Uses of ZLN-005 and Related Compounds

Abstract

Methods of treating diseases and disorders associated with impaired lysosomal acidification, increasing lysosomal acidity, and treating diseases and disorders associated with sepsis, infection, and V-ATPase dysfunction using ZLN-005 and other compounds of formula (I), and salts, hydrates, deuterated analogs, and fluorinated analogs thereof. [Formula 1] JPEG2025537881000065.jpg3345

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 426,426, filed November 18, 2022, and U.S. Provisional Application No. 63 / 482,384, filed January 31, 2023, the contents of which are incorporated herein by reference in their entireties. [Background technology]

[0002] Lysosomal acidification disorders can lead to phagocyte dysfunction. Lysosomal acidification disorders and phagocyte dysfunction are involved in numerous diseases and disorders, including several neurodegenerative disorders, diseases and disorders associated with V-ATPase dysfunction, sepsis, and infections (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).

[0003] There is a need for new treatments for treating diseases and disorders associated with impaired lysosomal acidification. Summary of the Invention

[0004] The present 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, increases the phagocytic activity of phagocytes in in vitro assays, promotes lysosomal acidification in in vitro assays, and reduces mortality in an in vivo sepsis model where mortality is known to be associated with impaired lysosomal acidification. ZLN-005 has the following structure:

[0005] [ka]

[0006] The assays indicate that ZLN-005 and related compounds can be used to increase lysosomal acidification in phagocytes, such as monocytes, of a subject, and (without being bound by theory) can be used to treat diseases and disorders associated with impaired lysosomal acidification, such as sepsis, infections, and diseases and disorders associated with V-ATPase dysfunction that involve suppressed innate immunity.

[0007] Thus, in one aspect, the disclosure provides a method of treating a subject having a disease or disorder associated with impaired lysosomal acidification, comprising administering to a subject a compound of formula (I): ZLN-005 or a compound of formula (I):

[0008] [ka] wherein Ar is

[0009] [ka] and W 1 But NR 1 , O, or S, or W 9 If N, then W 1 Further CR 50 may be W 2 But, CR 2 or N, W 3 But, CR 3 or N, W 4 But, CR 4 or N, W 5 But, CR 5 or N, W 6 But, CR 6 or N, W 7 But, CR 7 or N, W 8 But, CR 8 or N, W 9 is C or W 1 is CR 50 In the case of W 9 may 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 and R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C 1-4 ) alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxy, (C1-C4)alkoxycarbonylamino, carboxamido, (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, halo, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino; R 7 and R 9each independently represents hydrogen, deuterium, hydroxy, cyano, amino, halogen, halo(C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy,

[0010] [ka] and 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,

[0011] [ka] and R 30 However, (C1-C 10 ) hydrocarbyl, amino-substituted (C1-C 10 ) hydrocarbyl, (C1-C4) hydrocarbyl substituted (C1-C 10 ) hydrocarbyl, carboxy-substituted (C1-C 10 ) hydrocarbyl, carboxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkoxycarbonylamino, methylthio, heterocyclyl, (C1-C 10 ) Oxaalkyl, CHR 44 NHR 45 and guanidine, R 40 and R 41 are each independently hydrogen or (C-C)hydrocarbyl; R 42 is (C1-C5) alkyl; R 43 is (C1-C3) alkyl; R 44 is a naturally occurring amino acid side chain, R 45 is H, methyl or (C1-C4)alkoxycarbonyl, and R50 is H or (C1-C3) alkyl] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0012] In another aspect, the disclosure provides a method for increasing lysosomal acidity in a subject, comprising administering to the subject an amount of a drug effective to increase lysosomal acidity in the subject, wherein the drug is a compound of formula (I) (e.g., ZLN-005) or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0013] In another aspect, the present disclosure provides a method for treating a subject having sepsis, an infection, or a disease or disorder associated with V-ATPase dysfunction, comprising administering to the subject a therapeutically effective amount of a drug, wherein the drug 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.

[0014] Further features of compounds of Formula (I) and their salts, hydrates, deuterated analogs, and fluorinated analogs are described below in Section 5.2 and in numbered embodiments 1-3 and 90-103.

[0015] The compounds of Formula (I) and their salts, hydrates, deuterated analogs, and fluorinated analogs can be administered in pharmaceutical compositions comprising the compounds of Formula (I) or their salts, hydrates, deuterated analogs, or fluorinated analogs. Exemplary features of pharmaceutical compositions are described in Section 5.3 below.

[0016] Further features of the disclosed methods are described below in Section 5.4 and in numbered embodiments 1-106. [Brief explanation of the drawings]

