Mitochondrial enhancers for treating neurodegenerative disease

Abstract

A method of treating or preventing a neurodegenerative disease in a subject in need thereof is described. The method includes administering to the subject a therapeutically effective amount of a mitochondrial enhancer. Novel mitochondrial enhancer compounds are also described.

Description

MITOCHONDRIAL ENHANCERS FOR TREATING NEURODEGENERATIVEDISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 662,559, filed June 21, 2024, the disclosure of which is incorporated herein by reference.BACKGROUND

[0002] Normal mitochondrial function plays a crucial role in various cellular processes, including energy production, lipid metabolism, calcium buffering, and redox regulation. These functions are essential for cell growth, proliferation, and survival. Neurons, with their high energetic demands and limited capacity for regeneration, are particularly reliant on mitochondrial integrity. Disruptions in this integrity can lead to significant neuronal dysfunction. Problems such as dysregulation in mitochondrial fusion and fission, failure in biogenesis, or inefficient oxidative phosphorylation can cause a reduction in dendritic trees and axons, decreased neurotransmitter turnover, inflammatory responses, and ultimately, neuronal loss. This dysfunction is believed to contribute to the pathogenesis of neurodegenerative disorders like Huntington’s disease (HD) and Alzheimer’s disease (AD).

[0003] The inventors’ previous research identified CHIR99021 as a mitochondrial enhancer, effective in cell culture, iPSC-derived neurons, and transgenic mouse models of HD. While CHIR99021 is commonly known as a GSK3a / inhibitor in stem cell biology and cancer studies, our studies revealed its novel function in suppressing calpastatin degradation, independent of GSK3 inhibition, in HD. This action of CHIR99021 results in enhanced mitochondrial membrane potential, respiration, and network stability, and it also helps prevent the formation of mutant huntingtin aggregates and neuronal loss. Moreover, up-regulation of calpastatin via genetic modification seems to be effective in mitigating the progression of neuropathology in a spectrum of neurodegenerative diseases. Therefore, CHIR99021 analogs appear to be a promising agent for treating HD, and modulating calpastatin offers a new avenue for enhancing mitochondrial function and treating neurodegenerative diseases.SUMMARY OF THE INVENTION

[0004] Leveraging previously established mitochondrial-function-based screen platform, the inventors chemically optimized CHIR99021 and identified its lead molecule A36 as a putative mitochondrial enhancer. By directly targeting calpastatin, A36 treatment improved mitochondrial function and neuronal survival in HD neurons, and ameliorated neuropathology and motor deficits in HD mice, highlighting the pivot role of calpastatin downregulation during HD pathogenesis. Long-term administration of A36 also rescued the expression of calpastatin, and ameliorated tauoapthy and cognitive deficits in P301S Tau-expressing PS19 mouse. These proof-of-principle findings indicate: (1) mitochondrial enhancement via calpastatin modulation can be an effective therapeutic strategy for neurodegenerative disease; (2) A36 is a candidate lead with great promise of therapeutic development for neurodegenerative diseases.

[0005] In one aspect, the invention provides a method of treating or preventing a neurodegenerative disease in a subject in need thereof, comprising administering a therapeutically effective amount of a mitochondrial enhancer according to formula I:to the subject, wherein R1is a halogen, cycloalkyl, or cycloheteroalkyl group, R2-R5are hydrogen or halogen, X is a methylene, an oxygen, or an N-H group, Y is a cycloheteroalkyl or cycloheteroaryl group, and Z is a cycloheteroalkyl or cycloheteroaryl group, or a pharmaceutically acceptable salt thereof. In some embodiments, the neurodegenerative disease is Alzheimer’s disease or Huntington’s disease. In some embodiments, the mitochondrial enhancer is administered together with a pharmaceutically acceptable carrier.

[0006] Another aspect of the invention provides mitochondrial enhancer having a structure according to formula I:wherein R1is a halogen, cycloalkyl, or cycloheteroalkyl group, R2-R5are hydrogen or halogen, X is a methylene, an oxygen, or an N-H group, Y is a cycloheteroalkyl or cycloheteroaryl group, and Z is a cycloheteroalkyl or cycloheteroaryl group, or a pharmaceutically acceptable salt thereof.BRIEF DESCRIPTIONS OF THE DRAWINGS10007 ] The present invention may be more readily understood by reference to the following figures, wherein:

[0008] Figures 1A-1F provide a flowchart and graphs showing low-throughput screening identified A36 as a hit without GSK3 -interaction. A. Scheme of low-throughput screening to identify hit candidates, out of 132 chemical analogues of CHIR99021 that can improve mitochondrial membrane potential and cell survival in HdhQl l l cells. B. Quantification of mitochondrial membrane potential measurement in HdhQ7 / Ql l l cells treated with A36 at different doses. N=4. C. Calculation of ECso of A36 on mitochondrial membrane potential. D. Quantification of cell viability measurement in HdhQ7 / Ql 11 cells treated with A36 at different doses. N=4. E. Thermo-shift profiling assay to determine whether A36 directly binds to GSK3 a or p isofomrs in HdhQl 11 cells. F. Measurement of GSK3 enzyme activity in the presence of A36 at variable doses in vitro.

[0009] Figures 2A-2H provide graphs and images showing calpastatin is the direct binding target of A36. A. DARTS assay combined with proteomics analyses identified calpastatin as the top candidate for A36. B. Representative blots for in vitro DARTS assay using recombinant calpastatin incubated with variable concentrations of A36. C. Quantification of calpastatin intensity when incubating with A36 at different concentrations. Calculated ECso is 26.64 LIM (0-200 pM). D. Total cell lysates harvested from HdhQl 11 cells was incubated with A36 at different concentrations and subjected forpronase digestion, followed by western blot analyses.Shown are representative blots. N=4. E. Total cell lysates harvested from HdhQl l l cells was incubated with A36 and subjected for digestion by pronase at different concentrations, followed by western blot analyses. Shown are representative blots. N=4. F. Representative images of mitochondrial membrane potential measurement by using TMRM dye in vehicle (Veh) or A36- treated HdhQ7 and HdhQl l l cells expressing control (Consh) or calpastatin (CASTsh) shRNA. N=7. G. Measurement of cell viability in veh or A36-treated HdhQ7 and HdhQl l l cells expressing control (Consh) or calpastatin (CASTsh) shRNA. N=3. H. Representative blots for co-immunoprecipitation of calpastatin with calpain in veh- or A36-treated HdhQ7 and HdhQl l l cells. Histogram: quantification of relative intensity of calpain pulled down. N = 5.

[0010] Figures 3A-3F provide graphs and images showing A36 treatment improved mitochondrial function in neuronal HD striatal cells. A. Measurement of mitochondrial ROS production using mitoSOX dye in HdhQ7 / Ql 11 cells treated with veh or A36. Shown are the representative images of mitoSOX staining. Histogram shows the quantification of mitoSOX intensity. N=9. B. The half-inhibitory dose of A36 on mitoSOX production is 1.437 pM (0-10 pM). C. Measurement of mitochondrial oxygen consumption rate using Seahorse analyzer in HdhQ7 / Ql l l cells treated with veh or A36. D. Quantifications of mitochondrial respiration rates and ATP production from the Seahorse analyses. N=4. E. Immuno-fluorescence staining of mitochondrial Tom20 in HhdQ7 / l l l cells treated with veh or A36. Shown are the representative images of the cells. Histograms represent the quantification of percentage of cells with elongated, intermediate, or fragmented mitochondrial morphology. N=3. F. Total lysates were harvested from veh- or A36- treated HdhQ7 / l 11 cells and subjected for western blot analyses. Shown are the representative blots. Histogram represents the quantification of the immuno-density of the indicated protein bands. N=4.

[0011] Figures 4A-4E provide graphs and images showing A36 treatment is protective in neurons derived from HD patients’ iPSC. After 25-day differentiation, the neurons were treated with veh or A36 at luM for 5 consecutive days and subjected for the following biochemical analyses. (A) Immuno-fluorescence staining using anti-calpastatin and anti-MAP2 antibodies in WT and HD neurons treated with veh or A36. Shown are the representative images. (B) Immuno-fluorescence staining using anti-DARPP32 and anti-Tujl antibodies in WT and HD neurons treated with veh or A36. Shown are the representative images. (C) Total lysates were harvested and subjected for western blot analyses. Shown are the representative blots. (D)Histograms represent the quantification of the immuno-density of the indicated protein bands.N = 4.

[0012] Figures 5A-5I provide graphs and images showing A36 treatment mitigated HD- associated neuropathology in R6 / 2 mice. (A) Summary of the pharmacokinetics of A36 in the WT mice. (B) Survival curve of R6 / 2 and WT mice after 6-week treatment with veh or A36. (C) Quantification of the measurement of locomotor activity of R6 / 2 and WT littermates by open-field test after veh or A36 treatment. N = 10-15 mice per group. (D) Quantification of the hind-limb clasping score in R6 / 2 mice treated with veh or A36. (E) Immunohistochemistry staining of the brain slides from R6 / 2 and WT mice treated with veh or A36 by using anti- DARPP32 antibody. Shown are the representative images of dorsal striatum region. N = 3 mice each group. (F) Immunohistochemistry staining of the brain slides from R6 / 2 mice treated with veh or A36 by using anti-mtHtt antibody (EM48 clone). Shown are the representative images of dorsal striatum region. N = 6 mice each group. (G) Immuno- fluorescence staining of the brain slides from the WT and R6 / 2 mice treated with veh or A36 using anti-DARPP32, GFAP antibodies. Shown are the representative images of dorsal striatum region. N= 5 mice each group. (H) Immuno-fluorescence staining of the brain slides from the WT and R6 / 2 mice treated with veh or A36 using anti- DARPP32 and calpastatin antibodies. Shown are the representative images of dorsal striatum region. N= 4 mice each group. (I) striatum total lysates were harvested from R6 / 2 and WT mice treated with veh or A36 and subjected for western blot analyses using the indicated antibodies. Shown are the representative blots. Histogram represents the quantification of the relative density of the indicated protein bands. N =4 mice each group.

