WO2019094830A1 - Agents de modulation de mitofusine et leurs méthodes d'utilisation - Google Patents

Agents de modulation de mitofusine et leurs méthodes d'utilisation Download PDF

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Publication number
WO2019094830A1
WO2019094830A1 PCT/US2018/060177 US2018060177W WO2019094830A1 WO 2019094830 A1 WO2019094830 A1 WO 2019094830A1 US 2018060177 W US2018060177 W US 2018060177W WO 2019094830 A1 WO2019094830 A1 WO 2019094830A1
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mfn2
mitochondrial
mitofusin
group
triazol
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PCT/US2018/060177
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English (en)
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II Gerald W. DORN
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Washington University
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Priority claimed from PCT/US2018/028514 external-priority patent/WO2018200323A1/fr
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Publication of WO2019094830A1 publication Critical patent/WO2019094830A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/05—Hydrolases acting on acid anhydrides (3.6) acting on GTP; involved in cellular and subcellular movement (3.6.5)
    • C12Y306/05002—Small monomeric GTPase (3.6.5.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • the present disclosure generally relates to compositions and methods for treating mitochondria-associated diseases, disorders, or conditions. Also provided are methods for screening compositions.
  • One aspect of the present disclosure provides for a method of treating a mitochondria-associated disease, disorder, or condition.
  • the method comprises administering to a subject a therapeutically effective amount of a composition comprising one or more of a mitofusin modulating agent or a pharmaceutically acceptable salt thereof, wherein the mitofusin
  • modulating agent is a mitofusin agonist; the mitofusin modulating agent regulates mitochondrial fusion.
  • Another aspect of the present disclosure provides for a method of modulating mitofusin in a subject in need thereof.
  • the method comprises administering to a subject a composition comprising a mitofusin modulating agent or a pharmaceutically acceptable salt thereof; wherein, the mitofusin modulating agent is a mitofusin agonist; the mitofusin modulating agent regulates mitochondrial fusion; the subject has a mitochondria-associated disease, disorder, or condition; or the mitofusin modulating agent is not a compound of TABLE 4.
  • Another aspect of the present disclosure provides for a method of enhancing mitochondrial trafficking in nerve axons in a subject in need thereof.
  • the method comprises administering to a subject a composition comprising a mitofusin modulating agent or a pharmaceutically acceptable salt thereof; wherein, the mitofusin modulating agent is a mitofusin agonist; the mitofusin modulating agent regulates mitochondrial fusion; the subject has a mitochondria- associated disease, disorder, or condition; or the mitofusin modulating agent is not a compound of TABLE 4.
  • the mitofusin modulating agent is a small molecule mimetic of a Mfn2 peptide-peptide interface.
  • the mitofusin modulating agent has substantially similar functional potency and specificity of both 1-[2- (benzylsulfanyl)ethyl]-3-(2-methylcyclohexyl)urea (Cpd A) and 2- ⁇ 2-[(5-cyclopropyl-4- phenyl-4H-1,2,4-triazol-3-yl)sulfanyl]propanamido ⁇ -4H,5H,6H- cyclopenta[b]thiophene-3-carboxamide (Cpd B); targets at least two phosphorylated forms of MFN; or stimulates mitofusin activity (e.g., fusion and trafficking).
  • the mitofusin modulating agent enhances mitochondrial trafficking in nerve axons; increases microsomal stability; corrects cell and organ dysfunction caused by primary abnormalities in mitochondrial fission or fusion; reverses mitochondrial defects (e.g., dysmorphometry); restores, activates, regulates, modulates, promotes, or enhances the fusion, function, tethering, transport, trafficking (e.g., axonal mitochondrial trafficking), mobility, or movement of mitochondria (in, optionally, a nerve or a neuron); enhances mitochondrial elongation or mitochondrial elongation aspect ratio; disrupts intramolecular restraints in Mfn2; allosterically activates Mfn2; corrects mitochondrial dysfunction and cellular dysfunction; repairs defects in neurons with mitochondrial mutations; or targets Mfn1 or Mfn2.
  • the mitofusin modulating agent is selected from
  • R 1 is selected from the group consisting of C 1-8 alkyl, C 1-8 alkyl substituted with S, S, thiophene, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, thiophene, and thiophene carboxamide
  • R 2 is selected from the group consisting of C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, imidazole, thiophene, thiophene carboxamide, and triazole
  • R3 is absent or selected from the group consisting of hydrogen (H) and C1-8 alkyl
  • R4 is absent or selected form the group consisting of hydrogen (H) and C1-8 alkyl
  • R 5 is selected from the group consisting of C 1-8 alkyl, C 1-8 alkyl substituted with S, S, thiophene, C 3-8 cycloalkyl, C 3
  • the mitofusin modulating agent is selected from
  • R 2 is selected from the group consisting of
  • R 3 is selected from the group consisting of hydrogen (H) and C 1-8 alkyl
  • R 4 is selected form the group consisting of hydrogen (H) and C 1-8 alkyl
  • A is a bond, S, SO, SO 2 , C, or O;
  • R 1 , R 2 , R 3 , or R 4 are optionally substituted by one or more of: acetamide, C 1-8 alkoxy, amino, azo, Br, C 1-8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, or thiophene; and optionally further substituted with one or more acetamide, alkoxy, amino, azo, Br, C 1- 8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, or thioph
  • the compound is selected from the group consisting of:
  • the compound is selected from the group consistin of:
  • R 1 is selected from the group consisting of C 1-8 alkyl, C 1-8 alkyl substituted with S, S, thiophene, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, thiophene, and thiophene carboxamide
  • R 2 is selected from the group consisting of C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, imidazole, thiophene, thiophene carboxamide, and triazole
  • R3 is absent or selected from the group consisting of hydrogen (H) and C1- 8 alkyl
  • R4 is absent or selected form the group consisting of hydrogen (H) and C1-8 alkyl
  • R 5 is selected from the group consisting of C 1-8 alkyl, C 1-8 alkyl substituted with S, S, thiophene, C 3-8 cycloalkyl, C
  • R 1 and R 2 form a cyclic group
  • R 1 and R 4 form a cyclic group
  • R 2 and R 3 form a cyclic group
  • R 4 and R 3 form a cyclic group
  • R 8 and R 7 form a cyclic group, wherein, the bicyclononanone optionally comprises one or more N atoms; or formula (I), (II), or (III) is not a compound of TABLE 4,
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , or R 9 are optionally substituted by one or more of: acetamide, C 1-8 alkoxy, amino, azo, Br, C 1-8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, or thiophene; or optionally further substituted with one or more acetamide, alkoxy, amino, azo, Br, C 1-8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N
  • the compound is of formula
  • R 1 is selected from the group consisting of
  • R 2 is selected from the group consisting of ,
  • R 3 is selected from the group consisting of hydrogen (H) and C 1-8 alkyl
  • R 4 is selected form the group consisting of hydrogen (H) and C 1-8 alkyl
  • A is a bond, S, SO, SO 2 , C, or O;
  • R 1 , R 2 , R 3 , or R 4 are optionally substituted by one or more of: acetamide, C 1-8 alkoxy, amino, azo, Br, C 1-8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, or thiophene; and optionally further substituted with one or more acetamide, alkoxy, amino, azo, Br, C 1- 8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, or thioph
  • the compound is selected from:
  • the com ound is selected from:
  • the compound is a small molecule mimetic of a Mfn2 peptide-peptide interface.
  • the compound targets at least two phosphorylated forms of MFN; enhances mitochondrial trafficking in nerve axons; increases microsomal stability; corrects cell and organ dysfunction caused by primary abnormalities in mitochondrial fission or fusion; reverses mitochondrial defects (e.g., dysmorphometry); restores, activates, regulates, modulates, promotes, or enhances the fusion, function, tethering, transport, trafficking (e.g., axonal mitochondrial trafficking), mobility, or movement of mitochondria (in, optionally, a nerve or a neuron); enhances mitochondrial elongation or mitochondrial elongation aspect ratio; disrupts intramolecular restraints in Mfn2; allosterically activates Mfn2; corrects mitochondrial dysfunction and cellular dysfunction; repairs defects in neurons with mitochondrial mutations; or targets Mfn1 or Mfn2.
  • composition comprising a compound of formula (I), (II), or (III), optionally in combination with one or more therapeutically acceptable diluents or carriers.
  • the pharmaceutical composition comprises a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises a at least one compound selected from the group consisting of neuroprotectants, antiparkinsonian drugs, amyloid protein deposition inhibitors, beta amyloid synthesis inhibitors, antidepressants, anxiolytic drugs, antipsychotic drugs, anti-amyotrophic lateral sclerosis drugs, anti-Huntington’s drugs, anti-Alzheimer’s drugs, anti-epileptic drugs, or steroids.
  • Yet another aspect of the present disclosure provides for a method of treating a mitochondria-associated disease, disorder, or condition in a subject comprising administering to the subject a therapeutically effective amount of a mitofusin modulating agent comprising the compound of formula (I), (II), or (III).
  • the subject is diagnosed with or is suspected of having a mitochondria-associated diseases, disorders, or conditions.
  • the mitochondria-associated disease, disorder, or condition is selected from one or more of the group consisting of: a CNS or PNS injury or trauma, such as trauma to the CNS or PNS, crush injury, spinal cord injury (SCI), traumatic brain injury, stroke, optic nerve injury, or related conditions that involve axonal disconnection; a chronic neurodegenerative condition wherein mitochondrial fusion, fitness, or trafficking are impaired; a disease or disorder associated with mitofusin 1 (Mfn1) or mitofusin 2 (Mfn2) or mitochondrial dysfunction, fragmentation, or fusion; dysfunction in Mfn1 or Mfn2 unfolding; mitochondria dysfunction caused by mutations; a degenerative neurological condition, such as Alzheimer’s, Parkinson’s, Charcot Marie Tooth Disease, or Huntington’s diseases; or hereditary motor and sensory neuropathy, autism, autosomal dominant optic atrophy (ADOA), muscular dystrophy, Lou Gehrig’s disease, cancer, mitochondrial myopathy, Diabetes mellitus and deafness (DA).
  • NARP Neuropathy, ataxia, retinitis pigmentosa, and ptosis
  • MNGIE Myoneurogenic gastrointestinal encephalopathy
  • MRF Myoclonic Epilepsy with Ragged Red Fibers
  • Mitochondrial myopathy encephalomyopathy
  • lactic acidosis stroke-like symptoms
  • MELAS stroke-like symptoms
  • MNGIE neurogastrointestinal encephalomyopathy
  • PDCD/PDH pyruvate dehydrogenase complex deficiency
  • FIG.1A and FIG.1B are a series of hypothetical structures of human MFN2 modeled using I-TASSER. (top) MFN2 modeled in a closed configuration based on structural homology with Homo sapiens MFN1 and
  • FIG.1C and FIG.1D are a set of ribbon models depicting the human Mfn2 protein in its folded (inactive) conformation. Major domains are labeled on top. Note the magnified image displaying the interaction of the first and second heptad repeat domains, HR1 and HR2 is on the bottom (see e.g., Example 1).
  • FIG.2A, FIG.2B, FIG.2C, FIG.2D, FIG.2E, FIG.2F, FIG.2G and FIG.2H are a series of images and graphs showing MFN2 Ser 378 phosphorylation by PINK1 regulates mitochondrial fusion.
  • FIG.2A Amino acid sequence surrounding fusion-promoting MFN2 peptide (SEQ ID NO:6). Side chain characteristics (H, hydrophobic; +, basic; -, acidic) are above.
  • FIG.2B Mitochondrial fusion stimulated by N- and C-terminal minipeptides. Aspect ratio is mitochondrial long axis/short axis.
  • FIG.2C Alanine (A) scanning of minipeptide 374-384 fusion activity.
  • FIG.2D Ser378 substitution analysis of minipeptide 374-384 fusion activity. p values in D and E are vs parent minipeptide 374-384 (ANOVA).
  • FIG.2F Binding of Asp378 minipeptide to HR2 target sequence before (left) and after (right) Ala substitution for putative interacting amino acids.
  • FIG.2G Ion chromatograms from assigned MFN2 Ser378 phosphopeptide fragment ions after incubation with PINK1 kinase (top) and stable isotope-labeled synthetic counterpart (bottom); proportional intensities are in adjacent stack plots.
  • H Mitochondrial fusion promoted by MFN2 Ser378 mutants with and without PINK1 kinase; immunoblot of protein expression at bottom. p values are by ANOVA.
  • FIG.3A and FIG.3 B shows the purification of mitofusin agonist compounds A and B.
  • At the top are high performance liquid chromatography and mass spectra of compounds as they were obtained from the commercial vendor. On the bottom are spectra after in-house purification.
  • Cpd A expected m/z 306.18, exact mass found 307.3 [M + H] + ;
  • Cpd B expected m/z 453.15, exact mass found 454.3 [M + H] + .
  • FIG.4A, FIG.4B, FIG.4C, FIG.4D, FIG.4E and FIG.4F are a series of chemical structures and a bar graph that shows the structure and function of compounds A and B, more specifically, the mitofusin-dependent mitochondrial elongation provoked by prototype Mfn agonist peptide mimetics (compounds A and B).
  • FIG.4A shows 3D structures of 1-[2-(benzylsulfanyl)ethyl]-3-(2- methylcyclohexyl)urea, designated compound A, and 2- ⁇ 2-[(5-cyclopropyl-4-phenyl- 4H-1,2,4-triazol-3yl)sulfanyl] propanamido ⁇ -4H,5H,6H-cyclopenta[b]thiophene-3- carboxamide, designated compound B (FIG.4B).
  • FIG.4E is a schematic of compound B and the moieties subjugated to iterative modification.
  • FIG.4F is a bar graph showing the results of activity studies (increase in aspect ratio is mitochondrial elongation, reflecting increased Mfn-mediated mitochondrial fusion. Structures of derivatives are shown below the bar graph.
  • FIG.5A, FIG.5B, FIG.5C, and FIG.5D are a series of bar graphs, structures, graphs, and images showing that small molecule HR1 MP374-384 mimetics can be mitofusin agonists.
  • FIG.5B Representative confocal images from studies in (FIG.5A). Mitochondria were visualized with MitoTracker Orange. Cell viability was assessed simultaneously with mitochondrial aspect ratio - live cells have green cytoplasm (calcein AM) and dead cells lack calcein staining and have purple nuclei (red ethidium homodimer overlying blue Hoechst). Scale bars are 10 ⁇ m.
  • FIG.5C Initial dose-response relations of five fusogenic compounds from screening in (FIG.5A).
  • EC 50 values (indexed to the 100% maximal response elicited by the most effective compound, B1) are shown for the agonists with strong fusion- promoting activity; mean ⁇ SEM of 3 independent studies for each compound.
  • FIG. 5D Competition of the HR1 minipeptide at its MFN2 HR2 binding site by five fusogenic compounds from (FIG.5A).
  • IC 50 values are shown for agonists with >50% displacement (mean ⁇ SEM of 6 independent experiments per compound).
  • FIG.6A, FIG.6B, FIG.6C, FIG.6D and FIG.6E are a series of line and bar graphs, images, and structures showing compounds A and B synergistically promote mitochondrial fusion by acting upon different mitofusin conformational states and displaying the EC50 values of compounds A and B and a key phosphorylation site in Mfn2.
  • FIG.6A shows A+B synergism. More specifically, that the EC50 values of compounds A and B were each 100-200 nM. Note that when added in equal amounts, compounds A and B synergistically promoted mitochondrial elongation, with a combined EC50 of ⁇ 40 nM and a ⁇ 25% greater maximal increase in mitochondrial aspect ratio.
  • FIG.6B is a bar graph showing a negative charge conferred by Ser378 phosphorylation or Asp (D) substitution is essential for mini- peptide fusion promoting activity and shows how a S378D mutation, which mimics phosphorylation of this site, influenced Mfn2 conformation and function similarly to HR1 peptide 374-384 (see e.g., Example 2).
  • FIG.6C is a series of images showing representative confocal micrographs of cells treated with mini-peptides in compound B.
  • FIG.6D and FIG.6E are a series of images showing the structural consequences of Ser378 phosphorylation on the Mfn2 HR1-HR2 interacting face; His 380 rotates out and Leu379 rotates in.
  • FIG.7A, FIG.7B and FIG.7C are a series of structures and graphs showing the evaluation of chimeric small molecule mitofusin agonists.
  • FIG.7A Structures of compounds A and B and their chimeras.
  • FIG.7B Dose-response of compounds in (A) to promote mitochondrial fusion (increase in aspect ratio) in MFN2-deficient MEFs. Data for compounds A and B and chimera B-A/l in FIG.11B are re-plotted here for comparison.
  • FIG.7C Comparison of EC50 values calculated from studies in panel B. p values are from ANOVA with Tukey’s test.
  • FIG.8A and FIG.8B are a series of immunofluorescence images from cultured mouse neurons. Neurons with the human Charcot Marie Tooth disease mutation, Mfn2 T105M, exhibited increased mitochondrial fragmentation and neuronal pathology compared to control. Note how administration of compounds repaired the defects in mutant neurons (see e.g., Example 2).
  • FIG.9A, FIG.9B, FIG.9C, FIG.9D, FIG.9E, FIG.9F, FIG.9G and FIG.9H are a series of bar graphs and images showing mitofusin agonists correct mitochondrial damage induced by nonfunctioning MFN2 mutants by activating endogenous mitofusins.
  • FIG.9B Same as (FIG.9A) in MFN1 +/+ , MFN2 -/- cells.
  • FIG.9C Representative mitochondrial pathology in cultured neonatal mouse neurons expressing MFN2 R94Q and correction by mitofusin agonists. Immunoblot showing MFN2R94Q expression in individual mouse pups is above. Scale bars are 21 mm; expanded views are from white squares.
  • FIG.9D Group data for studies in (FIG.9C).
  • FIG.9E Results of similar studies in cultured neonatal mouse neurons expressing MFN2 T105M.
  • FIG.9F is a series of sequences and plots and bar graphs showing the identification of critical amino acids for Mfn antagonist peptido-mimetic design. Top, parent peptide (Ref 11) and depiction of N- and C- terminal fragments.
  • FIG.9G is a bar graph and a series of chemical compounds showing screening results and structures of mitofusin antagonists.
  • FIG.9H is a series of images showing intravenous mitofusin antagonist decreases brain infarct size after ischemia- reperfusion.
  • FIG.10 is illustration showing Mfn agonist peptide binding and its displacement by compounds A and B.
  • the schematic demonstrates components of the system, depicting FITC labeled peptide binding to its immobilized target (top) and displacement of the FITC peptide by competing small molecule.
  • FIG.11A, FIG.11B, FIG.11C, FIG.11D, FIG.11E, and FIG.11F are a series of images and graphs showing small molecule mimetics of MFN2 HR1 amino acid side chains that interact with HR2 are mitofusin agonists.
  • FIG.11A (top) Three dimensional representations of minipeptide conformations driven by Ser378 phosphorylation, and (bottom) their respective small molecule mimetics.
  • FIG.11D Restoration of MFN2 T105M-impaired mitochondrial fusion in MEFs by mitofusin agonists.
  • FIG.11E Selectivity of a class A, but not a class B, mitofusin agonist for Ser 378 -phosphorylated MFN2.
  • FIG.11F Impaired basal function, but normal proportional agonist responsiveness, of MFN2 mutations altering HR1-HR2 interacting amino acids.
  • FIG.12A, FIG.12B, FIG.12C and FIG.12D are a series of drawings that diagram the working model of Mfn2 conformation and function and Mfn folding/unfolding measured by FRET.
  • FIG.8 shows that HR1 and HR2 domain interaction can result in a folded conformation in which tethering to adjacent Mfn proteins is unfavorable. Disruption of HR1 and HR2 domains can result in an unfolded conformation in which tethering is favorable.
  • FIG.12A and FIG.12B are illustrations of change in FRET signaling evoked by Mfn conformation.
  • FIG.12D is a graph of a representative experiment with changes in ⁇ 80-275 Mfn2 FRET signal provoked by Mfn antagonist MP2 and agonist MP1.
  • This novel Forster resonance energy transfer (FRET) assay screens compounds that induce unfolding of a fluorescently tagged Mfn2 construct. Note that both mini-peptides 1 and 2 influenced the FRET signal, suggesting that they induced Mfn2 conformational changes (see e.g., Example 4).
  • FIG.13A, FIG.13B, FIG.13C, FIG.13D, FIG.13E, FIG.13F, FIG. 13G, FIG.13H, FIG.13I and FIG.13J shows a multi-species alignment of MFN2 amino acid sequence. Black highlighting shows identity with human MFN2 protein. (SEQ ID NOs: 6, and 29-65).
  • FIG.14A, FIG.14B and FIG.14C are a homology plot of MFN2 amino acid sequence by functional domain. Positions of HR1 MP374-384 (“fuse”) and its HR2 interacting site (“Binding”) are shown on exploded views below.
  • FIG.15A and FIG.15B is a series of images and a bar graph showing MFN2 Ser378 charge status determines fusion-promoting activity of HR1 MP374-384. Ser378 substitution analysis of mitochondrial fusion promoted by HR1 MP374-384. Representative confocal images of MitoTracker Green/TMRE (red) stained live cells are on the left; scale bars are 10 ⁇ m. Group mean data from FIG. 2D are to the right; p values are by ANOVA with Tukey’s post hoc comparison.
  • FIG.16A, FIG.16B, FIG.16C, FIG.16D, FIG.16E, FIG.16F and FIG.16G are a series of NMR spectroscopy images and calculated structures. NMR spectroscopy suggests a structural mechanism for effects of Ser378 phosphorylation on HR1372-384 minipeptide fusogenic function.
  • FIG.16A, FIG.16B Amide proton regions of 2D NOESY spectra of Ala371 to Arg384 fragment of hMFN2. left– unphosphorylated Ser378 peptide; right– peptide synthesized with phosphorylated Ser-378. Sequential cross peaks between amide groups indicative of ⁇ -helical secondary structure are labeled.
  • FIG.16C, FIG.16D Overlaid 15 N- 1 H heteronuclear single quantum coherence spectra of minipeptide backbone amides (bold highlights on covalent wire-model to the left). Red is Ser378 peptide; green is (p)-Ser378 peptide. # marks the positions of Ser378 and (p)-Ser378.
  • the amide signals for amino acids 379-382 shifted down-field (i.e. to higher values) after phosphorylation, as observed when amides within peptides form or strengthen hydrogen bonds.