[0017] [Figure 1-1] Figure 1A shows the study timeline for the cecal ligation and puncture (CLP) model of sepsis using C57BL / 6 mice treated intraperitoneally with vehicle (DMSO) or ZLN-005 every 24 hours after CLP (Figure 1A). The Kaplan-Meier survival rates of mice in the CLP model of sepsis treated with ZLN-005 or vehicle (n=9) (Figure 1B), the mRNA expression of Pgc1α and Tfam at 24 hours after CLP (n=6) (Figure 1C), and the mRNA expression of pro-inflammatory cytokines at 24 hours after CLP (n=6) (Figure 1D) (Examples 1 and 5). *P-values ​​**<0.0001, *0.0001-0.05. [Figure 1-2] Figure 1A shows the study timeline for the cecal ligation and puncture (CLP) model of sepsis using C57BL / 6 mice treated intraperitoneally with vehicle (DMSO) or ZLN-005 every 24 hours after CLP (Figure 1A). The Kaplan-Meier survival rates of mice in the CLP model of sepsis treated with ZLN-005 or vehicle (n=9) (Figure 1B), the mRNA expression of Pgc1α and Tfam at 24 hours after CLP (n=6) (Figure 1C), and the mRNA expression of pro-inflammatory cytokines at 24 hours after CLP (n=6) (Figure 1D) (Examples 1 and 5). *P-values ​​**<0.0001, *0.0001-0.05. [Figure 2] FIG. 1 shows the phagocytic activity of macrophages treated with ZLN-005 (Example 2). [Figure 3] FIG. 1 shows optical microscope images (top row) and fluorescent microscope images (bottom row) using a pH-sensitive dye of macrophages treated with ZLN-005 or vehicle (DMSO) (Example 3). [Figure 4] FIG. 1 shows lysosomal acidity in macrophages treated with ZLN-005 or vehicle (DMSO) (Example 3). [Figure 5] FIG. 1 shows optical microscopy images (top row) and fluorescence microscopy images (bottom row) of peritoneal cells collected from mice in the CLP model of sepsis treated with ZLN-005 or vehicle (DMSO) after incubation with fluorescent beads (Example 4). [Figure 6] FIG. 1 shows the relative phagocytic activity of peritoneal cells harvested from mice in the CLP model of sepsis treated with ZLN-005 or vehicle (DMSO) (Example 4). [Figure 7] FIG. 1 shows optical microscopy images (top row) and fluorescence microscopy images (bottom row) using a pH-sensitive dye of peritoneal cells from mice in the CLP model of sepsis treated with ZLN-005 or vehicle (DMSO) (Example 4). [Figure 8] FIG. 1 shows lysosomal acidity in peritoneal cells from mice in the CLP model of sepsis treated with ZLN-005 or vehicle (DMSO) (Example 4). [Figure 9] FIG. 1 shows peritoneal bacterial levels from mice in the CLP model of sepsis treated with ZLN-005 or vehicle (DMSO) (Example 4). [Figure 10-1] Figures showing the organ protective effect of ZLN-005 in a CLP model of sepsis (Example 5). Figure 10A: Ejection fraction, left ventricular diameter, and left ventricular diastolic wall thickness 24 hours after CLP (n=4). Figure 10B: Histological scores of the livers of mice 24 hours after CLP (n=3). Figure 10C: Histological scores of the lungs of mice 24 hours after CLP (n=3). P values ​​*0.0001-0.05. [Figure 10-2] Figures showing the organ protective effect of ZLN-005 in a CLP model of sepsis (Example 5). Figure 10A: Ejection fraction, left ventricular diameter, and left ventricular diastolic wall thickness 24 hours after CLP (n=4). Figure 10B: Histological scores of the livers of mice 24 hours after CLP (n=3). Figure 10C: Histological scores of the lungs of mice 24 hours after CLP (n=3). P values ​​*0.0001-0.05. [Figure 11-1]These figures show changes in oxygen consumption rate (OCR) (FIG. 11A) and extracellular acidification rate (ECAR) (FIG. 11B) of THP-1 cells in an inflammation model measured using flux analysis (Example 5). O: oligomycin, F: FCCP, A&R: antimycin and rotenone, G: glucose, 2-DG: 2-deoxy-D-glucose (n=5). Each indicator of glycolysis was calculated from the ECAR measurement results. Each indicator of mitochondrial respiratory function was calculated from the OCR, and glycolysis was calculated from the ECAR measurement results. P value **<0.0001, P value *0.0001-0.05. [Figure 11-2] These figures show changes in oxygen consumption rate (OCR) (FIG. 11A) and extracellular acidification rate (ECAR) (FIG. 11B) of THP-1 cells in an inflammation model measured using flux analysis (Example 5). O: oligomycin, F: FCCP, A&R: antimycin and rotenone, G: glucose, 2-DG: 2-deoxy-D-glucose (n=5). Each indicator of glycolysis was calculated from the ECAR measurement results. Each indicator of mitochondrial respiratory function was calculated from the OCR, and glycolysis was calculated from the ECAR measurement results. P value **<0.0001, P value *0.0001-0.05. [Figure 12-1]Figures showing changes in mitochondrial function by ZLN-005 (Example 5). Figure 12A: PGC1α and TFAM mRNA expression in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12B: Absolute copy number in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12C: MFI ratio of mtROS levels in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12D: MFI ratio of acidic mKeima Red signaling in THP-1 cells of an inflammatory model 0, 4, 8, and 24 hours after LPS administration (n=3). Figure 12E: MFI ratio of Δφ in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12F: Mitochondrial morphology in THP-1 cells of an inflammatory model 24 hours after LPS administration. All bars in the images represent 20 μm. The graph shows the mitochondrial footprint, junction pixel, and slab pixel area per cell (n = 5). Figure 12G: Evaluation of mitochondria-lysosome contact sites in THP-1 cells in an inflammation model at 0, 1, 2, 8, and 24 hours after LPS administration (n = 3). All bars in the images represent 20 µm. The graph shows the ratio of contact site area to total mitochondrial area per cell (n = 5). P value *0.0001-0.05. [Figure 12-2]Figures showing changes in mitochondrial function by ZLN-005 (Example 5). Figure 12A: PGC1α and TFAM mRNA expression in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12B: Absolute copy number in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12C: MFI ratio of mtROS levels in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12D: MFI ratio of acidic mKeima Red signaling in THP-1 cells of an inflammatory model 0, 4, 8, and 24 hours after LPS administration (n=3). Figure 12E: MFI ratio of Δφ in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12F: Mitochondrial morphology in THP-1 cells of an inflammatory model 24 hours after LPS administration. All bars in the images represent 20 μm. The graph shows the mitochondrial footprint, junction pixel, and slab pixel area per cell (n = 5). Figure 12G: Evaluation of mitochondria-lysosome contact sites in THP-1 cells in an inflammation model at 0, 1, 2, 8, and 24 hours after LPS administration (n = 3). All bars in the images represent 20 µm. The graph shows the ratio of contact site area to total mitochondrial area per cell (n = 5). P value *0.0001-0.05. [Figure 12-3]Figures showing changes in mitochondrial function by ZLN-005 (Example 5). Figure 12A: PGC1α and TFAM mRNA expression in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12B: Absolute copy number in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12C: MFI ratio of mtROS levels in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12D: MFI ratio of acidic mKeima Red signaling in THP-1 cells of an inflammatory model 0, 4, 8, and 24 hours after LPS administration (n=3). Figure 12E: MFI ratio of Δφ in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12F: Mitochondrial morphology in THP-1 cells of an inflammatory model 24 hours after LPS administration. All bars in the images represent 20 μm. The graph shows the mitochondrial footprint, junction pixel, and slab pixel area per cell (n = 5). Figure 12G: Evaluation of mitochondria-lysosome contact sites in THP-1 cells in an inflammation model at 0, 1, 2, 8, and 24 hours after LPS administration (n = 3). All bars in the images represent 20 µm. The graph shows the ratio of contact site area to total mitochondrial area per cell (n = 5). P value *0.0001-0.05. [Figure 12-4]Figures showing changes in mitochondrial function by ZLN-005 (Example 5). Figure 12A: PGC1α and TFAM mRNA expression in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12B: Absolute copy number in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12C: MFI ratio of mtROS levels in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12D: MFI ratio of acidic mKeima Red signaling in THP-1 cells of an inflammatory model 0, 4, 8, and 24 hours after LPS administration (n=3). Figure 12E: MFI ratio of Δφ in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12F: Mitochondrial morphology in THP-1 cells of an inflammatory model 24 hours after LPS administration. All bars in the images represent 20 μm. The graph shows the mitochondrial footprint, junction pixel, and slab pixel area per cell (n = 5). Figure 12G: Evaluation of mitochondria-lysosome contact sites in THP-1 cells in an inflammation model at 0, 1, 2, 8, and 24 hours after LPS administration (n = 3). All bars in the images represent 20 µm. The graph shows the ratio of contact site area to total mitochondrial area per cell (n = 5). P value *0.0001-0.05. [Figure 12-5]Figures showing changes in mitochondrial function by ZLN-005 (Example 5). Figure 12A: PGC1α and TFAM mRNA expression in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12B: Absolute copy number in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12C: MFI ratio of mtROS levels in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12D: MFI ratio of acidic mKeima Red signaling in THP-1 cells of an inflammatory model 0, 4, 8, and 24 hours after LPS administration (n=3). Figure 12E: MFI ratio of Δφ in THP-1 cells of an inflammatory model 24 hours after LPS administration (n=3). Figure 12F: Mitochondrial morphology in THP-1 cells of an inflammatory model 24 hours after LPS administration. All bars in the images represent 20 μm. The graph shows the mitochondrial footprint, junction pixel, and slab pixel area per cell (n = 5). Figure 12G: Evaluation of mitochondria-lysosome contact sites in THP-1 cells in an inflammation model at 0, 1, 2, 8, and 24 hours after LPS administration (n = 3). All bars in the images represent 20 µm. The graph shows the ratio of contact site area to total mitochondrial area per cell (n = 5). P value *0.0001-0.05. [Figure 13-1]Figures 13A-13D show changes in lysosomal function by ZLN-005 (Example 5). Figure 13A: mRNA expression of Tfeb and MFI ratio of lysosomal staining in peritoneal cells 24 hours after CLP (n=6). Figures 13B-13D: Assessment of phagocytosis (n=6) (Figure 13B), lysosomal acidification (Figure 13C), and cellular ROS (Figure 13D) in peritoneal cells 24 hours after CLP. Figure 13E: Degradation of pretreated DQ™ BSA in lysosomes in peritoneal cells 24 hours after CLP. The graph shows the MFI ratio of degraded DQ Green BSA and LysoTracker Red. Figures 13F-13G: mRNA expression of lysosomal hydrolases (Figure 13F) and autophagy-related genes (Figure 13G) 24 hours after CLP (n=6). Figure 13H: Western blotting analysis of autophagic flux in THP-1 cells exposed to LPS for 6 hours followed by treatment with autophagy inhibitors for 2 or 6 hours. The graph shows the expression ratio of LC3-II protein corrected for the expression level of LC3-I protein (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001-0.05. [Figure 13-2]Figures 13A-13D show changes in lysosomal function by ZLN-005 (Example 5). Figure 13A: mRNA expression of Tfeb and MFI ratio of lysosomal staining in peritoneal cells 24 hours after CLP (n=6). Figures 13B-13D: Assessment of phagocytosis (n=6) (Figure 13B), lysosomal acidification (Figure 13C), and cellular ROS (Figure 13D) in peritoneal cells 24 hours after CLP. Figure 13E: Degradation of pretreated DQ™ BSA in lysosomes in peritoneal cells 24 hours after CLP. The graph shows the MFI ratio of degraded DQ Green BSA and LysoTracker Red. Figures 13F-13G: mRNA expression of lysosomal hydrolases (Figure 13F) and autophagy-related genes (Figure 13G) 24 hours after CLP (n=6). Figure 13H: Western blotting analysis of autophagic flux in THP-1 cells exposed to LPS for 6 hours followed by treatment with autophagy inhibitors for 2 or 6 hours. The graph shows the expression ratio of LC3-II protein corrected for the expression level of LC3-I protein (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001-0.05. [Figure 13-3]Figures 13A-13D show changes in lysosomal function by ZLN-005 (Example 5). Figure 13A: mRNA expression of Tfeb and MFI ratio of lysosomal staining in peritoneal cells 24 hours after CLP (n=6). Figures 13B-13D: Assessment of phagocytosis (n=6) (Figure 13B), lysosomal acidification (Figure 13C), and cellular ROS (Figure 13D) in peritoneal cells 24 hours after CLP. Figure 13E: Degradation of pretreated DQ™ BSA in lysosomes in peritoneal cells 24 hours after CLP. The graph shows the MFI ratio of degraded DQ Green BSA and LysoTracker Red. Figures 13F-13G: mRNA expression of lysosomal hydrolases (Figure 13F) and autophagy-related genes (Figure 13G) 24 hours after CLP (n=6). Figure 13H: Western blotting analysis of autophagic flux in THP-1 cells exposed to LPS for 6 hours followed by treatment with autophagy inhibitors for 2 or 6 hours. The graph shows the expression ratio of LC3-II protein corrected for the expression level of LC3-I protein (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001-0.05. [Figure 13-4]Figures 13A-13D show changes in lysosomal function by ZLN-005 (Example 5). Figure 13A: mRNA expression of Tfeb and MFI ratio of lysosomal staining in peritoneal cells 24 hours after CLP (n=6). Figures 13B-13D: Assessment of phagocytosis (n=6) (Figure 13B), lysosomal acidification (Figure 13C), and cellular ROS (Figure 13D) in peritoneal cells 24 hours after CLP. Figure 13E: Degradation of pretreated DQ™ BSA in lysosomes in peritoneal cells 24 hours after CLP. The graph shows the MFI ratio of degraded DQ Green BSA and LysoTracker Red. Figures 13F-13G: mRNA expression of lysosomal hydrolases (Figure 13F) and autophagy-related genes (Figure 13G) 24 hours after CLP (n=6). Figure 13H: Western blotting analysis of autophagic flux in THP-1 cells exposed to LPS for 6 hours followed by treatment with autophagy inhibitors for 2 or 6 hours. The graph shows the expression ratio of LC3-II protein corrected for the expression level of LC3-I protein (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001-0.05. [Figure 14-1]Figure 14 shows the effect of ZLN-005 on Tfeb (Example 5). Figure 14A: Study design to evaluate Tfeb mRNA expression and MFI ratio of lysosomal staining in peritoneal cells 24 hours after CLP (n=6). Figures 14B-14D: Assessment of phagocytosis (n=6) (Figure 14B), lysosomal acidification (C), and cellular ROS (D) in peritoneal cells 24 hours after CLP. Figure 14E: Degradation of pretreated DQ™ BSA in lysosomes in peritoneal cells 24 hours after CLP. The graph shows the MFI ratio of degraded DQ™ Green BSA and LysoTracker™ 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 blotting analysis of autophagic flux in THP-1 cells exposed to LPS for 6 hours and then treated with autophagy inhibitors for 2 or 6 hours. The graph shows the expression ratio of LC3-II protein corrected for the expression level of LC3-I protein (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001-0.05. [Figure 14-2]Figure 14 shows the effect of ZLN-005 on Tfeb (Example 5). Figure 14A: Study design to evaluate Tfeb mRNA expression and MFI ratio of lysosomal staining in peritoneal cells 24 hours after CLP (n=6). Figures 14B-14D: Assessment of phagocytosis (n=6) (Figure 14B), lysosomal acidification (C), and cellular ROS (D) in peritoneal cells 24 hours after CLP. Figure 14E: Degradation of pretreated DQ™ BSA in lysosomes in peritoneal cells 24 hours after CLP. The graph shows the MFI ratio of degraded DQ™ Green BSA and LysoTracker™ 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 blotting analysis of autophagic flux in THP-1 cells exposed to LPS for 6 hours and then treated with autophagy inhibitors for 2 or 6 hours. The graph shows the expression ratio of LC3-II protein corrected for the expression level of LC3-I protein (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001-0.05. [Figure 14-3]Figure 14 shows the effect of ZLN-005 on Tfeb (Example 5). Figure 14A: Study design to evaluate Tfeb mRNA expression and MFI ratio of lysosomal staining in peritoneal cells 24 hours after CLP (n=6). Figures 14B-14D: Assessment of phagocytosis (n=6) (Figure 14B), lysosomal acidification (C), and cellular ROS (D) in peritoneal cells 24 hours after CLP. Figure 14E: Degradation of pretreated DQ™ BSA in lysosomes in peritoneal cells 24 hours after CLP. The graph shows the MFI ratio of degraded DQ™ Green BSA and LysoTracker™ 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 blotting analysis of autophagic flux in THP-1 cells exposed to LPS for 6 hours and then treated with autophagy inhibitors for 2 or 6 hours. The graph shows the expression ratio of LC3-II protein corrected for the expression level of LC3-I protein (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001-0.05. [Figure 14-4]Figure 14 shows the effect of ZLN-005 on Tfeb (Example 5). Figure 14A: Study design to evaluate Tfeb mRNA expression and MFI ratio of lysosomal staining in peritoneal cells 24 hours after CLP (n=6). Figures 14B-14D: Assessment of phagocytosis (n=6) (Figure 14B), lysosomal acidification (C), and cellular ROS (D) in peritoneal cells 24 hours after CLP. Figure 14E: Degradation of pretreated DQ™ BSA in lysosomes in peritoneal cells 24 hours after CLP. The graph shows the MFI ratio of degraded DQ™ Green BSA and LysoTracker™ 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 blotting analysis of autophagic flux in THP-1 cells exposed to LPS for 6 hours and then treated with autophagy inhibitors for 2 or 6 hours. The graph shows the expression ratio of LC3-II protein corrected for the expression level of LC3-I protein (n = 3). (Con: control, Baf: bafilomycin A1, CQ: chloroquine) P value **<0.0001, P value *0.0001-0.05. [Figure 15-1] Figure 15A shows Western blotting analysis of molecular pathways in peritoneal cells 24 hours after CLP (Figure 15A) and evaluation of lysosomal acidification in THP-1 cells 5 days after wortmannin administration (Figure 15B) (Example 5). The graph in Figure 15A shows the expression ratio of each protein corrected for the expression level of Gapdh protein (n = 3). In Figure 15B, the statistical significance of the difference between the two groups treated with and without ZLN-005 after wortmannin administration was evaluated using the Kolmogorov-Smirnov test. P value **<0.0001, P value *0.0001-0.05. [Figure 15-2]Figure 15A shows Western blotting analysis of molecular pathways in peritoneal cells 24 hours after CLP (Figure 15A) and evaluation of lysosomal acidification in THP-1 cells 5 days after wortmannin administration (Figure 15B) (Example 5). The graph in Figure 15A shows the expression ratio of each protein corrected for the expression level of Gapdh protein (n = 3). In Figure 15B, the statistical significance of the difference between the two groups treated with and without ZLN-005 after wortmannin administration was evaluated using the Kolmogorov-Smirnov test. P value **<0.0001, P value *0.0001-0.05. [Figure 16] 1 is a graphical representation showing the proposed mode of action of ZLN-005 in polymicrobial sepsis. The figure is for illustrative purposes only and is not intended to limit any aspect or embodiment disclosed herein to a particular mechanism. [Figure 17-1] Figure 17 shows the effect of ZLN-005 or vehicle (DMSO) on peritoneal bacterial levels from mice in a CLP model of sepsis (Example 6). Figure 17A shows the study design. Figure 17B shows images depicting the changes in bacterial colonies in the ascites of sham-treated, CLP and vehicle-treated, and CLP and ZLN-005-treated mice over 2 and 24 hours. Figure 17C shows bar graphs depicting the bacterial counts in the ascites of sham-treated, CLP and vehicle-treated, and CLP and ZLN-005-treated mice 2 hours after CLP. Figure 17D shows bar graphs depicting the bacterial counts in the ascites of sham-treated, CLP and vehicle-treated, and CLP and ZLN-005-treated mice 24 hours after CLP. [Figure 17-2]Figure 17 shows the effect of ZLN-005 or vehicle (DMSO) on peritoneal bacterial levels from mice in a CLP model of sepsis (Example 6). Figure 17A shows the study design. Figure 17B shows images depicting the changes in bacterial colonies in the ascites of sham-treated, CLP and vehicle-treated, and CLP and ZLN-005-treated mice over 2 and 24 hours. Figure 17C shows bar graphs depicting the bacterial counts in the ascites of sham-treated, CLP and vehicle-treated, and CLP and ZLN-005-treated mice 2 hours after CLP. Figure 17D shows bar graphs depicting the bacterial counts in the ascites of sham-treated, CLP and vehicle-treated, and CLP and ZLN-005-treated mice 24 hours after CLP. [Figure 18-1] Figure 18 shows the effect of ZLN-005 on v-ATPase assembly in a CLP mouse model of sepsis (Example 7). Figure 18A shows the study design. Figure 18B shows graphs of Western blotting results as relative protein expression levels of the proteins indicated above each graph. [Figure 18-2] Figure 18 shows the effect of ZLN-005 on v-ATPase assembly in a CLP mouse model of sepsis (Example 7). Figure 18A shows the study design. Figure 18B shows graphs of Western blotting results as relative protein expression levels of the proteins indicated above each graph. [Figure 19] Figure 19 shows the effect of TRPML1 inhibition on ZLN-005-mediated lysosomal acidification induced by LPS stimulation (Example 8). Figure 19A shows the study design. Figure 19B shows pHrod fluorescence levels in different treatment groups. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present disclosure provides novel uses of ZLN-005 and other compounds of Formula (I) (and salts, hydrates, deuterated analogs, and fluorinated analogs thereof) in, for example, methods of treating subjects with lysosomal acidification disorders, methods of increasing lysosomal acidity in subjects, methods of treating subjects with sepsis (e.g., subjects with suppressed innate immune function, e.g., associated with lysosomal acidification disorders), methods of treating subjects with infectious diseases, and methods of treating subjects with diseases or disorders associated with V-ATPase dysfunction. Exemplary compounds of Formula (I) and salts, hydrates, deuterated analogs, and fluorinated analogs thereof are described in Section 5.2. Exemplary pharmaceutical compositions comprising compounds of Formula (I) and salts, hydrates, deuterated analogs, and fluorinated analogs thereof are described in Section 5.3. Exemplary features of the methods of the present disclosure are described in Section 5.4.

[0019] 5.1.Definition 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. The following definitions are provided to provide a full understanding of the terms used herein.

[0020] As used herein and in the embodiments, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "an agent" includes multiple agents, including mixtures thereof.

[0021] Unless otherwise indicated, the "or" conjunction is intended to be used in its proper sense as a Boolean logic operator, encompassing both the selection of features in an alternative (A or B, where selection of A is mutually exclusive with B) and the selection of features in a combination (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 construed to mean that "or" is used to refer to mutually exclusive alternatives.

[0022] The term V-ATPase refers to vacuolar ATPases. V-ATPases acidify the lumen of various organelles, including lysosomes, endosomes, and secretory vesicles, and play a key role in the function of these organelles (Colacurio and Nixon, 2016, Ageing Rev. 2016 32:75-88). V-ATPases contain V1 and V0 domains. 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 the ATP6V0A1, ATP6V0A2, ATP6V0A3, ATP6V0A4, ATP6V0B, ATP6V0C, ATP6V0D1, ATP6V0D2, ATP6V0E1, and ATP6V0E2 genes.

[0023] The term "pathogenic mutation" refers to a mutation in a wild-type gene that is associated with a disease. Pathogenic mutations can be, for example, point mutations in which a single nucleotide change results in a codon encoding a different amino acid, nonsense mutations that introduce a stop codon into a gene sequence, the insertion of one or more nucleotides, or the deletion of one or more nucleotides. The term "wild-type" refers to the predominant gene sequence in a species, such as Homo sapiens.

[0024] A therapeutically effective amount of a drug or composition is an amount sufficient to achieve the desired therapeutic effect, and therefore does not require a cure or complete remission.

[0025] As used herein, the terms treat, treating, treatment, and grammatical variations thereof include reducing or ameliorating a disorder or dysfunction and / or its associated signs or symptoms, or slowing or halting its progression. It is understood that treating a disorder or dysfunction does not necessarily require, but would not eliminate, the disorder, dysfunction, or its associated symptoms completely. Treatment according to the present disclosure can be applied preventatively (e.g., to subjects at risk of developing a disease or dysfunction associated with a lysosomal acidification disorder), palliatively, or curatively. Preventative treatment can be administered to a subject before the onset of signs or symptoms, at the early onset of signs or symptoms (e.g., at the onset of early signs and symptoms), or after the established onset of signs or symptoms. Preventative administration can occur from several days to several years before the onset of symptoms.

[0026] 5.2. Compounds of Formula (I) The method of the present disclosure comprises reacting a compound of formula (I):

[0027] [ka] wherein Ar is

[0028] [ka] and W 1 But NR 1 , O, or S, or W 9 If N, then W 1 Further CR 50 may be W 2 But, CR 2 or N, W 3 But, CR 3 or N, W 4 But, CR 4 or N, W 5 But, CR 5 or N, W 6 But, CR 6 or N, W 7 But, CR 7 or N, W 8 But, CR 8 or N, W 9 is C or W 1 is CR 50 In the case of W 9 may 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 and R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C 1-4 ) alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxy, (C1-C4)alkoxycarbonylamino, carboxamido, (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, halo, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino; R 7 and R 9 each independently represents hydrogen, deuterium, hydroxy, cyano, amino, halogen, halo(C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy,

[0029] [ka] and 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,

[0030] [ka] and R 30 However, (C1-C 10 ) hydrocarbyl, amino-substituted (C1-C 10 ) hydrocarbyl, (C1-C4) hydrocarbyl substituted (C1-C 10 ) hydrocarbyl, carboxy-substituted (C1-C 10 ) hydrocarbyl, carboxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkoxycarbonylamino, methylthio, heterocyclyl, (C1-C 10 ) Oxaalkyl, CHR 44 NHR 45 and guanidine, R 40 and R 41 are each independently hydrogen or (C-C)hydrocarbyl; R 42 is (C1-C5) alkyl, R 43 is (C1-C3) alkyl; R 44 is a naturally occurring amino acid side chain, R 45is H, methyl or (C1-C4)alkoxycarbonyl, and R 50 is H or (C1-C3) alkyl] or a salt, hydrate, deuterated analog, or fluorinated analog thereof, to a subject.

