CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Serial No. 63/371,042, filed August 10, 2022, which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates in some aspects to methods of inhibiting ATP hydrolysis using epicatechin.

BACKGROUND

[0003] Mitochondrial ATP synthase (FiFo-ATPase, or complex V) can switch from an ATP producer to an ATP consumer (ATP hydrolysis) during cellular stress and pathologic processes. Under normal conditions, the electron transport chain (ETC) generates an electrochemical proton gradient that drives clockwise rotation of ATP synthase, resulting in ATP synthesis from ADP and phosphate. When this electrochemical proton gradient begins to fall, ATP synthase reverses direction to rotate counterclockwise, hydrolyzing ATP to actively transport protons from the mitochondrial matrix into the intermembrane space, thereby preventing membrane depolarization (Chinopoulos and Adam -Vizi (2010) Biochim Biophys Acta 1802(1): 221-227). As a result, less ATP is available for processes that are required for an adaptive stress response, because 1) mitochondria are no longer producing ATP efficiently, and 2) mitochondria are instead hydrolyzing imported cytosolic ATP (Classen et al. (1989) J Cell Physiol 141(1): 53-59; Grover et al. (2004) Am J Physiol Heart Circ Physiol 287(4): H1747-1755). Moreover, the hydrolytic adaptation can create overactive consumption of cytosolic ATP and in this scenario, the regulation of ATP synthase activity by the inhibitory protein ATPIF1 is fundamental (Garcia-Bermudez and Cuezva (2016) Biochim Biophys Acta 1857(8): 1167-1182). The role of ATP hydrolysis in stress conditions like heart ischemic injury and hypoxia/anoxia (Classen et al. (1989) J Cell Physiol 141(1): 53-59; St-Pierre et al. (2000) Proc Natl Acad Sci USA 97(15): 8670-8674) have been reported. However, long-term hydrolysis as a pathogenic mechanism, and experimental evidence of simultaneous synthase and hydrolase activity, are lacking.

[0004] Given that the key function of mitochondria are to perform in the adaptive stress response, and oxidative stress and respiratory failure are common features implicated in the initiation and progression of many diseases (such as mitochondrial diseases, muscular dystrophies, neurodegenerative diseases, diabetes, and cardiomyopathy), an evaluation of how the shift towards favoring ATP consumption over ATP synthesis is necessary to understand these pathological processes. Previous studies have sought to identify drugs that can modify mitochondrial function in mitochondrial deficient cells (Couplan et al. (2011) Proc Natl Acad Sci USA 108(29): 11989-11994; Garrido-Maraver et al. (2012) Br J Pharmacol 167(6): 1311-1328; Aiyar et al. (2014) Nat Commun 5: 5585; Sahdeo c/ a/. (2014) Mitochondrion 17: 116-125; Barrow et al. (2016) Afo/ Cell 64(1): 163-175; and, Chin et al. (2018) BMC Res Notes 11(1): 205). However, many of these drugs were specific for a particular mutation, had off-target effects and/or side effects, or were not specific for the disease model because they modulated both the control and the mutant metabolism.

[0005] When looking for a drug for treating mitochondrial disorders, therapeutic approaches with wide applicability would be preferable as well as targeting a specific mechanism malfunctioning in the disorder and not in a control situation. Taking advantage of a nature-developed mechanism for preventing overconsumption of ATP by ATP synthase, targeting the ATP Inhibitory Factor 1 (IF1 or ATPIF1), or ATP synthase selectively could be of clinical interest in pathological conditions. The present disclosure address this and other unmet needs.

BRIEF SUMMARY

[0006] Provided herein are methods of treating or preventing diseases or disorders resulting from mitochondrial ATP synthase (also referred to as “complex V”) dysfunction (e.g., inhibiting ATP hydrolysis, decreasing ATP hydrolysis, and/or preventing the dissociation of complex V into monomers) using epicatechin. Such methods are useful for the treatment of a variety of diseases or disorders, such as central nervous system (CNS) diseases or disorders and/or diseases or disorders associated with metabolic dysfunction.

[0007] One aspect of the present application provides a method of treating or preventing a disease or disorder that would benefit from inhibition of ATP hydrolysis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

[0008] In some embodiments, the inhibition of ATP hydrolysis does not block mitochondrial ATP synthesis.

[0009] In some embodiments, the disease or disorder causes metabolic dysfunction. In some embodiments, the disease or disorder causes impaired mitochondrial respiration. In some embodiments, the disease or disorder is selected from the group consisting of psychomotor delay, tubulopathy, renal disease, liver disease, and loss of synaptic density. [0010] In some embodiments, the epicatechin prevents the dissociation of Electron Transport Chain (ETC) complex V into monomers.

[0011] In some embodiments, the epicatechin prevents loss of mitochondrial ATP synthesis associated with the disease or disorder.

[0012] In other aspects, provided herein is a method of preventing Electron Transport Chain (ETC) complex V dissociation into monomers in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

[0013] In additional aspects, provided herein is a method of decreasing ATP hydrolysis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

[0014] In some embodiments, the subject has a disease or disorder selected from the group consisting of spinal cord injury or abnormality, liver disease, kidney disease, impaired cognition, neurodegenerative disease, dystonia, sarcopenia, cardiomyopathy of aging or other diseases associated with mitochondrial dysfunction, cardiomyopathy, ischemic vascular disease, immunodeficiency states, ataxia, pulmonary inflammation and fibrosis, infantile encephalomyopathy, epilepsy, Charcot-Marie-Tooth disease, exocrine pancreatic insufficiency, impaired wound healing, and growth of cancer cells. In some embodiments, the subject has a neurodegenerative disease selected from the group consisting of Alzheimer’s disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Leigh syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, and peripheral and central neuropathies.

[0015] In some embodiments, the epicatechin directly binds complex V, optionally wherein epticatechin directly binds in the ATP/ADP binding pocket of complex V. In some embodiments, the epicatechin binds to the ATP Inhibitory Factor 1 (ATPIF1) pocket of complex V.

[0016] In some embodiments, the epicatechin blocks Ca ²⁺ induced mitochondrial permeability transition pore (MPTP) opening.

[0017] In some embodiments, the epicatechin is administered in the form of a pharmaceutical composition comprising the epicatechin or the pharmaceutically acceptable salt, solvate, or co-crystal thereof, and a pharmaceutically acceptable excipient. In some embodiments, the epicatechin is in the form of a co-crystal.

[0018] In some embodiments, the epicatechin is administered orally, sublingually, subcutaneously, parenterally, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, or intraocularly. In some embodiments, the epicatechin is administered orally or intravenously.

[0019] In some embodiments, the epicatechin is administered in the form of a tablet, a capsule, a powder, a liquid, a suspension, a suppository, or an aerosol.

[0020] In some embodiments, the epicatechin is administered at a daily dosage of about 5 to about 500 mg. In some embodiments, the daily dosage is administered in a single dose or in 2, 3, or 4 divided doses. In some embodiments, the epicatechin is administered each day, every other day, weekly, every two weeks, every three weeks, or every four weeks.

[0021] In some embodiments, the epicatechin is (+)-epicatechin. In some embodiments, the epicatechin is (-)-epicatechin. In some embodiments, the epicatechin comprises at least 75% (+)-epicatechin. In some embodiments, the epicatechin comprises at least 75% (-)- epicatechin.

[0022] In some embodiments, the epicatechin is administered in combination with one or more additional therapeutic agents. In some embodiments, the epicatechin and the one or more additional therapeutic agents are administered concurrently. In some embodiments, the epicatechin and the one or more additional therapeutic agents are administered sequentially.

[0023] Also provided are kits and articles of manufacture comprising any one of the compositions described above and instructions for any one of the methods described above.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings illustrate certain embodiments of the features and advantages of this disclosure. These embodiments are not intended to limit the scope of the appended claims in any manner.

[0025] FIGS. 1 A-1N show that ATP synthesis and hydrolysis coexist in the same mitochondria, and can be differently modulated. FIG. 1 A shows representative Oxygen Consumption Rate (OCR) (top) and state 4 acidification rate (bottom) profiles of heart mitochondria respiring with the indicated substrates either in state 4 (no ADP) or state 3 (plus ADP) fueled by Pyruvate+Malate (Pyr+Mal). OLIGO refers to Oligomycin, which was used as control for inhibition of ATP synthesis and hydrolysis. Vertical dotted lines indicate time points at which ATP, FCCP, and oligomycin were added. FIG. IB shows extracellular acidification rate (ECAR) measurements in heart mitochondria respiring in state 3 (in the presence of ADP) or state 4 (no ADP) using Pyruvate and Malate (Pyr + Mai) under basal conditions and after ATP and FCCP sequential injections (n=4). FIG. 1C shows representative Oxygen Consumption Rate (OCR) (top) and state 4 acidification rate (bottom) profiles of heart mitochondria respiring with the indicated substrates either in state 4 (no ADP) or state 3 (plus ADP) fueled by Succinate and Rotenone (Succ+Rot). OLIGO refers to oligomycin, which was used as control for inhibition of ATP synthesis and hydrolysis. Vertical dotted lines indicate time points at which ATP, FCCP, and oligomycin were added. FIG. ID shows extracellular acidification rate (ECAR) measurements in heart mitochondria respiring in state 3 (in the presence of ADP) or state 4 (no ADP) using Succinate and Rotenone (Succ + Rot) under basal conditions and after ATP and FCCP sequential injections (n=4). FIG. IE shows State 4 acidification rates quantification in heart mitochondria under the indicated conditions (n>4). FIG. IF shows State 3 and ATP hydrolytic activity in fresh heart mitochondria untreated or treated with proteinase K (PK) (n=4). FIG. 1G shows quantification of the ratio of hydrolysis (ECAR rate on state 4 after ATP injection) versus synthesis (basal OCR rate on state 3) in heart mitochondria fueled with the indicated substrates (n>8). FIG. 1H shows representative Agilent Seahorse XF profiles measuring ATP hydrolytic activity in isolated heart mitochondria in the presence of the indicated concentrations of oligomycin (OLIGO). FIG. II shows a chart of the mean ± S.E.M. of the maximal ATP hydrolytic capacity normalized as a percentage of control cells for control versus patent-derived fibroblasts. FIG. 1J shows a Western blot of protein levels of ATPIF1 (IF 1), SDHB, ATP5A, and vinculin from control and patient-derived samples. FIG. IK shows the identification of binding sites in the IF-1 binding groove on the surface of Fl -ATP synthase (lohh, grey) using PELE: quercetin, (-)-catechin and (+)-epicatechin (left); (-)- epicatechin vs (+)-epicatechin (middle) and (+)-epicatechin and ATPIF1. FIG. IL shows representative Agilent Seahorse XF profiles measuring ATP hydrolytic activity in isolated heart mitochondria or cytosolic fraction in the presence on the indicated concentrations of (+)-epicatechin (EPI) using oligomycin (OLIGO) as a inhibitory control (left), and comparison between ATP hydrolytic activity in heart mitochondria and cytosol (right). FIG. IM shows maximal ATP hydrolysis capacity measured in frozen heart mitochondria in the presence of (+)-epicatechin (EPI) (100 nM) and using oligomycin (1 pM) as a negative control (n>7). FIG. IN shows the effects of increasing concentrations of (+)-epicatechin (EPI) in maximal Complex I, Complex II and ATP hydrolytic activity measured in frozen mouse heart mitochondria (n=4). Each point represents a biological replica sample. For each biological replicate, technical replicates were averaged. Data represent average ± SEM. Statistical analysis was performed with GraphPad Prism® 9.01 using one-way, two-way analysis of variance (ANOVA) or pair wise comparison as indicated in the FIG. legends. Corrections for multiple comparisons were made by Tukey posthoc test when appropriate. Differences were considered statistically different at p < 0.05. Statistical significance is denoted by p-value. Individual points in a graph denote individual cell preparations, mouse samples or biological replicates.

