Use of ZLN-005 and related compounds

Abstract

Methods of treating diseases and conditions associated with impaired lysosomal acidification, methods of increasing lysosomal acidity, and methods of treating sepsis, infections, and diseases and conditions associated with V-ATPase dysfunction using ZLN-005 and other compounds of formula (I) and salts, hydrates, deuterated analogs, and fluorinated analogs thereof. (I)

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 426,426, filed on November 18, 2022, and U.S. Provisional Application No. 63 / 482,384, filed on January 31, 2023, the contents of which are incorporated herein by reference in their entirety. Background Art

[0003] Impaired lysosomal acidification can lead to impaired phagocyte function. Impaired lysosomal acidification and impaired phagocyte function are associated with many diseases and conditions, including some neurodegenerative diseases, diseases and conditions related to V-ATPase dysfunction, sepsis and infection (Colacurio and Nixon, 2016, Ageing Rev. 2016 32:75-88; Chiswick et al., 2015, J. Immunol. 195(8):3793-802; Danikas et al., 2008, Clinical and Experimental Immunology 154:87-97).

[0004] New therapies are needed for the treatment of diseases and disorders associated with impaired lysosomal acidification. Summary of the Invention

[0005] This disclosure is based in part on the discovery that 2-(4-tert-butylphenyl)-1H-benzimidazole (ZLN-005; also known as TQS-168), an activator of Ppargc1α (PGC-1α) expression, enhances phagocytic activity of phagocytes in vitro, promotes lysosomal acidification in vitro, and reduces mortality in an in vivo sepsis model, in which mortality is known to be associated with impaired lysosomal acidification. ZLN-005 has the following structure:

[0006]

[0007] The trials indicate that ZLN-005 and related compounds may be used to increase lysosomal acidification, for example, in phagocytes (e.g., monocytes) in a subject, and (without being bound by theory) may be used to treat diseases and conditions associated with impaired lysosomal acidification, such as sepsis and infections associated with suppressed innate immunity, as well as diseases and conditions associated with V-ATPase dysfunction.

[0008] Thus, in one aspect, the present disclosure provides a method of treating a subject having a disease or condition associated with impaired lysosomal acidification, comprising administering to the subject a therapeutically effective amount of an agent that is ZLN-005 or other compound of Formula (I):

[0009]

[0010] Formula (I)

[0011] or a salt, hydrate, deuterated analog or fluorinated analog thereof, wherein:

[0012] Ar is , , ,or ;

[0013] W 1 NR 1 , O or S, or when W 9 When N, W 1 Can be CR 50 ;

[0014] W 2 CR 2 or N;

[0015] W 3 CR 3 or N;

[0016] W 4 CR 4 or N;

[0017] W 5 CR 5 or N;

[0018] W 6 CR 6 or N;

[0019] W 7 CR 7 or N;

[0020] W 8 CR 8 or N;

[0021] W 9 is C, or when W 1 CR 50 , W 9 Can be N;

[0022] R 1 H, (C1-C3) alkyl, CH2OC(=O)R 30 、CH2OP(=O)OR 40 OR 41 、C(=O)OR 42 , or C(=O)R 43 ;

[0023] R 2 、R 3 、R 4 and R 5 each independently represents hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C1-4)alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxyl, (C1-C4)alkoxycarbonylamino, formamido, (C1-C4)alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, mercapto, (C1-C4)alkylthio, aminosulfonyl, (C1-C4)alkylsulfonyl, or (C1-C4)acylamino;

[0024] R 6 and R 10 are each independently hydrogen, deuterium, halogen, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino;

[0025] R 7 and R 9 are independently hydrogen, deuterium, hydroxyl, cyano, amino, halogen, halogenated (C1-C4) alkyl, (C1-C4) alkoxy, halogenated (C1-C4) alkoxy, ,or ;

[0026] R 8 is hydrogen, deuterium, halogen, halo(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, ,or ;

[0027] R 30 (C1-C 10 ) alkyl, substituted with amino (C1-C 10 )alkyl, (C1-C4)alkyl substituted 10 ) alkyl, substituted with carboxyl (C1-C 10 )alkyl, carboxyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkoxycarbonylamino, methylthio, heterocyclic, (C1-C 10 )Oxaalkyl, CHR 44 NHR 45 and guanidine;

[0028] R 40 and R41 are each independently hydrogen or (C1-C6) hydrocarbon;

[0029] R 42 is (C1-C5)alkyl;

[0030] R 43 is (C1-C3) alkyl,

[0031] R 44 are naturally occurring amino acid side chains;

[0032] R 45 is H, methyl or (C1-C4)alkoxycarbonyl; and

[0033] R 50 is H or (C1-C3) alkyl.

[0034] In another aspect, the present disclosure provides a method for increasing lysosomal acidity in a subject, comprising administering to the subject an effective amount of an agent to increase lysosomal acidity in the subject, wherein the agent is a compound of Formula (I) (e.g., ZLN-005) or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0035] In another aspect, the present disclosure provides a method for treating a subject suffering from sepsis, infection, or a disease or condition associated with V-ATPase dysfunction, comprising administering to the subject a therapeutically effective amount of an agent, wherein the agent is a compound of Formula (I) (e.g., ZLN-005) or a salt, hydrate, deuterated analog, or fluorinated analog thereof. In some embodiments, the sepsis is polymicrobial sepsis. In other embodiments, the sepsis is monomicrobial sepsis.

[0036] Further features of the compounds of formula (I) and their salts, hydrates, deuterated analogs, and fluorinated analogs are described in Section 5.2 and in the embodiments numbered 1 to 3 and 90 to 103 below.

[0037] The compound of formula (I) and its salts, hydrates, deuterated analogs and fluorinated analogs can be administered in the form of a pharmaceutical composition comprising the compound of formula (I) or its salt, hydrate, deuterated analog or fluorinated analog. Exemplary features of the pharmaceutical composition are described in Section 5.3 below.

[0038] Further features of the disclosed methods are described in Section 5.4 and in the embodiments numbered 1 to 106 below. 4. Brief Description of the Figures

[0040] Figures 1A-1DShown is a study timeline of a cecal ligation and puncture (CLP) model of sepsis in C57BL / 6 mice, which were intraperitoneally administered with vehicle (DMSO) or ZLN-005 ( Figure 1A ) for treatment; Kaplan-Meier survival ratio of mice in the CLP sepsis model treated with ZLN-005 or vehicle (n=9) ( Figure 1B ); Pgc1α and Tfam mRNA expression 24 hours after CLP (n=6) ( Figure 1C ); and mRNA expression of proinflammatory cytokines 24 hours after CLP (n=6) ( Figure 1D ) (Example 1 and Example 5). P value ** < 0.0001, P value * 0.0001 to 0.05.

[0041] Figure 2 The phagocytic activity of macrophages treated with ZLN-005 is shown (Example 2).

[0042] Figure 3 Light microscopy images (upper row) and fluorescence microscopy images using a pH-sensitive dye (lower row) of macrophages treated with ZLN-005 or vehicle (DMSO) are shown (Example 3).

[0043] Figure 4 Lysosomal acidity of macrophages treated with ZLN-005 or vehicle (DMSO) is shown (Example 3).

[0044] Figure 5 Shown are optical microscopy images (upper row) and fluorescence microscopy images (lower row) of peritoneal cells collected from CLP sepsis model mice treated with ZLN-005 or vehicle (DMSO) after incubation with fluorescent beads (Example 4).

[0045] Figure 6 The relative phagocytic activity of peritoneal cells collected from CLP sepsis model mice treated with ZLN-005 or vehicle (DMSO) is shown (Example 4).

[0046] Figure 7 Shown are optical microscopy images (upper row) and fluorescence microscopy images using a pH-sensitive dye (lower row) of peritoneal cells collected from CLP sepsis model mice treated with ZLN-005 or vehicle (DMSO) (Example 4).

[0047] Figure 8 Lysosomal acidity of peritoneal cells collected from CLP sepsis model mice treated with ZLN-005 or vehicle (DMSO) is shown (Example 4).

[0048] Figure 9 Shown are peritoneal bacterial levels from CLP sepsis model mice treated with ZLN-005 or vehicle (DMSO) (Example 4).

[0049] Figures 10A-10C The organ protective effect of ZLN-005 in the CLP sepsis model was demonstrated (Example 5). Figure 10A : Ejection fraction, left ventricular diameter, and left ventricular diastolic wall thickness 24 hours after CLP (n=4). Figure 10B : Histological scoring of mouse liver 24 hours after CLP (n=3). Figure 10C : Histological scoring of mouse lungs 24 hours after CLP (n=3). P values ​​*0.0001 to 0.05.

[0050] Figures 11A-11B The oxygen consumption rate (OCR) of THP-1 cells in an inflammation model measured using flux analysis is shown ( Figure 11A ) and extracellular acidification rate (ECAR) ( Figure 11B ) (Example 5). O: oligomycin, F: FCCP, A&R: antimycin and rotenone, G: glucose, 2-DG: 2-deoxy-D-glucose (n = 5). Glycolysis indicators were calculated based on ECAR measurements. Mitochondrial respiratory function indicators were calculated based on OCR, and glycolysis was calculated based on ECAR measurements. P values ​​**<0.0001, P values ​​*0.0001 to 0.05.

[0051] Figures 12A-12G Alterations of mitochondrial function by ZLN-005 were demonstrated (Example 5). Figure 12A : mRNA expression of PGC1α and TFAM in THP-1 cells in the inflammation model 24 h after LPS administration (n=3). Figure 12B : Absolute copy number of THP-1 in the inflammation model 24 hours after LPS administration (n=3). Figure 12C : MFI ratio of mtROS levels in THP-1 cells in the inflammation model 24 h after LPS administration (n = 3). Figure 12D : MFI ratios of acidic mKeimaRed signaling in THP-1 cells in the inflammation model 0, 4, 8, and 24 h after LPS administration (n = 3). Figure 12E : MFI ratio of Δφ of THP-1 cells in the inflammation model 24 hours after LPS administration (n=3). Figure 12F Mitochondrial morphology of THP-1 cells in an inflammation model 24 hours after LPS administration. All bars in the image are 20 μm. The figure shows the mitochondrial footprint, junction pixel, and flat pixel area for each cell (n = 5). Figure 12GEvaluation of mitochondrial-lysosomal contact sites in THP-1 cells in an inflammation model at 0, 1, 2, 8, and 24 hours after LPS administration. All bars in the images are 20 μm. The graph shows the ratio of contact site area to total mitochondrial area per cell (n = 5). P values ​​range from 0.0001 to 0.05.

[0052] Figures 13A-13H Alterations in lysosomal function caused by ZLN-005 were shown (Example 5). Figure 13A : MFI ratio of Tfeb mRNA expression and lysosome staining in peritoneal cells 24 hours after CLP (n=6). Figures 13B-13D : Phagocytosis of peritoneal cells 24 hours after CLP (n=6) ( Figure 13B ), lysosomal acidification ( Figure 13C ) and cellular ROS ( Figure 13D ) assessment. Figure 13E : Lysosomal preload of DQ in peritoneal cells 24 hours after CLP TM Degradation of BSA. The graph shows the MFI ratio of degraded DQ Green BSA and LysoTracker Red. Figures 13F-13G : Lysosomal hydrolases 24 hours after CLP ( Figure 13F ) and autophagy-related genes ( Figure 13G ) mRNA expression (n=6). Figure 13H THP-1 cells were exposed to LPS for 6 hours and then treated with autophagy inhibitors for 2 or 6 hours. Western blotting was used to analyze autophagic flux. The figure shows the ratio of LC3-II protein expression normalized to LC3-I protein expression (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine). **P value < 0.0001, *P value 0.0001 to 0.05.

[0053] Figures 14A-14E The effect of ZLN-005 on Tfeb is shown (Example 5). Figure 14A : Study design to evaluate Tfeb mRNA expression and lysosomal staining MFI ratio in peritoneal cells 24 hours after CLP (n=6). Figures 14B-14D : Evaluation of phagocytosis of peritoneal cells 24 hours after CLP (n=6) ( Figure 14B ), lysosomal acidification (C) and cellular ROS (D). ( Figure 14E ) Lysosomes in peritoneal cells preloaded with DQ 24 hours after CLP TM The degradation of BSA. The figure shows the degradation of DQ TM Green BSA and LysoTracker TMMFI ratios for Red. Figures 14F-14G: mRNA expression of lysosomal hydrolases (Figure 14F) and autophagy-related genes (Figure 14G) 24 hours after CLP (n = 6). Figure 14H: Western blot analysis of autophagic flux in THP-1 cells following exposure to LPS for 6 hours and subsequent treatment with autophagy inhibitors for 2 or 6 hours. The figure shows the ratio of LC3-II protein expression normalized to LC3-I protein expression (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001 to 0.05.

[0054] Figures 15A-15B Western blot analysis of molecular pathways in peritoneal cells 24 hours after CLP is shown ( Figure 15A ) and assessment of lysosomal acidification in THP-1 cells 5 days after wortmannin administration ( Figure 15B ) (Example 5). Figure 15A The graph in Figure 4 shows the expression ratio of each protein corrected by the expression level of Gapdh protein (n=3). Figure 15B The Kolmogorov-Smirnov test was used to evaluate the statistical significance of the differences between the two wortmannin-administered groups (with or without ZLN-005). P values ​​were ** < 0.0001 and * 0.0001 to 0.05.

[0055] Figure 16 A schematic diagram of the proposed mode of action of ZLN-005 in polymicrobial sepsis is shown. This schematic diagram is for illustrative purposes only and does not limit any aspect or embodiment disclosed herein to a particular mechanism.

[0056] Figures 17A-17D The effects of ZLN-005 or vehicle (DMSO) on peritoneal bacterial levels in CLP sepsis model mice are shown (Example 6). Figure 17A Schematic diagram of the study design. Figure 17B Figures 2 and 24 show the changes in bacterial colonies in the ascites of sham-treated, CLP- and vehicle-treated, and CLP- and ZLN-005-treated mice. Figure 17C Bar graph showing bacterial counts in the ascitic fluid of sham-treated, CLP and vehicle-treated, and CLP and ZLN-005-treated mice 2 hours after CLP. Figure 17D Figure 5 is a bar graph showing bacterial counts in the ascitic fluid of mice treated with sham surgery, CLP plus vehicle, and CLP plus ZLN-005 at 24 hours after CLP.

[0057] Figures 18A-18B The effect of ZLN-005 on v-ATPase assembly in the CLP mouse sepsis model was shown (Example 7). Figure 18A Design diagram for the study. Figure 18B are graphs showing Western blotting results, expressed as relative protein expression levels of the proteins indicated above each graph.

[0058] Figures 19A-19B The effect of TRPML1 inhibition on ZLN-005-mediated lysosomal acidification caused by LPS stimulation was shown (Example 8). Figure 19A Design diagram for the study. Figure 19B pHrod fluorescence levels in different treatment groups are shown. 5. Detailed Description of the Invention

[0060] The present disclosure provides novel uses of ZLN-005 and other compounds of Formula (I) (and their salts, hydrates, deuterated analogs, and fluorinated analogs), such as methods for treating subjects with impaired lysosomal acidification, methods for increasing lysosomal acidity in a subject, methods for treating subjects with sepsis (e.g., when the subject's innate immune function is suppressed, such as associated with impaired lysosomal acidification), methods for treating subjects with infections, and methods for treating subjects with diseases or conditions associated with V-ATPase dysfunction. Exemplary compounds of Formula (I) and their salts, hydrates, deuterated analogs, and fluorinated analogs are described in Section 5.2. Exemplary pharmaceutical compositions comprising compounds of Formula (I) and their salts, hydrates, deuterated analogs, and fluorinated analogs are described in Section 5.3. Exemplary features of the disclosed methods are described in Section 5.4.

[0061] 5.1 Definition

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For a comprehensive understanding of the terms used in this specification, the following definitions are provided.