[0031] 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.

[0032] Exemplary compounds of formula (I) include the following compounds:

[0033] [ka]

[0034] In some embodiments, the drug is

[0035] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0036] In some embodiments, the drug is

[0037] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0038] In some embodiments, the drug is

[0039] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0040] In some embodiments, the drug is

[0041] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0042] In some embodiments, the drug is

[0043] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0044] In some embodiments, the drug is

[0045] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0046] In some embodiments, the drug is

[0047] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0048] In some embodiments, the drug is

[0049] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0050] In some embodiments, the drug is

[0051] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0052] In some embodiments, the drug is

[0053] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0054] In some embodiments, the drug is

[0055] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0056] In some embodiments, the drug is

[0057] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0058] In some embodiments, the drug 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.

[0059] In some embodiments, the drug is ZLN-005. In other embodiments, the drug is a salt of ZLN-005.

[0060] Pharmaceutical Compositions Compounds of formula (I) and their salts, hydrates, deuterated analogs, and fluorinated analogs can be prepared, for example, using techniques known in the art (e.g., Allen et al., eds., 2012, Remington: The Science and Practice of Pharmacy, 22 nd The formulation can be prepared for the intended route of administration according to the method described in the Pharmaceutical Press, London, UK (published in the Pharmaceutical Press, Edition, London, UK). Suitable routes of administration include, but are not limited to, intravenous administration and oral administration. Suitable routes of administration also include pulmonary administration, including inhalation. The most suitable route of administration may depend on the condition or disorder of the subject.

[0061] Compounds of formula (I), and salts, hydrates, deuterated analogs, and fluorinated analogs thereof, can be prepared by combining a compound of formula (I), or a salt, hydrate, deuterated analog, or fluorinated analog thereof, with one or more pharmaceutical excipients, such as those described in Handbook of Pharmaceutical Excipients, 8 th The pharmaceutical composition may be formulated with one or more excipients described in the Revised Ed. (2017). The pharmaceutical composition may be in unit dosage form.

[0062] 5.4. Uses of Compounds of Formula (I) The present invention provides methods of treating a subject with a compound of formula (I) (e.g., ZLN-005) and its salts, hydrates, deuterated analogs, and fluorinated analogs. In the methods of the present invention, the subject is preferably a mammal (e.g., a primate, or a rodent such as a mouse or rat), and most preferably a human.

[0063] In one aspect, the present disclosure provides a method for treating a subject with a disease or disorder associated with impaired lysosomal acidification, comprising administering a therapeutically effective amount of a drug to the subject, wherein the drug is a compound of Formula (I) (e.g., ZLN-005) or a salt, hydrate, deuterated analog, or fluorinated analog thereof. A therapeutically effective amount of a drug can be, for example, an amount effective to increase the acidity of lysosomes in a subject. Diseases and disorders associated with impaired lysosomal acidification include sepsis (e.g., a subject with intestinal perforation and / or abdominal infection), infectious diseases (including bacterial infections, such as multidrug-resistant bacterial infections, fungal infections, parasitic infections, and viral infections), and diseases and disorders associated with V-ATPase dysfunction, such as renal tubular acidosis, Zimmerman-Laband syndrome, cutis laxa type II or wrinkled skin syndrome, osteopetrosis, impaired glucose tolerance, diabetes, Parkinson's disease, Alzheimer's disease, and hearing loss.

[0064] In another aspect, the disclosure provides a method for increasing lysosomal acidity in a subject, comprising administering to the subject an amount of a drug effective to increase lysosomal acidity in the subject, wherein the drug is a compound of formula (I) (e.g., ZLN-005) or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

[0065] In another aspect, the present disclosure provides a method for treating a subject with sepsis (particularly a subject without systemic immune activation), an infection, or a disease associated with V-ATPase dysfunction, comprising administering a therapeutically effective amount of a drug to the subject, wherein the drug 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 has 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 another embodiment, the subject has monomicrobial sepsis (e.g., caused by a single pathogen species, such as bacteria or fungi). In some embodiments, the subject has early sepsis. Common (but not necessarily present) indicators of early sepsis include fever (e.g., above 38°C), a heart rate above 90 beats per minute, infection confirmed by a positive blood culture, and a rapid respiratory rate above 20 breaths per minute.

[0066] In some embodiments, when a subject has an infectious disease (e.g., bacterial, fungal, viral, parasitic, or a combination thereof), administering the drug can reduce bacterial burden. For example, administering the drug to a subject with a bacterial infection can reduce the bacterial burden of the subject. Methods for assessing pathogen (e.g., bacterial) burden levels are known in the art. For example, see Stranieri et al., 2018, Rev Inst Med Trop Sao Paulo. 60:e61.

[0067] Subjects, e.g., those with infection or sepsis, can be evaluated using the Sequential Organ Failure Assessment (SOFA) (Vincent et al., 1996, Intensive Care Med 22(7):707-10). SOFA scores range from 0 to 24, with higher scores correlated with higher mortality. In some embodiments, a subject treated according to a method of the present disclosure has 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) prior to treatment with a drug of the present disclosure (e.g., ZLN-005). In some embodiments, a subject has a SOFA score of 0 to 2 prior to treatment with a drug of the present disclosure (e.g., ZLN-005). In some embodiments, a subject has a SOFA score of 2 to 6 prior to treatment with a drug of the present disclosure (e.g., ZLN-005). In some embodiments, a subject treated according to a method of the present disclosure has a SOFA score of 7 to 9 (e.g., 7, 8, or 9) before treatment with a drug of the present disclosure (e.g., ZLN-005). In some embodiments, a subject treated according to a method of the present disclosure has a SOFA score of 10 to 12 (e.g., 10, 11, or 12) before treatment with a drug of the present disclosure (e.g., ZLN-005). In some embodiments, a subject treated according to a method of the present disclosure has a SOFA score of 13 to 14 (e.g., 13 or 14) before treatment with a drug of the present disclosure (e.g., ZLN-005). In some embodiments, a subject treated according to the methods of the present disclosure has 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) before treatment with a drug of the present disclosure (e.g., ZLN-005).

[0068] In some embodiments, the subject has at least one pathogen infection and one, two, three, or all four of the following: (1) lower than normal HLA-DR expression in peripheral blood; (2) higher than normal PD-1 expression in T cells; (3) lower than normal CD88 expression in neutrophils; and (4) lower than normal Th17 / Treg ratio. HLA-DR is strongly correlated with immune cell activation, and it has been reported that HLA-DR expression in the peripheral blood of sepsis patients is reduced compared to healthy individuals (Winkler et al., 2017, PLoS One 12:e0182427). Furthermore, it has been reported that a frequency of mononuclear cells expressing HLA-DR is less than 30% and indicates an immunosuppressed state (Misra et al., 2020, Crit Care Clin 36:167-176). Thus, in some embodiments, the subject has a frequency of mononuclear cells expressing HLA-DR of less than 30%. In T cells, increased expression of inhibitory signals, such as programmed cell death 1 (PD-1), correlates with poor prognosis in sepsis patients (Boomer et al., 2012, Crit Care 16:R112). Immune checkpoint inhibitors used in cancer treatment also showed significant efficacy in treating sepsis models (Huang et al., 2022, Mol Ther 30:1227-1238). This demonstrates the important role of Tregs in the immunosuppressive state of sepsis. Furthermore, the Th17 / Treg ratio was found to be a good indicator of the state of the immune system (Gupta et al., 2016, Cytokine 88:214-221). During the typical course of sepsis, this ratio increases early in sepsis and then declines, suggesting an immunosuppressive state consistent with a decline in HLA-DR (Xu et al., 2020, Scand J Immunol 91:e12813). In neutrophils, activated complement C5a inhibits the polymerization of RhoA, inactivating it and consequently reducing the expression of CD88, which has phagocytic function (Morris et al., 2011, Blood 117:5178-5188).It has been reported that reduced CD88 expression is a good reflection of an immunosuppressive state and is strongly correlated with secondary infection (Conway et al., 2018, Intensive Care Med 44:627-635).

[0069] In the method of the present disclosure, the amount of the drug can be an amount effective to increase the lysosomal acidity in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils).Lysosomal acidity can be measured by a lysosomal pH detection assay, for example, Dojindo Lysosomal Acidic pH Detection Kit (Dojindo product code L266).

[0070] In the methods of the present disclosure, the amount of drug can be an amount effective to increase lysosomal acidity in a subject's peritoneal cells, for example, peritoneal phagocytes (eg, macrophages, monocytes, or neutrophils).

[0071] In the methods of the present disclosure, the amount of the drug can be an amount effective to increase Tfeb (transcription factor EB) mRNA levels in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). In the methods of the present disclosure, the amount of the drug can be an amount effective to increase the activation (dephosphorylation) of TFEB in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). In the methods of the present disclosure, the amount of the drug can be an amount effective to increase the translocation of TFEB to the nucleus in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils).

[0072] In the methods of the present disclosure, the amount of drug can be an amount effective to increase the ratio of phosphorylated Akt (protein kinase B) to Akt (p-Akt / Akt) in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils).

[0073] In the methods of the present disclosure, the amount of drug may be an amount effective to increase the ratio of phosphorylated PI3K (phosphoinositide 3 kinase) to PI3K (p-PI3K / PI3K) in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils).

[0074] In the methods of the present disclosure, the amount of drug can be an amount effective to increase physical contact between mitochondria and lysosomes in phagocytic cells (e.g., macrophages, monocytes, or neutrophils) of the subject.

[0075] In the methods of the present disclosure, the amount of drug can be an amount effective to increase the spare respiratory capacity of phagocytic cells (eg, macrophages, monocytes, or neutrophils) of the subject.

[0076] In the methods of the present disclosure, the amount of drug can be an amount effective to increase glycolytic capacity in phagocytic cells (eg, macrophages, monocytes, or neutrophils) of the subject.

[0077] In the methods of the present disclosure, the amount of drug can be an amount effective to increase glycolytic reserve in phagocytic cells (eg, macrophages, monocytes, or neutrophils) of the subject.

[0078] In the methods of the present disclosure, the amount of drug can be an amount effective to increase lysosomal proteolysis in phagocytic cells (eg, macrophages, monocytes, or neutrophils) of the subject.

[0079] In the methods of the present disclosure, the amount of drug can be an amount effective to increase the mRNA levels of a hydrolase (e.g., Ctsd (cathepsin D)) and / or a membrane protein (e.g., Atp6v1A, Atp6v0d1, or Mcoln1 (mucolipin-1)) in a subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils).

[0080] In the methods of the present disclosure, the amount of the drug 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.

[0081] In some embodiments of the methods described herein, treatment may include reducing or alleviating one or more symptoms of the disease or dysfunction experienced by the subject.

[0082] In some embodiments of the methods of the present disclosure, 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 alpha. 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 as described in this paragraph can be measured by standard laboratory assays. Biomarker levels can be considered elevated if the measured value is above the upper limit of normal.

[0083] 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.

[0084] In some embodiments of the disclosed methods, the subject has suppressed innate immune function. Innate immune function can be measured by assaying the phagocytic activity of the subject's phagocytic cells (e.g., macrophages). If the phagocytic activity of the subject's phagocytic cells is lower than that of phagocytic cells of a healthy subject, the subject's innate immune function can be considered suppressed. Phagocytic activity assay kits are commercially available. An exemplary phagocytosis assay kit is the Cayman Chemical Phagocytosis Assay Kit (Cayman, product number 500290). In some embodiments, the amount of the drug administered is an amount effective to enhance the subject's innate immune function (e.g., as measured by the phagocytic activity of phagocytic cells such as macrophages).

[0085] In some embodiments of the methods of the present disclosure, the subject has sepsis (e.g., polymicrobial sepsis or monomicrobial sepsis) and (i) has suppressed innate immune function, (ii) has no systemic immune activation, or (iii) has suppressed innate immune function and no systemic immune activation. In some embodiments, the subject has an intestinal perforation.

[0086] In some embodiments of the disclosed methods, the subject has a bacterial infection, for example, an infection caused by a multidrug-resistant bacterium. Exemplary multidrug-resistant bacteria include vancomycin-resistant Enterococci (VRE), methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum β-lactamase (ESBL)-producing gram-negative bacteria, Klebsiella pneumoniae carbapenemase (KPC)-producing gram-negative bacteria, as well as Enterobacter species, E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. In other embodiments, the subject has a fungal infection (e.g., a Candida albicans infection). In other embodiments, the subject has a parasitic infection (e.g., a Plasmodium infection that causes malaria or a Trypanosoma cruzi infection that causes Chagas disease). In other embodiments, the subject has a viral infection (e.g., influenza). In some embodiments, the subject has an abdominal infection.

[0087] In some embodiments of the disclosed methods, the subject has a V-ATPase dysfunction (e.g., caused by a pathogenic mutation in a gene encoding a V-ATPase subunit or a V-ATPase accessory protein) and / or a disease or disorder associated with V-ATPase dysfunction. Exemplary diseases and disorders associated with V-ATPase dysfunction include, for example, renal tubular acidosis, Zimmerman-Laband syndrome, type II cutis laxa or wrinkled skin syndrome, osteopetrosis, impaired glucose tolerance, diabetes, Parkinson's disease, Alzheimer's disease, and hearing loss. Pathogenic ATP6V1B1 and ATP6V0A4 mutations can cause 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 Zimmerman-Laband syndrome (Kortum et al., 2015, Nat. Genet. 47(6):661-7). Pathogenic ATP6V0A2 mutations can cause cutis laxa type II or wrinkled skin syndrome (Kornak et al., 2008, Nat. Genet. 40(1):32-4). Pathogenic ATP6V0A3 mutations can cause osteopetrosis, which may be associated with neurological complications (Bhargava et al., 2012, JBC 287(32):26829-26839; Steward, 2003, Neuropathol Appl Neurobiol. 29(2):87-97). Defects in ATP6V1H are associated with impaired glucose tolerance and diabetes (Yang et al., 2022, Arch Biochem Biophys. 716:109116). Pathogenic mutations in ATP6AP2 (encoding a V-ATPase accessory protein) can cause Parkinson's disease, such as spastic X-linked Parkinson's disease (XPDS) (Korvatska et al., 2013, Hum Mol Genet. 22(16):3259-68).Pathogenic variants in PSEN1 (encoding presenilin-1, a protein involved in lysosomal targeting of V-ATPase) are associated with Alzheimer's disease (Lee et al., 2015, Cell Rep.12(9):1430-1444). Pathogenic variants in DMXL2 (encoding Dmx-like2, a protein involved in regulating V-ATPase activity) are associated with hearing loss (Chen et al., 2017, Genetics in Medicine 19:553-558).

[0088] In some embodiments of the methods of the present disclosure, the subject does not have a neurodegenerative disorder.

[0089] In the methods of the present disclosure, the drug can be administered by any suitable means, for example, enteral administration. In some embodiments, the drug is administered orally.