  • FIG.16E PepFold3 modeling of the HR1 minipeptide shows how different backbone structure provoked by Ser378 phosphorylation (see panel B) can alter Leu379 and His 380. * in (FIG.16B) and (FIG.16D) mark amino acids with the greatest changes between Ser378 and (p)-Ser378 peptides.
  • FIG.17A and FIG.17B show calculated structures from the modeling of HR1 MP374-384 conformation before (top) and after (bottom) S378 phosphorylation.
  • FIG.18A, FIG.18B, FIG.18C, FIG.18D, FIG.18E, FIG.18F, FIG. 18G and FIG.18H show mutagenesis analysis of MFN2-function based on Ser378 phosphorylation status and integrity of Met376 and His380 that are spatially regulated by Ser378 phosphorylation.
  • Group data and representative confocal images showing mitochondrial aspect ratio in mitofusin deficient cells (MFN1-/-, MFN2-/- MEFs) infected with adnoviri expressing ⁇ -galactosidase (negative control), wild-type (WT) MFN2 (positive control), or different single amino acid MFN2 mutants.
  • Fusogenic function was impaired in pseudo-phosphorylated MFN2 Ser378Asp (S378D) and alanine-substituted MFN2 Met376Ala (M376A) and His380Ala (H380A); non-phosphorylatable MFN2 Ser378Ala (S378A) and MFN2 Val372Ala (V372A, which is not in the HR1-HR2 interacting domain) retained full activity.
  • p values are by ANOVA with Tukey’s post hoc comparison. MEFs were stained as described in FIG. 15 legend. Scale bar is 10 ⁇ m.
  • FIG.19A, FIG.19B, FIG.19C, FIG.19D, FIG.19E, FIG.19F, FIG. 19G, FIG.19H, FIG.19I and FIG.19J are a series of high-resolution tandem mass spectra of peptides from a tryptic digest of PINK1-treated recombinant human MFN2.
  • the spectra of the phosphopeptide with the Ser-378 phosphorylation site (FIG.19A, FIG.19B), a stable isotope-labeled synthetic phosphopeptide (FIG.19C, FIG.19D), and the non-phosphorylated peptide (FIG.19E) are shown from a 4-hour in vitro PINK1 phosphorylation experiment.
  • FIG.19F and FIG.19G are like (FIG.19A) and (FIG.19D) after an overnight period for PINK1 phosphorylation.
  • the m/z values for the assigned ions are highlighted in the adjacent ion tables.
  • FIG.20A, FIG.20B, FIG.20C, FIG.20D and FIG.20E are a series of high-resolution mass spectra of PINK1-phosphorylated recombinant human MFN2 demonstrating phosphorylation of Thr111 and Ser442. These spectra were obtained in the study shown in FIG.19D and E. m/z values for assigned fragmentation ions are shown to the right.
  • FIG.21A, FIG.21B, FIG.21C, FIG.21D and FIG.21E are a series of high-resolution tandem mass spectra of peptides from tryptic digest of GRK- treated recombinant human MFN2.
  • FIG.21A Representative non-matching spectrum from the elution window of the Ser-378 phosphopeptide.
  • FIG.21B Representative non-matching spectrum from the elution window of the Ser-378 phosphopeptide.
  • FIG.22 is a series of representative live-cell confocal images and a bar graph from studies described in FIG.2H. Mitochondria of MFN1-/-, MFN2-/- MEFs infected with adenoviri expressing MFN2 mutants with or without adeno- PINK1 kinase were co-stained with MitoTracker Green (green) and TMRE (red); nuclei are stained blue with Hoechst. Scale bars are 10 ⁇ m. Quantitative group mean data to the right are reproduced from FIG.2H for comparison.
  • FIG.23 is a series of images and a bar graph showing effects of MFN2 mutations that prevent or mimic Ser378 phosphorylation on mitochondrial fusion measured as content exchange.
  • N 3 independent studies; p values are by ANOVA with Tukey’s post hoc comparison.
  • FIG.24A and FIG.24B show functional screening for fusogenic activity of mitofusin agonist pharmacophores.
  • A Mitochondrial fusogenicity measured as aspect ratio of MFN2 null MEFs after overnight treatment with 1 mM indicated library compound. Chemical details, structures, and commercial sources of these compounds are in TABLE 4.
  • Mock DMSO vehicle control. Horizontal dotted line indicates baseline value. Cells treated with 5 mM mitofusin agonist peptide HR1 367-384 (positive control) had aspect ratios of ⁇ 6.
  • correlation of rank order for initial model fit vs actual fusogenicity (r 0.214).
  • Red dots are compounds A10 and B1 that ranked 4 th and 2 nd for fusogenicity, but 22 nd and 31 st , respectively, for fit to the original pharmacophore model.
  • FIG.24B Cytotoxicity measured by live-dead assay. Compounds are ranked by fusogenicity as in A. Means ⁇ SEM of 3
  • FIG.25A, FIG.25B, and FIG.25C shows functional validation and dose-response relations of candidate fusogenic small molecules.
  • FIG.25A Chemical structures of 4 top candidate fusogenic compounds from initial screening (see e.g., FIG.24).
  • FIG.25B Dose relations with representative images of vehicle and 1 mM treated Mfn2 null MEFs for each of the compounds, only 3 of which were true positives. Cells are stained with Mitotracker orange, calcein AM (green; alive) and ethidium homodimer (red nucleus; dead). There are no dead cells. EC 50 values are provided for true positives; D9 showed no true fusogenic activity. Scale bars are 10 microns. Dose-response curves are means ⁇ SEM of 3 independent experiments.
  • FIG.25C Schematic depiction of pharmacophore model fit for the 3 true positive fusogenic compounds.
  • FIG.26A, FIG.26B, FIG.26C and FIG.26D are a graph and image showing the synergistic effects of a class A and class B mitofusin agonist.
  • Mitochondrial elongation in MFN2-deficient MEFs stimulated by equimolar concentrations of mitofusin agonists A and B.
  • Dose- response curve on the left is from 6 independent experiments. Peak aspect ratio achieved with A+B is ⁇ 25% greater than with either agonist alone (compare to SF10C).
  • Representative live-cell confocal images are on right. Scale bar is 10 mm.
  • FIG.27A, FIG.27B and FIG.27C are a series of graphs and an image showing the functional evaluation of structurally diverse mitofusin agonists.
  • FIG.27B Effects of cpds A and B or chimera B-A/l (1 ⁇ M) on dynamin- mediated endocytosis of Alexa-Fluor 594 Dextran. Dynasore is a dynamin inhibitor.
  • FIG.27C Cell viability assessed after overnight exposure to indicated
  • FIG.28A, FIG.28B, FIG.28C, FIG.28D, FIG.28E, FIG.28F and FIG.28G are a series of graphs and images showing mitofusin agonists restore axonal mitochondrial trafficking suppressed by CMT2A mutant MFN2 T105M.
  • FIG. 28A- FIG.28C Chimera B-A/l effects on mitochondrial mobility (FIG.28A), function (FIG.28B), and morphology (FIG.28C) in cultured CMT2A MFN2 T105M mouse neurons.
  • FIG.28D Kymograph of mitochondrial trafficking in a Ctrl mouse sciatic nerve.
  • FIG.28E Serial kymographs of mitochondria in a MFN2 T105M mouse sciatic nerve before and after chimera B-A/l.
  • FIG.28F Quantitative data for sciatic nerve mitochondrial motility studies.
  • FIG.28G Size of motile and static
  • FIG.29A FIG.29B and FIG.29C shows in vitro mouse
  • FIG.30 is a series of images showing mitofusin agonist chimera B- A/l reverses mitochondrial abnormalities induced by CMT2A mutant MFN2 T105M in cultured mouse neurons. Representative confocal images of living mouse neurons expressing MitoGFP and stained with TMRE and Hoescht from experiments reported in FIG.28B and FIG.28C. Scale bars are 21 ⁇ m; expanded views are from white squares. MFN2 T105M was induced by addition of adeno-Cre.
  • FIG.31 shows mitochondrial mobility in a neuronal axon of a control mouse sciatic nerve. Blue arrows represent the mitochondrial transport in the nerve.
  • FIG.32A and FIG.32B shows mitochondrial mobility in axons of a MFN2 T105M mouse sciatic nerve before and at serial 15 minute periods after application of chimera B-A/l. Blue arrows represent the mitochondrial transport in the nerve.
  • FIG.33 shows the synthetic route for preparation of Chimera B-A/l (compound 5).
  • FIG.34A and FIG.34B show RP-HPLC and HRMS of newly synthesized chimera B-A/l.
  • FIG.34A HPLC spectrum of chimera B-A/l. From top to bottom: UV Absorbance at 215nm; UV Absorbance at 254 nm; complete ionization mass selective detector (MSD) spectrum; evaporative light scattering detection spectrum. Chimera B-A/l was 99.99% pure.
  • FIG.34B HRMS chromatogram of compound B-A/l (C21H29N5OS) shows exact mass: [M + H] + : 400.2.
  • FIG.35A and FIG.35B show the proton and carbon-13 NMR of newly synthesized chimera B-A/l.
  • FIG.35A Full 1 H NMR spectrum (400 MHz) of compound B-A/l (DMSO-d 6 solvent) and expanded view of region ⁇ 0.0– 4.0 PPM.
  • FIG.35B 13 C NMR spectrum (126 MHz) of compound B-A/l (CDCl 3 solvent).
  • FIG.36 shows the synthetic route for preparation of chimera B-A/s (compound 3).
  • FIG.37A and FIG.37B show RP-HPLC and HRMS of newly synthesized chimera B-A/s.
  • FIG.37A HPLC spectrum of compound B-A/s. From top to bottom: UV Absorbance at 215nm; UV Absorbance at 254 nm; complete ionization MSD spectrum; evaporative light scattering detection spectrum. Chimera B-A/s was 99.99% pure.
  • FIG.37B HRMS chromatogram of compound B-A/s (C21H28N4OS) shows exact mass found: [M + H] + : 385.2.
  • FIG.38A and FIG.38B show the proton and carbon-13 NMR of newly synthesized chimera B-A/s.
  • FIG.38A Full 1 H NMR spectrum (500 MHz) of compound B-A/s (DMSO-d 6 solvent) and expanded view of region ⁇ 0.5– 4.8 PPM.
  • FIG.38B 13 C NMR spectrum (126 MHz) of compound B-A/s (DMSO-d 6 solvent) and expanded view of region ⁇ 5– 60 PPM.
  • FIG.39 shows the synthetic route for preparation of chimera A-B/l (compound 5).
  • FIG.40A and FIG.40B show RP-HPLC and HRMS of newly synthesized chimera A-B/l.
  • FIG.40A HPLC spectrum of newly synthesized chimera A-B/l. From top to bottom: UV Absorbance at 215nm; UV Absorbance at 254nm; complete ionization MSD spectrum; evaporative light scattering detection spectrum. Chimera A-B/l was 97.56% pure.
  • FIG.40B HRMS chromatogram of compound A- B/l (C18H21N3O2S2) shows exact mass found: [M + H] + : 376.0.
  • FIG.41A and FIG.41B show Proton and carbon-13 NMR of newly synthesized chimera A-B/l.
  • FIG.41A Full 1 H NMR spectrum (400 MHz) of newly synthesized chimera A-B/l (DMSO-d 6 solvent) and expanded view of region ⁇ 2.0– 4.1 PPM.
  • FIG.41b 13 C NMR spectrum (126 MHz) of chimera A-B/l (DMSO solvent).
  • FIG.42 is a schematic showing the synthetic route for preparation of chimera A-B/s (compound 3).
  • FIG.43A and FIG.43B show RP-HPLC and HRMS of newly synthesized chimera A-B/s.
  • FIG.43A HPLC spectrum of compound A-B/s. From top to bottom: UV Absorbance at 215 nm; UV Absorbance at 254 nm; complete ionization MSD spectrum; evaporative light scattering detection spectrum. Chimera A-B/s was 98.76% pure.
  • FIG.43B HRMS chromatogram of chimera A-B/s
  • FIG.44A and FIG.44B show the proton and carbon-13 NMR spectra of newly synthesized chimera A-B/s.
  • FIG.44A Full 1 H NMR spectrum (400 MHz) of chimera A-B/s (DMSO-d 6 solvent) and expanded view of region ⁇ 5.7– 8.2 PPM.
  • FIG.44B 13 C NMR spectrum (126 MHz) of chimera A-B/s (DMSO-d 6 solvent).
  • FIG.45 shows the initial PK studies of Chimera B-A/I, a.k.a.
  • Regeneurin-S In vitro pharmacokinetic profiling of Regeneurin-S reveals rapid degradation by liver mcirosomes. Chimera B-A/l from Rocha, et al Science 2018 was designated Regeneurin-S. Shown is its chemical structure and results of three independent pharmacokinetic (PK) assays performed months apart.
  • PK pharmacokinetic
  • FIG.46 is a series of structures showing structural considerations for chemical evolution of the lead mitofusin agonist.
  • (top left) Structural model of human Mfn2 HR1367-384 agonist peptide (ribbon) in context of Mfn2 HR1 domain from which it was derived (space-filling; from Franco Nature 2016); side chains of HR1-HR2 interacting amino acids Val372, Met376, and His380 are depicted.
  • (top right) Structure of HR1367-384 peptidomimetic Regeneurin-S (chimera B-A/l from Rocha Science 2018) is shown mimicking function-critical side chains from HR1367- 384. Modeled using Chimera UCSF.
  • (bottom) Functional groups of Regeneurin-S are depicted as conceived for chemical engineering: methylated cyclohexyl
  • FIG.47 is a series of structures and a graph showing backbone sulfur modifications or substitutions do not alter Regeneurin mitofusin agonist efficacy or affect its degradation by liver microsomes.
  • the backbone sulfur of the parent thioether was oxidized using hydrogen peroxide to generate the sulfoxide and sulfone, which are potential metabolites (top).
  • the ether and carbon variants and carbon variant with tetrahydropyran substituted for methylated cyclohexane were synthesized de novo (bottom). Red rectangles show substitutions.
  • T1 ⁇ 2 is for human liver microsomes, % bound is for human plasma. (All PK studies were not performed on all backbone variants.) Dose-response curves for mitochondrial elongation (bottom left) are similar for all compounds.
  • FIG.48A and FIG.48B is a bar graph and a series of structures showing functional screening of commercially available Cpd B triazol ring substitution variants.
  • FIG.49 is a series of structures and a graph showing dose- response and human liver microsomal stability data for fusogenic compounds from FIG.22.
  • EC 50 values are mean ⁇ SEM of 3 independent experiments assessing mitochondrial aspect ratio in Mfn2 null MEFs ; Group data dose-response curves are on the left.
  • T 1/2 values are from human liver microsome stability assay.
  • FIG.50 is a series of structures and a graph showing a mitolityn series of mitofusin agonists. The feature that distinguishes Mitolityns from
  • Regeneurins is replacement of the 2-phenyl group on the 2,4,5 triazol ring with ethyl or methyl groups.
  • Mitolityns 1 and 2 are 2-ethyl cyclohexane variants and Mitolityns 3 and 4 are 2-ethyl tetrahydropyran variants; Mitolityns 5 and 6 are like 3 and 4 with 2-methyl rather than 2-ethyl groups off the 2,4,5 triazol ring. Chemical differences from Mitolityn-1 are shown in red rectangles; molecular weights are in parentheses.
  • FIG.51A and FIG.51B shows dose-dependent mitochondrial fusion without cytotoxicity of structurally diverse mitofusin agonists.
  • FIG.52 shows the results of in vitro pharmacokinetic studies of Regeneurin-C, Regeneurin-C/O, and Mitolityn-4 mitofusin agonists.
  • FIG.53A and FIG.53B are a series of graphs and corresponding structures showing in vivo pharmacokinetics of Regeneurin-C, Regeneurin C/O and Mitolityn-4.
  • FIG.53A Three mice each were administered 1mg/kg agonist IV, IP, or IM. Graphs are mean plasma concentration for each administration route.
  • FIG.53B Results for individual mice were administered 1 mg/kg indicated agonist IM.
  • FIG.54 is a series of structures describing the ongoing chemical modifications and optimizations of Regeneurin C/O.
  • FIG.55 is a series of structures of the Fusogenin series of Mfn agonists currently being synthesized.
  • FIG.56A, FIG.56B and FIG.56C are a series of graphs describing Regeneurin-C (100 nM overnight) treatment of primary fibroblasts from human patients with genetically diverse neurodegenerative diseases.
  • FCCP treatment shows effects of complete mitochondrial uncoupling.
  • Ctrl are control primary human fibroblasts.
  • FIG.57A, FIG.57B, FIG.57C and FIG.57D are an illustration, traces, and bar graphs describing the initial phenotyping studies of CMT2A mouse (Mfn2 T105M flox-stop x H9B Cre).
  • FIG.57A Schematic depiction of nerve conduction studies; red arrows show positions of stimulating electrodes, blue arrows of sensing electrodes.
  • FIG.57B Representative CMAP tracings from normal control (top) and CMT2A Mfn2 T105M (bottom) mice. Posterior tibial tracings control for CMAP sensing, and are no different as expected. Note marked decrease in amplitude of Sciatic nerve tracing in T105M mouse.
  • FIG.57C Group data from ongoing CMAP studies; each n is a mouse. CMAP amplitude, but not conduction velocity (latency/length) is diminished after 20 weeks in CMT2A mice.
  • FIG.57D Group data from ongoing Rotarod studies suggest functional decline between 10 and 20 weeks.
  • FIG.58 is a series of images and a graph showing Regeneurin-C/O corrects CMT2A neuronal mitochondrial dymotility in vivo. 10 week old CMT2A MFN2 T105M mice were injected IM with Mfn agonist Regeneurin-C/O 2 mg/kg twice, or vehicle. Sciatic nerve mitochondrial motility was measured 4 hours later. Results for 2 CMT2A mice per group.
  • FIG.59A, FIG.59B, FIG.59C, FIG.59D, and FIG.59E show conformationally active mitofusins bind MIRO1 and are essential for MIRO1-Milton complex formation.
  • FIG.59B MFN2-MIRO1 co- immunoprecipitation assay. Fibroblasts were transfected with Flag-tagged WT or Met376Ala MFN2 and myc-MIRO1; immunoprecipitated (IP) with anti-Flag; and immunoblotted (IB). Blot to right is input cell homogenates.
  • FIG.59C Mitochondrial aspect ratio (length/width) in vehicle (DMSO) and mitofusin agonist Regeneurin S- treated (100 nM, 24 hours) Mfn1/Mfn2 null MEFs expressing wild-type (WT) or mutant MFN2. Decreased aspect ratio reflects impaired mitochondrial fusion.
  • FIG.59D MFN2-MIRO1 and MFN2-CAST co- immunoprecipitation. Regeneurin (Regen) S did not change MIRO1 binding to WT or Met376Ala MFN2.
  • FIG.59E MIRO1-Milton co-immunoprecipitation as a function of mitofusin activation with Regeneurin S.
  • FIG.60A, FIG.60B, FIG.60C, FIG.60D, FIG.60E and FIG.60F show mitofusin activation reverses mitochondrial defects in amyotrophic lateral sclerosis, Huntington disease, and CMT2A patient fibroblasts.
  • CMT2A Charcot-Marie-Tooth disease type 2A
  • PD Parkinson’s disease
  • Alzheimer’s disease Alzheimer’s disease
  • FIG.69-FIG.74 results by individual patient genotype are in FIG.69-FIG.74. Each point is the average value measured of ⁇ 15 mitochondria from a single cell; total numbers of cells assayed per treatment group in 4 or 5 independent experiments are shown below the horizontal axis. Error bars are mean ⁇ s.d.; p values are from 2-way ANOVA.
  • FIG.60D-FIG.60F results by individual patient genotype are in FIG.69-FIG.74.
  • FIG.61A, FIG.61B, FIG.61C, FIG.61D, FIG.61E and FIG.61F show mitofusin activation promotes mitochondrial trafficking and neuron growth in vitro.
  • FIG.61A Confocal micrographs (representative of >90 neurons/condition) of ⁇ - III tubulin (green), DAPI (blue) co-stained adult mouse DRG neurons expressing mitochondrial targeted RFP (red) 48 hours after dissociation axotomy; left is DMSO treatment and right is mitofusin agonist Regeneurin C (Regen) treatment. Scale bars, 44 ⁇ m; insets show enlarged axon termini.
  • FIG.61C Effect of Regeneurin C on mitochondrial localization to axon termini in regrowing adult mouse DRG neurons. Each dot is average result from one ⁇ -III-tubulin positive neuron; numbers of neurons are indicated below p values, which are from t-test. Error bars are mean ⁇ s.d.
  • FIG. 61D Effect of Regeneurin C on mitochondrial localization to axon termini in regrowing adult mouse DRG neurons. Each dot is average result from one ⁇ -III-tubulin positive neuron; numbers of neurons are indicated below p values, which are from t-test. Error bars are mean ⁇ s.d.
  • FIG. 61D Effect of Regeneurin C on mitochondrial localization to axon termini in regrowing adult mouse DRG neurons. Each dot is average result from one ⁇ -III-tubulin positive neuron; numbers of neurons are indicated below p values, which are from t-test. Error bars are mean ⁇ s.d
  • FIG.61F Relationship between axon regrowth (green confocal images on top) and mitochondrial motility (red kymographs on bottom) in vehicle, forskolin (Forsk)-, or Regeneurin-treated CMT (MFN2 T105M) mouse adult DRG neurons. Scale bars, 263 ⁇ m. Quantitative data are to the right. Each dot represents results from a single adult DRG neuron; n values are below the horizontal axes. P values are from ANOVA. Error bars are mean ⁇ s.d.
  • FIG.62A, FIG.62B, FIG.62C, FIG.62D, FIG.62E, FIG.62F and FIG.62G show mitofusin agonists promote regeneration of injured neuronal axons in vitro and in vivo.
  • FIG.62A Confocal images (representative of 6 independent preparations per group) of embryonic mouse cortical neurons cultured in
  • microgroove chambers in each image cell bodies are to the left, horizontal microgrooves containing axons in the middle, and axon termini to the right (labeled in first panel).
  • DIV axon termini were removed by suction (aspiration axotomy; second panel).
  • Vehicle (DMSO) or Regeneurin C (100 nM) were added after axotomy, and axon regeneration measured three days thereafter (panels 3 and 4, respectively).
  • FIG.62B Post axotomy axon regrowth (terminal axon area/microgroove channel). Each dot represents a single axon.