[0063] As used in the specification and embodiments, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "agent" includes a plurality of agents, including mixtures thereof.

[0064] Unless otherwise indicated, the conjunction "or" should be used in its proper Boolean logical operator sense, encompassing both alternative selections (A or B, where the selection of A is mutually exclusive with B) and combined selections (A or B, where both A and B are selected). In some places in the text, the term "and / or" is used for the same purpose, but this should not be interpreted as implying that "or" is used to represent mutually exclusive selections.

[0065] The term "V-ATPase" refers to a vacuolar ATPase. V-ATPase acidifies the lumen of various organelles, including lysosomes, endosomes, and secretory vesicles, and plays a key role in their function (Colacurio and Nixon, 2016, Ageing Rev. 2016 32:75-88). V-ATPase comprises a V1 domain and a V0 domain. Each domain contains multiple subunits, some of which have tissue-specific isoforms. Genes encoding the V1 subunit include ATP6V1A, ATP6V1B1, ATP6V1B2, ATP6V1C1, ATP6V1C2, ATP6V1D, ATP6V1E1, ATP6V1E2, ATP6V1F, ATP6V1G1, ATP6V1G2, ATP6V1G3, and ATP6V1H. Genes encoding the V0 subunit include ATP6V0A1, ATP6V0A2, ATP6V0A3, ATP6V0A4, ATP6V0B, ATP6V0C, ATP6V0D1, ATP6V0D2, ATP6V0E1, and ATP6V0E2 genes.

[0066] The term "pathogenic mutation" refers to an alteration from the wild-type gene that is associated with a disease. A pathogenic mutation can be, for example, a point mutation (in which a single nucleotide change results in a codon encoding a different amino acid), a nonsense mutation (introducing a stop codon into the gene sequence), an insertion of one or more nucleotides, or a deletion of one or more nucleotides. The term "wild-type" refers to the gene sequence that predominates in a species (e.g., humans).

[0067] A therapeutically effective amount of an agent or composition is an amount sufficient to achieve the desired therapeutic effect, thus not requiring a cure or complete remission.

[0068] As used herein, the term "treating" and grammatical variations thereof include alleviating or ameliorating a disease or disorder, and / or signs or symptoms associated therewith, or slowing or arresting its progression. It will be understood that treating a disease or disorder does not require complete elimination of the disease, disorder or symptoms associated therewith, although this does not exclude such a possibility. The treatments of the present disclosure may be applied prophylactically (e.g., to a subject at risk of developing a disease or disorder associated with impaired lysosomal acidification), palliatively, or remedially. Prophylactic treatment may be administered to a subject prior to the onset of signs or symptoms, early in the onset of signs or symptoms (e.g., at the onset of initial signs and symptoms), or after signs or symptoms have been established. Prophylactic administration may be performed from several days to several years before the onset of symptoms.

[0069] 5.2 Compounds of formula (I)

[0070] The methods of the present disclosure comprise administering to a subject an amount of an agent which is a compound of formula (I):

[0071]

[0072] Formula (I)

[0073] or a salt, hydrate, deuterated analog or fluorinated analog thereof, wherein:

[0074] Ar is , , ,or ;

[0075] W 1 NR 1 , O or S, or when W 9 When N, W 1 Can be CR 50 ;

[0076] W 2 CR 2 or N;

[0077] W 3 CR 3 or N;

[0078] W 4 CR 4 or N;

[0079] W 5 CR 5 or N;

[0080] W 6 CR 6 or N;

[0081] W 7 CR 7 or N;

[0082] W 8 CR 8 or N;

[0083] W 9 is C, or when W 1 CR 50 , W 9 Can be N;

[0084] R 1 H, (C1-C3) alkyl, CH2OC(=O)R 30 、CH2OP(=O)OR 40 OR 41 、C(=O)OR 42 , or C(=O)R 43 ;

[0085] R 2 、R 3 、R 4 and R 5 each independently represents hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C1-4)alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxyl, (C1-C4)alkoxycarbonylamino, formamido, (C1-C4)alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, mercapto, (C1-C4)alkylthio, aminosulfonyl, (C1-C4)alkylsulfonyl, or (C1-C4)acylamino;

[0086] R 6 and R 10 are each independently hydrogen, deuterium, halogen, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino;

[0087] R 7 and R 9 are independently hydrogen, deuterium, hydroxyl, cyano, amino, halogen, halogenated (C1-C4) alkyl, (C1-C4) alkoxy, halogenated (C1-C4) alkoxy, ,or ;

[0088] R 8 is hydrogen, deuterium, halogen, halo(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, ,or ;

[0089] R 30 (C1-C 10 ) alkyl, substituted with amino (C1-C 10 )alkyl, (C1-C4)alkyl substituted 10 ) alkyl, substituted with carboxyl (C1-C 10 )alkyl, carboxyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkoxycarbonylamino, methylthio, heterocyclic, (C1-C 10 )Oxaalkyl, CHR 44 NHR 45 and guanidine;

[0090] R40 and R 41 are each independently hydrogen or (C1-C6) hydrocarbon;

[0091] R 42 is (C1-C5)alkyl;

[0092] R 43 is (C1-C3) alkyl,

[0093] R 44 are naturally occurring amino acid side chains;

[0094] R 45 is H, methyl or (C1-C4)alkoxycarbonyl; and

[0095] R 50 is H or (C1-C3) alkyl.

[0096] Compounds of formula (I) are further described in PCT Publication No. WO 2021 / 262617, the contents of which are incorporated herein by reference in their entirety.

[0097] Exemplary compounds of formula (I) include the following:

[0098] , , , , , , , , , , ,and .

[0099] In some embodiments, the agent is

[0100] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0101] In some embodiments, the agent is

[0102] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0103] In some embodiments, the agent is

[0104] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0105] In some embodiments, the agent is

[0106] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0107] In some embodiments, the agent is

[0108] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0109] In some embodiments, the agent is

[0110] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0111] In some embodiments, the agent is

[0112] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0113] In some embodiments, the agent is

[0114] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0115] In some embodiments, the agent is

[0116] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0117] In some embodiments, the agent is

[0118] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0119] In some embodiments, the agent is

[0120] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0121] In some embodiments, the agent is

[0122] or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0123] In some embodiments, the agent is a compound of Formula (I) (eg, one of the specific compounds of Formula (I), whose structures are shown in this section) or a salt thereof.

[0124] In some embodiments, the agent is ZLN-005. In other embodiments, the agent is a salt of ZLN-005.

[0125] 5.3 Pharmaceutical Composition

[0126] The compound of formula (I) and its salt, hydrate, deuterated analog and fluorinated analog can be prepared according to the expected route of administration, for example according to techniques known in the art (e.g., Allen et al., ed., 2012, Remington: The Science and Practice of Pharmacy, 22nd Edition, Pharmaceutical Press, London, UK). Suitable routes of administration include, but are not limited to, intravenous administration and oral administration. Suitable routes also include pulmonary administration, including inhalation administration. The most suitable route may depend on the subject's condition or disorder.

[0127] The compound of formula (I) and its salt, hydrate, deuterated analog and fluorinated analog can be formulated as a pharmaceutical composition comprising the compound of formula (I) or its salt, hydrate, deuterated analog or fluorinated analog and one or more pharmaceutical excipients, such as one or more excipients described in the Handbook of Pharmaceutical Excipients, 8th revised edition (2017), which is incorporated by reference in its entirety. The pharmaceutical composition can be in unit dosage form.

[0128] 5.4 Uses of the Compounds of Formula (I)

[0129] The present disclosure provides methods for treating a subject using a compound of formula (I) (e.g., ZLN-005) and salts, hydrates, deuterated analogs, and fluorinated analogs thereof. In the methods of the present disclosure, the subject is preferably a mammal (e.g., a primate or rodent, such as a mouse or rat), most preferably a human.

[0130] In one aspect, the present disclosure provides a method for treating a subject suffering from a disease or condition associated with impaired lysosomal acidification, the method comprising administering to the subject a therapeutically effective amount of an agent, wherein the agent is a compound of Formula (I) (e.g., ZLN-005) or a salt, hydrate, deuterated analog, or fluorinated analog thereof. The therapeutically effective amount of the agent can be, for example, an amount effective to increase lysosomal acidity in the subject. Diseases and conditions associated with impaired lysosomal acidification include sepsis (e.g., in subjects with intestinal perforation and / or intra-abdominal infection), infections (including bacterial infections, e.g., multidrug-resistant bacterial infections, fungal infections, parasitic infections, and viral infections), and diseases and conditions associated with V-ATPase dysfunction, such as renal tubular acidosis, Zimmermann-Laband syndrome, cutis laxa type II or wrinkled skin syndrome, osteopetrosis, glucose intolerance, diabetes, Parkinson's disease, Alzheimer's disease, and hearing loss.

[0131] In another aspect, the present disclosure provides a method of increasing lysosomal acidity in a subject, comprising administering to the subject an amount of an agent effective to increase lysosomal acidity in the subject, wherein the agent is a compound of Formula (I) (e.g., ZLN-005) or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0132] In another aspect, the present disclosure provides a method for treating a subject suffering from sepsis (particularly in a subject without systemic immune activation), an infection, or a disease associated with V-ATPase dysfunction, comprising administering to the subject a therapeutically effective amount of an agent, wherein the agent is a compound of Formula (I) (e.g., ZLN-005) or a salt, hydrate, deuterated analog, or fluorinated analog thereof. In some embodiments, the subject suffers from polymicrobial sepsis (e.g., caused by a combination of two or more bacteria, a combination of two or more fungi, or a combination of one or more bacteria and one or more fungi). In other embodiments, the subject suffers from monomicrobial sepsis (e.g., caused by a single pathogenic species (e.g., bacteria or fungi)). In some embodiments, the subject suffers from early-stage sepsis. Common (but not necessarily present) indicators of early-stage sepsis include fever (e.g., greater than 38°C), a heart rate greater than 90 beats per minute, infection confirmed by a positive blood culture, and a rapid respiratory rate exceeding 20 breaths per minute.

[0133] In some embodiments, when a subject has an infection (e.g., bacterial, fungal, viral, parasitic, or a combination thereof), administration of the agent can result in a reduction in pathogen load. For example, administration of the agent to a subject with a bacterial infection can reduce the subject's bacterial load. Methods for assessing pathogen (e.g., bacterial) load levels are known in the art. See, for example, Stranieri et al., 2018, Rev Inst Med Trop Sao Paulo. 60:e61.

[0134] The Sequential Organ Failure Assessment (SOFA) can be used to assess subjects, such as subjects with infection or sepsis (Vincent et al., 1996, Intensive Care Med 22(7):707-10). SOFA scores range from 0 to 24, with higher scores indicating higher mortality. In some embodiments, subjects treated according to the methods of the present invention prior to treatment with an agent of the present invention (e.g., ZLN-005) have a SOFA score of 0 to 6 (e.g., 0 to 3, 1 to 4, 2 to 5, 0, 1, 2, 3, 4, 5, or 6). In some embodiments, subjects prior to treatment with an agent of the present invention (e.g., ZLN-005) have a SOFA score of 0 to 2. In some embodiments, subjects prior to treatment with an agent of the present invention (e.g., ZLN-005) have a SOFA score of 2 to 6. In some embodiments, the subject treated according to the methods of the invention prior to treatment with an agent of the invention (e.g., ZLN-005) had a SOFA score of 7 to 9 (e.g., 7, 8, or 9). In some embodiments, the subject treated according to the methods of the invention prior to treatment with an agent of the invention (e.g., ZLN-005) had a SOFA score of 10 to 12 (e.g., 10, 11, or 12). In some embodiments, the subject treated according to the methods of the invention prior to treatment with an agent of the invention (e.g., ZLN-005) had a SOFA score of 13 to 14 (e.g., 13 or 14). In some embodiments, the subject treated according to the methods of the invention prior to treatment with an agent of the invention (e.g., ZLN-005) had a SOFA score of 15 to 24 (e.g., 15 to 20, 20 to 24, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24).

[0135] In some embodiments, the subject is infected with at least one pathogen and has one, two, three, or all four of the following: (1) HLA-DR expression levels in peripheral blood are lower than normal; (2) PD-1 expression levels in T cells are higher than normal; (3) CD88 expression levels in neutrophils are lower than normal, and (4) Th17 / Treg ratio is lower than normal. HLA-DR is closely related to the activation of immune system cells. It has been reported that HLA-DR expression in the peripheral blood of patients with sepsis is reduced compared with healthy individuals (Winkler et al., 2017, PLoS One 12:e0182427). In addition, it has been reported that when the frequency of monocytes expressing HLA-DR is less than 30%, immunity is suppressed (Misra et al., 2020, Crit Care Clin 36:167-176). Therefore, in some embodiments, the frequency of monocytes expressing HLA-DR in the subject is less than 30%. In T cells, enhanced expression of inhibitory signals such as programmed cell death 1 (PD-1) is associated with poor prognosis in patients with sepsis (Boomer et al., 2012, Crit Care 16: R112). Immune checkpoint inhibitors for the treatment of cancer also have significant effects in the treatment of sepsis models (Huang et al., 2022, Mol Ther 30: 1227-1238). This shows that Treg plays an important role in the immunosuppressive state of sepsis. In addition, the Th17 / Treg ratio is found to be a good indicator of immune system status (Gupta et al., 2016, Cytokine 88: 214-221). In a typical course of sepsis, the ratio increases in the early stages of sepsis and then decreases, and the decline suggests an immunosuppressive state, which is consistent with the decline of HLA-DR (Xu et al., 2020, Scand J Immunol 91: e12813). In neutrophils, activated complement C5a inhibits RhoA polymerization, inactivating it and leading to reduced expression of CD88, which has phagocytic function (Morris et al., 2011, Blood 117:5178-5188). It is reported that reduced CD88 expression can well reflect the state of immunosuppression and is closely related to secondary infection (Conway et al., 2018, Intensive Care Med 44:627-635).

[0136] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase lysosomal acidity in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject. Lysosomal acidity can be measured by a lysosomal pH assay, such as the Dojindo Lysosomal Acid pH Assay Kit (Dojindo Product No. L266).

[0137] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase lysosomal acidity in a peritoneal cell (eg, a peritoneal phagocyte (eg, a macrophage, monocyte, or neutrophil)) of the subject.

[0138] In the methods of the present disclosure, the amount of the medicament may be an amount that effectively increases Tfeb (transcription factor EB) mRNA levels in the phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject. In the methods of the present disclosure, the amount of the medicament may be an amount that effectively increases TFEB activation (dephosphorylation) in the phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject. In the methods of the present disclosure, the amount of the medicament may be an amount that effectively increases TFEB nuclear migration in the phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject.

[0139] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase the ratio of phosphorylated Akt (protein kinase B) to Akt (p-Akt / Akt) in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

[0140] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase the ratio of phosphorylated PI3K (phosphoinositide 3-kinase) to PI3K (p-PI3K / PI3K) in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

[0141] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase physical contact between mitochondria and lysosomes in a phagocyte (eg, a macrophage, monocyte, or neutrophil) of the subject.

[0142] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase spare respiratory capacity in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

[0143] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase the glycolytic capacity in the subject's phagocytic cells (eg, macrophages, monocytes, or neutrophils).

[0144] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase the glycolytic reserve in the subject's phagocytic cells (eg, macrophages, monocytes, or neutrophils).

[0145] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase lysosomal protein breakdown in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

[0146] In the methods of the present disclosure, the amount of the agent can be an amount effective to increase the mRNA level of a hydrolase (e.g., Ctsd (cathepsin D)) and / or a membrane protein (e.g., Atp6v1A, Atp6v0d1, or Mcoln1 (mucolipin-1)) in phagocytes (e.g., macrophages, monocytes, or neutrophils) of a subject.