[0090] In some embodiments of the disclosed methods, the drug is administered at a dose ranging from 0.5 mg / kg to 1000 mg / kg per day, hi some embodiments, the dose is from 25 mg / kg to 1000 mg / kg per day. [Example]

[0091] [Example 1] 6.1. Example 1: ZLN-005 rescues septic animals in the cecal ligature puncture model A study was conducted to evaluate the ability of ZLN-005 to rescue mice from sepsis in a cecal ligament puncture (CLP) model.

[0092] C57BL / 6 mice approximately 10-20 weeks of age underwent cecal ligature puncture. 24 and 48 hours after cecal ligature puncture, mice were administered either ZLN-005 at 12 mg / kg (ip) or vehicle (DMSO) (Figure 1A).

[0093] The survival rates of mice administered ZLN-005 or vehicle are shown in Figure 1B. ZLN-005 increased the probability of survival compared to vehicle.

[0094] [Example 2] 6.2. Example 2: ZLN-005 increases macrophage phagocytic activity in vitro A study was conducted to evaluate the ability of ZLN-005 to enhance the phagocytic activity of macrophages.

[0095] THP-1 cells were differentiated into macrophages by incubation with 10 nM phorbol 12-myristate 13-acetate (PMA) for 48 hours (study hours 0–48). Cells were then incubated with 1 μM ZLN-005 or vehicle (DMSO) for 5 days (study hours 48–120), with or without 1 μg / mL LPS on the final day (study hours 96–120). Cells were then incubated with latex beads for 20 minutes, and phagocytic activity was assessed using a phagocytosis assay kit (IgG FITC) (Cayman Chemical, part number 500290).

[0096] The results are shown in Figure 2. ZLN-005 treatment increased the phagocytic activity of LPS-stimulated macrophages (see Figure 2, right bar).

[0097] [Example 3] 6.3. Example 3: ZLN-005 increases macrophage lysosomal acidity in vitro A study was conducted to evaluate the ability of ZLN-005 to increase lysosomal acidity in macrophages.

[0098] THP-1 cells were differentiated into macrophages by incubation with 10 nM phorbol 12-myristate 13-acetate (PMA) for 48 hours (study hours 0–48). Cells were then incubated with 1 μM ZLN-005 or vehicle (DMSO) for 5 days (study hours 48–120), with or without 1 μg / mL LPS on the final day (study hours 96–120). Cells were then stained for lysosomal acidity using a pH-sensitive dye.

[0099] Microscopic images of stained cells are shown in Figure 3. pH-sensitive dyes penetrate lysosomes based on pH, with fluorescence intensity increasing as acidity increases. ZLN-005 treatment was found to enhance lysosomal acidity in LPS-stimulated cells (see Figure 3, bottom right image). The MFI ratios for the various treatment groups are shown in Figure 4.

[0100] [Example 4] 6.4. Example 4: ZLN-005 increases phagocytic activity and lysosomal acidity in vivo A study was conducted to evaluate the ability of ZLN-005 to increase the phagocytic activity of phagocytes in vivo.

[0101] Mice underwent cecal ligature puncture at time 0 of the study. At the time of cecal ligature puncture, mice were administered either 12 mg / kg of ZLN-005 (ip) or vehicle (DMSO). 24 hours later, peritoneal cells were collected by peritoneal lavage, and phagocytic activity and lysosomal acidity were assessed. Peritoneal bacterial levels were also measured.

[0102] The results are shown in Figures 5 to 9. Mice treated with ZLN-005 were found to have increased phagocytic activity of phagocytes (Figures 5 to 6), lysosomal pH (Figures 7 to 8), and bactericidal activity (Figure 9) compared with mice treated with vehicle (DMSO).

[0103] [Example 5] 6.5. Example 5: ZLN-005 Improves Sepsis Survival 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 additional details related to materials and methods, as well as a detailed analysis of the study results.

[0104] 6.5.1 Materials and Methods 6.5.1.1. Sepsis model mouse C57BL / 6 mice were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan). Mice were maintained under specific pathogen-free conditions and provided with food and water ad libitum. Mice were anesthetized by inhalation of isoflurane (099-06571, Fujifilm Wako Pure Chemical Industries, Ltd., Tokyo, Japan). A midline incision was made, the cecum was exposed, ligated halfway between the distal pole and the base of the cecum, and punctured with a 21-gauge needle (NN-2116R, Terumo Corporation, Tokyo, Japan). Next, a small amount of fecal matter was gently squeezed out of the punctured cecum to confirm patency of the puncture site. The cecum was then repositioned, and the peritoneum and skin were sutured using 6 / 0 Ethicon Prolene sutures (Ethicon, Inc., Raritan, NJ, USA). Sham-operated mice underwent only incision and cecal exposure.

[0105] Intraperitoneal injection of ZLN-005 ZLN-005 (S7447, Selleck Chemicals, Houston, TX, USA) stock solution was prepared in dimethyl sulfoxide (DMSO) (046-21981, Fujifilm Wako Pure Chemical Industries, Ltd.) and adjusted to a concentration of 10 mM. Mice were injected daily with ZLN-005 (12 mg / kg) or the same amount of DMSO as the control group from day 0 to day 2. Mice were continuously monitored for survival up to day 6 after CLP.

[0106] 6.5.1.2. Isolation of Mouse Peritoneal Cells The outer peritoneal membrane was cut with scissors and gently pulled back to expose the endothelium lining the peritoneal cavity. The endothelium was punctured with an 18G Surflo IV catheter (SR-FS1851, Terumo Corporation, Tokyo), 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 1x 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 mixture was centrifuged at 800 × g for 5 minutes. The supernatant was discarded, and the cells were resuspended in PBS or culture medium.

[0107] 6.5.1.3. Peritoneal Cell Population Analysis Peritoneal cells were harvested 24 hours after CLP. Nonspecific Fc receptors were blocked with Fc blocking reagent (130-059-901, Miltenyi Biotec, Bergisch Gladbach, Germany) for 10 minutes at room temperature, followed by staining with PE anti-mouse / human CD11b antibody (101207, BioLegend, Inc., San Diego, California, USA) and FITC anti-mouse F4 / 80 antibody (123108, BioLegend, Inc.) or PE rat IgG2b, kappa isotype control antibody (400608, BioLegend, Inc.) and FITC rat IgG2a, kappa 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, Tokyo, Japan). Flow cytometry files were analyzed using FlowJo™ software (Ver. 10.8.1, Becton, Dickinson and Company).

[0108] 6.5.1.4. Echocardiography Chest hair was removed with cream the day before echocardiography was performed using a VisualSonics Vevo® 2100 (VisualSonics, Toronto, ON, Canada) equipped with an 18-38 MHz probe. Mice were anesthetized with isoflurane inhalation 24 h after CLP. The left ventricle was assessed in a parasternal short-axis view. Left ventricular end-systole and end-diastole were defined as the periods when the left ventricular cavity was shortest or most dilated, respectively. Diastolic left ventricular (LV) internal diameter, systolic LV internal diameter, diastolic LV anterior wall, and diastolic LV posterior wall were measured from LV M-mode tracings at the papillary muscle level. LV wall thickness was calculated as the average of the anterior and posterior wall thicknesses.

[0109] 6.5.1.5. Histology and Inflammation Scores A study was conducted to evaluate the ability of ZLN-005 to rescue mice from sepsis in a cecal ligament puncture (CLP) model.

[0110] The mouse heart, lungs, liver, right kidney, and spleen were harvested 24 hours after CLP and fixed in 4% paraformaldehyde (163-20145, Fujifilm Wako Pure Chemical Industries, Ltd.). All tissues were paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). Liver inflammation scores were assessed based on the severity of necrosis, hemorrhage, and infiltration in the liver using the method described by Shikuma et al., 2022, Front Immunol 13:825-171. Lung inflammation scores were assessed based on the severity of edema, intraalveolar cellular infiltration, congestion, and alveolar hemorrhage using the method described by An et al., 2019, Sci Rep 9:2836.

[0111] 6.5.1.6.Cell culture Human monocytic leukemia cell line THP-1 cells were cultured in Roswell Park Memorial Institute 1640 medium (11875-093, RPMI1640, Thermo Fisher Scientific Inc.) supplemented with 10% fetal bovine serum (FBS, 10270-106, Thermo Fisher Scientific Inc., Waltham, MA, USA). Cells were incubated at 37°C in a humidified 5% CO2 incubator. For the THP-1 inflammation model, cells were plated at 5 × 10 cells per well in a 12-well cell culture plate (353043, Corning Inc.). 5Cells were seeded at a concentration of 0.05% CI 0.01 to 0.05% in 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 h, the medium supernatant was carefully removed without removing the cells from the plate bottom and replaced with fresh medium containing 1 μM ZLN-005 or DMSO (0.1%) as a control. After 48 h, 1 μg / ml LPS (lipopolysaccharide, 125-05181, Fujifilm Wako Pure Chemical Industries, Ltd.) was added, and cells were harvested and used for studies at each time point. For PI3K inhibition, cells were seeded on plates and supplemented with 200 nM wortmannin (AG-CN2-0023-M001, Adipogen Life Sciences Inc.). For PGC1α gene knockdown, cells were nucleofected with 10 nmoles of siRNA-PGC1α (product number 4427037, Thermo Fisher Scientific Inc.) and 10 nmoles of siRNA-negative control (product number 4390844, Thermo Fisher Scientific Inc.) using Nucleofector™ 2b (Lonza, Walkersville, MD, USA) according to the manufacturer's protocol. For autophagic flux analysis, cells were stimulated with 1 μg / ml LPS for 6 hours, followed by 50 nM bafilomycin A1 (product number B0025, LKT Laboratories, Inc., Saint Paul, MN, USA) or 30 nM chloroquine (product number 08660-04, Nacalai Tesque, Kyoto, Japan) for 2 or 6 hours.

[0112] 6.5.1.7. RNA isolation, reverse transcription-PCR, and quantitative PCR Total RNA from cells and tissues was extracted using TRIzol™ (15596018, Thermo Fisher Scientific Inc.) and Direct-zol™ RNA MiniPrep Kit (R2052, Zymo Research, Irvine, CA, USA) with DNase I according to the manufacturer's recommendations. To perform qRT-PCR assays, 100 ng of total RNA was reverse transcribed using the PrimeScript™ RT Reagent Kit (RR036A, Takara Bio Inc., Shiga, Japan) and a T100 thermal cycler (Bio-Rad Laboratories, Inc.). qRT-PCR was performed on a CFX connect™ Real-Time System (Bio-Rad Laboratories, Inc.) using Kapa SYBR® Fast qPCR Kit Master Mix (2x) Universal (KK4602, Kapa Biosystems Ltd., Wilmington, MA, USA). Relative gene expression levels were normalized by the expression level of GAPDH (or Gapdh). mtDNA copy number (CN) was estimated from the ratio of 12S rRNA in mtDNA to ACTB (or Actb) in nuclear DNA using a relative quantification method based on the delta cycle threshold.

[0113] 6.5.1.8. Mitochondrial membrane potential (Δφ) 1 × 10 cells in culture medium containing 100 nM MitoTracker™ Green FM (MitoG, Thermo, M7514, Fisher Scientific, Inc.) and 100 nM Image-iT™ TMRM reagent (TMRM, T668, Thermo Fisher Scientific, Inc.). 5 Cells were resuspended at a concentration of 1 / ml and incubated at 37°C for 30 minutes. After staining, cells were immediately washed, resuspended in AutoMACS® running buffer, and evaluated using an SH800 cell sorter. Fluorescence intensity was analyzed using FlowJo™, and values ​​were calculated as the Δφ index by dividing the fluorescence intensity of TMRM by the fluorescence intensity of MitoG (TMRM / MitoG).

[0114] 6.5.1.9. Measurement of mitochondrial reactive oxygen species (mtROS) levels 1 × 10 cells in culture medium containing 5 μM MitoSOX™ Red mitochondrial superoxide indicator (MitoSOX, M36008, Thermo Fisher Scientific, Inc.). 5 The cells were resuspended at a concentration of 1 / ml and incubated for 30 min at 37°C. After staining, the cells were immediately washed, resuspended in AutoMACS® running buffer, and evaluated using an MA900 cell sorter (Sony Biotechnology Inc., Tokyo, Japan).

[0115] 6.5.1.1. Measurement of Intracellular Reactive Oxygen Species (ROS) 1 × 10 cells in culture medium containing 5 μM CellROX™ Deep Red (CellROX, C10491, Thermo Fisher Scientific, Inc.). 5 Cells were resuspended at a concentration of 1 / ml and incubated for 30 minutes at 37° C. After staining, cells were immediately washed, resuspended in AutoMACS® running buffer, and evaluated using an SH800 cell sorter.

[0116] 6.5.1.2. Mitophagy detection assay To detect mitophagy, THP-1 cells were transfected with a pMX retroviral vector incorporating monomeric Keima Red (mKeima Red). One week after retroviral introduction, mKeima Red-expressing cells were sorted using an SH800 cell sorter. Further sorting was performed to obtain more than 95% mKeima Red-expressing cells. The acidic mKeima Red signal was detected using an Attune (registered trademark) NxT flow cytometer (Thermo Fisher Scientific). mKeima Red was set to lasers at 488 nm (pH 7) and 561 nm (<pH 6) using emission filters of 590 / 40 nm and 615 / 20 nm, respectively. The mitophagy index was defined as the ratio of acidic (<pH 6) mKeima Red signal-positive cells to DMSO control cells at 0 h (Suzuki et al., 2017, Biochemical and Biophysical Research Communications 483:88-93).

[0117] 6.5.1.3. Phagocytosis assay Phagocytosis was evaluated using a phagocytosis assay kit IgG-FITC (500290, Cayman Chemical, Ann Arbor, MI, USA). Cells were suspended at a concentration of 3×10 5 in 1 ml of culture medium and stained with the latex bead-rabbit IgG-FITC complex of the kit at 37°C for 20 minutes. After staining, the cells were centrifuged at 400×g for 5 minutes, resuspended in 200 μl of autoMACS (registered trademark) running buffer, and evaluated using an SH800 cell sorter.

[0118] 6.5.1.4. Acidification of lysosomes Acidification of lysosomes was evaluated using pHrodo (trademark) Green dextran (P35368, Thermo Fisher Scientific, Inc.). Cells were suspended at a concentration of 3×10 5Peritoneal cells were suspended at a concentration of 0.01% and stained with 50 μg / ml pHrodo™ Green dextran from the kit for 20 minutes at 37° C. After staining, the cells were centrifuged at 400 × g for 5 minutes, resuspended in 200 μl of autoMACS® running buffer, and evaluated using an SH800 cell sorter.

[0119] 6.5.1.5. Lysosome staining Lysosomes were stained with LysoTracker™ Red DND-99 (LysoTracker Red, L7528, Thermo Fisher Scientific, Inc.). 3 × 10 cells were cultured in 1 ml of culture medium. 5 The cells were suspended at a concentration of 0.01% and stained with 50 nM LysoTracker™ Red from the kit for 15 minutes at 37° C. After staining, the cells were centrifuged at 400×g for 5 minutes, resuspended in 200 μl of autoMACS running buffer, and evaluated using an SH800 cell sorter.

