WO2018195491A1 - Compositions and methods for the treatment of amyotrophic lateral sclerosis - Google Patents

Compositions and methods for the treatment of amyotrophic lateral sclerosis Download PDF

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WO2018195491A1
WO2018195491A1 PCT/US2018/028665 US2018028665W WO2018195491A1 WO 2018195491 A1 WO2018195491 A1 WO 2018195491A1 US 2018028665 W US2018028665 W US 2018028665W WO 2018195491 A1 WO2018195491 A1 WO 2018195491A1
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seq
mitochondrial
peptide
als
composition
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French (fr)
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Amit Umesh JOSHI
Daria Mochly-Rosen
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The Board Of Trustees Of The Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • Embodiments of the present disclosure relate generally to the use of compositions which inhibit Dynamin-related protein 1 (Drpl), e.g., peptide compounds which inhibit translocation of Drpl into effector organelles such as mitochondria, as agents for the treatment of amyotrophic lateral sclerosis.
  • Drpl Dynamin-related protein 1
  • ALS Amyotrophic lateral sclerosis
  • MN motor neurons
  • SODl -associated ALS has been the most studied form of ALS, due to the early discovery of the disease-causing mutations and the availability of mouse models. Mutations in SOD gene are gain-of-function mutations that cause autosomal dominant inheritance of ALS. It is the toxicity of the mutant SODl protein, rather than a defect in the function of the normal SODl protein, that is thought to lead to the disease. See, US Pat. App. Pub. No. 2016/0082015. Previous studies have suggested that mutations in SODl cause various cellular events, including alteration of gene expression, abnormal protein interactions, dysfunction of mitochondria and cytoskeletal abnormalities (Sau et al, Human Molecular Genetics 16, 1604-1618 (2007)). However, the causal relationship between these events and the death of the motor neurons remains unclear.
  • mutant SODl binds preferentially to mitochondria, impairs respiration, decreases the Ca 2+ buffering capacity, blocks mitochondrial protein import, and induces apoptosis through Bcl-2 inhibition (Song et al , Neurobiology of Disease 51, 72- 81 (2013)).
  • Mutant SOD1G93A also affects mitochondrial dynamics; there is a significant decrease in mitochondrial length and an accumulation of round fragmented mitochondria (Tafuri et al, supra). The increase of fragmented mitochondria coincides with an arrest in both anterograde and retrograde axonal transport and increased cell death (Magrane et al , The Journal of Neuroscience, 32, 229-242 (2012)).
  • Mutant SOD1G93A induces a reduction in neurite length and branching that is accompanied with an abnormal accumulation of rounded mitochondria in growth cones of motor neurons (Rosen et al, Nature 362, 59-62 (1993); Magrane et al , Human Molecular Genetics 18, 4552-4564 (2009)).
  • Abnormal mitochondrial dynamics was also recently observed in skeletal muscle of the SODl G93A mice (Luo et al , PloS One 8, e82112 (2013), together indicating the importance of mitochondrial dynamics in ALS.
  • Mitochondria exist in the cells as highly dynamic entities, ranging from elaborate tubular networks to small organelles. These morphological changes are orchestrated through rapid and reversible fission and fusion processes. Mitochondrial fusion is mediated by mitochondrial large GTPase on the outer mitochondrial membrane; mitochondrial fission is mediated by the recruitment of dynamin-related protein 1 (Drpl or Dynamin-l-like protein DNM1L; UNIPROT Accession Nos.
  • dynamin-related protein 1 Drpl or Dynamin-l-like protein DNM1L
  • Drpl-mediated mitochondrial fission is important for cell survival and brain development, excessive Drpl -mediated fission causes mitochondrial fragmentation, mitochondrial membrane depolarization, increase in reactive oxygen production (ROS) and oxidative stress, a decrease in ATP production and other mitochondrial physiological functions (Babbar et al , Molecular & Cellular Pharmacology 5, 109-133 (2013); Youle et al , Science 337, 1062-1065 (2012); Wu et al , FEBS Journal, 278, 941-954 (2011)).
  • ROS reactive oxygen production
  • Drpl activity has been linked to Huntington's disease, Parkinson's disease, multiple sclerosis and stroke (Filichia et al , Scientific Reports 6, 32656 (2016); Guo et al , Biochem J. 461, 137-146 (2014); Guo et al , JCI, 123, 5371-5388 (2013)), its role in the pathogenesis of ALS and neurodegeneration of motor neurons was hitherto unknown. Additionally, these previous studies are silent with regard to usefulness of new inhibitors of Drpl in the prevention and treatment of neurological disorders such as ALS.
  • Riluzole does not prevent or stop the onset of ALS but is merely effective to slow the disease's progression in certain patients that have increased levels of glutamate in the brain.
  • Riluzole only acts by reducing an excitotoxic component of the disease, and while it prolongs life by 2 to 3 months, it provides little functional improvement (Miller et al, "Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND)." Cochrane Database Systemic Reviews, CD001447, 2007).
  • Embodiments disclosed herein provide for mitochondrial fission inhibitors (including constructs thereof) in inhibiting mitochondrial fragmentation and improving mitochondrial function.
  • Embodiments disclosed herein provide for mitochondrial fission inhibitors (including constructs thereof) in reducing mitochondrial oxidative stress, triggering of apoptotic pathways and signaling cascades that lead to cell lysis and/or death.
  • Particular embodiments relate to the use in vivo of mitochondrial fission inhibitors (including constructs thereof) in the treatment of ALS.
  • a method for the treatment of amyotrophic lateral sclerosis (ALS) in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a composition comprising a mitochondrial fission inhibitor or a derivative thereof.
  • ALS amyotrophic lateral sclerosis
  • the mitochondrial fission inhibitor inhibits the interaction of Drpl with mitochondrial fission 1 protein (Fisl). In other embodiments, the mitochondrial fission inhibitor is an inhibitor of Drpl/Fisl mediated fission of mitochondria. [0014] In some embodiments, the mitochondrial fission inhibitor is a mitochondrial fission inhibitor peptide.
  • the mitochondrial fusion inhibitor peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, or 99% sequence identity to a peptide selected from the group consisting of DLLPRGS (SEQ ID NO: 1), DLLPRGT (SEQ ID NO: 2), STQELLRFPK (SEQ ID NO: 3), KLSAREQRD (SEQ ID NO: 4), CSVEDLLKFEK (SEQ ID NO: 5), KGSKEEQRD (SEQ ID NO: 6), and ELLPKGS (SEQ ID NO: 7).
