Methods of disrupting interactions between MUNC13-4 and syntaxin 7 and compounds useful therein

Abstract

Disclosed herein are compounds that disrupt the protein-protein interaction between Muncl3-4 and syntaxin 7, methods of discovering such compounds, and methods of their use to treat systemic inflammation.

Description

[0001] TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0002] METHODS OF DISRUPTING INTERACTIONS BETWEEN MUNC13-4 AND SYNTAXIN 7

[0003] AND COMPOUNDS USEFUE THEREIN

[0004] CROSS-REFERENCE TO RELATED APPLICATION

[0005] This application claims priority to U.S. provisional patent application no. 63 / 734,373, filed December 16, 2024, the entirety of which is incorporated herein by reference.

[0006] GOVERNMENT SUPPORT

[0007] This invention was made with government support under R01AR070837 and R01HL088256 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] 1. BACKGROUND

[0009] There are over 100 known autoimmune diseases but few effective and specific therapies known to address them. Many drugs manage symptoms of autoimmune diseases, but few are disease-modifying therapies (DMTs) that can slow or prevent organ damage.

[0010] One example of a DMT is teplizumab, a CD3 -directed monoclonal antibody indicated for delaying the onset of stage 3 type 1 diabetes. Another is hydrochloroquine, which is used to protect vital organs in patients suffering from lupus. Hydrochloroquine functions by increasing the pH of lysosomes, thereby inhibiting endosomal Toll-like receptor activation. See, e.g., Peng-Cheng, L., et al., “Advancements on the impact of hydroxychloroquine in systemic lupus erythematosus” Heliyon 10 (2024) e30393. Unfortunately, drugs such as hydrochloroquine are not selective to the cells associated with lupus and a variety of adverse effects are associated with its use. Because Toll-like receptors play an essential role in innate immune response, a need exists for new, more selective approaches of affecting their signaling.

[0011] 2. SUMMARY

[0012] This invention is directed to compounds that disrupt the protein-protein interaction between Muncl3-4 and syntaxin 7 (STX7), methods of discovering such compounds, and methods of their use.

[0013] One embodiment of the invention is a method of discovering compounds that disrupt a proteinprotein interaction between Muncl3-4 and syntaxin 7 (STX7), which comprises: measuring a first amount of fluorescence from a first mixture; measuring a second amount of fluorescence from a second mixture; and comparing the first and second amounts; wherein: the first mixture comprises the compound, an antibody, calcium, modified STX7, and Muncl3-4 bound to a peptide tag; the second mixture comprises the compound, the antibody, calcium, modified STX7, and Muncl3-4 lacking amino acid residues necessary for Ca2+binding; the modified TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0014] STX7 is STX7 bound to a first fluorophore; the antibody recognizes the peptide tag and is bound to a second fluorophore; and the measured fluorescence is fluorescence from the first fluorophore, which fluorescence is caused by absorption of light by the second fluorophore.

[0015] Another embodiment of the invention encompasses compounds discovered or discoverable by the assay described herein. These compounds are referred to herein as “ENDOtollins” or “ENDOs”. Some ENDOtollins are of the formula:

[0016] A— L— B including pharmaceutically acceptable salts thereof, wherein:

[0017] A is an optionally substituted 5- to 10-membered carbocycle or heterocarbocycle, which optional substitution is with one or more R1;

[0018] B is an optionally substituted 5- to 10-membered carbocycle or heterocarbocycle, which optional substitution is with one or more R2;

[0019] L is -N=N-, -CH=CH-, -CH2-CH2-, -S-CH-, -CH-S-, O, -O-CH-, or -CH-O-; each R1is independently halo, OR1A, NR1AR1B, or optionally substituted C1-12 hydrocarbyl or C1-12 heterocarbyl which optional substitution is with one or more R1C;

[0020] R1Ais H or C1-6 alkyl;

[0021] R1Bis H or C1-6 alkyl; each R1Cis independently amino, halo, hydroxy, or oxo; each R2is independently halo, OR2A, NR2AR2B, or optionally substituted C1-12 hydrocarbyl or C1-12 heterocarbyl which optional substitution is with one or more R2C;

[0022] R2Ais H or C1-6 alkyl;

[0023] R2Bis H or C1-6 alkyl; and each R2Cis independently amino, halo, hydroxy, or oxo.

[0024] Another embodiment of the invention encompasses methods of using compounds that disrupt the protein-protein interaction between Muncl3-4 and STX7, such as methods of treating systemic inflammation and autoimmunity.

[0025] 3. DESCRIPTION OF THE DRAWINGS

[0026] Certain aspects of the invention may be understood with reference to the accompanying figures.

[0027] FIGS. 1A-F relate to decreased unmethylated cytosine-phosphorothioate-guanosine (CpG)- induced inflammation in Muncl3-4-KO mice. FIGS. 1A-D show inflammatory response to CpG. FIGS. 1E-F show neutrophil number (%) and MPO plasma levels, a marker of neutrophil-mediated systemic inflammation. For these data, mice were analyzed 6h after systemic (tail vein) administration of CpG type A or B. Each symbol indicates an independent mouse. #, outlier (Grubbs’, alpha=0.05). *, p<0.05; **, p<0.01; ns, not significant. Two-tailed Student’s t-test was used for two-group comparisons. TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0028] FIGS. 2A-C relate to a TR-FRET assay used for the identification of Muncl3-4-STX7 inhibitors. FIG. 2A show the mechanism of STX7 and Muncl3-4 regulation of endolysosome signaling, wherein Muncl3-4 binds to syntaxin 7 at the cytosolic side of the late endosome (LE). Muncl3-4 is in equilibrium between the free cytosolic and membrane -bound form, and specifically binds to membrane-bound STX7 in a calcium-dependent manner. Lysosome-LE fusion forms a degradative compartment where nucleic acid-sensing endosomal Toll-like receptors (eTLRs) are processed and activated. Specific endosomal TLR ligands lead to the upregulation of pro-inflammatory cytokines, which is inhibited in Muncl3-4 deficiency. FIG. 2B provides a schematic representation of the TR-FRET binding assay. Cell lysates expressing Flag-Muncl3-4 and GFP-syntaxin 7 (STX7) at late endosomes (LAMP1) are incubated with terbium (Tb)-conjugated anti-Flag antibody. The samples are excited at 340 nm. The emission peak of terbium (centered at 490 nm) overlaps with the excitation spectrum of GFP. FRET signal is measured by detecting GFP emission at 520 nm and results are expressed as the emission ratio of the acceptor (GFP, 520 nm) / donor (Tb, 490 nm, used as internal control). An increased emission ratio is indicative of specific binding. FIG. 2C provides results of a homologous competitive binding assay for STX7 using the TR-FRET assay. Specific binding was measured at a constant [GFP-STX7] (~50 nM) that was less than half of the IC50, in the presence of various concentrations of recombinant soluble rSTX7 (aal-236). The Xd (~2.7pM) was calculated using the homologous competitive binding curve fitted to a built-in equation of one-site competition. Mean ± SEM of three replicates.

[0029] FIGS. 3A-J relate to the effects of the small-molecules identified as inhibitors of the binding of MUNC13-4 to STX7 — i.e., “ENDOtollin compounds” some of which are members of a series of compounds beginning with “ENDO” — on CpG-induced TLR9 activation. FIG. 3A, ENDO3 inhibits STX7-MUNC-13-4 binding but is negative in TR-FRET counterscreens using MUNC13-4 and Rabl l or Rab27a. Four biological replicates, Mean ± SD. FIG. 3B, ENDO3 does not affect the binding of syntaxin 7 to the SNARE protein VAMP8. Three biological replicates, Mean ± SD. FIG. 3C shows the effect of ENDOtollins on TLR9 activation using HEK-Blue™-hTLR9 reporter cells, expressing the human TLR9 gene and an inducible SEAP (secreted embryonic alkaline phosphatase) reporter gene under the control of the IFN-P minimal promoter fused to five NF-KB and AP-1 -binding sites, stimulated with CpG for 24 hours. Eight biological replicates representative of 3 independent experiments. *, p<0.05; ****, p<0.0001. 2 -way ANOVA, Dunnett’s multiple comparison test. FIG. 3D shows results of a cell-based assay for the analysis of TLR9-mediated activation of plasmacytoid dendritic cells. pDCs (cell line CALI) were incubated with lOpM ENDO3, ENDO7, or vehicle (DMSO) for 1 hour and subsequently stimulated with CpG-ODN or vehicle. n=3 or 4 independent experiments. *, p<0.05, ns, not significant. One-Way ANOVA. FIG. 3E shows results of a dose-response study of ENDO3 inhibitor by analysis of CD40 mobilization in CAL-1 cells stimulated with CpG. Symbols (• and A) indicate two independent experiments. FIG. 3F-J pertain to ENDO3’s ability to specifically inhibit activation via CpG without inhibiting exocytosis via plasma membrane ligands. FIG. 3F, ENDO3 inhibits CpG-induced ERK TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 activation. n=3, Mean ± SEM. FIG. 3G, ENDO3 inhibits CpG-induced CD1 lb upregulation in neutrophils stimulated with the TLR9 ligand CpGB. n=3-6. Mean ± SEM. FIGS. 3H-J pertain to the mobilization of neutrophil secretory organelles in response to fMLF, which activates a plasma membrane receptor, is not affected by ENDO3. FIG. 3H, mobilization of secretory vesicles (CD1 lb). n=6. FIG. 31, secondary granules (CD66b), and FIG. 3 J, azurophilic granule exocytosis (MPO) n=9. CyTD, cytochalasin D; F, fMLF; ns, not stimulated. Each symbol represents an independent donor. Mean ± SEM. ns, not significant. *, p<0.05; **, p<0.01, ***, p<0.001; ****, p<0.0001. FIGS. 3F-G, ANOVA analysis followed by multiple comparisons test. FIGS. H-J, two-tailed Student’s / -test was used for two-group comparisons.

[0030] FIGS. 4A-G relate to ENDOs’ ability to decrease endolysosomal flux ex vivo and IL-6 production in vivo. FIG. 4A, ENDO3 inhibits endolysosomal flux manifested as increased late endosomes (LE) diameter. Each symbol represents an individual cell from three independent experiments. ****, p<0.0001. one-way ANOVA, multiple comparison test. FIG. 4B, analysis of endolysosomal dynamics. Acidic organelles of cells treated with ENDO3 or vehicle were labeled with LysoTracker and analyzed by TIRFM. Vesicle speed from vehicle (DMSO)- and ENDO3 (10 pM)-treated cells were segregated in 0.02 pm / sec increments and plotted as low-speed vs high-speed moving vesicles. The average speed for each speed-range is expressed as Mean ± SEM. *, p<0.05. n=14 (vehicle) and 13 (ENDO3) cells per condition. Two-tailed Student’s / -test. FIG. 4C, representative quantification of the analysis of the effect of ENDO3 on functional endolysosomes (cathepsin B activity) using the Anorogenic probe Magic Red. n=25 cells per group analyzed in two independent experiments. Mean ± SEM. ****, pO.OOOl, Two-tailed Student’s / -test. FIG. 4D pertains to the lack of effect of ENDOs on cell death. Analysis of the effect of ENDO3 and ENDO 12 on apoptosis (Annexin V) and cell death (Propidium Iodide) in human granulocytes by Row cytometry. n=6. FIG. 4E provides results showing that the compounds ENDO3 and ENDO 12 do not induce death of primary cells. Human neutrophils were incubated with the indicated concentrations of ENDO3 (E3), ENDO12 (E12) or vehicle (DMSO) and incubated for 2 hours at room temperature. Where indicated, neutrophils were incubated at 90°C for 10 minutes (heat-killed control). The cells were then labeled with the fluorescent probe Zombie Violet which only labels permeabilized, dead cells. n=3 independent healthy blood donors. FIGS. 4F and G, ENDO3 decreases IL-6 production in vivo. Mice treated with ENDO3 or vehicle (i.p.) were challenged with a single dose of the TLR9 ligand CpG-B or vehicle and analyzed 6 hours after insult. FIG. 4F, ENDO3 does not affect total white blood cells. FIG. 4G, ENDO3 treatment decreases CpG-mediated inflammation manifested as significantly decreased IL-6 production. n=6 mice per group performed in three independent experiments. Mean ± SEM. #, statistical outlier (Grubbs’ alpha=0.05). *, p<0.05; **, p<0.01; ns=not significant; One-way ANOVA, multiple comparison test.

[0031] FIGS. 5A-I pertain to the characterization of ENDO 12. FIG. 5A, TR-FRET analysis of the binding of Muncl3-4 to STX7, its four-point mutant R140A, N141A, L142A, W145A (RNLW-A), or the deletion mutant A129-148. FIG. 5B, expression of STX7 lacking the molecular interface (A129-148) TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 decreases its association with Muncl3-4 in living cells. n=4 independent experiments analyzed in Superplots, where each color represents an independent assay. FIG. 5C, analysis of SAR-derivative compounds in cell-based TLR9 activation assays. Compounds were tested using the HEK-Blue reporter cell-based assay. In this analysis, cells were treated with the indicated compound or vehicle for Ih before the addition of the TLR9 ligand CpG. ENDO 3 and ENDO 12, bars marked with Eight biological replicates. Mean ± SEM, ***, p<0.001; ****, p<0.0001. One-way ANOVA followed by Dunnett’s multiple comparisons test. FIG. 5D, analysis of the effect of the ENDO series on IRF signaling. IRF reporter cells were treated with the indicated compound or vehicle (DMSO) for Ih and subsequently stimulated with the TLR3 agonist Poly:IC. ***, p<0.001; ****, p<0.0001. n=3. One-way ANOVA followed by Dunnett’s multiple comparisons test. FIG. 5E, surface plasmon resonance analysis of ENDO 12 binding to recombinant STX7. Upper panel, representative binding curves from 3 independent experiments. Lower panel, individual and average Ku values for ENDO3 and ENDO 12 binding assays (n=3), indicating that ENDO 12 is more potent than ENDO3. FIG. 5F, dose-response analysis of the effect of the indicated compounds on TLR9 activity (A, ENDO12; • , ENDO3; ■, vehicle), n=3, mean ± SEM. FIG. 5G, ENDO 12 significantly decreases the colocalization of syntaxin 7 with Muncl3-4. FIGS. H and I, ENDO12 decreases the colocalization of TLR7 and TLR9 at LAMP1+ organelles. FIGS. 5G-I, Immunofluorescence analysis of endogenous proteins in RAW 264.7 murine macrophages. Where indicated, the cells were treated with ENDO3, ENDO12 (lOpM), or vehicle for 1 hour before analysis. n=4 (TLR9) or n=3 (TLR7, STX7). Mean ± SEM. Statistical analysis of Superplots was calculated based on the average values of each experiment (large symbols).

[0032] FIGS. 6A-L relate to ENDO12’s ability to decrease primary dendritic cells (DC) and in vivo responses to eTLR ligands without interfering with the host response to viral infection. FIGS. 6A-C, spleen-isolated primary DCs were treated with ENDO 12 or ENDO3 followed by stimulation with the eTLR ligands CpG (TLR9), CL097 (TLR7) and Poly:IC (TLR3) for 48h. FIG. 6A, CD40 surface expression, analyzed by flow cytometry showing that ENDO 12 is more effective than ENDO 3 in inhibiting primary DC activation. Each symbol represents an independent mouse. n=3 mice per condition. Two-way ANOVA. Mean ± SEM. *, p<0.05; **, p<0.01; ****, pO.OOOl. FIGS. 6B-C, IL-6 (B) and IFN-a (C) secretion by CD 1 lc+ DCs. Each symbol represents an independent mouse. n=5 mice per condition. Paired one-way ANOVA. Mean ± SEM. *, p<0.05; **, p<0.01; ns, not significant. FIGS. 6D-F show the effect of ENDO 12 on systemic inflammation. Mice treated with ENDO 12 or vehicle (i.p.) were challenged with a single dose of the TLR9 ligand CpG-A-ODN or vehicle and analyzed 6 hours after insult. FIGS. 6D and 6E show the effect of ENDO 12 on systemic levels of IL-6 and IFN-y. FIG. 6F, ENDO 12 decreases CpG-induced neutrophil-mediated systemic inflammation measured as plasma levels of myeloperoxidase (MPO). Each symbol represents an independent mouse. n=7-8 mice per condition. FIGS. 6G-L, ENDO 12 does not inhibit the in vivo response to LCMV infection. FIG. 6G-K, cytokine plasma levels of mice treated with ENDO12 (i.p. 15 mg / Kg) or vehicle (5% DMSO in PBS) and infected with LCMV or treated with PBS as control, were analyzed by multiplex technology. Myeloperoxidase TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 was analyzed by ELISA (FIG. 6L). Each symbol corresponds to an independent mouse. All data, Oneway ANOVA with multiple comparison tests. Mean ± SEM. *, p<0.05; **, p<0.0I; ***, p<0.001, ****, p<0.000I; ns, not significant.