[0120] Lysosomal Protein Degradation Cells were incubated with 0.1 mg / ml DQ™ Green BSA (DQ BSA, D12050, Thermo Fisher Scientific, Inc.) for 4 hours, washed twice, and incubated with fresh medium for 3 hours to allow DQ™ BSA to accumulate in lysosomes. Lysosomes were labeled with 50 nM Lysotracker™ 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.

[0121] 6.5.1.7. Measurement of Respiratory Function and Glycolysis Cellular respiratory function was measured using an XFe96™ extracellular flux analyzer (Agilent Technologies, Santa Clara, CA, USA). Cells were suspended in Seahorse™ XF RPMI medium (Agilent Technologies) containing 10 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine and plated at 1 × 10 cells per well in a Cell-Tak™ (CLS354240, Corning Inc.)-coated XFe96™ 96-well microplate (101085-004, Agilent Technologies). 5 Cells were seeded at a concentration of 0.1 μM. After seeding, cells were equilibrated for 20 minutes in a non-CO2 incubator before use. For respiratory function measurements, oligomycin (2 μM), carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 2 μM), and rotenone / antimycin A (0.5 μM) were prepared using reagents from the Seahorse™ XF Cell Mito Stress Test Kit (103015-100, Agilent Technologies) and added sequentially to each well after baseline measurements. Data are presented as oxygen consumption rate (OCR; pmol / min). Basal respiration, ATP production, maximal respiration, proton leak, spare respiratory capacity, non-mitochondrial oxygen (non-MTC), and coupling efficiency were calculated using Wave Controller™ 2.4 (Agilent Technologies). For glycolysis measurements, glucose (10 mM), oligomycin (1 μM), and 2-deoxy-D-glucose (2-DG, 50 mM) were prepared using reagents from the Seahorse™ XF Cell Glycolysis Stress Test Kit (103020-100, Agilent Technologies) and added sequentially to each well after baseline measurements. Data are presented as extracellular acidification rate (ECAR; mpH / min). Glycolysis, glycolytic capacity, and glycolytic reserve were calculated using Wave Controller™ 2.4.

[0122] 6.5.1.8. Peritoneal bacterial quantification Mice were euthanized using isoflurane 24 hours after CLP. After disinfection of the abdominal skin, an incision was made, and the peritoneal cavity was lavaged with 4 ml of sterile PBS containing 2 mM EDTA, taking care not to damage the muscle layer. The resulting peritoneal lavage solution was diluted 1:10,000 with PBS, and 40 μl of the diluted solution was applied to antibiotic-free LB agar (22700-025, Thermo Fisher Scientific, Inc.). After incubation at 37°C for 24 hours in a non-humidified incubator, the plates were photographed using a ChemiDoc Imaging System, and colony-forming units (CFUs) were counted using ImageJ™ (version 1.53t, National Institutes of Health, Bethesda, MD, USA). Results were presented in a 1 cm plate. 2 The results were expressed as CFU per 1000 cells.

[0123] 6.5.1.9. Subcellular isolation for Western blotting 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 Industries, Ltd.), pH 7.2) and homogenized by passing the cells 20 times through a 29-gauge needle. The lysate was kept on ice for 20 min and then centrifuged at 720 g for 5 min to separate the nuclei into a pellet and the cytoplasm, membranes, and mitochondria into a supernatant. The supernatant was centrifuged again at 12,000 g for 10 min. The cytoplasmic supernatant from the pellet was removed and transferred to a clean tube. The nuclear pellet was washed with 500 μL of fractionation buffer and centrifuged at 720 g for 10 min. The pellet was resuspended in fractionation buffer and then sonicated to shear genomic DNA and homogenize the lysate. These proteins were analyzed by Western blotting.

[0124] Western Blotting Cytoplasmic proteins were dissolved in RIPA buffer (182-02451, Fujifilm Wako Pure Chemical Industries, Ltd.), boiled for 10 min, electrophoresed on a 10% Mini-PROTEAN® TGX precast protein gel (4561036, Bio-Rad Laboratories Inc.), and 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, Abcam plc.), 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.). 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, IL-1) were incubated for 1 h.After washing, the membranes were 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. The blots were then developed using Clarity™ Western ECL Substrate (1705060, Bio-Rad Laboratories Inc.) or Clarity™ Max Western ECL Substrate (1705060, Bio-Rad Laboratories Inc.), and protein bands were visualized using a VersaDoc™ or ChemiDoc™ imaging system (Bio-Rad Laboratories Inc.). Protein levels were quantified using ImageJ™.

[0125] 6.5.1.11. Immunocytochemistry Cells were fixed in 4% paraformaldehyde solution for 15 minutes at 4°C in the presence of a protein blocking solution consisting of PBS supplemented with 5% normal goat serum (X090710-8, Agilent Technologies Inc., Santa Clara, CA, USA). Cells were incubated with anti-TFEB antibody (ab267351, Abcam plc.) in PBS overnight at 4°C. Cells were thoroughly washed in PBS and incubated with Alexa Fluor™ 488-tagged anti-rabbit IgG (H+L) antibody (Thermo Fisher Scientific, Inc.) for 30 minutes at room temperature. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1:500 dilution, #5748, Fujifilm Wako Pure Chemical Industries, Ltd.) in PBS for 30 minutes at room temperature. Fluorescent images were acquired using a Biorevo™ BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan). Identification of nuclear-translocated TFEB was performed using ImageJ™. First, multicolor images were separated into TFEB-stained and DAPI-stained images. These images were converted to binary images by thresholding, with foreground pixels assigned a maximum value of 255 and background pixels assigned the minimum possible value of 80. The overlapping area between the TFEB and DAPI regions was defined as nuclear TFEB. The percentage of cells containing nuclear TFEB in the image was calculated.

[0126] 6.5.1.12. Mitochondrial Network Analysis (MiNA) Mitochondrial morphology analysis was performed using the Mitochondrial Network Analysis (MiNA) toolset 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 masking and local contrast enhancement, are presented to the user through the MiNA interface. For analysis, images were first binarized by thresholding, assigning a maximum value of 255 to foreground pixels and a minimum value of 0 to background pixels. Next, using ImageJ™'s built-in skeletonization function, the binary image was converted into a skeleton, which represents the features of the original image as a wireframe of 1-pixel-wide lines. All pixels in the skeleton were classified into three categories: endpoint pixels, slab pixels, and junction pixels. The areas of the mitochondrial footprints, slab pixels, and junction pixels of individual cells were evaluated. The mitochondrial footprint was calculated by multiplying the number of pixels in the binary image containing signal by the pixel area, if calibration information was available.

[0127] 6.5.1.13. Mitochondrial-lysosome contact site analysis Analysis was performed using ImageJ™. First, multicolor images were separated into images stained with MitoTracker™ Green and LysoTracker™ Red. These images were converted to binary images by thresholding, with foreground pixels assigned a maximum value of 255 and background pixels assigned a minimum value of 100. Contact sites were defined as the overlapping areas of mitochondrial and lysosomal regions of interest (ROIs). The ratio of contact sites to mitochondrial area was assessed for individual cells.

[0128] 6.5.1.14.Statistical analysis Results are presented as mean ± standard deviation. Statistical differences between groups were assessed using unpaired parametric t-tests for bar graphs. The Mantel-Cox test was used for statistical analysis of Kaplan-Meier survival curve (Prism™ 9 software, GraphPad Prism Software Inc., San Diego, CA, USA) data sets. P < 0.05 was considered to indicate significance.

[0129] 6.5.2.Results 6.5.2.1. Improved Survival and Anti-inflammatory Effects of ZLN-005 in a CLP Sepsis Model 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 ligation site and the number of perforations. Ligation was performed at the base or middle of the cecum, and the severity was controlled by making one or two perforations with a 20-gauge needle beyond the ligation site. In a model in which the base of the cecum was ligated and two perforations were performed, the survival rate was 50% on postoperative day 2 (data not shown). The effect of ZLN-005 on naive THP-1 cells was observed over time at the mRNA and protein levels, with a two-fold increase in expression at 48 and 36 hours, respectively (data not shown). To examine the therapeutic effect of ZLN-005, CLP was performed on 10- to 20-week-old C57BL / 6 mice, followed by intraperitoneal injection of ZLN-005 for three consecutive days (Figure 1A).

[0130] In the sham-operated group, 25% of mice died after 24 hours, while none died in the ZLN-005 group. After 72 hours, all sham-operated mice had died, while 40% of ZLN-005-treated mice survived, and 30% remained alive 6 days later. Kaplan-Meier survival analysis showed significantly better survival in the ZLN-005 group (Figure 1B). Furthermore, just 2 hours after the first drug administration after CLP, the behavior of the animals was strikingly contrasting. In the sham-operated group, mice remained in the same place, were very unresponsive to stimuli, and barely moved. In contrast, in the ZLN-005 group, mice spontaneously moved around, rarely stayed in the same place, and quickly showed escape behavior in response to stimuli. These early behavioral changes suggest that ZLN-005 exerts a rapid effect on bacteria leaking into the peritoneal cavity, in addition to its mechanism of action as a PGC1α activator at the transcriptional level. In the CLP model, the peritoneal macrophage-monocyte lineage has been reported to exhibit a decisive response in the early stages of the disease. ZLN-005 has been reported to inhibit the expression of Pgc1α and Tfam, which plays an essential role in mitochondrial biogenesis, in peritoneal cells, including macrophages and monocytes. Both transcripts were significantly elevated (Figure 1C).

[0131] In the CLP model, inflammatory cytokines dramatically increased from the early stages, suggesting that excessive inflammatory cytokines play a key role in the pathogenesis (Figure 1D). In the sham group, TNF-α and II1β were expressed most highly in the liver, followed by the kidney, lung, and heart. II6 was most highly expressed in the kidney in the sham group, but was significantly downregulated by ZLN-005 treatment. TNF-α and II1β were also downregulated at levels similar to those in the sham group. Although the expression of INF-γ itself was lower than that of other cytokines, the inhibitory effect of ZLN-005 was significant in the heart and lung, where it was reduced to the expression level of the sham group. These results demonstrate the potent anti-inflammatory effect of ZLN-005.

[0132] 6.5.2.2. Organ Protection of ZLN-005 in the CLP Sepsis Model Echocardiography was performed 24 hours after CLP. The ejection fraction in the sham group ranged from 60% to 40%, whereas the ejection fraction in the ZLN-005-treated group remained in the 50% range (Figure 10A). Cardiac pathological examination revealed no significant findings such as cellular infiltration or hemorrhage, suggesting that dysfunction may be caused by humoral factors such as pro-inflammatory cytokines and coagulation factors, and that suppression of the cytokine storm by ZLN-005 plays a role in preserving cardiac function (data not shown). Similarly to the heart, the kidneys showed no significant changes (data not shown). However, the liver showed disorganized lobule structure, hemorrhagic lesions, and cellular infiltration in the sham group, whereas these findings were slightly improved in the ZLN-005-treated group (data not shown). Quantification revealed no statistically significant changes (Figure 10B). The spleen showed significant destruction of follicular structure in the sham group, whereas the follicular structure was well preserved in the ZLN-005-treated group (data not shown). The lungs showed the most prominent lesions, with cellular infiltration clearly suppressed, the interstitium preserved, and edema very mild in the ZLN-005 group (data not shown). Quantification also showed significantly less damage in the ZLN-005 group (Figure 10C).

[0133] We also investigated the effect of ZLN-005 on the clearance of intraperitoneal bacteria in a CLP model. Ascites fluid was collected 2 and 24 hours after CLP treatment, and colonies were counted by bacterial culture. At 2 hours, the number of colonies in the ZLN-005-treated group was already reduced by more than half, and by 24 hours, the number of colonies had further decreased by approximately one-quarter (data not shown). This indicates that ZLN-005 promotes the clearance of bacteria released into the peritoneal cavity. These results suggest that ZLN-005 was able to make the peritoneal monocyte-macrophage lineage more responsive to uncontrolled bacterial load. The rapid onset of action at 2 hours suggests that its mechanism of action is directly related to metabolism and digestion, rather than requiring transcription and translation processes.

[0134] 6.5.2.3.ZLN-005-induced changes in mitochondrial function In the CLP model, mitochondrial respiratory capacity and glycolytic metabolic capacity were examined in peritoneal cells 1 day after treatment. Peritoneal cells were harvested after CLP treatment and subjected to sequential administration of respiratory chain inhibitors and glycolysis inhibitors using Seahorse™ to obtain profiles of mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic capacity. While no significant changes in OXPHOS were observed in peritoneal cells with CLP on postoperative day 1, administration of ZLN-005 significantly reduced proton leakage, resulting in a significant increase in coupling efficiency. Furthermore, spare respiratory capacity significantly increased (Figure 11A). Meanwhile, similar to OXPHOS, CLP treatment did not significantly alter glycolysis on postoperative day 1, but administration of ZLN-005 significantly increased glycolytic capacity and glycolytic reserve (Figure 11B). This indicates that ZLN-005 is involved in improving the reserve capacity of both metabolic pathways.

[0135] The previously proposed mechanism of action of ZLN-005 was to enhance PGC1α at the transcriptional level; however, there were no significant changes in the OXPHOS profile other than reserve capacity, and no changes resulting in a dramatic improvement in peritoneal bactericidal activity were observed. Therefore, to investigate an alternative mechanism of action of ZLN-005 in this model, we investigated the mitochondrial function of ZLN-005 using the human macrophage line, THP-1. We examined the effect of ZLN-005 on THP-1 in an LPS-stimulated model 24 hours after stimulation. Pgc1α mRNA was significantly elevated at the transcriptional level with or without LPS stimulation with ZLN-005, whereas Tfam mRNA showed no significant change in either group (Figure 12A). However, although mtDNA copy number was reduced by LPS stimulation, ZLN-005 treatment significantly restored mtDNA copy number compared to the untreated group (Figure 12B). PGC1α is a key regulator of mitochondrial biogenesis, and mitochondrial DNA copy number was examined as a phenotype. ZLN-005 significantly increased copy number (Figure 12B). Mitochondrial mass was measured using MitoGreen and showed no significant changes after LPS stimulation or ZLN-005 treatment (data not shown). TMRM-based mitochondrial membrane potential (mtMP) (data not shown) was corrected for mitochondrial mass, and the mtMP / mtMass ratio increased with LPS stimulation and further increased with ZLN-005 treatment (Figure 12C). Mitochondrial ROS was significantly increased with LPS stimulation, but this increase was reversed by ZLN-005 (Figure 12D). To measure mitophagy, THP-1 cells constitutively expressing MitoKeima Red were engineered and the time-dependent effects of ZLN-005 under LPS stimulation were examined. Mild induction of mitophagy was detected as early as 2 hours after ZLN-005 administration, and no increase in mitophagy was observed over time (Figure 12E). This enhancement of mtDNA replication and increased mitophagy suggests an enhancement of mitochondrial turnover, although the extent of this enhancement was only slight under the conditions of this study. Without wishing to be bound by theory, it is possible that mitochondrial turnover does not significantly contribute to changes in cellular phenotype.