[0013] Figures 6A-6N provide graphs and images showing A36 treatment mitigate Tau- associated neuropathology and cognitive deficits in PS 19 mice. (A) Quantification for the measurement of short-term memory by Y-maze test in PS 19 and WT mice after 4- month treatment with veh or A36. N=10-12 mice each group. All male. After consecutive 3-day training, the long-term / spatial memory of WT and PS19 mice treated with veh or A36 were evaluated by Bamez maze test at (B) day 5, and (C) day 12. Representative images show the heat-track of the mice till escape or after 180s. Histograms summarize the escape timing. N = 10-12 mice each group. (D) Immunohistochemistry staining of the brain slides from PS 19 and WT mice treated with veh or A36 by using anti-phospho-Tau antibody (AT8 clone). Shown are the representative images of different hippocampus regions. (E) Histogram shows thequantification of the AT8 signal intensity as stained in (D). n=5 mice each group. (F) Immunofluorescence staining of the brain slides from PS 19 and WT mice treated with veh or A36 using anti- IBA1 and GFAP antibodies. Shown are the representative images of the hippocampus. Histograms show the quantification of (G) IBA1 and (H) GFAP. (I) Immuno-fluorescence staining of the brain slides from PS 19 and WT mice treated with veh or A36 using anti- calpastatin, anti- NeuN, and anti- phosphor-Tau antibodies. Shown are the representative images of the hippocampal CA3 regions. (J) Histogram shows the quantification of calpastatin intensity per NeuN cells within CA3 region, N=5 mice each group. (K) Hippocampal total lysates were harvested from PS 19 and WT mice treated with veh or A36 and subjected for western blot analyses. Show are the representative blots. (L) Histograms show the quantification of the intensity of indicated proteins. N = 4 mice each group. (M) Triton insoluble fractions were extracted using 2%SDS and subjected for western blot analyses. Shown are the representative blots. (N) Histograms show the quantification of intensity of tau and phosphor-tau in the insoluble fractions. N=4 mice each group.DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention provides method of treating or preventing a neurodegenerative disease in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a mitochondrial enhancer. The invention also provides novel mitochondrial enhancer compounds such as A36.Definitions

[0015] The terminology as set forth herein is for description of the embodiments only and should not be construed as limiting of the invention as a whole. As used in the description of the invention and the appended claims, the singular forms “a”, “an”, and “the” are inclusive of their plural forms, unless contraindicated by the context surrounding such.

[0016] As used herein, the term "organic group" is used to mean a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., alkaryl and aralkyl groups). In the context of the present invention, suitable organic groups for the compounds of this invention are those that do not interfere with the anti-cancer activity of the compounds. In the context of the present invention, the term "aliphatic group"means a saturated or unsaturated linear or branched hydrocarbon group. This term is used to encompass alkyl, alkenyl, and alkynyl groups, for example.

[0017] As used herein, the terms "alkyl", "alkenyl", and the prefix "alk-" are inclusive of straight chain groups and branched chain groups. Unless otherwise specified, these groups contain from 1 to 20 carbon atoms, with alkenyl groups containing from 2 to 20 carbon atoms. In some embodiments, these groups have a total of at most 10 carbon atoms, at most 8 carbon atoms, at most 6 carbon atoms, or at most 4 carbon atoms. Alkyl groups including 4 or fewer carbon atoms can also be referred to as lower alkyl groups. Alkyl groups can also be referred to by the number of carbon atoms that they include (z.e., Ci - C4 alkyl groups are alky groups including 1-4 carbon atoms).

[0018] Cycloalkyl, as used herein, refers to an alkyl group (z.f., an alkyl, alkenyl, or alkynyl group) that forms a ring structure. Cyclic groups can be monocyclic or polycyclic and preferably have from 3 to 10 ring carbon atoms. A cycloalkyl group can be attached to the main structure via an alkyl group including 4 or less carbon atoms. Exemplary cyclic groups include cyclopropyl, cyclopropylmethyl, cyclopentyl, cyclohexyl, adamantyl, and substituted and unsubstituted bornyl, norbomyl, and norbomenyl.

[0019] Unless otherwise specified, "alkylene" and "alkenylene" are the divalent forms of the "alkyl" and "alkenyl" groups defined above. The terms, "alkylenyl" and "alkenylenyl" are used when "alkylene" and "alkcnylcnc", respectively, arc substituted. For example, an arylalkylcnyl group comprises an alkylene moiety to which an aryl group is attached.

[0020] The term "haloalkyl" is inclusive of groups that are substituted by one or more halogen atoms, including perfluorinated groups. This is also true of other groups that include the prefix "halo-". Examples of suitable haloalkyl groups are chloromethyl, trifluoromethyl, and the like. Halo moieties include chlorine, bromine, fluorine, and iodine.

[0021] The term "aryl" as used herein includes carbocyclic aromatic rings or ring systems. Examples of aryl groups include phenyl, naphthyl, biphenyl, fluorenyl and indenyl. Aryl groups may be substituted or unsubstituted.

[0022] Unless otherwise indicated, the term "heteroatom" refers to the atoms O, S, or N. The term "heteroaryl" includes aromatic rings or ring systems that contain at least one ringheteroatom (e.g., O, S, N). In some embodiments, the term "heteroaryl" includes a ring or ring system that contains 2 to 12 carbon atoms, 1 to 3 rings, 1 to 4 heteroatoms, and O, S, and / or N as the heteroatoms. Suitable heteroaryl groups include furyl, thienyl, pyridyl, quinolinyl, isoquinolinyl, indolyl, isoindolyl, triazolyl, pyrrolyl, tetrazolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, benzofuranyl, benzothiophenyl, carbazolyl, benzoxazolyl, pyrimidinyl, benzimidazolyl, quinoxalinyl, benzothiazolyl, naphthyridinyl, isoxazolyl, isothiazolyl, purinyl, quinazolinyl, pyrazinyl, 1 -oxidopyridyl, pyridazinyl, triazinyl, tetrazinyl, oxadiazolyl, thiadi azolyl, and so on.

[0023] The terms "arylene" and "heteroarylene" are the divalent forms of the "aryl" and "heteroaryl" groups defined above. The terms "arylenyl" and "heteroarylenyl" are used when "arylene" and "heteroarylene", respectively, are substituted. For example, an alkylarylenyl group comprises an arylene moiety to which an alkyl group is attached.

[0024] The terms "hctcrocycloalkyl" and, interchangeably, "heterocyclyl," as used herein, alone or in combination, each refer to a saturated, partially unsaturated, or fully unsaturated monocyclic, bicyclic, or tricyclic heterocyclic radical containing at least one, preferably 1 to 4, and more preferably 1 to 2 heteroatoms as ring members, wherein each said heteroatom may be independently selected from the group consisting of nitrogen, oxygen, and sulfur, and wherein there are preferably 3 to 8 ring members in each ring, more preferably 3 to 7 ring members in each ring, and most preferably 5 to 6 ring members in each ring."Heterocycloalkyl" and "heterocyclyl" are intended to include sulfones, sulfoxides, N-oxides of tertiary nitrogen ring members, and carbocyclic fused and benzo fused ring systems; additionally, both terms also include systems where a heterocycle ring is fused to an aryl group, as defined herein, or an additional heterocycle group. Heterocyclyl groups of the invention are exemplified by aziridinyl, azetidinyl, 1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl, dihydrocinnolinyl, dihydrobenzodioxinyl, dihydro[l,3]oxazolo[4,5- b]pyridinyl, benzothiazolyl, dihydroindolyl, dihy-dropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl, isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl, tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like. The heterocyclyl groups may be optionally substituted unless specifically prohibited.

[0025] The term "optionally substituted" means the anteceding group may be substituted or unsubstituted. When substituted, the substituents of an "optionally substituted" group mayinclude, without limitation, one or more substituents independently selected from the following groups or a particular designated set of groups, alone or in combination: lower alkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl, lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lower haloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl, phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, lower acyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester, lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, lower alkylamino, arylamino, amido, nitro, thiol, lower alkylthio, arylthio, lower alkylsulfinyl, lower alkylsulfonyl, arylsulfinyl, arylsulfonyl, arylthio, sulfonate, sulfonic acid, trisubstituted silyl, N3, SH, SCH3, C(O)CH3, CO2CH3, CO2H, pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Two substituents may be joined together to form a fused five-, six-, or sevenmembered carbocyclic or heterocyclic ring consisting of zero to three heteroatoms, for example forming methylenedioxy or ethylenedioxy. An optionally substituted group may be unsubstituted (c.g., -CH2CH3), fully substituted (c.g., -CF2CF3), monosubstituted (c.g., - CH2CH2F) or substituted at a level anywhere in-between fully substituted and monosubstituted (e.g., -CH2CF3). Where substituents are recited without qualification as to substitution, both substituted and unsubstituted forms are encompassed. Where a substituent is qualified as "substituted," the substituted form is specifically intended. Additionally, different sets of optional substituents to a particular moiety may be defined as needed; in these cases, the optional substitution will be as defined, often immediately following the phrase, "optionally substituted with."

[0026] When a group is present more than once in any formula or scheme described herein, each group (or substituent) is independently selected, whether explicitly stated or not. For example, for the formula -C(O)-NR2 each R group is independently selected.

