ***, p = 0.0006 (CP-36, 60 mg / kg) and p = 0.0008 (CP-45, 60 mg / kg), and Viperin: p = 0.0122. * Indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001. FIGS.6-7 are tables of examples of nitroalkene compounds. FIGS.8-10 are tables of examples of nitroalkene compounds that incorporate an aromatic moiety. FIG.11. Endometriosis is reduced in STING KO mice. FIG.11A) Peritoneal endometriosis was established by injecting minced PMSG-treated EGFP uterine tissue into the peritoneum of STING KO and WT female mice and lesions were allowed to develop for 28 days. FIG.11B) Endometriotic lesions were visualized via in vivo imaging and quantified. Data are plotted as box and whiskers from minimum to maximum values. Data were analyzed by ANOVA followed by Bonferroni post hoc test, n = 8. ns: not significant, ***p <0.001; ****p <0.0001. C) Macroscopic quantification of endometriotic lesions was done at end-point analysis using a digital caliper. Data are expressed as diameter size (in mm). Mice with no observable lesions were included in the analysis (considered as zero). Data are shown as violin plots showing all points. Welch’s t-test **p=0.0014. FIG.12. STING KO mice display reduced levels of surrogate markers of pain. FIG.12A) IFNb and FIG.12B) Fractalkine levels, a bona fide marker of pain in endometriosis, in plasma and peritoneal fluid assessed by Multiplex assays. ****p<0.0001. nd: not detected. ns not significant. ANOVA followed by Bonferroni post hoc test. FIG.13: Loss-of-STING function in the uterine fragments transplanted in WT syngeneic mice reduces endometriosis lesion formation. (FIG.13A) Scheme depicting a reverse genetic approach in which endometriosis was established using WT and STING KO mice as donors transplanted in WT syngeneic mice. (FIG.13B) Lesion diameter was measured at end-point analysis (28 days) via digital caliper, demonstrate that lesions derived from STING KO mice are significantly reduced compared to WT. Welch’s t-test **p=0.0016. (FIG.13C) Pro-inflammatory CXCL10 / IP-10 expression levels in plasma and peritoneal fluid were quantified by Multiplex assays. Data points were plotted as violin plots. ns: not significant. FIG.14. NO2-CLA reduces endometriotic lesions in a syngeneic model of endometriosis. Peritoneal endometriosis was established using uterine fragments of PMSG-treated (3 days) EGFP 8123-112123-02 05 / 16 / 25 06666 female mice as donor (syngeneic model) and transplanted via i.p injection into C57BL / 6J female recipients (WT). Sham consisted of PBS i.p. injection (1ml). Mice were treated with NO2-CLA (2.5 mg / mice, s.c.) or vehicle (1% EtOH in PBS) daily for 28 days. FIG.14A) Donor-derived lesions were demonstrated using an AmiHTX imager in fluorescence mode (EGFP). FIG.14B) Lesions were quantified using Aura 4.0 imaging software. Data are plotted as box and whiskers. **p < .01, ***p < .001 vs. Sham; ^^^p < .001, vs. PBS. n = 4. ANOVA followed by Bonferroni post hoc test. NO2-CLA is a mixture of nitro-fatty acids derived from conjugated linoleic acids (CLA) and exists as a combination of various regio- and stereoisomers. Several of the most common NO2-CLA isomers are shown below: epithelial cells. FIG.15A) against level of cytotoxicity. IFNβ mRNA expression, endometriotic epithelial cells (12Z) were pre-treated with indicated CP-36 (1, 2, 4 and 8 μM) for 2 h, followed by cGAMP stimulation (4 μg / mL) for 4 h. Cytotoxicity in 12Z cells was measured by MTT assay, upon treatment with CP-36 in different concentrations (1, 2, 4, 8, 16, 32, 64, 128 μM) for 24 h. IFNβ mRNA expression is normalized to levels reached with cGAMP, expressed as percentage. Cytotoxicity was normalized to untreated cells. Data of the therapeuticindex represent the mean ± SEM of 3 replicates. FIG.15B) 12Z cells were grown in DMEM / F12 supplemented with 10%FBS, synchronized by 2% FBS overnight and treated as follows: cells were treated with cGAMP vehicle (Lipo3000) and treated with CP-36 or R-CP-36, prior to stimulation with STING agonist cGAMP (4ng / mL) for 6 h. Dose-dependent inhibition of viperin mRNA expression by CP-36 (2μM, 5μM) or R-CP-36, (n=4 biological replicates), One-way ANOVA, Bonferroni post hoc test, *p≤0.05, **p≤0.01; **p≤0.01; ***p≤0.001. FIG.16. Expression of STING in human eutopic endometrium and endometriotic lesions. Immunofluorescent localization of STING and EpCAM performed in tissue microarray (TMA)8123-112123-02 05 / 16 / 25 06666sections from endometriosis of endometrium, ovary and peritoneal tissues. Sections were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) to visualize all nuclei. Colocalization of STING and EpCAM is observed in normal and endometriotic endometrium, primarily at the epithelium whereas STING stromal expression was weaker (FIG.16A) STING expression is increased in ovarian and peritoneal endometriotic lesions from humans (FIG.16B). Size bar= 200μm. FIG.17. NO2-CLA inhibits STING activation in endometriotic stromal cells. EESC were obtained from eutopic biopsies of an endometriosis patient in the proliferative phase at Emory Midtown Hospital. NESCs were obtained from recruited parous women with regular menstrual cycles. Cells were grown in DMEM / F1210% FBS, synchronized with 2% FBS overnight and treated as follows: vehicle (Lipo3000), NO2-CLA (2µM) or CLA, prior stimulation with cGAMP (4ng / mL) for 6 h. Dose-dependent inhibition of CCL2 (FIG.17A) and IL6 (FIG.17B) mRNA expression by NO2-CLA (1μM, 2μM), 3h poststimulation with cGAMP. FIG.17C) Inhibition of STING activity (p- STING / tSTING) by NO2-CLA but not cLA (2μM), 6h after cGAMP, by western blot. Data shown as mean ± s.e.m (n=3 biological replicates), *p≤0.05, **p≤0.01 vs. Control; ††p≤0.01 vs cGAMP; ˄˄p≤0.01 vs. cLA. ANOVA, Bonferroni post-hoc. FIG.18: Oral NO2-CLA administration reduces syngeneic endometriotic lesions. FIG.18A) For oral delivery experiments, mice were randomized for treatment of either NO2-CLA, CLA (5 mg / kg / day), or vehicle (PEG) for 28 days. FIG.18B) Representative MS tracing and quantitation of NO2-CLA and its metabolite (DH-NO2-CLA) levels in plasma and peritoneal fluid (P.F). FIG.18C) Representative images of EGFP-derived lesions in response to each treatment. FIG.18D) EGFP emission at the peritoneal cavity was quantified using Aura 4.0 imaging. Data is plotted as box and whiskers from min. to max. values. Data analyzed by ANOVA followed by Bonferroni post hoc test. ns: not significant, **p<0.01; *p<0.05. FIG.18E) Macroscopic quantification of endometriotic lesions was done at end-point analysis (28 days) using a digital caliper. Data is expressed as diameter size (in mm). Animals with no observable lesions were included in the analysis (considered as zero). Data is shown as violin plots showing all points. ****p<0.0001, *p<0.05. FIG.19 Nitroalkene compound CP-36 and CP-45 inhibits STING-dependent activation in a model of primary Human Airway Epithelium. FIG.19A: Immunoblot analysis of whole-cell lysate of Human Airway Epithelium (HAE) cultured in an Air-Liquid Interphase (ALI) to evaluate levels of pSTING, STING, and pIRF3 after apical treatment with CP-36 and CP-45 (5 and 10 µM) for 1.5 h, followed by apical cAIM(PS)2Difluor (Rp / Sp) stimulation (6 µg / mL) for 3 h. Two different donors are shown. FIG 19B: 1) The HAE-ALI cultures are established on primary cells harvested by non- invasive nasal swabs of healthy donors (step 1). These are firstly, de-differentiated and expanded (step 2), before the basal cells are plated on Transwell membrane in a submerged condition (step 3). Next, the cultures are differentiated into a fully functional airway epithelium layer (step 4) with cilia beating and mucus production upon exposure to air on the apical side. This physiological relevant model consist of multiple cells types as ciliated cells, secretory cells such as club and goblet cells and basal8123-112123-02 05 / 16 / 25 06666cells, which are characterized by 2) Immunofluorescence staining of β-Tubulin (Ciliated cells, Red) and CC10 (Club cells, Green), 3) Scanning Electronic Microscopy (SEM) of ciliated cells, and 4) transverse sections of HAE-ALI cultures displays the stratified airway epithelium cell layer after H&E staining (top) and PAS staining (bottom). DETAILED DESCRIPTION Terminology The following explanations of terms and methods are provided to better describe the present compounds, compositions and methods, and to guide those of ordinary skill in the art in the practice of the present disclosure. It is also to be understood that the terminology used in the disclosure is for describing particular embodiments and examples only and is not intended to be limiting. “Administration” as used herein is inclusive of administration by another person to the subject or self-administration by the subject. The term “alkyl” refers to a branched or unbranched saturated hydrocarbon group, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like. Alkyl groups may be “substituted alkyls” wherein one or more hydrogen atoms are substituted with a substituent such as halogen, cycloalkyl, alkoxy, amino, hydroxyl, aryl, alkenyl, or carboxyl. For example, a lower alkyl or (C1-C6)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C3- C6)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C3-C6)cycloalkyl(C1- C6)alkyl can be cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, 2- cyclopropylethyl, 2-cyclobutylethyl, 2-cyclopentylethyl, or 2-cyclohexylethyl; (C1-C6)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy; (C2-C6)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1,-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1- hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5- hexenyl; (C2-C6)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1- pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1- hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5- hexynyl; (C1-C6)alkanoyl can be acetyl, propanoyl or butanoyl; halo(C1-C6)alkyl can be iodomethyl, bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl, 2-fluoroethyl, 2,2,2- trifluoroethyl, or pentafluoroethyl; hydroxy(C1-C6)alkyl can be hydroxymethyl, 1-hydroxyethyl, 2- hydroxyethyl, 1-hydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 1-hydroxybutyl, 4-hydroxybutyl, 1-hydroxypentyl, 5-hydroxypentyl, 1-hydroxyhexyl, or 6-hydroxyhexyl; (C1-C6)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, or hexyloxycarbonyl; (C1-C6)alkylthio can be methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, pentylthio, or hexylthio; (C2-C6)alkanoyloxy can be acetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy.8123-112123-02 05 / 16 / 25 06666The term “subject” includes both human and non-human subjects, including birds and non- human mammals, such as non-human primates, companion animals (such as dogs and cats), livestock (such as pigs, sheep, cows), as well as non-domesticated animals, such as the big cats. The term subject applies regardless of the stage in the organism’s life-cycle. Thus, the term subject applies to an organism in utero or in ovo, depending on the organism (that is, whether the organism is a mammal or a bird, such as a domesticated or wild fowl). A "therapeutically effective amount" refers to a quantity of a specified agent sufficient to achieve a desired effect in a subject being treated with that agent. Ideally, a therapeutically effective amount of an agent is an amount sufficient to inhibit or treat the disease or condition without causing a substantial cytotoxic effect in the subject. The therapeutically effective amount of an agent will be dependent on the subject being treated, the severity of the affliction, and the manner of administration of the therapeutic composition. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, or administering a compound or composition to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology or condition, or diminishing the severity of a pathology or condition. As used herein, the term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, an improvement in the overall health or well-being of the subject, or by other parameters well known in the art that are specific to the particular disease. The phrase “treating a disease” refers to inhibiting the full development of a disease, for example, in a subject who is at risk for a disease. “Preventing” a disease or condition refers to prophylactic administering a composition to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology or condition, or diminishing the severity of a pathology or condition. In certain embodiments, treating a disease refers to inhibiting metastasis of the disease. “Pharmaceutical compositions” are compositions that include an amount (for example, a unit dosage) of one or more of the disclosed compounds together with one or more non-toxic pharmaceutically acceptable additives, including carriers, diluents, and / or adjuvants, and optionally other biologically active ingredients. Such pharmaceutical compositions can be prepared by standard pharmaceutical formulation techniques such as those disclosed in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA (19th Edition). The compounds of the invention can exist in various isomeric forms, including configurational, geometric, and conformational isomers, as well as existing in various tautomeric forms, particularly those that differ in the point of attachment of a hydrogen atom. The term “isomer”8123-112123-02 05 / 16 / 25 06666is intended to encompass all isomeric forms of a compound of this invention, including tautomeric forms of the compound. Certain compounds described here may have asymmetric centers and therefore exist in different enantiomeric and diastereomeric forms. The compounds of the invention can be in the form of an optical isomer or a diastereomer. Accordingly, the invention encompasses compounds in the form of their optical isomers, diastereoisomers and mixtures thereof, including a racemic mixture. Optical isomers of the compounds of the invention can be obtained by known techniques such as asymmetric synthesis, chiral chromatography, simulated moving bed technology or via chemical separation of stereoisomers through the employment of optically active resolving agents. Unless otherwise indicated, “stereoisomer” means one stereoisomer of a compound that is substantially free of other stereoisomers of that compound. Thus, a stereomerically pure compound having one chiral center will be substantially free of the opposite enantiomer of the compound. A stereomerically pure compound having two chiral centers will be substantially free of other diastereomers of the compound. A typical stereomerically pure compound comprises greater than about 80% by weight of one stereoisomer of the compound and less than about 20% by weight of other stereoisomers of the compound, for example greater than about 90% by weight of one stereoisomer of the compound and less than about 10% by weight of the other stereoisomers of the compound, or greater than about 95% by weight of one stereoisomer of the compound and less than about 5% by weight of the other stereoisomers of the compound, or greater than about 97% by weight of one stereoisomer of the compound and less than about 3% by weight of the other stereoisomers of the compound. Overview Disclosed herein are nitroalkenes specifically designed to modulate the activity of stimulators of interferon genes (STING) for treating inflammatory diseases. STING plays a crucial role in the inflammatory response to cytosolic DNA, and its aberrant activation is associated with various inflammatory conditions. However, developing effective inhibitors for STING has been challenging. The compounds disclosed herein present a breakthrough in the form of nitroalkene-based compounds that effectively inhibit STING activity, thereby mitigating STING-driven inflammation. The incorporation of an aromatic moiety into nitroalkenes significantly enhanced their electrophilicity and reduced their degrees of freedom, leading to improved potency and specificity in inhibiting STING. The lead compounds CP-36 and CP-45, featuring a β-nitrostyrene moiety, potently inhibited STING activity in vitro and relieved STING-dependent inflammation in vivo. A library of novel nitroalkene compounds was built, including the lead compounds CP-36 and CP-45, characterized by a β-nitrostyrene moiety, demonstrating potent inhibition of STING activity in vitro. Furthermore, they were found to alleviate STING-dependent inflammation in vivo, validating their potential as drug candidates for the treatment of8123-112123-02 05 / 16 / 25 06666STING-driven inflammatory diseases. Mechanistically, by alkylating the reactive cysteines that are responsible for the palmitoylation of STING, nitroalkenes efficiently block this indispensable step in the STING signaling pathway, resulting in a significant reduction of STING-driven inflammation. This class of compounds was proven to inhibit both the wild-type STING and those with gain-of- function mutations, broadening the scope of its therapeutic application. The cyclic GMP-AMP (cGAMP) synthase (cGAS) is the primary cytosolic sensor of double- stranded DNA (dsDNA) in mammalian cells. Binding of dsDNA to cGAS prompts the synthesis of the second messenger molecule 2’3’-cGAMP (cGAMP), a potent activator of the intracellular adaptor molecule Stimulator of Interferon Genes (STING) (also known as MPYS, ERIS, MITA, TMEM173 or STING1). STING activation is an indispensable step for the induction of type I interferons (IFNs, i.e., IFNα and IFNβ) and pro-inflammatory cytokines. STING-mediated signaling comprises STING translocation from the endoplasmic reticulum (ER) through the ER-Golgi intermediate compartments (ERGIC) and Golgi to post-Golgi vesicles. STING exists as a constitutive dimer but undergoes stabilizing conformational changes upon binding of cGAMP. These changes involve a 180° rotation, “lid-closing” and the oligomerization of STING dimers. Additionally, posttranslational cysteine modifications through palmitoylation of residues (i.e., Cys88 / 91) modulate trafficking and activity. The C-terminal tail of STING provides a scaffold for the recruitment of the TANK-binding kinase 1 (TBK1) and TBK1 autophosphorylation as well as phosphorylation of STING at Ser366 and enhanced binding of interferon regulatory factor 3 (IRF3). The TBK1-promoted phosphorylation of IRF3 allows for IRF3 dimerization and translocation to the nucleus for the final induction of type I IFNs and interferon-stimulated genes (ISGs). In parallel, STING activation causes NFκB to upregulate several pro-inflammatory mediators as well as the induction of autophagy. A slew of autoimmune and inflammatory conditions, including systemic lupus erythematosus (SLE), Aicardi-Goutières syndrome (AGS), and COPA syndrome, are associated with aberrant STING activation and a pathological induction of type I IFNs and ISG signatures. Additionally, rare gain-of-function mutations in the STING-encoding gene TMEM173, result in ligand-independent STING activation by spontaneous conformational changes and translocation of STING. This STING overactivation causes the severe autoinflammatory syndrome STING-associated vasculopathy with onset in infancy (SAVI). SAVI patients show a clinical presentation of early-onset systemic inflammation, severe skin vasculopathy, and interstitial lung disease, causing pulmonary fibrosis and respiratory failure. Therapeutic interventions in the reported patient cases have so far proven unsatisfactory. The central role of the cGAS-STING pathway in pathogenic inflammation has invigorated extensive research into developing novel therapeutics modulating this pathway. Despite the intense work, no STING inhibitor has yet advanced into the clinical stage, calling for additional discovery and development efforts for the unmet clinical need of targeting STING-dependent inflammation.8123-112123-02 05 / 16 / 25 06666Designing an inhibitor that binds to the ligand-binding domain of the dimeric STING is challenging due to the large volume of its binding pocket, requiring a bulky ligand with unfavorable drug molecular properties. It is also uncertain whether such a ligand would effectively inhibit STING variants with gain-of-function mutations. To circumvent it, we hypothesized that developing covalent modifiers that alkylate Cys88 / 91 to block STING palmitoylation would be an effective strategy against both wild-type and mutated STINGs, regardless of the conformation of the cyclic dinucleotide (CDN)-binding site. Indeed, several Cys88 / 91 alkylating compounds have been reported in recent years. Using a library screen, the Ablasser group discovered nitrofuran analogs that covalently bind and inhibit STING. Two chemical entities, C-176 and C-178, inhibit murine STING specifically, whereas modified compounds, C-170, C-171, and H151, also inhibit human STING. These compounds inhibit STING signaling by covalently modifying Cys91 and preventing its palmitoylation. At that time, we discovered that nitrated fatty acids, a class of endogenous lipid signaling species, inhibit STING signaling by preventing STING palmitoylation through Cys88 and Cys91 post-translational modifications. More recently, the Cravatt group identified that electrophilic acrylamide analogs BPK-21 and BPK-25 inhibit STING by targeting palmitoylation at Cys91. These recent discoveries highlight that inhibiting STING palmitoylation is a relevant pharmacological target to reduce STING-dependent inflammation. The nitrated fatty acids are well-characterized bioactive lipids that exert a variety of anti- inflammatory and tissue-protective functions. These electrophilic compounds react reversibly with cellular nucleophiles (e.g., reactive cysteine and histidine-containing proteins) of targeted proteins via Michael addition. Nitroalkene-protein adducts will then transfer into glutathione (GSH) to be metabolized and excreted through the mercapturic acid pathway. We discovered that the nitrated fatty acids could covalently modify STING by nitro-alkylating Cys88 and Cys91, thus preventing the essential palmitoylation required for STING activation and translocation, and inhibiting the downstream STING signaling and the subsequent release of type I IFNs. Furthermore, nitrated fatty acids are consistently generated endogenously in the gastrointestinal tract before systemic distribution. They are also generated locally at inflammation sites, during ischemic events and in response to viral infections. This suggests that nitrated fatty acids constitute a naturally occurring feedback mechanism to restrain inflammation, and provide a scaffold to support the development of new-generation STING inhibitors. Nitrooleic acid (NO2-OA), a clinical candidate being evaluated in a Phase II clinical trial to treat obese asthmatics, became our prototypical nitrated fatty acids. Despite the high tolerance and promising efficacy, NO2-OA showed a limited half-life and was rapidly deactivated through loss or migration of the double bond to form non-electrophilic NO2-stearic acid or 10-NO2-octadec-8-enoic acid, respectively, limiting its half-life. Additionally, nitrated fatty acids are absorbed and distributed in chylomicrons esterified to triglycerides, leading to low ng / mL plasma concentrations of the free acid form, hence the bioactive form. We previously synthesized a small library of NO2-OA-derived8123-112123-02 05 / 16 / 25 06666mimetics and evaluated their activity in the Nrf2 and NF-κB pathways, revealing that the chemical scaffold (e.g., spatial orientation and positions of the nitrovinyl group) plays a role in their biological activity, and metabolic inactivation. Drawing from our prior experience and the hypothesis that chemical modifications of the nitrated fatty acid scaffold could enhance STING inhibition, we embarked on the design and synthesis of a novel series of chemical entities through iterative processes. These iterations involved constraining the degrees of freedom of the alkyl chain, adjusting the reactivity of the nitroalkene group, altering the hydrophobicity of the molecules, and tuning the steric hindrance of the ancillary moieties. The potential for inhibiting STING-dependent signaling was herein evaluated entirely in human cell models relevant to STING signaling. We tested if the lead nitroalkene compounds, CP-36, and CP-45, could modulate STING-dependent inflammation in vivo. The compounds showed a notable capacity to dampen both systemic and tissue-specific STING-dependent inflammation regardless of administration routes, with remarkable effects even after a single dose. In summary, nitroalkenes strongly target and inhibit STING-dependent signaling. Thus, our study provides structural insight into STING inhibition by nitroalkenes. This knowledge can guide the development of drug candidates for more effective STING inhibition for treating autoimmune and autoinflammatory diseases where STING is a central driver of devastating inflammation. Compounds and Pharmaceutical Compositions Disclosed herein are compounds having a structure of: Z-(CH2)x-C(NO2)=CH-A-(CH2)y-(CH=CH)z-C(=O)O-R1wherein x and y are each independently 0 to 10; z is 0 or 1; Z is CH3, cycloalkyl, substituted cycloalkyl, aryl, or substituted aryl; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl. In certain embodiments, Z is CH3. In certain embodiments, Z is phenyl. In certain embodiments, Z is phenyl, and x is 1. In certain embodiments, Z is cyclohexyl. In certain embodiments, Z is cyclohexyl, and x is 0. In certain embodiments, Z is adamantyl. In certain embodiments, Z is naphthyl. In certain embodiments, y is 0 and z is 1.8123-112123-02 05 / 16 / 25 06666In certain embodiments, z is 0. In certain embodiments, x and y are each independently 1 to 5. In certain embodiments, R1is C1-C6 alkyl. In certain embodiments, R1is C1-C6 alkenyl. In certain embodiments, R1is hydrogen. In certain embodiments, A is phenylene. Disclosed herein are compounds having a structure of: CH3-(CH2)x-C(NO2)=CH-A-(CH2)y-C(=O)O-R1wherein x and y are each independently 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl. In certain embodiments, x and y are each independently 1 to 5. In certain embodiments, R1is C1-C6alkyl. In certain embodiments, R1is C1-C6alkenyl. In certain embodiments, R1is hydrogen. In certain embodiments, A is phenylene. Disclosed herein are compounds having a structure of: Y-A-CH=C(NO2)-(CH2)x-(CH=CH)z-C(=O)O-R1wherein x is 1 to 10; z is 0 or 1; Y is CH3, halogen, alkyl, substituted alkyl, or hydroxyl; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl. In certain embodiments, z is 0. In certain embodiments, x is 1 to 5. In certain embodiments, R1is C1-C6alkyl. In certain embodiments, R1is C1-C6alkenyl. In certain embodiments, R1is hydrogen. In certain embodiments, A is phenylene.8123-112123-02 05 / 16 / 25 06666Disclosed herein are compounds having a structure of: CH3-A-CH=C(NO2)-(CH2)x-C(=O)O-R1wherein x is 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl. In certain embodiments, A is phenylene. In certain embodiments, A is pyridine. In certain embodiments, A is indole. In certain embodiments, x is 1 to 5. In certain embodiments, R1is C1-C6alkyl. In certain embodiments, R1is C1-C6alkenyl. In certain embodiments, R1is hydrogen. In some embodiments, one or more of the disclosed compounds (including compounds linked to a detectable label or cargo moiety) are mixed or combined with a suitable pharmaceutically acceptable carrier to prepare a pharmaceutical composition. Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any such carriers known to be suitable for the particular mode of administration. Remington: The Science and Practice of Pharmacy, The University of the Sciences in Philadelphia, Editor, Lippincott, Williams, & Wilkins, Philadelphia, PA, 21stEdition (2005), describes exemplary compositions and formulations suitable for pharmaceutical delivery of the compounds disclosed herein. In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients. Upon mixing or addition of the compound(s) to a pharmaceutically acceptable carrier, the resulting mixture may be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. Where the compounds exhibit insufficient solubility, methods for solubilizing may be used. Such methods are known and include, but are not limited to, using co- solvents such as dimethylsulfoxide (DMSO), using surfactants such as Tween®, using cyclodextrin inclusion complexes, using liposomes, using oils like triolein, using polyethylene glycol polymers, and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs may also be used in formulating effective pharmaceutical compositions. The disclosed compounds may also be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release8123-112123-02 05 / 16 / 25 06666formulations, such as, but not limited to, microencapsulated delivery systems. Formulations may also be obtained by dissolution with mid and long-chain natural oils. The disclosed compounds and / or compositions can be enclosed in multiple or single dose containers. The compounds and / or compositions can also be provided in kits, for example, including component parts that can be assembled for use. For example, one or more of the disclosed compounds may be provided in a lyophilized form and a suitable diluent may be provided as separated components for combination prior to use. In some examples, a kit may include a disclosed compound and a second therapeutic agent (such as an anti-retroviral agent) for co-administration. The compound and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the compound. The containers are preferably adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-filled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical administration. The pharmaceutical compositions may be in a dosage unit form such as an injectable fluid, an oral delivery fluid (e.g., a solution or suspension), a nasal delivery fluid (e.g., for delivery as an aerosol or vapor), a semisolid form (e.g., a topical cream), or a solid form such as powder, pill, tablet, or capsule forms. The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. A therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder. In some examples, a therapeutically effective amount of the compound is an amount that lessens or ameliorates at least one symptom of the disorder for which the compound is administered. Typically, the compositions are formulated for single dosage administration. The concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art. In some examples, about 1 mg to 1000 mg of a disclosed compound, a mixture of such compounds, or a physiologically acceptable salt or ester thereof, is compounded with a physiologically acceptable vehicle, carrier, excipient, binder, preservative, stabilizer, flavor, etc., in a unit dosage form. The amount of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained. The term “unit dosage form” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. In some examples, the compositions are formulated in a unit dosage form, each dosage containing from about 1 mg to about 1000 mg (for8123-112123-02 05 / 16 / 25 06666example, about 2 mg to about 500 mg, about 5 mg to 50 mg, about 10 mg to 100 mg, about 25 mg to 75 mg, or about 100 mg to 300 mg) of the one or more compounds. In other examples, the unit dosage form includes about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, or more of the disclosed compound(s). The disclosed compounds or compositions may be administered as a single dose, or may be divided into a number of smaller doses to be administered at intervals of time. The therapeutic compositions can be administered in a single dose delivery, by continuous delivery over an extended time period, in a repeated administration protocol (for example, by a multi-daily, daily, weekly, or monthly repeated administration protocol). It is understood that the precise dosage, timing, and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. In addition, it is understood that for a specific subject, dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only. When administered orally as a suspension, these compositions are prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners / flavoring agents. As immediate release tablets, these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants. If oral administration is desired, the compound is typically provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient. Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline8123-112123-02 05 / 16 / 25 06666cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring. When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors. When administered orally, the compounds can be administered in usual dosage forms for oral administration. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, it is preferred that they be of the sustained release type so that the compounds need to be administered only once or twice daily. In some examples, an oral dosage form is administered to the subject 1, 2, 3, 4, or more times daily. In additional examples, the compounds can be administered orally to humans in a dosage range of 0.1 to 70 mg / kg body weight in single or divided doses. One illustrative dosage range is 0.1 to 50 mg / kg body weight orally (such as 0.5 to 50 mg / kg body weight orally) in single or divided doses. For oral administration, the compositions may be provided in the form of tablets containing about 1 to 1000 milligrams of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 750, 800, 900, or 1000 milligrams of the active ingredient. It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy. Injectable solutions or suspensions may also be formulated, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer’s solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or di-, or triglycerides, and fatty acids, including oleic acid. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, olive oil, triolein, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium8123-112123-02 05 / 16 / 25 06666bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required. Where administered intravenously, suitable carriers include physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. The compounds can be administered parenterally, for example, by IV, IM, depo-IM, SC, or depo-SC. When administered parenterally, a therapeutically effective amount of about 0.1 to about 500 mg / day (such as about 1 mg / day to about 100 mg / day, or about 5 mg / day to about 50 mg / day) may be delivered. When a depot formulation is used for injection once a month or once every two weeks, the dose may be about 0.1 mg / day to about 100 mg / day, or a monthly dose of from about 3 mg to about 3000 mg. The compounds can also be administered sublingually. When given sublingually, the compounds should be given one to four times daily in the amounts described above for IM administration. The compounds can also be administered intranasally. When given by this route, the appropriate dosage forms are a nasal spray or dry powder. The dosage of the compounds for intranasal administration is the amount described above for IM administration. When administered by nasal aerosol or inhalation, these compositions may be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents. The compounds can be administered intrathecally. When given by this route, the appropriate dosage form can be a parenteral dosage form. The dosage of the compounds for intrathecal administration is the amount described above for IM administration. The compounds can be administered topically. When given by this route, the appropriate dosage form is a cream, ointment, or patch. When administered topically, an illustrative dosage is from about 0.5 mg / day to about 200 mg / day. Because the amount that can be delivered by a patch is limited, two or more patches may be used. The compounds can be administered rectally by suppository. When administered by suppository, an illustrative therapeutically effective amount may range from about 0.5 mg to about 500 mg. When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic8123-112123-02 05 / 16 / 25 06666glyceride esters of polyethylene glycols, which are solid at ordinary temperatures, but liquefy and / or dissolve in the rectal cavity to release the drug. It should be apparent to one skilled in the art that the exact dosage and frequency of administration will depend on the particular compounds administered, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular subject, and other medication the individual may be taking as is well known to administering physicians or other clinicians who are skilled in therapy of retroviral infections, diseases, and associated disorders. Methods of Treatment The compounds disclosed herein may be used for treating a (STING)-mediated inflammatory or autoimmune condition. Illustrative (STING)-mediated inflammatory or autoimmune conditions include an infectious diseases, systemic lupus erythematosus (SLE), psoriasis, endometriosis, primary biliary cholangitis, metabolic dysfunction-associated steatotic liver disease, alcoholic fatty liver disease, Sjogren's Syndrome, rheumatoid arthritis, type I diabetes, type 2 diabetes, Crohn’s disease, ulcerative colitis, Aicardi-Goutières syndrome (AGS), STING-Associated Vasculopathy with Onset in Infancy (SAVI), chronic viral infections such as HIV and hepatitis, pulmonary fibrosis, systemic sclerosis, multiple sclerosis, cardiac hypertrophy, coronary disease, atherosclerosis, neurodegeneration disease, neuroinflammation disease including Parkinson’s and Alzheimer’s disease, autoimmune thyroid disease, inflammatory myopathy, autoimmune skin disease gastrointestinal autoimmune disease, autoimmune neurological disease, vasculitis syndrome systemic sclerosis (scleroderma), ankylosing spondylitis, psoriatic arthritis, Behçet's disease, relapsing polychondritis, autoimmune eye disease, autoimmune myocarditis, autoimmune blood disorder autoimmune lung disease, autoimmune endocrine disorder, connective tissue disease, COPA syndrome, Mooren’s ulcer, prebyscusis, ischemia reperfusion injury (liver, heart, kidney, lung, brain) particularly those involving vascular and surgical interventions, which are known to trigger sterile inflammation mediated in part by the cGAS-STING pathway, dilated cardiomyopathy, heart failure, radiation-induced liver, lung and heart injury, Huntington’s disease, spinocerebellar ataxia type I, nerve regeneration, contrast-induced nephropathy, non-eosinophilic asthma, atopic dermatitis, chronic obstructive pulmonary disease, acute lung injury, renal fibrosis, APOL1 related kidney disease, intervertebral disk related pathologies, or pulmonary fibrosis. In certain embodiments, the (STING)-mediated inflammatory or autoimmune condition is endometriosis, including, but not limited to, ovarian endometriosis, pelvic endometriosis, peritoneal endometriosis, diaphragmatic endometriosis, thoracic endometriosis syndrome (TES), adenomyosis, uterine fibroids, polycystic ovary syndrome (PCOS), endometrial cancer and associated conditions or syndromes including, but not limited to, infertility and pregnancy complications, asthma, catamenial8123-112123-02 05 / 16 / 25 06666pneumothorax, catamenial hemothorax, catamenial hemoptysis, inflammatory bowel disease (IBD), and interstitial cystitis / bladder pain syndrome (IC / BPS). Illustrative embodiments are described below in the following numbered clauses: 1. A method comprising administering to a subject having a stimulator of interferon genes (STING)-dependent inflammatory or autoimmune condition a therapeutically effective amount of a compound that includes a nitrovinyl moiety conjugated to an aromatic moiety. 