[0033] 4. DETAILED DESCRIPTION

[0034] The activation of nucleic acid-sensing endosomal Toll-like receptors (TLRs) and late endosomal- initiated signaling are central mechanisms in innate immunity, inflammation, and autoimmunity1-7. These essential processes help shape appropriate cellular responses at infection sites but are also responsible for dysregulated activation of systemic inflammation and are potentially harmful to the host. Unrestricted activation of endosomal TLRs triggers inflammatory pathways leading to a broad range of diseases and pathological processes, including rheumatoid arthritis (RA)68, systemic lupus erythematosus (SLE)6’8’9, Juvenile idiopathic arthritis (JIA)10 11, autoimmune type 1 diabetes12and ischemia-reperfusion injury13.

[0035] Endosomal TLR activation is dependent on endosomal maturation, a process that facilitates the partial digestion of endosomal TLRs into their active forms14 15. We recently described a novel mechanism of endosomal maturation in primary inflammatory cells that involves the direct binding of the calcium sensor Muncl3-4 to a fusion modulator, the late endosomal SNARE protein syntaxin 7 (STX7)16. Using robust and unbiased approaches and rigorous statistical methods, we showed that calciumdependent binding of Muncl3-4 to syntaxin 7 is essential for endosomal maturation and signaling16. Thus, late endosomal defective phenotypes observed in Muncl3-4-deficient cells are rescued by wild-type Muncl3-4 but not by a calcium-binding-deficient mutant of Muncl3-4 that lacks affinity for syntaxin 716. We further identified the mechanism of endosomal maturation mediated by the binding of Muncl3-4 to syntaxin 7 as an essential process regulating endosomal TLR function. Interference with the binding of Muncl3-4 to syntaxin 7 by genetic approaches prevents late endosomal maturation16. This invention resulted, in part, from work done to explore whether small-molecules can be used to inhibit the Muncl3- 4-STX7 interaction and whether such inhibition would decrease inflammation by impairing the overactivation of endosomal TLR3, TLR7- and TLR9-dependent signaling pathways.

[0036] 4.1. DEFINITIONS

[0037] Unless otherwise indicated, the term “about” means ± 10% of the indicated range.

[0038] When used herein, the term “alkenyl” is accorded its conventional meaning. Examples of alkenyl moieties include straight-chain and branched C2-20, C2-12 and C2-6 alkenyl such as vinyl, allyl, 1-butenyl, 2- butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3 -methyl- 1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2- butenyl, 1 -hexenyl, 2-hexenyl, 3 -hexenyl, 1 -heptenyl, 2-heptenyl, 3 -heptenyl, 1 -octenyl, 2-octenyl, 3- octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1 -decenyl, 2-decenyl and 3 -decenyl.

[0039] The term “alkyl” is accorded its conventional meaning. Examples of alkyl moieties include straight-chain and branched C1-20 alkyl, C1-12 alkyl, C1-6 alkyl, C1-4 alkyl, and C1-3 alkyl, such as methyl, TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, and dodecyl. Unless otherwise indicated, the term “alkyl” encompasses cycloalkyl.

[0040] The term “alkynyl” is accorded its conventional meaning. Examples of alkynyl moieties include straight-chain and branched C2-20, C2-12 and C2-6 alkynyl, such as ethynyl and 2-propynyl (propargyl).

[0041] The term “aryl” refers to a single all-carbon-backbone aromatic ring or a multiple condensed all- carbon-backbone ring system wherein at least one of the rings is aromatic. Examples include C6-20, Ce-i4, C6-12, and C2-10 rings and multiple condensed carbon ring systems (e.g., ring systems comprising 2, 3 or 4 rings) having 9 to 20 carbon atoms in which at least one ring is aromatic and wherein the other rings may be aromatic or not aromatic. The rings of multiple condensed ring systems may be connected to each other via fused, spiro, or bridged bonds when valency allows. Examples of aryl moieties include anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, phenanthrenyl, and 1, 2, 3, 4- tetrahydronaphthy 1.

[0042] The term “carbocyle” refers to cyclic saturated or partially unsaturated all carbon-backbone ring having 3 to 20 carbon atoms (e.g., C3-20, C3-15, C3-7, C4-6 and C„ cycloalkyl). Examples include multicyclic carbocyles such as bicyclo [3.1.0]hexane and bicyclo[2.1.1]hexane, and polycyclic carbocycles, such as tricyclic and tetracyclic carbocycles. The rings of multiple condensed ring systems may be connected to each other via fused, spiro, and bridged bonds when valency allows. For example, multicyclic carbocycles may be connected to each other via a single carbon atom to form a spiro connection (e.g., spiropentane, spiro[4,5]decane), via two adjacent carbon atoms to form a fused connection (e.g., decahydronaphthalene, norsabinane, norcarane), or via two non-adjacent carbon atoms to form a bridged connection (e.g., norbomane, bicyclo[2.2.2]octane). Examples of cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptane, pinane, adamantane.

[0043] The term “cycloheteroalkyl” refers to a cyclic heteroalkyl moiety, defined below.

[0044] The term “halo” encompass fluoro, chloro, bromo, and iodo.

[0045] The term “heteroaryl” encompasses a single aromatic ring that has at least one atom other than carbon in the ring, wherein the atom is selected from the group consisting of oxygen, nitrogen and sulfur (e.g, single aromatic rings of from 1 to 6 carbon atoms and 1-4 heteroatoms ). The term also encompasses multiple condensed ring systems that have at least one such aromatic ring. Examles include acridinyl, benzimidazolyl, benzofuranyl, benzoisothiazolyl, benzoisoxazolyl, benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl, indazolyl, indolyl, isoquinolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, phthalazinyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl, pyrrolyl, quinolyl, quinoxalyl, quinazolinyl, quinolinyl, thienyl, tetrazolyl, thiadiazolyl, thiazolyl, triazinyl, and triazolyl.

[0046] The term “heterocarbyl” refers to a saturated or partially unsaturated moiety having 3- to 20-atom backbone of carbon and at least one heteroatom (e.g., O, N, S). Examples of heteroalkyl moieties include 2-8-membered, 2-6-membered, and 2-4-membered heteroalkyl moieties. Particular examples include TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 alkoxyl, acyl (e.g., formyl, acetyl, benzoyl), alkylamino (e.g, di-(Ci-3-alkyl)amino), arylamino, aryloxime, carbamates, carbamides, alkylcarbonyl, arylcarbonyl, aminocarbonyl, alkylaminocarbonyl, alkylsulfanyl, arylsulfanyl, alkylsulfmyl, arylsulfmyl, alkylsulfonyl, arylsulfonyl, alkylsulfonylamino, and arylsulfonylamino.

[0047] The term “heterocycle” refers to a cyclic (monocyclic or polycyclic) heterocarbyl moiety which may be aromatic, partially aromatic, or non-aromatic. Heterocycles include heteroaryls. Examples include 4-10-membered, 4-7-membered, 6-membered, and 5-membered heterocycles such as aziridinyl, azetidinyl, benzof l, 3]dioxolyl, benzoxazinyl, 1,3-benzodioxolyl, 1,4-benzodioxanyl, cinnolinyl, chromanyl, 2,3-dihydro-benzo[l,4]dioxinyl, dihydrooxazolyl, 1,2-dihydropyridinyl, 2,3- dihydrobenzofuranyl, 1,4-dioxane, dioxolane, butyrolactam, furanyl, homopiperidinyl, hydantoinyl, isoindolinyl-l-one, 2-oxa-6-azaspiro[3.3]heptanyl, hydantoin, imidazolidin-2-one, imidazolidine, imidazolidinone, morpholinyl, oxetanyl, oxiranyl, phthalimidyl, piperazinyl, piperidinyl, pyrrolidinonyl, piperidinyl, pyrrolidinyl, pyrazolidine, spiro[cyclopropane-l,l'-isoindolinyl]-3'-one, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, 1, 2,3,4- tetrahydroquinolyl, tetrahydrothiopyranyl, thiomorpholinyl, and valerolactamyl.

[0048] The term “hydrocarbyl” refers to a refers to a moiety having a saturated or partially unsaturated all carbon-backbone. The term encompasses alkenyl, alkyl, and alkynyl moieties.

[0049] The term “include” has the same meaning as “include, but are not limited to,” and the term “includes” has the same meaning as “includes, but is not limited to.” Similarly, the term “such as” has the same meaning as the term “such as, but not limited to.”

[0050] The term “pharmaceutically acceptable salt” refers to a salt that is generally recognized as safe to administer to a subject. Examples of pharmaceutically acceptable salts include acetate, chloride, diphosphate, hydrochloride, maleate, phosphate, potassium, sodium, and sulfate.

[0051] The terms “subject” and “patient” are used interchangeably. The terms “subject” and “subjects” refer to an animal, such as a non-primate mammal (e.g., cow, pig, horse, cat, dog, rat, and mouse) and a primate (e.g., monkey, a chimpanzee, human). Preferred subjects are human (e.g., adult humans).

[0052] A “therapeutically effective amount” of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition. A “therapeutically effective amount” of a compound means an amount, alone or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of the disease or condition. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces, or avoids symptoms or causes of a disease or condition, or enhances the therapeutic efficacy of another therapeutic agent.

[0053] The terms “treat,” “treating” and “treatment” contemplate an action that occurs while a patient is suffering from a specified disease or disorder, which reduces the severity of the disease or disorder or retards or slows the progression of the disease or disorder. TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0054] Unless otherwise indicated, an adjective before a string of nouns should be construed to apply to each. For example, the phrase “optionally substituted pyridyl, pyrazyl, or furanyl” means the same as “optionally substituted pyridyl, optionally substituted pyrazyl, or optionally substituted furanyl”.

[0055] A wavy line “ ■'vm” that intersects a bond in a chemical structure indicates the point of attachment of the bond that the wavy bond intersects in the chemical structure to the remainder of a molecule.

[0056] Compounds disclosed herein may exist as tautomeric isomers. Although only one delocalized resonance structure may be depicted, all such forms are contemplated within the scope of the invention.

[0057] Compounds disclosed herein may exist as zwitterions (e.g., at pharmacological pH). Unless otherwise indicated, it should be understood that a chemical drawing depicting the structure of such a compound encompasses all of its zwitterionic forms.

[0058] Some compounds exist as stereoisomers. When a stereoisomer of compound is defined by its name (e.g. , with the use of R or .S') or is depicted in a drawn structure (e.g. , using a bold, bold-wedge, dashed, or dashed-wedge to depict the relevant chemical bond), the enantiomeric excess (ee) of that compound, unless otherwise indicated, is to be understood to be at least 60, 70, 80, 90, 95, or 99%. A compound or composition enriched with one stereoisomer of a compound has that one stereoisomer in an amount measurably greater than the compound’s other stereoisomer(s). For example, a compound enriched with an R enantiomer will have an enantiomeric excess of that enantiomer vis-a-vis the S enantiomer.

[0059] Unless otherwise indicated, a composition described as comprising one stereoisomer of a compound should be understood as being substantially free of the compound’s other isomer(s). For example, if a composition is defined as comprising the R isomer of a racemic compound, that composition is substantially free of the compound’s S isomer, i.e., the composition does not contain a measurable amount of the S isomer or contains the R isomer in an enantiomeric excess of greater than 90 percent (e.g., greater than 95 or 99 percent). Similarly, a method that comprises the use (e.g., administration) of one stereoisomer of a compound is to be understood as a method of using an enantiomeric excess of that stereoisomer.

[0060] With regard to the claims, if a first claim recites a compound and / or a salt (e.g., a pharmaceutically acceptable salt) thereof, a dependent claim that recites a compound of the first claim should, unless otherwise indicated, be construed as encompassing both the compound and its salts.

[0061] 4.2. DISCUSSION

[0062] Muncl3-4 is a docking and fusion regulator that participates in the secretory pathway of several cellular systems17-20. Muncl3-4 regulates exocytosis in hematopoietic cells by a mechanism that includes its interaction with the small GTPases Rab27a19,21and Rabi I22. However, the regulation of late endosomal maturation by Muncl3-4 is independent of Rab27a, and syntaxin 7 is dispensable for controlling exocytosis by Muncl3-4. Instead, the exocytic syntaxins 3, 4, and 11 regulate Muncl3-4- TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 mediated exocytosis23. Significantly, Muncl3-4 has evolved to interact with various syntaxins by different mechanisms16. Thus, while endosomal recruitment and binding to STX7 is inhibited by deletion of the Muncl3-4 C2B in the C-terminal16, binding to STX3, 4 and 11 is mediated by the C2A domain of Muncl3-4 located in its N-terminal23. Thus, small -molecule inhibitors of Muncl3-4-syntaxin 7-binding are not expected to interfere with Muncl3-4's interaction with exocytosis mediators or its role in granule docking at the plasma membrane (shown in Figs. 3H-J).

[0063] Muncl3-4 is predominantly highly expressed in hematopoietic cells24but also in other cells with secretory function, including goblet cells of the bronchial epithelium, alveolar type II cells, kidney epithelial cells24, and HEK-293 cells25. Muncl3-4 deficiency causes familial hemophagocytic lymphohistiocytosis type 3 (FHL3) in humans, an immunodeficiency syndrome induced by defects in the exocytic machinery of cytotoxic T lymphocytes, natural killer cells, and neutrophils17. Deficiencies in Rab27a or syntaxin 11, partners of Muncl3-4 in exocytosis19’21’26, also induce hemophagocytic syndromes in humans27,28, consistent with the general knowledge that the defects observed in FHL3 are associated with the role that Muncl3-4 plays in exocytosis of lytic granules but are not linked to its function in late endosomal maturation17. Therefore, the targeted inhibition of the Muncl3-4-STX7 interaction is not expected to trigger hemophagocytic syndrome but is expected to inhibit endosomal-mediated systemic inflammation. Novel Muncl3-4 single -nucleotide polymorphism (SNP) haplotype is present in a relatively large population of individuals with systemic juvenile idiopathic arthritis (sJIA) with macrophage activation syndrome (MAS)29, an autoimmune disease with a strong systemic pro- inflammatory component. Similarly, a polymorphism in the transcription factor IRF5, which controls a pro-inflammatory TLR signaling pathway, is associated with MAS in patients with sJIA30, further linking endosomal Muncl3-4 to TLR-initiated systemic inflammation and disease.

[0064] Some of the results described herein result from the use of a unique high-throughput screening (HTS) approach that utilized a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay to measure the binding of syntaxin 7 to Muncl3-4 in subcellular preparations that preserve the integrity of intracellular organelles. The assay was used to identify small-molecule inhibitors of the pathway regulated by syntaxin 7 and Muncl3-4. Using in-silico design, structure-activity relationships, chemical optimization, cell-based assays, and in vivo experiments, we identified and characterized the first-in-kind small-molecule inhibitors of STX7-Muncl3-4-dependent endosomal maturation and signaling for use in immune cells that contribute to systemic inflammation. In vivo characterization of these small molecules supports that inhibition of syntaxin 7-Muncl3-4 binding and endosome maturation decreases eTLR-mediated systemic inflammation. TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0065] 4.2.1. Results

[0066] In vivo inflammatory response to endosomal TLR ligands

[0067] We previously showed that Muncl3-4 binding to STX7 regulates the endosomal TLR response to CpG16. Muncl3-4 polymorphism is associated with MAS, sJIA, and systemic inflammation, while chronic TLR9 stimulation in mice develops features characteristic of MAS, including elevated serum levels of IFN-y, interleukin 6 (IL-6) and hepatic inflammation10. Here, to study whether TLR9 activation is regulated by Muncl3-4 in vivo, we treated Muncl3-4-null (Muncl3-7,ra / J,ra) mice with a single CpG insult and analyzed the neutrophilic and cytokine-mediated inflammation. In these studies, cytokine production was analyzed 6 hours after CpG insult, which is a well-established time point of maximal cytokine production in mice challenged with CpG31. TLR9 stimulation of Muncl3-4‘ / !m: / ‘ / !m:mice results in reduced cytokine production, including IL-6, IFN-y, and IFN-a, compared to wild-type mice (FIGS. 1A- C). IL-12, although reduced, did not reach significance (FIG. ID). This differs from that observed in TLR4-initiated systemic inflammation in Muncl3-4-null mice32. Thus, the production of pro- inflammatory cytokines and tissue infiltration by myeloid cells were normal (similar to wild-type) in Muncl3-4-deficient mice when challenged, in vivo, with the TLR4 ligand LPS32. Despite normal neutrophil counts (FIG. IE), neutrophil activation measured as the plasma level of the azurophilic granule cargo myeloperoxidase (MPO) was also decreased in Muncl3-4-null mice challenged with CpG (FIG. IF). Our data indicate that the inflammatory response to in vivo challenge with the endosomal-TLR ligand CpG (Fig. 1) but not to the plasma membrane TLR ligand, LPS32, is impaired in Muncl3-4 deficiency. Based on these studies, we next tested the hypothesis that blocking Muncl3-4-STX7 binding, and therefore, the specific function of Muncl3-4 at late endosomes, is a potential approach to counteract endosomal signaling-induced systemic inflammation.