[0136] Because ZLN-005 significantly affected mitochondrial biogenesis and turnover under LPS stimulation, we investigated the effect of ZLN-005 on mitochondrial dynamics using THP-1 cells. Mitochondria were stained with TMRM, and mitochondrial morphology was quantified using ImageJ™. The footprint, which indicates mitochondrial mass, was more sensitive than the MitoGreen™ MFI measured by FACS (data not shown). ZLN-005 slightly increased mitochondrial mass in the unstimulated condition, but no change was observed in the LPS-stimulated condition (Figure 12F). No changes were observed in slab pixels and junction pixels, indicators of branching status, in the four groups (Figure 12F). The proximity of mitochondria and lysosomes was assessed by co-staining with dyes that stain the two organelles. The section with the largest lysosomal staining area was selected, and the co-stained area in that section was calculated. Although only one section was evaluated, a clear increase in co-stained area was observed after 24 hours of LPS exposure following exposure to ZLN-005. Without being bound by theory, these findings suggest the tethering of two organelles, suggesting that lysosomes may be involved in another mechanism of action of ZLN-005 (Figure 12G).

[0137] Lysosomal changes induced by ZLN-005 Subsequent studies were conducted to evaluate the hypothesis that phagocytic cells may have rapidly responded to a massive bacterial load by changing their phenotype and enhancing the process from phagocytosis to digestion. Peritoneal cells were harvested from the CLP model and used as material for studies of the phagocytosis-lysosome pathway. Lysosomes contain hydrolytic enzymes that digest pathogens and macromolecules. To process large amounts of pathogens, the phagolysosome's volume must expand and its lumen must be maintained at approximately pH 4.6, optimal for these hydrolytic enzymes. First, we examined the expression of Tfeb mRNA. It was significantly reduced by CLP alone and significantly increased by ZLN-005 treatment, but was still lower than that of the sham group (Figure 13A). Lysosomal mass increased with CLP alone and further increased with ZLN-005 treatment (Figure 13A). Peritoneal cells were incubated with fluorescent dextran, and the fluorescence intensity was used to assess the degree of phagocytosis. Phagocytosis was significantly elevated in the CLP group, and further elevated with ZLN-005, but the difference was not significant (Figure 13B). Next, we examined the final stage of bacterial killing and changes in the lysosomal lumen. Lysosomal pH is one of the most fundamental requirements for lysosomal function, and we measured increased acidity as an increase in fluorescence intensity using pHrod. Lysosomal acidity increased in the CLP group and was further significantly enhanced by ZLN-005 treatment (Figure 13C). However, intracellular ROS expression, measured by CellRox™, did not significantly increase during this period (Figure 13D). Acidification of lysosomal pH is associated with enhanced hydrolytic enzyme function, as demonstrated by the use of DQ™-BSA. Enhanced proteolysis releases fluorescent dyes from quenching inhibition, resulting in fluorescence excitation. Addition of DQ™-BSA resulted in some degree of proteolysis in unstimulated cells, but the extent of proteolysis was enhanced by LPS exposure and further enhanced by ZLN-005 treatment as observed by fluorescence microscopy (Figure 13E), and quantitative analysis by FACS showed that ZLN-005 significantly enhanced proteolysis (Figure 13E).Without being bound by theory, these results suggest that in this model, the oxidative burst does not significantly contribute to pathogen eradication 1 day after infection, but rather the hydrolysis process appears to be the major contributing factor.

[0138] The mRNAs of most lysosomal proteins with a CLEAR motif in their promoters, which are targets of TFEB, were elevated compared to those in the CLP group. In particular, the mRNAs of hydrolases such as Ctsd and membrane proteins such as Atp6v1A, Atp6v0d1, and Mcoln1 were all significantly elevated (Figure 13F). Regarding autophagy, Becn1 and Gabarap were only slightly elevated, and Rab7 was unchanged by ZLN-005 treatment, whereas Sqstm / p62 was significantly increased (Figure 13G). Without being bound by theory, analysis of these transcripts suggests that ZLN-005 may be more involved in phagolysosomal acidification than in xenophagy. Bacterial killing in phagocytes is achieved by phagosome-lysosome fusion or by xenophagy, in which the host induces autophagy when the phagosome is damaged by bacterial escape. In the THP-1 LPS model, we examined autophagic flux using LC3 conversion as an indicator in the presence of the ATPase inhibitor bafilomycin A1 and the autophagosome-lysosome fusion inhibitor chloroquine. Autophagy flux was measured after 6 hours of exposure to LPS and 2 or 6 hours of treatment with autophagy inhibitors. Neither LC3-II nor the LC3-II / I ratio increased with ZLN-005 treatment (Figure 13H). The effects of the two inhibitors were consistent in this system, significantly increasing LC3-II and the LC3-II / I ratio, but ZLN-005 did not significantly affect the increases in LC3-II and LC3-II / I ratios. Without wishing to be bound by theory, these results indicate that ZLN-005 does not significantly affect macroautophagy.

[0139] 6.5.2.5. TFEB Adjustment PGC1α knockdown (KD) did not significantly alter Tfeb mRNA levels in THP-1 cells stimulated with LPS alone (Figure 14B). Addition of ZLN-005 to LPS stimulation significantly increased Tfeb mRNA levels, and PGC1α KD caused an increase in Tfeb mRNA levels, although the rate of increase was significantly reduced (Figure 14B). Without being bound by theory, these results suggest that PGC1α plays a role in regulating TFEB at the transcriptional level after ZLN-005 treatment. Lysosomal acidification was not altered by PGC1α KD in LPS stimulation alone, but was significantly elevated when ZLN-005 was administered, although the increase in acidity was slightly reduced (Figure 14C). Without being bound by theory, this suggests that ZLN-005 may have a mechanism of action other than transcriptional regulation of Tfeb.

[0140] Alternatively, Tfeb is always present in the cytoplasm, and its nuclear translocation is regulated by phosphorylation, acting as a transcription factor. To investigate whether ZLN-005 might be involved in this pathway, we performed immunofluorescence staining of TFEB in THP-1 cells exposed to LPS and treated with ZLN-005 (Figure 14D). Quantification showed that nuclear translocation proceeded even without LPS exposure, but was further enhanced by LPS exposure with ZLN-005 (Figure 14D). To further confirm TFEB nuclear translocation, cells were harvested and separated into nuclear and cytoplasmic fractions, and Western blotting was performed to examine the presence of TFEB. Under LPS exposure, ZLN-005-induced nuclear translocation of TFEB was highly evident (Figure 14E). Finally, we investigated the molecular pathway of action of ZLN-005 and found that among the various factors promoting V-ATPase V0-V1 association, the PI3K / AKT axis is the pathway most closely involved in phagocytosis. We also investigated the involvement of mTORC1 downstream of this pathway, as well as AMPK, which is closely related to the regulation of both PGC1α and TFEB, using WB. We demonstrated that PI3K and AKT were significantly phosphorylated and activated by ZLN-005 treatment (Figure 15A). However, activation of S6 downstream of mTORC1 was not observed, and AMPK activity was barely altered by ZLN-005 administration (Figure 15A). Although both mTORC1 and AMPK are deeply involved in autophagy regulation, ZLN-005 did not activate either pathway in this study. Because the PI3K-AKT pathway was significantly activated by ZLN-005 and may play an important role in TFEB phosphorylation, we evaluated whether the PI3K inhibitor wortmannin could counteract the changes in lysosomal acidification. ZLN-005 induced lysosomal acidification in the absence of wortmannin, but not in its presence (Figure 15B). Without being bound by theory, these results suggest that ZLN-005 may cause lysosomal acidification via PI3K, which may promote V0-V1 association.

[0141] Mechanism of Action of ZLN-005 Without being bound by theory, the mechanism of action of ZLN-005 in bacterial infections is thought to be as follows: ZLN-005 acidifies the pH of lysosomes in macrophages, promoting lysosomal biogenesis and thereby facilitating the degradation of endocytosed bacteria (Figure 16). Regarding the V-ATPase, which consists of a V0 complex present in the lysosomal membrane and a V1 complex present in the cytoplasm or bound to the V0 complex, the V0-V1 association is promoted by ZLN-005. To drive V-ATPase, ZLN-005's promotion of mitochondrial biogenesis increases ATP production. Furthermore, the action of PI3K brings the two organelles into close proximity. Meanwhile, in the latter case, PI3K induces the conversion of phosphatidylinositides, dephosphorylating TFEB and translocating it to the nucleus using Ca as a second signal. This promotes lysosomal biogenesis and the production of various hydrolases present in the lysosomal membrane and lumen. All of these events contribute to the complete digestion of endocytosed bacteria.

[0142] 6.5.3. Discussion This example confirms that intraperitoneal administration of ZLN-005 has a therapeutic effect on polymicrobial sepsis. Without being bound by theory, this study suggests that the molecular mechanism underlying this effect is TFEB-mediated enhancement of lysosomal acidity and biogenesis. Again, without being bound by theory, it is possible that the upstream signal for TFEB activation is PI3K. This example demonstrates that ZLN-005 has dual effects on two intracellular organelles, mitochondria and lysosomes, and that these mechanisms of action may be coordinated. Furthermore, this example demonstrates that ZLN-005 has a mechanism of action that contributes to the improvement of pathological conditions by eliminating bacteria in the early stages of sepsis.

[0143] In this study, ZLN-005-induced Tfeb mRNA induction under LPS stimulation was suppressed by PGC1α knockdown, indicating that Tfeb mRNA is regulated by PGC1α. Meanwhile, immunofluorescence staining and Western blotting analysis of nuclear and cytoplasmic fractions revealed enhanced nuclear translocation of TFEB by ZLN-005 treatment, suggesting that ZLN-005 directly acts on TFEB nuclear translocation. The TFEB-mediated effect of ZLN-005 on lysosomes, in addition to the reciprocal enhancement of PGC1α at the transcriptional level, may be a mechanism for promoting TFEB nuclear translocation that persists for a certain period of time from the very early stage of pathological changes.

[0144] V-ATPase, a molecule involved in lysosomal acidification, has been shown to be regulated by the reversible degradation of V0-V1, but its role in pathology has primarily focused on cancer. Significantly, this example sheds light on the role of V-ATPase in sepsis. In sepsis, the inability of the lysosomal system to adequately respond to excessive bacterial load may contribute to early death, and dysfunction of V0-V1 ATPase association may be a molecular mechanism associated with this inability. In this example, ZLN-005 promoted bacterial killing and enhanced survival by increasing lysosomal acidity, suggesting that V0-V1 association is one of the molecular bases of innate immunity against sepsis.

[0145] Without being bound by theory, ZLN-005 may improve survival in the CLP model by promoting bacterial killing in the phagolysosome. The final stage of bacterial killing is thought to be directly carried out by lysosomal acidity, luminal hydrolases, and ROS. In this example, ZLN-005 did not significantly affect cellular ROS, and a significant increase and activation of hydrolases was observed, suggesting that bacterial elimination may be due to hydrolases rather than cellular ROS.

[0146] In this study, ZLN-005 strongly induced TFEB, a master gene that stimulates the production of membrane proteins and hydrolases in lysosomes, which then acts to increase lysosomal mass. TFEB is a transcriptional regulator that rapidly responds to environmental changes, primarily through post-translational modifications, and has a positive feedback loop that enhances PGC1α transcription. TFEB stability and translocation from the cytoplasm to the nucleus are regulated by phosphorylation by various kinases and dephosphorylation by phosphatases, and TFEB translocates to the nucleus to promote the expression of its own genes and genes involved in autophagy and lysosomal biogenesis. Many signal regulators, including mTORC1, negatively regulate TFEB, while calcineurin positively regulates it. PIKFYVE on the lysosome is a kinase that receives signals from AKT, which uses PI3P as a substrate to generate PI(3,5)P2. PI(3,5)P2 activates TRPML1, encoded by MCOLN1, which has been reported to release Ca2+ from lysosomes into the cytoplasm. Increased TRPML1 levels activate calcineurin, which subsequently dephosphorylates TFEB, acting as a transcription factor. The involvement of PI3K in the mechanism of action of ZLN-005 was indirectly demonstrated by the enhanced phosphorylation of PI3K and AKT in a CLP model. Furthermore, ZLN-005-induced lysosomal acidification was inhibited by the PI3K inhibitor wortmannin, directly demonstrating that ZLN-005 acts through PI3K. These factors, including PIKFYVE, TRPML1, and calcineurin, may be involved in and contribute to ZLN-005-induced TFEB activation.

[0147] Signals from PI3K are transmitted via AKT to mTORC1, which phosphorylates various factors, leading cells to anabolism, including proliferation. However, mTOR signaling inhibits all autophagic processes, not only initiation and nucleation of autophagy, but also autophagosome elongation, maturation, and termination. Some bacteria exploit this mTORC1-mediated inhibition of the autophagy process to evade innate immunity. Metabolism in sepsis causes a surge in energy demand, activating AMPK, which inhibits mTORC1 through Rheb-mediated phosphorylation of TSC2. Furthermore, AMPK maintains autophagy independently of mTOR through phosphorylation of ULK1. In this study, the S6 phosphorylation pathway downstream of mTORC1 was not involved in the pathogenesis of sepsis, and further activation of PI3K by ZLN-005 did not significantly alter downstream signaling of mTORC1. However, phosphorylation of AMPK, which acts antagonistically against mTORC1, is activated in sepsis, but no change was observed in this study. This is consistent with the fact that in this example, the expression of autophagy-related genes only slightly changed in the early stage of sepsis.

[0148] This example demonstrates that ZLN-005 increases mitochondrial-lysosome contact sites, enhancing OXPHOS. Physical contact between these two organelles has long been reported to occur during a mitophagy-like degradative process. However, recent studies have recognized that transient contact exists physiologically as a non-degradative process. This example demonstrates that LPS-stimulated THP-1 cells with ZLN-005 induces tethering of the two organelles from as early as 1 hour to as late as 24 hours. While the molecular mechanism by which ZLN-005 promotes coordination between the two organelles remains unclear, enhanced physical contact may (without wishing to be bound by theory) allow the dual functions of mitochondria and lysosomes to function more cohesively.

[0149] In sepsis, much effort has been focused on controlling the hyperimmune response. However, drug development to improve the efficiency of bacterial clearance in the early stages has stalled. This example demonstrates that ZLN-005 improves overall survival by ameliorating the pathophysiology of the disease in the early stages, addressing a long-standing need in the field. Furthermore, the dual effects of ZLN-005 on mitochondria and lysosomes demonstrated in the examples support the use of ZLN-005 in the treatment of neurodegenerative disorders based on lysosomal acidification deficiency, including Alzheimer's disease, Parkinson's disease, renal tubular acidosis, diabetes, Zimmerman-Laband syndrome (a condition associated with V1B2 mutations), and cutis laxa type II and wrinkled skin syndrome. Without being bound by theory, diseases with different pathologies from sepsis but whose molecular basis converges on lysosomal acidification deficiency are believed to be therapeutic targets for ZLN-005 and related compounds.

[0150] [Example 6] 6.6. Example 6: ZLN-005 reduces bacterial growth in a CLP-induced sepsis model This example further evaluates ZLN-005 and its ability to increase bacterial killing in a murine cecal ligament puncture (CLP) model.

[0151] Mice underwent CLP at study time 0 as described in Section 6.5.1.1. Mice were administered either 12 mg / kg ZLN-005 (ip) or vehicle (DMSO) at the time of cecal ligature puncture. Intraperitoneal ascites fluid was collected from each mouse at study time 2 and 24 hours, and peritoneal bacterial levels were assessed (Figure 17A).

[0152] The results are shown in Figures 17B-17D. The ascites fluid from vehicle-treated CLP mice showed significantly increased bacterial counts at 2 and 24 hours compared with that from sham-treated mice. The ascites fluid from ZLN-005-treated CLP mice showed a reduction in bacterial counts at both time points compared with that from vehicle-treated CLP mice.