  • FIG. 62C Representative live cell confocal images of axonal mitochondria pre- (top) and 1 hour post (middle and bottom) aspiration axotomy. Squares show areas enlarged to the right.
  • FIG.62D Quantitative group data for mitochondrial aspect ratio (left) and polarization status (right). Each dot represents aggregate data of 10-20
  • FIG.62E Top
  • CMAP quantitative group tibialis anterior compound muscle action potential
  • FIG.62F Representative (of five mice per group) tibialis anterior CMAP tracings 3 and 7 days after sciatic nerve crush injury;“uninjured” tracing is from contralateral leg of same mouse.
  • FIG.62G Immunohistological detection of neuronal regeneration marked by Superior Cervical Ganglion 10
  • FIG.63A, FIG.63B and FIG.63C show mitofusin agonist
  • FIG.63A FIG.63B.
  • Mitochondrial morphology (a, duplicated from Figure 1c for comparison) and polarization status (FIG.63B) in vehicle (DMSO) and mitofusin agonist
  • Regeneurin S-treated 100 nM, 24 hours) Mfn1/Mfn2 null MEFs expressing the MFN2 mutants in (FIG.63C).
  • Mitochondrial depolarization reflects loss of bioenergetic function that
  • Each point is the average value measured of ⁇ 20 mitochondria from a single cell from 4 or 5 independent
  • FIG.63C Confocal micrographs (representative of 20-90 cells/group) of MitoTracker Green, Tetramethylrhodamine, ethyl ester (TMRE; red) stained Mfn1/Mfn2 double-deficient MEFs transduced with adenoviri expressing wild-type (WT) or MFN2 mutants as indicated; Adeno ⁇ -gal is negative control.
  • FIG.64A, FIG.64B, FIG.64C and FIG.65D show different effects on mitochondrial fitness of functionally diverse MFN2 mutants expressed in normal MEFs.
  • FIG.64A Immunoblot analysis of MFN2 after viral vector-mediated expression of human MFN2 (MFN2) or indicated mutants in control C57BL/6J MEFs. ⁇ gal (adeno ⁇ -galactosidase) is negative control; anti- ⁇ -actin is protein loading control.
  • FIG.64B Mitochondrial length (aspect ratio: length/width) assessed by MitoTracker Green staining of MEFs 48 hours after viral-mediated expression.
  • FIG. 64C Mitochondrial length (aspect ratio: length/width) assessed by MitoTracker Green staining of MEFs 48 hours after viral-mediated expression.
  • Mitochondrial depolarization loss of the normal inner mitochondrial membrane electrochemical potential assessed as loss of TMRE fluorescence.
  • All 3 MFN2 loss- of-function mutants induced mitochondrial fragmentation (decreased mitochondrial aspect ratio), revealing dominant inhibition of mitochondrial fusion.
  • MFN2 Arg94Gln and Lys109Ala 2 GTPase-defective mutants provoked
  • FIG.64D Confocal micrographs
  • FIG.65A, FIG.65B and FIG.65C show Regeneurin S activates endogenous Mfn1 to overcome dominant inhibition of mitochondrial fusion by all MFN2 mutants.
  • FIG.65A Mitochondrial morphology (top) and polarization status (bottom) in vehicle (DMSO) or Regeneurin C treated (100 nM, 24 hours) Mfn2 null MEFs virally transduced with WT MFN2 or indicated MFN2 mutants; Mfn2 gene ablation does not reduce expression of endogenous Mfn1 (FIG.81). Each point is the average value from 15-20 mitochondria in a single cell; total numbers of cells assayed per treatment group in 4 or 5 independent experiments are shown below the horizontal axis.
  • FIG.65B Confocal micrographs (representative of >30 cells/group) of MitoTracker Green/TMRE (red) stained Mfn2 null wild-type MEFs transduced with adenoviri expressing wild-type (WT) or mutant MFN2 as indicated. Blue Hoechst stains nuclei. Scale bars, 10 ⁇ m.
  • FIG.66A, FIG.66B, and FIG.66C show Regeneurin S reverses mitochondrial abnormalities produced by MFN2 mutants in mouse neurons.
  • FIG. 66A Immunoblot analysis of MFN2 after viral vector-mediated expression of human MFN2 (MFN2) or indicated mutants in perinatal mouse hippocampal neurons. ⁇ gal is negative control; anti- ⁇ -actin shows protein loading.
  • FIG.66B Mitochondrial aspect ratio (top) and depolarization (bottom) assessed as in FIG.65. Each point is the average value from 15-20 mitochondria in a single cell; total numbers of cells assayed per treatment group in 4 or 5 independent experiments are shown below the horizontal axis. Error bars are mean ⁇ s.d.; p values are from 2-way ANOVA.
  • FIG.66C Confocal micrographs (representative of >70 neurons/group)
  • FIG.67A and FIG.67B shows structures and fusogenicities of mitofusin agonists Regeneurin S and Regeneurin C.
  • Regeneurin S (left) is identical to“chimera B-A/l” in reference 6.
  • Regeneurin C (right) is the carbon backbone variant, which was synthesized to eliminate the possibility of thioether oxidation to sulfoxide and sulfone in cells or tissues; the sulfur (S) and relevant carbon (C) in the two Regeneurin structures are indicated by arrows.
  • FIG.68A, FIG.68B, and FIG.68C show Mitofusin activation reverses mitochondrial defects in CMT2A MFN2 Thr105Met (T105M), Arg274Trp (R274W) and His361Tyr (H361Y) patient fibroblasts.
  • FIG.68A Mitochondrial dysmorphology (left), depolarization (center), and cellular autophagy (right) in genetically diverse CMT2A patient primary fibroblasts. Each point is the average value measured of ⁇ 15 mitochondria from a single cell; total numbers of cells assayed per treatment group in 4 or 5 independent experiments are shown above. Error bars are mean ⁇ s.d.; p values are from unpaired t-tests.
  • FIG.68B Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing reversal of mitochondrial fragmentation and depolarization by Regeneurin C (100 nM, 24 h).
  • FIG.68C Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing reversal of mitochondrial fragmentation and depolarization by Regeneurin C (100 nM, 24 h).
  • FIG.68C Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing reversal of mitochondrial fragmentation and depolarization by Regeneurin C (100 nM, 24 h).
  • FIG.68C Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple
  • LC3-green fluorescent protein GFP
  • Hoechst double-stained patient fibroblasts showing reduction of autophagy assessed as LC3-GFP dots per cell.
  • Scale bars 10 ⁇ m.
  • DMSO (1:1000) is the vehicle for Regeneurin C.
  • FIG.69A, FIG.69B, and FIG.69C show Mitofusin activation can improve mitochondrial fragmentation and depolarization in Parkinson’s disease LRRK2 Gly2019Ser (G2019S), PARK2 Arg275Trp (R275W) and PINK1 Ile368Asn (I368N) patient fibroblasts.
  • FIG.69A Mitochondrial dysmorphology (left)
  • FIG.69B Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing effects of Regeneurin C (100 nM, 24 h) on mitochondrial length and polarization status.
  • FIG.69C Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing effects of Regeneurin C (100 nM, 24 h) on mitochondrial length and polarization status.
  • FIG.69C Confocal micrographs of MitoTracker Green, TMRE,
  • FIG.70A, FIG.70B, and FIG.70C show Mitofusin activation has no effect on mitochondria from Alzheimer’s disease PSEN1 Met146Ile (M146I),
  • FIG.70A Glu184Asp (E184D), and Pro264Leu (P264L) patient fibroblasts.
  • FIG.70A Glu184Asp (E184D), and Pro264Leu (P264L) patient fibroblasts.
  • FIG.70B Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing effects of Regeneurin C (100 nM, 24 h) on mitochondrial length and polarization status.
  • FIG.70C Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing effects of Regeneurin C (100 nM, 24 h) on mitochondrial length and polarization status.
  • FIG.71A, FIG.71B, and FIG.71C show Mitofusin activation improves mitochondria from Huntington disease patient fibroblasts with HTT CAG repeat numbers of 40 (CAG#40), 57 (CAG#57), and 66 (CAG#66).
  • FIG.71A shows Mitofusin activation improves mitochondria from Huntington disease patient fibroblasts with HTT CAG repeat numbers of 40 (CAG#40), 57 (CAG#57), and 66 (CAG#66).
  • FIG.71A shows Mitofusin activation improves mitochondria from Huntington disease patient fibroblasts with HTT CAG repeat numbers of 40 (CAG#40), 57 (CAG#57), and 66 (CAG#66).
  • FIG. 71B Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing improvement in mitochondrial fragmentation and depolarization by Regeneurin C (100 nM, 24 h).
  • FIG.71C Confocal micrographs of LC3-GFP, Hoechst double-stained patient fibroblasts showing reduction of autophagy assessed as LC3-GFP punctae
  • FIG.72A, FIG.72B, and FIG.72C show Mitofusin activation reverses mitochondrial defects in amyotrophic lateral sclerosis SOD1 Leu38Val (L38V), Ile113Thr (I113T), and Leu144Pro (L144P) patient fibroblasts.
  • FIG.72A Mitochondrial dysmorphology (left), depolarization (center), and cellular autophagy (right) in genetically diverse amyotrophic lateral sclerosis patient primary fibroblasts. Each point is the average value measured of ⁇ 15 mitochondria from a single cell; total numbers of cells assayed per treatment group in 4 or 5 independent experiments are shown above.
  • FIG.72B Confocal micrographs of MitoTracker Green, TMRE, Hoechst triple-stained patient fibroblasts (representative of >40 cells/line) showing reversal of mitochondrial fragmentation and depolarization by Regeneurin C (100 nM, 24 h).
  • FIG.72C Confocal micrographs of LC3-GFP, Hoechst double-stained patient fibroblasts showing reversal of increased autophagy assessed as LC3-GFP punctae (fluorescent green dots) per cell. Scale bars, 10 ⁇ m. DMSO (1:1000) is the vehicle for Regeneurin C.
  • FIG.74 shows the peptide and small molecule peptidomimetic mitofusin agonists accelerate axon regrowth in cultured adult mouse DRG neurons.
  • Each point in the graphs is the number of axonal intersections computationally identified by blinded Sholl analysis of an individual ⁇ -III tubulin- positive cultured mouse dorsal root ganglion neuron.
  • FIG.75A, FIG.75B, and FIG.75C show Mitofusin agonists do not affect DRG neuronal survival in vitro.
  • FIG.75A Representative images (of >24 neurons/treatment group, 2 independent experiments) of cell viability studies in adult mouse DRG neurons treated with mitofusin agonist peptide or small molecule Regeneurin C or their respective vehicles.
  • FIG.76A, FIG.76B, FIG.76C, FIG.76D, FIG.76E, FIG.76F and FIG.76G show structure-function relations of Mitolityn series of mitofusin agonists.
  • FIG.76A Structures of Mitolityns 1-6.
  • FIG.76B Dose-response curves for Mitolityn series of mitofusin agonists; maximal fusogenicity (increase in mitochondrial aspect ratio) was defined as that promoted by 1 ⁇ M Regeneurin C (see Extended Data Fig. 5).
  • FIG.76C EC50 values for Mitolityns 1-6. Inactive is defined as less provoking less than a half-maximal fusogenic response at 1 ⁇ M concentration.
  • FIG.76D The structure-function relations of Mitolityn series of mitofusin agonists.
  • FIG.76A Structures of Mitolityns 1-6.
  • FIG.76B Dose-response curves for
  • FIG.76E Schematic depiction of how Mitolityn-4 structure mimics side chains of function-critical Val372, Met376 and His380 within the prototype MFN2 HR1367-384 mitofusin agonist peptide.
  • FIG.76F Dose-response curve for Mitolityn- 4 showing results of individual experiments; compare with FIG.63 for same data for Regeneurins S and C.
  • FIG.76G Mitolityn-4 stimulation of axonal outgrowth in normal mouse DRGs. Based on these studies Mitolityn-4 (referred to as Mitolityn in the manuscript text) was selected for further pharmacokinetic evaluation.
  • FIG.77 shows comparative pharmacokinetic properties of
  • Regeneurin S The protoype mitofusin agonist, Regeneurin S (Chimera B-A/l from reference 6), exhibited plasma protein binding that was too high, and liver microsome stability that was too low, for in vivo application. Substitution of the backbone sulfur of Regeneurin S with carbon in Regeneurin C, the lead molecule in current in vitro biological studies, made little difference in these factors.
  • FIG.78 shows kinetics of Mitolityn-4 release from a biocompatible protein gel matrix. Results of three independent experiments of Mitolityn-4 elution from Matrigel matrix. Drug concentrations were determined by LC-ESI/MS/MS.
  • FIG.79A, FIG.79B, FIG.79C and FIG.79D show a theoretical model for MFN2-MIRO-Milton interactions that promote mitochondrial trafficking.
  • FIG.79A A mitochondrion in its resting state is shown with inactive (closed conformation) MFN2 and MIRO1 at the outer mitochondrial membrane; Milton is in the cytosol.
  • FIG.79B Transitioning of MFN2 to is open conformation promotes its binding to MIRO1 on mitochondria, which modifies MIRO1 in as-yet unknown ways to enable its binding to Milton.
  • FIG.79C Milton-MIRO1 complexing either requires or provokes MFN2 separation; MFN2 is not part of a stable MIRO1-Milton complex.
  • FIG.79D Milton couples mitochondria to microtubules via its binding of
  • mitochondrial MIRO1 and Dynein or Kinesin molecular motors thus evoking mitochondrial trafficking.
  • FIG.80A and FIG.80B show Immunoblot analysis of MFN expression profiles in MEFs used for current studies.
  • FIG.80B Mfn2 immunoreactivity (top) with GAPDH loading control below. Representative of 2 independent experiments. DKO: Mfn1/Mfn2 double knockout.
  • FIG.81 shows the synthetic route for preparation of mitofusin agonist Regeneurin S.
  • FIG.82 shows the HPLC spectrum of compound Regeneurin S. From top to bottom: UV Absorbance at 220nm; UV Absorbance at 254nm; MS chromatogram. Regeneurin S was 97.6% pure.
  • FIG.83 shows High resolution mass spectroscopy (HRMS) chromatogram of Regeneurin S (C21H29N5OS) shows exact mass found: [M + H]+: 400.2
  • FIG.84 shows 1 H NMR spectrum (500MHz) of Regeneurin S (CDCl3 solvent).
  • FIG.85 shows 13 C NMR spectrum (126MHz) of Regeneurin S (CDCl3 solvent).
  • FIG.86 shows the synthetic route for preparation of mitofusin agonist Regeneurin C.
  • FIG.87 shows HPLC spectrum of compound Regeneurin C. From top to bottom: UV Absorbance at 220nm; UV Absorbance at 254nm; MS
  • FIG.88 shows HRMS chromatogram of Regeneurin C
  • FIG.89 shows 1 H NMR spectrum (500MHz) of Regeneurin C (CDCl3 solvent).
  • FIG.90 shows 13 C NMR spectrum (126MHz) of Regeneurin C (CDCl3 solvent).
  • FIG.91 shows the general synthetic route for preparation of Mitolityns1-4.
  • FIG.92 shows HPLC spectrum of compound Mitolityn-1. From top to bottom: UV Absorbance at 220nm; UV Absorbance at 254nm; MS chromatogram. Mitolityn-1 was 93.5% pure.
  • FIG.93 shows HRMS chromatogram of Mitolityn-1 (C18H31N5O) shows exact mass found: [M + H]+: 334.2.
  • FIG.94 shows 1 H NMR spectrum (400MHz) of Mitolityn-1 (CDCl3 solvent).
  • FIG.95 shows 13 C NMR spectrum (101MHz) of Mitolityn-1 (CDCl3 solvent).
  • FIG.96 shows HPLC spectrum of compound Mitolityn-2. From top to bottom: UV Absorbance at 220nm; UV Absorbance at 254nm; MS chromatogram. Mitolityn-2 was 98.0% pure.
  • FIG.97 shows HRMS chromatogram of Mitolityn-2 (C17H29N5O) shows exact mass found: [M + H]+: 320.1.
  • FIG.98 shows 1 H NMR spectrum (400MHz) of Mitolityn-2 (CDCl3 solvent). [00135] FIG.99 shows 13 C NMR spectrum (101MHz) of Mitolityn-2 (CDCl 3 solvent).
  • FIG.100 shows HPLC spectrum of compound Mitolityn-3. From top to bottom: UV Absorbance at 220nm; UV Absorbance at 254nm; MS chromatogram. Mitolityn-3 was 88.9% pure.
  • FIG.101A, FIG.101B and FIG.101C show HRMS chromatogram of Mitolityn-3 (C17H29N5O2) shows exact mass found: [M + H]+: 336.3.
  • FIG.102 shows 1 H NMR spectrum (400MHz) of Mitolityn-3 (CDCl 3 solvent).
  • FIG.103 shows 13 C NMR spectrum (101MHz) of Mitolityn-3 (CDCl 3 solvent).
  • FIG.104 shows HPLC spectrum of compound Mitolityn-4. From top to bottom: UV Absorbance at 220nm; UV Absorbance at 254nm; MS chromatogram. Mitolityn-4 was 99.9% pure.
  • FIG.105 shows HRMS chromatogram of Mitolityn-4
  • FIG.106 shows 1 H NMR spectrum (400MHz) of Mitolityn-4 (CDCl 3 solvent).
  • FIG.107 shows 13 C NMR spectrum (101MHz) of Mitolityn-4 (CDCl 3 solvent).
  • FIG.108 shows the synthetic route for preparation of Mitolityns 5 and 6.
  • FIG.109 shows HPLC spectrum of compound Mitolityn-5. From top to bottom: UV Absorbance at 220nm; UV Absorbance at 254nm; MS chromatogram. Mitolityn-5 was 94.8% pure.
  • FIG.110 shows HRMS chromatogram of Mitolityn-5
  • FIG.111 shows 1 H NMR spectrum (400MHz) of Mitolityn-5 (CDCl 3 solvent).
  • FIG.112 shows 13 C NMR spectrum (101MHz) of Mitolityn-5 (CDCl 3 solvent).
  • FIG.113 shows HPLC spectrum of compound Mitolityn-6. From top to bottom: UV Absorbance at 220nm; UV Absorbance at 254nm; MS chromatogram. Mitolityn-6 was 66.1% pure.
  • FIG.114 shows HRMS chromatogram of Mitolityn-6
  • FIG.115 shows 1 H NMR spectrum (400MHz) of Mitolityn-6 (CDCl 3 solvent).
  • FIG.116 shows 13 C NMR spectrum (101MHz) of Mitolityn-6 (CDCl 3 solvent).
  • FIG.117 shows characteristics and sourcing of primary human fibroblast lines derived from patients with genetically and mechanistically diverse neurodegenerative diseases.
  • the present disclosure is based, at least in part, on the discovery that modeling mini peptides can provide small molecule regulators of mitochondrial fusion for use in treating mitochondrial associated diseases, disorders, and conditions.
  • the present disclosure provides new compositions, uses, and techniques for regulating mitochondrial function, including mitochondrial tracking and fusion. These compositions and methods can be useful to correct cell and organ dysfunction caused by primary abnormalities in mitochondrial fission, fusion and subcellular motility/distribution. In particular, the compositions and methods can be useful to enhance mitochondrial trafficking facilitating regeneration of axons and nerves in CNS and PNS related injuries and traumas.
  • novel small molecules were designed that incorporated functional features (e.g., potency, specificity) of two mitofusin agonist peptidomimetic compounds identified from a functional screen (Cpds A and B) which were functionally synergistic because they acted on different phosphorylated forms of MFN (see e.g., Example 2).
  • MFN2 mitochondrial fusion and trafficking in CMT2A neurons.
  • Mitofusins MFNs promote fusion-mediated mitochondrial content exchange and subcellular trafficking.
  • CMT2A neurodegenerative Charcot Marie Tooth Disease type 2A
  • Mfn2 activity is determined by Met376 and His380 interactions with Asp725 and Leu727 and controlled by PINK1 kinase-mediated phosphorylation of adjacent Mfn2 Ser378.
  • CMT2A is the prototypical clinical disorder of defective mitochondrial fusion, but impaired mitochondrial trafficking may play as great a role as mitochondrial fragmentation in CMT2A axonal degeneration.
  • Individuals with CMT2A express one mutant MFN2 allele in combination with one normal MFN2 allele and harbor two normal MFN1 alleles.
  • a composition for the treatment of a mitochondria-associated disease, disorder, or condition can comprise a mitofusin modulating agent, such as a peptide mimetic (e.g., a small-molecule peptide mimetic).
  • a peptide mimetic e.g., a small-molecule peptide mimetic
  • a peptide mimetic can be a chemical peptide mimetic.
  • the peptide mimetic can mimic a mitofusin peptide.
  • mitofusin modulating agents can reverse mitochondrial defects.
  • mitofusin modulating agents can also have mitochondria transport activity.
  • a mitofusin modulating agent can modulate or enhance the transport (e.g., trafficking, mobility, or movement) of mitochondria, in for example, a nerve.
  • Example 5 shows that mitofusin agonists restore axonal mitochondrial trafficking (see e.g., FIG.28).
  • mitofusin agonists enhance mitochondrial elongation or mitochondrial elongation aspect ratio. Examples further show, pharmacological disruption of intramolecular restraints in Mfn2 by mitofusin modulating agents promotes mitochondrial fusion and trafficking in neurons.
  • the mitofusin modulating agents can increase mitochondrial trafficking without affecting or substantially affecting mitochondrial fusion or fission.
  • a peptide mimetic can be a mitofusin mini- peptide as described in US Provisional Patent Application US 62/397,110
  • a peptide mimetic can be a Mfn agonist (fusion-promoting) peptido-mimetic that competes with endogenous HR1-HR2 binding.
  • the Mfn agonist was designed based on the discovery that Mfn1 and Mfn2 share a common domain structure and structural homology with human Mfn1 and Arabidopsis thaliana dynamin-related protein. As described herein, Mfn1 and Mfn2 share a common domain structure that was modeled with I-TASSER and structural homology with bacterial dynamin-like protein, human Mfn1 and
  • Arabidopsis thaliana dynamin-related protein see e.g., FIG. 1.
  • the model shows how the first heptad repeat domain (HR1) interacts in an anti-parallel manner with the carboxyl terminal second heptad repeat (HR2) domain to restrain it and prevent its extension into the cytosol, which is currently believed to be necessary for mitochondrial tethering and fusion (see e.g., Example 1).
  • an Mfn agonist can inhibit or block HR1-HR2 binding or interaction.