[0068] High-throughput screening to identify small-molecule inhibitors

[0069] Late endosome-initiated pro-inflammatory signaling is a central mechanism in systemic inflammation and autoimmunity. We recently showed that this process is directly regulated by the novel interaction between the calcium-sensor Muncl3-4 and the late endosomal SNARE protein syntaxin 716(FIG. 2A). The affinity of STX7 for Muncl3-4, a calcium sensor, increases in the presence of Ca2+16. Expression of a calcium-binding-deficient mutant of Muncl3-4 fails to rescue LE defects16. Pharmacological modulators targeting the specific molecular pathways that regulate endosome-initiated inflammation would be beneficial for treating multiple human diseases, including autoimmune and autoinflammatory diseases. To address this clinical need, we developed an assay to screen molecular libraries to identify novel hits that can inhibit the specific binding between Muncl3-4 and syntaxin 7, two modulators of endolysosomal function necessary for eTLR activation. The assay is based on the principle of fluorescence resonance energy transfer (FRET) from a highly stable fluorescence donor, terbium cryptate (Tb), to green fluorescent protein (EGFP) (FIG. 2B). For the terbium-EGFP pair, the Forster TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 radius is approximately 43 A33. The FRET principle involves energy transfer from a terbium chelate with a long excited-state lifetime to a conventional fluorophore (EGFP) with a short excited-state lifetime. Emission is monitored 100 ms after excitation, and acceptor emission is only observed if energy transfer occurs. The time gating eliminates all fluorescence background, and less than 1% energy transfer is required for detection, making it a highly sensitive method for monitoring physical interaction34. Significantly, in these lanthanide -conjugated antibody-based assays, the conformational flexibility of the antibodies compensates for the more considerable distance between donor and acceptor, providing an abundant opportunity for energy transfer to occur during the extended detection window of the measurement, provided by the prolonged donor-excited state lifetime33. Our assay uses a Tb-conjugated anti -Flag antibody, which specifically binds to the tag in Flag-Muncl3-4 (FIG. 2B). TR-FRET signal increases by the specific binding of Flag-Muncl3-4 to EGFP-STX7. In this assay, the integrity of intracellular organelles is preserved and syntaxin 7 localizes at endosomes, its natural environment. This ensures that protein presentation recapitulates physiological cellular interactions, increasing the likelihood of finding biologically active compounds. The specificity of the reaction was demonstrated by competitive inhibition using recombinant STX7. Still, control proteins had no competitive effect (FIG. 2C). This high-throughput screening assay conforms to a “mix and measure” format and is easily automated. The assay is robust, highly reproducible, adaptable to low volumes and liquid handling devices, and has a high signal-to-background ratio.

[0070] For the identification of inhibitors of the interaction between Muncl3-4 and STX7, we screened the full Maybridge HitFinder 4 (MBHF4) and MBHF12 libraries; the latter has -50% compound diversity compared to the MBHF4 (-15,000 total compounds). Importantly, the Z’ -factor for the full MBHF screen was 0.76 ± 0.08, indicating high sensitivity, reproducibility, robustness, and low batch-to-batch and plate- to-plate variability and a hit rate = 0.28%. This screening approach led to the identification of ENDOtollins (ENDOs), inhibitors of MUNC13-4-STX7 interaction and endosomal TLR activation. The initial hits, shown below in Table 1, were confirmed as inhibitors of Muncl3-4-STX7 binding in validation assays and were selected for downstream analysis.

[0071] Table 1 TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0072] ENDOtollins based on the hits were then designed, synthesized, and tested. Some of these

[0073] ENDOtollins are shown below in Table 2. TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0074] Assay results obtained with the hit compounds (at 10 pM) are presented in Table 3. Assay results obtained with the designed compounds (at 10 pM) are shown in Table 4. Data is provided for CpG-TLR9 (HEK-blue Assay), Poly-IC (IRF3 Assay), CpG-CD40 (primary DCs, ex vivo), CpG-IFNa (primary DCs, TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 ex vivo), and CL097-INFa (primary DCs, ex vivo). In both tables: “Affinity” is the affinity constant for the binding pocket in the Muncl3-4 / MDH1-STX7-DR interface; “*” indicates mild inhibition; “**” indicates strong inhibition, and ” indicates that a value was not determined.

[0075] Table 3 Table 4

[0076] ENDO analogs are specific inhibitors of eTLR activation and endosomal signaling

[0077] Next, we studied the effect of the selected small molecules identified in the parent screen on the activation of endosomal TLR9. In this assay, we analyzed the impact of ENDO series on CpG-induced TLR9 activation using cells expressing the human TLR9 gene upstream of an inducible and secretable embryonic alkaline phosphatase (SEAP reporter system). The assay utilizes HEK-293 cells, which express Muncl3-4 and STX7 endogenously, as shown previously by proteomic analysis25and here by immunoblotting. Stimulating the reporter cells with the TLR9 ligand CpG oligodeoxynucleotide (CpG- TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0078] ODN) induces the production and secretion of alkaline phosphatase, which is detected in the culture medium by a colorimetric reaction. We show that one of the small molecules that was a positive hit in the parent TR-FRET assay (FIG. 3A), ENDO3, abolished CpG-mediated activation of TLR9 (FIG. 3C). This compound had minimal effect when tested in HEK293-null cells, which respond to TNFa using the same SEAP colorimetric reporter system used in HEK-Blue TLR9 cells, thus ruling out non-specific effects. ENDO7, and to a lesser extent, ENDO6, also exerted a partial but significant inhibition of TLR9 activation by CpG in this assay (FIG. 3C). Based on their performance in TR-FRET assays for the detection of Muncl3-4-STX7 binding and their inhibitory activity on TLR9 activation in cell-based assays, we selected ENDO3 and ENDO7 for initial downstream analysis and orthogonal validation.

[0079] Muncl3-4 interacts with syntaxin 7 in a Ca2+-dependent manner16(FIGS. 2A-C). Binding specificity was demonstrated by the inability of Muncl3-4 to pull down other syntaxins or to bind to the fusion regulator Vtilb16, by homologous competitive assays using recombinant STX7 (FIG. 2C) and by the use of Muncl3-4 inactive mutants. In addition to regulating late endosomal maturation through its interaction with syntaxin 7, Muncl3-4 regulates other critical cellular processes through interactions with the small GTPases Rab27a and Rabl l. Thus, Muncl3-4 and Rab27a regulate the last steps of lytic and azurophilic granule exocytosis in CTLs and neutrophils, respectively19,21. The Muncl3-4-Rabl 1 interaction regulates the docking of recycling endosomes at the plasma membrane and the production of reactive oxygen species22. To ensure that the compounds identified in our parent and pDC-based assays were specific inhibitors of the interaction between STX7 and Muncl3-4 but did not interfere with vesicle docking and exocytosis by inhibiting Rabs-Muncl3-4 complexes, ENDO compounds were counterscreened in TR-FRET assays using lysates expressing Flag-Muncl3-4 and EGFP-Rab27a or EGFP-Rabl l. Similar to the original TR-FRET screen (Muncl3-4-STX7), these counterscreens are robust and have excellent signal-to-background ratio22. In FIG. 3A, we show that ENDO3 specifically inhibits the interaction between Muncl3-4 and STX7 but does not inhibit the interactions of Muncl3-4 with either Rab27a or Rabl l, thus supporting the specificity of ENDO3 towards the endolysosomal function of Muncl3-4. We also show that ENDO3 does not interfere with the binding of STX7 to VAMP8 and therefore it is unlikely to block other STX7-mediated functions (FIG. 3B). These results further validate the approach to target protein-protein interactions rather than specific GTPases or effectors to increase specificity in pharmacological interventions to modulate trafficking pathways.

[0080] The inhibitory effect of ENDO compounds on the binding of Muncl3-4 to syntaxin 7 was confirmed by an orthogonal validation assay, consisting of the pull-down of Muncl3-4 with recombinant STX7 (not shown). We found that pre -incubation of lysates expressing Muncl3-4 with either the compound ENDO3 or the compound ENDO7 reduced the amount of Muncl3-4 detected in the pulldowns compared to controls incubated with vehicle alone (DMSO).

[0081] To analyze whether ENDO3 is a specific immunomodulator of TLR9 activation in immune cells, we analyzed the activation of TLR9 in the plasmacytoid dendritic cell (pDC)-like cell line CAL- 135,36stimulated with CpG. As readout, we studied the upregulation of CD40 at the plasma membrane by high- TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 throughput flow cytometry analysis. Of note, CD40 is a transmembrane glycoprotein surface receptor, a member of the Tumor Necrosis Factor Receptor superfamily (TNFRSF) and its activation is associated with increased effectiveness of antigen presentation and the upregulation of MHC class II and costimulatory molecules CD80 / CD86; thus, the level of CD40 plasma membrane is directly associated with pDC immune responses and autoimmune disease. Here, we show that ENDO3, but not ENDO7, significantly decreases the upregulation of CD40 in response to CpG in pDCs (FIG. 3D), further highlighting the inhibitory activity and biological relevance of this compound against cellular activation by TLR9 ligands. Interferon regulatory factor 7 (IRF7) is an interferon (IFN)-inducible transcription factor whose phosphorylation induces homodimerization and activation, and its dysregulation causes autoimmunity. Treatment with ENDO3 was found to decrease the detection of phosphorylated IRF7 in Cal 1 cells in a time-dependent manner, supporting the conclusion that the compound inhibits CpG-TLR9 downstream signaling. Finally, dose-response analysis of the effect of ENDO3 on Call cells indicates an IC50 of 1.17x1 O'7(FIG. 3E).

[0082] ENDO compounds specifically inhibit innate immune cell activation

[0083] Our previous studies show that CpG stimulation induces the rapid activation of neutrophil signal pathways, manifested as increased ERK phosphorylation and marked up-regulation of the integrin subunit CD1 lb at the plasma membrane in a Muncl3-4-STX7-dependent manner16. This process is inhibited by chloroquine treatment, indicating that CpG signals through the endosomal system16. Contrarily, CD1 lb upregulation in Muncl3-4-KO neutrophils was normal in response to the chemotactic peptide fMLF, a physiological stimulus that signals from plasma membrane receptors, further supporting that CD1 lb plasma membrane upregulation in response to CpG depends on TLR9 activation at endosomes16. We reason that treatment with ENDO3 would recapitulate the Muncl3-4-KO phenotype should the inhibitor operate through the inhibition of Muncl3-4-STX7 binding. Here, we first demonstrate that, similar to that shown for Muncl3-4-KO neutrophils, treatment with ENDO3 inhibits ERK phosphorylation in wild-type neutrophils stimulated with CpG (FIG. 3F). We further show that, as predicted, treatment with ENDO3 inhibits CpG-dependent CD1 lb upregulation in neutrophils (FIG. 3G).

[0084] The upregulation of neutrophil granule membrane proteins at the plasma membrane depends on the trafficking of intracellular granules, an essential mechanism in the neutrophil innate response. The trafficking and fusion of some of these organelles, including secondary and azurophilic granules, is regulated by Muncl3-4 through its interaction with Rab27a37but not by the interaction of Muncl3-4 with syntaxin 716. After establishing that ENDO3 does not interfere with Rab27a binding (FIG. 3A), we performed additional cell-based functional assays to rule out a possible off-target effect of ENDO3 in the mobilization of neutrophil granules, a Rab27a-dependent process. To this end, we analyzed the putative impact of ENDO3 on the mobilization of CD1 lb and CD66b from granules to the plasma membrane and on the secretion of myeloperoxidase (MPO) from azurophilic granules, using a plasma membrane receptor ligand. Cells were stimulated with the formylated peptide fMLF (formyl-Methionyl-Leucyl- TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0085] Phenylalanine), which signals through the plasma membrane receptor FRP 1 and activates neutrophil secretion independently of endosomal activation or TLR9 function. The assays were performed in the presence of cytochalasin D, which disrupts actin remodeling and facilitates the exocytosis of azurophilic granules. We show that ENDO3 does not inhibit the mobilization of either secretory vesicles (CD1 lb) (FIG. 3H) or specific granules (CD66b) (FIG. 31) in response to fMLF stimulation. Furthermore, MPO secretion, a mechanism also regulated by Muncl3-4 binding to Rab27a, was not affected by ENDO3 treatment (FIG. 3J) in neutrophils stimulated through plasma membrane receptors, further highlighting the specificity of the inhibitor towards endosomal Muncl3-4-syntaxin 7 binding and endosomal TLR9 activation by CpG.

[0086] ENDO3 inhibits endolysosomal flux in vitro and CpG-induced IL-6 production in vivo

[0087] Using a quantitative microscopy approach to analyze endolysosomal morphology, we previously showed that Muncl3-4 and syntaxin 7 regulate endosomal maturation. We also show that cells lacking Muncl3-4 expression have an accumulation of enlarged endosomes. The enlarged LAMP1+ compartment phenotype in Muncl3-4-KO was rescued by the expression of Muncl3-4 but not by the expression of Muncl3-4-C2A*C2B*, a mutant that disrupts its interaction with syntaxin 716. We hypothesize that inhibition of the Muncl3-4-STX7 interaction by ENDOtollins should recapitulate the impaired endosomal maturation phenotype observed in Muncl3-4 deficiency, manifested as an enlarged endolysosomal compartment. Here, we show that treatment of cells with ENDO3 for as short as two hours induces a significant increase in endolysosome size that recapitulates the phenotype observed in Muncl3-4-KO cells (FIG. 4A). In addition to enlarged late endosomes, Muncl3-4-deficient cells also showed defective late endosomal trafficking38. To directly analyze whether ENDO3 regulates the trafficking of endosomal acidic organelles, we labeled the cells with LysoTracker and quantitatively analyzed organelle dynamics using pseudo-TIRFM. This technique facilitates the study of organelles that may not necessarily be in areas adjacent to the plasma membrane39. Kinetic analyses showed that acidic organelles in ENDO3- treated cells have impaired trafficking compared to vehicle -treated controls (FIG. 4B), suggesting that, similar to Muncl3-4-deficient cells38, ENDO3 decreases endosomal vesicle dynamics. Next, because TLR9 activation requires endosomal maturation and Muncl3-4-deficiency was associated with reduced maturation and impaired endolysosomal degradative capacity16, we analyzed the effect of ENDO3 on endolysosomal activity. To this end, we utilized a cathepsin B substrate derived from cresyl violet (Magic Red). This membrane-permeable probe becomes fluorescent upon hydrolysis in endolysosomes, releasing membrane-impermeable fluorescent cresyl violet, which is trapped in endolysosomes. Magic Red becomes fluorescent in cathepsin-active endolysosomes but not in cathepsin-inactive terminal lysosomes40. Thus, Magic Red fluorescence requires endosomal maturation and the formation of a hybrid endolysosomal compartment. In FIG. 4C, we show that treatment with ENDO3 decreases endolysosomal activity, which recapitulates that observed in Muncl3-4-deficient cells16, further validating that the ENDO compounds inhibit Muncl3-4-mediated endosomal maturation. As an additional control, we analyzed a TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 possible negative impact of ENDOs on cell viability. To this end, we studied early apoptosis and cell death using Annexin V and propidium iodide, respectively, as described before41. We show that ENDO3 does not cause significant induction of apoptosis or cell death in immune TLR9-expressing human neutrophil-like cells, even at concentrations as high as 40 pM (FIG. 4D). We also show that ENDO3 does not cause cell death in primary neutrophils. Next, to analyze a possible beneficial role for ENDO3 against eTLR-mediated inflammation in vivo, we used a well-established model of CpG-ODN-induced systemic inflammation in mice42. In this model, cytokines are not detected until 4 hours of a single i.v. injection of CpG-ODN, a peak of cytokines is detected 6 h after the insult, and cytokines levels rapidly decreased to basal levels at 12 h42. A mild increase in the number of leukocytes in circulation was detected 6h after CpG-ODN insult in both vehicle and ENDO3 -treated mice (FIG. 4E). Next, we analyzed the effect of ENDO3 on CpG-induced production of IL-6. In FIG. 4F, we show that treatment with ENDO3 significantly prevents the increase in IL-6 plasma levels induced by CpG in mice.