[0147] In the methods of the present disclosure, the amount of the agent can be an amount effective to reduce the level of one or more inflammatory markers (e.g., in blood or serum). For example, the one or more inflammatory markers can include Tnfα, IL1β, IL6, IFNγ, or a combination thereof.

[0148] In some embodiments of the methods described herein, treating can include reducing or alleviating one or more symptoms of a disease or disorder experienced by the subject.

[0149] In some embodiments of the disclosed methods, the subject does not have systemic immune activation. Systemic immune activation may be associated with cytokine release syndrome (CRS). Thus, in some embodiments, the subject does not have CRS. Systemic immune activation and CRS are associated with elevated cytokines, including interleukin-6 (IL-6), interleukin-10 (IL-10), interferon (IFN)-γ, monocyte chemoattractant protein 1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), IL-1, IL-2, IL-2–receptor-α, IL-8, IL-17A, and IL-17F (see, e.g., Murthy et al., 2019 Immunotargets Ther. 8:43–52). Elevated C-reactive protein (CRP) is also observed in CRS. In some embodiments, the subject does not have elevated IL-6. In some embodiments, the subject does not have elevated IL-10. In some embodiments, the subject does not have elevated IFN-γ. In some embodiments, the subject does not have elevated MCP-1. In some embodiments, the subject does not have elevated GM-CSF. In some embodiments, the subject does not have elevated TNF. In some embodiments, the subject does not have elevated IL-1. In some embodiments, the subject does not have elevated IL-2. In some embodiments, the subject does not have elevated IL-2–receptor-α. In some embodiments, the subject does not have elevated IL-8. The subject does not have elevated IL-2. In some embodiments, the subject does not have elevated IL-17A. The subject does not have elevated IL-2. In some embodiments, the subject does not have elevated IL-17-F. In some embodiments, the subject does not have elevated CRP. In some embodiments, the subject does not have elevated D-dimer levels. Biomarker levels (such as those described in this paragraph) can be measured by standard laboratory tests. Biomarker levels are considered elevated when the measured value is above the upper limit of normal.

[0150] In some embodiments, the subject's pre-treatment serum IL-6 level is less than 200 pg / ml, 150 pg / ml, 100 pg / ml, 90 pg / ml, 80 pg / ml, 70 pg / ml, 60 pg / ml, 50 pg / ml, 40 pg / ml, 30 pg / ml, 20 pg / ml, 10 pg / ml, 5 pg / ml, 4 pg / ml, 3 pg / ml, or 2.5 pg / ml. In some embodiments, the subject's pre-treatment serum CRP level is less than 40 mg / L, 35 mg / L, 25 mg / L, 20 mg / L, 20 mg / L, 15 mg / L, 10 mg / L, 5 mg / L, or 2 mg / L.

[0151] In some embodiments of the disclosed methods, the subject's innate immune function is suppressed. Innate immune function can be measured by measuring the phagocytic capacity of the subject's phagocytes (e.g., macrophages). When the phagocytic capacity of the subject's phagocytes is lower than that of the phagocytes of a healthy subject, the subject's innate immune function is considered suppressed. Kits for measuring phagocytic activity are commercially available. An exemplary kit for measuring phagocytic activity is the Cayman Chemical phagocytic activity assay kit (Cayman Product No. 500290). In some embodiments, the amount of the administered agent is an amount effective to enhance the subject's innate immune function (e.g., as measured by phagocytic activity of phagocytes (e.g., macrophages)).

[0152] In some embodiments of the disclosed methods, the subject has sepsis (e.g., polymicrobial sepsis or monomicrobial sepsis) and (i) has suppressed innate immune function, (ii) does not have systemic immune activation, or (iii) has suppressed innate immune function and does not have systemic immune activation. In some embodiments, the subject has intestinal perforation.

[0153] In some embodiments of the disclosed methods, the subject suffers from bacterial infection, such as infection caused by multidrug-resistant bacteria. Exemplary multidrug-resistant bacteria include vancomycin-resistant enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), the gram-negative bacteria of extended-spectrum beta-lactamases (ESBL), the gram-negative bacteria of Klebsiella pneumoniae carbapenemase (KPC), and multidrug-resistant gram-negative bacteria (MDRGN), such as Enterobacter species, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. In other embodiments, the subject suffers from fungal infection (e.g., Candida albicans infection). In other embodiments, the subject suffers from parasitic infection (e.g., causing Plasmodium infection of malaria, or causing Trypanosoma cruzi infection of Chagas disease). In other embodiments, the subject suffers from viral infection (e.g., influenza). In some embodiments, the subject suffers from abdominal infection.

[0154] In some embodiments of the disclosed methods, the subject suffers from V-ATPase dysfunction (e.g., caused by pathogenic mutations in genes encoding V-ATPase subunits or V-ATPase accessory proteins) and / or a disease or condition associated with V-ATPase dysfunction. Exemplary diseases and conditions associated with V-ATPase dysfunction include renal tubular acidosis, Zimmermann-Laband syndrome, cutis laxa type II or rhytoid skin syndrome, osteosclerosis, glucose intolerance, diabetes, Parkinson's disease, Alzheimer's disease, and hearing loss. Pathogenic ATP6V1B1 and ATP6V0A4 mutations can lead to renal tubular acidosis (Uzak et al., 2013, Ren. Fail. 35(9):1281-1284; Stover et al., 2002, J. Med. Genet. 39(11):796-803). Pathogenic ATP6V1B2 mutations can cause Zimmermann-Laband syndrome (Kortum et al., 2015, Nat. Genet. 47(6):661-7). Pathogenic ATP6V0A2 mutations can cause cutis laxa type II or rhytoid skin syndrome (Kornak et al., 2008, Nat Genet. 40(1):32-4). Pathogenic ATP6V0A3 mutations can cause osteopetrosis and may also be associated with neurological complications (Bhargava et al., 2012, JBC 287(32):26829-26839; Steward, 2003, Neuropathol Appl Neurobiol. 29(2):87-97). ATP6V1H deficiency is associated with impaired glucose tolerance and diabetes (Yang et al., 2022, Arch Biochem Biophys. 716:109-116). Pathogenic mutations in ATP6AP2 (encoding a V-ATPase accessory protein) can cause Parkinson's disease, such as X-linked Parkinson's disease with spasticity (XPDS) (Korvatska et al., 2013, Hum Mol Genet. 22(16):3259-68). Pathogenic mutations in PSEN1 (encoding presenilin-1, a protein involved in V-ATPase targeting to lysosomes) are associated with Alzheimer's disease (Lee et al., 2015, Cell Rep. 12(9):1430-1444). Pathogenic mutations in DMXL2 (encoding Dmx-like 2, a protein involved in regulating V-ATPase activity) are associated with hearing loss (Chen et al., 2017, Genetics in Medicine 19:553-558).

[0155] In some embodiments of the disclosed methods, the subject does not have a neurodegenerative disease.

[0156] In the disclosed methods, the agent can be administered by any suitable route, such as enteral administration. In some embodiments, the agent is administered orally.

[0157] In some embodiments of the disclosed methods, the agent is administered at a dosage ranging from 0.5 mg / kg to 1000 mg / kg per day. In some embodiments, the dosage is from 25 mg / kg to 1000 mg / kg per day. Example

[0158] 6.1 Example 1: ZLN-005 rescues sepsis animals in the cecal ligation and puncture model

[0159] A study was conducted to evaluate the ability of ZLN-005 to rescue sepsis in mice in the cecal ligation and puncture (CLP) model.

[0160] C57BL / 6 mice, approximately 10 to 20 weeks old, were subjected to cecal ligation and puncture. At the time of cecal ligation and puncture, 24 hours later, and 48 hours later, mice were intraperitoneally injected with 12 mg / kg ZLN-005 or vehicle (DMSO). Figure 1A ).

[0161] Figure 1B Shown are the survival rates of mice given ZLN-005 or vehicle. ZLN-005 improved survival compared to vehicle.

[0162] 6.2 Example 2: ZLN-005 Enhances Macrophage Phagocytic Activity in Vitro

[0163] Studies were conducted to evaluate the ability of ZLN-005 to enhance the phagocytic activity of macrophages.

[0164] THP-1 cells were differentiated into macrophages by incubating with 10 nM phorbol 12-myristate 13-acetate (PMA) for 48 hours (study time 0 to 48 hours). The cells were then incubated with 1 μM ZLN-005 or vehicle (DMSO) for 5 days (study time 48 to 120 hours), with or without the addition of 1 μg / mL LPS on the final day (study time 96 to 120 hours). The cells were then incubated with latex beads for 20 minutes, and phagocytic activity was measured using a phagocytosis assay kit (IgG FITC) (Cayman Chemical, Product No. 500290).

[0165] The results are as follows Figure 2ZLN-005 treatment enhanced the phagocytic activity of LPS-stimulated macrophages (see Figure 2 right column chart).

[0166] 6.3. Example 3: ZLN-005 Increases Lysosomal Acidity in Macrophages in Vitro

[0167] Studies were conducted to evaluate the ability of ZLN-005 to increase lysosomal acidity in macrophages.

[0168] THP-1 cells were differentiated into macrophages by incubating with 10 nM phorbol 12-myristate 13-acetate (PMA) for 48 hours (study time 0 to 48 hours). The cells were then incubated with 1 μM ZLN-005 or vehicle (DMSO) for 5 days (study time 48 to 120 hours), with or without the addition of 1 μg / mL LPS on the final day (study time 96 to 120 hours). Lysosomal acidity was then stained using a pH-sensitive dye.

[0169] Microscope images of stained cells Figure 3 As shown. pH-sensitive dyes penetrate lysosomes according to pH, and fluorescence intensity increases with increasing acidity. ZLN-005 treatment was found to increase lysosomal acidity in LPS-stimulated cells (see Figure 3 The MFI ratios of the different treatment groups are shown in the lower right figure. Figure 4 shown.

[0170] 6.4. Example 4: ZLN-005 Enhances Phagocytic Activity and Lysosomal Acidity in Vivo

[0171] Studies were conducted to evaluate the ability of ZLN-005 to enhance the phagocytic activity of phagocytes in vivo.

[0172] Mice underwent cecal ligation and puncture at 0 hours after the start of the study. At the time of cecal ligation and puncture, mice were administered 12 mg / kg ZLN-005 (ip) or vehicle (DMSO). Twenty-four hours later, peritoneal cells were harvested by peritoneal lavage and assayed for phagocytic activity and lysosomal acidity. Peritoneal bacterial levels were also measured.

[0173] The results are as follows Figure 5-9 It was found that the phagocytic activity of phagocytes in mice treated with ZLN-005 was significantly increased compared with that in mice treated with vehicle (DMSO). Figure 5-6 ), lysosomal pH ( Figure 7-8 ) and bacterial killing ability ( Figure 9 ) have all improved.

[0174] 6.5. Example 5: ZLN-005 Improves Survival in Sepsis Patients

[0175] This example describes a study of ZLN-005 in sepsis. Some of the studies described in this example correspond to those described in previous examples, but this example includes more details regarding materials and methods, as well as a more detailed analysis of the study results.

[0176] Materials and methods

[0177] 6.5.1.1. Sepsis Model Mice

[0178] C57BL / 6 mice were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan). Mice were housed in a specific pathogen-free environment with free access to food and water. Mice were anesthetized by inhaled isoflurane (099-06571, FUJIFILM WakoPure Chemical Corporation, Tokyo, Japan). A midline incision was performed, followed by exteriorization. The cecum was ligated halfway between the distal end and the base of the cecum and punctured with a 21-gauge needle (NN-2116R, Terumo Corporation, Tokyo, Japan). Next, a small amount of feces was gently squeezed out of the punctured cecum to ensure puncture patency. The cecum was displaced, and the peritoneum and skin were sutured with 6 / 0 Ethicon PROLENE sutures (Ethicon, Inc., Raritan, NJ, USA). Mice in the sham-operated group underwent only the incision and exteriorization of the cecum.

[0179] 6.5.1.1. Intraperitoneal Injection of ZLN-005

[0180] A stock solution of ZLN-005 (S7447, Selleck Chemicals, Houston, TX, USA) was prepared in dimethyl sulfoxide (DMSO) (046-21981, FUJIFILM Wako Pure Chemical Corporation) and adjusted to a concentration of 10 mM. From day 0 to day 2, mice were injected daily with ZLN-005 (12 mg / kg) or an equivalent amount of DMSO as a control. Survival of mice was monitored continuously until 6 days after CLP.

[0181] 6.5.1.2. Isolation of Mouse Peritoneal Cells

[0182] The outer layer of peritoneal skin was cut open with scissors and gently pulled back to expose the skin lining the abdominal cavity. An 18G Surflo intravenous catheter (SR-FS1851, Terumo Corporation, Tokyo) was used to puncture the inner skin layer, and 4 ml of ice-cold PBS was injected. After injection, the abdomen was gently massaged to remove peritoneal cells. The collected fluid was filtered through a Falcon 40 μm cell strainer (352340, Corning Inc., Corning, NY, USA) and centrifuged at 800 × g for 5 minutes. The cell pellet was resuspended in 5 ml of 1× lysis buffer (555899, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) and incubated at room temperature for 5 minutes to lyse red blood cells. Then, 10 ml of PBS was added, and the cells were centrifuged at 800 × g for 5 minutes. The supernatant was discarded, and the cells were resuspended in PBS or culture medium.

[0183] 6.5.1.3. Analysis of peritoneal cell populations

[0184] Peritoneal cells were collected 24 hours after CLP. Nonspecific Fc receptors were blocked using Fc blocking reagent (130-059-901, Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes at room temperature, and then stained with PE anti-mouse / human CD11b antibody (101207, BioLegend, Inc., San Diego, CA, USA) and FITC anti-mouse F4 / 80 antibody (123108, BioLegend, Inc.) or PE rat IgG2b, k isotype control antibody (400608, BioLegend, Inc.) and FITC rat IgG2a, k isotype control antibody (400505, BioLegend, Inc.) for 30 minutes at 4°C. After staining, cells were immediately washed and resuspended in AutoMACS® running buffer (130-091-221, Miltenyi Biotec). Fluorescence data were collected using an SH800 cell sorter (Sony Biotechnology Inc., Tokyo, Japan). TM Flow cytometry data were analyzed using ELISA software (Ver. 10.8.1, Becton, Dickinson and Company).

[0185] 6.5.1.4. Echocardiographic measurements

[0186] One day before echocardiography, chest hair was removed with cream using a VisualSonics Vevo® 2100 (VisualSonics, Toronto, ON, Canada) equipped with an 18- to 38-MHz transducer. Twenty-four hours after CLP, mice were anesthetized by inhaled isoflurane. The left ventricle was assessed in the parasternal short-axis view. Left ventricular end-systole or end-diastole were defined as the period of minimal or maximal dilation of the left ventricular cavity, respectively. Left ventricular (LV) internal diameter at diastole, LV internal diameter at systole, anterior LV wall at diastole, and posterior LV wall at diastole were measured using LV M-mode tracings at the level of the papillary muscles. LV wall thickness was calculated as the average of the anterior and posterior wall thicknesses.

[0187] 6.5.1.5. Histology and Inflammatory Scoring

[0188] A study was conducted to evaluate the ability of ZLN-005 to rescue sepsis in mice in the cecal ligation and puncture (CLP) model.

[0189] The hearts, lungs, livers, right kidneys and spleens of mice were collected 24 hours after CLP and fixed with 4% paraformaldehyde (163-20145, FUJIFILM Wako Pure Chemical Corporation). All tissues were embedded in paraffin, sliced, and stained with hematoxylin-eosin (HE). According to the severity of liver necrosis, hemorrhage and infiltration, the liver inflammation score was assessed using the method described by Shikuma et al. (2022Front Immunol 13:825171). The lung inflammation score was assessed according to the severity of edema, alveolar cell infiltration, congestion and alveolar hemorrhage using the method described in An et al., 2019, Sci Rep 9:2836.