  • Met376, Ser378, His380, or Met 381 amino acids were discovered to be necessary for the HR1-HR2 interaction.
  • Mitofusin Modulating Agents Small Molecules to Target Mfn1 and/or Mfn2
  • the small molecule Mfn regulators as described herein are allosteric agonists.
  • An agonist can be a substance that fully activates the receptor that it binds to, and an antagonist can be a substance that binds to a receptor but does not activate and can block the activity of other agonists.
  • Mitofusin modulating agents are described herein (see e.g., Example 2).
  • Mitofusin modulating agents can be, of the formula:
  • R 1 is selected from the group consisting of C 1-8 alkyl, C 1-8 alkyl substituted with S, S, thiophene, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, thiophene, and thiophene carboxamide;
  • R 2 is selected from the group consisting of C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, imidazole, thiophene, thiophene carboxamide, and triazole;
  • R 3 is absent or selected from the group consisting of hydrogen (H) and C 1-8 alkyl
  • R 4 is absent or selected form the group consisting of hydrogen (H) and C 1-8 alkyl
  • R 5 is selected from the group consisting of C 1-8 alkyl, C 1-8 alkyl substituted with S, S, thiophene, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, thiophene, thiophene carboxamide, and triazole;
  • R 6 is selected from the group consisting of bicyclononanone, pyrrole, benzimidizole, pyrrole substituted pyrrole, and substituted benzimidizole;
  • R 7 is selected from the group consisting of C 1-8 alkyl, pyrrole, pyrrole substituted pyrrole, benzimidizole, and substituted benzimidizole;
  • R 8 is selected from the group consisting of hydrogen (H);
  • R 9 is selected from the group consisting of C 1-8 alkyl; pyrrole, substituted pyrrole, pyrrole substituted pyrrole, benzimidizole, and substituted benzimidizole;
  • X is selected from the group consisting of O, C, and N;
  • Y is selected from the group consisting of O, C, and N;
  • Z is a linker group selected from the group consisting of a bond or C 1-6 alkyl
  • R 1 and R 2 form a cyclic group
  • R 1 and R 4 form a cyclic group
  • R 2 and R 3 form a cyclic group
  • R 4 and R 3 form a cyclic group
  • R 8 and R 7 form a cyclic group
  • the bicyclononanone optionally comprises one or more N atoms.
  • the compound of formula (I), (II), or (III) is not a compound of TABLE 4, TABLE 5, TABLE 7, or the commercially sourced
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , or R 9 can be optionally substituted by one or more of acetamide, C 1-8 alkoxy, amino, azo, Br, C 1-8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, or thiophene and optionally further substituted with acetamide, alkoxy, amino, azo, Br, C 1-8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , or R 9 groups can be optionally substituted or further substituted with one or more groups independently selected from the group consisting of hydroxyl; C 1-10 alkyl hydroxyl; amine; C 1-10 carboxylic acid; C 1-10 carboxyl; straight chain or branched C 1-10 alkyl, optionally containing unsaturation; a C 2-8 cycloalkyl optionally containing unsaturation or one oxygen or nitrogen atom; straight chain or branched C 1-10 alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprising a phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms; unsubstituted phenyl ring; substituted phenyl ring; unsubstituted heterocyclyl; and substituted heterocyclyl, wherein the unsubstituted phenyl
  • heterocyclyl straight chain or branched C 1-10 alkyl amine; heterocyclic amine; and aryl comprising a phenyl; and heteroaryl containing from 1 to 4 N, O, or S atoms. Any of the above can be further optionally substituted.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , or R 9 are optionally substituted by one or more of: acetamide, alkoxy, amino, azo, Br, C 1-8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, nitrile, O, phenyl, S, sulfoxide, sulfur dioxide, or thiophene; and optionally further substituted with one or more acetamide, alkoxy, amino, azo, Br, C 1-8 alkyl, carbonyl, carboxyl, Cl, cyano, C 3-8 cycloalkyl, C 3-8 heteroaryl, C 3-8 heterocyclyl, hydroxyl, F, halo, indole, N, n
  • mitofusin modulating agent or agonists can be selected from the compounds below or the R 1 , R 2 , R 3 , R 4 , or R 5 groups or X or Y comprised in the below compounds can be selected independently and placed into formula (I), (II), or (III) (see e.g., TABLE 7, 70 commercially available compounds):
  • the mitofusin modulating agent can comprise a methylated cyclohexy, a backbone, and a substituted triazole ring (see e.g., FIG.46). In some embodiments, the mitofusin modulating agent can comprise one of the
  • The“imine” or“imino” group can be optionally substituted.
  • halogen and“halo”, as used herein, unless otherwise indicated, include a chlorine, chloro, Cl; fluorine, fluoro, F; bromine, bromo, Br; or iodine, iodo, or I.
  • acetamide is an organic compound with the formula CH3CONH2.
  • The“acetamide” can be optionally substituted.
  • aryl as used herein, unless otherwise indicated, include a carbocyclic aromatic group. Examples of aryl groups include, but are not limited to, phenyl, benzyl, naphthyl, or anthracenyl. The“aryl” can be optionally substituted.
  • The“amine” or“amino” group can be optionally substituted.
  • alkyl includes saturated monovalent hydrocarbon radicals having straight or branched moieties, such as but not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl groups, etc.
  • Representative straight-chain lower alkyl groups include, but are not limited to, -methyl, -ethyl, -n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n- octyl; while branched lower alkyl groups include, but are not limited to, -isopropyl, - sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl, 2-methylpentyl, 3- methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 2,2-dimethylpentyl, 2,3- dimethylpentyl, 3,3-dimethylpentyl, 2,3,4-trimethylpentyl, 3-methylhexyl, 2,2- dimethylhexyl, 2,4-dimethylhexyl, 2,5-di
  • The“carboxyl” can be optionally substituted.
  • alkenyl as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon double bond wherein alkyl is as defined above and including E and Z isomers of said alkenyl moiety.
  • An alkenyl can be partially saturated or unsaturated.
  • The“alkenyl” can be optionally substituted.
  • alkynyl as used herein, unless otherwise indicated, includes alkyl moieties having at least one carbon-carbon triple bond wherein alkyl is as defined above.
  • An alkynyl can be partially saturated or unsaturated.
  • The“alkynyl” can be optionally substituted.
  • acyl includes a functional group derived from an aliphatic carboxylic acid, by removal of the hydroxyl (–OH) group.
  • The“acyl” can be optionally substituted.
  • alkoxyl includes O-alkyl groups wherein alkyl is as defined above and O represents oxygen.
  • alkoxyl groups include, but are not limited to, -O-methyl, -O-ethyl, -O- n-propyl, -O-n-butyl, -O-n-pentyl, -O-n-hexyl, -O-n-heptyl, -O-n-octyl, -O-isopropyl, - O-sec-butyl, -O-isobutyl, -O-tert-butyl, -O-isopentyl, -O-2-methylbutyl, -O-2- methylpentyl, -O-3-methylpentyl, -O-2,2-dimethylbutyl, -O-2,3-dimethylbutyl, -O-2,2- dimethylpentyl, -O-2,3-dimethylpentyl, -O-3,3-dimethylpentyl, -O-2,3,
  • cycloalkyl includes a non-aromatic, saturated, partially saturated, or unsaturated, monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbon referred to herein containing a total of from 3 to 10 carbon atoms.
  • cycloalkyls include, but are not limited to, C 3-10 cycloalkyl groups include, but are not limited to, -cyclopropyl, - cyclobutyl, -cyclopentyl, -cyclopentadienyl, -cyclohexyl, -cyclohexenyl, -1,3- cyclohexadienyl, -1,4-cyclohexadienyl, -cycloheptyl, -1,3-cycloheptadienyl, -1,3,5- cycloheptatrienyl, -cyclooctyl, and -cyclooctadienyl.
  • cycloalkyl also includes -lower alkyl-cycloalkyl, wherein lower alkyl and cycloalkyl are as defined herein.
  • -lower alkyl-cycloalkyl groups include, but are not limited to, - CH 2 -cyclopropyl, -CH 2 -cyclobutyl, -CH 2 -cyclopentyl, -CH 2 -cyclopentadienyl, -CH 2 - cyclohexyl, -CH 2 -cycloheptyl, or -CH 2 -cyclooctyl.
  • The“cycloalkyl” can be optionally substituted.
  • heterocyclyl e.g., a“heteroaryl”
  • a“heteroaryl” includes an aromatic or non-aromatic cycloalkyl in which one to four of the ring carbon atoms are independently replaced with a heteroatom from the group consisting of O, S and N.
  • heterocycles include, but are not limited to, benzofuranyl, benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl, pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl, pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl, pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1,4)-dioxane, (1,3)-dioxolane, 4,5-dihydro-1H- imidazolyl, or tetrazolyl. Heterocycles can be substituted or unsubstituted.
  • Heterocycles can also be bonded at any ring atom (i.e., at any carbon atom or heteroatom of the heterocyclic ring).
  • a heterocyclic can be saturated, partially saturated, or unsaturated.
  • The“heterocyclic” can be optionally substituted.
  • indole is an aromatic heterocyclic organic compound with formula C8H7N. It has a bicyclic structure, consisting of a six- membered benzene ring fused to a five-membered nitrogen-containing pyrrole ring. The“indole” can be optionally substituted.
  • The“cyano” can be optionally substituted.
  • The“alcohol” can be optionally substituted.
  • solvate is intended to mean a solvate form of a specified compound that retains the effectiveness of such compound.
  • solvates include compounds of the invention in combination with, for example: water, isopropanol, ethanol, methanol, dimethylsulfoxide (DMSO), ethyl acetate, acetic acid, or ethanolamine.
  • DMSO dimethylsulfoxide
  • the term“mmol”, as used herein, is intended to mean millimole.
  • the term“equiv”, as used herein, is intended to mean equivalent.
  • the term“mL”, as used herein, is intended to mean milliliter.
  • the term“g”, as used herein, is intended to mean gram.
  • the term“kg”, as used herein, is intended to mean kilogram.
  • the term as used herein, is intended to mean micrograms.
  • the term“h”, as used herein, is intended to mean hour.
  • the term“min”, as used herein, is intended to mean minute.
  • the term“M”, as used herein, is intended to mean molar.
  • the term “ ⁇ L”, as used herein, is intended to mean microliter.
  • the term“ ⁇ M”, as used herein, is intended to mean micromolar.
  • the term“nM”, as used herein, is intended to mean nanomolar.
  • the term“N”, as used herein, is intended to mean normal.
  • the term “amu”, as used herein, is intended to mean atomic mass unit.
  • the term“°C”, as used herein, is intended to mean degree Celsius.
  • the term“wt/wt”, as used herein, is intended to mean weight/weight.
  • the term“v/v”, as used herein, is intended to mean volume/volume.
  • the term“MS”, as used herein, is intended to mean mass spectroscopy.
  • the term“HPLC”, as used herein, is intended to mean high performance liquid chromatograph.
  • the term“RT”, as used herein, is intended to mean room temperature.
  • the term “e.g.”, as used herein, is intended to mean example.
  • the term“N/A”, as used herein, is intended to mean not tested.
  • the expression“pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention.
  • Preferred salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate (i.e., 1,1'- methylene-bis-(
  • a pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion.
  • the counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound.
  • a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counterions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and/or one or more counterion.
  • the expression“pharmaceutically acceptable solvate” refers to an association of one or more solvent molecules and a compound of the invention.
  • solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine.
  • pharmaceutically acceptable hydrate refers to a compound of the invention, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of water bound by non-covalent intermolecular forces.
  • Mitochondria generate ATP that fuels neuronal activity.
  • Mitochondria must fuse in order to exchange genomes and promote mutual repair.
  • the initial stages of mitochondrial fusion proceed through the physiochemical actions of two closely related dynamin family GTPases, mitofusins (Mfn) 1 and 2.
  • the obligatory first step leading to mitochondrial fusion is molecular tethering of two mitochondria via homo- or hetero-oligomerization (in trans) of extended Mfn1 or Mfn2 carboxyl termini.
  • GTP binding to and hydrolysis by Mfn1 or Mfn2 promotes irreversible physical fusion of the organellar outer membranes.
  • Mitofusins belong to a class of highly conserved GTPases which are located on the outer membrane of mitochondria in mammals, flies, the worm and budding yeast. Each of Mfn1 and Mfn2, the mitofusins present in mammals, are anchored to the outer membrane by two transmembrane domains such that their N-terminus and C-terminus are exposed to the cytoplasm. Mitofusins on different organelles undergo transdimerization through anti-parallel binding of their extended carboxy terminal ⁇ -helical domains to form mitochondria-mitochondria tethers– the obligate initial step in mitochondrial fusion (Koshiba et al., 2004, Science, 305:858-861).
  • Mfn1 and Mfn2 share a common domain structure.
  • the amino terminal GTPase domain is followed by a coiled-coiled heptad repeat region (HR1), two adjacent small transmembrane domains, and a carboxyl terminal coiled heptad repeat region (HR2).
  • HR1 and Mfn2 varies by domain, being most highly conserved in the GTPase, transmembrane, and HR2 domains.
  • HR2 domains extending from Mfn1 molecules located on different mitochondria can bind to each other, forming inter-molecular HR2-HR2 interactions that link the molecules and tether the organelles (Koshiba et al. ibid).
  • HR2 can also bind to HR1 (Huang et al., 2011, PLoS One, 6:e20655), although there has been no determination of whether this is an inter- or intra-molecular interaction.
  • the amino acid sequence of human Mfn1 is SEQ ID NO:5.
  • the amino acid sequence of human MFN2 is SEQ ID NO: 6.
  • the amino acid sequence of human MFN2 is SEQ ID NO: 29.
  • the amino acid sequence of human MFN2 is SEQ ID NO: 30.
  • the amino acid sequence of chimpanzee MFN2 is SEQ ID NO: 31.
  • the amino acid sequence of gorilla MFN2 is SEQ ID NO: 32.
  • the amino acid sequence of monkey MFN2 is SEQ ID NO: 33.
  • the amino acid sequence of macaque MFN2 is SEQ ID NO: 34.
  • the amino acid sequence of marmoset MFN2 is SEQ ID NO: 35.
  • the amino acid sequence of bushbaby MFN2 is SEQ ID NO: 36.
  • the amino acid sequence of lemur MFN2 is SEQ ID NO: 37.
  • the amino acid sequence of gibbon MFN2 is SEQ ID NO: 38.
  • the amino acid sequence of elephant MFN2 is SEQ ID NO: 39.
  • the amino acid sequence of armadillo MFN2 is SEQ ID NO: 40.
  • the amino acid sequence of cat MFN2 is SEQ ID NO: 41.
  • the amino acid sequence of dog MFN2 is SEQ ID NO: 42.
  • the amino acid sequence of ferret MFN2 is SEQ ID NO: 43.
  • the amino acid sequence of boar MFN2 is SEQ ID NO: 44.
  • the amino acid sequence of dolphin MFN2 is SEQ ID NO: 45.
  • the amino acid sequence of sheep MFN2 is SEQ ID NO: 46.
  • the amino acid sequence of squirrel MFN2 is SEQ ID NO: 47.
  • the amino acid sequence of guinea pig MFN2 is SEQ ID NO: 48.
  • the amino acid sequence of rat MFN2 is SEQ ID NO: 49.
  • the amino acid sequence of mouse MFN2 is SEQ ID NO: 50.
  • the amino acid sequence of horse MFN2 is SEQ ID NO: 51.
  • the amino acid sequence of opossum MFN2 is SEQ ID NO: 52.
  • the amino acid sequence of collrd flyctchr MFN2 is SEQ ID NO: 53.
  • the amino acid sequence of human MFN2 is SEQ ID NO: 54.
  • the amino acid sequence of zebra finch MFN2 is SEQ ID NO: 55.
  • the amino acid sequence of chicken MFN2 is SEQ ID NO: 56.
  • the amino acid sequence of human MFN2 is SEQ ID NO: 57.
  • the amino acid sequence of turkey MFN2 is SEQ ID NO: 58.
  • the amino acid sequence of turtle MFN2 is SEQ ID NO: 59.
  • the amino acid sequence of pufferfish MFN2 is SEQ ID NO: 60.
  • the amino acid sequence of tilapia MFN2 is SEQ ID NO: 61.
  • the amino acid sequence of stickleback MFN2 is SEQ ID NO: 62.
  • the amino acid sequence of cod MFN2 is SEQ ID NO: 63.
  • the amino acid sequence of platyfish MFN2 is SEQ ID NO: 64.
  • the amino acid sequence of amazon molly MFN2 is SEQ ID NO: 65.
  • the amino acid sequence of spotted gar MFN2 is SEQ ID NO: 66.
  • the amino acid sequence of cave fish MFN2 is SEQ ID NO: 67.
  • the amino acid sequence of zebrafish MFN2 is SEQ ID NO: 68.
  • the amino acid sequence of coelacanth MFN2 is SEQ ID NO: 69.
  • the amino acid sequence of frog MFN2 is SEQ ID NO: 70.
  • the present disclosure provides for compositions and methods of treatment for treating mitochondria-related diseases, disorders, or conditions such as diseases or disorders associated with mitofusin 1 (Mfn1) and/or mitofusin 2 (Mfn2) and mitochondrial dysfunction.
  • a mitochondria-associated disease, disorder, or condition can be a disease associated with mitochondrial dysfunction, fragmentation, or fusion or associated with dysfunction in Mfn1 or Mfn2 unfolding.
  • Mitochondria dysfunction can be caused by mutations or can be caused by injury in the central nervous system (CNS) or peripheral nervous system (PNS).
  • the conditions associated with the tethering of mitochondria and ER can be cell death during reoxygenation after tissue ischemia in brain or heart infarcts (i.e., thrombotic stroke and myocardial infarction).
  • tissue ischemia the therapeutic goal of clinical percutaneous mechanical or thrombolytic vascular reperfusion
  • brain or heart infarcts i.e., thrombotic stroke and myocardial infarction
  • mitochondrial uptake of calcium released from ER paradoxically stimulates cell death and provokes infarct extension9, 10.
  • Preventing infarct extension is a major therapeutic goal in western countries, and could be accomplished by temporarily breaking the tethers between mitochondria and ER. Because there are currently no pharmacological Mfn antagonists this therapeutic approach has never been attempted.
  • Mitochondria transit within cells and undergo fusion to exchange genomes and promote mutual repair. Mitochondrial fusion and subcellular trafficking are mediated in part by mitofusins (Mfn) 1 and 2. Genetic mutations in Mfn2 that suppress mitochondrial fusion and motility cause Charcot Marie Tooth Disease 2A (CMT2A), the most common heritable axonal neuropathy. Mitochondrial
  • fragmentation, dysfunction, and dysmotility are also central features of other genetic neurodegenerative syndromes, such as amyotrophic lateral sclerosis, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease. Because no therapeutics exist that directly enhance mitochondrial fusion or trafficking, these diseases are unrelenting and irreversible.
  • mitochondria-related diseases, disorders, or conditions can be any disease disorder or condition that is related to mitochondrial dysfunction.
  • Mitochondrial dysfunction is implicated in chronic degenerative neurological conditions such as Alzheimer’s, Parkinson’s, and Huntington’s diseases.
  • the genetic neurodegenerative condition Charcot Marie Tooth Disease (type 2A) (CMT) or Hereditary motor and sensory neuropathy, is caused by multiple loss-of-function mutations of Mfn2.
  • CMT Charcot Marie Tooth Disease
  • Hereditary motor and sensory neuropathy is caused by multiple loss-of-function mutations of Mfn2.
  • Mfn2A Charcot Marie Tooth Disease
  • Hereditary motor and sensory neuropathy Hereditary motor and sensory neuropathy
  • Mitochondria-associated diseases, disorders, or conditions can be Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, Charcot Marie Tooth Disease (type 2A) (CMT), hereditary motor and sensory neuropathy, autism, autosomal dominant optic atrophy (ADOA), muscular dystrophy, Lou Gehrig’s disease, cancer, mitochondrial myopathy, Diabetes mellitus and deafness (DAD), Leber's hereditary optic neuropathy (LHON), Leigh syndrome, subacute sclerosing encephalopathy, Neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP), Myoneurogenic gastrointestinal encephalopathy (MNGIE), Myoclonic Epilepsy with Ragged Red Fibers (MERRF), Mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS), mtDNA depletion, mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), Dysautonom
  • Symptoms that can be treated with the methods as described herein can include poor growth, loss of muscle coordination, muscle weakness, visual problems, hearing problems, learning disabilities, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, autonomic dysfunction, and dementia.
  • mitofusin agonists e.g., chimera B-A/l
  • mitofusin agonists rapidly reverse mitochondrial dysmotility in sciatic nerve axons of a mouse model of Charcot Marie Tooth disease.
  • impaired mitochondrial fusion, fitness, and/or trafficking also contribute to neuronal degeneration in various neurodegenerative diseases (e.g., in Charcot Marie Tooth disease (CMT2A), Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease, and especially in Amyotrophic Lateral Sclerosis (ALS))
  • CMT2A Charcot Marie Tooth disease
  • Huntington’s disease Huntington’s disease
  • Parkinson’s disease Parkinson’s disease
  • Alzheimer’s disease e.g., Alzheimer’s disease
  • ALS Amyotrophic Lateral Sclerosis
  • a neurodegenerative disease, disorder or condition can be a disease of impaired neuronal mitochondrial dynamism or trafficking, such as a hereditary motor and sensory neuropathy (HMSN) (e.g., Charcot Marie Tooth (CMT) disease), CMT1 (a dominantly inherited, hypertrophic, predominantly demyelinating form), CMT2 (a dominantly inherited predominantly axonal form), Dejerine-Sottas (severe form with onset in infancy), CMTX (inherited in an X-linked manner), CMT4 (includes the various demyelinating autosomal recessive forms of Charcot-Marie-Tooth disease), hereditary sensory and autonomic neuropathy type IE, hereditary sensory and autonomic neuropathy type II, hereditary sensory and autonomic neuropathy type V, HMSN types 1A and 1B (e.g., dominantly inherited hypertrophic demyelinating neuropathies), HMSN type 2 (e.g.