[0088] Molecular optimization

[0089] Based on its performance in the original TR-FRET assay, validation binding assays, orthogonal cell-based assays, and counter screens, ENDO3 was selected for downstream molecular optimization. Using AlphaFold, we identified the most likely protein-protein-interaction domains of syntaxin 7 and Muncl3-4. The interface between Muncl3-4 and STX7 comprises the MDH1 domain of Muncl3-4 and residues S129-Q148 from the disordered region (DR) of STX7. This region is conserved in STX7 through species, but it is not present in other syntaxin family members. Ligand binding pocket prediction for the AFv3 complex using Discovery Studio v21. 1.0.20298 identified the pocket at the interface between Muncl3-4 MDH1-STX7-DR as the top-scoring binding site. This finding was validated by mutagenesis analysis. We show that replacing the R140, N141, L142and W145residues to alanine or deletion of the residues 129-148 of the disordered linker region of STX7, decreases binding to Muncl3-4 in TR-FRET assays (FIG. 5A). This modification in the disordered region of syntaxin 7 reduced the colocalization of STX7 with Muncl3-4 (FIG. 5B), further validating the importance of the STX7-DR in a cell-based assay.

[0090] Next, to generate ENDO3 analogs, we employed several independent Structure-Activity Relationship (SAR) strategies. We replaced the ENDO3 pyridine ring, azo moiety, bromine, and the hydroxyl group with alternative fragments (Maybridge). Molecular docking studies indicate that many of these analogs maintain high-affinity constants and engage the binding pocket in the Muncl3-4-MDHl- STX7-DR interface. Next, SAR derivative compounds were synthesized and vetted in functional assays consisting of the analysis of TLR9 activation using the HEK-Blue reporter cell line described above. In FIG. 5C we show that ENDO3 and ENDO 12 (Red bars), but not other derivatives, exert a potent inhibitory activity on TLR9 activation. In a yet additional cell -based assay for the analysis of eTLR activation, we analyzed the ability of ENDO analogs to inhibit IRF signaling stimulated with the TLR3 agonist poly-IC in Jurkat cells (FIG. 5D). ENDO3 and ENDO 12 were the most potent inhibitors also in this assay, further supporting their roles as specific inhibitors of endosomal TLR activation (FIG. 5D). TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0091] ENDOs 4, 5, 11 and 13 also showed inhibitory activity in this assay. Based on SAR, replacing ENDO3 pyridine ring with a heterocyclic scaffold, lH-imidazo[4,5-b] pyrazine (ENDO 14). or rearrangement of the bromine and methyl substituents on the pyridine ring (ENDO 17, ENDO 18, ENDO 19) did not improve activity. Further replacement of the azo group with ethylene (ENDO 16, ENDO 17, ENDO 19), vinylene (ENDO 14. ENDO18), or pyrazine (ENDO15), and Cl 1 hydroxyl with methoxy (ENDO18) did not result in improved activity. Replacing the bromine with a hydroxyl group (ENDO 13) and introducing a phenylacetamide (ENDO 11) at the diethylamino end of ENDO3 did not result in activity improvement (FIGS. 5C and D). The two most active ENDOtollins, ENDO3 and ENDO 12, are structurally similar. However, in ENDO 12, a fluorine group, generally considered more amenable for downstream med-chem applications than bromine43, replaces the bromine group present in ENDO3. Both ENDO3 and ENDO 12 are predicted to fit in the pocket formed by the STX7-Muncl3-4 interface, and are predicted to bind Argl40, but only ENDO12 forms an H-bond with Serl37 in the disordered region of STX7. Surface Plasmon Resonance analysis shows that both ENDO3 and ENDO 12 bind to STX7, albeit ENDO 12 binds with higher affinity (ENDO3: 3.1 ± 0.7 pM; ENDO12: 2.7 ± 0.7 pM, n=3; FIG. 5E). In agreement with this, ENDO 12 showed increased inhibitory activity in cell-based assays compared to ENDO3 (FIG. 5F). Thus, dose-response analyses confirms that ENDO12 is the most active compound with an IC50 of IxlO-7M (FIG. 5F). Despite this difference, both ENDO3 and ENDO 12 decrease the colocalization of STX7 with Muncl3-4 in living cells, validating their mode of action in intact cells (FIG. 5G). Both compounds also decreased the colocalization of TLR9 with LAMP1 (FIG. 5H), supporting that the interaction of Muncl3-4 with STX7 is necessary for the maturation of the TLR9 compartment. Also, ENDO12 significantly decreased the colocalization of TLR7 with LAMP1 (FIG. 51), but neither compound significantly altered the colocalization of TLR7 or TLR9 with early endosomes (Rab5) or Rab7+ late endosomes.

[0092] ENDO analogs 20-112, designed using computational biology from fragments compatible with medicinal chemistry and based on theoretical Kds, are shown below in Table 5, wherein “molLogP” is molecular logP (partition coefficient), “nof RotB” is the number of freely rotatable bonds, “nof HBA” is the number of hydrogen bond acceptors, “nof HBD” is the number of hydrogen bond donors, “Subst Score” is a substituent score, and “RTCNN Score” is the radial topological convolutional neural net score from MolSoft. See, e.g., Leeson, P., Springthorpe, B. “The influence of drug-like concepts on decisionmaking in medicinal chemistry” Nat Rev Drug Discov 6, 881-890 (2007); Ertl, P., “Cheminformatics Analysis of Organic Substituents: Identification of the Most Common Substituents, Calculation of Substituent Properties, and Automatic Identification of Drug -like Bioisosteric Groups” J. Chem. Inf. Comp. Sci. 2003 43 (2), 374-380; Vaid, T.M., et al., “Synergistic Inhibition Guided Fragment-Linking Strategy and Quantitative Structure-Property Relationship Modeling To Design Inhalable Therapeutics for Asthma Targeting CSF1R” ACS Omega 2023 8 (23), 20505-20512; De Magalhaes Pinheiro, I., et al. “Noncanonical roles of chemokine regions in CCR9 activation revealed by structural modeling and mutational mapping” Nat Commun 16, 7695 (2025). SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0093] Table 5

[0094] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0095] 22

[0096] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0097] 23

[0098] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0099] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0100] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0101] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0102] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0103] 28

[0104] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0105] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0106] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0107] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0108] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0109] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0110] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0111] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0112] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0113] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0114] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0115] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0116] SRI 2232.1PC / TSR3185P VHPM 18350.058W0

[0117] TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0118] ENDO12 treatment decreases eTLR ligand-initiated inflammation

[0119] CpG-ODN-initiated TLR9 signaling mediates the release of IL-6 and type I interferon-mediated response, which, if exacerbated, leads to systemic inflammation and autoimmunity but does not recapitulate a fully anti-viral response, which involves both eTLR-dependent and independent mechanisms. Here, to understand the ability of ENDOtollins to inhibit endosomal TLR activation, we first isolated mouse primary splenic CD1 lc+ DCs and analyzed their response to TLR3, TLR7, and TLR9 ligands ex vivo. We found that the activation of primary DCs by CpG, denoted by the surface expression of CD40, is inhibited by ENDO 12 and, to a lesser extent, by ENDO3 (FIG. 6A). The plasma membrane mobilization of CD40 in DCs stimulated with the TLR7 ligand CL097 was significantly inhibited by ENDO12 but not by ENDO3 (FIG. 6A). Poly:IC (TLR3) did not significantly stimulate the mobilization of CD40. In an orthogonal assay, we investigated the effect of these eTLR agonists on the production of IL-6 and IFN-a by splenic CD1 lc+ DCs. Of note, CpG and CL097 but not Poly:IC stimulated IL-6 secretion in this assay, while Poly:IC was the most efficient stimulus in inducing IFN-a secretion by CD1 lc+ DCs (FIG. 6B and C). We found that ENDO 12 significantly inhibited IL-6 production following CpG or CL097 stimulation (FIG. 6B) while ENDO3 had minimal inhibitory activity in this assay. IFN-a production was also inhibited by ENDO 12 when stimulated with eTLR ligands (FIG. 6C).

[0120] Next, we analyzed the effect of ENDO 12 on eTLR-mediated inflammation in vivo, using a mouse model of CpG-ODN-induced systemic inflammation. Here, we show that in vivo secretion of the pro- inflammatory mediators IL-6 and IFN-y in response to CpG was significantly decreased in the plasma of CpG-challenged mice after treatment with ENDO 12 (FIGS. 6D and E). We also found that treatment with ENDO 12 significantly decreases neutrophil secretion of azurophilic granule cargoes (myeloperoxidase, MPO) in response to CpG (FIG. 6F), an effect that is independent of the Rab27a- mediated Muncl3-4 function in secretion, but is instead mediated by the impact of ENDOs on the STX7- Muncl3-4 interaction regulating CpG-mediated endosomal activation. These studies highlight ENDOs, particularly ENDO 12, as new inhibitors of the endosomal function of Muncl3-4, demonstrating pharmacological complementation of the genetic phenotype, and support the notion that ENDO 12 is a potent inhibitor of eTLR both ex vivo and in vivo.

[0121] Finally, we investigated the potential impact of ENDO 12 on the host response to viral infection. In these assays, we used the lymphocytic choriomeningitis virus (LCMV) model of infection, a virus that activates both eTLR-dependent and independent mechanisms44. We show that the production of cytokines with anti-viral activity, including IL-6, IFN-a, and IFN-y, was not affected by ENDO 12 (FIGS. 6G-I). ENDO 12 only mildly decreased the production of the chemokines MIP-ip and MIP-2 (FIGS. 6J-K) in this viral infection model. All other cytokines and chemokines studied were not affected by ENDO 12 treatment. LCMV infection triggered a marked systemic neutrophil response, characterized by increased plasma levels of azurophilic granule cargoes. This neutrophil response to LCMV infection was also not TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 affected by ENDO12 (FIG. 6L). Altogether, our data support that END012 is a significant inhibitor of endosomal TLR activation, and it mediates anti-inflammatory activity in vivo in the setting of eTLR- mediated inflammation. However, ENDO 12 does not interfere with the host’s response to viral infection.

[0122] 4.2.2. Discussion

[0123] In this work, in vitro, in silico, and in vivo drug discovery approaches were used to develop novel inhibitors of endosomal maturation that impact eTLR activation. We strategically focused on developing inhibitors that specifically interfere with the interaction between the calcium-sensor Muncl3-4 with its partner, the endosomal SNARE, syntaxin 7. Inhibition of the protein-protein-interaction (PPI) of these two endosomal maturation mediators increases specificity by at least two independent mechanisms. First, because Muncl3-4 is mainly expressed in cells of the immune system, ENDOtollin-mediated inhibition of eTLRs and inflammation is a targeted and specific approach compared to, for instance, the use of chloroquine derivatives. Second, Muncl3-4 is a promiscuous effector that, in addition to binding to the SNARE STX7, binds to the Rab GTPases Rab27a and Rabi 1. Thus, because the interaction of Muncl3-4 with Rab GTPases is not perturbed by ENDOtollins (ENDOs), these compounds inhibit endosomal TLR activation without affecting exocytosis or endosomal recycling, therefore avoiding the potential pharmacological recapitulation of an FHL3-like phenotype characterized by defective secretory functions in immune cells17.

[0124] In this study, we validated the specificity of ENDOs using several cell-based approaches that include testing in primary neutrophils, DCs and pDC-like cell lines. In primary neutrophils, the Muncl3- 4-STX7 interaction controls the endosomal activation of TLR9 by CpG16and TLR7 by CL09745. This mechanism induces the mobilization of P2 integrins to the plasma membrane and has direct implications for neutrophil-mediated immunity and inflammation. CD1 lb mobilization was inhibited by ENDOs only in neutrophils stimulated with eTLR ligands but not when neutrophils were stimulated via plasma membrane receptors, thus supporting that ENDOs act by inhibiting endosomal pathways. Furthermore, exocytosis of gelatinase and azurophilic granules, both regulated by the interaction of Muncl3-4 with the secretory small GTPase Rab27a, were not affected by ENDO treatment when neutrophils are stimulated by ligands whose receptors operate at the plasma membrane, further supporting the specificity of these compounds against endosomal, Muncl3-4-dependent mechanisms mediated by STX7. This is also supported by our data showing that ENDO 12 inhibits the secretion of IL-6 and IFN-a by primary splenic DCs in response to not only TLR9, but also TLR3 and TLR7 ligands. In this context, it is highly relevant that, in addition to showing that ENDOs inhibit Muncl3-4-STX7 interaction when analyzed in cell lysates that maintain their natural subcellular environment, we also demonstrated direct binding of ENDOs to STX7 in biochemical (SPR) assays. Because the domain in STX7 that interacts with Muncl3-4 is conserved but uniquely present in STX7 and not in other syntaxins, it is predicted that ENDOs do not interfere with other SNAREs. This is also supported by data showing that ENDOs do not inhibit TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 neutrophil exocytosis, which requires SNAP-23, syntaxin-4, syntaxin-6, VAMP-1, and VAMP-246,47but not syntaxin-7. We also show that ENDOs do not interfere with the binding of STX7 to VAMP8 ruling out possible interference with other STX7-mediated functions. Our data highlight that Muncl3-4 binds syntaxin 7 via an intrinsically disordered domain in the linker region of STX7. Deletion of the C2B domain of Muncl3-4 prevents binding to STX7 when analyzed using intact endosomes16, likely reflecting the need for the C2B domain for Muncl3-4 recruitment to the endosomes. Here we show that the interaction between Muncl3-4 and syntaxin-7 requires residues R140, N141, L142and W145in the linker, disorder region of the SNARE protein and mutation of these residues decreases binding to Muncl3-4.

[0125] Although ENDO3 and ENDO 12 both bind to syntaxin 7 in biochemical assays and both demonstrated potent inhibition of endosomal signaling in several cellular systems, ENDO 12 showed increased potency in TLR9 inhibition assays, and increased affinity binding to STX7. A single dose of ENDO 12 decreased systemic inflammation induced by CpG in mice and the plasma levels of neutrophil secretory proteins were also reduced by ENDO 12. This finding is relevant not only in the context of ANCA-associated vasculitis, in which anti-MPO autoimmunity can be induced by TLR9-mediated dendritic cell activation50, but also of nonalcoholic steatohepatitis (NASH) in which hepatocyte mitochondrial DNA was shown to induce liver inflammation by activation of TLR9 in pro-inflammatory cells, including neutrophils51. Based on the inhibitory ex vivo and in vivo effects of ENDO 12 in response to CpG-mediated activation, it is likely that ENDOs would have a positive impact in the context of NASH.

[0126] As proof-of-concept that inhibitors of endosomal maturation have potential clinical applications, chloroquine (CQ) and hydroxychloroquine (HCQ), two lysosomotropic agents that prevent endosomal acidification, are widely used for the treatment of SLE52, RA53and to a lesser extent sJIA54. HCQ decreases the probability of flares accrual of damage, increases survival in SLE, and is generally well- tolerated52. Despite this, side effects, mainly gastrointestinal, cutaneous, and, to a lesser extent, retinal toxicity55, result in 15-30% of patients discontinuing CQ or HCQ treatment56,57, suggesting that new drugs and combinational therapies are needed to reduce adverse effects53. The mechanism of action of HCQ involves inhibition of endosomal TLR activation, reduction of ERK (extracellular signal -regulated kinase) signaling, decreased immune cell activity, and reduced intra-lysosomal protein processing58. While we show that inhibitors of STX7-Muncl3-4 have similar beneficial effects, Muncl3-4 is predominantly expressed in hematopoietic cells. Therefore, STX7-Muncl3-4 inhibitors constitute potent inhibitors of systemic inflammation predicted to have reduced off-target impact compared to HCQ. Because ENDOs are not only potent inhibitors of neutrophil activation but also inhibit eTLR-mediated activation of plasmacytoid dendritic cells, whose dysregulation is linked to autoimmune disease driven by type I interferon production by activated pDCs, it is proposed that the small-molecules identified in this work TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 has the potential to expand the repertoire of anti-inflammatory drugs that are beneficial in autoimmune disease.