[0027] As a means of simplifying the discussion and the recitation of certain terminology used throughout this application, the terms "group" and "moiety" are used to differentiate between chemical species that allow for substitution or that may be substituted and those that do not so allow for substitution or may not be so substituted. Thus, when the term "group" is used to describe a chemical substituent, the described chemical material includes the unsubstituted group and that group with nonperoxidic O, N, S, Si, or F atoms, for example, in the chain as well as carbonyl groups or other conventional substituents. Where the term "moiety" is used to describe a chemical compound or substituent, only an unsubstituted chemical material isintended to be included. For example, the phrase "alkyl group" is intended to include not only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tertbutyl, and the like, but also alkyl substituents bearing further substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl, halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, "alkyl group" includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls, hydroxy alky Is, cyanoalkyls, etc. On the other hand, the phrase "alkyl moiety" is limited to the inclusion of only pure open chain saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl, tert-butyl, and the like.

[0028] The invention is inclusive of the compounds described herein in any of their pharmaceutically acceptable forms, including isomers (e.g., diastereomers and enantiomers), tautomers, salts, solvates, polymoiphs, prodrugs, and the like. In particular, if a compound is optically active, the invention specifically includes each of the compound’s enantiomers as well as racemic mixtures of the enantiomers. It should be understood that the term "compound" includes any or all of such forms, whether explicitly stated or not (although at times, "salts" are explicitly stated).

[0029] A subject, as defined herein, is an animal such as a vertebrate or invertebrate organism. In other embodiments, the subject is a mammal such as a domesticated farm animal (e.g., cow, horse, pig) or pet (e.g., dog, cat). More preferably, the subject is a human.

[0030] Treat", "treating", and "treatment", etc., as used herein, refer to any action decreasing the rate of aging of a subject or providing a benefit to a subject having a neurodegenerative disease, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, etc.

[0031] As used herein, the term “prevention” includes either preventing or decreasing the risk of developing a neurodegenerative disease or disorder. This includes prophylactic treatment of those having an enhanced risk of developing a neurodegenerative disease or disorder. An elevated risk represents an above-average risk that a subject will develop a neurodegenerative disease or disorder, which can be determined, for example, through family history or the detection of genes causing a predisposition to develop a neurodegenerative disease or disorder. A subject can also have an increased risk of developing a neurodegenerative disease or disorder as a result of injury, exposure to toxins, or infection.

[0032] “Pharmaceutically acceptable” as used herein means that the compound or composition is suitable for administration to a subject for the methods described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

[0033] The terms “therapeutically effective’’ and “pharmacologically effective” are intended to qualify the amount of each agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies. The therapeutically effective amount may be administered in one or more doses.Methods of Treating or Preventing Neurodegenerative Disease

[0034] In one aspect, the present invention provides a method of treating or preventing a neurodegenerative disease in a subject in need thereof. The method includes administering a therapeutically effective amount of a mitochondrial enhancer according to formula I:

[0035] to the subject, wherein R1is a halogen, cycloalkyl, or heterocycloalkyl group, R2-R5arc hydrogen or halogen, X is a methylene, an oxygen, or an N-H group, Y is a hctcrocycloalkyl or heteroaryl group, and Z is a heterocycloalkyl or heteroaryl group, or a pharmaceutically acceptable salt thereof. In some embodiments, Y and Z are C3-C4 heterocycloalkyl or heteroaryl groups.

[0036] Neurodegeneration generally refers to the loss of structure or function of neurons, impairment of normal neuronal functions, and includes the death of neurons. Neurodegeneration results from various different causes including genetic mutation, mitochondrial dysfunction, and the inability to handle increasing levels of oxidative or nitrosative stress can also lead to the progression of neurodegeneration. Substantial evidence from many in vitro and in vivo studies suggests that there is a commonality of events for the progression of many neurodegenerative diseases of aging. Examples of neurodegenerativediseases include Parkinson’s disease, Huntington's disease, Amyotrophic Lateral Sclerosis (ALS), multiple sclerosis, and among the most common of the neurodegenerative disorders is Alzheimer's disease (AD). In some embodiments, the neurodegenerative disease being treated with a mitochondrial enhancer is Alzheimer’s disease or Huntington’s disease.

[0037] Defective mitochondrial proteostasis has been implicated in neurodegenerative disease. Douglas PM, Dillin A., J Cell Biol., 190:719-29 (2010). In addition, several proteins associated with early-onset neurodegenerative disease have been directly or indirectly connected to mitochondrial function. Lin et al., Nature, 443:787-95 (2006). Two genes associated with familial Parkinson’s disease, the E3 ubiquitin protein ligase Parkinson’s disease protein 2 (PARK2; also known as parkin) and PTEN-induced putative kinase 1 (PINK1), have a crucial role in mitophagy, partly by modulating mitochondrial fusion and fission. Likewise, the proteotoxic a-synuclein and the Parkinson’s disease-associated protein DJI (also known as PARK7) control mitochondrial morphology as well as fusion and / or fission events in the same pathway as parkin and PINK1. Kamp et l., EMBO J., 29:3571-89 (2010).

[0038] In some embodiments, the neurodegenerative disease is Alzheimer’s disease. Alzheimer's disease (AD) is a chronic neurodegenerative disease that results in the loss of neurons and synapses in the cerebral cortex and certain subcortical structures, resulting in gross atrophy of the temporal lobe, parietal lobe, and parts of the frontal cortex and cingulate gyrus. Wenk G., The Journal of Clinical Psychiatry. 64 Suppl 9: 7-10 (2003). Alzheimer's disease is usually diagnosed based on the person's medical history, history from relatives, and behavioral observations. The presence of characteristic neurological and neuropsychological features and the absence of alternative conditions is supportive. Advanced medical imaging with computed tomography (CT) or magnetic resonance imaging (MRI), and with single-photon emission computed tomography (SPECT) or positron emission tomography (PET) can be used to help exclude other cerebral pathology or subtypes of dementia.

[0039] In Alzheimer’s disease A[3 was shown to translocate to mitochondria, resulting in reduced ETC activity. This may occur following the induction of mitochondrial fragmentation, an increase in mitochondrial membrane viscosity and / or direct inhibition of complex IV activity, and involve mitochondria-associated endoplasmic reticulum membranes (MAMs) in the processing of amyloid precursor protein. Additional evidence supporting the potential involvement of mitochondria in Alzheimer’s disease has come from mtDNA haplogroupassociation studies, in which mtDNA haplogroups that have a greater likelihood of predisposing individuals to Alzheimer’s disease seem to be associated with defects in oxidative phosphorylation. Coskun et al., Biochim Biophys Acta., 1820:553-64 (2012).

[0040] The method includes administering a therapeutically effective amount of a mitochondrial enhancer according to formula I. In some embodiments, Y of formula I is a morpholine or cyanopyridine group. In other embodiments, R1of formula I is a piperidine group. In further embodiments, Z of formula I is a morpholine group. In yet further embodiments, R1and R3are halogen, and R2, R4, and R5are hydrogen.

[0041] In some embodiments, the mitochondrial enhancer has the structure:Mitochondrial Enhancer Compounds

[0042] In one aspect, the present invention provides a mitochondrial enhancer having a structure according to formula I:

[0043] wherein R1is a halogen, cycloalkyl, or heterocycloalkyl group, R2-R5are hydrogen or halogen, X is a methylene, an oxygen, or an N-H group, Y is a heterocycloalkyl or heteroaryl group, and Z is a heterocycloalkyl or heteroaryl group, or a pharmaceutically acceptable salt thereof. In some embodiments, Y and Z are C3-C4 heterocycloalkyl or heteroaryl groups.

[0044] The present invention provides mitochondrial enhancer compounds. Mitochondrial enhancing compounds can increase mitochondrial activity by increasing the activity of mitochondria themselves and / or the number of mitochondria. For a review of different methods to increase mitochondrial activity, see Andreux et al., Nat Rev Drug Discov., 12(6):465-483 (2013).

[0045] In some embodiments, Y of formula I is a morpholine or cyanopyridine group. In other embodiments, R1of formula I is a piperidine group. In further embodiments, Z of formula I is a morpholine group. In yet further embodiments, R1and R3are halogen, and R2, R4, and R5are hydrogen.

[0046] Additional mitochondrial enhancer compounds include the compounds A29, A47, A67, A54, A62, and A49, the structures of which are shown below:

[0047] In some embodiments, the mitochondrial enhancer has the structure:Formulation and Administration of Mitochondrial Enhancer Compounds

[0048] The present invention provides a method for administering one or more mitochondrial enhancer compounds in a pharmaceutical composition. Examples of pharmaceutical compositions include those for oral, intravenous, intramuscular, subcutaneous, or intraperitoneal administration, or any other route known to those skilled in the art, and generally involves providing a mitochondrial enhancer compound formulated together with a pharmaceutically acceptable carrier. Accordingly, in some embodiments the mitochondrial enhancer is administered together with a pharmaceutically acceptable carrier.

[0049] In some embodiments, the mitochondrial enhancer activator is administered orally. When preparing the compounds described herein for oral administration, the pharmaceutical composition may be in the form of, for example, a tablet, capsule, suspension or liquid. Thepharmaceutical composition is preferably made in the form of a dosage unit containing a particular amount of the active ingredient. Examples of such dosage units are capsules, tablets, powders, granules or a suspension, with conventional additives such as lactose, mannitol, com starch or potato starch; with binders such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins; with disintegrators such as com starch, potato starch or sodium carboxymethyl-cellulose; and with lubricants such as talc or magnesium stearate. The mitochondrial enhancer activator may also be administered by injection as a composition wherein, for example, saline, dextrose or water may be used as a suitable earner.

[0050] For intravenous, intramuscular, subcutaneous, or intraperitoneal administration, the compound may be combined with a sterile aqueous solution which is preferably isotonic with the blood of the recipient. Such formulations may be prepared by dissolving solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. The formulations may be present in unit or multi-dose containers such as sealed ampoules or vials.