  • DLLPRGS SEQ ID NO: 1
  • DLLPRGT SEQ ID NO: 2
  • STQELLRFPK SEQ ID NO: 3
  • KLSAREQRD SEQ ID NO: 4
  • CSVEDLLKFEK SEQ ID NO: 5
  • KGSKEEQRD SEQ ID NO: 6
  • ELLPKGS SEQ ID NO: 7
  • the mitochondrial fission inhibitor is a peptide selected from the group consisting of the peptides identified herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
  • the mitochondrial fission inhibitor comprises a salt, a solvate, a hydrate, a polymorph or a prodrug of the mitochondrial fission inhibitor peptide.
  • the mitochondrial fission inhibitor comprises a mitochondrial fission inhibitor peptide and a linker.
  • the linker is a poly-glycine linker comprising 2-6 glycine residues.
  • the mitochondrial fission inhibitor is administered as a composition comprising the mitochondrial fission inhibitor peptide and a carrier.
  • the carrier is a cationic lipid.
  • the carrier facilitates intracellular delivery of the mitochondrial fission inhibitor.
  • the carrier is a carrier peptide selected from the group consisting of YGRKKRRQRRR (SEQ ID NO: 8), RRRQRRKKRGY (SEQ ID NO: 9), RKKRRQRR (SEQ ID NO: 10), YARAAARQARA (SEQ ID NO: 11), THRLPRRRRRR (SEQ ID NO: 12), GGRRARRRRRR (SEQ ID NO: 13) and a combination thereof.
  • the mitochondrial fission inhibitor comprises a mitochondrial fission inhibitor peptide which is conjugated at its carboxyl-terminus or its amino-terminus, directly or via a linker, to the carrier.
  • the carrier is a carrier peptide and the mitochondrial inhibitor peptide is conjugated at its amino-terminus or its carboxyl-terminus to the carrier peptide by a polyglycine linker.
  • the polyglycine linker comprises 1-2, 1-3, 2-4, 2-3, 1-4, 1-5 or 1-6 glycine residues.
  • the method for treatment of ALS comprises administering to the subject a therapeutically effective amount of a composition comprising a compound comprising the structure PEP-L-CAR (Formula I), wherein, PEP is a peptide selected from the group consisting of peptides identified as SEQ ID NOs: 1-7, L is a linker selected from the group consisting of a peptide bond, GG, GGG, GGS, GGSG (SEQ ID NO: 14), GGSGG (SEQ ID NO: 15), GSGSG (SEQ ID NO: 16), GSGGG (SEQ ID NO: 17), GGGSG (SEQ ID NO: 18), and GSSSG (SEQ ID NO: 19), CAR is a carrier selected from the group consisting of sequences identified herein as SEQ ID NOs: 8-13, and where the position of PEP and CAR with respect linker L can be interchanged.
  • PEP is a peptide selected from the group consisting of peptides identified as SEQ ID NOs: 1-7
  • a method for treating ALS or for delaying progression of ALS is contemplated, where a composition comprising a therapeutically effective amount of a compound having the structure CARL-PEP (Formula II) or PEP-L-CAR (Formula I), wherein, PEP is a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a peptide selected from the group consisting of peptides identified as SEQ ID NOs: 1-7, L is a linker having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a linker selected from the group consisting of a peptide bond, GG, GGG, GGS, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and CAR is a carrier link
  • the structure of Formula I comprises a sequence selected from the group consisting of STQELLRFPKGGYGRKKRRQRRR (SEQ ID NO: 20),
  • the construct of Formula I comprises, consists or consists essentially of the sequence YGRKKRRQRRRGGDLLPRGT (SEQ ID NO: 27). In other embodiments, the construct of Formula I comprises, consists or consists essentially of the sequence YGRKKRRQRRRGGDLLPRGS (SEQ ID NO: 28).
  • the ALS is familial ALS.
  • the subject has a mutation in the superoxide dismutase (SOD) gene, fused-in-sarcoma (FUS1) gene, or trans active response DNA binding protein 43 kDa (TDP-43) gene, or a human homolog thereof.
  • the subject has a mutation in the superoxide dismutase 1 (SOD1) gene.
  • the subject is a human who has a G93A mutation in the human SOD1 gene.
  • the treatment results in improvement in at least one sign or symptom associated with ALS.
  • the treatment results in improvement in a histopathological trait, a behavioral trait, a physiological trait or a combination of traits associated with ALS.
  • the improvement in the histopathological trait associated with ALS comprises reduced oxidative stress, reduced cellular apoptosis, reduced death, or reduced degeneration of neuronal cells or muscle cells.
  • the improvement in the behavioral trait associated with ALS comprises improved general mobility, improved motor function, reduced phobia, reduced stereotypy, improved engagement time, improved engagement score or a combination thereof.
  • the improvement in general mobility includes improvement in the number of ambulatory episodes, improvement in the number of total ambulatory episodes, increase in moving time, increase in ambulatory time, an improvement in resting time or a combination thereof.
  • the improvement in motor function includes improved motor response to allodynia.
  • the improvement in the physiological trait associated with ALS comprises improved strength of grip.
  • the treatment with the inhibitor results in improvement in an outcome of the ALS disease in the subject.
  • the outcome is an epidemiological outcome, a histopathological outcome, or a physiological outcome, or a combination thereof.
  • the outcome is an epidemiological outcome selected from the group consisting of overall survival (OS), survival at clinical score 1 (SCS I), survival at terminal endpoint (STE), age at terminal endpoint (ATE), survival at clinical score 3 (SCS3), age at clinical score 3 (ACS3), total disease duration (TDD), duration between clinical score 2 to terminal endpoint (CS2T), time to progression of disease (TTP), time-to-death (TTD), and disease-free survival period (DFS), or a combination thereof.
  • OS overall survival
  • SCS I survival at clinical score 1
  • STE survival at terminal endpoint
  • ATE age at terminal endpoint
  • SCS3 survival at clinical score 3
  • ACS3 age at clinical score 3
  • TDD total disease duration
  • TTP time to progression of disease
  • TTD time-to-death
  • the outcome is selected from the group consisting of survival at clinical score 1 (SCSI), survival at terminal endpoint (STE), age at terminal endpoint (ATE), survival at clinical score 3 (SCS3), age at clinical score 3 (ACS3), total disease duration (TDD), and duration between clinical score 2 to terminal endpoint (CS2T) or a combination thereof.
  • SCSI survival at clinical score 1
  • STE survival at terminal endpoint
  • ATE age at terminal endpoint
  • SCS3 survival at clinical score 3
  • ACS3 age at clinical score 3
  • TDD total disease duration
  • CS2 comprises development of weakness/limpness in hind limbs
  • CS3 comprises development of paralysis of a hind limb.
  • the terminal endpoint includes paralysis in both hind limbs plus about a 20% or greater drop in the body weight or paralysis in both hind limbs plus a lack of righting reflex.