  • HMSN her
  • HMSN type 4 e.g., hypertrophic neuropathy [Refsum] associated with phytanic acid excess
  • HMSN type 5 associated with spastic paraplegia
  • HMSN type 6 e.g., with optic atrophy
  • a neurodegenerative disease, disorder or condition can be Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Alexander disease, Alpers' disease, Alpers-Huttenlocher syndrome, alpha-methylacyl-CoA racemase deficiency, Andermann syndrome, Arts syndrome, ataxia neuropathy spectrum, ataxia (E.g., with oculomotor apraxia, autosomal dominant cerebellar ataxia, deafness, and narcolepsy), autosomal recessive spastic ataxia of Charlevoix- Saguenay, Batten disease, beta-propeller protein-associated neurodegeneration, Cerebro-Oculo-Facio-Skeletal Syndrome (COFS), Corticobasal Degeneration, CLN1 disease, CLN10 disease, CLN2 disease, CLN3 disease, CLN4 disease, CLN6 disease,CLN7 disease, CLN8 disease, cognitive dysfunction, congenital insensitivity to pain with anhidros
  • Encephalopathies prion diseases
  • Wallerian-like degeneration Charcot Marie Tooth (CMT) Disease.
  • CMT disease is an example of a non-curable and currently untreatable neurodegenerative disease, disorder, or condition, which can be characterized by mutations of Mfn2 and/or axonal neuropathy.
  • mitochondrial transport not the conventional wisdom that mitochondria size, is implicated in CMT disease progression. It is shown here that the ability of mitochondria to get from point A to point B is the cause of progression.
  • CMT is a progressive disease, caused by mutation in Mfn2, and characterized by neuronal neuropathy. The disease affects the legs at 8 to 10 years of age, then upper limbs, muscle wasting, skeletal deformities, and results in being wheelchair bound.
  • the present disclosure provides for the discovery that the progression of CMT was not due to small mitochondria size but the length of mitochondrial travel. As such, this disclosure provides for the evaluation of mitochondrial trafficking as a route of therapy in the first mouse model of disease. It was discovered that the mitochondria in the legs do not move, but in the arm, there is mitochondria movement. As such, it was discovered that Mfn2 pays a role in mitochondria trafficking. Data showed that administration of a mitofusin modulating agent allowed for the mitochondria to move in a mouse model where mitochondria were not previously moving, which is applicable in any neuropathy (e.g.,
  • Huntington’s disease amyotrophic lateral sclerosis (ALS) or ALS-like sclerosis, Alzheimer’s disease).
  • ALS amyotrophic lateral sclerosis
  • ALS-like sclerosis Alzheimer’s disease
  • mitofusin agonists e.g., chimera B-A/l
  • mitofusin agonists rapidly reverses mitochondrial dysmotility in sciatic nerve axons of a mouse model of Charcot Marie Tooth disease. It is currently believed that impaired mitochondrial trafficking also contribute to neuronal degeneration in various neurological diseases (e.g., in Huntington’s, Parkinson’s, and Alzheimer’s diseases, and especially in Amyotrophic Lateral Sclerosis (ALS)).
  • a neurological disease, disorder, or condition can be Abulia; Agraphia; Alcoholism; Alexia; Alien hand syndrome; Allan–Herndon–Dudley syndrome;
  • Amnesia Amyotrophic lateral sclerosis (ALS); Aneurysm; Angelman syndrome; Anosognosia; Aphasia; Apraxia; Arachnoiditis; Arnold–Chiari malformation;
  • Asomatognosia Asperger syndrome; Ataxia; Attention deficit hyperactivity disorder; ATR-16 syndrome; Auditory processing disorder; Autism spectrum; Behcets disease; Bipolar disorder; Bell's palsy; Brachial plexus injury; Brain damage; Brain injury; Brain tumor; Brody myopathy; Canavan disease; Capgras delusion; Carpal tunnel syndrome; Causalgia; Central pain syndrome; Central pontine myelinolysis;
  • Centronuclear myopathy Cephalic disorder; Cerebral aneurysm; Cerebral arteriosclerosis; Cerebral atrophy; Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL); Cerebral dysgenesis– neuropathy–ichthyosis–keratoderma syndrome (CEDNIK syndrome); Cerebral gigantism; Cerebral palsy; Cerebral vasculitis; Cervical spinal stenosis; Charcot– Marie–Tooth disease; Chiari malformation; Chorea; Chronic fatigue syndrome;
  • CIDP Chronic inflammatory demyelinating polyneuropathy
  • Chronic pain Cockayne syndrome; Coffin–Lowry syndrome; Coma; Complex regional pain syndrome;
  • Compression neuropathy Congenital facial diplegia; Corticobasal degeneration; Cranial arteritis; Craniosynostosis; Creutzfeldt–Jakob disease; Cumulative trauma disorders; Cushing's syndrome; Cyclothymic disorder; Cyclic Vomiting Syndrome (CVS); Cytomegalic inclusion body disease (CIBD); Cytomegalovirus Infection;
  • Fibromyalgia Foville's syndrome; Fetal alcohol syndrome; Fragile X syndrome;
  • FXTAS Fragile X-associated tremor/ataxia syndrome
  • Gaucher's disease Gaucher's disease
  • polyneuritiformis Herpes zoster oticus; Herpes zoster; Hirayama syndrome;
  • Intracranial hypertension Isodicentric 15; Joubert syndrome; Karak syndrome;
  • Kearns–Sayre syndrome Kinsbourne syndrome; Kleine–Levin syndrome; Klippel Feil syndrome; Krabbe disease; Kufor–Rakeb syndrome; Lafora disease; Lambert– Eaton myasthenic syndrome; Landau–Kleffner syndrome; Lateral medullary
  • Mitochondrial myopathy Mobius syndrome; Monomelic amyotrophy; Morvan syndrome; Motor Neurone Disease - see amyotrophic lateral sclerosis; Motor skills disorder; Moyamoya disease; Mucopolysaccharidoses; Multi-infarct dementia;
  • Multifocal motor neuropathy Multiple sclerosis; Multiple system atrophy; Muscular dystrophy; Myalgic encephalomyelitis; Myasthenia gravis; Myelinoclastic diffuse sclerosis; Myoclonic Encephalopathy of infants; Myoclonus; Myopathy; Myotubular myopathy; Myotonia congenita; Narcolepsy; Neuro-Behçet's disease;
  • Neurofibromatosis Neuroleptic malignant syndrome; Neurological manifestations of AIDS; Neurological sequelae of lupus; Neuromyotonia; Neuronal ceroid
  • lipofuscinosis Neuronal migration disorders; Neuropathy; Neurosis; Niemann–Pick disease; Non-24-hour sleep–wake disorder; Nonverbal learning disorder; O'Sullivan- McLeod syndrome; Occipital Neuralgia; Occult Spinal Dysraphism Sequence; Ohtahara syndrome; Olivopontocerebellar atrophy; Opsoclonus myoclonus syndrome; Optic neuritis; Orthostatic Hypotension; Otosclerosis; Overuse syndrome; Palinopsia; Paresthesia; Parkinson's disease; Paramyotonia congenita;
  • Tetanus Tethered spinal cord syndrome; Thomsen disease; Thoracic outlet syndrome; Tic Douloureux; Todd's paralysis; Tourette syndrome; Toxic
  • Trichotillomania Trigeminal neuralgia; Tropical spastic paraparesis; Trypanosomiasis; Tuberous sclerosis; 22q13 deletion syndrome; Unverricht– Lundborg disease; Vestibular schwannoma (Acoustic neuroma); Von Hippel–Lindau disease (VHL); Viliuisk Encephalomyelitis (VE); Wallenberg's syndrome; West syndrome; Whiplash; Williams syndrome; Wilson's disease; Y-Linked Hearing Impairment; or Zellweger syndrome.
  • VHL Von Hippel–Lindau disease
  • VE Viliuisk Encephalomyelitis
  • Wallenberg's syndrome West syndrome; Whiplash; Williams syndrome; Wilson's disease; Y-Linked Hearing Impairment; or Zellweger syndrome.
  • the central nervous system includes the brain and the spinal cord and the peripheral nervous system (PNS) is composed of cranial, spinal, and autonomic nerves that connect to the CNS.
  • Damage to the nervous system caused by mechanical, thermal, chemical, or ischemic factors, can impair various nervous system functions such as memory, cognition, language, and voluntary movement. Most often, this is through crush or transection of nerve tracts. This results in the interruption of communication between nerve cell bodies and their targets. Other types of injuries can include disruption of the interrelations between neurons and their supporting cells or the destruction of the blood–brain barrier. Of all the types of injury, those to the CNS are among the most likely to result in death or permanent disability.
  • mitofusin agonists e.g., chimera B-A/l
  • mitofusin modulatory agents e.g., mitofusin agonists
  • the present disclosure provides for compositions and methods to treat an injury such as a crush injury.
  • an injury such as a crush injury.
  • nerve e.g., sciatic nerve, median nerve
  • mitochondria motility was implicated in neuropathy. It is believed that mitochondrial motility is also implicated in nerve injuries, especially in nerves that have not severed, such as a crush injury. After an accident or crush injury, nerves will regenerate or die.
  • the small molecule mitofusin modulatory agents, as described herein, can increase mitochondiral trafficking, enabling the nerve to regenerate after a crush injury.
  • heterologous DNA sequence refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also include non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • a "homologous" DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.
  • Expression vector, expression construct, plasmid, or recombinant DNA construct is generally understood to refer to a nucleic acid that has been generated via human intervention, including by recombinant means or direct chemical synthesis, with a series of specified nucleic acid elements that permit transcription or translation of a particular nucleic acid in, for example, a host cell.
  • the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
  • the expression vector can include a nucleic acid to be transcribed operably linked to a promoter.
  • A“promoter” is generally understood as a nucleic acid control sequence that directs transcription of a nucleic acid.
  • An inducible promoter is generally understood as a promoter that mediates transcription of an operably linked gene in response to a particular stimulus.
  • a promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter can optionally include distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
  • a "transcribable nucleic acid molecule” as used herein refers to any nucleic acid molecule capable of being transcribed into a RNA molecule. Methods are known for introducing constructs into a cell in such a manner that the
  • transcribable nucleic acid molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product.
  • Constructs may also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit translation of a specific RNA molecule of interest.
  • conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al.
  • The“transcription start site” or "initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions can be numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) can be denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.
  • a nucleic acid sequence or amino acid sequence (e.g., DNA, RNA, a genetic sequence, polynucleotide, oligonucleotide, primer, protein, polypeptide, peptide) can have about 80%; about 81%; about 82%; about 83%; about 84%; about 85%; about 86%; about 87%; about 88%; about 89%; about 90%; about 91%; about 92%; about 93%; about 94%; about 95%; about 96%; about 97%; about 98%; or about 99% sequence identity to a reference sequence or a naturally occurring sequence or contain at least one substitution modification to the reference sequence or naturally occurring sequence. Recitation of each of these discrete values is understood to include ranges between each value.
  • a nucleic acid sequence or an amino acid sequence can be operably linked to a heterologous promoter.
  • operably-linked refers preferably to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other.
  • a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably-linked to regulatory sequences in sense or antisense orientation.
  • the two nucleic acid molecules may be part of a single contiguous nucleic acid molecule and may be adjacent.
  • a promoter is operably linked to a gene of interest if the promoter regulates or mediates
  • a "construct” is generally understood as any recombinant nucleic acid molecule such as a plasmid, cosmid, virus, autonomously replicating nucleic acid molecule, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleic acid molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a nucleic acid molecule where one or more nucleic acid molecule has been operably linked.
  • a constructs of the present disclosure can contain a promoter operably linked to a transcribable nucleic acid molecule operably linked to a 3' transcription termination nucleic acid molecule.
  • constructs can include but are not limited to additional regulatory nucleic acid molecules from, e.g., the 3'- untranslated region (3' UTR).
  • constructs can include but are not limited to the 5' untranslated regions (5' UTR) of an mRNA nucleic acid molecule which can play an important role in translation initiation and can also be a genetic component in an expression construct.
  • These additional upstream and downstream regulatory nucleic acid molecules may be derived from a source that is native or heterologous with respect to the other elements present on the promoter construct.
  • transgenic refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.
  • Host cells containing the transformed nucleic acid fragments are referred to as “transgenic” cells, and organisms comprising transgenic cells are referred to as “transgenic organisms”.
  • Transformed refers to a host cell or organism such as a bacterium, cyanobacterium, animal or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome as generally known in the art and disclosed (Sambrook 1989; Innis 1995; Gelfand 1995; Innis & Gelfand 1999).
  • Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially mismatched primers, and the like.
  • the term "untransformed” refers to normal cells that have not been through the transformation process.
  • Wild-type refers to a virus or organism found in nature without any known mutation.
  • nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
  • Nucleotide and/or amino acid sequence identity percent is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • percent sequence identity X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
  • conservative substitutions can be made at any position so long as the required activity is retained.
  • So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr.
  • amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine).
  • Aliphatic amino acids e.g., Glycine, Alanine, Valine, Leucine, Isoleucine
  • Hydroxyl or sulfur/selenium-containing amino acids e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine
  • Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids.
  • Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
  • T m melting temperature of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize.
  • Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P.1988. Methods in Enzymology 167, 747-754). Such techniques include, but are not limited to, viral infection, calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, receptor-mediated uptake, cell fusion,
  • transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Exemplary nucleic acids which may be introduced to a host cell include, for example, DNA sequences or genes from another species, or even genes or sequences which originate with or are present in the same species, but are incorporated into recipient cells by genetic engineering methods. The term
  • exogenous is also intended to refer to genes that are not normally present in the cell being transformed, or perhaps simply not present in the form, structure, etc., as found in the transforming DNA segment or gene, or genes which are normally present and that one desires to express in a manner that differs from the natural expression pattern, e.g., to over-express.
  • exogenous gene or DNA is intended to refer to any gene or DNA segment that is introduced into a recipient cell, regardless of whether a similar gene may already be present in such a cell.
  • the type of DNA included in the exogenous DNA can include DNA which is already present in the cell, DNA from another individual of the same type of organism, DNA from a different organism, or a DNA generated externally, such as a DNA sequence containing an antisense message of a gene, or a DNA sequence encoding a synthetic or modified version of a gene.
  • Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif.41(1), 207–234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley- VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).
  • RNA interference e.g., small interfering RNAs (siRNA), short hairpin RNA
  • RNA micro RNAs
  • shRNA shRNA
  • miRNA micro RNAs
  • Fanning and Symonds (2006) Handb Exp Pharmacol.173, 289-303G describing hammerhead ribozymes and small hairpin RNA
  • Maher (1992) Bioassays 14(12): 807-15 describing targeting deoxyribonucleotide sequences
  • Lee et al. (2006) Curr Opin Chem Biol.10, 1-8 describing aptamers; Reynolds et al.
  • RNAi molecules are commercially available from a variety of sources (e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen).
  • siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iTTM RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research Computing). Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs. F ORMULATION
  • compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety.
  • Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • formulation refers to preparing a drug in a form suitable for administration to a subject, such as a human.
  • a “formulation” can include pharmaceutically acceptable excipients, including diluents or carriers.
  • pharmaceutically acceptable can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects.
  • examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Maryland, 2005 (“USP/NF”), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc. may also be used.
  • compositions can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents.
  • dispersion media can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents.
  • the use of such media and agents for pharmaceutical active substances is well known in the art (see generally Remington’s Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • a “stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0 oC and about 60 oC, for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.
  • the formulation should suit the mode of administration.
  • the agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal.
  • the individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents.
  • Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.
  • Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency.
  • Controlled-release preparations can also be used to affect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects.
  • Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time.
  • the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body.
  • the controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below.
  • therapies described herein one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.
  • T HERAPEUTIC M ETHODS T HERAPEUTIC M ETHODS
  • compositions and methods described herein can be used as a primary therapy for Charcot Marie Tooth, or adjunctive therapy for Huntington’s, Parkinson’s, and Alzheimer’s diseases or ALS to reverse or retard progression.
  • compositions and methods described herein can be used for the treatment of a physical injury.
  • a physical injury for example, as a primary therapy for any contusive injury involving the spine or peripheral nerves (perhaps even the brain, i.e. concussion), such as motor vehicle or sports injuries.
  • This therapy can help restore normal motor function by augmenting regeneration and repair of injured neurons.
  • a subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing a mitochondria-associated disease, disorder, or condition.
  • a determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art.
  • the subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, and humans.
  • the subject can be a human subject.
  • a safe and effective amount of a mitofusin modulating agent is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects.
  • an effective amount of a mitofusin modulating agent described herein can substantially inhibit mitochondria-associated disease, disorder, or condition, slow the progress of mitochondria-associated disease, disorder, or condition, or limit the development of mitochondria-associated disease, disorder, or condition.
  • a desired therapeutic effect can be a delay in peripheral neuropathy (e.g., over the course of three years) compared to placebo assessed by slower increase in modified composite CMT neuropathy score.
  • a desired therapeutic effect can be reversal or absence of progression of peripheral neuropathy compared to placebo, as indicated by lower or stable modified composite CMT neuropathy score.
  • a desired therapeutic effect can be reversal or absence of progression of dysregulated motor function or increased regeneration and repair of injured neurons.
  • administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
  • a therapeutically effective amount of a mitofusin modulating agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient.
  • the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to treat, prevent, or slow the progression of mitochondria-associated disease, disorder, or condition.
  • compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of
  • Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 , (the dose therapeutically effective in 50% of the
  • the dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD 50 /ED 50 , where larger therapeutic indices are generally understood in the art to be optimal.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al.
  • compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the
  • treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof.
  • treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms.
  • a benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.
  • Administration of a mitofusin modulating agent can occur as a single event or over a time course of treatment.
  • a mitofusin modulating agent can be administered daily, weekly, bi-weekly, or monthly.
  • the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
  • Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for treating, preventing, or slowing the progression of mitochondria-associated disease, disorder, or condition.
  • a mitofusin modulating agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an anti-inflammatory, or another agent.
  • a mitofusin modulating agent can be administered simultaneously with another agent, such as an antibiotic or an anti-inflammatory.
  • Simultaneous administration can occur through administration of separate compositions, each containing one or more of a mitofusin modulating agent, an antibiotic, an anti-inflammatory, or another agent.
  • Simultaneous administration can occur through administration of one composition containing two or more of mitofusin modulating agent, an antibiotic, an anti-inflammatory, or another agent.
  • a mitofusin modulating agent can be administered sequentially with an antibiotic, an anti- inflammatory, or another agent.
  • a mitofusin modulating agent can be administered before or after administration of an antibiotic, an anti-inflammatory, or another agent.
  • Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art.
  • the agents and composition can be used therapeutically either as exogenous materials or as endogenous materials.
  • Exogenous agents are those produced or
  • Endogenous agents are those produced or manufactured inside the body by some type of device
  • administration can be parenteral, pulmonary, oral, topical, transdermal (e.g., a transdermal patch) intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
  • transdermal e.g., a transdermal patch
  • intradermal intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
  • Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 ⁇ m), nanospheres (e.g., less than 1 ⁇ m), microspheres (e.g., 1-100 ⁇ m), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.
  • Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors.
  • an agent or composition can be administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site.
  • polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof.
  • a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
  • Agents can be encapsulated and administered in a variety of carrier delivery systems.
  • carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331).
  • Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.
  • Also provided are methods for screening (see e.g., Example 2, Example 3).
  • a FRET method for screening and evaluating small molecular regulators of mitochondrial tethering and fusion is provided.
  • a binding assay for screening and evaluating small molecular regulators of mitochondrial tethering and fusion is provided.
  • FRET fluorescence resonance energy transfer between molecules.
  • one fluorophore is able to act as an energy donor and the other of which is an energy acceptor molecule. These are sometimes known as a reporter molecule and a quencher molecule respectively.
  • the donor molecule is excited with a specific wavelength of light for which it will normally exhibit a fluorescence emission wavelength.
  • the acceptor molecule is also excited at this wavelength such that it can accept the emission energy of the donor molecule by a variety of distance-dependent energy transfer mechanisms.
  • the acceptor molecule accepts the emission energy of the donor molecule when they are in close proximity (e.g., on the same, or a neighboring molecule). See for example U.S. Pat.
  • assays can be designed and performed to screen candidate agents or molecules for specific compositions which can activate mitochondrial fusion.
  • identification of small molecule activators provides an alternate modulating composition which may be more efficient to synthesize and use.
  • Candidate agents encompass numerous chemical classes, typically synthetic, semi-synthetic, or naturally­ occurring inorganic or organic molecules.
  • Candidate agents include those found in large libraries of synthetic or natural compounds.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from commercial resources or are readily producible.
  • small molecule activators of mitochondrial fusion identified through these screening assays can become promising therapeutic agents for treating diseases or disorders associated with defects in mitochondrial fusion.
  • One screening assay can use the HR1 peptide or variant which has been shown to increase mitochondrial aspect ratio.
  • the Mfn2 protein or a fragment of the Mfn2 protein which contains the HR2 domain is immobilized to a solid substrate such as nitrocellulose or to the well surface of a high throughput screen plate or array substrate.
  • the immobilized protein or fragment is then incubated with the HR1 peptide or variant in a solution conducive to protein-protein interactions.
  • the HR1 peptide is conjugated to a detectable label such as FITC or other fluorescent dye, generating a signal in each well or array position.
  • Detectable labels are well-known in the art and include isotope, colorimetric, fluorescent, photochromic and electrochemical labels.
  • a candidate agent is assessed for its ability to compete with the HR1 peptide for binding to the solid phase-bound Mfn2 protein or HR2 domain.
  • An agent which can compete with the HR1 peptide for binding to Mfn2 protein or HR2 will reduce or eliminate the signal from the label.
  • a candidate agent able compete with the HR1peptide is an agent which can activate mitochondrial aspect ratio and/or mitochondrial fusion.
  • a method for identifying an agent or compound able to bind to the Mfn2 protein is provided.
  • the compound competes with the HR1peptide for binding to Mfn2 or to a fragment of Mfn2 comprising the HR2 domain.
  • a test compound is identified as active it if decreases the binding of the peptide, i.e., its effect on the extent of binding is above a threshold level. More specifically, if the decrease in binding of the labeled HR1 peptide to the solid phase bound Mfn2 protein or HR2 domain is a several-fold different between the control and experimental samples, the compound would be considered as having binding activity. Typically, a 2-fold or 4-fold threshold difference in binding between the test and control samples is sought. In some embodiments, this agent increases the mitochondrial aspect ratio when incubated in a cell.
  • an alternative assay is provided to identify a composition able to activate intermolecular binding of the HR2 domains of two Mfn proteins.
  • a first population of Mfn2 proteins is labeled with an acceptor fluorophore on its HR2 arm and a second population of Mfn2 proteins is labeled with a donor fluorophore on its HR2 arm.