[0127] During viral infections, the absence of Muncl3-4 or Rab27a result in a defective immunological synapse, leading to impaired cytotoxicity in CTLs and NK cells towards infected cells. The inability of Muncl3-4-deficient mice to respond to viral infections triggers a hemophagocytic-like syndrome characterized by increased cytokine production, as demonstrated by Crozat and Beutler59using Jinx (Muncl3-4-null) mice. ENDOs interfere with the binding of Muncl3-4 to syntaxin 7, but do not interfere with the Muncl3-4-Rab27a interaction. Therefore, ENDOs do not interfere with the ability of the host to respond to viral infections and therefore, these compounds do not trigger a hemophagocytic-like syndrome.

[0128] Equally important, dysregulation of IL-6 signaling has been linked to the pathogenic inflammatory mechanisms in autoimmune diseases, including rheumatoid arthritis and idiopathic juvenile arthritis60, and anti-IL-6R antibody therapies, like tocilizumab, have been successfully used in autoimmune diseases61. In this context, our observation that ENDOs decrease IL-6 in vivo is highly relevant. Cytokine release syndrome (CRS)62is a life-threatening condition caused by hyper-activation of the immune system commonly associated with viral infections, including SARS-Cov2, and is one of the most common adverse effects after CAR-T infusion therapy62. Of note, not only does IL-6 play an important role in CRS, but also neutrophil activation is proposed to contribute to CRS63,64. Anti-IL-6R therapy is also used for the prevention of CRS in CAR-T therapy62,65’66, but its effectiveness in treating COVID-19 patients has encountered contradicting results67-69. Thus, the dual effect of ENDO compounds in attenuating both cellular-mediated inflammation and IL-6 production / secretion underscores potential benefits and opens a window for considering their potential use for treating CRS in various pathological conditions. The ENDOtollins described herein constitute the first group of small-molecule inhibitors of endolysosomal signaling through the blockage of the Muncl3-4-STX7 interaction that attenuates inflammation. The inhibitors decrease endosomal flux through interference with the specific binding of Muncl3-4 to syntaxin 7 but do not affect other functions of Muncl3-4, highlighting that inhibition of protein-protein interactions markedly increases specificity. In vitro and in vivo data support the dual targeting of cellular and molecular inflammatory processes by ENDOtollins, whose potential applications include, but are not limited to, nucleic acid-induced systemic inflammation.

[0129] 4.3. METHODS OF USE

[0130] This invention is based, in part, on the discovery that small molecules can be used to disrupt the protein-protein interaction between Muncl3-4 and syntaxin 7 (STX7), which interaction plays an important role in immunity. Thus, one embodiment of this invention is method of disrupting a proteinprotein interaction between Muncl3-4 and STX7 in a cell, which method comprises contacting the cell with a compound of the invention (i.e., a compound disclosed herein). TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0131] Another embodiment is a method of inhibiting TLR3, TLR7, or TLR9 in a cell, which comprises contacting the cell with a compound of the invention.

[0132] Another embodiment is a method of inhibiting endolysosomal flux and decreasing endolysosomal cargo degradation in a cell, which comprises contacting the cell with a compound of the invention.

[0133] Another embodiment is a method of decreased nucleic acid-induced ERK signaling in neutrophils and IRF signaling in pDCs, which comprises contacting the cell with a compound of the invention.

[0134] Another embodiment is a method of reduced nucleic acid-induced systemic inflammation in a patient, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of the invention. In certain embodiments, the decreased systemic inflammation is manifested as decreased production and release of MPO, IL-6, or IFN-y.

[0135] Another embodiment is a method of treating inflammation associated with or caused by nonalcoholic steatohepatitis (NASH) or cytokine release syndrome (CRS), which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of the invention.

[0136] Another embodiment is a method of treating inflammation associated with or caused by an autoimmune disease, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of the invention. Examples of autoimmune diseases include ischemiareperfusion injury, juvenile idiopathic arthritis (JIA), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and type 1 diabetes (T1D).

[0137] 4.4. PHARMACEUTICAL FORMULATIONS

[0138] Compounds disclosed herein may be systemically administered in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable, edible carrier. They may be in the form of single unit dosage forms (e.g., enclosed in hard or soft shell gelatin capsules or compressed into tablets). For oral therapeutic administration, an active compound may be combined with one or more excipients in the form of ingestible tablets, buccal tablets, capsules, caplets, troches, elixirs, suspensions, syrups, and wafers.

[0139] Compounds may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of an active compound or its salts may be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0140] Pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride.

[0141] Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0142] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by fdter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-fdtered solutions.

[0143] 4.5. METHODS

[0144] 4.5.1. Human Neutrophil isolation

[0145] Human neutrophils were isolated from normal donor blood by Ficoll density centrifugation, as previously de scribed70All procedures involving human subjects were reviewed and approved by the Human Subjects Committee at The Scripps Research Institute and conducted according to its requirements and NIH guidelines. Informed consent was obtained from all donors.

[0146] 4.5.2. Mouse Neutrophil isolation

[0147] Bone marrow cells from experimental males and females, 6- to 10-week-old mice and sex and age-matched control mice were collected from the long bones of mouse legs by flushing the bones with 5 ml of phenol red-free RPMI (Life Technologies) using a 30 Gauge 'A” hypodermic needle and a BD Luer- Lok™ 10 ml syringe. Bone marrow-derived mature neutrophils were isolated by positive selection using Anti-Ly6G MicroBead Kit or Anti-Ly6G MicroBeads UltraPure purification kit (Miltenyi Biotec, 130- 120-337). Purity of Ly6G+ cells was -99% purity by flow cytometry70. The cells were then resuspended in phenol red-free RPMI and seeded into four-chamber 35 mm glass-bottom dishes (No. 1.5 borosilicate coverglass). The cells were incubated at 37°C in a tissue culture incubator (5% CO2) until analysis. Where indicated, cells were labeled for 5 min with LysoTracker (deep-red) before analyzed by TIRFM. TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0148] 4.5.3. Mouse models

[0149] In this work we used and Muncl3-4-deficient (Jinx) mice59an in bred control wild-type mice, as well as C57BL / 6Jfrom The Scripps Research Institute colony. Mice were maintained in a pathogen-free environment and had access to food and water ad libitum. Housing conditions included a 12-h dark / light cycle (light 6 a.m. to 6 p.m.), ambient temperature 24 °C, and humidity 50%. The mouse genotype was determined by Transnetyx. Animals were euthanized by over-anesthetization by standard and approved protocols. All animal studies were performed in compliance with the Department of Health and Human Services Guide for the Care and Use of Laboratory Animals. All studies were conducted according to National Institutes of Health and institutional guidelines and with the approval of the animal review boards at The Scripps Research Institute.

[0150] 4.5.4. In vivo analysis of CpG- and viral-induced induced inflammation

[0151] To analyze in vivo response to CpG-induced inflammation, mice were treated with a single i.v. injection of CpG-A or CpG-B as described previously71. Briefly, 2 pl of l g / pl CpG-A or CpG-B or vehicle were mixed with 98 pl of sterile PBS. CpG was mixed with 12pl DOTAP resuspended in 88 pl PBS, and the mix was incubated for 20 min at room temperature before proceeding with tail injection. Where indicated, mice were injected (i.p.) with 30 mg / Kg ENDOs (PBS in 5 % DMSO) or vehicle 1 hour before CpG i.v. administration. The peak for cytokine production in this model is detected at 6h42. After 6 hours, blood was obtained by cardiac puncture into EDTA-containing tubes. Cytokine analysis in plasma was performed by multiplex analysis (Millipore) or by ELISA (IL-6, R&D; IFN-alpha, pbl assay science, cat. 42115-1).

[0152] Where indicated, mice were treated with ENDO 12 or vehicle and subsequently infected with lymphocytic choriomeningitis (LCMV, Clone 13) virus. For these assays, LCMV was passaged on BHK- 21 cells as previously reported72. In these assays, 8-week-old mice were treated with a single i.p injection of ENDO3, ENDO 12 (15 mg / Kg), or vehicle (5% DMSO in PBS) in a total volume of 400 pl. After one hour, mice were challenged with LCMV (2xlOA6 PFU (plaque forming units), i.v. (via retro-orbital sinus) or PBS. After 16 h, blood was collected in EDTA-containing tubes. Plasma was collected by low-speed centrifugation to remove red and white blood cells. 10 pl of plasma was processed to perform 10-fold serial dilutions and viral quantified by focus forming assay on VeroE6 cells as previously described73. Plasma samples were analyzed for the presence of cytokines and chemokines by multiplex technology, and myeloperoxidase by ELISA (R&D).

[0153] 4.5.5. pDC differentiation

[0154] BM-derived pDCs were differentiated as described under Mutagenetix (https: / / mutagenetix.utsouthwestem.edu / protocol / protocol_rec. cfm?pid=21). Briefly, bone marrow cells from wild-type or Ra / ?27aash / ashmice were plated at 3-5 x 106cells / ml in the presence of hFLT3L at TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0155] 100 ng / mL. The cells were cultured for 9 days without disturbing the culture. Floating cells and loosely adherent cells were harvested by vigorous pipetting. Cultures were stained for CD11c, CD1 lb, and B220 and analyzed by FACS for CD1 lchl, CD1 lb10, B220hl. IFN-a production was analyzed by ELISA and immunofluorescence .

[0156] 4.5.6. Splenic DCs

[0157] Splenic dendritic cells (DCs) were isolated by manual and enzymatic tissue dissociation followed by magnetic bead-based positive selection. Briefly, mouse spleens were minced into small fragments using a scalpel. Tissues were incubated in enzyme mix (Miltenyi Biotec, Spleen Dissociation Kit #130- 095-926) at 37 °C for 15-30 min with agitation every 5 min to promote enzymatic digestion of extracellular matrix proteins. Samples were further dissociated mechanically by gentle pipetting with a 16G needle attached to a 3ml syringe and fdtered through a 70 pm cell strainer. The resulting cells were then subjected to magnetic bead -based positive selection using Pan DC MicroBeads (Miltenyi Biotec, 130-092-465). Cells were incubated with Pan DC MicroBeads for 15 min at 4 °C and, after washing, cells were applied to LS MACS Columns in a magnetic field; unlabeled cells passed through, while labeled DCs were retained and subsequently eluted. Positively selected splenic dendritic cells were stimulated with combinations of ENDO compounds and eTLR activators for 48 h at 37 °C and 5% CO2. Following incubation, cultures were centrifuged, and supernatants were collected for cytokines quantification by ELISA. Levels of IL-6 (R&D Systems, DY406-05) and IFN-a (VeriKine-HS, 42115-1) were measured according to manufacturer’s instructions. Cell pellets were blocked in 1% BSA and stained with anti CD40-APC (Miltenyi Biotec, 130-116-111) to assess surface CD40 expression by flow cytometry (NovoCyte). IFN-a production was analyzed by ELISA and immunofluorescence. IL-6 was analyzed by ELISA.

[0158] 4.5.7. Lysates preparation

[0159] Jump-In™ TI™ cells (Thermo, Cat#:M4454) containing two stable R4 integrase-specific recognition sites and an integration-activated blasticidin-resistant gene were transfected with retargeting vectors expressing EGFP-STX7, Flag-Muncl3-4 or Muncl3-4- mutants and with R4 integrase to generate stable cell lines expressing these proteins41. Blasticidin-selected clones expressing similar levels of protein were used. For screening, the individual components of the reaction were expressed in independent cell lines. The cells were resuspended in relaxation buffer (100 mM KC1, 3 mM NaCl, 3.5 mM MgCL, 1 mM ATP and 10 mM PIPES (pH 7.3) containing protease inhibitors (cOmplete EDTA-free, Roche, Indianapolis, IN). For lysis, we use nitrogen cavitation, a method that disrupts the plasma membrane but preserves intracellular organelles and minimizes lysosomal protease release74. Lysates were spun at 14,000 rpm for 1 min at 4°C to remove nuclei and tested in small-scale TR-FRET reactions, flash-frozen TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 using liquid nitrogen and stored at -80°C. The concentration of tagged proteins in each lysate was adjusted to 25 nM.

[0160] 4.5.8. TR-FRET, high-throughput screening (HTS) and counter screens

[0161] The HTS time-resolved fluorescence resonance energy transfer binding assay (TR-FRET) is based on using lysates obtained by non-denaturing methods, thus conserving the integrity of intracellular organelles. Cell lysates (5 pl) expressing FLAG-Muncl3-4 (or calcium-binding-deficient mutants lacking four aspartic acid residues necessary for Ca2+binding16(negative control) were dispensed into 384-well plates (Greiner cat. no. 788076). Test compounds (final concentration 10 pM) were added with a 50 nL pintool, and the plates were incubated for 30 minutes at 20° C. Next, 5 pL of EGFP-STX7 or EGFP (negative control) lysates were added, and samples were further incubated for 15 minutes. The reactions were complemented by adding the terbium cryptate-conjugated anti-Flag antibody (Cisbio, Bedford, MA) at 15 pg / pl in a final total volume of 11 pl and incubations were carried out for additional 10 min at 20°C. The reactions were carried out in the presence of 100 pM CaCE or 200 pM ethylene glycol tetraacetic acid (EGTA). The samples were read using a 2104 Envision plate reader with 340 nm excitation and 490 and 520 nm emission. The TR-FRET signal is measured just before and 5 min after calcium addition. For “% of inhibition” calculation, the differential signal activated by calcium in the presence of compounds is expressed in relationship to that observed in DMSO wells (100% activation) and to the GFP-STX7-Flag- Muncl3-4-C2A*B* mutant control (0% activation). An increased emission ratio is indicative of specific binding. High-throughput screening (HTS) for small-molecule inhibitors of the Muncl3-4-STX7 interaction were performed using the Maybridge HitFinder (MBHF) libraries (MH4 and MH 12) for a total of -32,000 compounds. For subsequent experiments, “solids” were obtained from Maybridge and resynthesized (Wuxi). The compounds were resuspended in DMSO at 10 mM, and stored at -20°C. Where indicated, GFP-Rab27a or GFP-Rabl 1 were used instead of GFP-STX7 and / or myc-VAMP8 was used in counterscreens control TR-FRET assays.

[0162] 4.5.9. Orthogonal cell-based validation assays of ENDO analogs

[0163] Plasmacytoid dendritic -like cells, Cal- 136were a generous gift from Dr Baccala. Cal-1 cells were cultured in suspension in complete growth medium RPMI 1640 GlutaMAX (Life Technologies Corp) supplemented with 10% FCS (Life Technologies Corp) and 1% penicillin / streptomycin (Life Technologies Corp). For analysis, Cal-1 cells were incubated with ENDO compounds or vehicle for 15 min to 16 h at the indicated concentrations. After activation using 5pM CpG (CpGB-ODN-2006-1, InvivoGen), Cal-1 activation was analyzed by cell surface expression of CD40 (clone 5C3, Biolegend) by flow cytometry. Where indicated, the effect of ENDOs on TLR9 activation was analyzed using HEK- Blue™ hTLR9 cells (InvivoGene). This cell line is engineered for the stable expression of the human TLR9 gene and expresses an inducible reporter gene for secreted embryonic alkaline phosphatase TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0164] (SEAP) downstream TLR9 activation. Cells were treated with ENDOs or vehicle and TLR9 activation in response to CpG ( pM) was analyzed by measuring SEAP activity, monitored using QUANTI-Blue™ (InvivoGene) following the manufacturer's recommendations. The effect of ENDOs on IRF3 activation was analyzed using human T- lymphocyte-derived Jurkat-Diial1 4Cells (InvivoGene) stimulated with Poly:IC (InvivoGene). In some experiments, HEK-Blue™ Null 1 is the parental cell line of HEK-Blue™ hTLR9 cells were used in control experiments in which cells pre-treated with ENDO3, ENDO 12 (1 or 10 pM) were stimulated with TNFa (100 ng / ml). Similar to HEK-Blue™ hTLR9, HEK-Blue™ Nulll cells (InvivoGene) express an inducible reporter gene for secreted embryonic alkaline phosphatase (SEAP).