[0051] Formulations suitable for parenteral administration conveniently comprise a sterile aqueous preparation of the active compound which is preferably made isotonic. Preparations for injections may also be formulated by suspending or emulsifying the compounds in nonaqueous solvent, such as vegetable oil, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol.

[0052] The dosage form and amount can be readily established by reference to known treatment or prophylactic regiments. The amount of therapeutically active compound that is administered and the dosage regimen for treating a disease condition with the compounds and / or compositions of this invention depends on a variety of factors, including the age, weight, sex, and medical condition of the subject, the severity of the disease, the route and frequency of administration, and the particular compound employed, the location of the unwanted proliferating cells, as well as the pharmacokinetic properties of the individual treated, and thus may vary widely. The dosage will generally be lower if the compounds arc administered locally rather than systemically, and for prevention rather than for treatment. Such treatments may be administered as often as necessary and for the period of time judged necessary by the treating physician. One of skill in the art will appreciate that the dosage regime ortherapeutically effective amount of the inhibitor to be administrated may need to be optimized for each individual. The pharmaceutical compositions may contain active ingredient in the range of about 0.1 to 2000 mg, preferably in the range of about 0.5 to 500 mg and most preferably between about 1 and 200 mg. A daily dose of about 0.01 to 100 mg / kg body weight, preferably between about 0.1 and about 50 mg / kg body weight, may be appropriate. The daily dose can be administered in one to four doses per day.

[0053] For example, the maximum tolerated dose (MTD) for mitochondrial enhancer compounds can be determined in tumor-free athymic nude mice. Agents are prepared as suspensions in sterile water containing 0.5% methylcellulose (w / v) and 0.1% Tween 80 (v / v) and administered to mice (7 animals / group) by oral gavage at doses of 0, 25, 50, 100 and 200 mg / kg once daily for 14 days. Body weights, measured twice weekly, and direct daily observations of general health and behavior will serve as primary indicators of drug tolerance. MTD is defined as the highest dose that causes no more than 10% weight loss over the 14-day treatment period.

[0054] The mitochondrial enhancer compounds can also be provided as pharmaceutically acceptable salts. The phrase “pharmaceutically acceptable salts” connotes salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically acceptable. Suitable pharmaceutically acceptable acid addition salts of the compounds may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic, and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, p- hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic, methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, y-hydroxybutyric, galactaric, and galacturonic acids. Suitable pharmaceutically acceptable base addition salts of the compounds described herein include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Alternatively, organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine)and procaine may be used form base addition salts of the compounds described herein. All of these salts may be prepared by conventional means from the corresponding compounds described herein by reacting, for example, the appropriate acid or base with the compound.Preparation of Compounds

[0055] Compounds of the invention may be synthesized by synthetic routes that include processes analogous to those well known in the chemical arts, particularly in light of the description contained herein. The starting materials are generally available from commercial sources such as Aldrich Chemicals (Milwaukee, Wisconsin, USA) or are readily prepared using methods well known to those skilled in the art (e.g., prepared by methods generally described in Louis F. Fieser and Mary Fieser, Reagents for Organic Synthesis, v. 1-19, Wiley, New York, (1967-1999 ed.) and similar texts known to those skilled in the art.

[0056] An example has been included to more clearly describe a particular embodiment of the invention and its associated cost and operational advantages. However, there are a wide variety of other embodiments within the scope of the present invention, which should not be limited to the particular example provided herein.EXAMPLEExample 1: Small molecule stabilization of calpastatin enhances mitochondrial function and mitigates neurodegeneration

[0057] To develop new therapeutic options, in this study, we modified CHIR99021 through medicinal chemistry to find analogs that retain mitochondrial protective effects but lack GSK3 kinase inhibition. As results, among 132 synthesized analogs, compound A36 met all our criteria. A36 treatment improves mitochondrial function and cell survival in HD neuron cultures, prevents the loss of medium spiny neurons and mutant huntingtin aggregation, reduces neuroinflammation and oligodendrocyte / myelin degeneration, and improves motor functions in R62 mouse models of HD. A36 also proved effective in iPSC-derived MSN and enhanced the expression of calpastatin. Furthermore, in the P301S Tau-expressing transgenic mice (PS 19) that show AD-like symptoms, daily treatment of A36 led to improvements in cognition and memory, locomotor activity, reduced levels of phosphorylated and aggregated Tau in the hippocampus, and decreased neuroinflammation. Our studies in both HD and ADmodels suggest that enhancing mitochondrial function through calpastatin modulation could be a useful therapeutic strategy. Compound A36 emerged as a leading candidate for treating a range of neurodegenerative diseases, underscoring its potential as a therapeutic agent.Results

[0058] A36 is an optimized lead showing mitochondrial protection without targeting GSK3. We employed advanced medicinal chemistry strategies to modify the molecular architecture of CHIR99021, specifically investigating the analogs that alter the left-hand side of the molecule. Our synthetic chemistry efforts resulted in the creation of 132 distinct compounds. These compounds underwent rigorous cell-based screening, utilizing mitochondrial membrane potential disruption as the primary assay and cell viability assessment as a secondary measure. The screening was earned out in HdhQ7 and QI 1 1 mouse striatal cells, which are established models for HD research. Furthermore, we conducted in vitro GSK3 kinase activity assay to identify compounds demonstrating minimal inhibition of this kinase, a key consideration in our selection process. Through a combination of these assays and a thorough investigation of doseresponse relationships, we narrowed our compound library to 10 promising analogs. Subsequently, we assessed their central nervous system (CNS) permeability using the MDCK- MDR1 permeability assay and in vitro metabolic stability, comparing their performance against the parent CHIR99021 molecule. Our collective screening efforts and in vitro evaluations highlighted compound A36 as the lead candidate, exhibiting optimal characteristics including mitochondrial and cellular protection under diseased conditions, the lack of GSK3 kinase inhibition, and significant brain peimeability. Consequently, we are advancing the A36 analogs into further phases of investigation, focusing on target engagement, mitochondrial functionality, and neuronal effect characterization both in vitro and in vivo.

[0059] Calpastatin is the direct target of A36. To extend our understanding of the mechanisms underlying A36-mediated enhancement of mitochondrial function as well as cell survival in HD neurons, we then determine the direct binding target of A36. Drug affinity responsive target stability (DARTS) is one of the most common methods to identify the direct protein target of small molecule, which is based on the fact that direct ligand binding can stabilize the protein and protect it from digestion by Pronase. To do so, we prepared the following samples for mass spectrometry: (1) cell total protein lysates: (2) cell total protein lysate digested by pronase: (3) cell total protein lysates digested by pronase after A36 incubation. The proteomics analysesrevealed 24 proteins with more than 50% reduction in their protein level upon pronase's digestion (Fig. 2A). A36 treatment prevented the pronase digestion of 15 proteins as their protein level was increased by at least 2-fold (Fig. 2A). As results of overlapping, there are only three proteins, Cast (calpastatin), Ybx3 (Y-box binding protein 3), and Eeal (early endosome antigen 1), showing significancy on the change of peptide levels, of which Cast has the lowest p-value (Fig. 2A).

[0060] Considering that CHIR99021, the parental compound of A36, can suppress calpastatin degradation in HD, we hypothesize that Cast is the direct binding target of A36. We then validated the direct interaction by DARTS using recombinant Calpastatin protein. In a dosedependent manner, direct incubation with A36 prevented proase-mediated digestion of Calpastatin in vitro, with EC50 at 26.6 pM (Fig. 2B and C). The direct interaction was further validated using cell protein lysates. While having little effect on EEA1, a cytosolic protein that was also significantly changed in the DARTS -proteomics analyses, A36 incubation preserved the level of calpastatin, in a dose-dependent manner (Fig. 2D). A36 treatment protected calpastatin, but not EEA1, from degradation by pronase at different doses (Fig. 2E). These results indicate that A36 selectively targets calpastatin.

[0061] To understand if calpastatin is required for A36-mediated improvement on mitochondrial function and cell viability, we treated calpastatin- knockdown Q7 and QI 11 striatal cells with A36 and examined mitochondrial membranes potential and cell viability. A36 treatment significantly improved mitochondrial membrane potential in Consh but not CASTsh QI 11 cells (Fig. 2F). Consistently, A36 treatment significantly improved the cell survival of Consh but not CASTsh QI 11 cells (Fig. 2G). These results again suggest that calpastatin is the molecule target of A36, and calpastatin is required for A36-confered protection. To understand whether the A36-mediated upregulation of calpastatin is associated with calpain inhibition, we then performed co-immunoprecipitation of calpastatin and calpain in Q7 / 111 cells upon A36 treatment. A36 treatment rescued the interaction between calpastatin and calpain in QI 11 cells, which inhibits calpain-induced mitochondrial impairment and cell damages (Fig. 2H). Together, these findings indicate A36 targets calpastatin that is required for mitochondrial protection and neuronal survival in HD cells.

[0062] A36 treatment rescued mitochondrial function and calpastatin expression in HD neuronal cells. We then assessed the effect of A36 treatment in HD striatal neuronal cultures.Comparing to Q7 control cells, the QI 11 cells exhibited accumulation of mitochondrial ROS as labeled by mitoSOX (Fig. 3A). In a dose-dependent manner, A36 treatment significantly reduced the level of mitochondrial ROS in QI 11 cells, with IC50 at 1.98uM (Fig. 3 A and B). By using Seahorse analyzer for measurement of mitochondrial respiration, we also observed that A36 treatment significantly improved mitochondrial ATP production and maximal respiration in QI 11 striatal cells (Fig. 3C and D), suggesting that A36 can also rescue mitochondrial respiratory capacity. Comparing to those in Q7 cells, the mitochondrial network in QI 1 1 cells exhibited as fragmented parts, implicating the functional impairment that leads to ROS production and respiratory deficits (Fig. 3E). Once again, in a dose-dependent manner, A36 treatment led to significant reduction of the percentage of fragmented mitochondrial and the elevation of the percentage of intermediate or elongated mitochondria in QI 11 cells (Fig. 3E). Of note, A36 had little effect on mitochondrial ROS, respiration nor the morphology in the WT Q7 cells. These results suggest that A36 can regulate and improve mitochondrial shape in accordance with mitochondrial respiration and ROS production.