  • fluorophore donors and complementary acceptor molecules for FRET analysis are well known (see, e.g., Jager et al., 2005, Protein Sci, 14:2059-2068; Jager et al, 2006, Protein Sci, 15:640- 646).
  • the free HR2 arm when an HR2 arm is liberated from the configuration in which it is interacting with the HR1 domain within the core of the Mfn protein, the free HR2 arm is able to interact with the free HR2 arm of a second Mfn2 to facilitate mitochondrial tethering and subsequent fusion.
  • each well or position in the array contains a test reaction mix which comprises a first population of Mfn2 proteins labeled at or near the HR2 arm with a donor fluorophore and a second population of Mfn2 proteins labeled at or near the HR2 arm with a acceptor fluorophore.
  • the fluorescence is measured in each test reaction mix and compared with a negative control reaction mix containing no HR2-binding peptide and a positive control reaction mix which contains an HR2-binding peptide and no candidate compound.
  • a fluorescence signal which is greater in a test reaction mix containing a candidate compound is identified the candidate compound as an activator of mitochondrial fusion.
  • a single Mfn2 protein is labeled with a single FRET donor and acceptor pair, wherein the donor is positioned at or near the HR1 domain and the acceptor is positioned at or near the HR2 domain, or vice versa.
  • Incubation of a peptide which inhibits mitochondrial fusion (decreases mitochondrial aspect ratio) (e.g., the 367-384Gly peptide or variant thereof) will cause the HR2 arm to extend, removing the quenching action of the FRET pair, resulting in fluorescence signal.
  • a library of candidate modulating molecules can be screened by mixing each with the Mfn2 protein labeled with a FRET donor acceptor pair. Any candidate molecule which increases fluorescence of the labeled Mfn2 protein by at least 50%, 60%, 70% compared to the labeled Mfn2 protein in the absence of a candidate molecule will be identified as an activator of mitochondrial fusion.
  • Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids,
  • polypeptides e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw
  • organic molecules or inorganic molecules including but not limited to salts or metals.
  • Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups.
  • the candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • a candidate molecule can be a compound in a library database of compounds.
  • One of skill in the art will be generally familiar with, for example, numerous databases for commercially available compounds for screening (see e.g., ZINC database, UCSF, with 2.7 million compounds over 12 distinct subsets of molecules; Irwin and Shoichet (2005) J Chem Inf Model 45, 177-182).
  • Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds.
  • a lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g.,
  • a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249).
  • Initial screening can be performed with lead-like compounds.
  • Fragment-based lead discovery also known as fragment- based drug discovery (FBDD) is a method that can be used for finding lead compounds as part of the drug discovery process. It is based on identifying small chemical fragments, which may bind only weakly to the biological target, and then growing them or combining them to produce a lead with a higher affinity.
  • FBLD can be compared with high-throughput screening (HTS).
  • HTS high-throughput screening
  • HTS high-throughput screening
  • libraries with up to millions of compounds, with molecular weights of around 500 Da are screened, and nanomolar binding affinities are sought.
  • libraries with a few thousand compounds with molecular weights of around 200 Da may be screened, and millimolar affinities can be considered useful.
  • ligand-observe nuclear magnetic resonance (NMR) methods such as water-ligand observed via gradient spectroscopy (waterLOGSY), saturation transfer difference spectroscopy (STD-NMR), 19F NMR spectroscopy and inter-ligand Overhauser effect (ILOE) spectroscopy
  • protein-observe NMR methods such as 1H-15N heteronuclear single quantum coherence (HSQC) that utilizes isotopically-labelled proteins, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) are routinely- used for ligand screening and for the quantification of fragment binding affinity to the target protein.
  • HSQC heteronuclear single quantum coherence
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • protein X-ray crystallography can be used to obtain structural models of the protein-fragment(s) complexes. Such information can then be used to guide organic synthesis for high-affinity protein ligands and enzyme inhibitors.
  • Advantages of screening low molecular weight fragment based libraries over traditional higher molecular weight chemical libraries can include:
  • hydrophilic ligand increases the chances that the final optimized ligand will not be too hydrophobic (log P ⁇ 5).
  • Fragments can be less likely to contain sterically blocking groups that interfere with an otherwise favorable ligand-protein interaction, increasing the combinatorial advantage of a fragment library even further.
  • kits can include an agent or
  • kits can facilitate performance of the methods described herein.
  • the different components of the composition can be packaged in separate containers and admixed immediately before use.
  • Components include, but are not limited to Mfn1, Mfn2, antagonist target peptides, agonist target peptides, or mitofusin modulating agents.
  • Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may, for example, comprise metal or plastic foil such as a blister pack.
  • Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
  • Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
  • reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
  • sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, and the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.
  • kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.
  • compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10:
  • numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term“about.”
  • the term“about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
  • the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.
  • the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • the terms“a” and“an” and“the” and similar references used in the context of describing a particular embodiment can be construed to cover both the singular and the plural, unless specifically noted otherwise.
  • the term“or” as used herein, including the claims is used to mean“and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
  • the terms“comprise,”“have” and“include” are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as“comprises,” “comprising,”“has,”“having,”“includes” and“including,” are also open-ended. For example, any method that“comprises,”“has” or“includes” one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that“comprises,”“has” or“includes” one or more features is not limited to possessing only those one or more features and can cover other unlisted features.
  • Mitochondria generate ATP that fuels neuronal activity.
  • Mitochondrial dysfunction is implicated in chronic degenerative neurological conditions such as Alzheimer’s, Parkinson’s, and Huntington’s diseases.
  • Mitochondria fuse in order to exchange genomes and promote mutual repair.
  • the initial stages of mitochondrial fusion proceed through the physiochemical actions of two closely related dynamin family GTPases, mitofusins (Mfn) 1 and 2.
  • the obligatory first step leading to mitochondrial fusion is molecular tethering of two mitochondria via homo- or hetero-oligomerization (in trans) of extended Mfn1 or Mfn2 carboxyl termini.
  • GTP binding to and hydrolysis by Mfn1 or Mfn2 promotes irreversible physical fusion of the organellar outer membranes.
  • CMT Charcot Marie Tooth Disease
  • Hereditary motor and sensory neuropathy is caused by multiple loss-of-function mutations of Mfn2.
  • Mfn2 The genetic neurodegenerative condition, Charcot Marie Tooth Disease (type 2A) (CMT) or Hereditary motor and sensory neuropathy.
  • CMT Charcot Marie Tooth Disease
  • Mfn2 Hereditary motor and sensory neuropathy
  • Mfn1 and Mfn2 share a common domain structure, which was modeled using I-TASSER and structural homology with bacterial dynamin-like protein, human Mfn1 and Arabidopsis thaliana dynamin-related protein (see e.g., FIG.1, top panel).
  • the model shows how the first heptad repeat domain (HR1) interacts in an anti-parallel manner with the carboxyl terminal second heptad repeat (HR2) domain to restrain it and prevent its extension into the cytosol, which can be necessary for mitochondrial tethering and fusion (see e.g., FIG.1, top panel).
  • Mitochondrial elongation evoked by compounds A and B was similar in cells expressing only Mfn2 (Mfn1 null) or only Mfn1 (Mfn2 null), but did not occur in the absence of both of their mitofusin targets
  • compound B is hydrophobic (phenyl) at one end and polar (carboxamide) at the other it mimics side chains presented by Val372/Met376/His381 (see e.g., FIG.1, top panel).
  • compound A is hydrophobic (phenyl and cyclohexyl) at both ends, which mimics the side chains presented to the Mfn2 hydrophobic core after the conformational shift to Val372/Met376/Leu379.
  • the following example describes a HR1-HR2 competition binding assay for screening and evaluating Mfn peptido-mimetic targeting and binding affinity.
  • Mfn-derived fusion-promoting and -inhibiting mini-peptides were modified from amino acid heptad repeats (HR) in the HR1 domain and predicted to interact with their counterparts in the carboxyl terminus HR2 domain within the Mfn stalk region (see e.g., FIG.1, top panel).
  • a high-throughput binding assay was designed whereby the target HR2 peptide sequences, modified to include amino terminal 6 x His tags and Gly linkers, were bonded to Ni-chelate resin (20 ⁇ g/ml) and used as immobilized“receptor” for amino-FITC-tagged Mfn2374-384 (agonist ligand) in which the Ser analogous to Ser378 was replaced with Asp to confer the negative charge essential for activity.
  • the ligand was amino- FITC-tagged Mfn2406-418.
  • the FITC peptide ligands are suspended at 1 mM in 30% DMSO, 70% water (to minimize spontaneous aggregation) and diluted into binding buffer (de-ionized water) to a final concentration of 25 ⁇ M in the presence or absence of competing compound.
  • Dose-dependent loss of resin-bound FITC signal (485 nm excitation/ 538 nm emission) measured in a 96 well spectrofluorometer represents binding of compound to its HR2 target (see e.g., FIG.10, FIG.11).
  • Either the ligand peptide or the receptor peptide can be modified to represent mutations, variations, or posttranslational modifications of Mfn2.
  • Target peptide-bound resin pre-incubated with FITC ligand in column form can be used for high throughput screening by monitoring FITC in the eluate.
  • SEQ ID NO: 1 (NH3) HHHHHH-GGGG-AAMNKKIEVLDSLQSKAKLLRNKA- GG (COOH) (receptor)
  • SEQ ID NO: 2 (NH3) FITC-GGGG-AVRGIMDDLHMAAR-GG (COOH) (amino FITC labeled ligand)
  • Amino acid sequences for Mfn antagonist peptido-mimetic binding assay SEQ ID NO: 3: (NH3) HHHHHH-GGGG-LHAFTGSLEQQVQHSCNSG-GG (COOH) (receptor)
  • SEQ ID NO: 4 (NH3) FITC-GGGG-KQLELLAQDYKLRIKQ-GG (COOH) (amino FITC labeled ligand)
  • the system can be modified to contain the respective Mfn1 sequences if specific interrogation of both Mfn isoforms is desired.
  • EXAMPLE 4 A FRET ASSAY FOR SCREENING AND EVALUATING MFN PEPTIDO- MIMETIC EFFECTS ON MFN CONFORMATION
  • the small molecules described herein enhance mitochondrial fusion by destabilizing the folded conformation of Mfn1 or Mfn2, thus promoting extension of HR2 carboxyl termini that mediate mitochondrial tethering by interacting in trans with similarly extended carboxyl termini of Mfn1 or Mfn2 on neighboring mitochondria (see e.g., FIG.12A).
  • the Forster resonance energy transfer (FRET) assay was designed to screen for and evaluate candidate agents of any chemical class, or molecules with specific alternate compositions, including large libraries of synthetic or natural compounds.
  • Mfn2 HR1 mini-peptides regulate Mfn1 and Mfn2 activity is by directing Mfns into either an unfolded active or folded inactive conformation, as demonstrated by a change in FRET signal of Mfn2 labeled with amino-terminal mCerulean and carboxyl terminal mVenus.
  • This FRET system was limited by low transfection efficiency as a plasmid, an unacceptably poor signal to noise ratio, and the confounding influences of Mfn GTPase activity.
  • GTPase domain eliminates the need for adding GTPase inhibitors such as GTP ⁇ S in the assay and increases the FRET signal to noise ratio.
  • the sequence-confirmed construct was sub-cloned into an adenoviral vector for expression in murine embryonic fibroblasts having different mitofusin expression profiles (wild-type, Mfn1 null, Mfn2 null, Mfn1/Mfn2 double null) or other cell types. Forty-eight hours after adenoviral transduction cells were pre- treated with the anti-fusion mini-peptide MP2 to increase FRET (1 ⁇ M, 1 hour; see e.g., FIG.12C).
  • FRET Fluorescence Reduction
  • mCerulean was excited at 436 nm with emission at 480 nm.
  • mVenus was excited at 500 nm with emission at 535 nm.
  • FRET was imaged with excitation at 436 nm and emission at 535 nm. Data are represented as FRET signal/ mCerulean signal.
  • Increased FRET reflects folded Mfn2; loss of FRET reflects Mfn2 unfolding that favors mitochondrial tethering and fusion.
  • the adeno-Mfn2 FRET ⁇ 80-275 is expressed at near 100% efficiency at 50 MOI in cultured murine embryonic fibroblasts (the cell of choice for functional screening of Mfn activity), and exhibits 5-fold greater signal/noise that the original Mfn2 FRET probe.
  • This system is useful in 96 or 384 well formats for high- throughput screening of Mfn agonists (extinguishing of HR1398-418 induced FRET) and antagonists (stimulation of baseline FRET or reversal of HR1374-384 FRET suppression; see e.g., FIG.12).
  • EXAMPLE 5 RATIONALLY DESIGNED MITOFUSIN AGONISTS REVERSE IN VITRO AND I N V IVO CMT2A M ITOCHONDRIAL D EFECTS
  • This example describes the reversal mitochondrial defects in preclinical models of Charcot Marie Tooth disease type 2A with MFN2 agonists and that pharmacological disruption of intramolecular restraints in MFN2 promotes mitochondrial fusion and trafficking in CMT2A neurons.
  • MFNs Mitofusins
  • CMT2A neurodegenerative Charcot Marie Tooth Disease type 2A
  • a mitofusin agonist normalized axonal mitochondrial trafficking within sciatic nerves of MFN2 Thr 105 ⁇ Met 105 mice, promising a therapeutic approach for CMT2A and other untreatable diseases of impaired neuronal mitochondrial dynamism and/or trafficking.
  • Mitochondria are organelles that generate a rich energy source for cells, which require their continuous subcellular redistribution via mitochondrial trafficking and mutual repair via mitochondrial fusion. Mitochondrial fusion and subcellular trafficking are mediated in part by mitofusin 1 (MFN1) and MFN2. Genetic mutations in MFN2 that suppress mitochondrial fusion and motility cause Charcot Marie Tooth Disease 2A (CMT2A), the most common heritable axonal neuropathy. Because no therapeutics exist that directly enhance mitochondrial fusion or trafficking, this disease is unrelenting and irreversible.
  • MFN1 mitofusin 1
  • CMT2A Charcot Marie Tooth Disease 2A
  • Amino acids controlling these events were identified, first by truncation analysis to define the smallest fusion promoting minipeptide (residues 374 to 384) (see e.g., FIG.2A-FIG.2B), and then through functional investigation of this minimal peptide by alanine (Ala) scanning.
  • MFN2 can be phosphorylated by mitochondrial PTEN-induced putative kinase 1 (PINK1). Targeted mass spectrometry demonstrated
  • MFN2 Ser 378 phosphorylation of MFN2 Ser 378 as well as MFN2 Thr 111 and Ser 442 by PINK1 kinase (see e.g., FIG.2G; FIG.19 and FIG.20; TABLE 3), but not by software-nominated G-protein receptor kinase 2 (see e.g., FIG.21).
  • MFN2 Ser 378 mutants were expressed with or without PINK1 in MFN1 and MFN2 doubly deficient (MFN1 -/- , MFN2 -/- ) cells.
  • Fusion-defective mitochondria in these cells were abnormally short at baseline, but forced expression of wild-type (Ser 378 ) MFN2 resulted in elongation from restoration of fusion (see e.g., FIG.2H, FIG.22).
  • MFN2 Ala 378 which cannot be phosphorylated promoted mitochondrial fusion resistant to PINK1 suppression (see e.g., FIG.2H, FIG.22).
  • the effects of MFN2 Ser 378 mutants were recapitulated in assays of fusion- mediated mitochondrial content exchange (see e.g., FIG.23).
  • a pharmacophore model was generated based on the interactions of HR1 and HR2 domains in the calculated structural model of Mfn2 in the closed conformation.
  • the key features included hydrophobic interactions involving Mfn2 HR1: Val372 and Met376, and aromatic interactions and hydrogen bonding involving Mfn2 HR1 His380.
  • the pharmacophore model did not structurally model mitofusin agonist minipeptide HR1 (367-384), it was noted that peptide residues Val6, Met10, and His14 correspond to Mfn2 HR1: Val372, Met376 and His380.
  • a library comprising ⁇ 14 million commercially available compounds was prepared in silico and evaluated using PHASE to fit these criteria.
  • Top ranked hits were clustered, and filtered based on pharmacological properties using Qikprop.
  • the top 55 (see TABLE 4) commercially available small molecules conforming to the model were selected for functional screening and purchased in 1 mg aliquots. Each compound was dissolved to a stock concentration of 10 mM in DMSO and applied to Mfn2 null MEFs overnight at a final concentration of 1 mM. Eleven of the library members were not soluble in DMSO at the required concentration.
  • a“mitofusin agonist” is a fusogenic compound that binds to the Mfn2 HR2 minipeptide target domain, promotes Mfn2 opening, and loses its fusogenic activity when endogenous mitofusin proteins are not present).
  • Molecular modeling of class A and B agonists assumed that the minipeptide a-helix is comprised of 3.6 amino acids per turn with a 1.4 A pitch advance per amino acid, resulting in a distance of ⁇ 5.4 A between amino acids of adjacent turns.
  • Aliphatic backbones assumed a distance between single bonded carbons of 1.54 A. Structures were created or edited using Marvin JS at the MolPort website and available chemical analogs (chemosimilars; TABLE 5) identified using the search function and a similarity parameter of 0.5.
  • Cpd A and B functionality were assimilated into a single molecule by creating Cpd A-B chimeras (see e.g., FIG.11, FIG.7, TABLE 6).
  • the novel chimeric compounds incorporated functional features (e.g., potency, specificity) of both Cpds A and B which were functionally synergistic because they acted on different phosphorylated forms of MFN (see e.g., Example 2).
  • mitofusin agonists required endogenous MFN1 or MFN2 to promote mitochondrial fusion, exhibited no promiscuous activity for structurally related dynamin, and did not compromise cell viability (see e.g., FIG.27).
  • FRET fluorescence resonance energy transfer
  • MFN1 -/- MFN2 -/- deficient murine embryonic fibroblasts
  • mitofusin agonists corrected mitochondrial dysmorphology and reversed mitochondrial hypopolarization induced by these MFN2 mutants when MFN1 was present
  • Mitofusin agonists also reversed mitochondrial fragmentation and hypopolarization in cultured neurons expressing (in addition to endogenous mitofusins) CMT2A mutants MFN2 R94Q (see e.g., FIG.9C, FIG.9D) or MFN2 T105M (see e.g., FIG.9E).
  • mitofusin agonists do not restore function of CMT2A MFN2 GTPase domain mutants. Rather, by destabilizing the fusion-permissive open conformation of endogenous MFN1 or MFN2, mitofusin agonists can overcome dominant suppression of mitochondrial fusion by these disease-causing dysfunctional proteins.
  • Chimera B-A/l reversed mitochondrial“clumping” formation of static mitochondrial aggregates
  • restored mitochondrial motility in cultured neurons expressing the CMT2A mutant MFN2 T105M see e.g., FIG.28A, FIG.29.
  • Mitochondrial hypopolarization and increased autophagy see e.g., FIG.28B, FIG.30
  • mitochondrial dysmorphology see e.g., FIG.28C, FIG.30
  • a small molecule mitofusin agonist enhanced organelle and cell fitness in CMT2A neurons by promoting mitochondrial fusion and subcellular transport.
  • B-A/l enhances mitochondrial structural defects, reduces mitochondrial ROS levels, and improves mitochondrial membrane potential in ALS and HD patient-derived fibroblasts and has no effect on fibroblasts from control subjects (see e.g., FIG.28).
  • CMT2A is the prototypical clinical disorder of defective mitochondrial fusion, but impaired mitochondrial trafficking may play as great a role as mitochondrial fragmentation in CMT2A axonal degeneration.
  • Individuals with CMT2A express one mutant MFN2 allele in combination with one normal MFN2 allele and harbor two normal MFN1 alleles.
  • Mitofusin agonists may also have therapeutic potential for neurological conditions other than CMT2A, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, wherein mitochondrial dysmotility and fragmentation are contributing factors.
  • Wild-type MEFs were prepared from E10.5 c57/bl6 mouse embryos.
  • SV-40 T antigen-immortalized Mfn1 null (CRL-2992), Mfn2 null (CRL-2993) and Mfn1/Mfn2 double null MEFs (CRL-2994) were purchased from ATCC.
  • MEFs were subcultured in DMEM (4.5g/L glucose) plus 10% fetal bovine serum, 1 ⁇ nonessential amino acids, 2 mM L-glutamine, 100U/ml penicillin and 100ug/ml streptomycin.
  • Mfn2-S378D-fw 5’-cgactcatcatggacgacctgcacatggcggc-3’ (SEQ ID NO: 7)
  • Mfn2-S378D-rv 5’-gccgccatgtgcaggtcgtccatgatgagtcg-3’ (SEQ ID NO: 8)
  • Mfn2-S378A-fw 5’ -gactcatggacgccctgcacatggcg-3’
  • Mfn2-S378A-rv 5’–cgccatgtgcagggcgtccatgatgagtc-3’ (SEQ ID NO: 10)
  • Mfn2 and its mutants were sub-cloned into adenoviral vector Type 5 (dE1/E3) with RGD-fiber modification (Vector Biolabs) using BamHI/XhoI. All constructs were verified by Sanger DNA sequencing.
  • Adeno-viral PINK1 was purchased from Vector Biolabs. Immunoblotting used mouse anti-Mfn2 (Abcam # ab56889, 1: 1000), anti-PINK1 (Sigma #P0076, 1: 500), and beta-actin (Santa Cruz Biotechnology #sc-81178, 1:1000). Protein detection and digital acquisition used peroxidase-conjugated anti mouse secondary antibody (Cell Signaling #7076S, 1:2500) and Western Lightning PLUS ECL substrate (Perkin Elmer 105001EA) on a Li-COR Odyssey instrument.
  • Mfn2367-384Gly peptides and Ala substituted variants of Mfn2374-384 were chemically synthesized and introduced into cells using TAT47–57 conjugation (ThermoFisher Scientific). Except when indicated, 1 mM stocks in sterile water were diluted into culture media 1:1000 to achieve a final concentration of 1 ⁇ M. Cells were treated overnight.
  • the helical secondary structure is often stabilized by a negatively charged group“capping” the positive N-terminal end of the helix dipole.
  • a phosphorylation of Ser 378 can produce H-bonding for the amide of Leu-379 and the negative phosphate can additionally stabilize the helical turns following 379, providing an explanation for observed down-field shifts (i.e., H-bonding induced) in amides of 380, 381 and 382.