[0026] FIGS. 2A-2M show that epicatechin ((+)-epicatechin), but not catechin, inhibits ATP hydrolysis without affecting ATP synthesis. FIG. 2A shows In Gel ATP hydrolytic activity in mouse heart mitochondria incubated in the presence of the indicated concentrations of epicatechin prior to freezing the mitochondrial preparation and lysing the mitochondria with digitonin. In-Gel activity is shown after 3 hours (left) and overnight incubation (middle). Coomassie staining was used as loading control. Oligomycin (oligo) was used as control for ATP synthesis and hydrolysis inhibition. FIG. 2B shows quantification of In Gel ATP hydrolytic under the indicated epicatechin ((+)-epicatechin) concentrations (n>5). FIG. 2C shows a representative blot showing complex V assembly by blue native gel electrophoresis in mouse heart mitochondria incubated in the presence of the indicated concentrations of epicatechin ((+)-epicatechin) prior to freezing the mitochondrial preparation and lysing the mitochondria with digitonin. FIG. 2D shows State 3 respiration driven by pyruvate plus malate (left) and succinate plus rotenone in isolated mitochondria from mouse heart. Epicatechin ((+)-epicatechin) was added in the respiration media at the indicated concentrations (n>3). FIG. 2E shows maximal respiration driven by Pyruvate and Malate (Pyr + Mai; left) and Succinate and Rotenone (Succ + Rot; right) in isolated mitochondria from mouse heart. Epicatechin ((+)-epicatechin) was added in the respiration media at the indicated concentrations (n=3). FIG. 2F shows In Gel ATP hydrolytic activity in mouse heart mitochondria incubated in the presence of the indicated concentrations of Catechin prior to freezing the mitochondrial preparation and lysing the mitochondria with digitonin. In Gel activity is shown after 3 hours (left) and overnight (O/N) incubation (middle). Coomassie staining was used as loading control. Oligomycin (oligo) was used as control for ATP synthesis and hydrolysis inhibition. FIG. 2G shows a quantification of In Gel ATP hydrolytic under the indicated Catechin (Cat) concentrations (n=4). FIG. 2H shows a representative blot showing Complex V assembly by blue native gel electrophoresis in mouse heart mitochondria incubated in the presence of the indicated concentrations of Catechin. FIG. 21 shows State 3 respiration driven by Pyruvate and Malate (Pyr+Mal; left) and Succinate and Rotenone (Succ+Rot; right)in isolated mitochondria from mouse heart. Catechin (Cat) was added in the respiration media at the indicated concentrations (n=3). FIG. 2J shows In Gel ATP hydrolysis in mouse heart lysed mitochondria, where epicatechin ((+)- epicatechin; EPI) and oligomycin (oligo) were added after mitochondrial complexes and supercomplexes were extracted from the membrane with digitonin. In Gel activity is shown after overnight incubation (left) and after stopping the activity with 50% methanol (middle). Coomassie staining was used as loading control. FIG 2K shows a quantification of In-Gel ATP hydrolysis in mouse heart lysed mitochondria, where epicatechin ((+)-epicatechin; EPI) was added after mitochondrial complexes and supercomplexes were extracted from the membrane with digitonin. FIG. 2L shows In-Gel ATP hydrolysis in mouse heart mitochondria, where epicatechin ((+)-epicatechin; EPI) and oligomycin (oligo) were added in the assay buffer after mitochondrial complexes and supercomplexes were separated by blue native gel electrophoresis. In-Gel activity is shown after overnight (O/N) incubation (left). Coomassie staining was used as loading control (middle). FIG. 2M shows a quantification of FIG. 2L, In-Gel ATP hydrolysis in mouse heart mitochondria, where epicatechin ((+)- epicatechin; EPI) and oligomycin (oligo) were added in the assay buffer after mitochondrial complexes and supercomplexes were separated by blue native gel electrophoresis (n=5).

[0027] FIGS. 3 A-3 Y show that epicatechin competitively binds complex V. FIG. 3 A shows State 3 respiration driven by Pyruvate and Malate (Pyr+Mal; left) and Succinate and Rotenone (Succ+Rot; right) in isolated mitochondria from mouse heart. BTB06584 (BTB) was added in the respiration media at the indicated concentrations (n=3). FIG. 3B shows maximal respiration driven by pyruvate plus malate (left) and succinate plus rotenone (right) in isolated mitochondria from mouse heart. BTB06584 (BTB) was added in the respiration media at the indicated concentrations (n=3). FIG. 3C shows maximal ATP hydrolysis capacity measured in frozen heart mitochondria in the presence of 100 pM BTB06584 (BTB) (n=10). FIG. 3D shows In-Gel ATP hydrolytic activity in mouse heart mitochondria incubated in the presence of the indicated concentrations of BTB06584 (BTB) prior to freezing the mitochondrial preparation and lysing the mitochondria with digitonin. In-Gel activity is shown after 3hrs (left) and overnight incubation (middle). Coomassie staining was used as loading control. Oligomycin (oligo) was used as control for ATP synthesis and hydrolysis inhibition. FIG. 3E shows a quantification of In-Gel ATP hydrolytic activity under the indicated BTB06584 (BTB) concentrations (n=4). FIG. 3F shows a representative blot showing complex V assembly by blue native gel electrophoresis in mouse heart mitochondria incubated in the presence of the indicated concentrations of BTB06584 (BTB). FIG. 3G shows ATPIF1-GFP binding assays to heart mitochondria and localization of ATPIF1-GFP in assembled complex V measured by F488 fluorescence in blue native gel electrophoresis. FIG. 3H shows complex V In-Gel activity in heart mitochondria incubated in the presence of different concentrations of ATPIF1-GFP. In-Gel activity is shown after 3hr incubation (left) and after stopping the activity with 50% methanol (middle). Coomassie staining was used as loading control. FIG. 31 shows complex V assembly in heart mitochondria incubated in the presence of different concentrations of ATPIF1-GFP. Complex II (SDHA) was used as loading control. FIG. 3 J shows quantification of ATPIF1 bound to complex V (left) and InGel activity (right) relative to complex V assembled levels when isolated mitochondria were incubated with increasing concentrations of recombinant ATPIF1 (n=3). FIG. 3K shows the effects of increasing concentrations of ATPIF1-GFP added to mitochondria in maximal conditions on acidification rate (left) and ATP hydrolytic capacity (right) as measured in frozen mouse heart mitochondria (n=4). FIG. 3L shows the effects of increasing concentrations of ATPIF1-GFP added to mitochondria in maximal Complex I, Complex II and Complex IV activity measured in frozen mouse heart mitochondria (n=4) by frozen respirometry. The left panel shows a representative Complex I and Complex IV Seahorse profile in previously frozen heart mitochondria where ATPIF1-GFP has been added at the indicated concentrations. The middle panel shows a representative Complex II and Complex IV Seahorse profile in previously frozen heart mitochondria where ATPIF1-GFP has been added at the indicated concentrations. The right panels shows the quantification of the maximal activities of Complex I, II and IV in the indicated conditions. FIG. 3M shows an ATPIF1-GFP and epicatechin ((+)-epicatechin; EPI) binding competition assay to complex V tetramer (CVt). F488 fluorescence (left) was used to detect the exogenously added ATPIF1 (ATPIF1-GFP) and ATP5A1 to detect complex V levels (right). FIG. 3N shows a quantification of the complex V assembly forms under the indicated treatments of FIG. 3M (n=4). FIG. 30 shows complex V In-Gel Activity in ATPIF1-GFP and epicatechin ((+)- epicatechin; EPI) binding competition assay in heart mitochondria. In-Gel Activity after 3hrs incubation (left) and after fixing (middle). Coomassie staining was used as loading control. FIG. 3P shows ATPIF1-GFP and epicatechin ((+)-epicatechin; EPI) binding competition assay in heart mitochondria. F488 fluorescence was used to detect the exogenously added ATPIF1 (ATPIF1-GFP). FIG. 3Q shows complex V assembly in heart mitochondria incubated in the presence of ATPIF1, epicatechin ((+)-epicatechin; EPI) or both. Complex II (SDHA) was used as loading control. FIG. 3R shows a quantification of ATPIF1 bound to complex V assembly forms under the indicated treatments (n=3). FIG. 3S shows expression levels of ATP5A1 and ATPIF1 in the indicated bovine complex V preparations. FIG. 3T shows In-Gel ATP hydrolysis in purified bovine complex V preparations under the indicated epicatechin ((+)-epicatechin; EPI) and oligomycin (oligo) concentrations for complex V monomer. FIG. 3U shows In-Gel ATP hydrolysis in purified bovine complex V preparations under the indicated epicatechin ((+)-epicatechin; EPI) and oligomycin (oligo) concentrations for complex V tetramer. FIG. 3 V shows In-Gel ATP hydrolysis in purified bovine complex V preparations under the indicated epicatechin ((+)-epicatechin; EPI) and oligomycin (oligo) concentrations for Oligomer Mix 1 AA205 (Davies G. et al. Mol Psychiatry. 2015 Feb;20(2): 183-92; Urbani A. et al. Nat Commun. 2019 Sep 25; 10(l):4341). FIG. 3W shows In-Gel ATP hydrolysis in purified bovine complex V preparations under the indicated epicatechin ((+)-epicatechin; EPI) and oligomycin (oligo) concentrations for Oligomer Mix 2 (Davies G. et al. Mol Psychiatry. 2015 Feb;20(2): 183-92; Urbani A. et al. Nat Commun. 2019 Sep 25; 10(l):4341). FIG. 3X shows expression levels of ATP5A1 and ATPIF1 in mouse tissue lysates. Vinculin is used as loading control. FIG. 3Y shows a plot illustrating the lack of correlation of ATPIF1 expression levels with epicatechin ((+)-epicatechin; EPI) effect in inhibiting ATP maximal hydrolytic activity. Each point represents a biological replica sample. For each biological replicate, technical replicates were averaged. Data represent average.

[0028] FIGS. 4A-4Z show that epicatechin ((+)-epicatechin; EPI) inhibits ATP hydrolysis in mitochondrial disease models. FIG. 4A shows blue native gel electrophoresis of ATPIF1 binding to ATP synthase (i.e., complex V) in control fibroblasts in glucose or galactose media, in the presence or absence of epicatechin ((+)-epicatechin; EPI). Compound II (SDHB) was used as control. FIG. 4B shows a quantification of ATPIF1 in assembled ATP synthase in glucose media from FIG. 4A. FIG. 4C shows confocal micrographs showing fibroblasts either untreated or treated with 50 nM epicatechin ((+)-epicatechin; EPI) for 24h, labelled with anti-ATPIFl + anti-ATP5A (PLA) antibodies, anti-TOMM20 antibodies, and DAPI. Maximum intensity projection is shown. Scale bars: 20 pm. FIG. 4D shows a quantification of ATPIF1 binding to the ATP synthase under physiologically-relevant conditions as shown by confocal microcopy. The chart shows mean ± S.E.M of PLA dots/pm ³ of mitochondria normalized in % of Ctrl. Two-way ANOVA followed by Tukey's multiple comparisons test shows statistical differences depicted by p-value (n=4). FIG. 4E shows confocal micrographs of control and patient fibroblasts overexpressing either wildtype ATPIF1 (left) or the constitutively-active H49K mutant ATPIF1 (right) FIG. 4F shows a blot of complex II (SDHA), complex V (ATP5A1), ATPIF1 (IF1), and hemagglutin (HA) in control and patient fibroblasts expressing endogenous ATPIF1 or overexpressing either the wild-type protein (OE IF1-WT) or the constitutively-active mutant (OE IF1-H49K). FIG. 4G shows ATP hydrolysis capacity in control cells, cells overexpressing wild-type ATPIF1 (middle), and cell overexpressing constitutively-active ATPIF1 (right) as a percentage of the maximal ATP hydrolytic capacity, defined in control cells grown in succinate plus rotenone (SR respiration). FIG. 4H shows mean ± S.E.M of tetramethylrhoadmine ethyl ester (TMRE) fluorescence intensity in the mitochondria area (MitoTracker Green (MTG)) normalized per % of control cells for control fibroblasts that were incubated for 30 minutes with varying concentrations of Antimycin A (AA) + 30 min of DMSO, 100 nM epicatechin ((+)- epicatechin; EPI) or 1 pM oligomycin. Two-way ANOVA followed by Tukey’s multiple comparison test shows statistical differences depicted by p-value (n=3 biological replicates). FIG. 41 shows the oxygen consumption rate (OCR) of control cells before and after administration of varying doses of Antimycin A (AA). FIG. 4J shows mean ± S.E.M of mitochondrial ATP content in the mitochondria area (MitoTracker Green (MTG)) normalized per % of control cells for control fibroblasts that were incubated for 30 min with varying concentrations of Antimycin A (AA) + 30 min of either DMSO or 100 nM epicatechin ((+)- epicatechin; EPI). Two-way ANOVA followed by Tukey’s multiple comparison test shows statistical differences depicted by p-value (n=3 biological replicates). FIG. 4K shows mean ± S.E.M of mitochondrial ATP content in the mitochondria area (MitoTracker Green (MTG)) normalized per % of control cells for control fibroblasts that were incubated for 1 hour with varying concentrations of Antimycin A (AA) + 30 min of either DMSO or 100 nM epicatechin ((+)-epicatechin. Two-way ANOVA followed by Tukey’s multiple comparison test shows statistical differences depicted by p-value (n=3 biological replicates). FIG. 4L shows the mean ± S.E.M of oxygen consumption rate OCR) of control cells before and after acute treatment with the indicated concentration of epicatechin ((+)-epicatechin; EPI). FIG. 4M shows a chart of mean ± S.E.M of mitochondrial ATP Fluorescence Intensity (F.I) in the mitochondria area (MitoTracker Green (MTG)) normalized per % of control cells, from control and mitochondrial disease patients’ derived skin fibroblasts that were incubated for 30 min with epicatechin ((+)-epicatechin; EPI) or DMSO. Two-way ANOVA followed by Dunnett’s multiple comparison test shows statistical differences depicted by p-value (n=3 biological replicates). FIG. 4N shows a chart of mean ± S.E.M of mitochondrial ATP content measured by luciferase assay and normalized by % of luminescence from control cells. Fibroblasts were treated for 24 h in glucose or galactose media with DMSO or 50 nM epicatechin ((+)-epicatechin; EPI). Two-way ANOVA followed by Tukey’s multiple comparison test shows statistical differences depicted by p-value (n=3). FIG. 40 shows a chart of mean ± S.E.M of total ATP content measured by luciferase assay and normalized by % of luminescence from control cells. Control and patients’ derived fibroblasts were treated for 24 h in glucose media with DMSO or 50 nM epicatechin ((+)-epicatechin; EPI). Two-way ANOVA followed by Tukey’s multiple comparison test shows statistical differences depicted by p-value (n=3). FIG. 4P shows a chart of mean ± S.E.M of total ATP content measured by luciferase assay and normalized by % of luminescence from control cells. Fibroblasts were treated for 24 h in glucose or galactose media with DMSO or 50 nM epicatechin ((+)- epicatechin; EPI). Two-way ANOVA followed by Tukey’s multiple comparison test shows statistical differences depicted by p-value (n=3). FIG. 4Q shows the doubling time of control and patient-derived fibroblasts grown in media containing glucose or galactose, untreated or with 50 nM of epicatechin ((+)-epicatechin; EPI). FIG. 4R shows a chart of mean ± S.E.M of total ATP content measured by luciferase assay and normalized by % change of luminescence from control cells, from control fibroblasts that were treated with 1 pM BTB06584 (BTB) or 1 pM oligomycin (Oligo) for 4 hours in glucose media. Two-way ANOVA followed by Dunnett’s multiple comparison test shows statistical differences depicted by p-value (n=3). FIG. 4S shows a chart of mean ± S.E.M of TMRE (fluorescent intensity (FI)) in the mitochondria area (MitoTracker Green (MTG)) normalized per % of control cells, in fibroblasts that were incubated for 30 min with DMSO, epicatechin ((+)- epicatechin; EPI), or oligomycin (Oligo). Two-way ANOVA followed by Tukey’s multiple comparison test shows statistical differences depicted by p-value (n=2 biological replicates). FIG. 4T shows a plot of determination of OXPHOS and glycolytic activity by monitoring oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), determined with an automatic flux analyzer (XF96, Seahorse/ Agilent). Cells were treated with 50 nM epicatechin ((+)-epicatechin; EPI) in glucose media for 24 h before measurement.