[0153] [Example 7] 6.7. Example 7: ZLN-005 Increases v-ATPase Association The vacuolar ATPase (v-ATPase) is an ATP-driven proton pump that functions to acidify intracellular compartments and transport protons across the plasma membrane. In lysosomes, v-ATPase functions to increase the acidity of the lysosomal lumen. V-ATPase activity has been shown to be regulated by the reversible degradation of V0-V1.

[0154] To evaluate the effect of ZLN-005 on v-ATPase assembly, mice underwent CLP at study time 0 as described in Section 6.5.1.1. Mice were administered 12 mg / kg ZLN-005 (ip) or vehicle (DMSO) at the time of cecal ligature puncture. Peritoneal cells were harvested at 24 hours. After cell fractionation, Western blotting was performed as described in Section 6.5.1.10 to determine the levels of V0 and V1 on the lysosomal membrane (Figure 18A).

[0155] Assembly of the v-ATPase involves the binding of the V0 and V1 subunits, where V0 is the membrane-bound subunit to which V1 binds to form the v-ATPase. Without being bound by theory, the level of V1 protein associated with the lysosomal membrane may indicate the amount of fully assembled v-ATPase on the lysosome. V1 protein levels in the cytosol were assessed by normalizing cytoplasmic V1A protein expression to cytoplasmic tubulin expression, and there was no significant difference between mock-treated and CLP + Vehicle or CLP + ZLN-005 mice (Figure 18B). V1A protein expression relative to VAPB, indicating the level of lysosome-associated V1, was significantly lower in CLP + Vehicle samples than in samples from mock-treated mice, suggesting a decrease in the number of fully assembled v-ATPase on the lysosomal membrane. This decrease was restored in samples from CLP mice treated with ZLN-005 (Figure 18B). No significant differences were observed in membrane-bound V0 protein between treatment groups (Figure 18B). Finally, V0 / V1 association was assessed by normalizing lysosome-associated V1 to cytosolic V1. CLP significantly reduced V0 and V1 association, but ZLN-005 treatment attenuated this reduction (Figure 18B).

[0156] [Example 8] 6.8. Example 8: ZLN-005-Mediated Lysosomal Acidification Under LPS Stimulation Is Abolished by TRPML1 Inhibition As described in Example 5, nuclear levels of TFEB regulate lysosomal biogenesis and autophagy. TRPML1 regulates cytosolic Ca2+, which leads to the nuclear translocation of TFEB. 2+ It is a protein that activates release.

[0157] The role of TRPML-1 in ZLN-005-associated increased 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 periods indicated in Figure 19A. Cells were sorted by FACS, and pHrod fluorescence was used as an indicator of lysosomal pH.

[0158] The results are shown in Figure 19B. ZLN-005 and LPS-treated cells showed higher pHrod fluorescence levels compared to LPS-only cells. ML-SI3 and LPS-treated cells showed pHrod fluorescence levels comparable to LPS-only cells. LPS-stimulated cells treated with both ZLN-005 and ML-SI3 showed a significant decrease in pHrod fluorescence.

[0159] 7. Specific Embodiments The present disclosure is illustrated by the following specific embodiments.

[0160] 1. A method of treating a subject having a disease or disorder associated with impaired lysosomal acidification, comprising administering to a subject a compound of formula (I):

[0161] [ka] wherein Ar is

[0162] [ka] and W 1 But NR 1 , O, or S, or W 9 If N, then W 1 Further CR 50 may be W 2 But, CR 2 or N, W 3 But, CR 3 or N, W 4 But, CR 4 or N, W 5 But, CR 5 or N, W 6 But, CR 6 or N, W 7 But, CR 7 or N, W 8 But, CR 8 or N, W 9 is C or W 1 is CR 50 In the case of W 9 may be N, R 1 is H, (C1-C3) alkyl, CH2OC(=O)R 30 , CH2OP(=O)OR 40 OR 41 , C(=O)OR 42 , or C(=O)R 43 and R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C 1-4 ) alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxy, (C1-C4)alkoxycarbonylamino, carboxamido, (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, halo, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino; R 7 and R 9 each independently represents hydrogen, deuterium, hydroxy, cyano, amino, halogen, halo(C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy,

[0163] [ka] and 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

[0164] [ka] and R 30 However, (C1-C 10 ) hydrocarbyl, amino-substituted (C1-C 10 ) hydrocarbyl, (C1-C4) hydrocarbyl substituted (C1-C 10 ) hydrocarbyl, carboxy-substituted (C1-C 10 ) hydrocarbyl, carboxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkoxycarbonylamino, methylthio, heterocyclyl, (C1-C 10 ) Oxaalkyl, CHR 44 NHR 45 and guanidine, R 40 and R 41 are each independently hydrogen or (C-C)hydrocarbyl; R 42 is (C1-C5) alkyl; R 43 is (C1-C3) alkyl; R 44 is a naturally occurring amino acid side chain, R 45 is H, methyl, or (C1-C4)alkoxycarbonyl, and R 50 is H or (C1-C3) alkyl] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 2. A method for increasing lysosomal acidity in a subject, comprising: an amount of a compound of Formula (I):

[0165] [ka] wherein Ar is

[0166] [ka] and W 1 But NR 1 , O, or S, or W 9 If N, then W 1 Further CR 50 may be W 2 But, CR 2 or N, W 3 But, CR 3 or N, W 4 But, CR 4 or N, W 5 But, CR 5 or N, W 6 But, CR 6 or N, W 7 But, CR 7 or N, W 8 But, CR 8 or N, W 9 is C or W 1 is CR 50 In the case of W 9 may 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 and R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C 1-4 ) alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxy, (C1-C4)alkoxycarbonylamino, carboxamido, (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, halo, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino; R 7 and R 9 each independently represents hydrogen, deuterium, hydroxy, cyano, amino, halogen, halo(C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy,

[0167] [ka] and 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,

[0168] [ka] and R 30 However, (C1-C 10 ) hydrocarbyl, amino-substituted (C1-C 10 ) hydrocarbyl, (C1-C4) hydrocarbyl substituted (C1-C 10 ) hydrocarbyl, carboxy-substituted (C1-C 10 ) hydrocarbyl, carboxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkoxycarbonylamino, methylthio, heterocyclyl, (C1-C 10 ) Oxaalkyl, CHR 44 NHR 45 and guanidine, R 40 and R 41 are each independently hydrogen or (C-C)hydrocarbyl; R 42 is (C1-C5) alkyl; R 43 is (C1-C3) alkyl; R 44 is a naturally occurring amino acid side chain, R 45 is H, methyl or (C1-C4)alkoxycarbonyl, and R 50 is H or (C1-C3) alkyl] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 3. A method of treating a subject having sepsis, infection, or a disease or disorder associated with V-ATPase dysfunction, comprising administering to a subject a compound of formula (I):

[0169] [ka] wherein Ar is

[0170] [ka] and W 1But NR 1 , O, or S, or W 9 If N, then W 1 Further CR 50 may be W 2 But, CR 2 or N, W 3 But, CR 3 or N, W 4 But, CR 4 or N, W 5 But, CR 5 or N, W 6 But, CR 6 or N, W 7 But, CR 7 or N, W 8 But, CR 8 or N, W 9 is C or W 1 is CR 50 In the case of W 9 may 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 and R 2 , R 3 , R 4 , and R 5 are each independently hydrogen, deuterium, halogen, perfluoro(C1-C4)alkyl, (C1-C4)alkyl, (C1-C4)alkoxy, perfluoro(C1-C4)alkoxy, (C1-C4)acyl, (C 1-4) alkoxy(C1-C4)alkyl, hydroxy(C1-C4)alkyl, hydroxy, carboxy, (C1-C4)alkoxycarbonylamino, carboxamido, (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, halo, (C1-C3)alkyl, perfluoro(C1-C3)alkyl, hydroxy, (C1-C3)alkoxy, perfluoro(C1-C3)alkoxy, or amino; R 7 and R 9 each independently represents hydrogen, deuterium, hydroxy, cyano, amino, halogen, halo(C1-C4)alkyl, (C1-C4)alkoxy, halo(C1-C4)alkoxy,

[0171] [ka] and 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,

[0172] [ka] and R 30 However, (C1-C 10 ) hydrocarbyl, amino-substituted (C1-C 10 ) hydrocarbyl, (C1-C4) hydrocarbyl substituted (C1-C 10 ) hydrocarbyl, carboxy-substituted (C1-C 10) hydrocarbyl, carboxy, (C1-C6) alkoxycarbonyl, (C1-C6) alkoxycarbonylamino, methylthio, heterocyclyl, (C1-C 10 ) Oxaalkyl, CHR 44 NHR 45 and guanidine, R 40 and R 41 are each independently hydrogen or (C-C)hydrocarbyl; R 42 is (C1-C5) alkyl; R 43 is (C1-C3) alkyl; R 44 is a naturally occurring amino acid side chain, R 45 is H, methyl or (C1-C4)alkoxycarbonyl, and R 50 is H or (C1-C3) alkyl] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 4. The method of any one of embodiments 1 to 3, wherein the amount of the drug is an amount effective to increase lysosomal acidity in the subject. 5. The method of any one of embodiments 1 to 4, wherein the amount of the drug is an amount effective to increase lysosomal acidity in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). 6. The method of any one of embodiments 1 to 5, wherein the amount of the drug is an amount effective to increase lysosomal acidity in peritoneal cells of the subject. 7. The method of any one of embodiments 1 to 6, wherein the amount of the drug is an amount effective to increase lysosomal acidity in the subject's peritoneal phagocytes (e.g., macrophages, monocytes, or neutrophils). 8. The method of any one of embodiments 1-7, wherein the amount of the drug is effective to increase Tfeb mRNA levels in the subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils). 9. The method of any one of embodiments 1-8, wherein the amount of the drug is an amount effective to increase Tfeb mRNA levels in the subject's peritoneal cells (e.g., phagocytes (e.g., macrophages, monocytes, or neutrophils)). 10. The method of any one of embodiments 1 to 9, wherein the amount of the drug is effective to increase dephosphorylation of TFEB in phagocytes (e.g., macrophages, monocytes, or neutrophils) of the subject. 11. The method of any one of embodiments 1 to 10, wherein the amount of the drug is effective to increase the translocation of TFEB to the nucleus in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). 12. The method of any one of embodiments 1 to 11, wherein the amount of the drug is effective to increase the ratio of phosphorylated Akt to Akt (p-Akt / Akt) in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). 13. The method of any one of embodiments 1 to 12, wherein the amount of the drug is effective to increase the ratio of phosphorylated PI3K to PI3K (p-PI3K / PI3K) in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). 14. The method of any one of embodiments 1 to 13, wherein the amount of the drug is an amount effective to increase physical contact between mitochondria and lysosomes in the subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils). 15. The method of any one of embodiments 1-14, wherein the amount of the drug is an amount effective to increase spare respiratory capacity in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). 16. The method of any one of embodiments 1-15, wherein the amount of the drug is effective to increase glycolytic capacity in phagocytic cells (e.g., macrophages, monocytes, or neutrophils) of the subject. 17. The method of any one of embodiments 1-16, wherein the amount of the drug is effective to increase glycolytic reserve in phagocytic cells (e.g., macrophages, monocytes, or neutrophils) of the subject. 18. The method of any one of embodiments 1 to 17, wherein the amount of the drug is an amount effective to increase lysosomal proteolysis in the subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils). 19. The method of any one of embodiments 1 to 18, wherein the amount of the drug is an amount effective to increase the mRNA levels of one or more hydrolases and / or one or more membrane proteins in the subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils). 20. The method of any one of embodiments 1-19, wherein the amount of the drug is effective to increase the mRNA level of Ctsd in the subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils). 21. The method of any one of embodiments 1 to 20, wherein the amount of the drug is an amount effective to increase Atp6v1A mRNA levels in the subject's phagocytic cells (e.g., macrophages, monocytes, or neutrophils). 22. The method of any one of embodiments 1 to 21, wherein the amount of the drug is an amount effective to increase the mRNA level of Atp6v0d1 in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). 23. The method of any one of embodiments 1 to 22, wherein the amount of the drug is effective to increase Mcoln1 mRNA levels in the subject's phagocytes (e.g., macrophages, monocytes, or neutrophils). 24. The method of any one of embodiments 1-22, wherein the amount of the drug is an amount effective to reduce the level of one or more inflammatory markers (e.g., in blood or serum). 25. The method of embodiment 24, wherein the one or more inflammatory markers comprise Tnfα, IL1β, IL6, IFNγ, or a combination thereof. 26. The method of any one of embodiments 1-25, wherein the subject does not have systemic immune activation. 27. The method of any one of embodiments 1-26, wherein the subject has suppressed innate immune function. 28. The method of embodiment 27, wherein the amount of the drug is an amount effective to enhance the subject's innate immune function. 29. The method of any one of embodiments 1-28, wherein the subject has suppressed macrophage phagocytic activity. 30. The method of embodiment 29, wherein the amount of the drug is an amount effective to enhance macrophage phagocytic activity in the subject. 31. The method of any one of embodiments 1-30, wherein the subject has an intestinal perforation. 32. The method of any one of embodiments 1-31, wherein the subject has sepsis. 33. The method of embodiment 32, wherein the subject has polymicrobial sepsis. 34. The method of embodiment 32, wherein the subject has monomicrobial sepsis. 35. The method of any one of embodiments 32-34, wherein the subject has early sepsis. 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, for example, 0 to 3, 1 to 4, 2 to 5, 0, 1, 2, 3, 4, 5, or 6, before treatment with the drug. 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, for example, 0, 1, or 2, prior to treatment with the drug. 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, for example, 2, 3, 4, 5, or 6, before treatment with the drug. 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, for example, 7, 8, or 9, before treatment with the drug. 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, for example, 10, 11, or 12, prior to treatment with the drug. 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, for example 13 or 14, before treatment with the drug. 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 drug. 43. The method of any one of embodiments 1-42, wherein the amount of the drug is effective to reduce the subject's SOFA score. 44. The method of any one of embodiments 1-42, wherein the amount of the drug is an amount effective to maintain the subject's SOFA score. 45. The method of any one of embodiments 1-44, wherein the subject has one, two, three, or four of the following (a)-(d): (a) lower than normal levels of HLA-DR expression in peripheral blood; (b) PD-1 expression on T cells is higher than normal; (c) lower than normal CD88 expression on neutrophils; (d) The Th17 / Treg ratio is lower than normal. 46. ​​The method of embodiment 45, wherein the subject has lower than normal levels of HLA-DR expression in peripheral blood. 47. The method of embodiment 45 or 46, wherein the frequency of HLA-DR-expressing mononuclear cells in the subject's peripheral blood is less than 30%. 48. The method of any one of embodiments 45-47, wherein the subject has higher than normal PD-1 expression in T cells. 49. The method of any one of embodiments 45-48, wherein the subject has lower than normal CD88 expression in neutrophils. 50. The method of any one of embodiments 45 to 49, wherein the subject has a lower than normal Th17 / Treg ratio. 51. The method of any one of embodiments 1 to 50, wherein the subject has suppressed innate immune function and / or does not have systemic immune activation. 52. The method of any one of embodiments 1-51, wherein the subject has an infection. 53. The method of embodiment 52, wherein the subject has an infection of the abdominal cavity. 54. The method of embodiment 52 or embodiment 53, wherein the infection is a bacterial infection. 55. The method of embodiment 54, wherein administering reduces the bacterial load in the subject. 56. The method of embodiment 54 or embodiment 55, wherein the bacterial infection is a multidrug-resistant bacterial infection. 57. The method of embodiment 52 or embodiment 53, wherein the infection is a fungal infection. 58. The method of embodiment 57, wherein administration reduces the fungal burden in the subject. 59. The method of embodiment 52 or embodiment 53, wherein the infection is a parasitic infection. 60. The method of embodiment 59, wherein administering reduces the parasite load in the subject. 61. The method of embodiment 52 or embodiment 53, wherein the infection is a viral infection. 62. The method of embodiment 61, wherein administering reduces the viral load in the subject. 63. The method of any one of embodiments 1-62, wherein the subject has a V-ATPase dysfunction. 64. The method of any one of embodiments 1-63, wherein the subject has a disease or disorder associated with V-ATPase dysfunction. 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, impaired glucose tolerance, diabetes, Parkinson's disease, Alzheimer's disease, or hearing loss. 66. The method of any one of embodiments 63-65, wherein the subject has a pathogenic mutation in a gene encoding a V-ATPase subunit. 67. The method of any one of embodiments 63-66, wherein the subject has a pathogenic mutation in the gene encoding V-ATPase subunit B. 68. The method of embodiment 67, wherein the subject has a pathogenic ATP6V1B1 mutation. 69. The method of embodiment 63 or embodiment 68, wherein the subject has renal tubular acidosis. 70. The method of any one of embodiments 63-66, wherein the subject has a pathogenic ATP6V1B2 mutation. 71. The method of embodiment 63 or embodiment 70, wherein the subject has Zimmerman-Laband syndrome. 72. The method of any one of embodiments 63-66, wherein the subject has a pathogenic mutation in the gene encoding V-ATPase subunit a. 73. The method of embodiment 72, wherein the subject has a pathogenic ATP6V0A4 mutation. 74. The method of embodiment 63 or embodiment 73, wherein the subject has renal tubular acidosis. 75. The method of embodiment 72, wherein the subject has a pathogenic ATP6V0A2 mutation. 76. The method of embodiment 63 or embodiment 75, wherein the subject has cutis laxa type II or wrinkled skin syndrome. 77. The method of embodiment 72, wherein the subject has a pathogenic ATP6V0A3 mutation. 78. The method of embodiment 63 or embodiment 77, wherein the subject has osteopetrosis. 79. The method of embodiment 78, wherein the subject has neurodegeneration. 80. The method of any one of embodiments 63-66, wherein the subject has a pathogenic ATP6V1H mutation. 81. The method of embodiment 63 or embodiment 80, wherein the subject has impaired glucose tolerance or diabetes. 82. The method of any one of embodiments 63-65, wherein the subject has a pathogenic mutation in a gene encoding a V-ATPase accessory protein. 83. The method of embodiment 82, wherein the V-ATPase accessory protein is ATP6AP2. 84. The method of embodiment 83, wherein the subject has Parkinson's disease, such as spastic X-linked Parkinson's disease (XPDS). 85. The method of any one of embodiments 63-65, wherein the subject has a pathogenic PSEN1 mutation. 86. The method of embodiment 63 or embodiment 85, wherein the subject has familial Alzheimer's disease. 87. The method of any one of embodiments 63-65, wherein the subject has a pathogenic DMXL2 mutation. 88. The method of embodiment 63 or embodiment 87, wherein the subject has hearing loss. 89. The method of any one of embodiments 1-64, wherein the subject has renal tubular acidosis, Zimmermann-Laband syndrome, cutis laxa type II or wrinkled skin syndrome, osteopetrosis, impaired glucose tolerance, diabetes, Parkinson's disease, Alzheimer's disease, or hearing loss. 90. Drugs,