  • the improved histopathological outcome comprises attenuation in the association of Drpl with mitochondria of spinal cord neurons in the subject treated with the inhibitor compared to an untreated subject. In other embodiments, the improved histopathological outcome comprises attenuation in the levels of p62 in the mitochondrial fraction.
  • the improved physiological outcome comprises increase in body weight compared to untreated subjects.
  • a method for identifying a test compound that promotes neuron survival and/or is useful for the treatment of ALS comprises (a) contacting a cell system or a cell-free system comprising Drpl and Fisl with a test compound; (b) detecting a parameter associated with mitochondrial dysfunction in the absence and in the presence of the test agent; and (c) selecting a test compound if it modulates the parameter associated with mitochondrial dysfunction.
  • the compound is selected if it modulates the parameter associated with mitochondrial dysfunction by at least 30% compared to a control.
  • the modulation is an increase or decrease in the parameter and the control comprises an untreated cell system or an untreated cell-free system.
  • the parameter associated with mitochondrial dysfunction comprises (a) increased mitochondrial interconnectivity or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death or cell lysis; (g) increased mitochondrial accumulation of mitophagy mediators selected from LC3- phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1); (h) increased c-Jun N-terminal kinase (INK) signaling.
  • MMP mitochondrial membrane potential
  • ROS reactive oxygen species
  • the increased c-Jun N-terminal kinase (JNK) signaling results in increased phosphorylated JNK levels and/or increased levels of downstream effectors XBP1, ATF6a, phosphorylated eIF2a, GRP78 and/or CHOP.
  • the parameter associated with mitochondrial dysfunction comprises (1) increased mitochondrial Drpl recruitment; (2) increased Drpl phosphorylation; or (3) increased mitochondrial accumulation of mitophagy mediators selected from LC3-II and p62.
  • a method of diagnosing a neurological disorder in a subject comprises: (a) obtaining a biological sample from the subject comprising a sample; (b) conducting at least one assay on the sample to detect mitochondrial dysfunction; and (c) diagnosing the subject as having the neurological disorder if the level of mitochondrial dysfunction in the patient sample is greater than the level of mitochondrial dysfunction in a control.
  • the mitochondrial dysfunction comprises (a) increased mitochondrial interconnectivity or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death or cell lysis; (g) increased mitochondrial accumulation of mitophagy mediators selected from LC3-phosphatidylethanolamine conjugate (LC3- II) and p62 (SQSTM1); or (h) increased c-Jun N-terminal kinase (JNK) signaling.
  • MMP mitochondrial membrane potential
  • ROS reactive oxygen species
  • JNK c-Jun N-terminal kinase
  • the increased c-Jun N-terminal kinase (JNK) signaling results in increased phosphorylated JNK levels and/or increased levels of downstream effectors XBP1, ATF6a, phosphorylated eIF2a, GRP78 and/or CHOP.
  • the mitochondrial dysfunction comprises (1) increased mitochondrial Drpl recruitment; (2) increased Drpl phosphorylation; or (3) increased mitochondrial accumulation of mitophagy mediators selected from LC3-II and p62.
  • FIGS. 1A-1I show mitochondrial structural and functional defects in ALS patient-derived fibroblasts are mediated by Drpl/ Fisl interaction.
  • FIG. 1A shows results of cell-staining experiments, wherein ALS-patient derived fibroblasts were treated with or without PI 10 (SEQ ID NO: 28) (1 ⁇ / 24 hours) for 48 hours in defined medium and then stained with anti-TOM20 (a marker of mitochondria, 1 :500 dilution). Side panels show enlarged areas of the boxed region. Scale bar: 0.5 ⁇ .
  • FIGS. 2A-2F show that the expression of SOD-1 G93A mutant in motor neurons induces cellular stress in a Drpl -dependent manner.
  • FIG. 2B shows levels of Drpl in mitochondrial fractions, as determined by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (1 ⁇ / 24 hours).
  • VDAC was used as a loading control.
  • FIG. 2C shows levels of Drpl phosphorylation in mitochondrial fractions, as determined by immunoblotting using anti-p-Drpl S616 or anti-p-Drpl S637 antibodies in hSODl-WT and hSODl- G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (1 ⁇ / 24 hours), ⁇ -actin was used as a loading control.
  • the protein levels were quantified and represented as fold-change of hSODl-WT (means ⁇ SD).
  • n 6. **P ⁇ 0.01; ****P ⁇ 0.0001.
  • FIG. 2D shows activities of various enzymes in homogenates of hSODl-WT and hSODl- G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (1 ⁇ / 24 hours).
  • Chymotrypsin-like activity was measured using fluorogenic substrate;
  • Suc- LLVY-AMC was used to measure proteasome activity.
  • FIG. 2E shows levels of Parkin and LC3BII in mitochondrial fractions as determined via immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 ⁇ / 24 hours).
  • VDAC was used as loading control.
  • FIG. 2F shows levels of phosphorylated- eIF2a, XBP1, and CHOP in total fractions, as measured by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 ⁇ / 24 hours), ⁇ - actin was used as a loading control.
  • FIGS. 3A-3H show inhibition of Drpl hyper-activation and its interaction with Fisl using PI 10 (SEQ ID NO: 28) improves behavioral outcomes in SOD1-G93A ALS mice.
  • FIG. 3A shows a cartoon of the treatment regime starting at day 90 with pumps replaced at day 120 as well as open field test regime.
  • FIG. 3B shows tracks of median control mice, SOD1 G93A + vehicle and SOD1 G93A + PI 10 (SEQ ID NO: 28) as measured in a 10-min open field test.
  • FIG. 3C shows ambulatory episodes as analyzed using activity chamber after 10 days or 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice (*P ⁇ 0.05; **P ⁇ 0.01; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
  • FIG. 3D shows ambulatory distances as analyzed using activity chamber after 10 days or 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice (*P ⁇ 0.05; **P ⁇ 0.01; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
  • FIG. 3E shows grip strength test was carried out to assess the on muscular degeneration after 25 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P ⁇ 0.05; ***p ⁇ 0.001 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post- hoc analysis).
  • FIG. 3F shows total resting time was measured using activity chamber after 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice (*P ⁇ 0.05; **P ⁇ 0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
  • FIG. 3G shows animal behavior outcomes associated in the context of fear; center zone entries were analyzed using activity chamber after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P ⁇ 0.05; **P ⁇ 0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
  • FIG. 3H shows PCA of the entire behavioral data shows behavioral separation between the three groups after treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice.
  • FIGS. 4A-4G show inhibition of Drpl hyperactivation and its association with Fisl in the symptomatic phase improves survival and slows disease progression.
  • FIGS. 4A-4B show Kaplan-Meier survival curve of G93A SOD1 (ALS) mice showing increased survival following PI 10 (SEQ ID NO: 28) treatment (dotted trace) as compared to the vehicle-treated control ALS mice (TAT; SEQ ID NO: 8) (solid trace) (FIG. 4A) and that the age at terminal endpoint was significantly improved in the P110-treated ALS mice (FIG. 4B).
  • Treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day using 28-day pumps with pumps replaced at 120 days Log-rank Mantel-cox test; p ⁇ 0.03; Mann Whitney test; p ⁇ 0.03).
  • FIGS. 4C-4D show the curve of time to clinical score 3, defined as a point wherein mice showed single leg paralysis.
  • PI 10 treatment (dotted trace) delayed the onset as compared to the vehicle treated control -treatment (TAT; SEQ ID NO: 8) (solid trace) (FIG. 4C).
  • FIG. 4D shows that the age at which mice reached the clinical score 3 was significantly improved in the PI 10 (SEQ ID NO: 28) treated ALS mice.
  • FIG. 4F is a Kaplan-Meier survival curve of G93A SOD1 (ALS) mice showing increased probability of survival post-paralysis following PI 10 (SEQ ID NO: 28) treatment (dotted trace) as compared to the vehicle-treated control ALS mice (TAT; SEQ ID NO: 8) (solid trace).
  • FIG. 4G shows spinal cord levels of Drpl in mitochondrial fractions, as determined by immunoblotting in WT and SOD1-G93A treated with either vehicle control or with PI 10 (SEQ ID NO: 28) at 3mg/kg/ day.
  • FIGS. 5A-5E show results of immunoblotting experiments.
  • FIG.6C shows levels of cytochrome c, BAX and Bcl-2 were examined in mitochondrial fractions by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 ⁇ / 24 hours); VDAC was used as a loading control.
  • FIG. 6D shows levels of phosphorylated- JNK, p62 and LC3BII in total fractions by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 ⁇ / 24 hours); beta-actin was used as a loading control for total fraction.
  • FIG. 6E shows levels of ATF6 and GRP78 in total fractions were measured by
  • FIG. 7A shows no significant difference in the onset of clinical score 1 between the two groups when the treatment was started.
  • FIG. 7C shows ambulatory time analyzed using activity chamber after 10 days or 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice (*P ⁇ 0.05; **P ⁇ 0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
  • FIG. 7D shows animal behavior outcome associated with motor function; jump time, analyzed using activity chamber after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P ⁇ 0.05; **P ⁇ 0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
  • FIG. 7E shows animal behavior outcome associated with motor function; jump count, analyzed using activity chamber after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P ⁇ 0.05; **P ⁇ 0.01; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
  • FIG. 7F shows animal behavior outcome associated in the context of fear; latency towards center, analyzed using activity chamber after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice.
  • FIG. 7G shows animal behavior outcome associated in the stereotypy analyzed using activity monitor after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice.
  • FIG. 7H shows animal behavior outcome associated in the total resting time analyzed using activity monitor after 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice.
  • FIGS. 8A-8B are a Western blot and analysis that show increased GFAP staining, which correlates with astrocytosis, and beta-actin staining, a marker of microglia, were blunted by treatment with PI 10 (SEQ ID NO: 28) in ALS mice relative to wild type mice or control (TAT, SEQ ID NO: 8) treated mice.
  • FIG. 8C shows the fold change in calcium-binding protein (SI 00b) in wild type mice and in ALS mice treated with vehicle control or with PI 10 (SEQ ID NO: 28).
  • FIGS. 8D-8G are bar graphs showing the tissue levels of several cytokines, interleukin- lbeta (FIG. 8D), interleukin 1 -alpha (FIG. 8E), interleukin-6 (FIG. 8F) and tumor necrosis factor- alpha (FIG.8G).
  • FIGS. 9A-9D shows results of treating a microglial cell line transfected with SOD1G93A mutant gene (WT) and treated with TAT (SEQ ID NO: 8) control or with PI 10 (SEQ ID NO: 28), FIG. 9A showing the mitochondrial aspect ratio, FIG. 9B the intracellular ATP, FIG. 9C the mitoSOX and FIG. 9D the total ROS in the transfected cells untreated (squares), control-treated (triangles) and PI 10-treated (inverted triangles).
  • Embodiments described herein reveal that mutations in SODl, FUS 1 or TDP-43 in fibroblasts derived from patients with ALS exhibited decreased mitochondrial function due to Drpl hyperactivation and thereby increasing mitochondrial fragmentation.
  • the data provided herein show that fibroblasts harboring mutations in any one of these three different genes all have a common pre- apoptotic mitochondrial defect trigger - Drpl-Fisl interaction.
  • Drpl association with the mitochondria was much greater relative to cells from healthy subjects, which was reduced by treatment with a selective peptide inhibitor of Drpl-Fisl interaction, exemplified by the peptide of SEQ ID NO: 1, and administered in the form of a peptide-carrier construct referred to as "PI 10" (SEQ ID NO: 28).
  • SEQ ID NO: 1 is exemplary and that the other peptides identified herein are contemplated and suitable, alone or in the form of a peptide-carrier construct, optionally with a linker.
  • Pl lO-mediated reduction in Drpl recruitment and activation at the mitochondrial axis was independent of the aforementioned mutations.
  • the instant disclosure establishes a link between SOD1 mutation and impairment in proteasome activity in the context of pathogenesis and progression of ALS.
  • the present disclosure reveals that Drpl inhibitors improve mitochondrial health, as evidenced by attenuated ROS production, reduced pro-apoptotic triggers, increased ATP production and reduced mitochondrial autophagy in two different in vitro model systems for ALS.
  • these findings point to a role of Drpl inhibitors in the diagnosis of mitochondrial dysfunction associated with many neurological disorders, e.g. , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc.
  • Drpl inhibitors such as the antagonist peptide in the PI 10 construct
  • the therapeutic utility of Drpl inhibitors was elucidated in an ALS mouse model Treating ALS-model mice with the inhibitor comprising a peptide of SEQ ID NO: 1 conjugated to a TAT deliver ⁇ ' moiety drastically improves clinical symptoms, which appear around day 90, such as reduced dragging hind feet/knuckles (Stage 1 ).
  • sustained treatment with the peptide-TAT conj ugate e.g. , SEQ ID NO: 28
  • the peptide-TAT conj ugate starting at the onset of the symptoms significantly improved behavioral outcomes, motor functioning and also increased survival.
  • embodiments disclosed herein provide the first evidence for the potential clinical and therapeutic utility of the peptides disclosed herein for the treatment of AL S patients.
  • the present disclosure relates to inhibitors of mitochondrial fission, particularly compositions which inhibit interaction between dynamin-related protein 1 (Drpl) and mitochondrial fission 1 protein (Fisl).
  • the mitochondrial fission inhibitor can be a small molecule (e.g., a hapten), a peptide, a protein, an antibody, an aptamer, a nucleic acid, or a combination thereof.