Mean±S.E.M of ECAR versus OCR per 10 ³ cells normalized by % of UT control cells (n=4). FIG. 4U shows a plot of determination of OXPHOS and glycolytic activity by monitoring oxygen consumption rates (OCR) and extracellular acidification rates (ECAR), determined with an automatic flux analyzer 6XF96, Seahorse/ Agilent). Cells were treated with 50 nM epicatechin ((+)-epicatechin; EPI) in glucose media for 24h before measurement. Mean ± S.E.M of ECAR versus OCR per 10 ³ cells normalized by % of UT control cells (n=4). FIG. 4V shows the mean of extracellular lactateas % of control cells, Cells were treated with 50 nM epicatechin ((+)-epicatechin; EPI) for 24 h in high glucose DMEM or galactose as indicated. Two-way ANOVA followed by Sidak's multiple comparisons test shows statistical differences depicted by p-value (n=4). FIG. 4W shows BN-PAGE showing the distribution of CIII and CV complexes and supercomplexes immunoblotted for UQCRC2, ATP5 A and SDHB antibodies in fibroblasts. Cells were treated with DMSO or epicatechin ((+)- epicatechin; EPI) for 24 h in glucose or galactose media. Representative image of three biological replicates. FIG. 4X shows charts of the mean ± S.E.M of CHE, CIII2+CIV and F0F1 relative intensity normalized per SDHB and represented as % of control cells (n=3) from blue native gel electrophoresis analysis of FIG. 4W. FIG. 4Y shows a blue native gel electrophoresis analysis of ATP synthase complex formation in control and patient-derived cells. FIG. 4Z shows a quantification of the analysis from FIG. 4Y, of F0F1 complex formation normalized to complex II formation in control and patient-derived cells.

[0029] FIGS. 5A-5H show that epicatechin ((+)-epicatechin; EPI) replaces ATPIF1 to block ATP hydrolysis in mitochondrial disease models. FIG. 5A shows an BNGE-page analysis of ATPIF1, SDHB, and ATP5A in control and patient-derived fibroblasts. FIG. 5B shows a chart of the mean ± S.E.M. of ATPIF1 relative intensity normalized per SDHB/Vinculin and represented as % of control cells (n=3). FIG. 5C shows confocal micrographs showing patient fibroblasts untreated or treated with 50 nM epicatechin ((+)- epicatechin; EPI) for 24 h, labelled with anti-ATPIFl + anti-ATP5A (PLA) and anti- TOMM20 antibodies. Maximum intensity projection is shown. Scale bars: 20 pm.. FIG. 5D shows a chart of the mean ± S.E.M of PLA dots/pm ³ of mitochondria normalized in % of control cells for control and patient fibroblasts treated with 50 nM epicatechin ((+)- epicatechin; EPI). Two-way ANOVA followed by Tukey's multiple comparisons test shows statistical differences depicted by p-value (n=2). FIG. 5E shows a chart of the mean ± S.E.M of PLA dots/pm ³ of mitochondria normalized in % of control cells for patient-derived fibroblasts treated with the indicated concentrations of epicatechin ((+)-epicatechin; EPI). Two-way ANOVA followed by Tukey's multiple comparisons test shows statistical differences depicted by p-value (n=2). FIG. 5F shows a chart of the mean of extracellular acidification rate (ECAR) in 25 pg of protein normalized by Succinate Rotenone (SR) respiration as a percentage of control cells, showing maximal ATP hydrolysis capacity. Cells stably express ATPIF1-H49K and were incubated with 50 nM epicatechin ((+)-epicatechin; EPI) for 24h in high glucose DMEM. Two-way ANOVA followed by Sidak's multiple comparisons test shows statistical differences depicted by p-value (n=4). FIG. 5G shows a chart of the mean ± S.E.M of mitochondrial ATP in the mitochondria area (MitoTracker Green (MTG)) normalized per % of control cells in patient-derived fibroblasts either untreated or treated for 24 h with 100 nM of epicatechin and expressing the different IF1 variants ((+)-epicatechin; EPI). FIG. 5H shows a chart of the mean ± S.E.M of total ATP content in patient-derived fibroblasts that were either untreated or treated for 24 h with 50 nM of epicatechin.

[0030] FIGS. 6A-6H show that ATP hydrolysis is increased in the Mdx eccentric injury model. FIG. 6A shows maximal ATP hydrolysis capacity per mitochondria measured in frozen gastrocnemius homogenate in wild-type and Mdx mice after 1 h (top) or 24 h (bottom) of eccentric injury. FIG. 6B shows a representative western blot (left) showing complex V (ATP5A1) and complex II (SDHA) levels in gastrocnemius homogenate in wild-type and Mdx mice after 1 h (top) or 24 h (bottom) of eccentric injury. Quantification of protein levels (right) shows the mean ± S.E.M. of the indicated protein. Vinculin was used as loading control. FIG. 6C shows maximal ATP hydrolysis capacity per total complex V measured in frozen gastrocnemius homogenate in wild-type and Mdx mice after 1 h (top) or 24 h (bottom) of eccentric injury. FIG. 6D shows analysis of OPA1 levels and isoforms (left) and quantification (right) in gastrocnemius homogenate in wild-type and Mdx mice after 1 h (top) or 24 h (bottom) of eccentric injury. FIG. 6E shows frozen respirometry to measure complex I (left), complex II (middle), and complex IV (right) oxygen consumption in gastrocnemius homogenate in wild-type and Mdx mice after Ihr (top) or 24 h (bottom) of eccentric injury. FIG. 6F shows the ratio between complex I and II respiratory capacity in wild-type and Mdx gastrocnemius homogenate at the indicated time post injury. FIG. 6G shows analysis of Cytochrome c release in gastrocnemius supernatants of wild-type and Mdx at the indicated time post injury measured by immunoblot. FIG. 6H shows analysis of Cytochrome c release in gastrocnemius supernatants of wild-type and Mdx at the indicated time post injury measured by ELISA. From FIGS. 6A-6H, n=4. For each biological replicate, technical replicates were averaged. Data represent average mean ± S.E.M. [0031] FIGS. 7A-7M show that epicatechin ((+)-epicatechin; EPI) prevents ATP hydrolysis in Mdx eccentric injury model. FIG. 7A shows maximal force in gastrocnemius muscle in vehicle and epicatechin ((+)-epicatechin; EPI) treated Mdx mice at the indicated doses, SID indicates once daily dosing and BID indicates twice daily dosing. Doses are indicated as mg/kg/day. Wild-type vehicle was used as control. FIG. 7B shows a quantification of muscle force after eccentric injury under the indicated conditions. FIG. 7C shows quantification of percentage of force loss induced by eccentric injury under the indicated conditions. FIG. 7D shows a representative immunoblot (top) showing complex V (ATP5A1) and complex II (SDHA) levels in gastrocnemius homogenate in Mdx vehicle- or epicatechin-treated mice 24 h after eccentric injury. Quantification of the protein levels (bottom) shows the mean ± S.E.M. of the indicated protein. Vinculin was used as loading control. FIG. 7E shows maximal ATP hydrolysis capacity per total complex V measured in frozen gastrocnemius homogenate in Mdx vehicle- or epicatechin ((+)-epicatechin; EPI)-treated mice 24 h after eccentric injury. FIG. 7F shows the correlation between muscle force post injure and maximal ATP hydrolytic activity per amount of complex V. Drawn line separates all epicatechin ((+)-epicatechin; EPI)-treated data points from all vehicle-treated. FIG. 7G shows analysis of OPA1 levels and isoforms (left) and quantification thereof (right) in gastrocnemius homogenate in Mdx mice, evaluated 24 hours post-eccentric injury. FIG. 7H shows Cytochrome c release in gastrocnemius supernatants of Mdx vehicle or epicatechin ((+)-epicatechin; EPI) treated 24 h after eccentric injury measured by immunoblot (left) and quantification of the protein levels therein (right). FIG. 71 shows Cytochrome c release in gastrocnemius supernatants of Mdx vehicle- or epicatechin ((+)-epicatechin; EPI)-treated mice 24 h after eccentric injury measured by ELISA. FIG. 7J shows the results of frozen respirometry to measure complex I, complex II, and complex IV oxygen consumption in gastrocnemius homogenate in Mdx mice in the indicated conditions. FIG. 7K shows the ratio between complex I and II respiratory capacity in Mdx gastrocnemius homogenate from vehicle- compared to epicatechin ((+)-epicatechin; EPI)-treated mice (15 mg/kg, twice daily) at 24 hours post-injury. FIG. 7L shows Principal Component Analysis (PCA) to determine the influence of epicatechin ((+)-epicatechin; EPI) treatment in injury recovery of gastrocnemius after eccentric injury. FIG. 7M shows Principal Component Analysis (PCA) to determine the influence of epicatechin ((+)-epicatechin; EPI) treatment in injury recovery of gastrocnemius after eccentric injury. DETAILED DESCRIPTION

[0032] The mitochondrial ATP synthase reverses its function to hydrolyze ATP in response to mitochondrial depolarization. However, whether the reversal of mitochondrial ATP synthase contributes to the pathogenesis of diseases showing impaired respiration remains uncharacterized; to date, no compounds or regulatory factors have been identified that selectively block the reversal of mitochondrial ATP synthase, without simultaneously inhibiting ATP synthesis.

[0033] By integrating in-silico screening and docking analyses with biochemical assays using purified ATP synthase, isolated mitochondria, and total cell lysates, the inventors of the presently claimed methods identified that the flavonoid epicatechin competitively binds the mitochondrial ATP synthase in a small area of the ATPIF1 binding pocket, to selectively block ATP hydrolysis but not ATP synthesis. In live cells and tissues, epicatechin treatment blocked ATP hydrolysis and reversed metabolic dysfunction induced by mutations in respiratory complexes as well as by stress induced mitochondrial dysfunction.

[0034] Thus, in one aspect the present application provides methods of treating or preventing a disease or disorder that would benefit from inhibition of ATP hydrolysis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

I. Definitions

[0035] As used herein and in the appended claims, the singular forms “a”, “an” and “the” include plural forms, unless the context clearly dictates otherwise.

[0036] As used herein, and unless otherwise specified, the terms “about” and “approximately,” when used in connection with doses, amounts, molar percent, or weight percent of ingredients of a composition or a dosage form, mean a dose, amount, molar percent, or weight percent that is recognized by those of ordinary skill in the art to provide a pharmacological effect equivalent to that obtained from the specified dose, amount, molar percent, or weight percent. Specifically, the terms “about” and “approximately,” when used in this context, contemplate a dose, amount, molar percent, or weight percent within 15%, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%, or within 0.5% of the specified dose, amount, molar percent, or weight percent.

[0037] As used herein, “therapeutically effective amount” indicates an amount that results in a desired pharmacological and/or physiological effect for the condition. The effect may be prophylactic in terms of completely or partially preventing a condition or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for the condition and/or adverse effect attributable to the condition.

[0038] As used herein, the term “pharmaceutically acceptable excipient,” and cognates thereof, refers to adjuvants, binders, diluents, etc. known to the skilled artisan that are suitable for administration to an individual (e.g., a mammal or non-mammal). Combinations of two or more excipients are also contemplated. The pharmaceutically acceptable excipient(s) and any additional components, as described herein, should be compatible for use in the intended route of administration (e.g., oral, parenteral) for a particular dosage form, as would be recognized by the skilled artisan.

[0039] As used herein, the term "co-crystal" denotes crystalline molecular complexes, encompassing hydrates and solvates. "Co-crystals" are composed of multi-component, stoichiometric and neutral molecular species, each existing as a solid under ambient conditions. Co-crystals exhibit properties different from free drugs or salts. The solid form influences relevant physico-chemical parameters such as solubility, dissolution rate of the drug, chemical stability, melting point, and hygroscopicity, which can result in solids with superior properties.

[0040] The terms “treat,” “treating,” and “treatment” are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or to slowing the progression, spread or worsening of a disease, disorder or condition or of one or more symptoms thereof. Often, the beneficial effects that a subject derives from a therapeutic agent do not result in a complete cure of the disease, disorder or condition.

[0041] The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), monkey, cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms “subject” and “patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human.

[0042] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. [0043] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0044] The following statements provide a summary of some aspects of the inventive nucleic acids and methods described herein.

II. Methods of use

[0045] The present application provides methods of using epicatechin. Epicatechin may competitively bind the mitochondrial ATP synthase in a small area of the ATPIF1 binding pocket. Importantly, the epicatechin may selectively block ATP hydrolysis but not ATP synthesis. As described in the working examples of the present disclosure, in live cells and tissues, epicatechin treatment blocked ATP hydrolysis and reversed metabolic dysfunction induced by mutations in respiratory complexes as well as by stress induced mitochondrial dysfunction.