2. The method of clause 1, wherein the nitrovinyl moiety conjugated to an aromatic moiety is a β-nitrostyrene moiety. 3. The method of clause 1 or 2, wherein the (STING)-mediated inflammatory or autoimmune condition are infectious diseases, systemic lupus erythematosus (SLE), psoriasis, primary biliary liver disease, non-alcoholic fatty liver disease, Sjogren's Syndrome, rheumatoid arthritis, type I diabetes, type 2 diabetes, Crohn’s disease, ulcerative colitis, Aicardi-Goutières syndrome (AGS), STING-Associated Vasculopathy with Onset in Infancy (SAVI), chronic viral infections such as HIV and hepatitis, pulmonary fibrosis, systemic sclerosis, multiple sclerosis, cardiac hypertrophy, coronary disease, atherosclerosis, neurodegeneration and neuroinflammation including Parkinson’s and Alzheimer’s disease, autoimmune thyroid diseases like Hashimoto's Thyroiditis and Graves' Disease, inflammatory myopathies like polymyositis, dermatomyositis and inclusion body myositis, Autoimmune Skin Diseases like Pemphigus Vulgaris, Bullous Pemphigoid, Gastrointestinal Autoimmune Diseases like celiac disease, autoimmune hepatitis, Primary Biliary Cholangitis, Primary Sclerosing Cholangitis, Microscopic Colitis and Autoimmune Gastritis, Autoimmune Neurological Diseases like Myasthenia Gravis, Guillain-Barré Syndrome, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Vasculitis Syndromes like Giant Cell Arteritis, Granulomatosis with Polyangiitis (Wegener's Granulomatosis), Microscopic Polyangiitis, Kawasaki Disease, other autoimmune disorders like systemic sclerosis (Scleroderma), Ankylosing Spondylitis, Psoriatic Arthritis, Behçet's Disease, Relapsing Polychondritis, Autoimmune Eye Diseases like Uveitis and Scleritis, autoimmune myocarditis, Autoimmune Blood Disorders like Autoimmune Hemolytic Anemia, Immune Thrombocytopenic Purpura (ITP) and Autoimmune Neutropenia, Autoimmune Lung Diseases like Granulomatosis with Polyangiitis and Churg-Strauss Syndrome, Autoimmune Endocrine Disorders like Addison's Disease and Autoimmune Pancreatitis, Connective Tissue Diseases like mixed connective tissue disease (MCTD) and Ehlers-Danlos Syndrome, COPA syndrome, Mooren’s ulcer, prebyscusis, ischemia reperfusion injury (liver, heart, kidney, lung, brain) dilated cardiomyopathy, heart failure, radiation-induced liver, lung and heart injury, Huntington’s disease, spinocerebellar ataxia type I, nerve regeneration, chronic obstructive pulmonary disease,8123-112123-02 05 / 16 / 25 06666acute lung injury, renal fibrosis, APOL1 related kidney disease, intervertebral disk related pathologies, or pulmonary fibrosis. 4. The method of clause 1 or 3, wherein the compound that includes the nitrovinyl moiety conjugated to an aromatic moiety has a structure of: CH3-(CH2)x-C(NO2)=CH-A-(CH2)y-C(=O)O-R1wherein x and y are each independently 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl. 5. The method of clause 1 or 3, wherein the compound that includes the nitrovinyl moiety conjugated to an aromatic moiety has a structure of: CH3-A-CH=C(NO2)-(CH2)x- C(=O)O-R1wherein x is 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl. 6. The method of any one of clauses 2 to 5, wherein Ar is phenylene. 7. The method of clause 2 or 3, wherein the compound that includes a β-nitrostyrene moiety is (E)-3-(3-(2-nitrohept-1-en-1-yl)phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof. 8. The method of clause 2 or 3, wherein the compound that includes a β-nitrostyrene moiety is methyl (E)-4-nitro-5-(p-tolyl)pent-4-enoate, or a pharmaceutically salt thereof. 9. A compound, or a pharmaceutically acceptable salt thereof, having a structure of: CH3-(CH2)x-C(NO2)=CH-A-(CH2)y-C(=O)O-R1wherein x and y are each independently 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and8123-112123-02 05 / 16 / 25 06666R1is hydrogen, an alkyl, or a substituted alkyl. 10. The compound, or a pharmaceutically acceptable salt thereof, of clause 9, wherein A is phenylene. 11. The compound, or a pharmaceutically acceptable salt thereof, of clause 10, wherein the compound, or a pharmaceutically acceptable salt thereof, is (E)-3-(3-(2-nitrohept-1-en-1- yl)phenyl)propanoic acid. 12. A compound, or a pharmaceutically acceptable salt thereof, having a structure of: CH3-A-CH=C(NO2)-(CH2)x- C(=O)O-R1wherein x is 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl. 13. The compound, or a pharmaceutically acceptable salt thereof, of clause 12, wherein A is phenylene. 14. The compound, or a pharmaceutically acceptable salt thereof, of clause 13, wherein the compound, or a pharmaceutically acceptable salt thereof, is methyl (E)-4-nitro-5-(p- tolyl)pent-4-enoate. Examples Comprehensive structure-activity relationship study of nitroalkene inhibition of STING Oleic acid nitration products, 9- and 10-nitrooleic acids, have been shown to inhibit STING. To optimize the nitrated fatty acid chemical scaffold for STING inhibition, we first evaluated the inhibition of STING-dependent signaling using a small library of analogs in human cells. The in- house library is composed of nitroalkene acyl acids and fatty alcohols with varying chain lengths (14 to 24 carbons), and the nitrovinyl group is placed at distances ranging from as near as three carbons from the carboxyl group to the ω-end of the fatty acid chain (CP-1 to CP-16 and CP-18 to CP-21. Nitroalkenes were first tested using NF-κB and IRF reporter assays in THP-1 dual cells featuring NF-8123-112123-02 05 / 16 / 25 06666κB-SEAP and IRF-Lucia luciferase. This allows simultaneous evaluation of the NF-κB and IRF- pathways by measuring SEAP and secreted luciferase, respectively. Several structure-activity relationship features were identified: a) the inhibition was abolished with fatty acids nitroalkenes shorter than 17 carbons (CP-1 to CP-3) and longer than 20 carbons (CP-13 to CP-16); b) the distance between the nitrovinyl group and the carboxyl group influences the activities with 7-NO2 to 9-NO2 alkenes providing the optimal spacing; and c) a hydrophobic tail was essential for STING inhibition (FIG.1A). We next tested the inhibition of CXCL-10, an interferon-induced cytokine, expression in THP-1 cells. Like the results obtained in the NF-κB and IRF reporter activity assay, CP-8 and CP-10 demonstrated a marked CXCL-10 reduction (FIG.1B). As negative controls, oleic (OA) and linoleic acid (LA) did not suppress the response, showing that the activity was dependent on the presence of the nitroalkene group (FIG.1A). As one of the most consistent STING inhibitors, we synthesized and evaluated individual isomers of CP-8 (7-NO2 nonadecenoic acid (CP-8a) and 8-NO2 nonadecenoic acid (CP-8b)). Both isomers demonstrated dose-dependent CXCL-10 inhibition, comparable to H151 at the highest concentration tested (FIG.1C). Nitrated fatty acids are largely metabolized to nitrodicarboxylates in humans, dogs, rats, and mice due to sequential ω- oxidation and various degrees of β-oxidation. To evaluate whether dicarboxylate metabolites would retain the ability to inhibit STING, we synthesized a series of nitroalkene dicarboxylate esters (CP-23 to CP-30, to promote cellular uptakes dicarboxylates are less cell-permeable. We found that the presence of an additional carboxyl group abolished the suppression of STING, suggesting that an aliphatic tail could be essential for STING nitroalkylation. To establish if other electrophilic compounds could inhibit STING, we tested the α,β-unsaturated keto electrophile, kynurenine-carboxyketoalkene (kyn-CKA, here just termed CKA). CKA, a non-nitroalkene endogenous electrophile, recently reported to exert anti-inflammatory activities, also failed to elicit STING inhibition (FIG.1C). Next, we sought to establish that the anti-inflammatory effects were correlated to the suppression of the STING signaling pathway. The lead compounds, CP-6, CP-8a, CP-8b, and CP-10, efficiently suppressed phosphorylation of STING, TBK1, and IRF3, in a dose-dependent manner (FIG.1D). On the contrary, the unrelated electrophilic species, CKA, and the nitroalkene diesters derived from di- COOH-C3 did not retain this activity, suggesting the importance of the structural components of the compounds beyond their electrophilic warhead. Notably, no suppression on pSTING and pIRF3 was observed by H151 either (FIG.1D). As a result of IFN inhibition, NO2OA and the CP-8s also impaired the induction of interferon-stimulated genes (ISGs), including IFIT1 and ISG15. In this case, the effect was mirrored by H151 (FIG.1E). Besides their ability to inhibit wild-type STING, we also evaluated the potential of the lead nitroalkene compounds for blocking the STING activity in SAVI patient-derived fibroblasts bearing the gain-of-function mutation (N154S). By blocking the palmitoylation of STING, the critical step of STING oligomerization and translocation, the selected nitroalkenes (i.e. CP-8s, CP-10, and CP-12) efficiently suppressed type I IFN release in a dose-8123-112123-02 05 / 16 / 25 06666dependent manner in SAVI fibroblast (FIG.1F). This effect was again correlated with the suppression of pSTING, TBK1, and IRF3 in SAVI cells (FIG.1G). Although H151 also reduced IFN expression, the effect was not STING-dependent as pSTING, TBK1, or IRF3 was not altered in SAVI patient- derived fibroblasts. Collectively, several lead compounds in this generation (i.e., CP-6, CP-8, CP-10, CP-12) robustly reduced CXCL-10 and type I IFN levels prominently by inhibiting STING activity. Conjugated nitroalkenes with favorable physicochemical properties amplified the STING inhibition With the critical structural features learned from the initial screen, we engaged in compound optimization efforts, focusing on improving the nitroalkene electrophilicity and achieving greater specificity by reducing the degree of freedom in the molecules. Thus, a series of conjugated nitroalkenes (CP-33 to CP-58) were designed to make a more rigid molecular scaffold. Conjugated nitroalkenes, including aromatic rings, are reported to be strong electrophiles. Additionally, the embedded aromatic ring was introduced to limit β-oxidation, a primary metabolic pathway. We also tested NO2-eleostearic acid (NO2-EA), a naturally occurring and endogenously formed compound recently found in human urine. To assess the improved inhibition profiles and pharmacological characteristics of the second-generation nitroalkenes, we evaluated the in vitro therapeutic index based on the ability to suppress cGAMP-induced CXCL-10 release and cytotoxicity in THP-1s. Based on this assay, several conjugated nitroalkene compounds (i.e., CP-36, CP-45, CP-46, CP-50, CP-51, and NO2-EA) strongly inhibited the cGAMP-induced CXCL-10 release while showing minimal cytotoxicity evaluated by MTT measurements (FIG.2). The STING inhibitory effects of these selected conjugated nitroalkene compounds were further evaluated in SAVI patient-derived fibroblasts bearing the gain-of-function mutation (N154S). CP-36, CP-45, CP-46, and CP-50 dose-dependently reduced significantly IFN expression while showing minimum cytotoxicity (FIG.3A). The selected conjugated nitroalkenes (i.e., CP-36, CP-45, and CP-46) also efficiently suppressed the NF-κB and IRF reporter activity in THP-1 dual cells induced by cGAMP in a dose-dependent manner (FIG.3B). Benchmarked against CP-8b, CP-36, and CP-50 also dose-dependently inhibited STING signaling, indicated by reduced STING, TBK1, and IRF3 phosphorylation in SAVI cells (FIG.3C). To further characterize the physicochemical properties of the lead CP-36 and CP-45 compounds (nitroalkenes as in CP-8b have previously been characterized (44)), we evaluated their reactivity towards glutathione (GSH). The compounds have absorption peaks shifted to the right (323 nm and 333 nm for CP-36 and CP-45, respectively) compared to nitrooleic acid (268 nm) because of the increased conjugation conferred by the adjacent aromatic ring (FIG.3D). Reaction with GSH, the most abundant intracellular thiol, resulted in a decrease in peak absorbance and a single isosbestic point formed at 275 nm for CP-36 and 279 nm for CP-45, suggesting that no intermediates accumulate and that the reaction proceeds in only one kinetically relevant step (FIG.3D). To characterize the reaction, we performed a stopped-flow kinetic8123-112123-02 05 / 16 / 25 06666analysis of the reaction following the loss of absorbance at the compound’s peak wavelength. The plots of the pseudo-first-order exponential constant (kobs) against GSH concentration were linear, resulting in kon second-order rate constants of 50.7 ± 0.6 M-1s-1and 164 ± 1 M-1s-1(25 °C, pH 7.4) for CP-36 and CP-45, respectively (FIG.3E-F). A possible explanation for the lower rate constant of CP- 36 is the presence of a negative charge at the reaction pH, which could slow the reaction with the