  • the mitochondrial fission inhibitor is a peptide (PEP), which optionally comprises a linker (L) and a carrier moiety (CAR).
  • PEP peptide
  • L linker
  • CAR carrier moiety
  • the mitochondrial fission inhibitor comprises the inhibitor peptide (PEP) compound only.
  • the mitochondrial fission inhibitor comprises the inhibitor peptide compound (PEP) and the carrier moiety (CAR), wherein the inhibitor peptide is linked, either covalently or non-covalently, to the carrier moiety.
  • PEP inhibitor peptide compound
  • CAR carrier moiety
  • the mitochondrial fission inhibitor comprises the inhibitor peptide compound (PEP) and both the linker (L) and the carrier moiety (CAR), wherein the inhibitor peptide is linked, either covalently or non-covalently, to the linker and the linker is linked, either covalently or non-covalently, to the carrier.
  • PEP inhibitor peptide compound
  • L linker
  • CAR carrier moiety
  • the mitochondrial fission inhibitor is a construct comprising the structure PEP-L-CAR (Formula I), wherein, PEP is the inhibitor peptide, L is a linker which is either present or absent, and CAR is a carrier moiety.
  • the mitochondrial fission inhibitor comprises the structure CAR-L-PEP (Formula II), wherein CAR, L and PEP are as described above.
  • the peptide inhibitor is linked to the linker, which is linked to a carrier via a covalent bond.
  • the PEP, L and CAR groups in the construct of Formula I or Formula II are linked together via a peptide bond.
  • a "peptide bond” is formed by the condensation reaction between two amino acids, wherein the acid moiety of one reacts with the amino moiety of the other to produce a peptide bond (-CO-NH-) between the two amino acids.
  • a mitochondrial fission inhibitor peptide construct comprises, in order from NH 2 (amino) terminus to COOH (carboxy) terminus: (a) inhibitor peptide (PEP); (b) optionally a linker (L); and (c) carrier (CAR).
  • a mitochondrial fission inhibitor peptide comprises, in order from NH 2 (amino) terminus to COOH (carboxyl) terminus: (a) carrier (CAR); (b) optionally a linker (L); and (c) inhibitor peptide (PEP).
  • the various components of the constructs of Formula I or Formula II are linked together via one or more peptide bonds.
  • Peptide (PEP) Peptide
  • the disclosure relates to a mitochondrial fission inhibitor peptide (PEP), which inhibits mitochondrial fission in a cell under pathological conditions, but does not inhibit mitochondrial fission in normal control cells.
  • PEP mitochondrial fission inhibitor peptide
  • a mitochondrial fission inhibitor peptide of the present disclosure is useful for inhibiting aberrant (pathological) mitochondrial fission.
  • Representative mitochondrial fission inhibitor peptides are disclosed in U.S. Patent Nos. 9,243,232 and 9,243,232, the disclosures in which are incorporated by reference in their entirety.
  • the mitochondrial fission inhibitor peptide can have a length of from about 7 amino acids to about 50 amino acids, e.g. , from about 7 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, or from about 45 amino acids to about 50 amino acids, or longer than 50 amino acids.
  • a mitochondrial fission inhibitor peptide can have a length of from about 7 amino acid residues to about 20 amino acid residues, e.g. , a mitochondrial fission inhibiting peptide can have a length of 7 residues, 8 residues, 9 residues, 10 residues, 11 residues, 12 residues, 13 residues, 14 residues, 15 residues, 16 residues, 17 residues, 18 residues, 19 residues, or 20 residues, or more.
  • peptide includes a natural peptide comprising a linear chain or branched amino acids, peptidomimetics, as well as pharmaceutically acceptable salts thereof.
  • a peptide comprises a plurality of amino acid residues, e.g. , 2, 3, 4, 5, 6, 8, 10, or more amino acid residues which are bonded to each other via covalent bonds, e.g., a peptide bond.
  • amino acid residue means the individual amino acid units incorporated into the peptides of the disclosure.
  • amino acid means a naturally occurring or synthetic amino acid, as well as amino acid analogs, stereoisomers, and amino acid mimetics that function similarly to the naturally occurring amino acids. Included by this definition are natural amino acids such as: (1) histidine (His; H) (2) isoleucine (He; I) (3) leucine (Leu; L) (4) Lysine (Lys; K) (5) methionine (Met; M) (6) phenylalanine (Phe; F) (7) threonine (Thr; T) (8) tryptophan (Tip; W) (9) valine (Val; V) (10) arginine (Arg; R) (11) cysteine (Cys; C) (12) glutamine (Gin; Q) (13) glycine (Gly; G) (14) proline (Pro; P) (15) serine (Ser; S) (16) tyrosine (Tyr; Y) (17) alanine (Ala;
  • the mitochondrial fission inhibitor peptide comprises the amino acid sequence set forth in the sequences identified herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a variant thereof or a derivative thereof.
  • the term "derivative” includes salts, amides, esters, enol ethers, enol esters, acetals, ketals, acids, bases, solvates, hydrates, polymorphs or prodrugs of the individual amino acids or the aforementioned inhibitor peptides. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization.
  • the derivatives suitable for use in the methods described herein may be administered to animals or humans without substantial toxic effects and either are biologically active or are prodrugs.
  • the derivatives comprise salts of the amino acids or the inhibitor peptide.
  • salt includes salts derived from any suitable of organic and inorganic counter ions well known in the art and include, by way of example, hydrochloric acid salt or a hydrobromic acid salt or an alkaline or an acidic salt of the aforementioned amino acids.
  • the derivative can, in addition or alternatively, be a solvent addition forms, e.g., a solvate or alcoholate.
  • Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water; alcoholates are formed when the solvent is alcohol. Solvates of compounds described herein can be conveniently prepared or formed using routine techniques. Polymorphs refer to alternate crystal forms of the compounds described herein.
  • Polymorphic purity of drug samples can be checked using techniques such as powder X-ray diffraction, IR/Raman spectroscopy, and utilizing the differences in their optical properties in some cases (Thomas et al , Chemical Communications, 48: 10559-10561 (2012)).
  • the derivative can further comprise amides or esters of the amino acids and/or isomers (e.g. , tautomers or stereoisomers) of the amino acids, as desired.
  • the mitochondrial fission peptide is a variant of the peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.
  • variant refers to a biomolecule (e.g., polypeptide or nucleic acid) whose sequence that differs from that of a parent sequence by virtue of at least one modification or amino acid substitution. Accordingly, variant peptides comprise at least one modification or substitution of an amino acid residue.
  • Variant peptides can have at least 1 amino acid substitution compared to the parent polypeptide, e.g. , from about 1 to 10 ten amino acid substitutions, 1 to about 5 amino acid substitutions compared to the parent, or 1, 2, 3, 4 or 5 amino acid substitutions, e.g., differing in amino acid sequence by one, two, three, four, or five amino acids, compared to amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.