A. Epicatechin

[0046] Epicatechin is flavonoid that is a more active isomer of catechin. Catechin is a flavan-3-ol, a type of natural phenol and antioxidant. Epicatechin and catechin are abundant flavanols in fruits, chocolate, red wine, and tea. In some aspects, provided herein is a method comprising use of a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. The term “epicatechin” as used herein refers to epicatechin in its various forms, including but not limited to epicatechin, a co-crystal of epicatechin, a therapeutically effective amount of epicatechin, a pharmaceutically acceptable salt of epicatechin, a pharmaceutical composition comprising epicatechin, or any combination thereof.

[0047] The epicatechin of the present disclosure may bind Electron Transport Chain (ETC) complex V (e.g., ATP synthase). In some embodiments, the epicatechin binds complex V. In some embodiments, the epicatechin directly binds complex V. In some embodiments, the epicatechin binds the Fl head of complex V. In some embodiments, the B-ring catechol hydroxyls of epicatechin hydrogen bond with the side chains of E454 and Q456 of complex V. In some embodiments, the A-ring resorcinol hydroxyl of epicatechin hydrogen bonds with the side chain of H451 of complex V. In some embodiments, the epicatechin binds the ATPIF1 (a complex V inhibitor) binding pocket of complex V. In some embodiments, the epicatechin acts as a partial ATPIF1 mimic. In some embodiments, the dihydropyran heterocycle and B-ring benzyl of epicatechin bind near L452 and P453 that form the ATPIF1 binding groove.

[0048] Epicatechin, when administered as part of the methods provided herein, can regulate ATP hydrolysis by complex V. ATP hydrolysis by complex V can become increased in various diseases. Additionally, ATPIF1 can become downregulated in various diseases. ATP hydrolysis is reported to happen in stress conditions like heart ischemic injury and hypoxia/anoxia in order to maintain the mitochondrial membrane potential by ATP synthase. [0049] In some embodiments, the epicatechin inhibits ATP hydrolysis by complex V. In some embodiments, the epicatechin partially inhibits ATP hydrolysis by complex V (e.g., decreased ATP hydrolysis occurs). In some embodiments, the epicatechin decreases the ATP hydrolysis of complex V by between about 1% and about 50%, such as between about 1% and about 25%, between about 10% and about 40%, or between about 20% and about 50%. In some embodiments, the epicatechin decreases the ATP hydrolysis by complex V by greater than about 1%, such as greater than any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In some embodiments, the epicatechin decreases the ATP hydrolysis by complex V by less than about 50%, such as less than any of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less. In some embodiments, the epicatechin completely inhibits ATP hydrolysis (e.g., no ATP hydrolysis occurs) by complex V. In some embodiments, the epicatechin inhibits ATP hydrolysis by binding complex V. In some embodiments, the epicatechin inhibits ATP hydrolysis by complex V by binding the ATPIF1 binding pocket of complex V. In some embodiments, the epicatechin inhibits ATP hydrolysis by directly binding the ATPIF1 binding pocket of complex V.

[0050] In some embodiments, the epicatechin does not inhibit ATP synthesis by complex V. In some embodiments, the epicatechin decreases and/or inhibits ATP hydrolysis by complex V without inhibiting ATP synthesis by complex V. In some embodiments, the epicatechin does not inhibit electron transfer by complex V. In some embodiments, the epicatechin decreases and/or inhibits ATP hydrolysis by complex V without the epicatechin does not inhibit electron transfer by complex V. In some embodiments, the epicatechin optimizes complex V activity. Complex V activity may be optimized by decreasing ATP hydrolysis without inhibiting ATP synthesis. [0051] In some embodiments, the epicatechin blocks the opening of the mitochondrial permeability transition pore (MPTP). In some embodiments, the epicatechin blocks the Ca ²⁺ induced mitochondrial permeability transition pore (MPTP) opening.

[0052] In some embodiments, the epicatechin prevents the dissociation of complex V into monomers. In some embodiments, the epicatechin maintains complex V in its assembled (e.g., duplexed) state.

[0053] In some embodiments, the epicatechin modulates mitochondrial function. In some embodiments, the epicatechin inhibits cell death. In some embodiments, the epicatechin inhibits stress induced cell death. Stress induced cell death may occur due to disease or disorders associated with mitochondrial dysfunction, including CNS disorders, mitochondrial diseases, and/or muscle dystrophies.

[0054] In some embodiments, epicatechin is enantiomerically pure or enantiomerically enriched. In some embodiments, the epicatechin is enatiomerically pure or enantiomerically enriched (+) epicatechin. In other embodiments, the epicatechin is enantiomerically pure or enantiomerically enriched (-) epicatechin. The purity of the enantiomerically pure or enantiomerically enriched (+)/(-) epicatechin is at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or 100%. In some embodiments, the epicatechin comprises at least about 75% (+)-epicatechin, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or greater, (+)-epicatechin. In some embodiments, the epicatechin comprises at least about 75% (-)-epicatechin, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, 99.9%, or greater, (-)-epicatechin.

[0055] In some embodiments, the epicatechin is in the form of a co-crystal. Co-crystals of epicatechin have been previously described, for example, in US2021/0380535, which is hereby incorporated by reference in its entirety. Co-crystals described herein can have a purity of at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 99.9%. In some embodiments, the co-crystal is a co-crystal of (+) epicatechin : trigonelline. In some embodiments, the co-crystal is a co-crystal of (-) epicatechin : trigonelline. In some embodiments, the co-crystal is a co-crystal of (+) epicatechin : D-proline. In some embodiments, the co-crystal is a co-crystal of (+) epicatechin : L-proline. B. Methods

[0056] In some aspects, provided herein is a method of treating or preventing a disease or disorder that would benefit from inhibition of ATP hydrolysis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. In some embodiments, the inhibition of ATP hydrolysis does not block mitochondrial ATP synthesis (e.g., complex V ATP synthesis). [0057] The vast majority of the body's need for ATP is supplied through the process of oxidative phosphorylation, carried out in the mitochondria in all tissues. There are 5 protein complexes, known as the Electron Transport Complexes that effect ATP synthesis. ETC complex I, II, III, and IV mediate electron transport. ETC I, III, and IV also function as proton pumps that maintain an electrochemical gradient necessary for activity of ETC V, the ATP synthase enzyme that makes ATP from ADP. Complex T, also known as cytochrome c oxidase, (COX), consists of 14 subunits whose assembly into a functional complex requires an additional 30 protein factors. In some embodiments, the disease or disorder causes impaired function of any of the complexes of the ETC, or a combination thereof. In some embodiments, disease or disorder causes impaired function of ETC complex V.

[0058] In some embodiments, the disease or disorder causes metabolic dysfunction. In some embodiments, the disease or disorder causes impaired mitochondrial respiration. In some embodiments, the disease or disorder causes mitochondrial toxicity. In some embodiments, the mitochondrial toxicity is identified based on or associated with one or more biological effects, which include, but are not limited to, abnormal mitochondrial respiration, abnormal oxygen consumption, abnormal extracellular acidification rate, abnormal mitochondrial number, abnormal lactate accumulation, and abnormal ATP levels. In some embodiments, the mitochondrial toxicity is identified based on or associated with one or more physiological manifestations, which include, but are not limited to, elevations in markers known to relate to injury to the heart, liver, and/or kidney, elevated serum liver enzymes, elevated cardiac enzymes, lactic acidosis, elevated blood glucose, and elevated serum creatinine. Methods for assessing such biological effects or markers are known in the art and may be used in connection with the embodiments described herein. In some embodiments, the disease or disorder deceases mitochondrial ATP synthesis. In some embodiments, the disease or disorder increases ATP hydrolysis.

[0059] The diseases or disorders for treatment or prevention may be any disease or disorder for which epicatechin is indicated, including, without limitation, any of the disease or conditions described in US 11,154,546, US 9,187,448, US 11,273,144, US2018/0193306, US 9,975,869, US 10,898,465, US2019/0262347, and US2021/0380535, each of which is hereby incorporated by reference in its entirety. In some embodiments, the disease or disorder is selected from the group consisting of psychomotor delay, tubulopathy, renal disease, liver disease, and loss of synaptic density.

[0060] In some embodiments, the epicatechin prevents loss of mitochondrial ATP synthesis associated with the disease or disorder. In some embodiments, the epicatechin does not affect mitochondrial ATP synthesis. In some embodiments, the mitochondrial ATP synthesis after administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof is the same as the mitochondrial ATP synthesis before administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof.

[0061] Other aspects of the present application provide methods of preventing Electron Transport Chain (ETC) complex V dissociation into monomers in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. In some embodiments, the epicatechin prevents the dissociation of complex V into monomers. In some embodiments, the epicatechin holds complex V in a duplexed form.

[0062] Further aspects of the present application provide methods of decreasing ATP hydrolysis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. In some embodiments, the ATP hydrolysis is decreased by greater than any of about 0.05- fold, 0.1-fold, 0.5-fold, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, or more, compared to the ATP hydrolysis prior to administering the therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. In some embodiments, the ATP hydrolysis is decreased by less than any of about 5-fold, 4-fold, 3-fold, 2-fold, 1-fold, 0.5-fold, 0.1-fold, 0.05-fold, or less, compared to the ATP hydrolysis prior to administering the therapeutically effective amount of epicatechin or a pharmaceutically acceptable salt thereof. In some embodiments, the ATP hydrolysis is decreased by between about 1% and about 50%, such as between about 1% and about 25%, between about 10% and about 40%, or between about 20% and about 50%. In some embodiments, the ATP hydrolysis is decreased by greater than about 1%, such as greater than any of about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In some embodiments, the ATP hydrolysis is decreased by less than about 50%, such as less than any of about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or less. In some embodiments, the ATP hydrolysis is completely inhibited (e.g., no ATP hydrolysis occurs).

[0063] In some embodiments, the subject in need of a method of decreasing ATP hydrolysis has a disease or disorder. The disease or disorder may be any disease or disorder for which epicatechin is indicated, including, without limitation, any of the disease or conditions described in US 11,154,546, US 9,187,448, US 11,273,144, US2018/0193306, US 9,975,869, US 10,898,465, US2019/0262347, and US2021/0380535, each of which is hereby incorporated by reference in its entirety. In some embodiments, the subject has a disease or disorder selected from the group consisting of spinal cord injury or abnormality, liver disease, kidney disease, impaired cognition, neurodegenerative disease, dystonia, sarcopenia, cardiomyopathy of aging or other diseases associated with mitochondrial dysfunction, cardiomyopathy, ischemic vascular disease, immunodeficiency states, ataxia, pulmonary inflammation and fibrosis, infantile encephalomyopathy, epilepsy, Charcot-Marie-Tooth disease, exocrine pancreatic insufficiency, impaired wound healing, and growth of cancer cells. In some embodiments, the subject has a neurodegenerative disease selected from the group consisting of Alzheimer’s disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), Leigh syndrome, Duchenne muscular dystrophy, Becker muscular dystrophy, and peripheral and central neuropathies.

III. Pharmaceutical compositions and dosage

[0064] According to the methods described herein, the epicatechin or pharmaceutical composition thereof may be administered at a therapeutically effective dosage, e.g., a dosage sufficient to provide treatment for the disease state. While human dosage levels have yet to be optimized for the chemical entities described herein (e.g., epicatechin, co-crystals of epicatechin, etc.), generally, a daily dose ranges from about 0.01 to 100 mg/kg of body weight; in some embodiments, from about 0.05 to 10.0 mg/kg of body weight, and in some embodiments, from about 0.10 to 1.4 mg/kg of body weight. Thus, for administration to a 70 kg person, in some embodiments, the dosage range would be about from 0.7 to 7000 mg per day; in some embodiments, about from 3.5 to 700.0 mg per day, and in some embodiments, about from 7 to 100.0 mg per day.

[0065] In some embodiments, the epicatechin is administered each day, every other day, weekly, every two weeks, every three weeks, or every four weeks. The amount of the chemical entity administered will be dependent, for example, on the subject and disease state being treated, the severity of the affliction, the manner and schedule of administration and the judgment of the prescribing physician. For example, an exemplary dosage range for oral administration is from about 5 mg to about 500 mg per day, and an exemplary intravenous administration dosage is from about 5 mg to about 500 mg per day, each depending upon the pharmacokinetics. In some embodiments, the epicatechin is administered at a daily dosage of between about 5 mg to about 500 mg, such as between about 5 mg and about 100 mg, about 50 mg and about 200 mg, about 100 mg and about 300 mg, about 200 mg and about 400 mg, or about 300 mg and about 500 mg. In some embodiments, the epicatechin is administered at a daily dosage of less than about 500 mg, such as less than any of about 450 mg, 400 mg, 350 mg, 300 mg, 250 mg, 200 mg, 150 mg, 100 mg, 50 mg, 40 mg, 30 mg, 20 mg, 10 mg, 5 mg, or less. In some embodiments, the epicatechin is administered at a daily dosage of greater than about 5 mg, such as greater than any of about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, or greater.

[0066] A daily dose is the total amount administered in a day. A daily dose may be, but is not limited to be, administered each day, every other day, each week, every 2 weeks, every month, or at a varied interval. In some embodiments, the daily dose is administered for a period ranging from a single day to the life of the subject. In some embodiments, the daily dose is administered once a day. In some embodiments, the daily dose is administered in multiple divided doses, such as in 2, 3, or 4 divided doses. In some embodiments, the daily dose is administered in 2 divided doses.