[0190] 6.5.1.6. Cell culture

[0191] THP-1 cells, a human monocytic leukemia cell line, were cultured in Roswell Park Memorial Institute 1640 medium (11875-093, RPMI 1640, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, 10270-106, Thermo Fisher Scientific Inc.). Cells were incubated at 37°C in a humidified incubator with 5% CO2. To establish the THP-1 inflammation model, cells were seeded in 12-well cell culture plates (353043, Corning Inc.) at a density of 5 × 10 cells per well in growth medium. 5Cells were grown in a growth medium containing 10 nM phorbol 12-myristate 13-acetate (PMA, AG-CN2-0010-M001, Adipogen Life Sciences Inc., San Diego, CA, USA). After 48 hours, the supernatant was carefully removed to prevent cells adhering to the bottom of the culture plate from falling off, and the culture medium was replaced with fresh medium containing 1 μM ZLN-005 or DMSO (0.1%) as a control. After 48 hours, 1 μg / ml LPS (lipopolysaccharide, 125-05181, FUJIFILM Wako PureChemical Corporation) was added, and cells were collected at each time point for study. For PI3K inhibition experiments, cells were seeded in culture plates and 200 nM wortmannin (AG-CN2-0023-M001, Adipogen LifeSciences Inc.) was added. For PGC1α gene knockdown, a Nucleofector was used. TM 2b (Lonza, Walkersville, MD, USA) cells were nucleofected with 10 nmole siRNA-PGC1α (4427037, Thermo Fisher Scientific Inc.) and 10 nmole siRNA-negative control (4390844, Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. For autophagic flux analysis, cells were stimulated with 1 μg / ml LPS for 6 h and then with 50 nM bafilomycin A1 (B0025, LKT Laboratories, Inc., Saint Paul, MN, USA) or 30 nM chloroquine (08660-04, Nacalai Tesque Inc., Kyoto, Japan) for 2 or 6 h.

[0192] 6.5.1.7. RNA Isolation, Reverse Transcription-PCR, and Quantitative PCR

[0193] Using TRIzol TM (15596018, Thermo Fisher Scientific Inc.) and Direct-zol TM Total RNA was extracted from cells and tissues using the RNA MiniPrep Kit (R2052, Zymo Research, Irvine, CA, USA) with DNase I according to the manufacturer's recommendations. For qRT-PCR, PrimeScriptTM Reverse transcription was performed on 100 ng of total RNA using the RT Reagent Kit (RR036A, Takara Bio, Shiga, Japan) and a T100 thermal cycler (Bio-Rad Laboratories, Inc.). qRT-PCR was performed using Kapa SYBR® Fast qPCR Kit Master Mix (2×) Universal (KK4602, Kapa Biosystems Ltd., Wilmington, MA, USA) on a CFX connect TM The assay was performed using a real-time system (Bio-Rad Laboratories, Inc.). Relative gene expression levels were normalized to GAPDH (or Gapdh) expression. The mtDNA copy number (CN) was estimated using the ratio of 12S rRNA on mtDNA to ACTB (or Actb) on nuclear DNA using a delta cycle threshold-based relative quantification method.

[0194] 6.5.1.8. Mitochondrial membrane potential (Δφ)

[0195] The cells were plated at 1×10 5 The density of 100 μg / ml was resuspended in 100 nM MitoTracker TM Green FM (MitoG, Thermo, M7514, Fisher Scientific, Inc.) and 100nM Image-iT TM The cells were incubated at 37°C for 30 minutes in a culture medium containing TMRM Reagent (TMRM, T668, Thermo Fisher Scientific, Inc.). Immediately after staining, the cells were washed, resuspended in AutoMACS® running buffer, and evaluated using an SH800 cell sorter. FlowJo was used to analyze the cells. TM Fluorescence intensity was analyzed, and the value was calculated by dividing the fluorescence intensity of TMRM by the fluorescence intensity of MitoG (TMRM / MitoG), which was used as an indicator of Δφ.

[0196] 6.5.1.9. Measurement of mitochondrial reactive oxygen species (mtROS) levels

[0197] The cells were plated at 1×10 5 The cells were resuspended in 5 μM MitoSOX at a density of / ml. TMIn the medium containing Red mitochondrial superoxide indicator (MitoSOX, M36008, Thermo Fisher Scientific, Inc.), incubate at 37°C for 30 minutes. After staining, wash the cells immediately, resuspend them in AutoMACS® running buffer, and evaluate using a MA900 cell sorter (Sony Biotechnology Inc., Tokyo, Japan).

[0198] 6.5.1.1. Measurement of cellular reactive oxygen species (ROS)

[0199] Resuspend the cells at a density of 1×10 5 / ml in the medium containing 5 μM CellROX TM Deep Red (CellROX, C10491, Thermo Fisher Scientific, Inc.), incubate at 37°C for 30 minutes. After staining, wash the cells immediately, resuspend them in AutoMACS® running buffer, and evaluate using a SH800 cell sorter.

[0200] 6.5.1.2. Mitophagy detection assay

[0201] To detect mitophagy, the pMX retroviral vector carrying monomeric Keima Red (mKeima Red) was transfected into THP-1 cells. One week after retroviral transfection, cells expressing mKeima Red were screened using a SH800 cell sorter. After re-sorting, more than 95% of the cells expressed mKeima Red. The acidic mKeima Red signal was detected by an Attune® NxT flow cytometer (Thermo Fisher Scientific). The lasers for mKeima Red were set at 488 nm (pH 7) and 561 nm (pH < 6), and the emission filters were 590 / 40 nm and 615 / 20 nm, respectively. The mitophagy index was defined as the ratio of the number of cells with positive acidic (<pH 6) mKeima Red signal at hour 0 to the DMSO control cells (Suzuki et al., 2017, Biochemical and Biophysical Research Communications 483:88-93).

[0202] 6.5.1.3. Phagocytosis assay

[0203] Phagocytosis was assessed using the IgG-FITC phagocytosis assay kit (500290, Cayman Chemical, Ann Arbor, MI, USA). Cells were plated at 3 × 10 5 Cells were suspended in 1 ml of culture medium at a concentration of 1:1 and stained with the latex beads-rabbit IgG-FITC complex provided in the kit for 20 minutes at 37°C. After staining, cells were centrifuged at 400 x g for 5 minutes, resuspended in 200 μl of autoMACS® running buffer, and evaluated using an SH800 cell sorter.

[0204] 6.5.1.4. Lysosomal acidification

[0205] Using pHrodo TM Lysosomal acidification was assessed using Green dextran (P35368, Thermo Fisher Scientific, Inc.). Peritoneal cells were suspended in 1 ml of culture medium at a concentration of 3 × 10 5 , using 50 μg / ml pHrodo TM Green dextran staining was performed at 37°C for 20 minutes. After staining, cells were centrifuged at 400 × g for 5 minutes, resuspended in 200 μl of autoMACS® running buffer, and evaluated using an SH800 cell sorter.

[0206] 6.5.1.5. Lysosomal staining

[0207] LysoTracker for lysosomes TM Red DND-99 (LysoTracker Red, L7528, Thermo Fisher Scientific, Inc.) staining was performed. The cells were plated at 3 × 10 5 The concentration was suspended in 1 ml of culture medium and 50 nM LysoTracker TM Red staining was performed for 15 minutes at 37°C. After staining, cells were centrifuged at 400 × g for 5 minutes, resuspended in 200 μl of autoMACS running buffer, and evaluated using an SH800 cell sorter.

[0208] 6.5.1.6. Lysosomal proteolysis

[0209] Cells were incubated with 0.1 mg / ml DQ TMThe cells were incubated with DQ Green BSA (DQ BSA, D12050, Thermo Fisher Scientific, Inc.) for 4 hours, washed twice, and then incubated in fresh medium for 3 hours to allow DQ TM BSA accumulates in lysosomes. TM Lysosomes were labeled with ELISA Red for 15 minutes. After staining, cells were centrifuged at 400 × g for 5 minutes, resuspended in 200 μl of autoMACS® running buffer, and evaluated using an SH800 cell sorter.

[0210] 6.5.1.7. Measurement of respiratory function and glycolysis

[0211] Using XFe96 TM Cellular respiration was measured using an extracellular flux analyzer (Agilent Technologies, Santa Clara, CA, USA). Cells were suspended in Seahorse PBS containing 10 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine. TM XF RPMI medium (Agilent Technologies) and 1 × 10 5 Cells were seeded at a density of 100 cells per well on a plate coated with Cell-Tak TM (CLS354240, Corning Inc.) XFe96 TM 96-well microplates (101085-004, Agilent Technologies). After seeding, cells were equilibrated in a CO2-free incubator for 20 minutes before use in the assay. To measure respiratory function, after baseline measurement, oligomycin (2 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 2 μM), and rotenone / antimycin A (0.5 μM) were sequentially added to each well. These reagents were prepared using Seahorse TM The reagents in the XF Cell Mitochondrial Stress Test Kit (103015-100, Agilent Technologies) were adjusted. Data are expressed as oxygen consumption rate (OCR; pmol / min). Wave Controller TM2.4 (Agilent Technologies) Calculation of basal respiration, ATP production, maximal respiration, proton leak, spare respiratory capacity, non-mitochondrial oxygen (non-MTC), and coupling efficiency. To measure glycolysis, glucose (10 mM), oligomycin (1 μM), and 2-deoxy-D-glucose (2-DG, 50 mM) were sequentially added to each well after baseline measurement. These were performed using a Seahorse TM The reagents in the XF Cell Glycolysis Stress Test Kit (103020-100, Agilent Technologies) were adjusted. Data are expressed as the extracellular acidification rate (ECAR; mph / min). Wave Controller TM 2.4 Software calculates glycolysis, glycolytic capacity, and glycolytic reserve.

[0212] 6.5.1.8. Peritoneal bacterial quantification

[0213] Mice were euthanized with isoflurane 24 hours after CLP. After disinfection, the abdominal skin was incised and the peritoneal cavity was flushed with 4 ml of sterile PBS containing 2 mM EDTA without damaging the muscle layer. The peritoneal lavage fluid obtained was diluted in PBS at a ratio of 1:10,000, and 40 μL of the dilution was inoculated on LB agar (22700-025, ThermoFisher Scientific, Inc.) without any antibiotics. After incubation in a non-humidified incubator at 37°C for 24 hours, the culture dish was photographed using the ChemiDoc imaging system and the images were taken using ImageJ. TM Colony-forming units (CFU) were counted using the ELISA kit (Version 1.53t, National Institutes of Health, Bethesda, MD, USA). Results are expressed as CFU per square centimeter.

[0214] 6.5.1.9. Subcellular fractionation for Western blotting

[0215] THP-1 cells were resuspended in fractionation buffer (20 mM HEPES, 10 mM KCl, 2 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 M DTT, 1 / 100 protease inhibitor cocktail set I (FUJIFILM Wako Pure Chemical Corporation), pH 7.2) and homogenized by puncturing 20 times with a 29-gauge needle. The lysate was placed on ice for 20 minutes and then centrifuged at 720 g for 5 minutes to separate into a pellet containing nuclei and a supernatant containing cytoplasm, membranes, and mitochondria. The supernatant was centrifuged again at 12,000 g for 10 minutes. The cytoplasmic supernatant from the pellet was transferred to a clean tube. The nuclear pellet was washed with 500 μL of fractionation buffer and centrifuged again at 720 g for 10 minutes. The pellet was resuspended in fractionation buffer and sonicated to shear genomic DNA and homogenize the lysate. These proteins were analyzed by Western blotting.

[0216] 6.5.1.10. Western blotting

[0217] Cytoplasmic proteins were dissolved in RIPA buffer (182-02451, FUJIFILM Wako Pure Chemical Corporation), boiled for 10 min, electrophoresed through a 10% Mini-PROTEAN® TGX precast protein gel (4561036, Bio-Rad Laboratories Inc.), and then electroblotted onto a PVDF transfer membrane (IPVH00010, Merck KGaA, Darmstadt, Germany). The membrane was blocked with PBS containing 5% skim milk and 0.05% Tween 20 (P1379, Merck KGaA) and incubated with PGC1α (sc-517380, Santa Cruz Biotechnology, Dallas, TX, USA), TFEB (ab267351, Abcamplc.), phospho-Akt (Ser473) (9271, Cell Signaling Technology, Inc.), PI3 kinase p85 (19H8) (4257, Cell Signaling Technology, Inc.), phospho-PI3 kinase p85 (Tyr458) / p55 (Tyr199) (4228, Cell Signaling Technology, Inc.), S6 ribosomal protein (5G10) (2217, Cell Signaling Technology, Inc.), phospho-S6 ribosomal protein (Ser235 / 236) (2211, Cell Signaling Technology, Inc.), AMPKα (23A3) (2603, Cell Signaling Technology, Inc.), phospho-AMPKα (Thr172) (40H9) (2535, Cell Signaling Technology, Inc.), LC3 (0231-100BIOTIN / LC3-5F10, nanotools GmbH, Teningen, Germany), GAPDH (MAB374, Merck KGaA), and α-tubulin (66031-1-Ig, Proteintech Group, Inc., Rosemont) were incubated for 1 hour.After washing, the membrane was incubated with anti-mouse IgG (7076S, Cell Signaling Technology, Inc.) or anti-rabbit IgG HRP-linked antibody (7074S, Cell Signaling Technology, Inc.) diluted 1:5000 in blocking buffer. Subsequently, Clarity was used. TM Western ECL substrate (1705060, Bio-Rad Laboratories Inc.) or Clarity TM Max Western ECL substrate (1705062, Bio-Rad Laboratories Inc.), the blot was developed, and the VersaDoc TM or ChemiDoc TM Protein bands were visualized using ImageJ. TM Protein levels were quantified.

[0218] 6.5.1.11. Immunocytochemistry

[0219] Cells were fixed at 4°C for 15 minutes in 4% paraformaldehyde in the presence of a protein blocking solution (X090710-8, Agilent Technologies Inc., Santa Clara, CA, USA) consisting of PBS supplemented with 5% normal goat serum. Cells were incubated overnight with anti-TFEB antibodies (ab267351, Abcam plc.) in PBS at 4°C. The cells were thoroughly washed with PBS and then incubated for 30 minutes at room temperature with anti-rabbit IgG (H+L) antibodies labeled with Alexa Fluor™ 488 (Thermo Fisher Scientific, Inc.). The nuclei were counterstained with 4', 6-diamidino-2-phenylindole (DAPI; diluted 1:500, #5748, FUJIFILM Wako Pure Chemical) in PBS at room temperature for 30 minutes. Using Biorevo TM Fluorescence images were acquired using a BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan). TMTo identify TFEB that migrates to the nucleus, multicolor images were first separated into TFEB- and DAPI-stained images. These images were converted to binary images using thresholding, where foreground pixels were assigned a maximum value of 255 and background pixels were assigned a minimum possible value of 80. Regions where TFEB and DAPI areas overlapped were defined as nuclear TFEB. The percentage of cells in the image with nuclear TFEB was calculated.

[0220] 6.5.1.12. Mitochondrial Network Analysis (MiNA)

[0221] Mitochondrial morphology analysis was performed using the Mitochondrial Network Analysis (MiNA) toolset, which can be downloaded from github.com / stuartlab (Valente et al., 2017, Acta Histochem 119:315-326). To obtain accurate results, image quality was first improved. Image preprocessing options, including unsharp filters and enhancing local contrast, were presented to the user through the MiNA interface. For analysis, the image was first binarized by thresholding, assigning a maximum value of 255 to foreground pixels and a minimum value of 0 to background pixels. Next, the image was analyzed using ImageJ. TM The built-in skeletonization function converts a binary image into a skeleton, which represents the features of the original image as a one-pixel-wide wireframe. All pixels within the skeleton are then classified into three categories: endpoint pixels, flat pixels, and connected pixels. The mitochondrial footprint, flat pixels, and connected pixel areas of individual cells were assessed. If calibration information is available, the mitochondrial footprint is calculated as the number of pixels in the binary image containing signal multiplied by the pixel area.