also negatively charged GSH. In contrast, CP-45 is neutral. The y-intercepts of the linear regression of the kobs versus GSH concentration plots were slightly below 0 for both compounds. While values below 0 are not expected from a kinetic perspective, the data likely indicate that the reaction of both compounds with GSH is irreversible or has a very small negligible koff(FIG.3F). This strongly contrasts with other prototypical nitroalkenes like nitrooleic acid and conjugated nitrolinoleic acid that display a reversible reaction with thiols. Nitrooleic acid has a lower kon reaction constant (64 M-1s-1) than CP-45 and a higher koff (0.006 s-1). It could be speculated, that another distinctive characteristic of CP-36 and CP-45 is that they likely are not esterified to triglycerides and packed into chylomicrons for distribution, as reported for nitrated fatty acids. The selected lead compounds in this generation were further evaluated in acute STING animal models. Intraperitoneal administration of CP-36 dampens STING-dependent inflammation in vivo. To assess the STING inhibition of CP-36 in vivo, we injected a synthetic cGAMP analog, cAIM(PS)2Difluor (Rp / Sp), intraperitoneally into mice to induce STING-dependent systemic inflammation. Both CP-8b and CP-36 were initially selected for testing because of their ability to modulate STING activity and low toxicity in SAVI patient-derived fibroblasts. Intraperitoneal administration of CP-8b was not well tolerated in mice. In contrast, CP-36 (both at low and high doses) was well tolerated and allowed for determining tissue-specific STING-dependent anti- inflammatory effects, as evaluated by Ifnb1 gene expression and ISGs induction in various tissues. CP-36 administration improved protection against STING activation by dampening Ifnb1 expression in several tissues, including the liver, lung, spleen, and heart. While there appears to be a discernible trend in the protective effects, the large variability observed in the responses to the cGAMP analog undermined the ability to establish significant protection. A significantly larger n would be required to establish a statistical significance in Ifnb1 expression. Nonetheless, CP-36 markedly decreased the expression of Ifnb1 in the liver. In addition, given the role of STING in lung inflammatory processes, we specifically evaluated the dampening of STING signaling pathways in the liver and lung. Here, we found statistically significant pTBK1, TBK1, and Viperin reductions in lung tissue (FIG.4A, C) and STAT1 in liver tissue (FIG.4B, C) with the 30 mg / kg CP-36 administration. As reported earlier, nitro fatty acids rely on their electrophilicity to modulate STING activity, and nitroalkylating Cys88 / 91 blocks STING palmitoylation. Therefore, we also synthesized a reduced CP-36 (R-CP-36) by saturating the nitroalkene into a nitroalkane as a negative control. The loss of electrophilicity in R-CP-8123-112123-02 05 / 16 / 25 0666636, implying loss of reactivity toward the Cys88 / 91, completely abolished the STING inhibition observed with CP-36 (FIG.4D – F). CP-36 significantly decreased both Ifnb1 and Cxcl-10 expression in BDMDs, whereas R-CP-36 was completely ineffective (FIG.4F). Additionally, only CP-36, rather than R-CP-36, exerts significant reductions of Isg15 and Ifit1 expression. Moreover, the cGAMP induction of Ifnb, Cxcl-10, Isg15, and Ifit1 expression is dependent on STING, while no response upon cGAMP stimulation was seen in bone marrow-derived macrophages (BMDMs) from STING knockout mice. This indicates that the inhibition is specific to STING and requires the electrophilicity of the nitroalkene group. Collectively, the conjugated nitroalkene, represented by CP-36, significantly mitigated the tissue-specific inflammation in STING-activated animals and relied on the electrophilic nitroalkene warhead to exert the STING inhibitory effects. Oral administration of nitroalkene compounds CP-36 and CP-45 dampens STING-dependent inflammation in vivo. Oral bioavailability is a key characteristic in drug development as it significantly increases the reach of the therapeutic approach. To compensate for any loss in drug absorption and bioavailability compared to intraperitoneal (i.p.), we elevated the oral dosage of the administered nitroalkene compounds to 40mg / kg and 60mg / kg. Given that CP-8b i.p. administration was not well tolerated, we incorporated CP-45 alongside CP-36 in subsequent in vivo experiments using oral delivery. CP-45 demonstrated a favorable therapeutic index in earlier patient-derived SAVI cell evaluations, and it is structurally distinctive from CP-36, with the carboxyl group positioned on the same side as the nitroalkene. Furthermore, as a methyl ester, CP-45 is expected to exhibit improved passive intestinal absorption compared to its carboxylic parent compound. First, we assessed if CP-36 and CP-45 were absorbed and systemically distributed. For CP- 45, only the de-methylated metabolite was assessed, as the determination of the methyl ester by HPLC-MS / MS and GC-MS / MS posed challenges, including very low sensitivity due to the lack of ionizable groups (LC) and enhanced degradation during work-up and heating (GC). Consequently, the CP-45 levels were significantly lower than CP-36, likely indicating limited hydrolysis of the ester. Similar to nitrated fatty acids, both CP-36 and CP-45 were found to be deactivated by a reductase in vivo, potentially prostaglandin reductase 1 (a phase II metabolic enzyme), to give rise to their reduced forms (dihydro-CP-36, R-CP-36 and dihydro-CP-45, R-CP-45). Next, we examined the capability of CP-36 and CP-45 to inhibit tissue-specific STING- dependent inflammation by assessing both the STING signaling pathway and the induction of interferon-stimulated genes (ISGs). Our findings revealed a strong and statistically significant reduction in STAT1 levels in lung tissue (FIG.5A, B). We also observed a significant effect on STING and phosphorylated STING upon administration of CP-45, whereas Viperin was mostly8123-112123-02 05 / 16 / 25 06666reduced by 40 mg / kg CP-36. Notably, no variance in ISG15 levels was detected (FIG.5A, B). Moreover, we observed a comparable trend of inhibition of the STING-dependent inflammation in the liver tissue. Then, we wanted to evaluate if CP-36 and CP-45 could systemically inhibit STING- dependent inflammation. We observed an overall reduction of relevant inflammatory cytokines and chemokines (e.g., for IFN-α, IFN-β, MCP-1, and MIP-1β) upon administration of 60 mg / kg CP-36 and CP-45, though a high animal variability in the responses to the activator (FIG.5C). We also observed an overall suppression of TNF-α and CXCL-10 upon administering 40 mg / kg CP-36. Lastly, a statistically significant reduction was seen in MCP-1 levels with 40 mg / kg CP-36 and in TNF-α levels with 60 mg / kg CP-45 (FIG.5C). Collectively, single doses of CP-36 and CP-45 dampened STING-dependent inflammatory cytokines and chemokines systemically and demonstrated substantial protection against cGAMP-induced STING-dependent inflammation. Aberrant activation of STING signaling is associated with a range of autoimmune and autoinflammatory conditions such as STING-associated vasculopathy with onset in infancy (SAVI). SAVI patients present with severe symptoms, including early-onset systemic inflammation, severe skin vasculopathy and interstitial lung disease, ultimately causing pulmonary fibrosis and respiratory failure. Currently, there are no clinically available therapeutics directly targeting the STING signaling, let alone treatment options for SAVI patients or those experiencing other inflammatory conditions resulting from gain-of-function STING mutations. Many STING inhibitors have been developed to target the cyclic dinucleotide (CDN)-binding domain to prevent cGAMP binding, thereby reducing the downstream signaling of the cGAS-STING pathway. However, the V155M and N154S mutations of SAVI patients are assumed to cause STING to adopt a more closed conformation and become constitutively active, regardless of CDN binding. Therefore, the inhibitors may not be effective for STING with gain-of-function mutations. Thus, inhibiting palmitoylation of Cys88 / 91, which facilitates STING translocation and oligomerization, could represent a more robust strategy to suppress STING signaling in both normal and SAVI conditions. In recent years, several compounds targeting the essential STING palmitoylation event at Cys88 / 91 have been identified, such as inhibitors containing reactive electrophilic nitroalkene, nitrofuran, indole urea, and acrylamide groups. These compounds have been tested in diverse in vivo and ex vivo models to evaluate their pre-clinical treatment potential in inhibiting inflammation such as STING-dependent inflammatory processes. Recent discoveries of additional compounds, such as BB-Cl-amidine targeting STING oligomerization through Cys148 modification, underscore the growing interest in inhibiting STING and the potential and effectiveness of covalent modulators. Here, we screened nitroalkene compounds for their potential to inhibit STING-dependent signaling and inflammation. The in vitro therapeutical potential of the nitroalkene compounds was determined by measuring their ability to suppress type I IFN and CXCL-10 release, NFκB / IRF activation, and STING signaling, in addition to assessing levels of cytotoxicity. This process enabled8123-112123-02 05 / 16 / 25 06666us to identify several lead nitroalkene compounds capable of inhibiting STING signaling, with conjugated nitroalkenes emerging as potent inhibitors while displaying minimal cytotoxicity. The structure-activity findings allowed us to identify optimal inhibitors for blocking STING activation. Furthermore, we tested the ability of lead nitroalkene compounds, CP-36 and CP-45, to modulate STING-dependent inflammation in vivo. First, CP-36, when administered intraperitoneally, effectively mitigated tissue-specific STING-dependent inflammation through its electrophilic properties, as no effect was observed with the deactivated analog R-CP-36. Subsequently, both lead nitroalkene compounds, CP-36 and CP-45, significantly dampened tissue-specific and systemic STING-dependent inflammation in vivo when administrated orally. Notably, the inhibitory effect was observed after a single dose and was independent of the administration route, highlighting the pharmacological potential of nitroalkene-based drugs as oral STING inhibitors. Overall, we observed a dose-dependent damping effect upon intraperitoneal and oral administration. One of the pharmacological characteristics of electrophilic modifiers is that they exhibit a bell-shaped dose-response curve, losing efficacy at high doses. This phenomenon has been widely described and has also been observed in preclinical models using nitro fatty acids. Adequate dosing of electrophiles such as nitroalkene compounds is a critical component for successful in vivo studies and should be considered in future evaluation of the lead compounds identified in this study. In a recent study, a very limited therapeutical effect of H-151 was observed. The authors of that study speculate that an extremely low oral bioavailability (F = 0.6 %) and a very short half-life of H-151 could be the reason. Our findings revealed that the H151 could strongly reduce the expression of CXCL-10, type I IFN, and ISGs in vitro. However, its impact on STING signaling components such as pSTING, pTBK1, and pIRF3 seemed minimal in THP-1s and SAVI fibroblasts. This disparity might stem from H-151's potential inability to achieve or sustain adequate concentrations in the cytoplasm for effective inhibition of STING activation and downstream signaling. Another plausible explanation is that H-151 may exhibit varying affinities or specificities for different STING variants or mutants, resulting in diverse responses to the inhibitor. Moreover, both CP-36 and CP-45 reacted with GSH with a high degree of irreversibility in comparison to nitrated fatty acids. However, our study clearly demonstrates that these compounds inhibit STING activity, suggesting a higher reaction rate constants with STING cysteines and / or preferential binding of the nitroalkene compounds to STING. Thus, our study underscores the substantial therapeutic potential of the nitroalkene compounds CP-36 and CP-45 as potent STING inhibitors with improved pharmacokinetic properties compared to H-151. The results presented in FIGURES 11–18 provide a comprehensive evaluation of the role of STING signaling in endometriosis pathophysiology and its modulation by therapeutic agents developed in this application. Genetic deletion of STING significantly reduced endometriotic lesion formation in mice. STING knockout (KO) female mice developed smaller and fewer lesions compared to wild-type (WT) controls, accompanied by lower levels of surrogate markers of8123-112123-02 05 / 16 / 25 06666inflammation and pain, including interferon-β (IFNβ) and fractalkine. A reverse genetic approach further demonstrated that uterine tissues lacking STING, when transplanted into WT