[0067] Administration of the epicatechin (e.g., epicatechin, a pharmaceutical composition comprising the epicatechin, and/or a co-crystal of the epicatechin) can be via any accepted mode of administration for therapeutic agents including, but not limited to, oral, sublingual, subcutaneous, parenteral, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, or intraocular administration. In some embodiments, the epicatechin or pharmaceutical composition thereof is administered orally, sublingually, subcutaneously, parenterally, intravenously, intranasally, topically, transdermally, intraperitoneally, intramuscularly, intrapulmonarily, vaginally, rectally, or intraocularly. In some embodiments, the epicatechin or pharmaceutical composition thereof is administered orally or intravenously. In some embodiments, the epicatechin or pharmaceutical composition thereof is administered orally.

[0068] Pharmaceutically acceptable compositions include solid, semi-solid, liquid and aerosol dosage forms, such as tablet, capsule, powder, liquid, suspension, suppository, and aerosol forms. In some embodiments, the epicatechin is administered in the form of a tablet, a capsule, a powder, a liquid, a suspension, a suppository, or an aerosol. The epicatechin or pharmaceutical composition thereof can also be administered in sustained or controlled release dosage forms (e.g., controlled/sustained release pill, depot injection, osmotic pump, or transdermal (including electrotransport) patch forms) for prolonged timed, and/or pulsed administration at a predetermined rate. In some embodiments, the epicatechin or pharmaceutical composition thereof are provided in unit dosage forms suitable for single administration of a precise dose.

[0069] The epicatechin or pharmaceutical composition thereof can be administered either alone or in combination with one or more conventional pharmaceutical carriers or excipients (e.g., mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin, sucrose, magnesium carbonate). If desired, the pharmaceutical composition can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like (e.g., sodium acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate). Generally, depending on the intended mode of administration, the pharmaceutical composition will contain about 0.005% to 95%, or about 0.5% to 50%, by weight of a compound disclosed and/or described herein. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0070] In some embodiments, the epicatechin or pharmaceutical composition thereof will take the form of a pill or tablet and thus the composition may contain, along with epicatechin (e.g., an epicatechin co-crystal), one or more of a diluent (e.g., lactose, sucrose, dicalcium phosphate), a lubricant (e.g., magnesium stearate), and/or a binder (e.g., starch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives). Other solid dosage forms include a powder, marume, solution or suspension (e.g., in propylene carbonate, vegetable oils or triglycerides) encapsulated in a gelatin capsule.

[0071] Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing or suspending etc. a co-crystal disclosed and/or described herein and optional pharmaceutical additives in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycols, ethanol or the like) to form a solution or suspension. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, as emulsions, or in solid forms suitable for dissolution or suspension in liquid prior to injection. The percentage of the epicatechin contained in such parenteral compositions depends, for example, on the physical nature of the co-crystal, the activity of the epicatechin, and the needs of the subject. However, percentages of active ingredient of 0.01% to 10% in solution are employable, and may be higher if the composition is a solid which will be subsequently diluted to another concentration. In some embodiments, the composition will comprise from about 0.2 to 2% of epicatechin in solution.

[0072] Pharmaceutical compositions of epicatechin may also be administered to the respiratory tract as an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the pharmaceutical composition may have diameters of less than 50 microns, or in some embodiments, less than 10 microns.

[0073] In addition, pharmaceutical compositions can include epicatechin, epicatechin cocrystals, and one or more additional medicinal agents, pharmaceutical agents, adjuvants, and the like. In some embodiments, the epicatechin is administered in the form of a pharmaceutical composition comprising the epicatechin or the pharmaceutically acceptable salt, solvate, or co-crystal thereof, and a pharmaceutically acceptable excipient.

[0074] The epicatechin may be administered in combination with one or more additional therapeutic agents. In some embodiments, the epicatechin and the one or more additional therapeutic agents are administered concurrently. In some embodiments, the epicatechin and the one or more additional therapeutic agents are administered sequentially.

IV. Kits

[0075] Also provided are articles of manufacture and kits containing epicatechin, such as epicatechin, a co-crystal of epicatechin, or pharmaceutical compositions thereof, provided herein. The article of manufacture may comprise a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. The container may hold a pharmaceutical composition provided herein. The label on the container may indicate that the pharmaceutical composition is used for preventing, treating or suppressing a condition described herein, and may also indicate directions for either in vivo or in vitro use.

[0076] In one aspect, provided herein are kits containing epicatechin, such as epicatechin, a co-crystal of epicatechin, or pharmaceutical compositions thereof, described herein and instructions for use. The kits may contain instructions for use in the treatment of any disorder, disease, or condition, provided herein in a subject in need thereof. A kit may additionally contain any materials or equipment that may be used in the administration of the epicatechin, such as epicatechin, a co-crystal of epicatechin, or pharmaceutical compositions thereof, such as vials, syringes, or IV bags. A kit may also contain sterile packaging.

EXAMPLES

[0077] The invention will be more fully understood by reference to the following examples. They should not, however, be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Example 1: Protocol to determine the fraction of mitochondrial ATP synthase working in forward and reverse mode, as well as maximal ATP hydrolytic capacity

[0078] This Example demonstrates that a portion of mitochondrial ATP synthases (used interchangeably herein with “complex V” and “CV”) are functioning in reverse under normal conditions (e.g., hydrolyzing ATP rather than synthesizing ATP).

[0079] To assess whether the fraction of synthetic and reverse hydrolytic activities of mitochondrial ATP synthase can be measured in the same preparation, a protocol was developed to monitor changes in oxygen consumption and proton release in response to changes in mitochondrial ATP synthase activity. The proton released and the concomitant changes in the pH can be monitored using the ECAR (extracellular acidification) channel in the Agilent Seahorse XF analyzer using isolated mitochondria, to ensure that the changes in pH cannot be attributed to glycolysis or other contributors to ECAR.

[0080] Mice were anesthetized with isoflurane followed by a cervical dislocation, and the heart was immediately removed and placed in ice cold relaxation buffer (5 mM sodium pyrophosphate, 100 mM KC1, 5 mM EGTA, 5 mM HEPES; pH 7.4). The heart was squeezed with tweezers to remove blood, minced with scissors, and then placed in a glass-glass Dounce homogenizer with 3 mL of HES homogenization buffer (250 mM sucrose, 5 mM HEPES, 1 mM EDTA; pH to 7.2, adjusted with KOH). The heart tissue was homogenized first with the loose pestle and followed by the tight pestle. The homogenized tissue was placed in a prechilled 15 mL conical tube and centrifuged at 900 x g (4 °C) for 10 minutes (min). The supernatant was removed, placed in a new tube and centrifuged again at 900 x g for 10 min. The supernatant was then transferred to 2 mL microcentrifuge tubes and centrifuged at 10,000 x g (4 °C) for 10 min. The mitochondrial pellets were re-suspended in ice cold HES buffer and mitochondrial protein was measured with a BCA assay (Pierce). The concentrated mitochondrial pellet was stored on ice.

[0081] Heart mitochondria (0.75-1.5 pg) were loaded into a Seahorse XF96 microplate in 20 pL of MAS (70 mM Sucrose, 220 mM Mannitol, 5 mM KH2PO4, 5 mM MgC12, 1 mM EGTA, 2 mM HEPES; pH 7.2) plus 1% free fatty acid BSA containing substrates. The loaded plate was centrifuged at 2,000 g for 5 min at 4 °C (no brake) and an additional 130 pL of MAS was added to each well. When assessing compounds effect on respirometry, the compounds were added at this point at the indicated concentration in MAS buffer. To avoid disrupting mitochondrial adherence to the bottom of the plate, MAS was added using a multichannel pipette pointed at a 45° angle to the top of the well-chamber, as instructed by the manufacturer. Substrate concentrations in the well when assay was starting in state 4 were as follow: (i) 5 mM pyruvate + 5 mM malate or ii) 5 mM succinate + 2 pM rotenone. Substrate concentrations in the well when assay was starting in state 3 were as follow: (i) 5 mM pyruvate + 5 mM malate + 4 mM ADP or ii) 5 mM succinate + 2 pM rotenone + 4 mM ADP. Injections were performed as indicated in the figure descriptions at the following final concentration in the well: oligomycin (3.5 pM), FCCP (4 pM), Antimycin A (2 pM).

Compounds were added at the indicated concentration.

[0082] ATP hydrolysis capacity or state 4 acidification was measured using Seahorse XF96 as described in Divakaruni et al. (2018) Anal Biochem 552:60-65 and Acin-Perez et al. (2021) Life 11 (9):949, in MAS (70 mM Sucrose, 220 mM Mannitol, 5 mM KH2PO4, 5mM MgCh, 1 mM EGTA, 2 mM HEPES; pH 7.2). Plates were loaded with 0.75-1.5 pg of mouse heart mitochondria, or 25 pg of cell lysate. Cell lysates were prepared by subjecting the samples to 4 cycles of free thaw (liquid nitrogen-37°C water bath) before measuring protein concentration. When assessing compounds effect on respirometry, the compounds were added in MAS after sample centrifugation. Initial respiration of the samples was sustained by the addition of 5 mM succinate + 2 pM rotenone in the MAS after centrifugation. Injections were performed at the following final concentration in the well: Antimycin A (2 pM), oligomycin (5 pM), FCCP (1 pM), ATP (20 mM). To assess maximal ATP concentration, ATP was injected consecutively.

[0083] Oxygen consumption was measured in isolated fresh intact mitochondria from mouse heart, as oxygen consumption is linked to maximal ATP synthesis and proton release when mitochondria are respiring either in the presence of substrates and ADP (ATP synthesizing mitochondria or state 3) or in the presence of substrates but not ADP (state 4). Oxygen consumption was fueled by Pyruvate and Malate (Pyr+Mal, FIG. 1 A and FIG. IB) or Succinate and Rotenone (Succ+Rot, FIG. 1C and FIG. ID). Proton release in isolated mitochondria is mostly a result of ATP hydrolysis (FIGS. 1A-1D). As shown in FIGS. 1 A- 1D, mitochondria showed normal state 3 - ATP synthesis linked respiration and were responsive to an injection of ATP, which induced a decrease in oxygen consumption and an increase in proton release or state 4 ATP hydrolysis. This response was similar in mitochondria starting in state 3 or state 4 (FIGS. 1 A-1D). These results indicate that ATP hydrolysis can happen independently if the mitochondria begin in an ADP excess state and have sufficient ATP to decrease respiration, or if they begin in state 4 with no ADP.

[0084] When FCCP, a potent mitochondrial oxidative phosphorylation uncoupler, was added after ATP, the ATP hydrolytic activity was increased to its maximal (FIG. IE).

[0085] To exclude the possibility that the ATP hydrolysis observed prior to FCCP injection was caused by a fraction of broken mitochondria as a result of the isolation procedure or contaminant extramitochondrial ATPases, the mitochondrial fractions were treated with proteinase K. When state 3 respiration and ATP hydrolysis were measured in mitochondrial treated with proteinase K, no changes were observed in their respective activities, suggesting that the mitochondria were intact. These results indicate that the ATP hydrolytic activity detected was not stemming from contaminant ATPase outside the mitochondria (FIG. IF). Based on these results, the percentage of mitochondrial ATP synthase acting in reverse (state 4 ATP hydrolysis) versus synthesis (state 3 respiration) was determined to be around 20% in isolated heart mitochondria (FIG. 1G).

[0086] An additional assay was developed to determine the maximal ATP hydrolysis capacity in frozen mitochondria. Maximal ATP hydrolytic capacity was assessed in frozen heart mitochondria where mitochondrial respiration is inhibited by Antimycin A and maximal ATP hydrolysis is driven by co-injection of FCCP and ATP, followed by inhibition in the presence of oligomycin. Addition of oligomycin at different concentrations in the assay media revealed a dose dependent inhibition of ATP hydrolysis (FIG. 1H), demonstrating the specificity of the assay. Example 2: Increased ATP hydrolysis by ATP synthase and ATPIF1 downregulation is a common compensatory mechanism is mitochondrial diseases

[0087] This Example demonstrates the prevalence of ATP synthase and ATPIF1 misregulation in fibroblasts derived from samples from patients with mitochondrial diseases. [0088] The reversal capacity of the mitochondrial ATP synthase was quantified in fibroblasts derived from patients with mitochondrial diseases. Fibroblasts with mutations causing dysfunction in complex I (CI; NDUFA6), complex III (CIII; BCS1L and TTC19), complex V itself (CV; TMEM and ATP6), and MICOS (MIC26) were analyzed. An increase in the maximal ATP hydrolytic capacity (FIG. II) of mitochondrial ATP synthase, determined using the protocol in Example 1, was observed for CIII mutants (BCS1L and TTC19), MICOS deficiency (MIC26), and mitochondrial ATP synthase (ATP6).

As ATPIF1 is understood to modulate ATP synthesis and hydrolysis in live cells, it was hypothesized that ATPIF1 can contribute to the pathological mechanism in mitochondrial disease models, and changes in total ATPIF1 content could be a common compensatory mechanism in mitochondrial deficiencies. Total ATPIF1 protein levels were measured by immunoblot. ATPIF1 protein levels were decreased in skin fibroblasts derived from mitochondrial disease patients (FIG. 1 J), suggesting that excessive ATP hydrolysis could be a pathogenic process exacerbating their metabolic dysfunction.

[0089] Overall, these data indicate that downregulation of ATPIF1, together with increasing ATP hydrolytic capacity, is an adaptive mechanism in mitochondrial diseases. This mechanism may allow better consumption of cytosolic ATP and keep the membrane potential, thereby avoiding apoptotic cell death.