[0222] 6.5.1.13. Analysis of Mitochondria-Lysosome Contact Sites

[0223] Using ImageJ TM First, the multicolor image was separated into TM Green and LysoTracker TM Red-stained images. These images were converted to binary images by thresholding, where the maximum value of foreground pixels was assigned to 255 and the minimum possible value of background pixels was assigned to 100. Contact sites were defined as areas where the regions of interest (ROIs) of mitochondria and lysosomes overlapped. The ratio of contact sites to the area of ​​mitochondria in individual cells was assessed.

[0224] 6.5.1.14. Statistical analysis

[0225] The results are presented as mean ± standard deviation. The statistical significance of the differences between the groups was assessed using parametric unpaired t-tests with bar graphs. The Kaplan-Meier survival curve dataset was statistically analyzed using the Mantel-Cox test (Prism TM 9 software, GraphPad Prism Software Inc., San Diego, CA, USA). P < 0.05 was considered significant.

[0226] Results

[0227] 6.5.2.1. ZLN-005 Improves Survival and Anti-Inflammatory Effects in a CLP Sepsis Model

[0228] Cecal ligation and puncture (CLP) has been shown to be the most suitable animal model for polymicrobial sepsis and was used in this study. The severity of CLP can be controlled by the location of the ligature and the number of punctures. Ligation is performed at the base or middle of the cecum, and the severity is controlled by one or two punctures with a 20-gauge needle outside the ligature. In the model with cecal base ligation and two punctures, the survival rate on the second day after surgery was 50% (data not shown). Over time, the effect of ZLN-005 on naive THP-1 was observed at the mRNA and protein levels, with its expression increasing by 2-fold at 48 hours and 36 hours, respectively (data not shown). To investigate the therapeutic effect of ZLN-005, 10-20 week old C57BL / 6 mice were subjected to CLP and then intraperitoneally injected with ZLN-005 for three consecutive days ( Figure 1A ).

[0229] 25% of the mice in the sham-operated group died after 24 hours, while no mice in the ZLN-005 group died. At 72 hours, all mice in the sham-operated group died; however, 40% of the mice treated with ZLN-005 survived, and 30% were still alive after 6 days. Kaplan-Meier survival analysis showed that the survival rate of the ZLN-005 group was significantly better ( Figure 1B). In addition, just 2 hours after the first administration of the drug after CLP, the animals' behavior showed a sharp contrast. The sham-operated mice stayed in one place, were very unresponsive to stimuli, and rarely moved. However, the mice in the ZLN-005 group moved around spontaneously, rarely stayed in one place, and they quickly exhibited escape behavior when stimulated. These early behavioral changes suggest that in addition to its mechanism of action as a PGC1α activator at the transcriptional level, ZLN-005 also has an immediate effect on bacteria leaked into the peritoneal cavity. In the CLP model, the intraperitoneal macrophage-monocyte lineage has been reported to show a decisive response in early pathogenesis, and the expression of Pgc1α (which has been reported to respond to ZLN-005) and Tfam (which plays an important role in mitochondrial biogenesis) in intraperitoneal cells (including macrophages and monocytes) was examined. Both transcripts were significantly elevated (Figure 1C).

[0230] In the CLP model, inflammatory cytokines increase dramatically from the early stage, and excessive inflammatory cytokines play an important role in the pathogenesis ( Figure 1D In the sham-operated group, TNF-α and Il1β were most highly expressed in the liver, followed by the kidney, lung, and heart. Il6, which was most highly expressed in the kidney in the sham-operated group, was significantly downregulated after ZLN-005 treatment. TNF-α and Il1β were also significantly downregulated, reaching levels similar to those in the sham-operated group. Although Infγ expression itself was lower than that of other cytokines, ZLN-005 had a significant inhibitory effect in the heart and lung, reducing its expression to levels comparable to those in the sham-operated group. These results suggest that ZLN-005 has a potent anti-inflammatory effect.

[0231] 6.5.2.2. Organ Protective Effects of ZLN-005 in the CLP Sepsis Model

[0232] Echocardiography was performed 24 hours after CLP. The ejection fraction in the sham group was in the range of 60% to 40%, while the ejection fraction in the ZLN-005-treated group remained in the range of 50% ( Figure 10A ). No significant changes were found in cardiac pathological examination, including cellular infiltration or hemorrhage, suggesting that dysfunction may be caused by fluid factors such as proinflammatory cytokines and coagulation factors, and that ZLN-005's inhibition of cytokine storm may help maintain cardiac function (data not shown). The kidneys, like the heart, showed no significant changes (data not shown). However, the sham group had disordered liver lobule structure, hemorrhagic lesions, and cellular infiltration, while the examination results of the ZLN-005 treatment group were slightly improved (data not shown). Quantitative analysis did not reveal statistically significant changes ( Figure 10BThe splenic follicular structure was severely damaged in the sham-operated group, while it was well preserved in the ZLN-005-treated group (data not shown). Lung lesions were most pronounced, with cell infiltration significantly inhibited in the ZLN-005 group, the interstitial tissue well preserved, and mild edema (data not shown). Quantitative analysis also showed that the ZLN-005 group had significantly less damage ( Figure 10C ).

[0233] The effect of ZLN-005 on intraperitoneal bacterial elimination in the CLP model was also investigated. Ascitic fluid was collected 2 and 24 hours after CLP treatment, and colony counts were measured by bacterial culture. At 2 hours, the colony count in the ZLN-005-treated group had been reduced by more than half, and by 24 hours, the colony count had been further reduced by approximately one-quarter (data not shown). This suggests that ZLN-005 promotes the elimination of bacteria released into the peritoneal cavity. These results suggest that ZLN-005 can make the peritoneal monocyte-macrophage lineage more resilient to uncontrolled bacterial loads. The early onset of action at 2 hours suggests that its mechanism of action is directly related to metabolism and digestion, without the need for transcription and translation processes.

[0234] 6.5.2.3. ZLN-005-induced changes in mitochondrial function

[0235] In the CLP model, the respiratory capacity of mitochondria and the metabolic capacity of the glycolytic system were examined 1 day after intraperitoneal cell treatment. TM Respiratory chain complex inhibitors and glycolytic system inhibitors were used sequentially to obtain curves of mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic capacity. Intraperitoneal cells on the first day after surgery showed no significant changes in OXPHOS with CLP, but the administration of ZLN-005 reduced proton leak, resulting in a significant increase in coupling efficiency. In addition, spare respiratory capacity was significantly increased ( Figure 11A On the other hand, in the glycolysis system, like OXPHOS, CLP treatment did not cause significant changes on the first day after surgery, but ZLN-005 administration significantly increased glycolytic capacity and glycolytic reserve ( Figure 11B This suggests that ZLN-005 is responsible for increasing the reserve capacity of both metabolic pathways.

[0236] The currently proposed mechanism of action of ZLN-005 is to enhance PGC1α at the transcriptional level, but apart from the reserve capacity, no significant changes in the OXPHOS spectrum occurred, and no changes were found that led to a significant improvement in the peritoneal sterilization effect. Therefore, this study used the human macrophage cell line THP-1 to test the mitochondrial function of ZLN-005 to explore an alternative mode of action of ZLN-005 in this model. In the LPS stimulation model, the effect of ZLN-005 on THP-1 was tested 24 hours after stimulation. Regardless of whether ZLN-005 was stimulated with LPS, Pgc1α mRNA was significantly increased at the transcriptional level, while Tfam mRNA did not show significant changes in either group ( Figure 12A However, mtDNA copy number decreased under LPS stimulation, but significantly recovered to the level of the untreated group after ZLN-005 treatment ( Figure 12B PGC1α is a major regulator of mitochondrial biogenesis, and mitochondrial DNA copy number was examined as a phenotype. A significant increase in copy number was observed after treatment with ZLN-005 ( Figure 12B Mitochondrial mass was measured by MitoGreen, and no significant changes were observed after LPS stimulation or ZLN-005 administration (data not shown). Mitochondrial membrane potential (mtMP) measured by TMRM (data not shown) was corrected for mitochondrial mass. LPS stimulation resulted in an increase in mtMP / mtMass, and ZLN-005 administration further resulted in an increase in mtMP / mtMass ( Figure 12C LPS stimulation leads to a significant increase in mitochondrial ROS, but ZLN-005 can reverse this increase ( Figure 12D To measure mitophagy, we constructed THP-1 cells that continuously express MitoKeima Red and examined the effects of ZLN-005 over time under LPS stimulation. Mild induction of mitophagy was detected within 2 hours of ZLN-005 administration, and mitophagy did not increase over time ( Figure 12E Increased mtDNA replication and mitophagy suggest enhanced mitochondrial turnover, but under the conditions of this study, this enhancement was minimal. Without being bound by theory, mitochondrial turnover may not significantly affect cellular phenotypic changes.

[0237] Since ZLN-005 significantly affects mitochondrial biogenesis and turnover under LPS stimulation, its effect on mitochondrial dynamics was studied using THP-1 cells. Mitochondria were stained with TMRM and analyzed using ImageJ. TM Quantification of mitochondrial morphology. Footprint ratio indicating mitochondrial mass from MitoGreen by FACS TMThe MFI footprint obtained was more sensitive (data not shown) and was slightly increased by ZLN-005 in the unstimulated state but remained unchanged in the LPS-stimulated state ( Figure 12F ). None of the four groups showed changes in Slub pixels and Junction pixels, which are indicators of branch status ( Figure 12F ). Co-staining with dyes that stain both organelles was performed to assess the proximity of mitochondria and lysosomes. The cross-section with the largest lysosomal staining area was selected, and the co-stained area of ​​that cross-section was calculated. Although only one cross-section was evaluated, a significant increase in the co-stained area was observed upon exposure to ZLN-005 within 24 hours after LPS exposure. Without being bound by theory, these findings suggest that tethering exists between the two organelles and suggest that lysosomes may be involved in another mechanism of action of ZLN-005 ( Figure 12G ).

[0238] 6.5.2.4. Lysosomal changes induced by ZLN-005

[0239] Subsequent studies were designed to evaluate the hypothesis that phagocytes may alter their phenotype by rapidly responding to large bacterial loads, thereby enhancing the process from phagocytosis to digestion. Intraperitoneal cells were collected from the CLP model and used as material for studies from phagocytosis to lysosomes. Although lysosomes contain hydrolases that digest pathogens and large molecules, in order to process large numbers of pathogens, the volume of the phagolysosomes must be expanded and their lumen maintained at a pH of approximately 4.6 (the optimal pH for these hydrolases). First, the expression of Tfeb mRNA was detected, and the results showed that CLP treatment alone significantly reduced the expression of Tfeb mRNA, while ZLN-005 treatment significantly increased the expression of Tfeb mRNA, but it was still lower than that of the sham group ( Figure 13A ). CLP treatment alone increased lysosomal mass, while ZLN-005 treatment further enhanced lysosomal mass ( Figure 13A The cells in the peritoneal cavity were incubated with fluorescent dextran, and the fluorescence intensity was used to evaluate the degree of phagocytosis. The phagocytosis of the CLP group was significantly enhanced, and ZLN-005 showed further enhancement, but no significant difference was observed ( Figure 13B Next, we examined the final stages of bacterial killing and the changes within the lysosomal lumen. Lysosomal pH is one of the most basic requirements for lysosomal function, and increased acidity was measured using pHrod as an increase in fluorescence intensity. Lysosomal acidity increased in the CLP group, and this was further enhanced after ZLN-005 treatment ( Figure 13C However, using CellRox TM The measured intracellular ROS expression did not increase significantly during this period ( Figure 13D ). The acidic shift in lysosomal pH is associated with enhanced hydrolase function, as demonstrated by the use of DQ TMAs shown in the case of BSA, when protein degradation is enhanced, the fluorescent dye is released from the quenched inhibition state and fluorescence is excited. TM -BSA, a certain degree of proteolysis occurs, but LPS exposure enhances the extent of proteolysis, and ZLN-005 administration further enhances the extent of proteolysis, as observed by fluorescence microscopy ( Figure 13E ), FACS quantitative analysis also showed that ZLN-005 promoted protein hydrolysis, and there was a significant difference ( Figure 13E Without being bound by theory, these results suggest that in this model, the oxidative burst is not significantly involved in pathogen clearance 1 day after infection, and hydrolysis is believed to be the major contributor.

[0240] Compared with the CLP group, the mRNAs of most lysosomal proteins with CLEAR motifs (targets of TFEB) in their promoters were elevated. In particular, the mRNAs of all hydrolases (e.g., Ctsd) and membrane proteins (e.g., Atp6v1A, Atp6v0d1, and Mcoln1) were significantly elevated ( Figure 13F In terms of autophagy, Becn1 and Gabarap were only slightly elevated, and Rab7 was unchanged by ZLN-005 administration, although a significant increase was observed in Sqstm / p62 ( Figure 13G ). Without being bound by theory, analysis of these transcripts suggests that ZLN-005 may be more involved in the acidification of phagolysosomes rather than xenophagy. Bacterial killing in phagocytes can be achieved by fusion of phagosomes with lysosomes or xenophagy, in which the host triggers autophagy when the phagosome is damaged by the escape behavior of bacteria. In the LPS model of THP-1, autophagic flux was detected in the presence of the ATPase inhibitor bafilomycin A1 and the autophagosome-lysosome fusion inhibitor chloroquine, using LC3 conversion as an indicator. Autophagic flux was measured after 6 hours of exposure to LPS and then 2 or 6 hours of treatment with autophagy inhibitors. Neither LC3-II nor the LC3-II / I ratio increased after treatment with ZLN-005 ( Figure 13H Both inhibitors had a general effect in this system, with significant increases in LC3-II and the LC3-II / I ratio. ZLN-005 had no significant effect on the increases in LC3-II and LC3-II / I ratio observed with either inhibitor. Without being bound by theory, these results suggest that ZLN-005 does not significantly affect macroautophagy.

[0241] 6.5.2.5. Regulation of TFEB

[0242] In THP-1 cells stimulated with LPS alone, PGC1α knockdown (KD) did not significantly alter the mRNA level of Tfeb ( Figure 14B When ZLN-005 was added to LPS stimulation, Tfeb mRNA levels increased significantly, while PGC1α knockdown led to an increase in Tfeb mRNA levels, but the rate of increase was significantly reduced ( Figure 14B Without being bound by theory, these results suggest that PGC1α plays a role in the regulation of TFEB at the transcriptional level following treatment with ZLN-005. PGC1α knockdown did not alter lysosomal acidification when stimulated with LPS alone, whereas lysosomal acidification was significantly increased following ZLN-005 administration, but the increase in acidity was slightly reduced ( Figure 14C Without being bound by theory, this suggests that ZLN-005 may have other mechanisms of action other than Tfeb transcriptional regulation.