[0063] We demonstrated, previously, that calpastatin was downregulated in QI 11 HD striatal cells. To understand whether A36 can rescue the expression of calpastatin, we ran western blot and found that the protein level of calpastatin was improved upon A36 treatment in QI 11 cells, whereas calpastatin was not changed in Q7 cells (Fig. 3F). In parallel, the level of cleaved aSpectrin-II, a substrate of calpain, was reduced upon A36 treatment in QI 11 cells, suggesting calpain inhibition by calpastatin (Fig. 3F). Calpastatin downregulation led to Drpl-mediated mitochondrial fission and thus mitochondrial impairment. As previously reported, while the protein level of S616 phosphorylated- Drpl was increased in QI 11 cells, A36 treatment led to significant decrease of the protein level of pS616-Drpl (Fig. 3F), consistent with reduced level of mitochondrial Drpl, which could explain the restoration of mitochondrial tubular shape by A36. Overall, these results suggest that A36 treatment protect mitochondrial respiration and morphology by mediating calpastatin-calpain interaction.

[0064] A36 treatment rescued neuronal function and calpastatin expression in MSN-derived from HD iPSC. HD is defined by the selective loss of striatal GABAergic MSNs. We previously differentiated HD patient-induced pluripotent stem (iPS) cells into GABAergic striatal and MSNs; their mtHtt had the same number of CAG repeats as cells from source HD patients. These cells exhibited short neurites and were sensitive to stressors. Mitochondrial depolarization, mitochondrial fragmentation, and neuronal cell death were also evident in thesepatient neurons. Thus, this patient-derived model represented a platform to validate smallmolecule enhancers of mitochondrial function and survival in human neurons with HD phenotypes caused by an HD genotype. MAP2+ Neurons differentiated from HD patients’ iPSC had lower expression of calpastatin, which was improved upon A36 treatment (Fig.4A). Consistent with previous findings, the HD neurons exhibited shorter branches labeled by Tuj 1 , and lower immuno-intensity of DARPP32, an MSN marker, suggesting the loss / degeneration of MSN (Fig.4B). A36 treatment not only improved the length of neuronal branches, but also rescued the expression DARPP32 (Fig. 4A and B), which indicates the protective effect of A36 on neuronal health and survival. The protective effect of A36 treatment on HD neurons was further validated using western blot analyses. We detected a consistent decrease in DARPP32 protein level in HD neurons, which was rescued upon A36 treatment (Fig.4C and D). A36 treatment also improved the protein level of PSD95, a synaptic marker, in HD neurons (Fig.4C and D). Moreover, in accordance with calpastatin down-regulation, calpain-induced aSpectrin II cleavage and p25 production and Drpl phosphorylation at Serine 616 were all significantly up-regulated in the HD neurons (Fig.4C and D). A36 treatment rescued the expression of calpastatin and suppressed calpain activity and Drpl phosphorylation (Fig.4C and D). Together, these results indicate that A36 treatment rescued neuronal health via modulating calpastatin-calpain signaling.

[0065] A36 administration mitigated HD-associated neuropathology and motor deficits in HD R6 / 2 mice

[0066] We next determined whether A36 treatment affects disease progression in HD mouse model. We used HD R6 / 2 transgenic mice which express a fragment of human mtHtt protein and aggressively develop HD-associated pathologies, including mtHtt accumulation, striatal degeneration, and motor deficits. This model has been widely used as a primary screening model for HD drug candidates. Because A36 is a novel molecule without any in vivo characterization, we first evaluated the pharmacokinetics of A36 and test whether it can cross the blood-brain barrier after intraperitoneal injection (i.p.) (Fig.5A). These results indicate that A36 has a great brain penetrance capacity, supporting the use as an CNS drug.

[0067] Then we treated HD R6 / 2 and WT mice by i.p. injection with the molecule (10 mg / kg / day, five days / week) starting at 6 weeks old to determine whether A36 administration prevented rapid and severe progression of HD-related pathology. A 6-week treatment with A36improved the survival of HD R6 / 2 mice when compared with vehicle-treated counterparts (Fig. 5B). A36 administration significantly reduced deficits in mouse locomotor activity as measured by an open-field activity chamber (Fig.5C) and attenuated the clasping behavior of HD R6 / 2 mice which reflect motor deficits (Fig.5D). Moreover, the 6-week treatment regimen increased the immunodensity and protein levels of DARPP32 (Fig. 5E and G), and BDNF - a neurotrophic factor for MSN survival (Fig.S2B) - and also reduced mtHtt aggregates (Fig.5F), a hallmark of HD pathology, in the striatum of HD R6 / 2 mice when compared with vehicle- treated HD mice. Additionally, the fluorescence intensity of calpastatin in MSN, which was reduced in the dorsal striatum of R6 / 2 mice, was rescued upon A36 treatment (Fig.5G). A36 administration also suppressed microglial activation and rescued the oligodendrocyte number and the myelin volume around dorsal striatal region. Western blot analyses further show that A36 treatment rescued the expression of calpastatin and inhibited calpain-mediated cleavage of aSpectrin II and P35 in the dorsal striatum of R6 / 2 mice (Fig.51). These data suggested a protective effect of A36 in HD R6 / 2 mice. Importantly, this dosing regimen was not toxic: A36 treatments had no effects on behavioral status, body weight, and survival rates of WT mice over the 6-week treatment period (Fig. 5).

[0068] A36 administrate mitigated tauopathy and cognitive deficits in P301S-Tau expressing mice. The pathological role of calpain hyper-activation and the protective effects of calpain inhibition via the genetic enhancement of CAST have been observed in several other neurodegenerative diseases such as AD, PD, and ALS. Thus, we sought to understand if A36 can be a general intervention for a broad spectrum of neurodegenerative diseases. Previous findings that genetic upregulation of calpastatin mitigated the neuropathology and cognitive deficits in tauopathy prompt us to examine the effect of A36 treatment in the transgenic PSI 9 mice. The PS 19 transgenic mice express human mutant P301S tau driven by the mouse prion protein (Prnp) promoter, which leads to development of neuronal loss and brain atrophy mainly in the hippocampus. They develop widespread neurofibrillary tangle-like inclusions in the neocortex, amygdala, hippocampus, brain stem, and spinal cord. Tangle pathology is accompanied by microgliosis and astrocytosis. Behaviorally, PS 19 mice display signs of age- associated cognitive impairment, including selective deficits in spatial learning and memory ability. Same as the regime for HD R6 / 2 mice, we started i.p. injection of A36 (10 mg / kg / day, five days / week) in the PS 19 mice and the W littermates from 4 months to 8 months old to determine whether A36 administration prevents the progression of Tau-associated pathology. A 4-month treatment with A36 improved the memory of the PS 19 mice when compared withvehicle-treated counterparts as measured by the Y-maze and Barnez maze tests (Fig.6A-C). Pathologically, A36 administration significantly reduced the expression of phosphor-Tau in hippocampus in PS 19 mice (Fig.6D and E). Moreover, the 4-month treatment regime significantly reduced the immuo-density of GFAP and IB Al, markers for astrocyte and microglia, in the hippocampus of PS 19 mice when comparing with the veh- treated PS 19 mice (Fig.6F-H). Additionally, the fluorescence intensity of calpastatin in hippocampal CA3 region where it is mainly expressed, which was reduced in the veh-treated PS 19 mice, was rescued in A36-treated PS 19 mice (Fig.61 and J). Consistent with previous findings, we observed calpastatin downregulation in accordance with the upregulation of cleaved a-spectrin II and p25 and pS616-Drpl in the veh-treated PS19 mice, whereas A36 administration rescued calpastatin protein level and subsequently suppressed calpain activation (Fig. 6H and L). Furthermore, while the protein level of soluble Tau was not affected in the A36-treated PS 19 mice, the level of insoluble Tau as well as the level of both soluble and insoluble phosphor- Tau (AT8) were all significantly decreased in the A36-trcatcd PS 19 mice, when compared to the veh-treated PS 19 mice (Fig.6M and N). Together, these results indicated that A36 treatment mitigated Tau-related neuropathology and cognitive deficits.Discussion

[0069] Considering the causative role of mitochondrial dysfunction in the pathogenesis of HD, we previously built up a mitochondrial function-based screening platform and identified a putative mitochondrial enhancer, CHIR99021, showing strong protection in multiple models of HD. Nevertheless, our results indicated an off-target effect of CHIR99021 by suppressing calpastatin degradation rather than inhibiting GSK3 activity. Thus, we speculate that eliminating its GSK3 interaction could enhance the efficacy of CHIR99021 towards mitochondrial protection. To improve the efficacy of CHIR99021 to reach pre-clinical / clinical potential, in this study, we did medicinal chemistry by synthesizing 132 structural analogues of CHIR99021, and, by leveraging our established screening platform, identified A36 as a top lead. We demonstrated that A36 directly binds to calpastatin without any GSK3 inhibition. Mechanistically, A36 treatment significantly reduced mitochondrial ROS production and improved mitochondrial morphology, mitochondrial bioenergetics, and calpastatin expression in HD cell culture model. We further validated the neuroprotection of A36 by using MSN derived from HD patients’ iPSC, and the results showed that A36 treatment improved the neuronal morphology, survival and the protein level of calpastatin in HD MSN. In addition,A36 administration significantly mitigated the HD-related neuropathology and motor deficits, and rescued the expression of calpastatin in R6 / 2 mouse model of HD. Last, we demonstrated that A36 treatment significantly mitigated cognitive deficits and tau aggregates, and improved calpastatin expression in P301S Tau mice (PS19). Therefore, our findings suggested that improving mitochondrial function by modulating calpastatin could be a potential strategy, and small molecules like A36 are potential therapeutic leads for HD and AD.