Example 3: Binding of catechins to mitochondrial ATP synthase

[0090] This Example demonstrates that epicatechin has a high binding affinity to the head of ATP synthase, and binds in the same pocket of ATP synthase as the inhibitory protein ATPIF1. These results in a mitochondrial disease cell model highlights the need for pharmacological intervention by modulating complex V activity.

[0091] To find possible mitochondrial ATP synthase interactors that selectively regulate forward activity, (i.e. ATP synthesis) or reverse activity (i.e. ATP hydrolysis), an insilico screening was performed of 7441 natural or synthetic derivatives compounds from CoconutDB (Sorokina et al. (2021) J Cheminform 13( 1 ):2) ) by categorizing them by antioxidant and free radical scavenging capabilities, as well as creatine kinase (CK) inhibition and docking capabilities to mitochondrial ATP synthase. Ligands with molecular weight between 80 and 800 kDa were filtered from the CoconutDB database using mongoDB (Sorokina, M el al. J Cheminform 13, 2 (2021)). To reinforce the potential mitochondrial activity, PASS (Prediction of Activity Spectra for Substances) analysis, as described in Filimonov et al. (2014) Chemistry of Heterocyclic Compounds 50(3):444-457, was used for prediction of the three-biological activity parameters: creatine kinase inhibition, antioxidant scavenging capability, and free radical scavenging capability. In situ ligand screening was carried out using Autodock Vina 4.2, as described in Trott and Olson (2010) J Comput Chem 31(2):455-461, against the bovine ATP synthase (PDB: 6ZQN) as a receptor. PYMOL structure viewer was used for visualization and selection of coordinates. The selected region contained the Fl head of the ATP synthase, more specifically with the Alpha and Beta subunits around the central stalk where ATPIF1 may be interacting with ATP synthase. [0092] From the initial 7441 compounds, 6827 ligands were removed from additional analysis due to the lack of selected function. A total of 614 ligands (data not shown) were used for direct docking with the Fl head of ATP synthase, with 445 ligands having equal or greater than -8.5 Kcal/mol of affinity to the selected region. From this subset of ligands, flavonoids had the highest affinity. One group of 28 compounds belonging to the family of catechins, listed in Table 1, had particularly high affinity to the Fl head of ATP synthase, where ATPIF1 inhibits the enzyme function.

Table 1: In silico screening results for the Catechin family

[0093] All simulations for the interaction of (+)- and (-)-epicatechin, quercetin, and (-)- catechin with Fl -ATP synthase and ATPIF1 were started from the x-ray structure of Fl- ATPase complexed with ATPIF1 corresponding to Protein Data Bank ID: lohh. Structures of the epicatechins, quercetin and (-)-catechin were retrieved from Pubchem (CID: 182232: (+)- epicatechin; 72276: (-)-epicatechin; 5280343: quercetin; 73160: (-)-catechin). Structures were prepared using YASARA (methods described in Land and Humble (2018) Methods Mol Biol 1685:43-67), adding hydrogen atoms, checking the protonation states of side chains, and optimizing the hydrogen-bond network. If necessary, loops were closed using YASARA. Simulations were carried out with ATPIF1 present and absent. PELE (Protein Energy Landscape Exploration) webserver was used to identify potential epicatechin binding sites on Fl -ATP synthase, using default webserver settings. Resulting poses were analyzed for ligand affinities and energies using AutoDock Vina (methods described in Trott and Olson (2010) J Comput Chem 31(2): 455-461). Visualizations were analyzed using UCSF Chimera (methods described in Pettersen et al. (2004) J Comput Chem 25(13): 1605-1612).

[0094] This analysis was run using the ligands identified in the CoconutDB study and also included epicatechin. PELE identified a low energy binding site for quercetin and (-)-catechin and another member of the catechin family, whereas (+)-epicatechin bound in the vicinity of the proposed ATPIF1 binding groove on the surface of Fl -ATP synthase (FIG. IK). The three compounds bound with similar affinities (AutoDock Vina binding score: -5.3 for (+)- epicatechin, -5.7 for quercetin and -5.5 for (-)-catechin (FIG. IK, left panel).

[0095] When comparing the different epicatechin isomers, it was observed that the resulting pose of (+)-epicatechin indicated the B-ring catechol hydroxyls hydrogen bond with the side chains of E454 and Q456 and the A-ring resorcinol hydroxyl hydrogen bonds with the side chain of H451. The remainder of the interactions were hydrophobic in nature with the dihydropyran heterocycle and B-ring benzyl near L452 and P453 that form the ATPIF1 binding groove. The 3 -OH of (+)-epicatechin positions away from the surface unlike the same group in (-)-epicatechin which sterically hinders the interaction. The failure of (-)- epicatechin to bind may explain the selectivity of (+)-epicatechin in inhibiting ATPIF1 binding to the ATP synthase (FIG. IK, middle panel). Finally, the data shows that ATPIF1 and (+)-epicatechin are aligning at the same position and orientation in the ATPIF1 binding groove (FIG. IK, right panel).

[0096] Taken together, these results suggest that ATP hydrolysis and synthesis can happen at the same time.

Example 4: Epicatechin decreases the ATP hydrolytic capacity of mitochondrial ATP synthase, without affecting ATP synthesis capacity or electron transfer

[0097] This Example demonstrates that epicatechin (e.g., (+)-epicatechin, also referred to as EPI) has an inhibitory effect on mitochondrial ATP synthase.

[0098] Based on the binding score in silico results described in Examples 3 and 4, the effects of (+)-epicatechin (EPI) on maximal ATP hydrolysis capacity of mitochondrial ATP synthase were studied. Contrary to oligomycin, EPI only inhibited about 30% of the hydrolytic activity (FIG. IM). The ATP hydrolytic activity was specific to mitochondrial ATP synthase and was absent in the cytosolic fraction (FIG. IL). To further understand whether epicatechin could affect the activity of other electron transport chain complexes, maximal activity of electron transport chain (ETC) complex I and II was measured. As shown in FIG. IN, epicatechin specifically inhibits ATP hydrolytic activity without affecting either complex I or II activities.

[0099] To determine whether epicatechin effects on ATP hydrolysis were mediated by direct action on the mitochondrial ATP synthase, epicatechin was added either to intact mitochondria, broken mitochondria, or directly to a gel. First, assembled ATP synthase was separated by blue native gel electrophoresis (BNGE) and its In Gel ATP hydrolytic activity measured under increasing concentrations of epicatechin. Mitochondria derived from tissues were permeabilized with either 8 mg digitonin/mg protein. Digitonin incubation was performed on ice for 5 minutes and then centrifuged at 20,000 x g for 30 min as previously described (Acin-Perez et al. (2008) Afo/ Cell 32(4):529-539; Acin-Perez et al. (2020) Methods Cell Biol 155: 181-197). Cells preparations for BN-PAGE were performed as described in Fernandez- Vizarra et al. (2021) FEBS Lett 595(8): 1062-1106. Supernatant containing mitochondrial complexes and super complexes were mixed with Blue Native sample buffer (5% Blue G dye in 1 M 6-amiohexanoic acid), then were loaded and run on a 3-12% native precast gel (Invitrogen). Gels were run until the blue front was run off of the gel. Complex V In Gel activity was performed as described in Acin-Perez et al. (2020) Methods Cell Biol 155: 181-197. Complex V In Gel Activity was stopped in 50% methanol. Finally, gels were stained with Coomassie to correct for loading. Imaging was performed at different stages: after 3 h and overnight In Gel Activity, after fixing with 50% methanol, and after Coomassie staining.

[0100] To assess protein levels, gel electrophoresis was employed as described above, and the proteins were transferred to methanol-activated PVDF membrane in xCell SureLock Mini-Cells under 45 V constant voltage for 1-1.5 hours at 4 °C. Coomassie was completely washed off of blue native blots using 100% methanol. Blots were blocked with 3% BSA in PBST (1 mL/L Tween-20/PBS) and incubated with primary antibody diluted in 1% BSA/PBST overnight at 4°C. The next day, blots were washed three times for 10 min each time in PBST, probed with IRDye 680RD or HRP conjugated secondary antibodies diluted in blocking solution for 1 h at room temperature, and rinsed again three times for 10 min each time in PBST. Detection was achieved using a ChemiDoc Molecular Imager (BioRad). Band densitometry was quantified using ImageJ Gel Plugin (NIH). Primary antibodies used were ATP5A1 and SDHA, SDHB, Cytochrome c and vinculin, ATPIF1 (D6P1Q), and UQCRC2. [0101] EPI decreased In Gel ATP hydrolytic activity in a dose dependent manner (FIGS. 2A-2B), without altering the assembly of mitochondrial ATP synthase (FIG. 2C). These data suggest that EPI is not changing complex V structure but its hydrolytic activity.

[0102] Next it was tested whether EPI could modulate respiration driven by mitochondrial ATP synthesis, using the protocol from Example 1. EPI at 100 nM promoted a slight increase in state 3 respiration driven by both Pyruvate and Malate (Pyr + Mai) or Succinate and Rotenone (Succ+Rot) (FIG. 2D) without any effects in maximal respiration (MRR) (FIG. 2E). Changes in state 3 but not in MRR confirm that epicatechin is targeting ATP synthase. Unlike EPI, catechin did not promote any inhibition of ATP hydrolytic activity or changes in state 3 respirometry (FIGS. 2F-2I).

[0103] To assess if EPI needed to be imported into mitochondrial matrix to inhibit ATP hydrolysis or whether it could bind directly to complex V moieties in the intermembrane space, EPI was added in digitonin-lysed mitochondria prior to protein separation by BNGE. As shown in FIGS. 2J-2K, EPI inhibits hydrolysis to the same extent in lysed mitochondria as when added to intact mitochondria. These results indicate that the effect of EPI is not limited by the rate of its diffusion into the mitochondrial matrix.

Example 5: In Gel treatment confers direct effect of epicatechin on FIFO

[0104] This Example demonstrates that epicatechin (e.g., (+)-epicatechin, also referred to as EPI) directly inhibits complex V by binding the Fl head.

[0105] EPI was added to the reaction buffer when assessing hydrolysis, after sample mitochondria were run on BNGE. In Gel treatment by EPI decreased ATP hydrolysis (FIGS. 2L-2M). These results indicate that the effect of EPI is not occurring by breaking the ATP synthase complex or the dimer, as could happen in intact mitochondria, but instead that EPI directly inhibits complex V by directly interacting with Fl.

Example 6: Epicatechin competes with ATPIF1 to bind purified mitochondrial ATP synthase

[0106] This Example demonstrates that epicatechin (e.g., (+)-epicatechin, also referred to as EPI) competes with ATPIF1 for the binding pocket on ATP synthase. Furthermore, EPI is able to bind to ATP synthase in the absence of ATPIF1, and is effective in inhibiting ATP hydrolysis in tissues regardless of their level of ATPIF1 expression. [0107] Similar experiments to those in Example 4 were performed to determine any differences in the mechanism of action between EPI and ATPIF1, however instead of adding epicatechin, ATPIF1 activity was blocked with the specific inhibitor BTB06584 (BTB). In contrast with the effects of EPI, inhibiting ATPIF1 with BTB affected both ATP synthesis and hydrolysis, and supported the assembly of dimers and tetramers (FIGS. 3 A-3F).

[0108] Recombinant ATPIF1 (ATPIF1-GFP) was used to monitor ATPIF1 binding at the mitochondrial ATP synthase of heart mitochondria by BNGE using fluorescence at 488 nm, an indicator of ATPIF1-GFP presence in the gel. ATPIF1-GFP bound to both monomeric and dimeric mitochondrial ATP synthase was detected (FIG. 3G). The more ATPIF1 was bound to the ATP synthase, the less ATP hydrolytic activity was detected in the absence of changes in ATP synthase assembly (FIGS. 3H-3J). Next, ATPIF1 hydrolytic inhibition was validated by measuring maximal ATP hydrolysis capacity by monitoring proton release in frozen mitochondrial fractions (FIG. 3K). Similar to the results observed when the experiments were performed in the presence of EPI (FIGS. IL- IM), ATPIF1-GFP binding to mitochondria inhibited ATP hydrolytic activity without changing electron transport chain complexes activity (FIGS. 3K-3L). These data confirm that GFP fusion does not change ATPIF1 activity.

[0109] Experiments were then performed to determine if the target of EPI is ATPIF1 (i.e., epicatechin recruits ATPIF1) or complex V (i.e., epicatechin mimics ATPIF1 when binding complex V). Binding competition assays were performed between EPI and ATPIF1-GFP using both purified ATP synthase tetramers (FIGS. 3M-3N) and heart mitochondria (FIGS. 3O-3R). Briefly, recombinant ATPIF1 was added to bovine mitochondrial complex V tetramer or isolated mouse heart mitochondria at a protein/protein ratio 1 :5 in MAS. Binding assays were performed at room temperature for 10 min, after which samples were prepared to be loaded in Native gels. Immunoblotting was performed as described in Example 4.

[0110] In both instances, EPI was able to reduce the amount of ATPIF1-GFP bound to mitochondria ATP synthase in all of its assembly forms (FIG. 3R and FIG. 3N), and preserved the absence of ATP hydrolysis activity (FIG. 30). These data demonstrate that EPI displaces ATPIF1 bound to mitochondrial ATP synthase.