[0243] Alternatively, Tfeb is always present in the cytoplasm, its nuclear translocation is regulated by phosphorylation, and it functions as a transcription factor in the cytoplasm. We considered whether ZLN-005 might interfere with this pathway and performed fluorescent immunostaining of TFEB in THP-1 cells after LPS exposure and ZNL-005 administration ( Figure 14D Quantitative analysis showed that in the absence of LPS exposure induced by ZLN-005, the cell nucleus migrated, and when LPS was exposed with ZLN-005, the migration process of the cell nucleus was further enhanced ( Figure 14D To further confirm the nuclear migration of TFEB, cells were collected and divided into nuclear and cytoplasmic fractions, and Western blotting was performed to detect the presence of TFEB; under LPS exposure conditions, ZLN-005-induced TFEB nuclear migration was very obvious ( Figure 14E Finally, the molecular pathways of ZLN-005 were examined, and it was found that among the many factors that promote the assembly of V-ATPase V0-V1, the PI3K / AKT axis is the pathway most closely related to phagocytosis. In addition, Western blot was used to study the involvement of mTORC1 downstream of this pathway and AMPK, which is closely related to the regulation of PGC1α and TFEB. The results showed that PI3K and AKT were significantly phosphorylated and activated under ZLN-005 treatment ( Figure 15A However, no activation of S6 downstream of mTORC1 was observed, and ZLN-005 administration barely altered AMPK activity ( Figure 15A). Both mTORC1 and AMPK are deeply involved in autophagy regulation, and ZLN-005 did not activate either of them in this study. Because the PI3K-AKT pathway is strongly activated by ZLN-005, and this pathway may play a major role in the phosphorylation of TFEB, it was evaluated whether the PI3K inhibitor wortmannin can offset the changes in lysosomal acidification. In the absence of wortmannin, ZLN-005 did cause lysosomal acidification, but not in the presence of wortmannin ( Figure 15B Without being bound by theory, these results suggest that ZLN-005 causes lysosomal acidification via PI3K, thereby promoting V0-V1 assembly.

[0244] 6.5.2.6. Mode of Action of ZLN-005

[0245] Without being bound by theory, it is believed that the mechanism of action of ZLN-005 in bacterial infection is as follows:

[0246] ZLN-005 promotes the degradation of incorporated bacteria by acidifying lysosomal pH in macrophages and promoting lysosomal biogenesis ( Figure 16 In the former, the V-ATPase consists of a V0 complex, located in the lysosomal membrane, and a V1 complex, located in the cytosol or associated with the V0 complex. The presence of ZLN-005 promotes the assembly of the V0-V1 complex. To drive the V-ATPase, ZLN-005's mitochondrial biogenesis-stimulating effect increases ATP production. The action of PI3K also brings these two organelles into close proximity. In the latter, on the other hand, PI3K induces the conversion of phosphatidylinositol, dephosphorylating TFEB and translocating it into the nucleus using Ca as a secondary signal. This promotes lysosomal biogenesis and the production of various hydrolases in the lysosomal membrane and lumen. All of these events contribute to the complete digestion of incorporated bacteria.

[0247] Discussion

[0248] This example demonstrates that intraperitoneal administration of ZLN-005 has a therapeutic effect on multibacterial sepsis and suggests (without being bound by theory) that the molecular mechanism of this effect is through enhanced lysosomal acidification and biogenesis via TFEB. Also without being bound by theory, the upstream signaling pathway for TFEB activation may be PI3K. This example demonstrates that ZLN-005 has dual effects on two intracellular organelles (mitochondria and lysosomes), and that their mechanisms of action can be coordinated. This example further demonstrates that ZLN-005 has a mechanism of action that contributes to disease-modifying bacterial clearance in the early stages of sepsis.

[0249] In this example, under LPS stimulation, ZLN-005 induction of Tfeb mRNA was inhibited by PGC1α knockdown, indicating that Tfeb mRNA is regulated by PGC1α. Furthermore, ZLN-005 treatment enhanced TFEB migration to the nucleus, as observed by immunofluorescence staining and Western blot analysis of nuclear and cytoplasmic fractions, suggesting that ZLN-005 directly affects TFEB nuclear migration. The action of ZLN-005 on lysosomes via TFEB, combined with the reciprocal enhancement of PGC1α at the transcriptional level, may be a mechanism that promotes TFEB nuclear translocation, beginning with very early pathological changes and persisting for a period of time.

[0250] V-ATPase, the molecule responsible for lysosomal acidification, has been shown to be regulated by the reversible disassembly of V0-V1, but its role in pathogenesis has primarily focused on cancer. This example reveals the role of V-ATPase in sepsis, which is of great significance. In sepsis, the inability of the lysosomal system to adequately cope with excessive bacterial loads may be a factor in early mortality, and dysfunction of V0-V1 ATPase assembly may be the molecular mechanism leading to this inability. In this example, ZLN-005 promoted bacterial killing and improved survival by enhancing lysosomal acidity, indicating that V0-V1 assembly is one of the molecular bases of innate immunity in sepsis.

[0251] Without being bound by theory, ZLN-005 appears to improve survival in the CLP model by promoting bacterial killing within the phagolysosome. Regarding factors directly executing this final stage of bacterial killing, lysosomal acidity, intraluminal hydrolases, and ROS need to be considered. ZLN-005 had no significant effect on cellular ROS, while a significant increase and activation of hydrolases was observed in this example, suggesting that bacterial clearance may be attributable to hydrolases rather than cellular ROS.

[0252] In this example, ZLN-005 strongly induced TFEB, a master gene that stimulates the production of membrane proteins and hydrolases in lysosomes, which in turn contributes to an increase in lysosomal mass. TFEB is a transcriptional regulator with a positive feedback loop that rapidly responds to environmental changes primarily through post-translational modifications and transcriptionally enhances PGC1α. TFEB's stability and translocation from the cytoplasm to the nucleus are regulated by phosphorylation by various kinases and dephosphorylation by phosphatases. It translocates to the nucleus to promote the expression of its own genes as well as genes involved in autophagy and lysosomal biogenesis. TFEB is negatively regulated by many signaling regulators, including mTORC1, but is positively regulated by calcineurin. PIKFYVE, a kinase on the lysosome that receives signals from AKT, uses PI3P as a substrate to generate PI(3,5)P2. It has been reported that PI(3,5)P2 activates TRPML1, which is encoded by MCOLN1, and TRPML1 releases Ca2+ from lysosomes into the cytoplasm. Increased levels of PI(3,5)P2 activate calcineurin, leading to dephosphorylation of TFEB, thereby acting as a transcription factor. PI3K is implicated in the mechanism of action of ZLN-005, as indirectly demonstrated by enhanced phosphorylation of PI3K and AKT in the CLP model. Furthermore, ZLN-005-induced lysosomal acidification can be inhibited by the PI3K inhibitor wortmannin, directly demonstrating that ZLN-005 acts through PI3K. These factors, including PIKFYVE, TRPML1, and calcineurin, may contribute to and contribute to ZLN-005's activation of TFEB.

[0253] PI3K signaling is transmitted through AKT to mTORC1, which phosphorylates multiple factors, leading to cellular anabolic metabolism, including proliferation. However, mTOR signaling inhibits all autophagic processes, not only the initiation and nucleation of autophagy, but also the elongation, maturation, and termination of autophagosomes. Some bacteria exploit mTORC1's inhibition of autophagy to evade innate immunity. The metabolic surge in sepsis leads to activation of AMPK, which inhibits mTORC1 through phosphorylation of TSC2 via Rheb. Furthermore, AMPK supports autophagy through phosphorylation of ULK1 (independent of mTOR). In this example, the S6 phosphorylation pathway downstream of mTORC1 is not implicated in the pathogenesis of sepsis, and further activation of PI3K by ZLN-005 did not significantly alter downstream mTORC1 signaling. However, phosphorylation of AMPK, which antagonizes mTORC1, is activated in sepsis but remained unchanged in this example. This is consistent with the fact that expression of autophagy-related genes was only slightly altered in the early stages of sepsis in this example.

[0254] This example demonstrates that ZLN-005 increases mitochondrial-lysosomal contact sites and enhances OXPHOS. While physical contact between these two intracellular organelles has long been reported to occur during a degradation process similar to mitophagy, it has recently been recognized that transient contact is a physiologically non-degradative process. This example demonstrates that LPS-stimulated THP-1 cells produced by ZLN-005 lead to the connection of the two organelles from as early as one hour to as late as 24 hours. While the molecular mechanism by which ZLN-005 promotes coordination between the two organelles remains to be elucidated, it is possible (without being bound by theory) that enhanced physical contact allows the dual functions of mitochondria and lysosomes to function more organically.

[0255] For sepsis, much effort has focused on controlling the hyperimmune response. However, the development of drugs that improve early bacterial clearance has been stagnant. This example demonstrates that ZLN-005 improves overall survival by ameliorating the pathophysiology of the disease early in its course, addressing a long-standing and pressing need in the field. Furthermore, the dual effects of ZLN-005 on mitochondria and lysosomes demonstrated in this example support its potential use in treating neurodegenerative diseases characterized by lysosomal acidification failure, including Alzheimer's disease, Parkinson's disease, renal tubular acidosis, diabetes, Zimmermann-Laband syndrome (a condition associated with V1B2 mutations), and cutis laxa type II and scaly skin syndrome. Without being bound by theory, it is believed that diseases with lysosomal acidification dysfunction as a molecular basis, despite having distinct pathologies from sepsis, are therapeutic targets for ZLN-005 and related compounds.

[0256] 6.6. Example 6: ZLN-005 reduces bacterial growth in a CLP-induced sepsis model

[0257] This example further evaluated ZLN-005 and its ability to enhance bacterial killing in the mouse cecal ligation and puncture (CLP) model.

[0258] Mice were subjected to CLP at study hour 0 as described in Section 6.5.1.1. At the time of cecal ligation and puncture, mice were administered 12 mg / kg (ip) ZLN-005 or vehicle (DMSO). Ascites was collected from each mouse at study hour 2 and 24, and peritoneal bacterial levels were determined ( Figure 17A ).

[0259] The results are as follows Figures 17B-17D As shown in Figure 3, the bacterial counts in the ascitic fluid of vehicle-treated CLP mice were significantly increased at 2 and 24 hours compared to the ascitic fluid of sham-operated mice. ZLN-005-treated CLP mice showed a decrease in bacterial counts in the ascitic fluid compared to vehicle-treated CLP mice at both time points.

[0260] 6.7. Example 7: ZLN-005 Increases v-ATPase Assembly

[0261] The vacuolar ATPase (v-ATPase) is an ATP-driven proton pump that acidifies intracellular compartments and transports protons across the plasma membrane. Within lysosomes, v-ATPase increases the acidity of the lysosomal lumen. V-ATPase activity has been shown to be regulated by the reversible decomposition of V0-V1.

[0262] To assess the effect of ZLN-005 on v-ATPase assembly, mice were subjected to CLP at 0 hours after study initiation, as described in Section 6.5.1.1. At the time of cecal ligation and puncture, mice were administered 12 mg / kg (ip) of ZLN-005 or vehicle (DMSO). Peritoneal cells were harvested 24 hours later. Following cell fractionation, Western blot analysis was performed as described in Section 6.5.1.10. to determine the levels of V0 and V1 on lysosomal membranes. Figure 18A ).

[0263] The assembly of v-ATPase involves the association of the V0 and V1 subunits, with V0 being the membrane-bound subunit and V1 binding to it to form the v-ATPase. Without being bound by theory, the level of V1 protein bound to the lysosomal membrane may indicate the amount of fully assembled v-ATPase on the lysosome. Cytosolic V1 protein levels were assessed by normalizing the expression of cytosolic V1A protein with that of cytosolic tubulin. No significant differences were found between sham treatment and either CLP + vehicle or CLP + ZLN-005 ( Figure 18B The expression of V1A protein relative to VAPB (indicating lysosome-associated V1 levels) was significantly lower in CLP + vehicle samples than in samples collected from sham-operated mice, suggesting a reduction in the amount of fully assembled v-ATPase on lysosomal membranes. This reduction was reversed in samples from CLP mice treated with ZLN-005 ( Figure 18B There was no significant difference in membrane-bound V0 protein between the treatment groups ( Figure 18B Finally, assessment of V0 / V1 assembly by normalizing lysosome-associated V1 with cytosolic V1 revealed that CLP significantly reduced the association of V0 and V1, whereas ZLN-005 treatment alleviated this reduction ( Figure 18B ).

[0264] 6.8. Example 8: Abolishing ZLN-005-mediated lysosomal acidification under LPS stimulation by inhibiting TRPML1

[0265] As described in Example 5, nuclear levels of TFEB regulate lysosomal biogenesis and autophagy. TRPML1 is a protein that activates cytosolic Ca2+ release, leading to TFEB nuclear translocation.

[0266] The role of TRPML-1 in ZLN-005-associated increases in lysosomal acidification was assessed in THP-1 cells. After macrophage differentiation, cells were treated with the indicated concentrations of PMA, ZLN-005, the TRPML1 inhibitor ML-SI3, and LPS for the indicated times. Figure 19A Cells were sorted by FACS, where pHrod fluorescence was used as an indicator of lysosomal pH.

[0267] The results are as follows Figure 19B As shown in Figure 3, pHrod fluorescence levels were higher in cells treated with both ZLN-005 and LPS compared to cells treated with LPS alone. pHrod fluorescence levels in cells treated with both ML-SI3 and LPS were comparable to those in cells treated with LPS alone. pHrod fluorescence in LPS-stimulated cells treated with both ZLN-005 and ML-SI3 decreased significantly.

[0268] 7. Specific implementation plan

[0269] The present disclosure is illustrated by the following specific embodiments.

[0270] 1. A method for treating a subject suffering from a disease or disorder associated with impaired lysosomal acidification, comprising administering to the subject a therapeutically effective amount of an agent which is a compound of formula (I):

[0271]

[0272] Formula (I)

[0273] or a salt, hydrate, deuterated analog or fluorinated analog thereof, wherein:

[0274] Ar is , , ,or ;

[0275] W 1 NR 1 , O or S, or when W 9 When N, W 1 Can be CR 50 ;

[0276] W 2 CR 2 or N;

[0277] W 3CR 3 or N;

[0278] W 4 CR 4 or N;

[0279] W 5 CR 5 or N;

[0280] W 6 CR 6 or N;

[0281] W 7 CR 7 or N;

[0282] W 8 CR 8 or N;

[0283] W 9 is C, or when W 1 CR 50 , W 9 Can be N;

[0284] R 1 H, (C1-C3) alkyl, CH2OC(=O)R 30 、CH2OP(=O)OR 40 OR 41 、C(=O)OR 42 , or C(=O)R 43 ;

[0285] R 2 、R 3 、R 4 and R 5 each independently represents hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C1-4)alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxyl, (C1-C4)alkoxycarbonylamino, formamido, (C1-C4)alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, mercapto, (C1-C4)alkylthio, aminosulfonyl, (C1-C4)alkylsulfonyl, or (C1-C4)acylamino;

[0286] R 6 and R 10are each independently hydrogen, deuterium, halogen, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino;

[0287] R 7 and R 9 are independently hydrogen, deuterium, hydroxyl, cyano, amino, halogen, halogenated (C1-C4) alkyl, (C1-C4) alkoxy, halogenated (C1-C4) alkoxy, ,or ;

[0288] R 8 is hydrogen, deuterium, halogen, halo(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, ,or ;

[0289] R 30 is (C1-C 10 ) alkyl, substituted with amino (C1-C 10 )alkyl, (C1-C4)alkyl substituted 10 ) alkyl, substituted with carboxyl (C1-C 10 )alkyl, carboxyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkoxycarbonylamino, methylthio, heterocyclic, (C1-C 10 )Oxaalkyl, CHR 44 NHR 45 and guanidine;

[0290] R 40 and R 41 are each independently hydrogen or (C1-C6) hydrocarbon;

[0291] R 42 is (C1-C5)alkyl;

[0292] R 43 is (C1-C3) alkyl,

[0293] R 44 are naturally occurring amino acid side chains;

[0294] R 45 is H, methyl or (C1-C4)alkoxycarbonyl; and

[0295] R 50 is H or (C1-C3) alkyl.