[0070] Identifying shared molecular hubs with essential pathogenic impact may shed light in the development of common intervention strategy for a broad spectrum of neurodegenerative diseases. Calpain is the cysteine protease mainly in neurons and is typically maintained in an inactive precursor state until activation by micro-molar calcium levels. Calpain activation is associated with the cleavage of prion-like proteins as well as their aggregation, which plays a pivotal role in the pathogenesis of neurodegenerative diseases, including Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis. Under physiological conditions, calpain limits cellular proteolysis and is tightly regulated by its endogenous proteinaceous calpain inhibitor, calpastatin. The development of inhibitors directly targeting calpains has proven challenging due to poor specificity and potential toxicity. As an alternative, the selective inhibition of calpain activity via elevation of calpastatin may be attractive. Indeed, the genetic upregulation of calpastatin is one of few universal approaches, to our knowledge, that shows beneficial effects in the animal models of neurodegenerations. For instances, calpastatin overexpression in fly and mouse HD models ameliorated HD- associated neuropathology and behavioral deficits. Calpastatin knock-in suppressed calpain- mediated cleavage of Tau and thus tauopathy, and cognitive deficits in PS 19 mice. Neuronspecific overexpression of calpastatin inhibited the calpain-CDK5 signaling axis and thus reduced the production of toxic Tau and SOD1 oligomer in the SOD1 G93A mouse model of ALS. Moreover, calpastatin overexpression reduced calpain- mediated a-synuclein cleavage and aggregation, astrogliosis, and synaptic impairment in A30P SNCA mouse model of PD. These preponderances of evidence suggest that calpastatin upregulation may be a potential common therapeutic strategy for neurodegenerative diseases. In this study, we elucidated that the lead molecule A36 can directly binds to calpastatin and modulate its protein level in models of HD and AD. Our evidence that A36 treatment rescued mitochondrial function and cell viability in a calpastatin-dependent manner further highlight the strong selectivity of A36 on calpastatin. Therefore, A36 is a novel calpastatin modulator with great promise for developing therapeutics for the neurodegenerative diseases. Future studies on the assessment of A36’sefficacy in the preclinical models of other neurodegenerative diseases, such as ALS and PD, will provide clue on the potency of A36. Although our PK analyses demonstrated the penetrance of A36 across blood-brain barrier, efforts are needed to refine the chemical configurations to improve its solubility as well as half-life in vivo before moving forward to the clinical evaluation.

[0071] Calpain activation and CAST loss have been linked to mitochondrial dysfunction. The presence of calpain and CAST on mitochondria has been well documented. While calpain accumulation on mitochondria leads to mitochondrial depolarization, oxidative stress, and energy depletion, CAST overexpression attenuates calpain-mediated mitochondrial damage in response to various stimuli. Mitochondria are dynamic organelles that are balanced by fusion and fission processes. Enhanced fission and fragmentation result in mitochondrial depolarization and bioenergetic failure, contributing to the pathogenesis of neurodegenerative diseases, including HD and AD. The calpain-mediated cleavage of p35 to p25 activates CDK5 which then binds to and phosphorylates Drpl, leading to Drpl recmitment to the mitochondria and resultant fragmentation. Activated calpain may also directly cleave Dipl, possibly contributing to mitochondrial fragmentation and neuronal damage in AD models. The inhibition of calpain by CAST appears to preserve mitochondrial morphology by reducing the mitochondrial fission proteins, Drpl and Fisl, and protecting neurons against excitotoxic cell death. Additionally, CAST also prevents the mitochondrial translocation of Drpl by inhibiting calpain-mediated calcineurin activation. A recent study reported that CAST was also distributed on the mitochondria-ER-associated membrane and was transported along neuronal axons by the mitochondrial fusion related protein, mitofusin 2, to prevent synaptic elimination during ALS. Thus, the protection of CAST from neuronal death is involved in the inhibition of mitochondrial impairment by balancing mitochondrial fission and fusion processes. In this study, we further showed that A36 treatment suppressed Drpl hyperactivation and resultant mitochondrial fragmentation. Our previous work revealed CAST-dependent protection on mitochondrial dynamics upon CHIR99021 treatment; and that CAST loss completely abolished CHIR99021 -mediated inhibition of Drpl activity and improved mitochondrial morphology. Given that A36 is a chemical analogue of CHIR99021, with much higher selectivity on CAST, CAST may be a key molecule upstream of Drpl that mediates mitochondrial protection and neuroprotection induced by A36 in HD models.

[0072] In summary, we did chemical modifications on CHIR99021 to identify a potent lead, A36, as a putative mitochondrial enhancer by directly targeting and stabilizing calpastatin. Our proof-of-principles ex vivo and in vivo evaluation demonstrated the efficacy of A36 in mitigating HD associated neurodegeneration and neuropathology, highlighting the pivotal role of calpastatin loss during HD pathogenesis. Furthermore, A36 treatment efficiently ameliorated the tauopathy as well as cognitive deficits in PS 19 mouse model of AD. The protective effects of calpain inhibition via the genetic enhancement of CAST have been observed in several other neurodegenerative diseases such as AD, PD, and ATS. Thus, A36 holds plausibility to become a universal treatment for neurodegenerative diseases. Further investigations on the pharmaceutical optimization of A36 in aspects of solubility and stability may accelerate the development of therapeutic agents against neurodegenerative diseases.Material and methodsReagents and antibodies

[0073] Protein phosphatase inhibitor cocktail (P5726), protease inhibitor cocktails (8340) were purchased from MilliporeSigma (Burlington, MA, USA). CHIR99021 (S2924) was from Selleckchem (Houston, TX, USA). MG132 (13697), lactacystin (70980), epoxomicin (10007806), bafilomycin Al (BFA, 11038), tunicamycin (11445), thapsigargin (10522) were purchased from Cayman Chemical (Ann Arbor, MI, USA). N-acetyl-calpastatin (184-210) (J66680MCR) was purchased from Alfa Acsar (Haverhill, MA, USA). The antibodies used in the study are listed below: Anti-DARPP-32 (ab40801, Abeam, Cambridge, UK, 1:3000), anti- BDNF (ab 108319, Abeam, 1:1000), anti-CDK5 (ab40773, Abeam, 1 : 1000), anti-ATPB (17247-1 -AP, Proteintech, 1 :3000), anti-GSK3 (05-412, MilliporeSigma, 1 : 1000), anti- huntingtin protein (MAB5374, clone EM48, MilliporeSigma, 1:1000), anti-[3-actin (A1978, MilliporeSigma, 1:10000), anti-DLPl (611113, BD Bioscience, Franklin Lakes, NJ, USA, 1:2000), anti-PSD95 (2507, Cell Signaling, Danvers, MA, USA, 1:5000), anti-Drpl-pS616 (3455S, Cell Signaling, 1:1000), anti-p35 / 25 (2680, Cell Signaling, 1: 1000), anti-CAST (4146, Cell Signaling, 1 :2000), anti-calpain I (2556, Cell Signaling, 1 :1000), anti-spectrin-a II (sc- 48382, Santa Cruz Biotechnology, Dallas, TX, USA, 1 :500), and anti-enolase (sc-15343, Santa Cruz Biotechnology, 1:2000), HRP-conjugated anti-rabbit or anti-mouse IgG (31430 / 31460, ThermoFisher Scientific, 1:5000).Cell culture

[0074] Striatal neuron- like WT HdhQ7 and HD mutant HdhQl 11 cells were obtained from the Cure Huntington’s Disease Initiative (CHDI) Foundation and were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 mg / mL penicillin, 100 mg / mL streptomycin, and 400 pg / mL G418. Cells were grown at 33°C in a 5% CO2 environment and were used within 14 passages for studies.

[0075] Induced pluripotent stem (iPS) cells from normal subjects and patients with HD were differentiated into neurons using a protocol from our previous study14, 18. Briefly, iPS cells were plated onto 6-well plates pre-coated with 2.5% matrigel (Corning) and allowed reach 90% confluence in feeder-free medium (mTeSR™ Plus, 100-0276, STEMCELL Technologies, Vancouver, Canada). For the first 10 days, cells were treated with SB431542 (10 mM; Tocris Bioscience, Bristol, UK) and Noggin (100 ng / mL; R&D systems, Minneapolis, MI, USA) in neural medium containing ncurobasal™ -A medium (ThermoFisher Scientific, Waltham, MA) and DMEM / F12 GlutaMAX™ (ThemioFisher Scientific) (1: 1), B27 supplement minus vitamin A (50X, Invitrogen, Carlsbad, CA, USA), N2 supplement (100X, Invitrogen), GlutaMax (100X, Invitrogen), human recombinant fibroblast growth factor-basic (FGF2, 20 ng / mL, PEPROTECH, Rocky Hill, NJ, USA) and human recombinant epidermal growth factor (EGF, 20 ng / mL, MilliporeSigma), 100 units / mL penicillin and 100 pg / mL streptomycin. For the next 10 days, cells were treated with human recombinant Sonic hedgehog (SHH, 200 ng / mL, PEPROTECH), human recombinant DKK1 (100 ng / mL, PEPROTECH) and human recombinant brain derived neurotrophic factor (BDNF, 20 ng / mL, PEPROTECH), and 10 mM Y27632 (MilliporeSigma) in neuronal differentiation medium containing neurobasal™ -A medium and DMEM / F12 Glutamax™ medium (1 :3), B27, N2, GlutaMax, and 50 units / mL penicillin and 50 pg / mL streptomycin. Cells were then switched to treatment with BDNF (20 ng / mL), ascorbic acid (200 mM, MilliporeSigma), dibutyryl cAMP (0.5 mM, MilliporeSigma), and Y27632 (10 pM) in neuronal differentiation medium. Twenty days after differentiation initiation, neurons (approximately 5,000 cells) were plated onto 12-mm poly-D-lysine (MilliporeSigmaj / laminine (ThermoFisher Scientific)-coated coverslips and grown in 24-well plates in neuronal differentiation medium. After a 5-day treatment with CHIR99021 (Selleckchem, Houston, TX, USA) at 1 pM, cells were fixed in 4% paraformaldehyde and subjected to immunostaining. To measure cell survival, the same number of neurons were plated in 96- well plates. After a 4-day treatment with 1 pM CHIR99021, BDNF was withdrawnfrom the medium and cell viability measured after 12 h using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Roche, Indianapolis, IA, USA) following manufacturer’s instructions.Mitochondrial membrane potential measurements