  • MFN2 FRET for conformational studies
  • Mfn2 FRET probes contained N-termini-ceruleum and C-termini- mVenus fused to the human (h) mitofusin protein as previously described. FRET analyses were performed either on mitochondria isolated from Mfn1/Mfn2 null MEFs expressing the WT hMfn2 FRET-hMfn2 protein or intact Mfn1/Mfn2 null MEFs expressing WT or mutant Mfn2 FRET proteins (50 MOI).
  • organelle protein was used for each reaction in a total volume of 100 ⁇ l diluted in 10 mM Tris-MOPS (pH 7.4), 10mM EGTA/Tris, and 200 mM sucrose.1 ⁇ M of mitofusin agonist in DMSO was added simultaneously with 2 ⁇ M mitofusin antagonist peptide, incubated in dark at room-temperature for 30 minutes, and FRET signal corrected for Cerulean signal analyzed using a Tecan Safire II multi-mode plate reader in polystyrene 96 well assay plate (Costar 3916).
  • Mfn1/Mfn2 double null MEFs at 70% confluence were infected with adenoviri expressing FRET-hMfn2, FRET-hMfn2 (S378A) or FRET hMfn2 (S378D) at 50 MOI.
  • HEK293 cells were transfected with wild-type or mutant MFN2 having an amino terminal FLAG epitope tag using Lipofectamine 3000 (Invitrogen) per the manufacturer’s instructions. After 48 hours cells mitofusin agonists or DMSO vehicle were added at the indicated concentrations for 1 hour (37°C). Cells were harvested and proteins extracted using Invitrogen cell extraction buffer
  • Target HR2 peptide sequence modified to include amino terminal 6 x His tags and Gly linkers were bonded to Ni-NTA resin (4.4 ⁇ g/ml) (Quiagen) and used as immobilized“receptor” for amino-FITC-tagged Mfn2374-384 (ligand) in which the Ser analogous to Ser378 was replaced with Asp to confer the negative charge essential for activity.
  • FITC peptide ligands were suspended at 1 mM in 30% DMSO, 70% water (to minimize spontaneous aggregation) and diluted into binding buffer (de-ionized water). For the displacement binding, 2.5 nmol of FITC labeled agonist peptide was used in the presence or absence of different amounts of competing compounds.
  • Resin-bound FITC signal (485 nm excitation/ 538 nm emission) measured in a 96 well spectrofluorometer (Spectramax M5e, Molecular Devices) represented binding to HR2 target. Competition binding isotherms were plotted and IC 50 values calculated using Prism 7 (GraphPad).
  • Sequences for binding assay components are:
  • the hypothetical structures of human Mfn2 were developed using the I-TASSER Suite package.
  • the putative closed conformation is based on structural homology with bacterial dynamin-like protein (PDB: 2J69), human Mfn1 (PDB:5GNS), and Arabidopsis thaliana dynamin-related protein (PDB: 3T34).
  • the putative open conformation was based on structural homology with human Opa1, retrieved from the following structures: rat dynamin (PDB: 3ZVR), human dynamin 1- like protein (PDB: 4BEJ), and human myxovirus resistance protein 2 (PDB: 4WHJ).
  • Mfn2 orthologous sequences were retrieved from the Ensembl project database. Protein alignments were performed using Clustal Omega.
  • GRK2/bARK1 was the top hit (score of 31.595), and GRK isoforms comprised 5 of the top 7 hits; ROCK kinase (score 15.919) and PKCa (score 11.48) were the other two hits.
  • PINK1 kinase is not represented at this site, and no other sites reported any likely kinases for Mfn2 Ser378.
  • coli Ubiquigent: 66-0043-050
  • 10 mg human GRK2 10 mg
  • kinase buffer 20 mM Hepes pH 7.4, 10 mM DTT, 0.1 mM EGTA, 0.1 mM ATP and 10 mM MgCl2
  • Peptides were prepared using a modified filter-aided sample preparation method: dried sample was dissolved in 60 ⁇ L of Tris buffer, pH 7.6 that contained 4% SDS and 100 mM DTT and denatured by heating (95oC) for 5 min. The sample was then alkylated with 50 mM iodoacetamide (Sigma, A3221) for 1 h at room temperature in the dark. After the addition of 1 ml of 50 mM ammonium bicarbonate buffer (pH 8.5) containing 8M urea (UA) and vortexing, equal volumes of the samples were transferred to two YM-30 filter units (Millipore, Ref No.
  • Nano-LC-MS/MS Analysis of Phosphopeptides The samples were loaded (2.5 ⁇ L) at a constant pressure of 700 bar at 100% of mobile phase solvent A (0.1%FA) onto a 75 ⁇ m i.d. ⁇ 50 cm Acclaim® PepMap 100 C18 RSLC column (Thermo-Fisher Scientific) using an EASY nanoLC (Thermo Fisher
  • the maximum injection time was 60 ms for parent-ion accumulations and 60 ms for product-ion analysis.
  • the parent ions that were selected for MS2 were dynamically excluded for 20 sec.
  • the automatic gain control was set at a target ion value of 1e6 for MS1 scans and 1e5 for MS2 acquisition. Peptide ions with charge states of one or > 8 were excluded for CID acquisition.
  • Phosphopeptide data from the PINK kinase reactions were also acquired in targeted mode.
  • the CID spectra were acquired at resolving power of 17,500 with maximum table time of 120 ms.
  • the loop count was set to 4 and the isolation width was 2 Da.
  • the acquisition of CID spectra were triggered by an inclusion list of four m/z values for the +2 and +3 charge state of the natural abundance phosphorylated and non-phosphorylated peptide (see e.g., TABLE 3, above, for values).
  • An AGC target value of 3e6 was used for MS scans and 2e5 for MS/MS scans.
  • the unprocessed LC-MS data were analyzed using SKYLINE (version 3.6.9).
  • Wild-type MEFs (100,000 cells) were grown on cover slips. When they reached 60% confluency they were washed with serum-free DMEM.
  • Step A 5-Cyclopropyl-4-phenyl-4H-1,2,4- triazole-3-thiol (1) (1 mmol) was dissolved in 1 mL of CH 3 OH/H 2 O (50:50), then NaOH (1 mmol) was added, stirred for 10 min, and 2-(boc-amino)ethyl bromide (2) (1 mmol) was added at 25 oC. The reaction was allowed to stir for 3 hours then poured into 10 mL water. The precipitate was filtered and dried to get a solid.
  • Step B 2-((5-Cyclopropyl-4-phenyl-4H- 1,2,4-triazol-3-yl)thio)ethan-1-amine (3) (0.5 mmol) and 1,1′-carbonyldiimidazole (CDI) (1 mmol) were dissolved in 0.6 ml CH 3 CN, the mixture was kept at a temperature of 70 oC for 1 h, and then the 2-methyl-cyclohexylamine (4) (0.5 mmol) was added. The mixture was heated for 2 hours at 70 oC, then filtered, and evaporated.
  • CDI 1,1′-carbonyldiimidazole
  • Step A Under an argon atmosphere, into a reaction vessel of 2-amino-5,6- dihydro-4H-cyclopenta[b]thiophene-3-carboxamide (1) (1.0 mmol), potassium iodide (0.8 mmol), potassium carbonate (1.0 mmol), N,N-dimethylformamide (DMF) 1 mL and 2,2,2-trifluoroethyl chloroformate (2) (1.0 mmol) were added.
  • the reaction vessel was heated to 80 °C, and the mixture was stirred for 12 hours.
  • the reaction vessel was cooled to room temperature, and ethyl acetate 100 mL was added.
  • the organic layer was washed with water (50 mL), saturated brine (50 mL), and dried over sodium sulfate. The sodium sulfate and the solvent were distilled off.
  • Step B To a solution of 2 mmol of a 2,2,2-trifluoroethyl (3-carbamoyl-5,6-dihydro-4H-cyclopenta[b]thiophen-2- yl)carbamate (3) and 2 mmol of an 2-(benzylthio)ethan-1-amine (4) in 2 mL of acetonitrile, 0.2 mmol of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) was added. The reaction mixture was heated at 80 °C for 4 h. Then 0.5-2 mL of water was added to the hot reaction mixture.
  • DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
  • LC/MS analysis was carried out using Agilent 1100 Series LC/MSD system with DAD ⁇ ELSD and Agilent LC ⁇ MSD VL (G1956A), SL (G1956B) mass- spectrometer or Agilent 1200 Series LC/MSD system with DAD ⁇ ELSD and Agilent LC ⁇ MSD SL (G6130A), SL (G6140A) mass-spectrometer. All the LC/MS data were obtained using positive/negative mode switching.
  • the compounds were separated using a Zorbax SB-C181.8 ⁇ m 4.6x15mm Rapid Resolution cartridge (PN 821975- 932) under a mobile phase ( ⁇ – acetonitrile, 0.1% formic acid; ⁇ – water (0.1% formic acid)).
  • Flow rate 3ml/min; Gradient 0 min– 100% B; 0.01 min– 100% B; 1.5 min - 0% B; 1.8 min - 0% B; 1.81 min - 100% B; Injection volume 1 ⁇ l; Ionization mode atmospheric pressure chemical ionization (APCI); Scan range m/z 80-1000.
  • Neonatal mouse hippocampal neurons were cultured from brains of one day old Mfn2 T105M or non-transgenic sibling mouse pups as described. After 10 days of differentiating culture neurons were infected with Adeno-Cre to induce Mfn2 T105M expression or Adeno- ⁇ gal as a control (50 MOI). After an additional 72 hours mitofusin agonists or DMSO vehicle were added.
  • neuronal mitochondria were labeled with adenoviral-expressed mitoGFP plus TMRE. Autophagy was measured by LC3 aggregation in neurons infected with adenoviral LC3-GFP. For time-lapse studies of mitochondrial trafficking bi-cistronic Adeno- Cre/GFP marked Cre expression and mitochondria were labeled with adeno- mitoDsRed. Confocal live cell images were acquired with a time-lapse of
  • Mitofusin agonist chimera B- A/l was added after the first 10 minute imaging period (final concentrations of 1 or 5 mM) and nerve axons imaged for another 40 minutes. Because there was no difference in mitochondrial trafficking or response to mitofusin agonist between male and female mice, the data were combined.
  • HD patient-derived fibroblast cell lines (GM04693 from 33-year-old male patient and GM05539 from 10-year-old male patient) were purchased from Invitrogen.
  • ALS patient-derived fibroblasts (ALS 1: ND29509 from 55-year old male patient having SOD1 mutation; ALS 2: ND30327 having FUS1 mutation) and fibroblasts of control healthy individuals were purchased from Coriell Institute, USA. All fibroblast cultures were maintained in MEM supplemented with 15% (v/v) FBS and 1% (v/v) penicillin/streptomycin at 37°C in 5% CO2-95% air.
  • Galactose media was used, to increase mitochondria-dependent metabolism oxidative
  • Human fibroblasts were switched to grow for 48 h in DMEM deprived of glucose and containing galactose (4.5 g/l), 1 % FBS, 5 mM sodium pyruvate and 2 mM L-glutamine for the studies.
  • TMRM tetra-methyl-rhodamine methyl ester
  • Plasma stability of 2 mM compound in clarified freeze-thawed plasma was assessed by LC-MS/MS of supernatants after protein precipitation; 120 min data are reported for studies including 0, 10, 30, 60, and 120 min.
  • Microsome stability of 1 mM compound in liver microsomes (0.5 mg/ml) after 0, 5, 10, 20, 30, 60 min. incubation was assessed by LC/MS/MS of reaction extracts.
  • Regeneurin-S was considered to have three functional domains corresponding to amino acid side chains of the prototype mitofusin agonist minipeptide it was designed to mimic: the methylated cyclohexane group
  • tetrahydropyran substituted for the cyclohexyl group exhibited enhanced microsomal stability and improved (decreased) plasma protein binding.
  • Mitolityn-3 and–4 with methyl rather than ethyl groups on the triazol ring were synthesized (see e.g., FIG.50).
  • the Mitolityn series of compounds were then assayed for fusogenicity (increase in aspect ratio) and liver microsome stability (see e.g., FIG.50).
  • Mitolityn-4 exhibited the reciprocal of these features, with low plasma protein binding, stability in the liver microsome assay, no passive diffusion across lipid membrane and therefore no reverse transport by P-gp. These features were also not conducive to central and peripheral nervous system delivery. Regeneurin C/O, however, had intermediate features, being modestly bound to plasma proteins, stable in the liver microsome assay, and exhibiting intermediate passive permeability and P-gp mediated reverse transport. [0328] Preliminary in vivo pharmacokinetic studies revealed that the presently disclosed mitofusin agonists compounds are eliminated from the circulation within 2 hours of IV, IP, or IM administration (see e.g., FIG.53); IM administration provided the longest plasma half-time and bioavailability.
  • mitochondrial trafficking, fusion, and polarization status would improve mitochondria fitness in primary fibroblasts from human patients with a variety of mutations causing Amyotrophic lateral sclerosis (3 SOD1 gene mutations) Huntington’s (HD gene CAG repeat numbers 40, 57, and 66), Parkinson’s (Parkin, PINK, and LRRK2 gene mutations), and Alzheimer’s diseases (3 PSEN1 gene mutations), as well as CMT2A (3 Mfn2 gene mutations).
  • SOD1 gene mutations Huntington’s (HD gene CAG repeat numbers 40, 57, and 66), Parkinson’s (Parkin, PINK, and LRRK2 gene mutations), and Alzheimer’s diseases (3 PSEN1 gene mutations), as well as CMT2A (3 Mfn2 gene mutations).
  • mitofusin agonists markedly enhance or improve mitochondrial function (e.g., trafficking) in ALS and confer a modest benefit in Huntington’s disease (see e.g., FIG.56, TABLE 11
  • Standard mouse models for ALS suitable for in vivo efficacy studies are available from Jackson Labs (JAX).
  • a target product profile is shown in TABLE 12, below. TABLE 12.
  • Crush injury models are well known in the art. Any model for crush injury can be used (see e.g., Dobek et al. Comp Med.2013 Jun;63(3):227-32; Jager et al., BioMed Research International, Volume 2014 (2014)).
  • Mature neurons typically fail to regenerate after injury. Reduced mitochondrial motility and energy deficits in injured axons are intrinsic mechanisms controlling regrowth in mature neurons (see e.g., Zhou et al.2016 J. Cell Biol. Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits). Axotomy induces acute mitochondrial depolarization and ATP depletion in injured axons. Thus, mature neuron-associated increases in mitochondria-anchoring protein syntaphilin (SNPH) and decreases in mitochondrial transport cause local energy deficits. Enhancing mitochondrial transport via administration of mitofusion modulatory agents is expected to facilitate regenerative capacity by replenishing healthy mitochondria in injured axons, thereby rescuing energy deficits. An in vivo sciatic nerve crush study is expected to show that enhanced mitochondrial transport in accelerates axon regeneration.
  • SNPH mitochondria-anchoring protein syntaphilin
  • mitofusin modulatory agents e.g., peptides, peptide mimetics, agonists
  • enhancing mitochondrial transport is one of the potential new therapeutic strategies to stimulate axon regeneration in the injured CNS and PNS.
  • Activated mitofusin mobilizes mitochondria in damaged axons to promote nerve regeneration
  • Mitochondria generate chemical fuel to sustain neuronal activity. Bioenergetic support of neuronal repair after physical or genetic damage requires active mitochondrial transport through axons to sites of injury or degeneration 1,2 . Impaired mitochondrial trafficking may therefore contribute to the unique
  • MFN allosteric mitofusin
  • CMT Charcot Marie Tooth
  • ALS amyotrophic lateral sclerosis
  • mitochondrial MIRO proteins bind to the adaptor protein Milton to anchor organelles to kinesin or dynein on microtubules 7,8 . Although the significance is unclear, MIRO proteins also form complexes with outer mitochondrial membrane mitofusins (MFN) 1 and 2 3 , so designated because they promote mitochondrial fusion 9 .
  • MFN mitochondrial membrane mitofusins
  • the fusogenic activity of mitofusins depends in part upon conformational shifts controlled by intra-molecular peptide-peptide interactions 10 .
  • Allosteric MFN activation with competing minipeptides or small molecule peptidomimetics disrupts the critical peptide-peptide interactions that maintain mitofusins in an inactive state, thereby augmenting both mitochondrial fusion and trafficking 6,10 .
  • Glu184Asp, and Pro264Leu mutations ALS (SOD1 Leu38Val, Ile113Thr, and Leu144Pro mutations), and CMT2 (MFN2 Thr105Met, Arg274Trp, and His361Tyr mutations) (FIG.117).
  • mouse cells expressing recombinant human CMT2 MFN2 mutants FIG.63, FIG.64
  • pharmacological mitofusin activation reversed mitochondrial fragmentation and depolarization in primary human CMT2 fibroblasts (FIG.60A-FIG.60F; FIG.67).
  • Mitochondrial dysmorphology was mild and not meaningfully affected by Regeneurin in Parkinson’s and Alzheimer’s disease fibroblasts (FIG.60A-FIG.60F; FIG.69 and FIG.70) (although mitofusin activation improved mitochondrial depolarization in PINK1 and PARK2 mutant Parkinson’s disease cells; FIG.69).
  • mitofusin activation improved mitochondrial depolarization in PINK1 and PARK2 mutant Parkinson’s disease cells; FIG.69.
  • modest mitochondrial dysmorphology and depolarization in Huntington disease cells, and severe mitochondrial fragmentation and loss of polarization in ALS cells were reversed by mitofusin activation (FIG.60A-FIG.60F; FIG.71 and FIG.72).
  • DRG root ganglion
  • Mitochondrial localization at axon termini and an increase in the number and aspect ratio of axonal mitochondria in normal (FIG.61C, 3d) and CMT (MFN2 T105M) mouse DRGs (FIG.61E) suggested that mitofusin activation improved mitochondrial transport within regenerating axons. Indeed, Regeneurin treatment stimulated both axonal outgrowth and mitochondrial motility in mitochondrial dynamism-impaired CMT2A DRG neurons (FIG.61F).
  • CMT2A and ALS are rare, each having an estimated prevalence of no more than 0.01%. By contrast, 1-3% of all individuals suffering trauma from vehicle accidents, sports injuries, or occupational mishaps will suffer peripheral nerve damage requiring therapeutic intervention 19,20 .
  • mitofusin activation enhances neuronal regeneration to the in vivo context it was necessary to chemically modify Regeneurin to retard hepatocellular degradation and reduce plasma protein binding, thus producing the structurally distinct but equipotent mitofusin agonist
  • Mitolityn (FIG.76 and FIG.77Mitolityn was slowly released from a biocompatible protein matrix (FIG.78), providing for extended local release of the mitofusin agonist at the site of nerve injury.
  • Peripheral neurons are uniquely handicapped by elongated axonal
  • Rosa-STOP-mMFN Thr105Met (T105M) mice C57BL/6 Gt(ROSA)26 Sortm1 (CAG-MFN2*T105M)Dple/J) from The Jackson Laboratory (Bar Harbor, Maine, USA; Stock No: 025322) were crossed to HB9-Cre mice (B6.129S1-Mnx1tm4(cre)Tmj/J) from The Jackson Laboratory (Stock No: 006600) to generate motor neuron-targeted MFN2 T105M mice as described 6 .
  • SOD1-Gly93Ala (G93A) transgenic mice B6SJL- Tg(SOD1*G93A)1Gur/J were obtained from The Jackson Laboratory (Stock No: 002726). All surgical and experimental procedures were approved by the Washington University in St. Louis, School of Medicine Animal Studies Committee; protocol number 20160276.
  • MEFs Normal mouse embryonic fibroblasts
  • Mfn2 null and Mfn1/Mfn2 double null MEFs and NIH 3T3 fibroblasts were purchase from American Type Culture Collection (ATCC Manassas, Virginia, USA) (CRL-2994, CRL-2993, and CRL-1658 respectively).
  • MEFs were cultured at 37°C, 5% CO2-95% air in Dulbecco’s minimal essential medium (DMEM) containing glucose (4.5 g/l) with 10% (v/v) fetal bovine serum (FBS; Gibco,
  • DMEM minimal essential medium
  • FBS fetal bovine serum
  • Neonatal mouse cortical neurons were isolated from individual postnatal day 1 C57BL/6J Gly93Ala mice by papain digestion and mechanical dispersion as described 24 . Briefly, mouse brain cortices were isolated under a dissecting microscope and sliced into 0.5-1 mm thick sections in Leibovitz’s L-15 Medium (Gibco Cat:#11415-064) containing BSA (0.23mg/ml, Sigma Cat:#A7030 ). Papain (1mg/ml, Sigma Cat:#P4762) was added and the tissue digested for 20 min at 37°C. The papain solution was replaced and micropipettes used to triturate the solution until no more tissue was visible. Culture of cortical cells in microfluidic neuron XonaChip chambers and aspiration axotomy methodology is described below.
  • DRG adult mouse dorsal root ganglion
  • HBSS Hank's Balanced Salt Solution
  • Neurons were dissociated by step-wise papain digestion (1mg/ml, Worthington Biochemical, Lakewood, NJ, USA Cat:# LS003126) for 20 min at 37°C and collagenase digestion (14.5 mg/ml, Worthington Biochemical, Lakewood, NJ, USA Cat:# 41J12861) for 20 min at 37°C, followed by trituration through a P1000 pipette tip ⁇ 30 times.
  • Dissociated cells were plated on 12-well slide dishes (BD Bioscience, Missisauga, ON, Canada) coated with Poly-d-Lysine (Sigma Aldrich Cat:# P7886) and laminin (Sigma Aldrich Cat:# L2020) and cultured in Gibco Neurobasal-A medium supplemented with 2% B-27. DRG neurons were distinguished from non- neuronal cells by positive staining with anti- ⁇ -III tubulin.
  • Thr105Met and MFN2 His361Tyr were from Dr. Robert Baloh (Department of Neurology, Cedars-Sinai Medical Center, Los Angeles, CA USA).
  • Primary human fibroblasts from a CMT2A patient (MFN2 Arg274Trp) 26 were from Dr. Barbara Zablocka (Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Tru).
  • CTL5:ND34770 were purchased from the NINDS Human Cell and Data Repository, National Institutes of Health, USA. Human fibroblasts were maintained in DMEM supplemented with 15% (v/v) FBS and 1% (v/v) penicillin/streptomycin at 37°C in 5% CO2-95% air. Prior to live-cell studies fibroblasts were plated on glass coverslips and transitioned to no glucose DMEM (Thermo Fisher Scientific, Waltham,
  • Ad vectors expressing human FLAG-MFN2 17 , rat Mfn2 Lys109Ala 10 human FLAG-MFN2 Met 376Ala 6 , LC3-GFP 6 , and lentivirus expressing MFN2 Arg94Gln 21 have been previously described.