[0296] 2. A method of increasing lysosomal acidity in a subject, comprising administering to the subject an agent in an amount effective to increase lysosomal acidity in the subject, wherein the agent is a compound of formula (I):

[0297]

[0298] Formula (I)

[0299] or a salt, hydrate, deuterated analog or fluorinated analog thereof, wherein:

[0300] Ar is , , ,or ;

[0301] W 1 NR 1 , O or S, or when W 9 When N, W 1 Can be CR 50 ;

[0302] W 2 CR 2 or N;

[0303] W 3 CR 3 or N;

[0304] W 4 CR 4 or N;

[0305] W 5 CR 5 or N;

[0306] W 6 CR 6 or N;

[0307] W 7 CR 7 or N;

[0308] W 8 CR 8 or N;

[0309] W 9 is C, or when W 1 CR 50 , W 9 Can be N;

[0310] R 1 H, (C1-C3) alkyl, CH2OC(=O)R 30 、CH2OP(=O)OR 40 OR41 、C(=O)OR 42 , or C(=O)R 43 ;

[0311] R 2 、R 3 、R 4 and R 5 each independently represents hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C1-4)alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxyl, (C1-C4)alkoxycarbonylamino, formamido, (C1-C4)alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, mercapto, (C1-C4)alkylthio, aminosulfonyl, (C1-C4)alkylsulfonyl, or (C1-C4)acylamino;

[0312] R 6 and R 10 are each independently hydrogen, deuterium, halogen, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino;

[0313] R 7 and R 9 are independently hydrogen, deuterium, hydroxyl, cyano, amino, halogen, halogenated (C1-C4) alkyl, (C1-C4) alkoxy, halogenated (C1-C4) alkoxy, ,or ;

[0314] R 8 is hydrogen, deuterium, halogen, halo(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, ,or ;

[0315] R 30 is (C1-C 10 ) alkyl, substituted with amino (C1-C 10 )alkyl, (C1-C4)alkyl substituted 10 ) alkyl, substituted with carboxyl (C1-C 10 )alkyl, carboxyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkoxycarbonylamino, methylthio, heterocyclic, (C1-C 10 )Oxaalkyl, CHR 44NHR 45 and guanidine;

[0316] R 40 and R 41 are each independently hydrogen or (C1-C6) hydrocarbon;

[0317] R 42 is (C1-C5)alkyl;

[0318] R 43 is (C1-C3) alkyl,

[0319] R 44 are naturally occurring amino acid side chains;

[0320] R 45 is H, methyl or (C1-C4)alkoxycarbonyl; and

[0321] R 50 is H or (C1-C3) alkyl.

[0322] 3. A method of treating a subject suffering from sepsis, infection, or a disease or disorder associated with V-ATPase dysfunction, comprising administering to the subject a therapeutically effective amount of an agent which is a compound of formula (I):

[0323]

[0324] Formula (I)

[0325] or a salt, hydrate, deuterated analog or fluorinated analog thereof, wherein:

[0326] Ar is , , ,or ;

[0327] W 1 NR 1 , O or S, or when W 9 When N, W 1 Can be CR 50 ;

[0328] W 2 CR 2 or N;

[0329] W 3 CR 3 or N;

[0330] W 4 CR 4 or N;

[0331] W 5 CR5 or N;

[0332] W 6 CR 6 or N;

[0333] W 7 CR 7 or N;

[0334] W 8 CR 8 or N;

[0335] W 9 is C, or when W 1 CR 50 , W 9 Can be N;

[0336] R 1 H, (C1-C3) alkyl, CH2OC(=O)R 30 、CH2OP(=O)OR 40 OR 41 、C(=O)OR 42 , or C(=O)R 43 ;

[0337] R 2 、R 3 、R 4 and R 5 each independently represents hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C1-4)alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxyl, (C1-C4)alkoxycarbonylamino, formamido, (C1-C4)alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, mercapto, (C1-C4)alkylthio, aminosulfonyl, (C1-C4)alkylsulfonyl, or (C1-C4)acylamino;

[0338] R 6 and R 10 are each independently hydrogen, deuterium, halogen, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino;

[0339] R 7 and R 9 are independently hydrogen, deuterium, hydroxyl, cyano, amino, halogen, halogenated (C1-C4) alkyl, (C1-C4) alkoxy, halogenated (C1-C4) alkoxy, ,or ;

[0340] R 8 is hydrogen, deuterium, halogen, halo(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, ,or ;

[0341] R 30 (C1-C 10 ) alkyl, substituted with amino (C1-C 10 )alkyl, (C1-C4)alkyl substituted 10 ) alkyl, substituted with carboxyl (C1-C 10 )alkyl, carboxyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkoxycarbonylamino, methylthio, heterocyclic, (C1-C 10 )Oxaalkyl, CHR 44 NHR 45 and guanidine;

[0342] R 40 and R 41 are each independently hydrogen or (C1-C6) hydrocarbon;

[0343] R 42 is (C1-C5)alkyl;

[0344] R 43 is (C1-C3) alkyl,

[0345] R 44 are naturally occurring amino acid side chains;

[0346] R 45 is H, methyl or (C1-C4)alkoxycarbonyl; and

[0347] R 50 is H or (C1-C3) alkyl.

[0348] 4. The method of any one of embodiments 1 to 3, wherein the amount of the agent is an amount effective to increase lysosomal acidity in the subject.

[0349] 5. The method of any one of embodiments 1 to 4, wherein the amount of the agent is an amount effective to increase lysosomal acidity in a phagocyte (e.g., a macrophage, monocyte, or neutrophil) of the subject.

[0350] 6. The method of any one of embodiments 1 to 5, wherein the amount of the agent is an amount effective to increase lysosomal acidity in peritoneal cells of the subject.

[0351] 7. The method of any one of embodiments 1 to 6, wherein the amount of the agent is an amount effective to increase lysosomal acidity in peritoneal phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject.

[0352] 8. The method of any one of embodiments 1 to 7, wherein the amount of the agent is an amount effective to increase the level of Tfeb mRNA in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject.

[0353] 9. The method of any one of embodiments 1 to 8, wherein the amount of the agent is an amount effective to increase the level of Tfeb mRNA in a peritoneal cell (e.g., a phagocyte (e.g., a macrophage, monocyte, or neutrophil)) of the subject.

[0354] 10. The method of any one of embodiments 1 to 9, wherein the amount of the agent is an amount effective to increase TFEB dephosphorylation in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

[0355] 11. The method of any one of embodiments 1 to 10, wherein the amount of the agent is an amount effective to increase TFEB nuclear translocation in a phagocyte (e.g., a macrophage, monocyte, or neutrophil) of the subject.

[0356] 12. The method of any one of embodiments 1 to 11, wherein the amount of the agent is an amount effective to increase the ratio of phosphorylated Akt to Akt (p-Akt / Akt) in a phagocyte (e.g., a macrophage, monocyte, or neutrophil) of the subject.

[0357] 13. The method of any one of embodiments 1 to 12, wherein the amount of the agent is an amount effective to increase the ratio of phosphorylated PI3K to PI3K (p-PI3K / PI3K) in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject.

[0358] 14. The method of any one of embodiments 1 to 13, wherein the amount of the agent is an amount effective to increase physical contact between mitochondria and lysosomes in a phagocyte (e.g., a macrophage, monocyte, or neutrophil) of the subject.

[0359] 15. The method of any one of embodiments 1 to 14, wherein the amount of the agent is an amount effective to increase the spare respiratory capacity in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils).

[0360] 16. The method of any one of embodiments 1 to 15, wherein the amount of the agent is an amount effective to increase the glycolytic capacity in the subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils).

[0361] 17. The method of any one of embodiments 1 to 16, wherein the amount of the agent is an amount effective to increase the glycolytic reserve in the subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils).

[0362] 18. The method of any one of embodiments 1 to 17, wherein the amount of the agent is an amount effective to increase lysosomal protein breakdown in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject.

[0363] 19. The method of any one of embodiments 1 to 18, wherein the amount of the agent is an amount effective to increase the mRNA level of one or more hydrolases and / or one or more membrane proteins in a phagocyte (e.g., a macrophage, a monocyte, or a neutrophil) of the subject.

[0364] 20. The method of any one of embodiments 1 to 19, wherein the amount of the agent is an amount effective to increase the mRNA level of Ctsd in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject.

[0365] 21. The method of any one of embodiments 1 to 20, wherein the amount of the agent is an amount effective to increase the level of mRNA for Atp6v1A in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

[0366] 22. The method of any one of embodiments 1 to 21, wherein the amount of the agent is an amount effective to increase the level of mRNA of Atp6v0d1 in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

[0367] 23. The method of any one of embodiments 1 to 22, wherein the amount of the agent is an amount effective to increase the level of Mcoln1 mRNA in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

[0368] 24. The method of any one of embodiments 1 to 22, wherein the amount of the agent is an amount effective to reduce the level of one or more inflammatory markers (eg, in blood or serum).

[0369] 25. The method of embodiment 24, wherein the one or more inflammatory markers include Tnfα, IL1β, IL6, IFNγ, or a combination thereof.

[0370] 26. The method of any one of embodiments 1 to 25, wherein the subject does not have systemic immune activation.

[0371] 27. The method of any one of embodiments 1 to 26, wherein the subject has suppressed innate immune function.

[0372] 28. The method of embodiment 27, wherein the amount of the agent is an amount effective to enhance the innate immune function of the subject.

[0373] 29. The method of any one of embodiments 1 to 28, wherein the subject has suppressed macrophage phagocytic activity.

[0374] 30. The method of embodiment 29, wherein the amount of the agent is an amount effective to enhance the phagocytic activity of the subject's macrophages.

[0375] 31. The method of any one of embodiments 1 to 30, wherein the subject has intestinal perforation.

[0376] 32. The method of any one of embodiments 1 to 31, wherein the subject has sepsis.

[0377] 33. The method of embodiment 32, wherein the subject has polymicrobial sepsis.

[0378] 34. The method of embodiment 32, wherein the subject has monomicrobial sepsis.

[0379] 35. The method of any one of embodiments 32 to 34, wherein the subject has early-stage sepsis.

[0380] 36. The method of any one of embodiments 1 to 35, wherein the subject has a Sequential Organ Failure Assessment (SOFA) score of 0 to 6, e.g., 0 to 3, 1 to 4, 2 to 5, 0, 1, 2, 3, 4, 5, or 6 prior to treatment with the agent.

[0381] 37. The method of any one of embodiments 1 to 35, wherein the subject has a Sequential Organ Failure Assessment (SOFA) score of 0 to 2, e.g., 0, 1, or 2, prior to treatment with the agent.

[0382] 38. The method of any one of embodiments 1 to 35, wherein the subject has a Sequential Organ Failure Assessment (SOFA) score of 2 to 6, e.g., 2, 3, 4, 5, or 6, prior to treatment with the agent.

[0383] 39. The method of any one of embodiments 1 to 35, wherein the subject has a Sequential Organ Failure Assessment (SOFA) score of 7 to 9, e.g., 7, 8, or 9, prior to treatment with the agent.

[0384] 40. The method of any one of embodiments 1 to 35, wherein the subject has a Sequential Organ Failure Assessment (SOFA) score of 10 to 12, e.g., 10, 11, or 12, prior to treatment with the agent.

[0385] 41. The method of any one of embodiments 1 to 35, wherein the subject has a Sequential Organ Failure Assessment (SOFA) score of 13 to 14, e.g., 13 or 14, prior to treatment with the agent.

[0386] 42. The method of any one of embodiments 1 to 35, wherein the subject has a Sequential Organ Failure Assessment (SOFA) score of 15 to 24, e.g., 15 to 20, 20 to 24, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 prior to treatment with the agent.

[0387] 43. The method of any one of embodiments 1 to 42, wherein the amount of the agent is an amount effective to reduce the subject's SOFA score.

[0388] 44. The method of any one of embodiments 1 to 42, wherein the amount of the agent is an amount effective to maintain the subject's SOFA score.

[0389] 45. The method of any one of embodiments 1 to 44, wherein the subject has one, two, three, or four of (a)-(d):

[0390] (a) Lower than normal level of HLA-DR expression in peripheral blood;

[0391] (b) higher than normal PD-1 expression in T cells;

[0392] (c) lower than normal CD88 expression in neutrophils;

[0393] (d) Lower than normal Th17 / Treg ratio.

[0394] 46. ​​The method of embodiment 45, wherein the subject has lower than normal levels of HLA-DR expression in peripheral blood.

[0395] 47. The method of embodiment 45 or 46, wherein the frequency of monocytes expressing HLA-DR in the subject's peripheral blood is less than 30%.

[0396] 48. The method of any one of embodiments 45 to 47, wherein the subject has higher than normal PD-1 expression in T cells.

[0397] 49. The method of any one of embodiments 45 to 48, wherein the subject has lower than normal expression of CD88 in neutrophils.

[0398] 50. The method of any one of embodiments 45 to 49, wherein the subject has a lower than normal Th17 / Treg ratio.

[0399] 51. The method of any one of embodiments 1 to 50, wherein the subject has suppressed innate immune function and / or lacks systemic immune activation.

[0400] 52. The method of any one of embodiments 1 to 51, wherein the subject has an infection.

[0401] 53. The method of embodiment 52, wherein the subject has an intra-abdominal infection.

[0402] 54. The method of embodiment 52 or embodiment 53, wherein the infection is a bacterial infection.

[0403] 55. The method of embodiment 54, wherein the administering reduces the bacterial load in the subject.

[0404] 56. The method of embodiment 54 or embodiment 55, wherein the bacterial infection is a multidrug-resistant bacterial infection.

[0405] 57. The method of embodiment 52 or embodiment 53, wherein the infection is a fungal infection.

[0406] 58. The method of embodiment 57, wherein the administering reduces the fungal load in the subject.

[0407] 59. The method of embodiment 52 or embodiment 53, wherein the infection is a parasitic infection.

[0408] 60. The method of embodiment 59, wherein said administering reduces the parasite load in the subject.

[0409] 61. The method of embodiment 52 or embodiment 53, wherein the infection is a viral infection.

[0410] 62. The method of embodiment 61, wherein the administering reduces the viral load in the subject.

[0411] 63. The method of any one of embodiments 1 to 62, wherein the subject has V-ATPase dysfunction.

[0412] 64. The method of any one of embodiments 1 to 63, wherein the subject has a disease or disorder associated with V-ATPase dysfunction.

[0413] 65. The method of embodiment 64, wherein the disease or disorder associated with V-ATPase dysfunction is renal tubular acidosis, Zimmermann-Laband syndrome, cutis laxa type II or wrinkled skin syndrome, osteopetrosis, glucose intolerance, diabetes mellitus, Parkinson's disease, Alzheimer's disease, or hearing loss.

[0414] 66. The method of any one of embodiments 63 to 65, wherein the subject has a pathogenic mutation in a gene encoding a V-ATPase subunit.

[0415] 67. The method of any one of embodiments 63 to 66, wherein the subject has a pathogenic mutation in the gene encoding V-ATPase subunit B.

[0416] 68. The method of embodiment 67, wherein the subject has a pathogenic ATP6V1B1 mutation.

[0417] 69. The method of embodiment 63 or embodiment 68, wherein the subject has renal tubular acidosis.

[0418] 70. The method of any one of embodiments 63 to 66, wherein the subject has a pathogenic ATP6V1B2 mutation.

[0419] 71. The method of embodiment 63 or embodiment 70, wherein the subject has Zimmermann-Laband syndrome.

[0420] 72. The method of any one of embodiments 63 to 66, wherein the subject has a pathogenic mutation in the gene encoding V-ATPase subunit a.

[0421] 73. The method of embodiment 72, wherein the subject has a pathogenic ATP6V0A4 mutation.

[0422] 74. The method of embodiment 63 or embodiment 73, wherein the subject has renal tubular acidosis.

[0423] 75. The method of embodiment 72, wherein the subject has a pathogenic ATP6V0A2 mutation.

[0424] 76. The method of embodiment 63 or embodiment 75, wherein the subject has cutis laxa type II or wrinkled skin syndrome.

[0425] 77. The method of embodiment 72, wherein the subject has a pathogenic ATP6V0A3 mutation.

[0426] 78. The method of embodiment 63 or embodiment 77, wherein the subject has osteopetrosis.