[0076] Cells seeded on 96-well plate (8000 cells / well) were washed in PBS (pH 7.4) and incubated with 0.25 uM TMRM and 5 pg / mL Hoechst for 20 min at 33°C for mouse striatal cells. Cells were then washed in PBS for three times and directly imaged by Keyence fluorescence microscope (BZX-710). At least three images were taken for each well, image quantification was performed using ImageJ software. TMRM fluorescence density was normalized to the total number of cells.

[0077] Cells cultured on coverslips were washed in PBS (pH 7.4) and incubated with 0.25 M TMRM and 5 pg / mL Hoechst for 20 min at 33°C for mouse striatal cells and 37°C for other cells. Images were visualized by confocal microscopy (Olympus, Tokyo, Japan; Fluoview FV3000) and image quantification was performed using ImageJ software. At least 100 cells / group were counted for analysis. TMRM fluorescence density was normalized to the total number of cells.Measurement of mitochondrial respiratory capacity

[0078] Mouse striatal HdhQ7 / Ql 11 cells were seeded in XFp 8- well miniplates (103025-100, Agilent, Santa Clara, CA, USA) at 3,000 cells / well in 100 pL growth medium. Two days after treatment with 3 pM CHIR99021, mitochondrial respiration activity in intact cells was analyzed using a Seahorse Bioscience XFp Extracellular Flux analyzer (Agilent). Briefly, 1 h prior to measuring oxygen consumption, cell culture media was replaced with XF assay medium and maintained in a non-CCb incubator for 1 h at 33°C. Sensor cartridges were placed in the XFp analyzer according to manufacturer’s instructions from the Mito Stress Test kit (Agilent, 103010-100). Mitochondrial function was determined by the sequential injection of oligomycin A (1 pM), FCCP (1 pM) and rotenone / antimycin A (0.5 pM). The total protein content in each well was determined after respiration measurement, and all results were normalized to the total protein content.Mitochondrial ROS measurement

[0079] Mouse striatal cells cultured on coverslips were washed in PBS (pH 7.4) and incubated with 5 M MitoSOX™ Red (Invitrogen, M36008), a mitochondrial superoxide indicator, and 5 pg / rnL Hoechst for 10 min at 33°C. Images were visualized by confocal microscopy (Olympus, Fluoview FV3000) and image quantification performed using ImageJ software. At least 100 cells / group were counted for analysis. MitoSOX fluorescence density was normalized to the total number of cells.Mouse models

[0080] All animal studies were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of Case Western Reserve University, and were performed based on the NIH Guide for the Care and Use of Laboratory Animals. Sufficient procedures were employed for reducing pain or discomfort of mice during the experiments. All mice were maintained with a 12-h light / dark cycle (on at 06:00 hours and off at 18:00 hours).

[0081] Male R6 / 2 mice and their WT littermates (4 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA; B6CBA-TgN [HD exon 1]); JAX stock number: 006494). R6 / 2 mice (C57BL / 6 and CBA genetic background) are transgenic for the 5’ end of the human HD gene carrying 100-150 glutamine (CAG) repeats. Male R6 / 2 mice at 6-12 weeks were used in the study.

[0082] Male Tau P301S transgenic mice (PS 19) and their WT litter mates (8 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA; B6; C3Tg (Prnp- MAPT*P301S) PS19Vle / J, JAX: 008169). Male Tau P301S mice at 4—9 months were used in the study.A36 treatment in vivo

[0083] All randomization and compound treatments were prepared by a researcher not associated with behavioral and neuropathological analyses. For R6 / 2 mice, male hemizygous HD R6 / 2 mice and their age-matched WT littermates were given an intraperitoneal (i.p.) injection of A36 (10 mg / kg dissolved in 10% Solutol (MedChemExpress, NJ, USA)) or vehicle once a day for 5 days / week starting at 6 weeks old. At 12 weeks, the treatment was terminated.For Tau P301S mice, male hemizygous Tau P301S mice and their age-matched WT littermates were given an i.p. injection of A36 (10 mg / kg dissolved in 10% Solutol or vehicle once a day for 5 days / week starting at 4 months old.Behavioral analysis in HD mice

[0084] All behavioral analyses were conducted by a researcher blinded to genotypes and treatment groups, as we previously described. Gross locomotor activity was assessed in R6 / 2 mice and age-matched WT littermates at 6, 9 and, 12 weeks old. In an open-field activity chamber (Omnitech Electronics Inc., Columbus, OH, USA), mice were placed in the center of the chamber and allowed to explore while being tracked by an automated beam system (Vertax, Omnitech Electronics Inc.,). Horizontal and vertical distances and rearing activities were all recorded. Because R6 / 2 mice were sensitive to changes in the environment and handling, we only conducted a 1 h locomotor activity analysis for these mice and WT littermates. Hind-limb clasping was assessed by a tail suspension test once a week from 9-12 weeks old. Briefly, mice were suspended for 20 sec and hindlimb latency or four paw clasping was recorded using a scoring system: clasping over 10 s, score 3: 5-10 s, score 2; 0-5 s, score 1: and 0 s, score 0. Body weights and survival rates of R6 / 2 mice and WT littermates were recorded throughout the study period.RNA interference

[0085] Glycogen synthase kinase 3 (GSK3) siRNA (1320001) and control siRNA were purchased from Fisher Scientific (Hampton, NH, USA). Cyclin-dependent kinase (CDK5) siRNA (L-040544-00-0005) was purchased from Dharmacon (Lafayette, Co, USA). CAST short hairpin RNA (shRNA) (TRCN0000080114), Calpain I shRNA (TRCN0000030664) and control shRNA were purchased from MilliporeSigma. For GSK3 and CDK5 siRNA delivery, cells were transfected with TransIT-TKO transfection reagent (Minis Bio) and maintained for 3 days before studies. Cells were infected with lentiviral particles containing CAST shRNA or Calpain I shRNA for 2 days and selected using puromycin (2 pg / mL) (Corning) to generate a stable CAST or Calpain I knock-down line.

[0086] To knock down CAST in neurons differentiated from human iPSCs, lentiviral particles containing control or CAS T shRNA (Origene, Rockville, MD, USA) were added into medium (MOI 12:1) with polybrene (4 pg / mL) (MilliporeSigma). Medium was changed after 12 hinfection. Neurons were infected again 24 h after the first infection, and subjected for studies after 48 h.Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) and western blotting

[0087] Protein concentrations were determined by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Protein (15-25 pg) was resuspended in 5X Laemmli buffer, boiled at 100°C for 5 min, and subjected to SDS-PAGE. Separated proteins were then transferred to nitrocellulose membranes (Bio-Rad Laboratories) and blocked for 1 h in 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST). Membranes were then probed overnight with the primary antibodies. After washing three times in TBST, membranes were incubated for 1 h at room temperature with secondary anti-rabbit or anti-mouse IgG (31430 / 31460, ThermoFisher Scientific, 1:5000), followed by visualization with enhanced chemiluminescence. Representative blots have been cropped for presentation.Immunohistochemistry10088 ] Mice were deeply anesthetized and trans-cardially perfused with 4% paraformaldehyde in PBS. Brains were processed for paraffin embedding. Brain sections (10 pm, coronal) were hydrated and de-paraffinized by sequential incubation in xylene (three times), 100% ethanol (twice), 95% ethanol, 70% ethanol, 50% ethanol, and water (twice). After antigen retrieval in 0.01 M sodium citrate buffer plus 0.05% Tween 20 (pH 6.0), slides were incubated with 3% hydrogen peroxide (H2O2) in methanol to quench endogenous peroxidase. They were then treated with 5% normal goat serum (Invitrogen) in TBST for 1 h at room temperature. Sections were then incubated with anti-DARPP-32 (1710-1, Epitomics, Burlingame, CA, USA; 1:500) and anti-huntingtin protein (MAB5374, clone EM48, MilliporeSigma, 1 :500) in a humidified chamber overnight at 4°C. The next day, slides were incubated with biotin-conjugated secondary antibody (goat anti-mouse / rabbit) and streptavidin-conjugated HRP using an immunohistochemistry select HRP / DAB kit (MilliporeSigma, DAB 150) and DAB solution following manufacturer’s instructions. Slides were mounted after sequential dehydration in water, 50% ethanol, 70% ethanol, 95% ethanol, 100% ethanol, and xylene. Images were captured using a digital microscope (VHX-7000, Keyence, Osaka, Japan). Quantitation of DARPP-32 and mutant huntingtin (mtHtt) aggregates / 100 pm2immunostained area was conducted using imaged software. The same image exposure times and threshold settings wereused for all group sections. A researcher blinded to experimental groups conducted quantification analyses.Immunocy tochemi stry