mice, gave rise to significantly smaller lesions and did not elevate CXCL10 / IP-10 levels, highlighting a cell-intrinsic role for STING in lesion development and associated inflammation. Pharmacological targeting of STING with the nitrated fatty acid derivative NO2-CLA also resulted in robust therapeutic effects. Daily subcutaneous administration of NO2-CLA for 28 days significantly reduced lesion burden in a syngeneic mouse model of endometriosis. In vitro, CP-36, a synthetic STING inhibitor, showed favorable therapeutic indices in 12Z human endometriotic epithelial cells, suppressing STING-driven IFNβ and viperin mRNA expression with low cytotoxicity. Human relevance was confirmed by immunofluorescence analysis showing STING expression localized primarily in the epithelial compartments of eutopic and ectopic endometrial tissues, including ovarian and peritoneal lesions. NO2-CLA inhibited cGAMP-induced expression of proinflammatory cytokines (CCL2 and IL6) and STING activation (phosphorylation) in primary human endometrial stromal cells (Figure 17), confirming that STING is active in endometriosis- relevant cell types. Finally, oral administration of NO2-CLA significantly reduced lesion development in mice, with corresponding systemic and peritoneal exposure demonstrated by HPLC-mass spectrometry, supporting its translational potential as an orally bioavailable STING modulator for endometriosis therapy. The safety and proven anti-inflammatory properties of nitroalkenes, as supported by the concluded (NCT03422510, NCT04053543, and NCT03449524) and ongoing (NCT03762395) Phase II clinical trials underscore their potential as therapeutic compounds. These clinical studies predominantly focused on the anti-inflammatory actions of nitrated fatty acids, particularly their roles in NFκB inhibition and Nrf2 activation. The positive safety and tolerability outcomes of nitrated fatty acids in clinical testing further highlight the therapeutic promise of nitroalkene compounds, providing a strong foundation for the preclinical development of the conjugated nitroalkenes identified as lead compounds in this study. In summary, our study concludes that conjugated nitroalkene compounds, distinguished by their electrophilicity and pharmacokinetics, robustly target and inhibit STING-dependent signaling. Through this research, we have advanced our understanding of the optimal structure-activity relationship for designing covalent modifiers that block STING palmitoylation. These findings serve as a foundation for the research and development of clinical STING inhibitors, offering potential treatments for several autoimmune and autoinflammatory diseases where STING plays a pivotal role. Methods Cells and culture conditions. Human acute monocytic leukemia cell line, THP-1s wt (ATCC, RRID:CVCL_0006) and NF-κB-SEAP and IRF-Lucia luciferase Reporter Monocytes, THP-1-Dual8123-112123-02 05 / 16 / 25 06666cells (InvivoGen, RRID:CVCL_X599) were cultured in RPMI 1640 (Sigma-Aldrich) supplemented 10 % heat inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich), 100 Units / mL penicillin, 100 μg / mL streptomycin, and 292 μg / mL glutamine (P-S-G, Gibco) (hereafter termed complete RPMI). The THP-1-Dual cells were further supplemented with 100 µg / mL Normocin in addition to 10 µg / mL Blasticidin, and 100 µg / ml Zeocin at every second passage (InvivoGen). To differentiate THP-1s wt into adherent macrophages, cells were stimulated with 100 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich) for 24 h in complete RPMI before the medium was replaced with complete RPMI without PMA and cells were allowed to further differentiate for one additional day before experiments. HEK-Blue IFN-α / β cells (InvivoGen, RRID:CVCL_KT26) were cultured in DMEM supplemented with 10 % heat-inactivated FBS (Sigma-Aldrich) and P-S-G (Gibco) (hereafter termed complete DMEM) further supplemented with 30 µg / mL Blasticidin, 100 µg / mL Normocin and 100 µg / mL Zeocin (InvivoGen). Fibroblasts isolated from a patient suffering from STING-associated vasculopathy with onset in infancy (SAVI) bearing the mutation in the coding region, c.461A>G, of STING (TMEM173) leading to an Asn to Ser substitution in protein, p.N154S were kindly provided by Raphaela Goldbach-Mansky (RGM, NIH, Bethesda, USA) and cultured in complete DMEM using a 50 % media change at passing and seeded 48 h prior to experiments. Passing of HEK-Blue IFN-α / β cells and SAVI fibroblasts was done using 0.25 % Trypsin (Gibco) supplemented with 0.3 mM EDTA (Invitrogen). All cell cultures were kept at 37 °C, 5 % CO2, and cell lines were regularly tested for mycoplasma contamination by sequencing from GATC Biotech (Germany). SAVI Fibroblasts. Primary fibroblasts from the SAVI patient were obtained from superficial skin biopsies. The patient with genetically confirmed SAVI was enrolled into the protocol (clinicaltrials.gov: NCT02974595) at the NIH between 2008 and 2015. The protocol was approved by the National Institute of Allergy and Infectious Diseases IRB at the NIH. Written informed consent was obtained from the patient or their legal guardians (RGM). Animals. For in vivo experiments with intraperitoneally (i.p.) administration, nine weeks old pathogen-free C57BL / 6JRj (WT) male mice were bred at the animal vendor Janvier Labs (France). Experiments were carried out at Aarhus University. Prior to experiments, mice were allowed to acclimatize, and animals received proper care in agreement with animal protocols approved by Animal Welfare Bodies at Health, Aarhus University and conducted with ethical permission from the Animal Experiments Inspectorate, Danish Veterinary and Food Administration (permission#: 2016- 15-0201-01072) to perform i.p. injections. The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals, EEC Council Directive 2010 / 63 / EU. For in vivo experiments with oral gavage administration, 12 weeks old pathogen-free C57BL / 6J male mice (RRID: IMSR_JAX:000664) were maintained in a temperature- and light-8123-112123-02 05 / 16 / 25 06666controlled environment (12-hour light / dark cycle) at Morehouse School of Medicine Center for Laboratory Animal Resources (CLAR) and were fed standard diet (PicoLab rodent diet 5LOD; LabDiet) and water ad libitum. Animal experiments were approved by the Institutional Animal Care and Use Committee of Morehouse School of Medicine (21-06) and performed in accordance with institutional guidelines. For the BMDM experiments, male and female C57BL / 6J (RRID: IMSR_JAX:000664) and STING knockout mice (RRID:IMSR_JAX:025805) (12 weeks old) were maintained at Morehouse School of Medicine CLAR under above-described conditions and regulations. Animals for endometriosis protocols. C57BL / 6J (wild-type (WT), IMSR_JAX:000664), STING knockout mice (IMSR Cat# JAX:025805), and C57BL / 6J-Tg(CAG-EGFP) (EGFP transgenic mice, IMSR_JAX:006567) were purchased from the Jackson Laboratory. Mice were housed at the Center for Laboratory Animal Resources (CLAR) facilities and acclimated for 5 days. All mice were maintained in a temperature- and light-controlled environment and were fed standard diet (PicoLab rodent diet 5LOD; LabDiet) and water ad libitum. Animal experiments were approved by the Morehouse School of Medicine Institutional Animal Care and Use Committee (protocol 21-06) and performed in accordance with institutional guidelinesSyngeneic EGFP Endometrial Peritoneal Injection Model. The syngeneic endometriosis model was generated according to well-established models. Briefly, at 8 weeks of age, female C57BL / 6J-Tg(CAG-EGFP), C57BL6 / J / J (Wildtype), and STING Knockout mice were treated with 200uL of 2IU Pregnant Mare Serum Gonadotrophin (PMSG) to synchronize the estrous cycle and stimulate uterine growth via peritoneal injection for 72 hours. Donor C57BL6-Tg(CAG-EGFP) mice were euthanized, and the abdomen area was disinfected with 70% ethanol. An abdominal incision was performed for en bloc isolation of the uterus. Uterine horns were collected, rinsed in cold PBS, and minced in preparation of uterine fragments for injection into the peritoneal cavity of STING WT (C57BL / 6J) or STING KO recipient mice at a 2:1 donor-to- recipient ratio. Sham control mice were prepared by injection of equivalent volumes of ~200uL PBS only. Endometriosis was allowed to progress for 28 days. For some experiments, mice were randomized for the treatment with NO2-CLA via intraperitoneal injection or oral gavage as described in detail below. BMDMs. For the bone marrow-derived macrophages (BMDMs), the mouse L-929 (RRID:CVCL_0462) conditioned medium was used as a source of colony-stimulating factor activity. The L-929 cells were grown in RPMI supplemented with 10% FBS with fresh medium added every 3 days (20% v / v). After 10 days, the culture medium was removed, filtered (0.22 pm filter), and used for BMDM cultures. The BMDMs were obtained from bone marrow cells from C57BL / 6J and STING knockout mice by flushing the marrow from femurs with RPMI containing 10% FBS. The marrow plugs were disrupted into a single cell suspension, cultured in RPMI containing 10% FBS8123-112123-02 05 / 16 / 25 06666supplemented with 15% L-929 cell-conditioned medium at a density of 1 × 106cells. After 7 days, adherent BMDM cells were washed with PBS and medium replaced with RPMI, 1% FBS, 15% L-929 cell-conditioned medium overnight. Screening compounds. The cellular screening used both commercially available compounds and in- house synthesized novel nitroalkene. The commercially available compounds included STING inhibitor H151(InvivoGen), nitrated oleic acids 9-NO2OA and 10-NO2OA, along with non-nitrated control lipids oleic acid (OA) and linoleic acid (LA) (Cayman Chemicals). Whereas the in-house synthesized novel nitroalkenes, listed in FIGS.6-10 in addition to reduced nitroalkene as negative controls. All compounds were dissolved in DMSO (CKA in MetOH). Cells were pre-treated with the testing compounds for 2 h before stimulation. Appropriate control vehicles consisting of EtOH, DMSO or MetOH were used. cGAMP stimulation. The STING agonist, 2’3’-cGAMP (InvivoGen) was used for intracellular stimulation using Lipofectamine2000 (Lipo, Invitrogen). cGAMP and Lipo were diluted in serum-free medium with a ratio of Lipo / cGAMP of 1:1 and the final concentration of cGAMP was 4 ug / mL and lipo alone was used as mock treatment. For BMDM experiments, the STING activation was done using 2’3’-cGAMP sodium salt (Tocris) and Lipoefectamine2000 (ThermoFischer Scientific). In vivo administration of compounds and cGAMP. For in vivo administration of compounds, the synthesized nitroalkenes were gently warmed to obtain an oily form, weighed out, and further mixed with Poly-ethylene glycol (PEG400, Sigma-Aldrich) (2 / 3 of final volume) and next 1 / 3 volume of saline was added before being administrated. For intraperitoneally (i.p.) injections, the compounds (10 or 30 mg / kg) were formulated in the above described PEG400 / saline mixture. The same formulation of testing compounds (40 or 60 mg / kg) was applied for oral administration. The PEG400 / saline mixture was also used for mock treatment in both the i.p. and oral administration. For in vivo activation of STING, the Rp / Sp-isomers of the bisphosphorothioate derivative of cAIMP, an analog of the bacterial cyclic dinucleotide 3'3'-cGAMP (cAIM(PS)2 Difluor (Rp / Sp), CL656, InvivoGen)(69) were diluted in saline (InvivoGen) and administrated by i.p. injections at 0.2 mg / kg and mock treatment was performed with saline. One mouse in the 10 mg / kg CP-36 group was excluded from all downstream analyses due to inappropriate STING activation based on Viperin protein levels in the lung. Sample collection and preparation. After sacrifice, whole blood was harvested to Microvette 500, EDTA K3 tubes (Sarstedt) and stored at room temperature (RT) until processing. Plasma was harvested after 1500 x g centrifugation for 15 min and cryopreserved at -80 °C until analysis. Next, the heart was perfused with saline to rinse the heart and lungs before tissue harvest. Both whole8123-112123-02 05 / 16 / 25 06666organs or smaller organ pierces were snap-frozen using dry ice and stored at -80 °C until further processing. MTT assay. To evaluate cytotoxicity, the colorimetric Cell Proliferation Kit I (MTT-based) (Roche) was used according to the manufacturer’s recommendations. The absorbance of the formazan product was measured at 550 nm on Synergy HTX multimode reader (BioTEK). Cytokine and Type I IFN measurements. Human CXCL-10 was quantified by DuoSet ELISA Development System (R&D Systems) according to the manufacturer’s recommendations and absorbance was read at 450 nm on Synergy HTX multimode reader (BioTEK). Human type I IFN was quantified using the reporter cell line HEK-Blue™ IFN-α / β reporter cells by the manufacturer´s instructions (InvivoGen, RRID:CVCL_KT26). Using QUANTI-Blue the SEAP levels were determined by reading the OD at 630 nm on Synergy HTX multimode reader (BioTEK). Multiplex cytokine analysis by Meso Scale Discovery. Two customized, high-sensitive 3- and 4- cytokine U-plex panels (Meso Scale Discover (MSD) were used to analyze plasma levels of IFNα, IFNβ, MIP-1β and TNFα (4-plex) and CXCL-10, MCP-1, and IL-6 (3-plex) in the cryopreserved plasma according to the manufacturer´s protocol. Data were acquired using a QuickPlex SQ 120 instrument (MSD, Rockville, MD, USA). The lower limit of quantification of individual assays was as follows IFN-α: 490.19 pg / ml; IFN-β: 2.898 pg / ml, MIP-1β: 19.81 pg / ml, TNF-α: 0.1234 pg / ml; CXCL-10: 0.643 pg / ml; MCP-1: 1.973 pg / ml, and IL-6: 2.18 pg / ml. NF-κB / IRF reporter assay. The NF-κB-SEAP (secreated embryonic alkaline phosphatase) and IRF- Lucia luciferase Reporter Monocytes, THP-1-Dual cells (InvivoGen, RRID:CVCL_X599) were used according to the manufacturer’s recommendation. NF-κB and IRF activation were assessed by measuring the levels of SEAP and Lucia luciferase using QUANTI-Blue and QUANTI-Luc, respectively. Levels of SEAP were determined by reading the OD at 630 nm, and those of Lucia luciferase were determined by measuring the relative light units (RLUs) in a luminometer. Both readings were conducted on the Synergy HTX multimode reader (BioTEK). Stopped flow kinetic studies. Kinetics studies were performed using an Applied Photophysics SX20 stopped-flow approaches. CP-36 or CP-45 were individually mixed with increasing concentrations of GSH (0.001–0.0035 M) in phosphate buffer (0.1 M, pH 7.4, DTPA 0.1 mM) at 25 °C. Absorbance was recorded over time at 323 and 333 nm for CP-36 and CP-45, respectively, as these are the maximal absorbances in phosphate buffer for each compound. As a control, no changes in absorbance8123-112123-02 05 / 16 / 25 06666were observed in the absence of GSH, except for slight photobleaching. Single exponential plus linear functions were fit to the reaction time courses. The exponential pseudo-first order rate constants kobs were plotted against GSH concentration and the second-order rate constants were determined from the slopes. GSH concentration was verified by titration with 5,5’-dithiobis(2-nitrobenzoate) using an absorption coefficient at 412 nm of 14,150 M-1cm-1for the product(70). GSH stocks were prepared in phosphate buffer (0.1 M, pH 7.4, DTPA 0.1 mM) and the pH was adjusted with KOH to the same pH of the buffer. Western Blot. In vitro preparation of samples was as follows, 400,000 THP-1s wt or 100,000 SAVI fibroblasts were lysed in 100 or 50 µl, respectively, ice-cold Pierce RIPA lysis buffer (Thermo Fischer Scientific) supplemented with 10mM NaFlouride, 1X cOmplete EDTA-free Protease Inhibitor Cocktail (Roche) and 50 units / mL Benzonase (Sigma-Aldrich) (hereafter termed complete lysis buffer). Similarly, a small piece of tissue was first homogenized in complete lysis buffer using steel beads and the TissueLyser II (Qiagen). Lysates were pelleted and the supernatants were sonicated using the Bioruptor (Diagenode). The protein concentration was determined using Pierce BCA protein assay kit (Thermo Fischer Scientific). Whole-cell or tissue lysates were denatured for 3 min at 95 °C in the presence of XT Sample Buffer and XT reducing agent (BioRad). A total of 10 μg of reduced samples was separated by SDS-PAGE on 4–20% Criterion TGX precast gradient gels (BioRad). Each gel was run initially for 15 min at 70 V, followed by 45 min at 110 V. Transfer onto midi 0.2 µM PVDF membranes (BioRad) was done using a Trans-Blot Turbo Transfer system (BioRad) for 7 min. Membranes were blocked for 1 h with 5% skim milk (Merck Millipore) at RT in PBS supplemented with 0.05% Tween-20 (PBS-T). Membranes were fractionated in smaller pieces and probed overnight at 4 °C with any of the following specific primary antibodies were used 1:1000 in PBS-T with 0.05 % NaAzide for the in vitro samples: anti-STING (Cell Signaling Technology (CST) Cat# 13647, RRID:AB_2732796), anti-phospho-STING (CST Cat# 19781, RRID:AB_2737062), anti-TBK1 / NAK (CST Cat# 3013, RRID:AB_2199749), anti-phospho-TBK1 / NAK (CST Cat# 5483, RRID:AB_10693472), anti-IRF3 (CST Cat# 11904, RRID:AB_2722521), anti-phospho-IRF3 (Ab76493, Abcam, RRID:AB_1523836), anti-IFIT1(CST Cat# 14769, RRID:AB_2783869), anti- ISG15 (CST Cat# 2758, RRID:AB_2126200) and anti-Vinculin CST Cat# 18799, CST, RRID:AB_2714181) as loading control. In addition to the above listed, the following specific primary antibodies were used 1:1000 in PBS-T with 0.05 % NaAzid for the in vivo samples: anti-Viperin (Millipore Cat# MABF106, RRID:AB_11203644), anti-STAT1 (CST cat# 14994, RRID:AB_2737027), anti-phopho-STAT1 (CST Cat# 14994, RRID:AB_2737027). After three washes in PBS-T, secondary antibodies, Peroxidase AffiniPure F(ab’)2 Donkey anti-mouse IgG (H+L) or peroxidase AffiniPur F(ab’)2 Donkey anti-rabbit IgG (H+L) (Jackson ImmunoResearch) were added used 1:10,000 in PBS-T 1% milk for 1 h at room temperature. All membranes were8123-112123-02 05 / 16 / 25 06666washed three times in PBS-T and exposed using either the SuperSignal West Pico PLUS Chemiluminescent Substrate or the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fischer Scientific) and Image Quant LAS4000 mini-imager (GE Healthcare) or Azure 300 imager (Azure Biosystems). For the BMDM experiments, cells were lysed in Tissue Protein Extraction Reagent (T-PER, Thermo Scientific) supplemented with a protease inhibitor cocktail (Roche Applied Science). Protein extracts were resolved in 10% SDS–PAGE gels and transferred to nitrocellulose membranes (Bio-Rad). After blocking in TBST containing 5 % non-fat dry milk at room temperature for 1 hour, membranes were immunoblotted with primary antibodies at 4 °C overnight. The following antibodies were used: Phospho-STING (D8F4W) (CST Cat# 72971, RRID:AB_2799831; 1:500); Phospho-IRF-3 (4D4G) (CST Cat# 4947, RRID:AB_823547; 1:500), STING (D2P2F) (CST Cat# 13647, RRID:AB_2732796, 1:1000); IRF3 (D83B9) (CST Cat# 4302, RRID: AB_1904036, 1:1000); GAPDH (14C10) (CST Cat# 2118, RRID: AB_561053, 1:1000). The membranes were washed by TBST before incubated with Goat Anti-Rabbit IgG (H L)-HRP Conjugate secondary antibody (RRID: AB_11125142, 1:5000) at room temperature for 1h followed by signal detection using Clarity Western ECL Substrate (Bio-Rad). Western blots were scanned and quantified using an iBright FL1500 Imager (ThermoFisher Scientific). Nitrocellulose membranes were stripped from primary and secondary antibodies using Restore™ Western Blot Stripping Buffer (ThermoFisher Scientific) for 15 min and re-probed following the above antibody order. The levels of proteins were quantified by densitometry using the AzureSpot Pro software (Azure Biosystems) or NIH Image J. qPCR analysis. For in vivo experiments, smaller pieces of tissues (approx.25 mg) were homogenized in Buffer RLT Plus (Qiagen) using steel beads and TissueLyser II (Qiagen). Lysates were pelleted and RNA isolated from supernatants using qPCR RNeasy Mini kit with either on-column DNase digestion or gRNA eliminator spin columns (Qiagen) following manufacturer’s protocol. RNA quality was assessed by NanoDrop spectrometry (Thermo Fischer Scientific). Gene expression was determined by real-time quantitative PCR (qPCR) using and TaqMan detection systems (Applied Biosystems). RNA levels for murine Ifnb1 (Mm00439552_s1) and Actb (Mm02619580_g1) were analyzed using premade TaqMan assays and the RNA-to-Ct-1-Step kit according to manufacturer’s recommendations. Samples were analyzed in technical duplicates and appropriate controls were included. Expression levels were quantified relative to beta-actin expression, using the 2−ΔΔCt method (71) and normalized to the cGAMP group as a fold change. For BMDM experiments, total RNA was extracted using Aurum Total RNA Mini Kit (Bio-Rad). RNA quantity was determined using NanoDrop spectrometry (ThermoFisher Scientific). RNA was reverse transcribed into cDNA using random primers with GoScript Reverse Transcriptase System (Promega). Specific transcript levels for murine Ifnb1 (forward primer: 5’ CAG CTC CAA GAA AGG ACG AAC 3’, reverse primer: 5’ GGC AGT GTA ACT CTT CTG CAT 3’), Cxcl-10 (forward primer: 5’ CCA AGT GCT GCC GTC ATT TTC 3’, reverse primer: 5’ TCC CTA TGG CCC TCA TTC TCA 3’), Ifit1 (forward primer: 5’ CTG AGA TGT CAC TTC ACA TGG AA 3’, reverse primer: 5’ GTG CAT CCC CAA TGG GTT CT 3’),8123-112123-02 05 / 16 / 25 06666Isg15 (forward primer: 5’ GGT GTC CGT GAC TAA CTC CAT 3’, reverse primer: 5’ TGG AAA GGG TAA GAC CGT CCT 3’) and 18S (forward primer: 5’ GGA AGG GCA CCA CCA GGA GT 3’, reverse primer: 5’ TGC AGC CCC GGA CAT CTA G 3’) were assessed by a CFX Connect Real- Time PCR Detection System (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad) and the ΔΔCt threshold cycle method of normalization. Gene expression levels were quantified relative to the expression of 18S and normalized to the cGAMP group as a fold change. For in vivo administration of NO2-CLA in the syngeneic endometriosis model, the pure synthesized nitroalkene was warmed gently to obtain an oily form, weighted out and further mixed with 2 / 3 volume of poly(ethylene glycol) (PEG400, Sigma-Aldrich) and next 1 / 3 volume of saline before being administered. PEG in saline was prepared for a negative control / vehicle. For subcutaneous (s.c.) injections, the NO2-CLA was given at 2.5mg / mice, whereas for oral gavage, a daily dose of 5mg / kg / day was provided. Equal doses of pure CLA (5mg / kg / day) were used as non-nitrated controls. Assessment of EGFP-Donor-Derived Endometriosis Lesions In Vivo. Visualization of EGFP-donor- derived lesions was obtained using an AmiHTX imager (Spectral imaging systems) in fluorescence mode to assess EGFP intensity of donor-derived lesions within the peritoneal cavity. Endometriotic lesions were quantified using Aura 4.0 imaging software and the diameter of lesions was obtained using a digital caliper. Data was plotted as box and whisker plots of minimum to maximum values. Significant values were determined by Two-tailed P value, non-parametric Mann-Witney test, n = 8. Assessment of WT and STING Knockout Donor-Derived Endometriosis Lesions In Vivo. The peritoneal cavity of euthanized mice was assessed to locate endometriotic lesions for isolation. Confirmed lesions were quantified and then size of lesions (mm) was measured using a caliper. Human Endometriosis Tissue Microarray (TMA). A deidentified archived formalin-fixed paraffin- embedded endometrium and endometriosis tissue microarray was obtained from the Moffitt Cancer Center (Moffitt) Tissue Core. Eutopic endometrial samples from women with endometriosis and controls in secretory phase were included in the TMA. Control endometrial samples were obtained from women undergoing hysterectomy for benign gynecologic conditions such as uterine fibroids of dysfunctional bleeding, who had normal cycling endometrium (not hyperplastic, not menopausal, in secretory phase) as per pathologic analysis. Immunostaining for STING, EpCAM, and DAPI was conducted according to standardized protocols and normalized antibody dilutions and predetermined experimental conditions. Immunostaining intensity of STING (Cell Signaling #13647) conjugated to Alexa Fluor 488 (Invitrogen A32731), EpCAM (ThermoFisher 14-9326-82) conjugated to alexa fluor 594 (Invitrogen A-11005) and DAPI was assessed in the glands and stroma of the tissues of the TMA using an Olympus Fluorescent microscope. Data were analyzed as differences in positive staining for8123-112123-02 05 / 16 / 25 06666STING, EpCAM, and DAPI (BD Pharmingen 564907) expression among the groups (lesion type and secretory endometrium from cases and controls). Endometrial Stromal Cell (ESC) Cultures. Primary endometrial stromal cells (ESCs) from human eutopic endometrial biopsies from women with (EESC) and without evidence of endometriosis (NESC) were prepared according to the previously published method. Cells (passages 3–5) were cultured and routinely maintained in Dulbecco’s Modified Eagle’s Medium / Ham’s Nutrient Mixture F-12 (DMEM / Ham’s F-12; Life Technologies, Inc.-BRL) supplemented with 12% fetal bovine serum (FBS; Thermo Fisher Scientific, Grand Island, NY, USA), 1% non-essential amino acids, 1% sodium pyruvate, and 1% penicillin–streptomycin (Penstrep, Sigma-Aldrich, St Louis, MO, USA), within a 5% CO2 atmosphere at 37 °C in a humidified incubator. Cells were grown to 80% confluency in 100- mm plates (Corning, NY, USA). The culture media was replaced with low serum-containing media overnight before any experiments. Treatment of Normal Endometrial Stromal Cells (NESCs). NESCs were grown up to 80% confluency in in 6-well plates. Cells were grown in DMEM / F12 supplemented with 10%FBS, synchronized by 2% FBS overnight and treated as follows: C) control cells were treated with cGAMP vehicle (Lipo3000, Invitrogen) and treated with NO2-CLA (2μM) or CLA, prior to stimulation with STING agonist cGAMP (4ng / mL, Tocris) for 6 h. Cells were harvested for the mRNA and protein expression. In view of the many possible embodiments to which the principles of the disclosed compositions and methods may be applied, it should be recognized that the illustrated embodiments are only preferred examples and should not be taken as limiting the scope of the invention.