[0427] 79. The method of embodiment 78, wherein the subject has neurodegeneration.

[0428] 80. The method of any one of embodiments 63 to 66, wherein the subject has a pathogenic ATP6V1H mutation.

[0429] 81. The method of embodiment 63 or embodiment 80, wherein the subject has glucose intolerance or diabetes.

[0430] 82. The method of any one of embodiments 63 to 65, wherein the subject has a pathogenic mutation in a gene encoding a V-ATPase accessory protein.

[0431] 83. The method of embodiment 82, wherein the V-ATPase accessory protein is ATP6AP2.

[0432] 84. The method of embodiment 83, wherein the subject has Parkinson's disease, e.g., X-linked Parkinson's disease with spasticity (XPDS).

[0433] 85. The method of any one of embodiments 63 to 65, wherein the subject has a pathogenic PSEN1 mutation.

[0434] 86. The method of embodiment 63 or embodiment 85, wherein the subject has familial Alzheimer's disease.

[0435] 87. The method of any one of embodiments 63 to 65, wherein the subject has a pathogenic DMXL2 mutation.

[0436] 88. The method of embodiment 63 or embodiment 87, wherein the subject has hearing loss.

[0437] 89. The method of any one of embodiments 1 to 64, wherein the subject has renal tubular acidosis, Zimmermann-Laband syndrome, cutis laxa type II or wrinkled skin syndrome, osteopetrosis, glucose intolerance, diabetes, Parkinson's disease, Alzheimer's disease, or hearing loss.

[0438] 90. The method of any one of embodiments 1 to 89, wherein the agent is , , , , , , , , , , , or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0439] 91. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0440] 92. The method of embodiment 91, wherein the agent is or a salt thereof.

[0441] 93. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0442] 94. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0443] 95. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0444] 96. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0445] 97. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0446] 98. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0447] 99. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0448] 100. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0449] 101. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0450] 102. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0451] 103. The method of embodiment 90, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

[0452] 104. The method of any one of embodiments 1 to 103, wherein the agent is administered enterally.

[0453] 105. The method of embodiment 104, wherein the agent is administered orally.

[0454] 106. The method of any one of embodiments 1 to 103, wherein the agent is administered orally (po).

[0455] 8. References

[0456] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated as being incorporated by reference. In the event of any inconsistency between the teachings of one or more references cited herein and the present disclosure, the teachings of this specification control.

Claims

1. A method for treating a subject suffering from a disease or disorder associated with impaired lysosomal acidification, comprising administering to the subject a therapeutically effective amount of an agent which is a compound of formula (I): Formula (I) or a salt, hydrate, deuterated analog or fluorinated analog thereof, wherein: Ar is 、 、 or ; W 1 NR 1 , O or S, or when W 9 When N, W 1 Can be CR 50 ; W 2 CR 2 or N; W 3 CR 3 or N; W 4 CR 4 or N; W 5 CR 5 or N; W 6 CR 6 or N; W 7 CR 7 or N; W 8 CR 8 or N; W 9 is C, or when W 1 CR 50 When W 9 Can be N; R 1 H, (C1-C3) alkyl, CH2OC(=O)R 30 、CH2OP(=O)OR 40 OR 41 、C(=O)OR 42 , or C(=O)R 43 ; R 2 、R 3 、R 4 and R 5 each independently represents hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C1-4)alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxyl, (C1-C4)alkoxycarbonylamino, formamido, (C1-C4)alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, mercapto, (C1-C4)alkylthio, aminosulfonyl, (C1-C4)alkylsulfonyl, or (C1-C4)acylamino; R 6 and R 10 are each independently hydrogen, deuterium, halogen, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino; R 7 and R 9 are independently hydrogen, deuterium, hydroxyl, cyano, amino, halogen, halogenated (C1-C4) alkyl, (C1-C4) alkoxy, halogenated (C1-C4) alkoxy, ,or ; R 8 is hydrogen, deuterium, halogen, halo(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, ,or ; R 30 is (C1-C 10 ) alkyl, substituted with amino (C1-C 10 )alkyl, (C1-C4)alkyl substituted 10 ) alkyl, substituted with carboxyl (C1-C 10 )alkyl, carboxyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkoxycarbonylamino, methylthio, heterocyclic, (C1-C 10 )Oxaalkyl, CHR 44 NHR 45 and guanidine; R 40 and R 41 are each independently hydrogen or (C1-C6) hydrocarbon; R 42 is (C1-C5)alkyl; R 43 is (C1-C3) alkyl, R 44 are naturally occurring amino acid side chains; R 45 is H, methyl or (C1-C4)alkoxycarbonyl; and R 50 is H or (C1-C3) alkyl.

2. A method of increasing lysosomal acidity in a subject, comprising administering to the subject an agent in an amount effective to increase lysosomal acidity in the subject, wherein the agent is a compound of formula (I): Formula (I) or a salt, hydrate, deuterated analog or fluorinated analog thereof, wherein: Ar is 、 、 or ; W 1 NR 1 , O or S, or when W 9 When N, W 1 Can be CR 50 ; W 2 CR 2 or N; W 3 CR 3 or N; W 4 CR 4 or N; W 5 CR 5 or N; W 6 CR 6 or N; W 7 CR 7 or N; W 8 CR 8 or N; W 9 is C, or when W 1 CR 50 , W 9 Can be N; R 1 H, (C1-C3) alkyl, CH2OC(=O)R 30 、CH2OP(=O)OR 40 OR 41 、C(=O)OR 42 , or C(=O)R 43 ; R 2 、R 3 、R 4 and R 5 each independently represents hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C1-4)alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxyl, (C1-C4)alkoxycarbonylamino, formamido, (C1-C4)alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, mercapto, (C1-C4)alkylthio, aminosulfonyl, (C1-C4)alkylsulfonyl, or (C1-C4)acylamino; R 6 and R 10 are each independently hydrogen, deuterium, halogen, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino; R 7 and R 9 are independently hydrogen, deuterium, hydroxyl, cyano, amino, halogen, halogenated (C1-C4) alkyl, (C1-C4) alkoxy, halogenated (C1-C4) alkoxy, ,or ; R 8 is hydrogen, deuterium, halogen, halo(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, ,or ; R 30 is (C1-C 10 ) alkyl, substituted with amino (C1-C 10 )alkyl, (C1-C4)alkyl substituted 10 ) alkyl, substituted with carboxyl (C1-C 10 )alkyl, carboxyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkoxycarbonylamino, methylthio, heterocyclic, (C1-C 10 )Oxaalkyl, CHR 44 NHR 45 and guanidine; R 40 and R 41 are each independently hydrogen or (C1-C6) hydrocarbon; R 42 is (C1-C5)alkyl; R 43 is (C1-C3) alkyl, R 44 are naturally occurring amino acid side chains; R 45 is H, methyl or (C1-C4)alkoxycarbonyl; and R 50 is H or (C1-C3) alkyl.

3. A method of treating a subject suffering from sepsis, infection, or a disease or disorder associated with V-ATPase dysfunction, comprising administering to the subject a therapeutically effective amount of an agent which is a compound of formula (I): Formula (I) or a salt, hydrate, deuterated analog or fluorinated analog thereof, wherein: Ar is , , ,or ; W 1 NR 1 , O or S, or when W 9 When N, W 1 Can be CR 50 ; W 2 CR 2 or N; W 3 CR 3 or N; W 4 CR 4 or N; W 5 CR 5 or N; W 6 CR 6 or N; W 7 CR 7 or N; W 8 CR 8 or N; W 9 is C, or when W 1 CR 50 , W 9 Can be N; R 1 H, (C1-C3) alkyl, CH2OC(=O)R 30 、CH2OP(=O)OR 40 OR 41 、C(=O)OR 42 , or C(=O)R 43 ; R 2 、R 3 、R 4 and R 5 each independently represents hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C1-4)alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxyl, (C1-C4)alkoxycarbonylamino, formamido, (C1-C4)alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C1-C4)alkylamino, di(C1-C4)alkylamino, mercapto, (C1-C4)alkylthio, aminosulfonyl, (C1-C4)alkylsulfonyl, or (C1-C4)acylamino; R 6 and R 10 are each independently hydrogen, deuterium, halogen, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino; R 7 and R 9 are independently hydrogen, deuterium, hydroxyl, cyano, amino, halogen, halogenated (C1-C4) alkyl, (C1-C4) alkoxy, halogenated (C1-C4) alkoxy, ,or ; R 8 is hydrogen, deuterium, halogen, halo(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, ,or ; R 30 is (C1-C 10 ) alkyl, substituted with amino (C1-C 10 )alkyl, (C1-C4)alkyl substituted 10 ) alkyl, substituted with carboxyl (C1-C 10 )alkyl, carboxyl, (C1-C6)alkoxycarbonyl, (C1-C6)alkoxycarbonylamino, methylthio, heterocyclic, (C1-C 10 )Oxaalkyl, CHR 44 NHR 45 and guanidine; R 40 and R 41 are each independently hydrogen or (C1-C6) hydrocarbon; R 42 is (C1-C5)alkyl; R 43 is (C1-C3) alkyl, R 44 are naturally occurring amino acid side chains; R 45 is H, methyl or (C1-C4)alkoxycarbonyl; and R 50 is H or (C1-C3) alkyl.

4. The method of any one of claims 1 to 3, wherein the amount of the agent is an amount effective to increase lysosomal acidity in the subject.

5. The method of any one of claims 1 to 4, wherein the amount of the agent is an amount effective to increase lysosomal acidity in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

6. The method of any one of claims 1 to 5, wherein the amount of the agent is an amount effective to increase lysosomal acidity in peritoneal cells of the subject.

7. The method of any one of claims 1 to 6, wherein the amount of the agent is an amount effective to increase lysosomal acidity in peritoneal phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject.

8. The method of any one of claims 1 to 7, wherein the amount of the agent is an amount effective for: (a) increasing the level of Tfeb mRNA in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (b) increasing the level of Tfeb mRNA in peritoneal cells (e.g., phagocytes (e.g., macrophages, monocytes, or neutrophils)) of the subject; (c) increasing dephosphorylation of TFEB in phagocytic cells (e.g., macrophages, monocytes, or neutrophils) of the subject; (d) increasing TFEB nuclear translocation in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (e) increasing the ratio of phosphorylated Akt to Akt (p-Akt / Akt) in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (f) increasing the ratio of phosphorylated PI3K to PI3K (p-PI3K / PI3K) in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (g) increasing physical contact between mitochondria and lysosomes in a phagocytic cell (e.g., a macrophage, monocyte, or neutrophil) of the subject; (h) increasing spare respiratory capacity in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (i) increasing the glycolytic capacity in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (j) increasing glycolytic reserve in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (k) increasing lysosomal protein breakdown in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (1) increasing the mRNA level of one or more hydrolases and / or one or more membrane proteins in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (m) increasing the mRNA level of Ctsd in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (n) increasing the level of Atp6v1A mRNA in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (o) increasing the mRNA level of Atp6v0d1 in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject; (p) increasing the level of Mcoln1 mRNA in phagocytes (eg, macrophages, monocytes, or neutrophils) of the subject; or (q) reducing the level of one or more inflammatory markers (e.g., in blood or serum).

9. The method of any one of claims 1 to 8, wherein the subject does not have systemic immune activation.

10. The method of any one of claims 1 to 9, wherein the subject has suppressed innate immune function, optionally wherein the amount of the agent is an amount effective to enhance the subject's innate immune function.

11. The method of any one of claims 1 to 10, wherein the subject has suppressed macrophage phagocytic activity, optionally wherein the amount of the agent is an amount effective to enhance the macrophage phagocytic activity of the subject.

12. The method of any one of claims 1 to 11, wherein the subject suffers from intestinal perforation.

13. The method of any one of claims 1 to 12, wherein the subject has sepsis, eg, polymicrobial sepsis or monomicrobial sepsis.

14. The method of claim 13, wherein the subject has early-stage sepsis.

15. The method of any one of claims 1 to 14, wherein the subject has an infection.

16. The method of claim 15, wherein the infection is a bacterial infection, a fungal infection, a parasitic infection, or a viral infection.

17. The method of any one of claims 1 to 16, wherein the subject has V-ATPase dysfunction.

18. The method of any one of claims 1 to 17, wherein the subject suffers from a disease or disorder associated with V-ATPase dysfunction.

19. The method of claim 18, wherein the disease or disorder associated with V-ATPase dysfunction is renal tubular acidosis, Zimmermann-Laband syndrome, cutis laxa type II or wrinkled skin syndrome, osteopetrosis, glucose intolerance, diabetes, Parkinson's disease, Alzheimer's disease, or hearing loss.

20. The method of any one of claims 17 to 19, wherein the subject has a pathogenic mutation in a gene encoding a V-ATPase subunit.

21. The method of any one of claims 17 to 20, wherein the subject has a pathogenic mutation in the gene encoding V-ATPase subunit B, optionally wherein the subject has a pathogenic ATP6V1B1 mutation.

22. The method of claim 21, wherein the subject has a pathogenic ATP6V1B1 mutation.

23. The method of claim 17 or claim 22, wherein the subject has renal tubular acidosis.

24. The method of any one of claims 17 to 20, wherein the subject has a pathogenic ATP6V1B2 mutation.

25. The method of claim 17 or claim 24, wherein the subject has Zimmermann-Laband syndrome.

26. The method of any one of claims 17 to 20, wherein the subject has a pathogenic mutation in a gene encoding V-ATPase subunit a.

27. The method of claim 26, wherein the subject has a pathogenic ATP6V0A4 mutation.

28. The method of claim 17 or claim 27, wherein the subject has renal tubular acidosis.

29. The method of claim 26, wherein the subject has a pathogenic ATP6V0A2 mutation.

30. The method of claim 17 or claim 29, wherein the subject has cutis laxa type II or wrinkled skin syndrome.

31. The method of claim 26, wherein the subject has a pathogenic ATP6V0A3 mutation.

32. The method of claim 17 or claim 31 , wherein the subject has osteopetrosis.

33. The method of claim 32, wherein the subject suffers from neurodegeneration.

34. The method of any one of claims 17 to 20, wherein the subject has a pathogenic ATP6V1H mutation.

35. The method of claim 17 or claim 34, wherein the subject suffers from glucose intolerance or diabetes.

36. The method of any one of claims 17 to 19, wherein the subject has a pathogenic mutation in a gene encoding a V-ATPase accessory protein.

37. The method of claim 36, wherein the V-ATPase accessory protein is ATP6AP2.

38. The method of claim 37, wherein the subject has Parkinson's disease, eg, X-linked Parkinson's disease with spasticity (XPDS).

39. The method of any one of claims 17 to 19, wherein the subject has a pathogenic PSEN1 mutation.

40. The method of claim 17 or claim 39, wherein the subject has familial Alzheimer's disease.

41. The method of any one of claims 17 to 19, wherein the subject has a pathogenic DMXL2 mutation.

42. The method of claim 17 or claim 41, wherein the subject suffers from hearing loss.

43. The method of any one of claims 1 to 18, wherein the subject has renal tubular acidosis, Zimmermann-Laband syndrome, cutis laxa type II or wrinkled skin syndrome, osteopetrosis, glucose intolerance, diabetes, Parkinson's disease, Alzheimer's disease, or hearing loss.

44. The method of any one of claims 1 to 43, wherein the agent is 、 、 、 、 、 、 、 、 、 、 、 or a salt, hydrate, deuterated analog or fluorinated analog thereof.

45. The method of claim 44, wherein the agent is or a salt, hydrate, deuterated analog or fluorinated analog thereof.

46. ​​The method of claim 45, wherein the agent is or a salt thereof.

47. The method of any one of claims 1 to 46, wherein the agent is administered enterally.

48. The method of claim 47, wherein the agent is administered orally.

49. The method of any one of claims 1 to 46, wherein the agent is administered orally (po).

Images