  • the variant mitochondrial fission inhibitor peptide may comprise a sequence which is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, identical to, for example, one of the following polypeptide sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
  • conservative amino acid substitutions in the context of a mitochondrial fission inhibitor peptide are selected so as to preserve activity of the peptide. Residues that are semi- conserved may tolerate changes that preserve charge, polarity, and/or size.
  • a mitochondrial fission inhibitor peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 may have 1, 2 or 3 amino acid substitutions, at position 1, 2, 3, 4, 5, 6, and/or 7, wherein the substituted amino acid may be any one of the known 20 amino acids, wherein the inhibitor peptide maintains a mitochondrial fission inhibiting function.
  • the mitochondrial fission inhibitor peptide of Formula I or Formula II can include a linker which joins or links a carrier moiety or peptide to a mitochondrial fission inhibitor peptide.
  • the linker may be a peptide having any of a variety of amino acid sequences.
  • a linker which is a spacer peptide can be of a flexible nature, although other chemical linkages are not excluded.
  • a linker peptide can have a length of about 1, 2, 3, 4, 5 amino acids or from about 1 to 2, 1 to 3, 2 to 4, 2 to 5, 1 to 5, 5 to 10, 10 to 20, 20 to 30, or 30 to 40 amino acids in length. Peptide linkers with a degree of flexibility can be used.
  • the linking peptides may have virtually any amino acid sequence, where in some embodiments the linker peptide will have a sequence that results in a generally flexible peptide.
  • the use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art.
  • Various linkers are commercially available and are considered suitable for use.
  • Exemplary flexible linkers which can be used to join or link a carrier moiety to a mitochondrial fission inhibitor peptide, for example, via peptide bonds, include glycine polymers (G)n, (e.g., where n is an integer from 1 to about 20); glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 29) and (GGGS)n (SEQ ID NO: 30), where n is an integer of between 1 and 10, e.g. , 1, 2, 3, 4, 5, 6, 7, or more), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art.
  • G glycine polymers
  • Glycine and glycine-serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components. Glycine polymers are used in some embodiments. See Scheraga et al, in Reviews in Computational Chemistry, K. B. Lipkowitz and D. B. Boyd, Eds., VCH Publishers, New York, 1992, Vol. 3, pp. 73-142 (1992).
  • Exemplary flexible linkers include, but are not limited to GG, GGG, GGS, GGSG (SEQ ID NO: 14), GGSGG (SEQ ID NO: 15), GSGSG (SEQ ID NO: 16), GSGGG (SEQ ID NO: 17), GGGSG (SEQ ID NO: 18), GSSSG (SEQ ID NO: 19), and the like.
  • the linker is non-peptide linker.
  • Non-peptide linker moieties can also be used to join or link a carrier moiety to a mitochondrial fission inhibitor peptide.
  • the linker molecules are generally about 6-50 atoms long.
  • the linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof.
  • Other linker molecules which can bind to polypeptides may be used in light of this disclosure.
  • the inhibitor peptide may be linked to the carrier peptide by a disulfide bond.
  • the disulfide bond is formed between two cysteines, two cysteine analogs or a cysteine and a cysteine analog.
  • both the modulatory peptide and the carrier peptide contain at least one cysteine or cysteine analog.
  • the cysteine residue or analog may be present as the N-terminal or C-terminal residue of the peptide or as an internal residue of the inhibitor peptide and of the carrier peptide.
  • the disulfide linkage is then formed between the sulfur residues on each of the cysteine residues or analogs.
  • the disulfide linkage may form between, for example, the N-terminus of the inhibitor peptide and the N-terminus of the carrier peptide, the C- terminus of the inhibitor peptide and the C-terminus of the carrier peptide, the N-terminus of the inhibitor peptide and the C-terminus of the carrier peptide, the C-terminus of the inhibitor peptide and the N-terminus of the carrier peptide, or any other such combination including at any internal position within the inhibitor peptide and/or the carrier peptide.
  • the peptide inhibitor containing an additional amino acid comprising a reactive side chain e.g., SH group of cysteine may be coupled to the carrier via click chemistry. See Liang et al. , J. Angew. Chem., Int. Ed., 48, 965 (2009).
  • the construct of Formula I or Formula II may include a carrier moiety.
  • Carrier moiety refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane.
  • a carrier moiety attached to another molecule facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle.
  • a carrier moiety facilitates crossing the blood-brain barrier.
  • a carrier moiety is covalently linked to the amino terminus of a mitochondrial fission inhibiting peptide.
  • a carrier moiety is covalently linked to the carboxyl terminus of a mitochondrial fission inhibiting peptide.
  • the carrier moiety is a carrier peptide and is covalently linked to a fission inhibiting peptide, e.g. , via a peptide bond.
  • the carrier peptide can be a peptide having a length of from about 5 to 50 amino acids, e.g., from about 5 to 10, 5 to 15, 10 to 15, 10 to 20, 15 to 20, 10 to 25, 20 to 25, 20 to 30, or 30 to 40 amino acids.
  • Exemplary carriers which may be linked to the mitochondria fission inhibitor peptide include but are not limited to a minimal undecapeptide protein transduction domain corresponding to residues 47-57 of human immunodeficiency virus-1 (HIV-1) TAT (GENBANK Acc. No. AEB53027;
  • YGRKKRRQRRR SEQ ID NO: 8
  • RRRQRRKKRGY SEQ ID NO: 9
  • a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al , Cancer Gene Ther. 9(6):489-96 (2002)); a Drosophila Antennapedia protein transduction domain (Noguchi et al. Diabetes 52,1732-1737 (2003)); a truncated human calcitonin peptide (Trehin et al. Pharm. Research 21,1248-1256 (2004)); polylysine (Wender et al. , PNAS USA 97,13003-13008 (2000)); RRQRRTSKLMKR (SEQ ID NO: 31); transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 32);
  • RQIKIWFQNRRMKWKK (SEQ ID NO: 34).
  • exemplary carriers include but are not limited to, YGRKKRRQRRR (SEQ ID NO: 8); RRRQRRKKRGY (SEQ ID NO: 9); RKKRRQRRR (SEQ ID NO: 10); an arginine homopolymer of from 3 arginine residues to 50 arginine residues; a TAT (48-60) polypeptide comprising the sequence GRKKRRQRRRPPQ (SEQ ID NO: 35); R9 peptide comprising the sequence RRRRRRRRR (SEQ ID NO: 36); a penetratin peptide comprising the sequence
  • RQIKIWFQNRRMKWKK (SEQ ID NO: 34); a pentratin-arginine fusion peptide (Pen-Arg) comprising the sequence RQIRIWFQNRRMRWRR (SEQ ID NO: 37); a pVEC peptide comprising the sequence LLIILRRRIRKQAHAHSK (SEQ ID NO: 38); an M918 peptide comprising the sequence MVTVLFRRLRIRRACGPPRVRV (SEQ ID NO: 39); a TP10 peptide comprising the sequence AGYLLGKINLKALAALAKKIL (SEQ ID NO: 40).