[0165] 4.5.10. Cell death and metabolic assays

[0166] For apoptosis and cell death assays we used the FITC Annexin V Apoptosis Detection Kit I (BD Biosciences). Human granulocytes (1 x 106cells) were incubated with the indicated ENDO for 4 h. The cells were stained according to manufacturer instructions and analyzed by flow cytometry. In some experiments cell death was analyzed by flow cytometry using the probe Zombie Violet (BioLegend, cat# 423113), an amine-reactive fluorescent dye that is non-permeant to live cells but permeant to the cells with compromised membranes.

[0167] 4.5.11. Pull-down assays

[0168] Pull-down assays were performed as described previously41. Briefly, 5pg of GST-STX7 or GST (control) were bound to 30 pl of prewashed glutathione-Sepharose 4B (Amersham Biosciences). Beads were washed with PBS and utilized to pull down Muncl3-4 by incubation with 293T cell lysates expressing FLAG-Muncl3-4. Recombinant proteins and lysates were rotated overnight at 4°C in binding buffer, containing protease inhibitors (cOmplete EDTA-free ; Roche, Indianapolis, IN). Were indicated, compounds (lOpM) or vehicle were added in the reaction media. Beads were washed three times with wash buffer (PBS containing 0.05% Tween-20) and once with ice-cold PBS. The proteins in the pulldown were resuspended in sample buffer, samples were resolved by NuPAGE gel electrophoresis, and JFC1 and GST were detected by Western blot.

[0169] 4.5.12. Western blot analysis, ERK and IRF7 signaling

[0170] Mouse neutrophils were treated with 10 ng / ml GM-CSF for 30 min before stimulation with 5 pM CpG for the indicated times. The cells were lysed with RIPA buffer supplemented with protease inhibitor cocktail (Roche) and phosphatase inhibitors (Calbiochem, 524-624 and 524-625) and subsequently subjected to Western blotting using the indicated antibodies as described before16. Briefly, gel electrophoresis was carried out using 4-12% gradient or 12% Bolt Bis-Tris Plus gels (Life Technologies). Proteins were transferred onto 0.45-pm nitrocellulose membranes (GVS Filter Technology, 1-215-484) and the membranes were incubated overnight at 4°C in the indicated primary antibodies, followed by TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 incubation with HRP-conjugated secondary antibodies (Bio-Rad Laboratories, goat anti-mouse HRP, 170-6516; or goat anti-rabbit HRP, 170-6515). The blots were developed using SuperSignal West Pico (Thermo Scientific, 34-580), clarity ECL Western blotting substrate (Bio-Rrad, 1-709-060), or Femto chemiluminescence substrate systems (Thermo Fisher Scientific, 34-094). For IRF7 analysis, blots were incubated with anti-p-(Ser477) IRF7 (PA5-64834, Thermo Fisher Scientific) followed by fluorescently label secondary antibodies (Li Cor) and visualized using azure Biosystems 600. ERK phosphorylation was quantified using ImageJ. The phosphorylated ERK intensity values were normalized to total ERK. For the analysis of the expression of endogenous Muncl3-4 and STX7 in HEK-293T cells, 10 pg of protein were resolved by gel electrophoresis carried out using 4-12% gradient or 12% Bolt Bis-Tris Plus gels (Life Technologies). Proteins were transferred to nitrocellulose, total protein was assessed using FastGreen fluorescence labelling and Muncl3-4 and STX7 were detected using anti-Muncl3-4 (R&D, MAB89661) and anti-STX7 (R&D, AF5478) antibodies, respectively.

[0171] 4.5.13. Neutrophil stimulation, ELISA, cytokine analysis, and flow cytometry analysis

[0172] Neutrophils (1 x 106) were resuspended in phenol red-free RPMI and either stimulated with CpG- B (5 M) (ODN 1826; InvivoGen) or vehicle for the indicated time. Where indicated, cells were primed with Cytochalasin D (lOpg / ml, SIGMA) or vehicle for 30 min at 37°C before stimulation with the formylated peptide fMLF (SIGMA). The cells were spun down, and the supernatants were collected for ELISA (MPO, R&D)70or Multiplex analysis70. Plasma membrane expression of CD1 lb (clone MI / 70; BD Biosciences, San Jose, CA) and anti-human CD66b (FITC, BioLegend, cat# 305104, clone G10F5) were analyzed by flow cytometry as we described previously41, using aNovoCyte 3000 flow cytometer with BD FACS Diva 6 software, and the data were processed using FlowJo software (Ashland, OR).

[0173] 4.5.14. Total Internal Reflection Fluorescence (TIRF) Microscopy analysis of vesicular trafficking and size

[0174] TIRFM experiments were performed using a 100x / 1.45 NA TIRF objective (Nikon Instruments, Melville, NY) on a Nikon TE2000U microscope custom-modified with a TIRF illumination module as described75. Images were acquired on a 14-bit, cooled charge -coupled device (CCD) camera (Hamamatsu) controlled through NIS-Elements software. After placing the cells on the stage, the position of the individual laser beams was adjusted with the TIRF illuminator to impinge on the coverslip at an angle to yield a calculated evanescent field depth of 100 or 400 nm for TIRFM and pseudo-TIRFM modes, respectively. For TIRFM, the objective is pre -calibrated for Z-position assignment and for chromatic shift between the channels using 100 nm Tetraspeck beads as specified by Nikon. For pseudo-TIRFM, the calculation of the penetration depth was performed by measuring the position at which the beam is exiting from the center of the objective and subsequently correlated to the penetration depth vs the displacement of the beam from the center using the exponential decay depth equation described76and incorporated into TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 the Nikon software. The penetration depth for different wavelengths is estimated at plus or minus 20 nm for a given beam position. For live experiments, the images were recorded using 200-300 ms exposures, depending on the sample's fluorescence intensity. Vesicle dynamics were tracked using surface rendering or spot modules in IMARIS. For vesicle size, images were analyzed using Image. / software to measure late endosome diameter, as we described before16,39. Specifically, the late endosome diameter was drawn manually using the Straight-Line tool, and the length was measured using the "Measure" tool.

[0175] 4.5.15. Confocal microscopy image acquisition and colocalization analysis

[0176] All 12-bit / l 6-bit images were acquired using the full dynamic intensity range (0-4096 / 0-65536) of the specified fluorophores using an optimal frame size of 1024 x 1024. Images were processed for quantitative colocalization using Zen Pro in 2D (Zeiss) using colocalization modules. Samples stained for secondary antibodies and unlabelled controls alone were used to define thresholds of real signal above background and non-specific signal. Regions of interest (ROIs) were drawn around each cell, previously defined minimum thresholds were input, two fluorescent channel signals were selected. The software automatically calculated pixel intensity spatial overlap coefficients between them (both Mander’s and Pearson’s coefficients are scored). Mander’s overlap coefficient (MOC) was primarily used and is defined as MOC = 22i(Ri x Gi)^iRi2 x 22) G i 2 where Ri is the intensity of the first fluorophore in an individual pixel, whereas Gi is the corresponding intensity for the second fluorophore in the same pixel. Areas of overlap were pseudo-coloured in white (binary) to mathematically and spatially defined colocalized zones in cells.

[0177] 4.5.16. Functional endolysosomal analysis

[0178] Magic Red (Immunochemistry Technologies, #6134) was used to measure Cathepsin B activity following the manufacturer’s instructions. Briefly, 250,000 cells were seeded in 4-chamber p35 plates until 80% confluence. After the indicated treatment, cells were incubated with Magic Red staining solution for 60 min at 37°C protected from light. Cathepsin B activity was determined by measuring Magic Red fluorescence intensity Excitation / emission filters (590 / 620nm) using a Zeiss LSM 880 laserscanning confocal microscope attached to a Zeiss Observer Z1 microscope using the 60 x oil Plan Apo, 1.4 numerical aperture infinity-corrected optics at 21°C. Images were processed using ImageJ.

[0179] 4.5.17. Muncl3-4-Syntaxin 7 small molecule docking strategy

[0180] AlphaFold2-Multimer was used to generate the Muncl3-4-STX7 complex by submitting full- length human protein sequences of Muncl3-4 (Uniprot ID: Q70J99) and STX7 (Uniprot ID: 015400) with default settings to create five relaxed models. The tool applies MMseqs2 and HHsearch for multiple sequence alignment (MSA) and template search. The MSAs and templates were further utilized to predict the interaction and spatial arrangement of the amino acids and 3D structure of the protein complex. In the TSRI 2232.1PC / TSR3185P VHPM 18350.058W01 absence of an experimental reference structure, the model’s quality was assessed through the pLDDT score, predicted alignment error (PAE) plot, and ChimeraX-based analysis. The top-ranked model was selected for further analysis using UCSF ChimeraX vl.6.1 and Molsoft-Chemist v3.9. Prediction and visualization of the ligand binding pocket were performed using Molsoft Chemist v3.9, and the docking binding energy calculations were conducted using PyRx vO.9.9 with Autodock Vina.

[0181] 4.5.18. Surface Plasmon Resonance (SRP)

[0182] The SPR based ligand binding analysis was conducted by using Biacore S200 system (Cytiva) at the Scripps Research Biophysics and Biochemistry Core Facility. The His-tagged syntaxin-7 recombinant protein (Uniprot ID: 070257) was purchased from Synaptic Systems (Catalog No. 110-7P). For the protein immobilization step, a high-density streptavidin chip (SCBS SAHC1500M, XanTec bioanalytics GmBH) was employed. Briefly, for immobilized protein surface preparation, syntaxin-7 was diluted to 1 pM and subsequently injected over the surface for 120 sec at 10 pL / min. This approach resulted in an immobilized signal gain of 2,000 response units (RU). The running buffer for all immobilization assays was IX PBS-P+ (20 mM phosphate 137 mM NaCl, 2.7 mM KC1, pH 7.4, 0.05% Surfactant 20) and 2% DMSO. The assays were performed using the compound concentration range of 31.25 nM - 3 pM in IX PBS P+ buffer, and 2% DMSO as running buffer. All runs were performed at least in triplicates.

[0183] 4.5.19. Mutagenesis

[0184] Syntaxin 7 ws cut from Pmixip GFP-STX7 (ADDGENE 45921) using EcoRI and BamHI, and ligated into EGFP-C1. Mutagenesis of EGFP-C1-STX7 was performed by GenScript. The vector was linearized using Agel and BamHI. To generate the inserts, two PCRs were performed using F1R1 and F2R2 proprietary (GenScript) primers. The resulting fragments were then ligated into the linearized vector.

[0185] 4.5.20. Statistical analysis and reproducibility

[0186] Data are presented as mean, and error bars correspond to standard errors of the means (SEM) unless otherwise indicated. Statistical significance was determined using the Students’ t-test or nonparametric Mann-Whitney or Wilcoxon signed rank test fortwo-group comparisons. We used one-way ANOVA with multiple comparisons test. Analyses were performed using GraphPad InStat (version 3) or Excel software, and graphs were made using GraphPad Prism (version 9.2) software. All measurements were taken from distinct samples. All methods were analyzed using two-tailed analyses unless indicated. A p-value <0.05 was considered statistically significant. Assays were repeated at least three times, including those shown as representative data unless otherwise stated in the Figure Legends. The number of samples, cells, or mice per group is indicated in Figure Legends. Statistical analysis of Superplots was calculated based on the average values of each experiment (large symbols). TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0187] 4.6. REFERENCES

[0188] 1 Bird, P. I., Trapani, J. A. & Villadangos, J. A. Endolysosomal proteases and their inhibitors in immunity. Nat Rev Immunol 9, 871-882 (2009). https: / / doi.org / 10.1038 / nri2671

[0189] 2 Deane, J. A. & Bolland, S. Nucleic acid-sensing TLRs as modifiers of autoimmunity. J Immunol 177, 6573-6578 (2006).

[0190] 3 Krieg, A. M. & Vollmer, J. Toll-like receptors 7, 8, and 9: linking innate immunity to autoimmunity. Immunol Rev 220, 251-269 (2007). https: / / doi.Org / 10.l 11 l / j, 1600-065X.2007.00572.x

[0191] 4 Baccala, R., Hoebe, K., Kono, D. H., Beutler, B. & Theofilopoulos, A. N. TLR- dependent and TLR-independent pathways of type I interferon induction in systemic autoimmunity. Nat Med 13, 543-551 (2007). https: / / doi.org / 10.1038 / nml590

[0192] 5 Lee, B. L. & Barton, G. M. Trafficking of endosomal Toll-like receptors. Trends Cell Biol 24, 360-369 (2014). https: / / doi.Org / 10.1016 / j.tcb.2013.12.002

[0193] 6 Santegoets, K. C., van Bon, L., van den Berg, W. B., Wenink, M. H. & Radstake, T. R. Toll-like receptors in rheumatic diseases: are we paying a high price for our defense against bugs? FEBS Lett 585, 3660-3666 (2011). https: / / doi.Org / 10.1016 / j.febslet.2011.04.028

[0194] 7 Leadbetter, E. A. et al. Chromatin-IgG complexes activate B cells by dual engagement of IgM and Toll-like receptors. Nature 416, 603-607 (2002). https: / / doi.org / 10.1038 / 416603a

[0195] 8 Hennessy, E. J., Parker, A. E. & O'Neill, L. A. Targeting Toll-like receptors: emerging therapeutics? Nat Rev Drug Discov 9, 293-307 (2010). https: / / doi.org / 10.1038 / nrd3203

[0196] 9 Guiducci, C. et al. TLR recognition of self nucleic acids hampers glucocorticoid activity in lupus. Nature 465, 937-941 (2010). https: / / doi.org / 10.1038 / nature09102

[0197] 10 Behrens, E. M. et al. Repeated TLR9 stimulation results in macrophage activation syndrome-like disease in mice. J Clin Invest 121, 2264-2277 (2011). https: / / doi.org / 10.1172 / JCI43157

[0198] 11 Mellins, E. D., Macaubas, C. & Grom, A. A. Pathogenesis of systemic juvenile idiopathic arthritis: some answers, more questions. Nat Rev Rheumatol 7, 416-426 (2011). https: / / doi.org / 10. 1038 / nrrheum.2011.68

[0199] 12 Nair, A., Wolter, T. R., Meyers, A. J. & Zipris, D. Innate immune pathways in virus- induced autoimmune diabetes. Ann N Y Acad Sci 1150, 139-142 (2008). https: / / doi.org / 10. 1196 / annals. 1447.004

[0200] 13 Bamboat, Z. M. et al. Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury. Hepatology 51, 621-632 (2010). https: / / doi.org / 10.1002 / hep.23365

[0201] 14 Ewald, S. E. et al. The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456, 658-662 (2008). https: / / doi.org / 10.1038 / nature07405

[0202] 15 Li, Y ., Berke, I. C. & Modis, Y. DNA binding to proteolytically activated TLR9 is sequence-independent and enhanced by DNA curvature. EMBO .731 . 919-931 (2012). https: / / doi.org / 10.1038 / emboj.2011.441 TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0203] 16 He, J. et al. Muncl3-4 interacts with syntaxin 7 and regulates late endosomal maturation, endosomal signaling, and TLR9-initiated cellular responses. Mol Biol Cell 27, 572-587 (2016). https: / / doi.org / 10.1091 / mbc.E15-05-0283

[0204] 17 Feldmann, J. et al. Muncl3-4 is essential for cytolytic granules fusion and is mutated in a form of familial hemophagocytic lymphohistiocytosis (FHL3). Cell 115, 461-473 (2003).

[0205] 18 Brzezinska, A. A. et al. The Rab27a Effectors JFCl / Slpl and Muncl3-4 Regulate Exocytosis of Neutrophil Granules. Traffic. 9, 2151-2164 (2008).

[0206] 19 Johnson, J. L., Hong, H., Monfregola, J., Kiosses, W. B. & Catz, S. D. MUNC13-4 restricts motility of RAB27A-expressing vesicles to facilitate lipopolysaccharide-induced priming of exocytosis in neutrophils. J Biol.Chem. 286, 5647-5656 (2010).

[0207] 20 Elstak, E. D. et al. The muncl3-4-rab27 complex is specifically required for tethering secretory lysosomes at the plasma membrane. Blood 118, 1570-1578 (2011).

[0208] 21 Neeft, M. et al. Muncl3-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells. Mol.Biol.Cell 16, 731-741 (2005).