[0173] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 91. Drugs,

[0174] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 92. Drugs,

[0175] [ka] or a salt thereof. 93. Drugs,

[0176] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 94. Drugs,

[0177] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 95. Drugs,

[0178] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 96. Drugs,

[0179] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 97. Drugs,

[0180] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 98. Drugs,

[0181] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 99. Drugs

[0182] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 100. Drugs,

[0183] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 101. Drugs,

[0184] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 102. Drugs,

[0185] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 103. Drugs,

[0186] [ka] or a salt, hydrate, deuterated analog, or fluorinated analog thereof. 104. The method of any one of embodiments 1-103, wherein the drug is administered enterally. 105. The method of embodiment 104, wherein the drug is administered orally. 106. The method of any one of embodiments 1-103, wherein the drug is administered orally (po).

[0187] 8. Citation of References All publications, patents, patent applications, and other documents cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. In the event of a conflict between the teachings of one or more references incorporated herein and the present disclosure, the teachings of the present disclosure are intended.

Claims

1. 1. A method of treating a subject having a disease or disorder associated with impaired lysosomal acidification, comprising administering to a subject a compound of formula (I): 【Chemistry 1】 wherein Ar is 【Chemistry 2】 and W １ But N-R １ , O, or S, or W ９ If N, then W １ Further, C-R ５０ may be W ２ But, C-R ２ or N, W ３ But, C-R ３ or N, W ４ But, C-R ４ or N, W ５ But, C-R ５ or N, W ６ But, C-R ６ or N, W ７ But, C-R ７ or N, W ８ But, C-R ８ or N, W ９ is C, or W １ is C-R ５０ In the case of W ９ may be N, R １ But, H, (C １ -C ３ ) alkyl, CH ２ OC(=O)R ３０ , C.H. ２ OP(=O)OR ４０ OR ４１ , C(=O)OR ４２ or C(=O)R ４３ and R ２ , R ３ , R ４ , and R ５ are each independently hydrogen, deuterium, halogen, perfluoro(C １ -C ４ ) alkyl, (C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, perfluoro(C １ -C ４ ) alkoxy, (C １ -C ４ ) acyl, (C １－４ ) alkoxy(C １ -C ４ ) alkyl, hydroxy (C １ -C ４ ) alkyl, hydroxy, carboxy, (C １ -C ４ ) alkoxycarbonylamino, carboxamide, (C １ -C ４ ) alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C １ -C ４ ) alkylamino, di(C １ -C ４ ) alkylamino, mercapto, (C １ -C ４ ) alkylthio, aminosulfonyl, (C １ -C ４ ) alkylsulfonyl, or (C １ -C ４ ) acylamino, R ６ and R １０ are each independently hydrogen, deuterium, halo, (C １ -C ３ ) alkyl, perfluoro(C １ -C ３ ) alkyl, hydroxy, (C １ -C ３ ) alkoxy, perfluoro(C １ -C ３ ) alkoxy, or amino; R ７ and R ９ are each independently hydrogen, deuterium, hydroxy, cyano, amino, halogen, halo(C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, halo (C １ -C ４ ) alkoxy, 【Transformation 3】 and R ８ However, hydrogen, deuterium, halogen, halo (C １ -C ４ ) alkyl, (C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, halo (C １ -C ４ ) alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, 【Chemistry 4】 and R ３０ However, (C １ -C １０ ) hydrocarbyl, amino-substituted (C １ -C １０ ) hydrocarbyl, (C １ -C ４ ) hydrocarbyl-substituted (C １ -C １０ ) hydrocarbyl, carboxy-substituted (C １ -C １０ ) hydrocarbyl, carboxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkoxycarbonylamino, methylthio, heterocyclyl, (C １ -C １０ ) oxaalkyl, CHR ４４ NHR ４５ and guanidine, R ４０ and R ４１ are each independently hydrogen or (C １ -C ６ ) hydrocarbyl, R ４２ However, (C １ -C ５ ) alkyl, R ４３ However, (C １ -C ３ ) alkyl, R ４４ is a naturally occurring amino acid side chain, R ４５ is H, methyl or (C １ -C ４ ) alkoxycarbonyl, and R ５０ is H or (C １ -C ３ ) alkyl] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

2. 1. A method of increasing lysosomal acidity in a subject, comprising: an amount of Formula (I): effective to increase lysosomal acidity in the subject; 【Transformation 5】 wherein Ar is 【Transformation 6】 and W １ But N-R １ , O, or S, or W ９ If N, then W １ Further, C-R ５０ may be W ２ But, C-R ２ or N, W ３ But, C-R ３ or N, W ４ But, C-R ４ or N, W ５ But, C-R ５ or N, W ６ But, C-R ６ or N, W ７ But, C-R ７ or N, W ８ But, C-R ８ or N, W ９ is C or W １ is C-R ５０ In the case of W ９ may be N, R １ But, H, (C １ -C ３ ) alkyl, CH ２ OC(=O)R ３０ , C.H. ２ OP(=O)OR ４０ OR ４１ , C(=O)OR ４２ or C(=O)R ４３ and R ２ , R ３ , R ４ , and R ５ are each independently hydrogen, deuterium, halogen, perfluoro(C １ -C ４ ) alkyl, (C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, perfluoro(C １ -C ４ ) alkoxy, (C １ -C ４ ) acyl, (C １－４ ) alkoxy(C １ -C ４ ) alkyl, hydroxy (C １ -C ４ ) alkyl, hydroxy, carboxy, (C １ -C ４ ) alkoxycarbonylamino, carboxamide, (C １ -C ４ ) alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C １ -C ４ ) alkylamino, di(C １ -C ４ ) alkylamino, mercapto, (C １ -C ４ ) alkylthio, aminosulfonyl, (C １ -C ４ ) alkylsulfonyl, or (C １ -C ４ ) acylamino, R ６ and R １０ are each independently hydrogen, deuterium, halo, (C １ -C ３ ) alkyl, perfluoro(C １ -C ３ ) alkyl, hydroxy, (C １ -C ３ ) alkoxy, perfluoro(C １ -C ３ ) alkoxy, or amino; R ７ and R ９ are each independently hydrogen, deuterium, hydroxy, cyano, amino, halogen, halo(C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, halo (C １ -C ４ ) alkoxy, 【Transformation 7】 and R ８ However, hydrogen, deuterium, halogen, halo (C １ -C ４ ) alkyl, (C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, halo (C １ -C ４ ) alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, 【Transformation 8】 and R ３０ However, (C １ -C １０ ) hydrocarbyl, amino-substituted (C １ -C １０ ) hydrocarbyl, (C １ -C ４ ) hydrocarbyl-substituted (C １ -C １０ ) hydrocarbyl, carboxy-substituted (C １ -C １０ ) hydrocarbyl, carboxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkoxycarbonylamino, methylthio, heterocyclyl, (C １ -C １０ ) oxaalkyl, CHR ４４ NHR ４５ and guanidine, R ４０ and R ４１ are each independently hydrogen or (C １ -C ６ ) hydrocarbyl, R ４２ However, (C １ -C ５ ) alkyl, R ４３ However, (C １ -C ３ ) alkyl, R ４４ is a naturally occurring amino acid side chain, R ４５ is H, methyl or (C １ -C ４ ) alkoxycarbonyl, and R ５０ is H or (C １ -C ３ ) alkyl] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

3. 1. A method of treating a subject having sepsis, infection, or a disease or disorder associated with V-ATPase dysfunction, comprising administering to a subject a compound of formula (I): 【Chemistry 9】 wherein Ar is 【Chemistry 10】 and W １ But N-R １ , O, or S, or W ９ If N, then W １ Further, C-R ５０ may be W ２ But, C-R ２ or N, W ３ But, C-R ３ or N, W ４ But, C-R ４ or N, W ５ But, C-R ５ or N, W ６ But, C-R ６ or N, W ７ But, C-R ７ or N, W ８ But, C-R ８ or N, W ９ is C or W １ is C-R ５０ In the case of W ９ may be N, R １ But, H, (C １ -C ３ ) alkyl, CH ２ OC(=O)R ３０ , C.H. ２ OP(=O)OR ４０ OR ４１ , C(=O)OR ４２ or C(=O)R ４３ and R ２ , R ３ , R ４ , and R ５ are each independently hydrogen, deuterium, halogen, perfluoro(C １ -C ４ ) alkyl, (C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, perfluoro(C １ -C ４ ) alkoxy, (C １ -C ４ ) acyl, (C １－４ ) alkoxy(C １ -C ４ ) alkyl, hydroxy (C １ -C ４ ) alkyl, hydroxy, carboxy, (C １ -C ４ ) alkoxycarbonylamino, carboxamide, (C １ -C ４ ) alkylaminocarbonyl, cyano, acetoxy, nitro, amino, (C １ -C ４ ) alkylamino, di(C １ -C ４ ) alkylamino, mercapto, (C １ -C ４ ) alkylthio, aminosulfonyl, (C １ -C ４ ) alkylsulfonyl, or (C １ -C ４ ) acylamino, R ６ and R １０ are each independently hydrogen, deuterium, halo, (C １ -C ３ ) alkyl, perfluoro(C １ -C ３ ) alkyl, hydroxy, (C １ -C ３ ) alkoxy, perfluoro(C １ -C ３ ) alkoxy, or amino; R ７ and R ９ are each independently hydrogen, deuterium, hydroxy, cyano, amino, halogen, halo(C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, halo (C １ -C ４ ) alkoxy, 【Chemistry 11】 and R ８ However, hydrogen, deuterium, halogen, halo (C １ -C ４ ) alkyl, (C １ -C ４ ) alkyl, (C １ -C ４ ) alkoxy, halo (C １ -C ４ ) alkoxy, cyano, phenyl, phenoxy, benzyloxy, amino, 【Chemistry 12】 and R ３０ However, (C １ -C １０ ) hydrocarbyl, amino-substituted (C １ -C １０ ) hydrocarbyl, (C １ -C ４ ) hydrocarbyl-substituted (C １ -C １０ ) hydrocarbyl, carboxy-substituted (C １ -C １０ ) hydrocarbyl, carboxy, (C １ -C ６ ) alkoxycarbonyl, (C １ -C ６ ) alkoxycarbonylamino, methylthio, heterocyclyl, (C １ -C １０ ) oxaalkyl, CHR ４４ NHR ４５ and guanidine, R ４０ and R ４１ are each independently hydrogen or (C １ -C ６ ) hydrocarbyl, R ４２ However, (C １ -C ５ ) alkyl, R ４３ However, (C １ -C ３ ) alkyl, R ４４ is a naturally occurring amino acid side chain, R ４５ is H, methyl or (C １ -C ４ ) alkoxycarbonyl, and R ５０ is H or (C １ -C ３ ) alkyl] or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

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

5. 5. The method of any one of claims 1 to 4, wherein the amount of drug is effective to increase lysosomal acidity in phagocytic cells (e.g., macrophages, monocytes, or neutrophils) of the subject.

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

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

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

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

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

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

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

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

14. 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. 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 a V-ATPase dysfunction.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

35. 35. The method of claim 17 or claim 34, wherein the subject has impaired glucose tolerance or diabetes.

36. 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 accessory protein.

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

38. 38. The method of claim 37, wherein the subject has Parkinson's disease, e.g., spastic X-linked Parkinson's disease (XPDS).

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

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

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

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

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

44. The drug is 【Chemistry 13】 or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

45. The drug is 【Chemistry 14】 or a salt, hydrate, deuterated analog, or fluorinated analog thereof.

46. The drug is 【Chemistry 15】 or a salt thereof.

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

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

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

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