  • Preferred carrier sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 8); RRRQRRKKRGY (SEQ ID NO: 9); RKKRRQRR (SEQ ID NO: 10); YARAAARQARA (SEQ ID NO: 11); THRLPRRRRRR (SEQ ID NO: 12); and GGRRARRRRRR (SEQ ID NO: 13).
  • the carrier is a short amphipathic peptide carrier, Pep-1, which can facilitate rapid cellular uptake of various peptides, proteins, and even full-length antibodies with high efficiency and less toxicity. See Morris et al, Nat. Biotechnol., 19, 1173-1176, 2001.
  • the left-most amino acid residue is normally the N-terminal end of the carrier peptide, which is conjugated to the C-terminal end of the mitochondrial fission inhibitor peptide (PEP) or the C-terminal end of the linker (L), e.g., via a covalent bond, particularly a peptide bond.
  • PEP mitochondrial fission inhibitor peptide
  • L linker
  • the orientation of the carrier may be interchanged as long as the resulting construct possesses the desired activity, e.g. , inhibit the interaction between Drpl and Fisl, prevent mitochondrial fission, and/or attenuate cellular apoptosis or death due to mitochondrial injury.
  • the carrier peptide (CAR) is conjugated to the N-terminal end of the mitochondrial fission inhibitor peptide (PEP) or the linker (L), e.g., to form a structure CAR-PEP or CAR-L-PEP
  • a reverse orientation of the conjugation i.e. , C-terminal end of the carrier peptide (rightmost amino acid) is conjugated to the N-terminal end of the linker (L) or the N-terminal end of the mitochondrial fission inhibitor peptide (PEP)
  • a reverse orientation of the conjugation i.e. , C-terminal end of the carrier peptide (rightmost amino acid) is conjugated to the N-terminal end of the linker (L) or the N-terminal end of the mitochondrial fission inhibitor peptide (PEP)
  • CAR carrier peptide
  • L mitochondrial fission inhibitor peptide
  • the carrier is a non-peptide molecule, e.g. , cationic lipids as described in Zuris et al., Nat Biotechnol. 33(l):73-80 (2015)).
  • Non-limiting examples of a mitochondrial fission inhibitor peptide construct of Formula I include the following fusion peptides:
  • KLSAREQRD-GG-YGRKKRRQRRR SEQ ID NO: 21
  • KGSKEEQRD-GG-YGRKKRRQRRR SEQ ID NO: 25
  • the construct is a linear construct. In another embodiment, the construct is cyclic. In one embodiment, the construct is no more than 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, or 65 amino acids in length.
  • embodiments further include variants which comprise an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, or 99% amino acid sequence identity to the constructs identified herein.
  • the mitochondrial fission inhibitor peptide can comprise an amino acid sequence differing in amino acid sequence by 1, 2, 3, 4 or 5 amino acids, compared to the core structure of Formula I or Formula II.
  • the variant peptide can comprise additional amino acids at the C-terminal end (e.g. , the arginine R residue in the aforementioned compounds), which allow functionalization or derivatization.
  • the variant peptide can comprise additional amino acids at the linker region (e.g. , the glycine G residues in the middle portion of aforementioned compounds), which confer desired length, hydrophilicity and other physiochemical properties to the variant peptides.
  • additional amino acids at the linker region e.g. , the glycine G residues in the middle portion of aforementioned compounds
  • variant mitochondrial fission inhibitor peptides comprising a mutation in the core peptide sequence set forth in STQELLRFPK (SEQ ID NO: 3), KLSAREQRD (SEQ ID NO: 4), DLLPRGS (SEQ ID NO: 1), DLLPRGT (SEQ ID NO: 2), CSVEDLLKFEK (SEQ ID NO: 5), KGSKEEQRD (SEQ ID NO: 6) or ELLPKGS (SEQ ID NO: 7).
  • the mutation is a substitution, deletion, addition of 1, 2, 3, 1-2, or 1-3 amino acids, wherein the mutation(s) does (do) not change the in vivo or in vitro activity of the inhibitor peptide construct as described herein.
  • a subject peptide comprises one or more modifications.
  • a mitochondrial fission inhibitor construct or peptide can be cyclized.
  • a subject peptide can have one or more amino acid modifications.
  • a subject mitochondrial fission inhibitor construct or peptide can include one or more D-amino acids.
  • Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g. , acetylation, or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are peptides that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
  • mitochondrial fission inhibitor peptides or a construct of Formula I or Formula II
  • Analogs of such peptides include those containing residues other than naturally occurring L-amino acids, e.g. , D-amino acids or non-naturally occurring synthetic amino acids.
  • a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes.
  • various post-translational modifications may be achieved.
  • one may provide famesylation or prenylation.
  • a mitochondrial fission inhibitor construct or peptide can be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as a liposome.
  • Formula I or Formula II include, e.g. , (1) end-cappings of the terminal of the peptides, such as amidation of the C-terminus and/or acetylation or deamination of the N-terminus; (2) introducing peptidomimetic elements in the structure; and (3) cyclization, in which the cyclization of the peptide can occur through natural amino acids or non-naturally-occurring building blocks.
  • a modified mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be a peptoid (N-substituted oligoglycines), e.g.
  • a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can include naturally-occurring and non-naturally occurring amino acids.
  • a subject mitochondrial fission inhibitor construct or peptide can comprise D-amino acids, a combination of D- and L-amino acids, and various "designer" amino acids (e.g., ⁇ -methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids, etc.) to convey special properties to peptides.
  • a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be a cyclic peptide.
  • a subject mitochondrial fission inhibitor construct or peptide can include non-classical amino acids in order to introduce particular conformational motifs. Any known non-classical amino acid can be used.
  • Non-classical amino acids include, but are not limited to, l,2,3,4-tetrahydroisoquinoline-3-carboxylate; (2S,3S)-methylphenylalanine, (2S,3R)-methyl- phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine; 2- aminotetrahy dronaphthalene-2-carboxylic acid; hydroxy- 1 ,2,3,4-tetrahy droisoquinoline-3-carboxylate; ⁇ -carboline (D and L); HIC (histidine isoquinoline carboxylic acid); and HIC (histidine cyclic urea).