[0209] 22 Johnson, J. L. et al. Muncl3-4 is a Rabi 1-binding protein that regulates Rabi 1 -positive vesicle trafficking and docking at the plasma membrane. The Journal of biological chemistry (2015). https: / / doi.org / 10.1074 / jbc.M115.705871

[0210] 23 Boswell, K. L. et al. Muncl3-4 reconstitutes calcium-dependent SNARE-mediated membrane fusion. J Cell Biol. 197, 301-312 (2012).

[0211] 24 Koch, H., Hofmann, K. & Brose, N. Definition of Munc 13 -homology-domains and characterization of a novel ubiquitously expressed Muncl3 isoform. Biochem.J. 349, 247-253 (2000).

[0212] 25 Huttlin, E. L. et al. Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 184, 3022-3040 e3028 (2021). https: / / doi.Org / 10.1016 / j.cell.2021.04.011

[0213] 26 Ameson, L. N. et al. Cutting edge: syntaxin 11 regulates lymphocyte-mediated secretion and cytotoxicity. J. Immunol. 179, 3397-3401 (2007).

[0214] 27 Menasche, G. et al. Mutations in RAB27A cause Griscelli syndrome associated with haemophagocytic syndrome. Nat.Genet. 25, 173-176 (2000).

[0215] 28 zur Stadt, U. et al. Linkage of familial hemophagocytic lymphohistiocytosis (FHL) type-4 to chromosome 6q24 and identification of mutations in syntaxin 11. Hum Mol Genet 14, 827-834 (2005). https: / / doi.org / 10.1093 / hmg / ddi076

[0216] 29 Zhang, K. et al. Macrophage activation syndrome in patients with systemic juvenile idiopathic arthritis is associated with MUNC13-4 polymorphisms. Arthritis Rheum 58, 2892-2896 (2008). https: / / doi.org / 10.1002 / art.23734

[0217] 30 Yanagimachi, M. et al. Association of IRF5 polymorphisms with susceptibility to macrophage activation syndrome in patients with juvenile idiopathic arthritis. J Rheumatol 38, 769-774 (2011). https: / / doi.org / 10.3899 / jrheum.100655 TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0218] 31 Gotoh, K. et al. Selective control of type I IFN induction by the Rac activator DOCK2 during TLR-mediated plasmacytoid dendritic cell activation. J Exp Med 207, 721-730 (2010). https: / / doi.org / 10.1084 / jem.20091776

[0219] 32 Johnson, J. L., Hong, H., Monfregola, J. & Catz, S. D. Increased survival and reduced neutrophil infdtration of the liver in Rab27a- but not Muncl3-4-deficient mice in lipopolysaccharide- induced systemic inflammation. Infect.Immun 79, 3607-3618 (2011).

[0220] 33 Riddle, S. M., Vedvik, K. L., Hanson, G. T. & Vogel, K. W. Time-resolved fluorescence resonance energy transfer kinase assays using physiological protein substrates: applications of terbiumfluorescein and terbium-green fluorescent protein fluorescence resonance energy transfer pairs. Anal.Biochem. 356, 108-116 (2006).

[0221] 34 Hu, L. A., Zhou, T., Hamman, B. D. & Liu, Q. A homogeneous G protein-coupled receptor ligand binding assay based on time-resolved fluorescence resonance energy transfer. Assay. Drug Dev.Technol. 6, 543-550 (2008).

[0222] 35 Yu, J. et al. Defective endomembrane dynamics in Rab27a deficiency impairs nucleic acid sensing and cytokine secretion in immune cells. Cell Rep 43, 114598 (2024). https: / / doi.Org / 10.1016 / j.celrep.2024.l 14598

[0223] 36 Maeda, T. et al. A novel plasmacytoid dendritic cell line, CAL-1, established from a patient with blastic natural killer cell lymphoma. Int J Hematol 81, 148-154 (2005). https: / / doi.org / 10.1532 / ijh97.04116

[0224] 37 Brzezinska, A. A. et al. The Rab27a Effectors JFCl / Slpl and Muncl3-4 Regulate Exocytosis ofNeutrophil Granules. Traffic (Copenhagen, Denmark) 9, 2151-2164 (2008). https: / / doi.org / 10. 111 l / j,1600-0854.2008.00838.x

[0225] 38 Zhang, J. et al. Cross-regulation of defective endolysosome trafficking and enhanced autophagy through TFEB in UNC13D deficiency. Autophagy, 1-19 (2019). https: / / doi.org / 10.1080 / 15548627.2019.1596475

[0226] 39 Johnson, J. L., Pestonjamasp, K., Kiosses, W. B. & Catz, S. D. Super-Resolution Microscopy and Particle-Tracking Approaches for the Study of Vesicular Trafficking in Primary Neutrophils. Methods Mol Biol 2233, 193-202 (2021). https: / / doi.org / 10.1007 / 978-l-0716-1044-2_13

[0227] 40 Bright, N. A., Davis, L. J. & Luzio, J. P. Endolysosomes Are the Principal Intracellular Sites of Acid Hydrolase Activity. Curr Biol 26, 2233-2245 (2016). https: / / doi.Org / 10.1016 / j.cub.2016.06.046

[0228] 41 Johnson, J. L. et al. Identification ofNeutrophil Exocytosis Inhibitors (Nexinhibs), Small Molecule Inhibitors ofNeutrophil Exocytosis and Inflammation: DRUGGABILITY OF THE SMALL GTPase Rab27a. J Biol Chem 291, 25965-25982 (2016). https: / / doi.org / 10.1074 / jbc.M116.741884

[0229] 42 Gotoh, K. et al. Selective control of type I IFN induction by the Rac activator DOCK2 during TLR-mediated plasmacytoid dendritic cell activation. Journal of Experimental Medicine 207, 721- 730 (2010). https: / / doi.org / 10.1084 / jem.20091776 TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0230] 43 Shah, P. & Westwell, A. D. The role of fluorine in medicinal chemistry. J Enzyme Inhib Med Chem 22, 527-540 (2007). https: / / doi.org / 10.1080 / 14756360701425014

[0231] 44 Gonzalez-Quintial, R. et al. Lupus acceleration by a MAVS-activating RNA virus requires endosomal TLR signaling and host genetic predisposition. PLoS One 13, e0203118 (2018). https : / / do i . org / 10. 1371 / j oumal .pone .0203118

[0232] 45 Ramadass, M., Johnson, J. L. & Catz, S. D. Rab27a regulates GM-CSF-dependent priming of neutrophil exocytosis. JLeukoc Biol (2016). https: / / doi.Org / 10.l 189 / jlb.3AB0416-189RR

[0233] 46 Logan, M. R. et al. A critical role for vesicle-associated membrane protein-7 in exocytosis from human eosinophils and neutrophils. Allergy 61, 777-784 (2006). https: / / doi.org / 10. 1111 / j.1398-9995 ,2006.01089.x

[0234] 47 Smolen, J. E., Hessler, R. J., Nauseef, W. M., Goedken, M. & Joe, Y. Identification and cloning of the SNARE proteins VAMP -2 and syntaxin-4 from HL-60 cells and human neutrophils. Inflammation 25, 255-265 (2001).

[0235] 48 Hodgetts, K. J., Combs, K. J., Elder, A. M. & Harriman, G. C. in Annual Reports in Medicinal Chemistry Vol. 45 (ed John E. Macor) 429-448 (Academic Press, 2010).

[0236] 49 Shah, P. & Westwell, A. D. The role of fluorine in medicinal chemistry. Journal of Enzyme Inhibition and Medicinal Chemistry 22, 527-540 (2007). https: / / doi.org / 10.1080 / 14756360701425014

[0237] 50 Ford, S. L. et al. Toll-like Receptor 9 Induced Dendritic Cell Activation Promotes Anti- Myeloperoxidase Autoimmunity and Glomerulonephritis. IntJMol Sci 24 (2023). https : / / doi. org / 10.3390 / ijms24021339

[0238] 51 Garcia-Martinez, I. et al. Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9. J Clin Invest 126, 859-864 (2016). https: / / doi.org / 10.1172 / JCI83885

[0239] 52 Alarcon, G. S. et al. Effect of hydroxychloroquine on the survival of patients with systemic lupus erythematosus: data from LUMINA, a multiethnic US cohort (LUMINA L). Ann Rheum Dis 66, 1168-1172 (2007). https: / / doi.org / 10.1136 / ard.2006.068676

[0240] 53 Rainsford, K. D., Parke, A. L., Clifford-Rashotte, M. & Kean, W. F. Therapy and pharmacological properties of hydroxychloroquine and chloroquine in treatment of systemic lupus erythematosus, rheumatoid arthritis and related diseases. Inflammopharmacology 23, 231-269 (2015). https: / / doi.org / 10.1007 / sl0787-015-0239-y

[0241] 54 Tynjala, P. et al. Aggressive combination drug therapy in very early polyarticular juvenile idiopathic arthritis (ACUTE-JIA): a multicentre randomised open-label clinical trial. Ann Rheum Dis IQ, 1605-1612 (2011). https: / / doi.org / 10.1136 / ard.2010.143347

[0242] 55 Wolfe, F. & Marmor, M. F. Rates and predictors of hydroxychloroquine retinal toxicity in patients with rheumatoid arthritis and systemic lupus erythematosus. Arthritis Care Res (Hoboken) 62, 775-784 (2010). https: / / doi.org / 10.1002 / acr.20133 TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0243] 56 Ruiz-Irastorza, G., Ramos-Casals, M., Brito-Zeron, P. & Khamashta, M. A. Clinical efficacy and side effects of antimalarials in systemic lupus erythematosus: a systematic review. Ann

[0244] Rheum Dis 69, 20-28 (2010). https: / / doi.org / 10.1136 / ard.2008.101766

[0245] 57 Wang, C. et al. Discontinuation of antimalarial drugs in systemic lupus erythematosus. J Rheumatol 26, 808-815 (1999).

[0246] 58 Kyburz, D., Brentano, F. & Gay, S. Mode of action of hydroxychloroquine in RA- evidence of an inhibitory effect on toll-like receptor signaling. Nat Clin Pr act Rheumatol 2, 458-459 (2006) . https: / / doi.org / 10.1038 / ncprheum0292

[0247] 59 Crozat, K. et al. Jinx, an MCMV susceptibility phenotype caused by disruption of Uncl3d: a mouse model of type 3 familial hemophagocytic lymphohistiocytosis. J.Exp.Med. 204, 853- 863 (2007).

[0248] 60 Grebenciucova, E. & VanHaerents, S. Interleukin 6: at the interface of human health and disease. Front Immunol 14, 1255533 (2023). https: / / doi.org / 10.3389 / fimmu.2023.1255533

[0249] 61 Choy, E. H. et al. Translating IL-6 biology into effective treatments. Nat Rev Rheumatol 16, 335-345 (2020). https: / / doi.org / 10.1038 / s41584-020-0419-z

[0250] 62 Morris, E. C., Neelapu, S. S., Giavridis, T. & Sadelain, M. Cytokine release syndrome and associated neurotoxicity in cancer immunotherapy. Nat Rev Immunol 22, 85-96 (2022). https: / / doi.org / 10.1038 / s41577-021-00547-6

[0251] 63 Yang, S. et al. Neutrophil activation and clonal CAR-T re-expansion underpinning cytokine release syndrome during ciltacabtagene autoleucel therapy in multiple myeloma. Nat Commun 15, 360 (2024). https: / / doi.org / 10.1038 / s41467-023-44648-3

[0252] 64 Leclercq, G. et al. Dissecting the mechanism of cytokine release induced by T-cell engagers highlights the contribution of neutrophils. Oncoimmunology 11, 2039432 (2022). https: / / doi.org / 10.1080 / 2162402X.2022.2039432

[0253] 65 Si, S. & Teachey, D. T. Spotlight on Tocilizumab in the Treatment of CAR-T-Cell- Induced Cytokine Release Syndrome: Clinical Evidence to Date. Ther Clin Risk Manag 16, 705-714 (2020). https: / / doi.org / 10.2147 / TCRM.S223468

[0254] 66 Le, R. Q. et al. FDA Approval Summary: Tocilizumab for Treatment of Chimeric Antigen Receptor T Cell -Induced Severe or Life-Threatening Cytokine Release Syndrome. Oncologist 23, 943-947 (2018). https: / / doi.org / 10.1634 / theoncologist.2018-0028

[0255] 67 Xu, X. et al. Effective treatment of severe COVID-19 patients with tocilizumab. Proc Natl Acad Sci USA 117, 10970-10975 (2020). https: / / doi.org / 10.1073 / pnas.2005615117

[0256] 68 Rosas, I. O. et al. Tocilizumab in Hospitalized Patients with Severe Covid-19 Pneumonia. N Engl JMed 384, 1503-1516 (2021). https: / / doi.org / 10.1056 / NEJMoa2028700

[0257] 69 Rossi, B. et al. Effect of Tocilizumab in Hospitalized Patients with Severe COVID-19 Pneumonia: A Case-Control Cohort Study. Pharmaceuticals (Basel) 13 (2020). https : / / doi .org / 10.3390 / ph 13100317 TSRI 2232.1PC / TSR3185P VHPM 18350.058W01

[0258] 70 Johnson, J. L. et al. Differential dysregulation of granule subsets in WASH-deficient neutrophil leukocytes resulting in inflammation. Nature Communications 13 (2022). https: / / doi.org / ARTN 5529 10.1038 / s41467-022-33230-y

[0259] 71 Tabeta, K. et al. Toll-like receptors 9 and 3 as essential components of innate immune defense against mouse cytomegalovirus infection. Proc Natl Acad Sci USA 101, 3516-3521 (2004). https: / / doi.org / 10.1073 / pnas.0400525101

[0260] 72 Welsh, R. M. & Seedhom, M. O. Lymphocytic choriomeningitis virus (LCMV): propagation, quantitation, and storage. Curr Protoc Microbiol Chapter 15, Unit 15A 11 (2008). https: / / doi.org / 10.1002 / 9780471729259.mcl5a01s8

[0261] 73 Battegay, M. et al. Quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24- or 96-well plates. J Virol Methods 33, 191-198 (1991). https: / / d0i.0rg / l 0. 1016 / 0166-0934(91)90018-u

[0262] 74 Klempner, M. S., Mikkelsen, R. B., Corfrnan, D. H. & ndre-Schwartz, J. Neutrophil plasma membranes. I. High-yield purification of human neutrophil plasma membrane vesicles by nitrogen cavitation and differential centrifugation. J Cell Biol. 86, 21-28 (1980).

[0263] 75 Johnson, J. L., Monfregola, J., Napolitano, G., Kiosses, W. B. & Catz, S. D. Vesicular trafficking through cortical actin during exocytosis is regulated by the Rab27a effector JFCl / Slpl and the RhoA-GTPase-activating protein Gem-interacting protein. Mol Biol Cell 23, 1902-1916 (2012). https: / / doi.org / 10.1091 / mbc.El l-12-1001

[0264] 76 Axelrod, D. Cell-substrate contacts illuminated by total internal reflection fluorescence. J Cell Biol. 89, 141-145 (1981).

[0265] All publications (e.g., patents and patent applications) cited herein are incorporated herein by reference in their entireties.

Claims

1. TSRI 2232.1PC / TSR3185P VHPM 18350.058W01CLAIMSWhat is claimed is:

1. A method of identifying a compound that disrupts a protein-protein interaction between Muncl3-4 and syntaxin 7 (STX7), which method comprises: measuring a first amount of fluorescence from a first mixture; measuring a second amount of fluorescence from a second mixture; and comparing the first and second amounts; wherein: the first mixture comprises the compound, an antibody, calcium, modified STX7, and Muncl3-4 bound to a peptide tag; the second mixture comprises the compound, the antibody, calcium, modified STX7, and Muncl3-4 lacking amino acid residues necessary for Ca2+binding; the modified STX7 is STX7 bound to a first fluorophore; the antibody recognizes the peptide tag and is bound to a second fluorophore; and the measured fluorescence is fluorescence from the first fluorophore, which fluorescence is caused by absorption of light by the second fluorophore.