[OHl] The effect of EPI on ATP hydrolysis in bovine purified preparations of mitochondrial ATP synthase enriched in oligomeric, tetrameric, and monomeric forms that contain different amount of ATPIF1 was evaluated (FIG. 3S). The amount of ATPIF1 did not change the level of ATP synthase inhibition by EPI(FIGS. 3T-3W). Interestingly, both EPI and oligomycin inhibited ATP hydrolysis, not only in the fully assembled complex V form and its oligomers, but also in the Fl subcomplex, where ATPIF1 is not present (FIGS. 3T- 3W). These results support that EPI can inhibit complex V in the absence of ATPIF1, and that ATPIF1 may not be necessary for the inhibition of complex V by EPI.

[0112] Finally, to demonstrate that EPI actions on ATP hydrolysis are independent of the presence and expression of ATPIF1, ATP hydrolytic activity was analyzed in tissues expressing different amounts of ATPIF1 (FIG. 3X). In tissues with low ATPIF1 expression, EPI had the same efficacy blocking ATP hydrolysis (FIG. 3 Y). These results indicate that the inhibitory action of EPI on ATP hydrolytic activity is independent of the levels of ATPIF1.

Example 7: Epicatechin replaces ATPIF1 and blocks hydrolysis in live cells

[0113] This Example demonstrates that epicatechin (e.g., (+)-epicatechin, also referred to as EPI) competes with ATPIF1 and inhibits ATP synthase hydrolysis in live cells.

[0114] EPI competition with ATPIF1 in live cells was investigated. BNGE was used to quantify the amount of ATPIF1 bound to ATP synthase in control fibroblasts grown in glucose and galactose media (to force the ATP synthase to work only on forward mode) (FIG. 4A). No differences were observed in cells grown in galactose, while a tendency for the reduction of ATPIF1 content in assembled ATP synthase was observed in cells grown in glucose (FIG. 4B).

[0115] To confirm the changes in ATPIF1 binding to the ATP synthase under more physiologically relevant conditions, the Duolink® Proximity Ligation Assay (PLA) was used to detect binding between endogenous ATPIF1 and the alpha subunit (ATP5A) of the mitochondrial ATP synthase Fl head (FIG. 4C). PLA will only detect signal when molecules are in close contact of below 10 nm distance. Cells were fixed with 4% Paraformaldehyde in Phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized (0.1% Triton X-100, 0.05% sodium deoxy cholate in PBS), blocked, and stained with primary and secondary antibodies in blocking solution (5% donkey serum). Coverslips were mounted with Mowiol4-88 (Calbiochem). Coverslips were fixed, permeabilized, and blocked as described above. The manufacturer’s protocol was followed using the Duolink In Situ Red Starter Kit Mouse/Rabbit (Sigma Aldrich). After the last wash step, coverslips were incubated overnight with primary antibodies in order to detect immunofluorescence. The antibodies used were: Mouse monoclonal anti-ATP5A (1 : 1000), Mouse monoclonal anti-SDHA (1 : 1000), Mouse monoclonal anti-SDHB (1 : 1000), Rabbit polyclonal anti-UQRC2 (1 : 1000), Rabbit polyclonal anti-ATPIFl (1 : 1000), Rabbit monoclonal anti-TOMM20 Alexa Fluor 488 (1 : 100), Mouse monoclonal anti-Vinculin (1 : 1000), Mouse monoclonal anti-OPAl (1 : 1000), Mouse monoclonal anti-cytochrome c (1 :5000). Image acquisition was performed using a Zeiss LSM880 Confocal system with Airyscan, equipped with a Zeiss Pla-Apochromat 40x/1.2 and 63x/1.4 N.A. objective. 3D image stacks were acquired at optimal Z-distance and reconstructed using Zen black vx.x software. Quantification of PLA dots and mitochondria volume was performed in aleatorily acquired images using Aivia v.10 software. Pixel classification was used to segment mitochondria and PLA dots. EPI treatment decreased ATPIF1 binding in control cells (FIG. 4D) for all concentrations tested, demonstrating that EPI replaces ATPIF1 in control cells in order to inhibit the ATP hydrolysis.

[0116] To validate whether the effects of EPI on hydrolysis were susceptible to competition with ATPIF1 in live cells, EPI effects were evaluated on control fibroblasts overexpressing ATPIF1 wild-type, and its constitutively active mutant H49K (FIGS. 4E-4F). Primary fibroblasts were plated at 6000/well 48 hours prior to the assay. 24 h after plating, standard maintenance media was replaced with high glucose (4.5 g/L) DMEM supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10% FBS, and antibiotic-antimycotic, and cells were treated with 50 nM (+)epicatechin (EPI). On the day of the assay, the medium was replaced with assay medium composed of DMEM (Sigma #D5030) with 5 mM HEPES, pH 7.4, supplemented with 2 mM glutamine and 1 mM sodium pyruvate. OCR and ECAR were measured in a Seahorse XF96 analyzer under basal conditions as well as after injection of 2 pM oligomycin, two sequential additions of 1.5 pM FCCP, followed by 1 pM rotenone with 2 pM antimycin A. Respiratory parameters were calculated according to standard protocols, and all rates were corrected for non-mitochondrial respiration/background signal by subtracting the oxygen consumption rate insensitive to rotenone plus antimycin A.

[0117] Both ATPIF1-WT and ATPIF1-H49K expression reduced maximal ATP hydrolytic capacity in control fibroblasts, consistent with the effect of EPI on the maximal hydrolytic capacity in control cells with endogenous ATPIF1 levels (FIG. 4G). In cells overexpressing either ATPIF1-WT or H49K, EPI was no longer able to reduce the ATP hydrolytic capacity.

Example 8: Epicatechin prevents hydrolysis and protects the ATP pool in vitro

[0118] This Example demonstrates that epicatechin (e.g., (+)-epicatechin, also referred to as EPI) is able to prevent ATP hydrolysis in vitro in fibroblast cells. In particular, this Example demonstrates that EPI prevents ATP hydrolysis after the addition of drugs that decrease proton motive force and normally increase ATP hydrolysis.

[0119] ATP hydrolysis was shown to be a common compensatory mechanism in mitochondrial diseases (FIG. II) with subsequent downregulation of ATPIF1 levels (FIG. 1J). The replacement of ATPIF1 by EPI (FIG. 4D) to reduce hydrolytic capacity in control cells indicates the possibility of rescue in disease models where ATP hydrolysis is contributing to disease progression. To explore the implications of blocking hydrolysis in cellular models, and to understand if together with a mitochondrial dysfunction the ATP hydrolysis can be pathogenic, the effect of EPI in different cellular models with increased ATP hydrolysis was explored.

[0120] ATP synthase is the main force to provide the proton motive force (A m and ApH) and the directionality of F0-F1 is mostly controlled by changes in A m. Therefore, cells were treated with Antimycin A (AA) to decrease A m and induce hydrolysis in live cells (FIG. 4H).

[0121] Control fibroblasts were treated with different concentrations of AA and AFm was measured by staining with TMRE (tetramethylrhodamine ethyl ester) and MTG (MitoTracker® Green FM) to obtain the mean fluorescence intensity in a region of interest created by MTG area. TMRE is a dye that reports changes in A m real time, and MTG is a cumulative dye that cannot report real time changes on the A m, and thus is used to define the area of the mitochondrial network. Cells were incubated for 1 h with 200 nM MitoTracker green (MTG), 200 nM TMRE (Biotium), and 1 pg/ml Hoeschst, washed twice with PBS, and incubated with 200 nM in medium at 37 °C (5% CO2). Imaging was performed in triplicates and in two focus planes with a PerkinElmer Operetta CLS high content system (20x objective) at 37 °C and 5% CO2. Analysis was performed with Harmony ® 4. Isoftware by measuring mean TMRE fluorescence intensity inside a region of interest generated by MTG area.

[0122] Treating the cells with different concentrations of AA for 30 min showed that depolarization was AA concentration dependent (FIG. 4H). Concentrations below 500 nM of AA failed to promote significant depolarization, while concentrations of 5 pM AA were sufficient. Oxygen consumption (OCR) before and after the injection of AA at different concentrations showed complete blockage of OCR by AA at all concentrations tested (FIG. 41). These data indicate that the absence of depolarization at lower concentrations was caused by lower sensitivity of membrane potential measurements to detect changes in mitochondrial function. Adding 1 pM oligomycin further depolarized mitochondria, demonstrating that under AA, mitochondrial ATP synthase was working in reverse. No changes were observed when EPI was incubated with AA, which is consistent with the hypothesis that EPI will only act in the molecules of ATP synthase working in reverse and where ATPIF1 could be displaced.

[0123] To test whether EPI can block AA-induced hydrolysis in cellular models, mitochondrial ATP (mtATP) content was measured using an ATP fluorogenic dye targeted to the mitochondria (BioTracker™). Cells were incubated for 1 h with 200 nM MitoTracker green (MTG) and 1 pg/ml Hoeschst, washed twice with PBS, and incubated for 15 min with 5 pM BioTracker™ ATP-red dye (Millipore) in medium at 37 °C (5% CO2). Before measurement, the cells were washed twice with medium and fresh medium containing 2.5 pM ATP-red was added. Imaging was performed in triplicates, and in two focus planes with a Perkin Elmer Operetta system (20x objective) at 37 °C and 5% CO2. Analysis was performed with Harmony® 4.1 software by measuring mean ATP red fluorescence intensity inside a region of interest generated by MTG area.

[0124] mtATP was depleted within the first 30 min of AA treatment (FIG. 4 J), while an increase in mtATP was observed after 1 h of AA treatment (FIG. 4K). Treatment with EPI protected against the consumption of the internal ATP pool (occurs following 30 min AA treatment), while no effect was observed after Adenine nucleotide translocator (ANT) reversal (occurs following 1 h AA treatment) (FIGS. 4J-K). The lowest concentration of AA- induced mtATP depletion resulted in almost complete elimination of OCR (FIG. 41).

[0125] No differences were observed in OCR of intact cells after EPI injection, indicating that the prevention of mtATP depletion in cells from control subjects following acute EPI treatment is not due to changes in OCR (FIG. 4L).

Example 9: Epicatechin inhibits ATP hydrolysis and improves total ATP pool in mitochondrial disease models in vitro

[0126] This Example demonstrates that epicatechin (e.g., (+)-epicatechin, also referred to as EPI) inhibits ATP hydrolysis and increases the total ATP content of fibroblasts derived from patients with mitochondrial diseases, while not interfering with the ECAR or complex formation.

[0127] Primary fibroblasts derived from patients with mitochondrial diseases were acquired. Cells were maintained in EMEM (Eagle's Minimum Essential Medium; ATCC #30-2003) and supplemented with 15% FBS and antibiotic-antimycotic in a humidified atmosphere at 37 °C and 5% CO2. During the treatment, media was changed to free phenol red DMEM (Dulbecco's Modified Eagle Medium; ThermoFisher #A1443001) containing 4.5 g/L glucose or galactose supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 10% FBS, and antibiotic-antimycotic. Compounds were prepared in DMSO and added directly to the growth media at the specified concentration. Lentiviral infection of pLentiATPIFl-WT and pLentiATPIFl-H49K (produced by Welgen, Inc.) was performed using 8 mg/mL polybrene in EMEM for 30 h, and positive selection was performed using 1 pg/mL puromycin.

[0128] To determine whether EPI treatment was effective in decreasing ATP hydrolysis in the cellular models of mitochondrial disease, patient-derived fibroblasts were treated with varying concentrations of EPI. All mutant cells responded to 30 min of EPI treatment, showing an increase in mtATP content in a concentration-dependent manner (FIG. 4M). Control cells showed increased mtATP after 24 h in galactose (FIG. 4N), indicating that OXPHOS-deficient cells have a compromised mtATP pool and depend on total ATP content for survival. No difference in mtATP content was observed after 24 h treatment with EPI, neither in glucose nor galactose, suggesting a successful readjustment of the mtATP pools after ATP hydrolysis blockage (FIG. 4N). These results suggest that mitochondrial ATP hydrolysis is a common response to electron transport dysfunction. Therefore, EPI (an ATP hydrolysis inhibitor) can be used as a tool to determine whether the reversal of the mitochondrial ATP synthase is adaptive or maladaptive.

[0129] The response of total ATP content of these mutant fibroblasts was measured after 24 h treatment with EPI, as total ATP content adjustment can be an integrated measure of metabolic health and cellular proliferation. Cells were washed twice with PBS and lysed using 0.5% Triton-XlOO in PBS. After 3 min of centrifugation at 13,000 RPM, 5 pL of total lysate was used in triplicates for an assessment of total ATP content using the ATP Determination Kit (#A22066 Thermo Fisher) and manufacturer’s instructions. Luminescence was monitored at ~ 560 nm using a Tecan Spark luminometer.

[0130] Total ATP content was decreased in almost all the mutants tested when compared with control cells (FIGS. 4O-4P). Interestingly, several of the models tested, including a CIII deficiency due to BCS1L mutations, a CV deficiency due to ATP6 mutations, as well as mutants in MIC26 and TMEM70, showed an increased amount of total ATP after 24 h of EPI treatment (FIG. 4O-P), and the doubling time for the cells with the BCS1L significantly decreased with EPI treatment (FIG. 4Q). Oligomycin and BTB decreased total ATP content in most mutants tested (FIG. 4R), further supporting that this metabolic improvement was due to the selective blockage of ATP hydrolysis. Furthermore, oligomycin increased A m in most mutants, while no changes were induced by EPI, further supporting its selectivity of action to ATP synthase molecules working in reverse (FIG. 4S).