  • Ad-FLAG-MFN2 Arg94Gln was generated by PCR mutagenesis of human FLAG-MFN2 using (mutant nucleotide underlined):
  • MFN2 Arg94Gln forward primer 5’- gtgaggtgctggctcagaggcacatgaaagt -3’
  • MFN2 Arg94Gln reverse primer 5’- actttcatgtgcctctgagccagcacctcac -3’
  • Arg94Gln vector was prepared at Vector Biolabs (Malvern, PA, USA).
  • Adenoviri expressing ⁇ -galactosidase (Ad-CMV- ⁇ -Gal; #1080), human Milton1 (Ad-h-TRAK1; #ADV-226338) and human Miro1 (Ad-h-RHOT1;
  • Ad-mito-GFP mitochondrial-targeted green fluorescent protein
  • Ad-LC3-GFP was a gift from Dr. Lorrie Kirshenbaum, University of Manitoba, Canada.
  • Live adenovirus titers were determined using the AdEasy Viral Titer Kit (#972500; Agilent Technologies, Cedar Creek, TX, USA). Unless otherwise stated, viral vectors were added to cells at a multiplicity of infection (MOI) of 50.
  • MOI multiplicity of infection
  • Mouse monoclonals anti-mitofusin 2 (ab56889 - 1:1000), anti- VDAC1 (ab14734 - 1:1000), and anti MIRO1 (ab1880291:1000) were from AbCAM (Cambridge, MA, USA). Monoclonal mouse anti- ⁇ -actin (sc-47778 - 1:2000) and anti-mitofusin 1 (sc-50330) were from Santa Cruz Biotechnology (Dallas, TX, USA). Rabbit anti-TRAK1 (Milton) was from Sigma-Aldrich (HPA005853). Rabbit polyclonal anti-GAP 43 (Cat:# AB5200) was from EMD Millipore (Burlington, MA, USA). Rabbit polyclonasl anti-Stathmin-2 (SCGN10; cat # NBP1-49461) was from Novus
  • Mitochondria of cultured neurons were visualized after transduction with ad-mito-GFP 48 hours prior to study and TMRE staining 30 minutes prior to imaging. Autophagy was assessed in primary human fibroblasts after transduction with Ad- LC3-GFP 48 hours prior to visualization.
  • mice DRG axonal outgrowth used a randomized (JA) treatment scheme, and both the investigators performing confocal neuron imaging (AF) and performing Sholl analysis of those images (WK) were blinded as to treatment group.
  • AF confocal neuron imaging
  • WK Sholl analysis of those images
  • adult mouse DRGs were fixed in 4% paraformaldehyde and 4% sucrose in phosphate-buffered saline (PBS) for 10 min., permeabilized with 0.1% Triton X-100 in PBS for 15 min., and blocked in 10% goat serum (Jackson Immunoresearch, West Grove, PA, USA.
  • Sholl analyses were preformed using ImageJ as described 27 using an open source Sholl analysis plugin (https://imagej.net/Sholl_Analysis). Briefly, a starting radius was set to encompass the soma of tubulin III-positive DRG neurons. After a scale was set to convert pixel units into microns for the given images a point was placed in the center of the starting radius corresponded to the center of the soma. Next, an ending radius of 40 microns was set, establishing a circle with 40 micron radius. Concentric circles between the starting and ending radii were established at 10 micron increments.
  • the Scholl analysis plugin was initiated and numbers of axon and radii intersections were totaled for all circles to derive intersection number, corresponding to axonal branching. Maximal axon length per cell was determined as the larges radius step size with any intersections. Special attention was given to ensure that there was uniform staining along all parts of the DRG soma and axons so that the plugin was able to accurately assess the number of intersections accurately. Tubulin III-positive cells with no axon/dendrite growth were excluded.
  • Cell lysates for different mitofusin mutants in MEFs were prepared in Cell Extraction Buffer according to manufacturer instructions (Invitrogen, Thermo Fisher Scientific Cat:# FNN0011). To enrich mitochondrial proteins, cells were washed in ice-cold phosphate buffer (PBS) and centrifuged (500 x g at 4C) for 5 min. The whole-cell pellet was disrupted by dounce homogenization in isolation buffer (200 mM sucrose, 1 mM EGTA-Tris and 10 mM Tris-MOPS, pH 7.4) and the homogenate clarified by centrifugation at 800 ⁇ g for 10 min at 4 degrees C.
  • isolation buffer 200 mM sucrose, 1 mM EGTA-Tris and 10 mM Tris-MOPS, pH 7.4
  • the supernatant containing cell elements except nuclei and intact cells was recovered and a mitochondria-rich fraction obtained by centrifugation for 10 min at 8,000 ⁇ g at 4 degrees C.
  • the mitochondrial pellet was resuspended in NaCl 150 mM, EDTA 1mM, Triton 1%, PMSF 1mM, Tris 50 mM (pH 7.5) for 10 min on ice, and
  • Chemiluminescence Substrate (Thermo Scientific #32132) were used for signal detection. Quantification of immunoreactive proteins was performed on a LI-COR Odyssey infrared detection system (Lincoln, NE, USA, version 1.0.17).
  • HEK293 cells (#CRL-1573, ATCC Manassas, Virginia, USA) were subcultured onto 6 wells dishes at a density of 50,000 cells/well; after 2 days cells were at ⁇ 70% confluency. Cells were transfected with wild-type FLAG-MFN2 17 , FLAG-MFN2 Arg94Gln, or FLAG-MFN2 Met376Ala 6 plus myc-Miro1 3 as indicated using Lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer instructions.
  • Flag-MFN2–MIRO1 co-immunoprecipitation was performed essentially as described 30 . Briefly, 25 ⁇ l of anti-FLAG M2 resin (#A-2220, Sigma- Aldrich) was equilibrated in PBS and gently and continuously mixed with the clarified lysate (1mg) in a total volume of 700 ⁇ l at 4°C overnight.
  • the resin beads were washed with 1ml PBS and recovered by brief centrifugation (1,000xg at 4 ⁇ C for 1min), boiled for 10 minutes in 100 ⁇ l 2x SDS sample loading buffer containing 50mM dithiothreitol (DTT) to dissociate affinity bound protein complexes, and centrifuged for 10min at 25,000 x g.
  • FLAG-resin bound proteins in the supernatants were size- separated by SDS page electrophoresis on 10% reducing mini-gels (Biorad) and electrically transferred at 4 ⁇ C overnight for one hour with constant electrical potential of 110V using semi-dry blotting to PVDF membranes.
  • Membranes were
  • anti-mitofusin 2 (ab56889 - 1:1000) or anti MIRO1 (ab188029 1:1000) and visualized using peroxidase-conjugated anti-mouse IgG (#7076S - 1:2500) from Cell Signaling (Danvers, MA, USA).
  • Wild-type or Mfn1/Mfn2 double null MEFs were infected with Ad-h- RHOT1 (adenovirus encoding MIRO1) and Ad-h-TRAK1 (adenovirus encoding Milton) for 72 hours. Two hours prior to cell extraction Regeneurin S (1 ⁇ M) or vehicle (DMSO) were added. Proteins were extracted and quantified as described above.
  • Milton immunoprecipitation was performed using 50 ⁇ l of Protein A agarose (cat # 20333, Thermo Fisher Scientific, Waltham, MA, USA) previously equilibrated in 1ml phosphate-buffered saline, to which was added 600 ⁇ g of protein lysate and anti-TRAK1 antibody (HPA005853, Sigma, St. Louis, MO, USA)at 4°C overnight. Agarose beads were washed in PBS and sedimented at 1,000xg at 4 ⁇ C followed by boiling for 10 minutes in 100 ⁇ l 2x SDS sample loading buffer containing 50mM dithiothreitol. Immunocomplexes were resolved by SDS-PAGE electrophoresis, transferred to PVDF membranes, and proteins identified by immunoblotting for MIRO1, Milton, MFN2. Equal input protein was verified by GAPDH immunoblotting.
  • Mfn1/Mfn2 double-null MEFs were transduced with ad-MFN2 (wild- type), lenti-MFN2 Arg94Gln, ad-Mfn2 Lys109Aala, ad-MFN2 Met376Ala, or ad- ⁇ -gal (as a negative control) and 72 hours later mitochondria were isolated as described 31 .
  • penicillin/streptomycin (Gibco #15070-063) and 0.3% glucose (Sigma G 5767) (5-5 media) were added to microfluidic neuron XonaChips with 450 ⁇ m microgroove barriers (#XC450; Xona Microfluidics, Temecula, CA, USA) coated with 0.5mg/ml Poly(D)lysine (Sigma #P7280). After 10 minutes, 150 ⁇ l of 5-5 media supplemented with 0.5 ⁇ l Insulin-Transferrin-Sodium Selenite (Sigma I 1884) was added to each well and the neurons cultured under standard conditions (37 degrees C, 5%CO 2 /95% air).
  • Aspiration axotomy and post-axotomy regrowth analyses were performed as described 27 . Briefly, vacuum aspiration axotomy of DIV (days in vitro) neurons was followed by application of fresh neuron feeding media containing either mitofusin agonist Regeneurin C (100 nM final concentration) or vehicle (Me 2 SO, 1:1,000). Cells were fixed in situ; axonal outgrowth and post-axotomy regrowth were analyzed by confocal analysis of ⁇ -III tubulin positive cells.
  • DRG neurons from lumbar vertebrae 1-6 were isolated from ⁇ 8 week old MFN2 Thr105Met flox-stop transgenic 6 , SOD1 Gly93Ala transgenic 18 , or normal C57BL/6J mice using papain/collagenase dissociation as described above and in reference 25. Upon plating in 12-well Laminin-coated dishes, MFN2
  • Thr105Met floxed-stop DRG neurons were infected with Adeno-Cre to induce MFN2 Thr105Met expression.
  • DRG neurons were immediately treated with Regeneurin C (100 nM), its vehicle Me 2 SO 4 (1:1000), or forskolin (5 ⁇ M; as a positive control for axonal outgrowth) and infected with Adeno-mitoDsRed2.48 hours after plating DRGs were co-stained with Calcein AM (0.5uM; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA Cat:# C3100MP) for 30 minutes at 37°C to visualize axons.
  • Calcein AM 0.5uM; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA Cat:# C3100MP
  • Time-lapse images were acquired at 37°C, 5%CO 2 on a Nikon A1Rsi Confocal Microscope using a 40x oil objective at a frame rate of 5 seconds per frame for a total of 15 minutes (180 total frames).
  • Adeno-mitoDsRed2 was excited at 561 nm and emission monitored at 585nm.
  • Calcein was excited at 488 nm and its emission monitored at 525 nm.
  • Kymographs and quantitative data were generated using an Image-J plug-in as previously described 6 .
  • Mitolityn-4 matrix for local depo application: All procedures were performed under sterile conditions. A stock solution of 25 mg/ml Mitolityn-4 in ME 2 SO was diluted 1:10 (final concentration 2.5 mg/ml) in 0.9% w/v saline (Winchester Laboratories, Sulzbach-Laufen, Germany) and sterile-filtered (0.22 ⁇ m PVDF, #SLGV033RS, Millipore, Cork, Ireland).
  • Pre-cooled Matrigel ® solution (#354234; Corning; Corning, NY, USA) was combined with Mitolityn-4 or ME 2 SO (vehicle control) at a 3:1 ratio, immediately transferred in 0.2 ml aliquots to 1ml tuberculin syringes (#309659; Becton Dickinson, Franklin Lakes, NJ, USA) and polymerized at room temperature for 1 hour prior to administration.
  • ThermoScientificTM ThermoScientificTM.
  • the samples were loaded (2.5 ⁇ L) onto a 75 ⁇ m i.d. ⁇ 50 cm Acclaim PepMap 100 RP column.
  • the analytes were eluted at a flow rate of 300 nL/min with an acetonitrile (ACN) gradient in aqueous formic acid (FA) (1%) as mobile phase A.
  • ACN acetonitrile
  • FA aqueous formic acid
  • the full-scan mass spectra were acquired over mass-to-charge (m/z) range of 100 to 1500 to a resolution of 70,000 in the orbitrap mass analyzer.
  • the maximum injection time was 60 ms for parent-ion analysis with an automatic gain control target value of 1e6.
  • the instrument files were imported into SKYLINE (version 4.1.1.18179) for signal identification and
  • Optimal stimulating electrode position was determined that gave the greatest (CMAP) amplitude, and 3-4 independent events were recorded using a Viasys Healthcare Nicolet Biomedical instrument (Middleton, WI, USA Cat:# OL060954) running Viking Quest version 11.2 software. Three or four independent events were acquired for each study and the average data values (amplitude, latency, duration) reported. Studies were performed at 3 and 7 days after surgery. In two mice (one vehicle- treated and one Mitolityn-treated) optimal CMAP tracings could not be obtained. The contralateral non-injured leg was used as control.
  • Sciatic nerve immunohistology was performed 3d after crush injury using in vivo perfusion fixation of anesthetized mice. Mice were perfused via left ventricular puncture with ice cold phosphate buffered saline (PBS) saline, and perfusion-fixed with ice-cold 4% paraformaldehyde (PFA) in PBS. Sciatic nerves were dissected free intact from spine to ankle, fixed in PFA for 2 additional hours, and transferred to 30% sucrose/PBS overnight at 4 degrees C. The following day nerves were imbedded in optimal cutting temperature (OCT, Tissue-TEK Cat: 4583) medium and maintained thereafter at– 80 degrees C. Immunostaining was performed on 10 ⁇ m cryostat sections.
  • OCT optimal cutting temperature
  • Mfn2-deficient MEFs were seeded on day1 in 6 well plates at a density of 1x10 4 cells/ml. The next day, 4 ⁇ l of each dissolved small molecule was added to each well (to achieve a final concentration between 0 and 20 ⁇ M) and allowed to incubate over night. The solvent used for dissolving the small molecules (DMSO) was used as vehicle (0 ⁇ M). The next day, mitochondria were stained with MitoTracker Orange (100 nM; M7510; Invitrogen, Carlsbad, CA, USA).
  • NIH 3T3 fibroblasts were seeded on day1 in 6 well plates at a density of 2.5x10 4 cells/ml. The next day, 4 ⁇ l of each dissolved small molecule was added to each well (to achieve a final concentration between 0 and 100 ⁇ M) and allowed to incubate over night. The solvent used for dissolving the small molecules (DMSO) was used as vehicle (0 ⁇ M). As a positive control for cell death, 10 ⁇ M doxorubicin (D1515, Sigma, St. Louis, MO, USA) was added. The next day, live cells were stained with 0.5 ⁇ M calcein AM and 1 ⁇ M ethidium homodimer-1 (L3224; Invitrogen, Carlsbad, CA, USA)
  • One hundred ⁇ M dosing solution was prepared by diluting 10 ⁇ L of the intermediate solution (1 mM) with 90 ⁇ L 45%methanol/H2O.
  • Ninety eight ⁇ L of blank plasma was spiked with 2 ⁇ L of dosing solution (100 ⁇ M) to achieve 2 ⁇ M of the final concentration in duplicate and samples were Incubated at 37°C in a water bath.
  • 400 ⁇ L of stop solution was added to precipitate protein and mixed thoroughly.
  • 200 ng/mL tolbutamide plus 200 ng/mL labetalol in 50% ACN/MeOH were used.
  • Sample plates were centrifuged at 4,000 rpm for 10 min. An aliquot of supernatant (50 ⁇ L) was transferred from each well and mixed with 100 ⁇ L ultra pure water. The samples were shacked at 800 rpm for about 10 min before submitting to LC-MS/MS analysis.
  • test compound concentration was determined using HPLC with UV detection.
  • Binding to human and CD-1 mouse plasma proteins was measured using equilibrium dialysis. Pooled individual frozen EDTA anticoagulated plasma mouse and human samples were used as test matrix. Warfarin was used as a positive control. The test compounds were spiked into blank matrix at the final concentration of 2 ⁇ M. A 150 ⁇ L aliquot of matrix sample was added to one side of the chamber in a 96-well equilibrium dialyzer plate (HTD dialysis) and an equal volume of dialysis buffer was added to the other side of the chamber. An aliquot of matrix sample was harvested before the incubation and used as T0 samples for recovery calculation. The incubations were performed in triplicate. The dialyzer plate was placed in a humidified incubator and rotated slowly for 4 hours at 37°C.
  • Agonist in vitro stability was measured in human and mouse liver microsomes.
  • An intermediate solution (100 ⁇ M of small molecule) was initially prepared in methanol and subsequently used to prepare the working solution. This was achieved by a 10-fold dilution step of the intermediate solution in 100 mM potassium phosphate buffer.
  • Ten ⁇ L compound or control working solution was added to all wells of a 96-well plate for the time points (minutes): T0, T5, T10, T20, T30, T60, NCF60, except matrix blank.
  • microsome solution (680 ⁇ L/well) (#452117, Corning; Woburn, MA, USA; #R1000, Xenotech; Kansas City, Kansas, USA and #M1000, Xenotech; Kansas City, Kansas, USA) was dispersed to 96-well plate as reservoir according to the plate map. Then, 80 ⁇ L/well was added to every plate by ADDA (Apricot Design Dual Arm, Apricot Designs, Inc., Covina, CA, USA), and the mixture of microsome solution and compound were allowed to incubate at 37°C for about 10 min. Next, 10 ⁇ L 100 mM potassium phosphate buffer/well was added to NCF60 and incubated at 37°C (timer 1H was started).
  • ADDA Apricot Design Dual Arm, Apricot Designs, Inc., Covina, CA, USA
  • NADPH NADPH
  • regenerating system 90 ⁇ L/well of NADPH (#00616, Sigma, Aldrich, St. Louis, Missouri, USA) regenerating system was dispensed to 96-well plate as reservoir according to the plate map. Then 10 ⁇ L/well was added to every plate by ADDA to start reaction. To terminate the reaction, 300 ⁇ L/well of stop solution (cold in 4°C, including 100 ng/mL tolbutamide and 100 ng/mL labetalol as internal standards) was used, and sampling plates were shacked for approximately 10 min. The samples were next centrifuged at 4000 rpm for 20 min at 4°C and supernatants were analyzed by LC-MS/MS.
  • PAMPA Parallel artificial membrane permeability assay
  • T0 sample 20 ⁇ L donor solution was transferred to new well followed by the addition of 250 ⁇ L PBS (DF: 13.5) and 130 ⁇ L of acetonitrile (ACN) (containing internal standard) as T0 sample.
  • ACN acetonitrile
  • acceptor sample The plate was removed from incubator and 270 ⁇ L solution was transferred from each acceptor well and mixed with 130 ⁇ L ACN (containing internal standard) as acceptor sample.
  • donor sample 20 ⁇ L solution was transferred from each donor well and mixed with 250 ⁇ L PBS (DF: 13.5), 130 ⁇ L ACN (containing internal standard) as donor sample.
  • the acceptor samples and donor samples were analyzed by LC-MS/MS.
  • Example 10 Chemical synthesis, purification and analyses of small molecule mitofusin agonists
  • Regeneurin S (1-(2-((5-cyclopropyl-4-phenyl-4H-1,2,4-triazol-3- yl)thio)ethyl)-3-(2-methylcyclohexyl)urea) was synthesized as a racemic mixture (FIG.83).
  • Regeneurin C (1-(3-(5-cyclopropyl-4-phenyl-4H-1,2,4-triazol-3- yl)propyl)-3-(2-methylcyclohexyl)urea) was synthesized as a racemic mixture (FIG. 88).
  • Mitolityn-1 (1-(3-(5-cyclopropyl-4-ethyl-4H-1,2,4-triazol-3-yl)propyl)- 3-(2-methylcyclohexyl)urea) was synthesized as a racemic mixture (FIG.93).
  • compound 1-1 (245 mg, 2.16 mmol, 285 uL) and TEA (625 mg, 6.18 mmol, 860 uL) in DCM (10.0 mL) was added CDI (368 mg, 2.27 mmol) and the reaction mixture was stirred at 5 ⁇ 10 °C for 3 h.
  • Mitolityn-2 (1-cyclohexyl-3-(3-(5-cyclopropyl)-4-ethyl-4H-1,2,4- triazol-3-yl)propyl)urea) was synthesized as a racemic mixture (FIG.98).
  • compound 1-2 (215 mg, 2.17 mmol, 248 uL) and TEA (626 mg, 6.19 mmol, 861 ⁇ L) in DCM (10.0 mL) was added CDI (368 mg, 2.27 mmol) and the reaction mixture was stirred at 5 ⁇ 10°C for 3 h.

Abstract

L'invention concerne des compositions comprenant des régulateurs modulateurs à petites molécules (agents modulateurs de mitofusine). Ces agents modulateurs de mitofusine sont utiles pour traiter des maladies ou des troubles associés à une maladie, un trouble ou un état pathologique associés aux mitochondries, tels que des maladies ou des troubles associés à la mitofusine 1 (Mfn1) et/ou à la mitofusine 2 (Mfn2), ou un dysfonctionnement mitochondrial. La présente invention concerne également des procédés de traitement, des formules pharmaceutiques et des procédés de criblage permettant d'identifier des composés qui régulent la fonction mitochondriale.
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US11760733B2 (en) 2017-04-23 2023-09-19 Washington University Small molecule regulators of mitochondrial fusion and methods of use thereof
WO2021046368A1 (fr) * 2019-09-05 2021-03-11 The Board Of Trustees Of The Leland Stanford Junior University Procédés et composés modifiant la fonction mitochondriale
CN114929242A (zh) * 2019-09-05 2022-08-19 小利兰·斯坦福大学托管委员会 修饰线粒体功能的方法和化合物
CN114716386A (zh) * 2021-01-04 2022-07-08 北京和理咨询有限公司 一种新型外泌体抑制剂及其制备方法
WO2023196340A1 (fr) * 2022-04-06 2023-10-12 Dorn Ii Gerald W Activateurs de mitofusine comportant un groupe carbonyle lié endocyclique et leurs méthodes d'utilisation
CN117285533A (zh) * 2023-11-24 2023-12-26 四川省医学科学院·四川省人民医院 CD47/SIRPα小分子抑制剂及其应用和抗肿瘤药物组合物
CN117285533B (zh) * 2023-11-24 2024-01-30 四川省医学科学院·四川省人民医院 CD47/SIRPα小分子抑制剂及其应用和抗肿瘤药物组合物

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