2. The method of claim 1, wherein the first fluorophore is green fluorescent protein.

3. The method of claim 1 or 2, wherein the second fluorophore is a chelated lanthanide (e.g., chelated Tb).

4. A compound of the formula:A— L— B or a pharmaceutically acceptable salt thereof, wherein:A is an optionally substituted 5- to 10-membered carbocycle or heterocarbocycle, which optional substitution is with one or more R1;B is an optionally substituted 5- to 10-membered carbocycle or heterocarbocycle, which optional substitution is with one or more R2;L is -N=N-, -CH=CH-, -CH2-CH2-, -S-CH-, -CH-S-, O, -O-CH-, or -CH-O-; each R1is independently halo, OR1A, NR1AR1B, or optionally substituted C1-12 hydrocarbyl or C1-12 heterocarbyl which optional substitution is with one or more R1C;R1Ais H or C1-6 alkyl;R1Bis H or C1-6 alkyl; each R1Cis independently amino, halo, hydroxy, or oxo; each R2is independently halo, OR2A, NR2AR2B, or optionally substituted C1-12 hydrocarbyl or C1-12 heterocarbyl which optional substitution is with one or more R2C;TSRI 2232.1PC / TSR3185P VHPM 18350.058W01R2Ais H or Ci-6 alkyl;R2Bis H or Ci-6 alkyl; and each R2Cis independently amino, halo, hydroxy, or oxo.

5. The compound of claim 4, wherein A is optionally substituted phenyl, pyridyl, or 1H- imidazo [4, 5 -b]pyrazyl .

6. The compound of claim 5, wherein A is substituted with at least one R1.

7. The compound of any of claims 1-5, wherein B is optionally substituted phenyl.

8. The compound of claim 7, wherein B is substituted with at least one R2.

9. The compound of claim 1, which is of the formula:

10. The compound of any of claims 1-9, wherein each R1is independently halo, hydroxy, or Ci-3 alkyl.

11. The compound of claim 10, wherein at least one R1is bromo or fluoro.

12. The compound of any of claims 1-11, wherein each R2is independently OR2AorNR2AR2B.

13. The compound of claim 12, wherein R2Ais H or C1-3 alkyl.

14. The compound of claim 13, wherein R2Bis C1-3 alkyl.

15. The compound of claim 12, wherein at least one R2is N(CH2CH3)2.

16. The compound of any of claims 4-15, wherein L is -N=N-, -CH=CH-, or -CH2-CH2-.

17. A compound, which compound is: ethyl 4-(3-oxoisothiazol-2(3H)-yl)benzoate;1-(4-(4-chlorophenyl)-3-methylthiazol-2(3H)-ylidene)-3-phenethylthiourea;(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -(diethylamino)phenol;2-(2-undecyl-4,5-dihydro-lH-imidazol-l-yl)ethan-l-ol;N-allyl-2-((4-chlorobenzyl)thio)-7-methyl-5,6,7,8-tetrahydropyrido[4',3':4,5]thieno[2,3- d] pyrimidin-4-amine ; l-phenyl-3-(6-(3-(trifluoromethyl)phenoxy)pyridin-3-yl)urea;4-(4-bromophenyl)-l,3-dihydro-2H-imidazole-2-thione; l,4-dioxane-2,3-diyl bis(4-nitrobenzoate);1 -(benzo [d] [ 1 ,3]dioxol-5 -yloxy)-3 -(4-(6-chlorobenzo [d]thiazol-2-yl)- 1 ,4-diazepan- 1 -yl)propan-2- ol;TSRI 2232.1PC / TSR3185P VHPM 18350.058W01(E)-l-((4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)(ethyl)amino)-3,3-dimethylbutan-2- one;(E)-2-((4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)(ethyl)amino)-l-phenylethan-l-one;(E)-5-(diethylamino)-2-((5-fluoropyridin-2-yl)diazenyl)phenol;(E)-6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-ol;(E)-2-(2-(5-bromo-lH-imidazo[4,5-b]pyrazin-2-yl)vinyl)-5-(diethylamino)phenol;2-(5 -(6-bromopyridin-3 -yl)pyrazin-2-yl)-5 -(diethylamino)phenol;2-(2-(5-bromopyridin-2-yl)ethyl)-5-(diethylamino)phenol;2-(2-(5 -bromo-6-methylpyridin-2-yl)ethyl)-5 -(diethylamino)phenol;(E)-4-(2-(5-bromo-4-methylpyridin-2-yl)vinyl)-N,N-diethyl-3-methoxyaniline;2-(2-(3 -bromo-6-methylpyridin-2-yl)ethyl)-5 -(diethylamino)phenol; or a pharmaceutically acceptable salt thereof.

18. A compound, which compound is:(E)-2-((5 -(3 -cyclopropylprop-2-yn- 1 -yl)pyridin-2-yl)diazenyl)-5 -(diethylamino)phenol;(E)-5-(diethylamino)-2-((5-(5,5-dimethylcyclopent-l-en-l-yl)pyridin-2-yl)diazenyl)phenol;2-((E)-(5 -(( 1 S,4R)-2 -azabicyclo [2.2.2] oct-5 -en-2-yl)pyridin-2-yl)diazenyl)-5 - (diethylamino)phenol;(E)-5-(diethylamino)-2-((5-(2-(pent-4-yn-l-yloxy)ethyl)pyridin-2-yl)diazenyl)phenol;(S,E)-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)(spiro[2.3]hexan-l- yl)methanone;5 -(diethylamino)-2-((E)-(5 -(( 1 S,5R)-6,6-dimethylbicyclo [3.1.1 ]hept-2-en-3 -yl)pyridin-2- yl)diazenyl)phenol;((lS,6S)-bicyclo[4.1.0]heptan-7-yl)(6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin- 3-yl)methanone;(S,Z)-2-((6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3- yl)methylene)quinuclidin-3-ol;(6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)((2S,5S)-hexahydro-2,5- methanopentalen-3 a( 1 H) -yl)methanone ;(E)-N-((6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)methyl)-N- phenylmethanesulfonamide;(S,E)-2-((6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)methyl)-N-(3-hydroxy- 2,2-dimethylpropyl)butanamide ;(E)-N-((6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)methyl)-2- (phenylthio)acetamide ;5-(diethylamino)-2-((E)-(5-((E)-2-iodostyryl)pyridin-2-yl)diazenyl)phenol;(S,E)-2-cyclohexyl-2-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)acetamide;TSRI 2232.1PC / TSR3185P VHPM 18350.058W01(E)-N-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)-2, 2,3,3- tetramethylcyclopropane - 1 -carboxamide ;5-(diethylamino)-2-((E)-(5-((lR,5S)-l,8,8-trimethyl-3-azabicyclo[3.2.1]octan-3-yl)pyridin-2- yl)diazenyl)phenol ;(6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)methyl (2R,3as,5S,6as)- hexahydro-2, 5 -methanopentalene-3 a( 1 H)-carboxylate ;((lR,2R,4S)-bicyclo[2.2.1]heptan-2-yl)(6-((E)-(4-(diethylamino)-2- hydroxyphenyl)diazenyl)pyridin-3 -yl)methanone ;(2S,3S,4R,6R)-6-((6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)oxy)-4- methoxy-2,4-dimethyltetrahydro-2H-pyran-3-ol;(S,E)-5-(diethylamino)-2-((5-(((2-methylbutyl)thio)methyl)pyridin-2-yl)diazenyl)phenol;(lS,3S,6R)-3-((6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)methyl)-3- azabicyclo[4.

2. l]nonan-3-ium;(E)-6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)nicotinaldehyde O-isobutyl oxime;5-(diethylamino)-2-((E)-(5-((3aR,7aR)-3a,4,5,6,7,7a-hexahydrobenzo[d]isoxazol-3-yl)pyridin-2- yl)diazenyl)phenol ;(E)-2-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)nicotinoyl)-4-methylthiazole-5-carboxylic acid;(E)-2-(2-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)thiazol-5-yl)-2- methylpropanoic acid;(E)-5-(diethylamino)-2-((5-((thiophen-2-ylmethoxy)methyl)pyridin-2-yl)diazenyl)phenol;(E)-l-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)-N- phenylmethanesulfonamide ;(lR,3r,5S,8r)-3-((6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)nicotinoyl)oxy)-8-methyl- 8-azabicyclo[3.

2. l]octan-8-ium;(E)-4-((6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)amino)-2, 2,6,6- tetramethylpiperidin- 1 -ol;(R,E)-5-(diethylamino)-2-((5-((4,4-dimethylpentan-2-yl)oxy)pyridin-2-yl)diazenyl)phenol;1 -(6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3 -y 1) -3 -((E)- 1 -methylpyrrolidin- 2-ylidene)urea;(E)- 1 -(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3 -y 1) -3 -(methylsulfonyl)propan- 1 - one;(E)-3 -(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3 -yl)propyl (furan-2- ylmethyl)carbamate;(E)-3-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)-N-(6- hydroxyhexyl)propenamide ;TSRI 2232.1PC / TSR3185P VHPM 18350.058W01(S,E)-l-acetyl-N-((6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3- yl)methyl)pyrrolidine-2-carboxamide;(E)-N-((6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)methyl)-N- isopropylcyclohexanecarboxamide;(E)-2-((5-(2-(benzofuran-4-yl)ethyl)pyridin-2-yl)diazenyl)-5-(diethylamino)phenol;(E)-5-(diethylamino)-2-((5-(2-(5-methoxybenzo[b]thiophen-3-yl)ethyl)pyridin-2- yl)diazenyl)phenol ;(E)-2-(((6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)methyl)thio)-N,N- dimethylbenzamide ;(E)-l-(3-chlorophenyl)-3-(6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3- yl)propan-l-one oxime; methyl (E)-(cyclopentylmethyl)(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3- yl)carbamate;(E)-5-(diethylamino)-2-((5-(2-(2,3-dihydrobenzofuran-7-yl)ethyl)pyridin-2-yl)diazenyl)phenol;(E)-l-((6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)amino)-4-((furan-2- ylmethyl)thio)butan-2-one ;(E)-l-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)-N-(pyridin-4- ylmethyl)methanesulfonamide;(E)- 1 -(2-bromophenyl)-2-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3 -yl)ethan- 1 - one;(E)-2-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3-yl)-3-methyl-4H-chromen-4- one;(E)-2-(6-((4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3 -yl)- 1 -(3 ,4-dihydroquinolin-1 (2H)-yl)ethan- 1 -one ;(E)-2-((5 -(2-(4H-imidazo [4,5 -b]pyridin-4-yl)ethyl)pyridin-2-yl)diazenyl)-5 - (diethylamino)phenol;(3aS,6aS)-2-(3-(6-((E)-(4-(diethylamino)-2-hydroxyphenyl)diazenyl)pyridin-3- yl)propyl)tetrahydrocyclopenta[c]pyrrole-l,3(2H,3aH)-dione;(E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(cyclopropylethynyl)phenol;(S,E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(l-(methoxyamino)ethyl)phenol;2-((E)-(5-bromopyridin-2-yl)diazenyl)-5-((ls,3s)-3-(hydroxymethyl)cyclobutyl)phenol;(S,E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(hexan-2-yl)phenol;S,E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(3-hydroxy-2-methylbutan-2-yl)phenol;(E)-3 -(4-((5 -bromopyridin-2-yl)diazenyl)-3 -hydroxyphenyl)-N-hydroxypropanamide ;(R,E)-3-(4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxybenzyl)pyrrolidin-l-ium;(E)-3-(4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)-N-methylcyclobutan-l-aminium;(E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(cyclopentylethynyl)phenol;TSRI 2232.1PC / TSR3185P VHPM 18350.058W01(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -( 1 -(methyl (prop-2-yn- 1 -yl)amino)vinyl)phenol;(E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(phenylethynyl)phenol;(E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(5-hydroxy-2-methylpentan-2-yl)phenol;(E)-6-(4-((5 -bromopyridin-2-yl)diazenyl)-3 -hydroxyphenyl)hex-5 -yn- 1 -aminium;(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -(6-hydroxyhex- 1 -yn- 1 -yl)phenol;(E)-N-(3-(4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)prop-2-yn-l-yl)acetamide;(S,E)-N-(4-(4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)but-3-yn-2-yl)acetamide;(E)-2-((5-bromopyridin-2-yl)diazenyl)-5 -(3 -(2 -methoxyethoxy )prop-l-yn-l-yl)phenol;(3aR,6aS)-5-(4-((E)-(5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)octahydro- cyclopenta[c]pyrrol-2-ium;(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -( 1 -ethynylcyclohexyl)phenol;(E)-l-(3-(4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)cyclobutyl)azetidin- 1-ium;(R,E)-4-((4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)amino)azepan-l-ium;(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -(4-(prop- 1 -yn- 1 -yl)thiophen-3 -yl)phenol;(E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(l-(furan-2-yl)-2-methylpropan-2-yl)phenol;(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -(3 -hydroxyphenethyl)phenol;(E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(2-(pyrimidin-4-ylamino)ethyl)phenol;(E)-2-(4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)ethane-l-sulfonohydrazide;(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -(spiro [3.5]nonan-7 -yl)phenol;5 -(( 1 r,5r)-bicyclo [3.3.1 ]nonan- 1 -yl)-2-((E)-(5 -bromopyridin-2-yl)diazenyl)phenol;(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -( 1 -(prop-2 -yn- 1 -yl)piperidin-4-yl)phenol;2-((E)-(5-bromopyridin-2-yl)diazenyl)-5-((2s,3aR,5s,6aS)-hexahydro-2,5-methanopentalen- 2(lH)-yl)phenol;(S,E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(indolin-2-yl)phenol;(R,E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -(3 -hydroxy-3 -( 1 -hydroxy cyclobutyl)prop- 1 -yn- 1 - yl)phenol;(E)-2-((5 -bromopyridin-2-yl)diazenyl)-5 -(3 -(phenylamino)prop- 1 -yn- 1 -yl)phenol;(S,E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(3-hydroxy-3-phenylprop-l-yn-l-yl)phenol;(E)-4'-((5-bromopyridin-2-yl)diazenyl)-3'-hydroxy-4-methoxy-[l,r-biphenyl]-2 -carbonitrile;(S,E)-l-(4-(4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)but-3-yn-2-yl)-3-methylurea;(E)-5 -( 1 -( 1 -aminoethyl)piperidin-4-yl)-2-((5 -bromopyridin-2-yl)diazenyl)phenol;2-((E)-(5-bromopyridin-2-yl)diazenyl)-5-((lS,2S)-2-propylcyclohexyl)phenol;(R,E)-2-(4-((5-bromopyridin-2-yl)diazenyl)-3-hydroxyphenyl)-2,3-dihydro-lH-inden-4-ol;(S,E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(4-(l-hydroxyethyl)piperidin-l-yl)phenol;(E)-2-((5-bromopyridin-2-yl)diazenyl)-5-(4-fluorobicyclo[2.2.2]octan-l-yl)phenol;(Z)-2-(2-(5-bromopyridin-2-yl)-2-fluorovinyl)-5-(diethylamino)phenol;(Z)-2-(2-(5-bromopyridin-2-yl)-2-fluorovinyl)-5-(diethylamino)phenol;TSRI 2232.1PC / TSR3185P VHPM 18350.058W01(Z)-2-(2-(5-bromopyridin-2-yl)-2-hydroxyvinyl)-5-(diethylamino)phenol; or is a pharmaceutically acceptable salt thereof.

19. A pharmaceutical composition comprising a compound of any of claims 4-18 and a pharmaceutically acceptable excipient.

20. A method of disrupting a protein-protein interaction between Muncl3-4 and syntaxin 7 (STX7) in a cell, which method comprises contacting the cell with a compound of any of claims 4-18.

21. A method of inhibiting TLR3, TLR7, or TLR9 in a cell, which comprises contacting the cell with a compound of any of claims 4-18.

22. A method of inhibiting endolysosomal flux and decreasing endolysosomal cargo degradation in a cell, which comprises contacting the cell with a compound of any of claims 4-18.

23. A method of decreased nucleic acid-induced ERK signaling in neutrophils and IRF signaling in pDCs in a cell, which comprises contacting the cell with a compound of any of claims 4-18.

24. A method of reduced nucleic acid-induced systemic inflammation in a patient, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of any of claims 4-18 or a pharmaceutical composition of claim 19.

25. The method of claim 24, wherein the systemic inflammation is manifested as decreased production and release of MPO, IL-6, or IFN-y.

26. A method of treating inflammation associated with or caused by nonalcoholic steatohepatitis (NASH) or cytokine release syndrome (CRS), which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of any of claims 4-18 or a pharmaceutical composition of claim 19.

27. A method of treating inflammation associated with or caused by an autoimmune disease, which comprises administering to a patient in need thereof a therapeutically effective amount of a compound of any of claims 4-18 or a pharmaceutical composition of claim 19.

28. The method of claim 27, wherein the autoimmune disease is ischemia-reperfusion injury, juvenile idiopathic arthritis (JIA), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), or type 1 diabetes (TIB).

Images