[0131] EPI has been previously shown to increase mitochondrial mass with a corresponding elevation of respiration in all states. Thus, it was next determined whether an indirect improvement in respiration, downstream of ATP hydrolysis blockage, could contribute to the restoration in ATP content induced by long term (24 h) EPI treatment. To determine mitochondrial mass, muscle homogenate (8 pg) in 20pl of MAS (70 mM Sucrose, 220 mM Mannitol, 5 mM KH2PO4, 5mM MgC12, 1 mM EGTA, 2 mM HEPES; pH 7.2) was placed in a clear-bottom 96-well microplate. Then, 130 pl of a 1 :2000 dilution of Mitotracker Deep Red FM (MTDR, ThermoFisher) was added and incubated for 10 min at 37° C. Plates were centrifuged at 2,000 g for 5 min at 4°C (no brake), and supernatant was carefully removed. Finally, 100 pl of PBS was added per well and MTDR fluorescence measured (^excitation ⁼ 625 nmj /-emission ⁼ 670 nm). Mitochondrial content was calculated as MTDR signal (minus blank) per microgram of protein.

[0132] Fibroblasts from control subjects showed an increase in both oxygen consumption (OCR) and media acidification (ECAR) in response to EPI, but for the mutants, OCR values were more variable in response to EPI (FIGS. 4S-4T). However, the effect of increasing ECAR was present for almost all the cells tested, which can be a result of either increased lactate release from glycolysis or increased CO2 release by the TCA cycle. To measure lactate, IxlO ⁶ cells were plated and treated for 24 h in media. Cells were fixed and media were frozen at -80 °C until further processing. Media was thawed on ice and diluted 1 :70 in PBS, and 5ul was used in triplicate for assessment of lactate using the Lactate-Glo Assay (#J5021 Promega) following manufacturer’s instructions. Luminescence was recorded using a Tecan Spark luminometer.

[0133] An increase in lactate was observed for untreated CIII-BCS1L and CV-ATP6 mutants when compared to the control cells (FIG. 4V). Control cells in galactose decreased lactate accumulation, as no glycolysis is occurring and they have a functional ETC. EPI treatment decreased levels of lactate in CIII-BCS1L, and did not further increase lactate release in CV-ATP6 cell lines, suggesting that the increase in ECAR could be a result of elevated TCA cycle activity. The decrease in lactate in CIII-BCS1L mutant is another proof of the beneficial effect of ATP hydrolysis inhibition. [0134] BNGE analysis was utilized to investigate CIII and CV assembly using digitonin solubilized mitochondria (FIGS. 4V-4Y). As BCS1L protein mediates the incorporation of UQCRFS1 to pre-CIII2 in human and mouse mitochondria, the labeling of UQCRC2 showed an accumulation of dysfunctional CIII2 and CIII2+CIV compared to control cells (FIGS. 4V- 4W). No differences were observed in the assembly of these complexes after 24 h incubation of EPI. These results show that blocking ATP synthase hydrolysis does not impact CIII assembly as no differences were observed in control cells, but also that the amelioration of phenotype in CIII-BCS1L patient cells was due to better regulation of ATP hydrolytic activity. In CV-ATP6 mutant cells, there is an accumulation of the Fl head and F0F1 compared to the control, but no significant difference was observed with EPI treatment in either of the control or mutant cells, showing a specific modulation of complex activity, not complex amount, in these cells (FIG. 4Y-Z).

Example 10: Epicatechin binds mitochondrial ATP synthase and replaces ATPIF1 in vitro

[0135] This Example demonstrates that epicatechin (e.g., (+)-epicatechin, also referred to as EPI) competes with ATPIF1 to bind ATP synthase in mitochondrial disease models. In particular, this Example demonstrates that unlike ATPIF1, the inhibition of hydrolysis by EPI protects the ATP pool (e.g., does not interfere with ATP synthesis).

[0136] BNGE was used to quantify the amount of ATPIF1 bound to ATP synthase in control, CIII-BCS1L mutated, and CV-ATP6 mutated fibroblasts (FIGS. 5A-5B). As shown in FIGS. 5A-5B, around 50% reduction of ATPIF1 content in assembled ATP synthase was observed in CIII-BCS1L mutant cells. In contrast, CV-ATP6 mutant patient fibroblasts showed no difference in the amount of ATPIF1 bound to mitochondrial ATP synthase compared to controls. EPI treatment for 24 h decreased ATPIF1 binding to the ATP synthase in CV-ATP6 but not in CIII-BCS1L cells. These results indicate that ATPIF1 displacement can only be detected if sufficient ATPIF1 is expressed and bound to ATP synthase.

[0137] The Duolink® Proximity Ligation Assay (PLA) was used to confirm the changes in ATPIF1 binding to the ATP synthase in patient-derived fibroblasts (FIG. 5C). PLA revealed that ATPIF1 binding to the ATP synthase was decreased in mutant fibroblasts compared with control cells (FIG. 5D). EPI treatment further decreased ATPIF1 binding in control and CV- ATP6 mutant cells, while the already low content of ATPIF1 bound in CIII-BCS1L cells was unchanged by EPI treatment (FIG. 5E). Taken together, the PLA assays confirm the displacement of ATPIF1 and binding of EPI to ATP synthase to inhibit ATP hydrolysis. [0138] To validate whether the effect of EPI on hydrolysis is susceptible to competition with ATPIF1 in live cells, the same EPI treatment conditions were used on CIII-BCS1L mutant fibroblasts with restored ATPIF1 expression, as the CIII-BCS1L cells have low endogenous levels of ATPIF1. Similar to control cells (FIG. 4G) the wild-type ATPIF1 or its constitutively active mutant H49K were stably expressed in CIII-BCS1L cells. CIII-BCS1L cells presented higher maximal hydrolytic capacity than control cells (FIG. II), and both ATPIF1-WT and ATPIF1-H49K expression reduced the maximal ATP hydrolytic capacity similarly to under EPI treatment conditions (FIG. 5F). In cells overexpressing ATPIF1-WT or ATPIF1-H49K, high levels of overexpressed ATPIF1 abolished the blocking of ATP hydrolysis by EPI (FIG. 4E).

[0139] These data indicate that the effect of EPI on ATP synthase hydrolysis is dependent on ATPIF1 levels and displacement. Therefore, the effects of EPI on mtATP and total ATP in the ATPIF1 -overexpressed CIII-BCS1L mutant cell were assayed. In cells with endogenous levels of ATPIF1, an increase in mtATP was observed after acute treatment with EPI (FIG. 4M) and an increase in total ATP content was observed after 24 h treatment with EPI (FIG. 40). In agreement with the competitive effects between EPI and ATPIF1 on ATP hydrolytic capacity, there was no addictive effect of EPI increasing mtATP content (FIG. 5G) nor in total ATP content in the presence of ATPIF1 (FIG. 5H). Overexpression of ATPIF1 protein does not rescue the ATP pool in the patient cells, unlike EPI.

[0140] Overall, EPI selectively inhibited ATP hydrolysis without blocking mitochondrial ATP synthesis, thus rescuing the ATP pool in these cells.

Example 11: Epicatechin administration modulates ATP synthase and reverses muscle injury in mice with muscular dystrophy associated with mitochondrial dysfunction.

[0141] This Example demonstrates that epicatechin (e.g., (+)-epicatechin, also referred to as EPI) treatment improves mitochondrial function and recovery from muscle injury in a mouse model for Duchenne Muscular Dystrophy (DMD) by modulating ATP synthase.

[0142] To determine EPI efficacy in an in vivo disease model with cellular ATP depletion, mice with DMD were treated with EPI. The mdx mice, used to study DMD, have a mutation in the dystrophin protein and show a combination of ATP depletion and mitochondrial dysfunction. The mdx mutation on a DBA/2J genetic background (D2.mdx) induces a severe phenotype, as loss of the dystrophin protein destabilizes the muscle cell membrane, leading to membrane damage and calcium influx. In this context, no pharmacological approaches targeting the mitochondria have been tested to date to treat DMD, nor the role of mitochondrial ATP hydrolysis in the development of DMD.

[0143] To study the contribution of ATP hydrolysis in DMD pathology, 12-week old D2.mdx mice were subjected to gastrocnemius eccentric injury (as described in Blaauw et al. (2010) J Appl Physiol (1985) 108(1): 105-111; Khairallah et al. (2012) Sci Signal 55(236):ra56; Call and Lowe (2016) Methods Mol Biol 1460:3-18) and analyzed the effect on ATP hydrolysis in untreated wildtype and mdx mice after the injury. In every mouse, the right leg was subjected to eccentric injury and the left leg was the sham control, with muscles being collected 1 h or 24 h post injury (pi). Mouse gastrocnemius muscles were homogenized in 0.75 mL MAS (70 mM Sucrose, 220 mM Mannitol, 5 mM KH2PO4, 5mM MgCh, 1 mM EGTA, 2 mM HEPES; pH 7.2) using a glass-glass Dounce homogenizer. 30 strokes with the tight pestle were done per sample. The homogenate was centrifuged at 1,530 rpm to pellet non-broken material and the supernatant was stored at -80 °C until further use.

[0144] Respirometry in previously frozen samples was performed as described in Acin- Perez et al. (2020) EMBO J 39(13):el04073 and Osto et al. (2020) Curr Protoc Cell Biol 89(1): e 116. For respiratory assays, 8 pg of total mouse gastrocnemius homogenates was loaded. For ATP hydrolysis capacity or state 4 acidification measurements, 20 pg of mouse gastrocnemius homogenate was added to a Seahorse XF96, and the protocol from Example 1 was followed.

[0145] ATP hydrolysis in gastrocnemius homogenates was increased in mdx 1 h pi, whereas there were no changes in mdx at 24 h pi or in wild-type mice at any time measured (FIG. 6A). To determine whether the change in ATP hydrolysis capacity was caused by changes in total mitochondrial ATP synthase, SDS-PAGE was used to measure ATP synthase content. 15-30 pg of protein from total tissue homogenates or cell RIPA buffer lysates were loaded into 4-12% Bis-Tris precast gels and gel electrophoresis was performed in xCell SureLock Mini-Cells (Novex) under constant voltage of 80 V for 15 min (to clear stacking) and 120 V for 60 min. Immunoblotting was performed as in Example 4. Cytochrome c (Cyt c) release was measured in mitochondrial supernatants of gastrocnemius homogenates. 50 pg of gastrocnemius homogenate was centrifuged at 10,000 x g for 10 min. Supernatant was collected and Cytochrome c levels were measured by immunoblot and ELISA (R&D Systems) using 10 pg and 3 pg of supernatant, respectively.

[0146] Total ATP synthase decreased 24 h pi only in injured muscles from mdx mice. Normalizing ATP hydrolysis by the total content of mitochondrial ATP synthase showed that both at 1 h and 24 h pi, mdx mice showed elevated ATP hydrolysis only in the injured muscle (FIG. 6C). As a consequence of injury, the mitochondrial inner membrane was damaged and 0PA1 was cleaved (FIG. 6D), affecting complex I maximal respiration (FIGS. 6E-6F) and resulting in Cytochrome c release from the mitochondria to the cytosol (FIGS. 6G-6H). [0147] The effects of EPI versus vehicle treatment in mdx mice on their muscular performance and mitochondrial function was then studied. Maximal force production of the plantarflexor muscle group was measured in vivo with a 305C muscle lever system (Aurora Scientific Inc., Aurora, CAN), as previously described in Khairallah et al. (2012) Sci Signal 5(236):ra56 and Call and Lowe (2016) Methods Mol Biol 1460:3-18. Animals were anesthetized via inhalation (~2% isoflurane, SomnoSuite, Kent Scientific), placed on a thermostatically controlled table with anesthesia maintained via nose-cone (~2% isoflurane), the knee fixed with a pin pressed against the tibial head, and the foot firmly fixed to a footplate on the motor shaft. Contractions were elicited by percutaneous electrical stimulation of the tibial nerve (0.2 ms pulse, 500 ms train duration) at increasing frequencies. Following assessment of isometric torque, susceptibility to injury was assayed with 25 eccentric contractions as previously described in Khairallah et al. (2012) Sci Signal 5(236):ra56 and Call and Lowe (2016) Methods Mol Biol 1460:3-18, at maximal isometric torque (150 ms duration, 0.2-ms pulse train at 150 Hz). Eccentric contractions were achieved by translating the footplate 38° backward at a velocity of 800°/s after the first 100 ms of the isometric contraction. The decrease in the peak isometric force before the eccentric phase was taken as an indication of muscle damage. Blue Native Gel Electrophoresis was performed as described in Example 4, except that 3 mg digitonin/mg protein was used.

[0148] EPI was administered twice a day for 2 weeks, resulting in a decrease of the percentage in loss force as compared to vehicle treated mdx mice (FIGS. 7A-7C). ATP synthase content was decreased in vehicle mice with injury (FIG. 7D), reproducing the data of non-treated mdx mice (FIG. 6B). While EPI treatment only induced a trend to increase mitochondrial ATP synthase in injured mdx mice (FIG. 7D), EPI was sufficient to decrease ATP hydrolysis in injured muscle (FIG. 7E). These effects were concurrent with an improved preservation of muscular force in mdx injured mice treated with EPI (FIG. 7F). Additionally, EPI treatment blocked OPA1 cleavage (FIG. 7G), Cytochrome c release (FIGS. 7H-7I), and the drop in complex I respiration (FIGS. 7J-7K), all of which demonstrate a preserved mitochondrial cristae and function. Principal Component Analysis (PCA) was carried out in R studio (R version 4.1.1) using Singular value decomposition to determine the principal components and the eigenvectors for the analyzed variables. PCA of the different inputs showed how EPI treatment improved muscular function in injured mdx mice (FIGS. 7L-7M). [0149] Overall, these data show that an early event in muscle dysfunction is the reversal of ATP synthase to hydrolyze ATP, which then triggers OPA1 cleavage and decreases respiration. These pathological mechanisms can be moderated by treatment with EPI.