Claims

8123-112123-02 05 / 16 / 25 06666What is claimed is:

1. A method comprising administering to a subject having a stimulator of interferon genes (STING)-dependent inflammatory or autoimmune condition a therapeutically effective amount of a compound that includes a nitrovinyl moiety conjugated to an aromatic moiety.

2. The method of claim 1, wherein the nitrovinyl moiety conjugated to an aromatic moiety is a β-nitrostyrene moiety.

3. The method of claim 1 or 2, wherein the (STING)-mediated inflammatory or autoimmune condition is an infectious disease, systemic lupus erythematosus (SLE), psoriasis, primary biliary liver disease, non-alcoholic fatty liver disease, Sjogren's Syndrome, rheumatoid arthritis, type I diabetes, type 2 diabetes, Crohn’s disease, ulcerative colitis, Aicardi-Goutières syndrome (AGS), STING-Associated Vasculopathy with Onset in Infancy (SAVI), chronic viral infections such as HIV and hepatitis, pulmonary fibrosis, systemic sclerosis, multiple sclerosis, cardiac hypertrophy, coronary disease, atherosclerosis, neurodegeneration disease, neuroinflammation disease, autoimmune thyroid disease, inflammatory myopathy, autoimmune skin disease gastrointestinal autoimmune disease, autoimmune neurological disease, vasculitis syndrome systemic sclerosis (scleroderma), ankylosing spondylitis, psoriatic arthritis, Behçet's disease, relapsing polychondritis, autoimmune eye disease, autoimmune myocarditis, autoimmune blood disorder autoimmune lung disease, autoimmune endocrine disorder, connective tissue disease, COPA syndrome, Mooren’s ulcer, prebyscusis, ischemia reperfusion injury, (liver, heart, kidney, lung, brain) dilated cardiomyopathy, heart failure, radiation-induced liver, lung and heart injury, Huntington’s disease, spinocerebellar ataxia type I, nerve regeneration, chronic obstructive pulmonary disease, acute lung injury, renal fibrosis, APOL1 related kidney disease, intervertebral disk related pathologies, or pulmonary fibrosis.

4. The method of claim 1 or 2, wherein the (STING)-mediated inflammatory or autoimmune condition is endometriosis.

5. The method of claim 4, wherein the endometriosis is ovarian endometriosis, pelvic endometriosis, peritoneal endometriosis, diaphragmatic endometriosis, thoracic endometriosis syndrome (TES), adenomyosis, uterine fibroids, polycystic ovary syndrome (PCOS), or endometrial cancer.8123-112123-02 05 / 16 / 25 066666. The method of claim 1 or 2, wherein the wherein the (STING)-mediated inflammatory or autoimmune condition is infertility and pregnancy complications, asthma, catamenial pneumothorax, catamenial hemothorax, catamenial hemoptysis, inflammatory bowel disease (IBD), interstitial cystitis / bladder pain syndrome (IC / BPS), primary biliary cholangitis, metabolic dysfunction-associated steatotic liver disease, alcoholic fatty liver disease, Parkinson’s disease, Alzheimer’s disease, contrast-induced nephropathy, non-eosinophilic asthma, or atopic dermatitis.

7. The method of claim 1, 3, 4, 5 or 6, wherein the compound that includes the nitrovinyl moiety conjugated to an aromatic moiety has a structure of: Z-(CH2)x-C(NO2)=CH-A-(CH2)y-(CH=CH)z-C(=O)O-R1wherein x and y are each independently 0 to 10; z is 0 or 1; Z is CH3, cycloalkyl, substituted cycloalkyl, aryl, or substituted aryl; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.

8. The method of claim 7, wherein Z is CH3, phenyl, cyclohexyl, adamantyl, or naphthyl.

9. The method of claim 7 or 8, wherein y is 0 and z is 1.

10. The method of claim 7 or 8, wherein z is 0.

11. The method of any one of claims 7 to 10, wherein A is phenylene.

12. The method of claim 1, 3, 4, 5 or 6, wherein the compound that includes the nitrovinyl moiety conjugated to an aromatic moiety has a structure of: CH3-(CH2)x-C(NO2)=CH-A-(CH2)y-C(=O)O-R1wherein x and y are each independently 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.

13. The method of claim 10, wherein A is phenylene.8123-112123-02 05 / 16 / 25 0666614. The method of claim 1, 3, 4, 5 or 6, wherein the compound that includes the nitrovinyl moiety conjugated to an aromatic moiety has a structure of: Y-A-CH=C(NO2)-(CH2)x-(CH=CH)z-C(=O)O-R1wherein x is 1 to 10; z is 0 or 1; Y is CH3, halogen, alkyl, substituted alkyl, or hydroxyl; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.

15. The method of claim 14, wherein Y is CH3.

16. The method of claim 14 or 15, wherein A is phenylene.

17. The method of claim 1, 3, 4, 5 or 6, wherein the compound that includes the nitrovinyl moiety conjugated to an aromatic moiety has a structure of: CH3-A-CH=C(NO2)-(CH2)x-C(=O)O-R1wherein x is 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.

18. The method of claim 17, wherein A is phenylene.

19. The method of claim 3, 4, 5 or 6, wherein the compound is (E)-3-(3-(2-nitrohept-1- en-1-yl)phenyl)propanoic acid, or a pharmaceutically acceptable salt thereof.

20. The method of claim 3, 4, 5 or 6, wherein the compound is methyl (E)-4-nitro-5-(p- tolyl)pent-4-enoate, or a pharmaceutically salt thereof.

21. A method comprising administering to a subject having endometriosis a therapeutically effective amount of a composition that includes a mixture of nitro-fatty acids derived from conjugated linoleic acids.8123-112123-02 05 / 16 / 25 0666622. A compound, or a pharmaceutically acceptable salt thereof, having a structure of: Z-(CH2)x-C(NO2)=CH-A-(CH2)y-(CH=CH)z-C(=O)O-R1wherein x and y are each independently 0 to 10; z is 0 or 1; Z is CH3, cycloalkyl, substituted cycloalkyl, aryl, or substituted aryl; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.

23. The compound of claim 22, wherein Z is CH3, phenyl, cyclohexyl, adamantyl, or naphthyl.

24. The compound of claim 22 or 23, wherein y is 0 and z is 1.

25. The compound of claim 22 or 23, wherein z is 0.

26. The compound of any one of claims 22 to 25, wherein A is phenylene.

27. The compound of any one of claims 22 to 26, wherein Z is CH3.

28. A compound, or a pharmaceutically acceptable salt thereof, having a structure of: CH3-(CH2)x-C(NO2)=CH-A-(CH2)y-C(=O)O-R1wherein x and y are each independently 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.

29. The compound of claim 28, wherein A is phenylene.

30. The compound of claim 28 or 29, wherein x and y are each independently 1 to 5.

31. The compound of any one of claims 28 to 30, wherein R1is C1-C6 alkyl.

32. The compound of any one of claims 28 to 30, wherein R1is C1-C6 alkenyl.8123-112123-02 05 / 16 / 25 0666633. The compound of any one of claims 28 to 30, wherein R1is hydrogen.

34. The compound of claim 28, wherein the compound, or the pharmaceutically acceptable salt thereof, is (E)-3-(3-(2-nitrohept-1-en-1-yl)phenyl)propanoic acid.

35. A compound, or a pharmaceutically acceptable salt thereof, having a structure of: Y-A-CH=C(NO2)-(CH2)x-(CH=CH)z-C(=O)O-R1wherein x is 1 to 10; z is 0 or 1; Y is CH3, halogen, alkyl, substituted alkyl, or hydroxyl; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.

36. The compound of claim 35, wherein z is 0.

37. The compound of claim 35 or 36, wherein x is 1 to 5.

38. The compound of any one of claims 35 to 37, wherein R1is C1-C6 alkyl.

39. The compound of any one of claims 35 to 37, wherein R1is C1-C6 alkenyl.

40. The compound of any one of claims 35 to 37, wherein R1is hydrogen.

41. The compound of any one of claims 35 to 40, wherein A is phenylene.

42. A compound, or a pharmaceutically acceptable salt thereof, having a structure of: CH3-A-CH=C(NO2)-(CH2)x-C(=O)O-R1wherein x is 1 to 10; A is arylene, substituted arylene, heteroarylene, or substituted heteroarylene; and R1is hydrogen, an alkyl, or a substituted alkyl.8123-112123-02 05 / 16 / 25 0666643. The compound of claim 42, wherein A is phenylene.

44. The compound of claim 42 or 43, wherein x is 1 to 5.

45. The compound of any one of claims 42 to 44, wherein R1is C1-C6 alkyl.

46. The compound of any one of claims 42 to 44, wherein R1is C1-C6 alkenyl.

47. The compound of any one of claims 42 to 44, wherein R1is hydrogen.

48. The compound of claim 42, wherein the compound is methyl (E)-4-nitro-5-(p- tolyl)pent-4-enoate.

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