[0089] Cells cultured on coverslips were washed in PBS (pH 7.4), fixed in 4% paraformaldehyde, and permeabilized in 0.1 % Triton X- 100. After incubation with 2% normal goat serum, fixed cells were incubated overnight at 4°C with the following primary antibodies: anti-Tau (T1308-1, rPeptide, Athens, Georgia, USA 1 :200), anti-DARPP-32 (ab40801, Abeam, 1:500), anti-MAP2 (4542, Cell Signaling, 1 :500), anti-GAD67 (MAB5406, MilliporeSigma, 1 :300), anti-Tubulin P 3 (Tujl) (801201, BioLegend, San Diego, CA, USA, 1:500), and anti-Tom20 (11802-1-AP, Proteintech. 1:1000). Cells were washed in PBS (pH 7.4) and incubated with Alexa Fluor goat anti-mouse / rabbit 568 or 488 secondary antibodies (ThermoFisher Scientific, 1:1000), followed by incubation with Hoechst dye (1 :10,000). Coverslips were mounted and slides were imaged by confocal microscopy (Olympus). MAP2+neurite length, TauVTuj 1+axonal length, and mitochondrial morphology were quantified using ImageJ software.Preparation of total lysates

[0090] Cells were washed in cold PBS (pH 7.4) and incubated on ice for 30 min in total lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 % Triton X- 100, protease inhibitors cocktail, and phosphatase inhibitors cocktail (MilliporeSigma)). Mouse brains were minced and homogenized in lysis buffer and placed on ice for 30 min. Cells or tissues were centrifuged at 12,000 g for 10 min at 4°C to generate total lysate supernatants.Immunoprecipitation

[0091] Cells were harvested and lysed in total lysis buffer for 30 mins on ice and centrifuged at 12,000 x g for 10 min at 4 °C. The supernatants were incubated with anti-CAST antibody overnight at 4 °C, followed by incubation with protein A / G beads (sc-2003, Santa Cruz Biotechnology) for 2 h at 4 °C. The immunoprecipitates were washed with lysis buffer three times for a total of 30 min and then subject to western blotting.Isolation of subcellular fractions

[0092] Cells were washed in cold PBS and incubated on ice for 30 min in subcellular lysis buffer (250 mM sucrose, 20 mM HEPES-NaOH, pH 7.5, 10 mM KC1, 1.5 mM MgCb, 1 mM EDTA, protease and phosphatase inhibitor cocktails (MilliporeSigma)). Mouse brains were minced and homogenized in lysis buffer and placed on ice for 30 min. Cells or tissues were disrupted 20 times by repeated aspiration through a 25-gauge needle, followed by a 30-gauge needle. Homogenates were centrifuged at 800 g for 10 min at 4°C and resulting supernatants further centrifuged at 10,000 g for 20 min at 4°C. Pellets were washed in lysis buffer and recentrifuged at 10,000 g for 20 min at 4°C. Final pellets were suspended in lysis buffer containing 1% Triton X-100 and were assigned as mitochondrial-rich lysate fractions. Supernatants were designated as cytosolic fractions.Label-free proteomics

[0093] HhdQl 11 and Q7 cells were harvested and lysed with total lysis buffer. Following lysis, the samples were processed using a filter-aided sample preparation clean-up protocol with Amicon Ultra molecular weight cut-off (MWCO) 3K filters (Millipore, Billerica, MA, USA). Samples were reduced and alkylated on filters using 10 mM dithiothreitol (Acres, Fair Lawn, NJ, USA) and 25 mM iodoacetamide (Acres), respectively, and then concentrated to a final volume of 40 pL in 8 M urea. Protein concentrations were measured using the Bradford method according to manufacturer’s instructions (Bio-Rad).

[0094] Following reduction and alkylation, total protein (10 pg) was subjected to enzymatic digestion. The urea concentration was adjusted to 4 M using 50 mM Tris (pH 8) and proteins digested using mass spectrometry-grade lysyl endopeptidase (Wako Chemicals, Richmond, VA, USA) at an enzyme / substrate ratio of 1:40 for 2 h at 37°C. Then, the urea concentration was further adjusted to 2 M using 50 mM Tris (pH 8) and lysyl peptides digested overnight 37°C in sequencing-grade trypsin (Promega, Madison, WI, USA) at an enzyme / substrate ratio of 1 :40. Finally, samples were diluted in 0.1% formic acid (Thermo Scientific, Rockford, IL, USA) before liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis.

[0095] The 300 ng of lysed protein from each of group were loaded onto a column in a 3 pL injection volume with blanks in between for a total of four LC / MS / MS runs. Data were acquired with an Orbitrap Velos Elite mass spectrometer (Thermo Electron, San Jose, CA,USA) equipped with a Waters nanoACQUITY LC system (Waters, Taunton, MA, USA). Peptides were desalted on a trap column (180 pm x 20 mm, packed with Cl 8 Symmetry, 5 pm, 100 A, Waters) and subsequently resolved on a reversed -phase column (75 pm x 250 mm nano column, packed with C18 BEH130, 1.7 pm, 130A (Waters)). LC was conducted at an ambient temperature at a flow rate of 300 nL / min using a gradient mixture of 0.1 % formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The gradient ranged from 4%- 44% solvent B over 210 min. Peptides eluting from the capillary tip were introduced into the nanospray mode at a capillary voltage of 2.4 kV. A full scan was obtained for eluted peptides in the range of 380-1800 atomic mass units, followed by 25 data-dependent MS / MS scans. MS / MS spectra were generated by collision-induced dissociation of peptide ions at a normalized collision energy of 35% to generate a series of b- and y-ions as major fragments. In addition, a 1 h wash was included between samples. The proteins were identified with Mascot (Matrix Sciences, London, UK). Key search parameters were Trypsin for enzyme; a maximum of 1 missed cleavage; peptide charge states of +2 to +3; peptide tolerance of 10 ppm; and MS / MS tolerance of 0.8 Da. Oxidation of methoinine was a variable modification. Identifications were merged, the spectra summed and then quantified using spectral counting (selecting normalized total spectra) in Scaffold version 4.4.0 (Proteome Software, Inc. Portland, Oregon). The protein probability was determined using a protein threshold of 99%, two peptides and a peptide threshold of 95%. A false discovery rate (FDR) of 0.86% was calculated using ProteinProphet algorithm for the 56,902 spectra examined. Both MASCOT and Scaffold strategies utilized the Uniprot human database from June 2016 (20,199 sequences).Quantification and statistical analysis

[0096] Sample sizes were determined by a power analysis based on pilot data collected in our laboratory or from published studies. For animal studies, we used n = 10-15 mice / group for behavioral tests, n = 3-6 mice / group for biochemical analyses, and n = 3-10 mice / group for pathology studies. In cell culture studies, each experiment was independently conducted at least three times. For animal studies, we ensured randomization and blinded evaluations. For imaging studies, a blinded observer performed quantification analyses. No samples or animals were excluded from our analysis.

[0097] Data were analyzed using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The un-paired student’s / -test was used for comparisons between two groups. Comparisons between three or more independent groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test. Comparisons of the effect of independent variables on a response variable were performed using two-way ANOVA. Survival rate was analyzed by Log-rank (Mantel-Cox) test. All values are reported as the mean ± standard error of the mean (SEM). Data are representative of at least three independent experiments. Statistical parameters were presented in each figure legend. We considered p < 0.05 as statistically significant.

[0098] The complete disclosure of all patents, patent applications, and publications, and electronically available materials cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. In particular, while various theories are presented describing possible mechanisms through with the compounds are effective, the compounds are effective regardless of the particular mechanism employed and the inventors are therefore not bound by theories described herein. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

CLAIMSWhat is claimed is:

1. A method of treating or preventing a neurodegenerative disease in a subject in need thereof, comprising administering a therapeutically effective amount of a mitochondrial enhancer according to formula I:to the subject, wherein R1is a halogen, cycloalkyl, or hetercycloalkyl group, R2-R are hydrogen or halogen, X is a methylene, an oxygen, or an N-H group, Y is a heterocycloalkyl or hctcroaryl group, and Z is a hctcrocycloalkyl or hctcroaryl group, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the neurodegenerative disease is Alzheimer’s disease or Huntington’s disease.

3. The method of claim 1, wherein Y is a morpholine or cyanopyridine group.

4. The method of claim 1, wherein R1is a piperidine group.

5. The method of claim 1, wherein Z is a morpholine group.

6. The method of claim 1, wherein R1and R3are halogen, and R2, R4, and R5are hydrogen.

7. The method of claim 1, wherein the mitochondrial enhancer has the structure:

8. The method of claim 1, wherein the mitochondrial enhancer is administered together with a pharmaceutically acceptable carrier.

9. The method of claim 1, wherein the subject is human.

10. A mitochondrial enhancer having a structure according to formula I:wherein R1is a halogen, cycloalkyl, or heterocycloalkyl group, R2-R5are hydrogen or halogen, X is a methylene, an oxygen, or an N-H group, Y is a heterocycloalkyl or heteroaryl group, and Z is a heterocycloalkyl or heteroaryl group, or a pharmaceutically acceptable salt thereof.

11. The mitochondrial enhancer of claim 10, wherein Y is a morpholine or cyanopyridine group.

12. The mitochondrial enhancer of claim 10, wherein R1is a piperidine group.

13. The mitochondrial enhancer of claim 10, wherein Z is a morpholine group.

14. The mitochondrial enhancer of claim 10, wherein R1and R3are halogen, and R2, R4, and R5are hydrogen.

15. The mitochondrial enhancer of claim 1, wherein the mitochondrial enhancer has the structure:

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