  • Amino acid analogs and peptidomimetics can be incorporated into a subject mitochondrial fission inhibitor construct or peptide to induce or favor specific secondary structures, including, but not limited to, LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a ⁇ -turn inducing dipeptide analog; ⁇ -sheet inducing analogs; ⁇ -turn inducing analogs; a-helix inducing analogs; ⁇ -turn inducing analogs; Gly-Ala turn analog; amide bond isostere; tretrazol; and the like.
  • LL-Acp LL-3-amino-2-propenidone-6-carboxylic acid
  • a ⁇ -turn inducing dipeptide analog ⁇ -sheet inducing analogs
  • ⁇ -turn inducing analogs a-helix inducing analogs
  • ⁇ -turn inducing analogs Gly-Ala turn analog
  • a non-naturally occurring amino acid can provide for linkage to a polymer, a second polypeptide, a scaffold, etc.
  • a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) linked to a water-soluble polymer can be made by reacting a water-soluble polymer (e.g. , poly(ethylene glycol) (PEG)) that comprises a carbonyl group to an the subject mitochondrial fission inhibitor construct or peptide that comprises a non-naturally encoded amino acid that comprises an aminooxy, hydrazine, hydrazide or semicarbazide group.
  • PEG poly(ethylene glycol)
  • a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) is linked (e.g. , covalently linked) to a polymer (e.g. , a polymer other than a polypeptide).
  • a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be linked to a water-soluble polymer (e.g., PEG).
  • Suitable polymers can have an average molecular weight in a range of from 500 Da to 50,000 Da, e.g. , from 5000 Da to 40,000 Da, from 25,000 to 40,000 Da, or from 40,000 to 60,000 Da.
  • a mitochondrial fission inhibitor peptide can be isolated and purified in accordance with conventional methods of recombinant synthesis.
  • a lysate may be prepared of the expression host and the lysate purified.
  • the compositions which are used will comprise at least 80% by weight of the desired product, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. The percentages can be based upon total protein.
  • a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) may be prepared by in vitro (e.g. , cell-free) synthesis, using conventional methods as known in the art.
  • Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, Calif, Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
  • various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface, or provide some other desired property such as increased solubility, increased resistance to proteolysis, increased in vivo half-life, and the like.
  • One or more cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.
  • a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) as described herein may be in the form of a pharmaceutically acceptable salt.
  • Pharmaceutically acceptable salts include acid addition salts, such as hydrochloride, hydrobromide, sulfurate, nitrate, phosphorate, acetate, propionate, glycolate, pyruvate, oxalate, malate, malonate, succinate, maleate, fumarate, tartarate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, p- toluene-sulfonate, salicylate and the like, and base addition salts, such as sodium, potassium, calcium, magnesium, lithium, aluminum, zinc, ammonium, ethylenediamine, arginine, piperazine, etc.
  • compositions comprising a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II).
  • the composition can comprise, in addition to a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II), one or more of: a salt, e.g., NaCl, MgCl, KC1, MgS0 4 , etc.; a buffering agent, e.g., a Tris buffer, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-( ⁇ - ⁇ ⁇ 1 ⁇ ) ⁇ 3 ⁇ 68 ⁇ 1 ⁇ acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.;
  • a salt
  • Compositions comprising a mitochondrial fission inhibitor peptide may include a buffer, which is selected according to the desired use of the peptide, and may also include other substances appropriate to the intended use. Those skilled in the art can readily select an appropriate buffer, a wide variety of which are known in the art, suitable for an intended use.
  • a composition comprising the mitochondrial fission inhibitor peptide is a pharmaceutical composition containing a pharmaceutically acceptable carrier.
  • a pharmaceutical composition can be administered to a subject in need thereof (e.g., a subject in need of inhibition of abnormal (e.g. , pathological) mitochondrial fission).
  • a subject pharmaceutical composition comprises: a) a mitochondrial fission inhibitor construct or peptide; and b) a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein.
  • Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, "Remington: The Science and Practice of Pharmacy", 19th Ed.
  • kits or other articles of manufacture which contains one or more of the mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) or a composition comprising the same, together with instructions for formulating and/or using the composition, e.g. , in the treatment of ALS.
  • Kits or other articles of manufacture may include a container, a syringe, vial and any other articles, devices or equipment useful in administration (e.g. , intravenous, subcutaneous, or inhalation).
  • Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), ampules, cartridges, reservoirs, pumps, or lyo-jects.
  • the container may be formed from a variety of materials such as glass or plastic.
  • a container is an osmotic pump, e.g. , ALZET pump, DUROS pump (see, Rohloff et al , J Diabetes Sci Technol., 2, 461-467 (2008)) or Intarcia Pump 650 (Intarcia Therapeutics, Boston, MA, USA).
  • Suitable pre- filled syringes include, but are not limited to, borosilicate glass syringes with baked silicone coating, borosilicate glass syringes with sprayed silicone, or plastic resin syringes without silicone.
  • the container may hold one or more formulations and a label on, or associated with, the container that may indicate directions for reconstitution and/or use.
  • the label may indicate that the formulation is reconstituted to concentrations as described above.
  • the label may further indicate that the formulation is useful or intended for, for example, subcutaneous administration.
  • a container may contain a single dose of a stable formulation containing the peptide inhibitor or the construct of Formula I or Formula II.
  • a single dose of the stable formulation is present in a volume of less than about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml, or less.
  • a container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the formulation.
  • Kits or other articles of manufacture may further include a second container comprising a suitable diluent (e.g., BWFI, saline, buffered saline).
  • the final peptide concentration in the reconstituted formulation will generally be at least 1 ⁇ g/ml (e.g. , at least 5 ⁇ g/ml, at least 10 ⁇ g/ml, at least 20 ⁇ g/ml, at least 50 ⁇ g/ml, at least 100 ⁇ g/ml, at least 300 ⁇ g/ml, at least 500 ⁇ g/ml, at least 1 mg/ml, at least 3 mg/ml, at least 10 mg/ml or more).
  • 1 ⁇ g/ml e.g. , at least 5 ⁇ g/ml, at least 10 ⁇ g/ml, at least 20 ⁇ g/ml, at least 50 ⁇ g/ml, at least 100 ⁇ g/ml, at least 300 ⁇ g/ml, at least 500 ⁇ g/ml, at least 1 mg/ml, at least 3 mg/ml, at least 10 mg/ml or more.
  • Kits or other articles of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • kits or other articles of manufacture may include an instruction for self-administration.
  • a subject synthetic nucleic acid comprises a nucleotide sequence encoding a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II). Included herein are nucleic acids encoding the mitochondrial fission inhibitor peptides identified herein by SEQ ID NOs: 1-7, or a variant thereof having 1, 2, 3, 4, or 5 amino substitutions, the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof.
  • nucleic acids encoding the following mitochondrial fission inhibitor peptide constructs of Formula I or Formula II, including the constructs identified herein by SEQ ID NOs: 21-28, or a variant thereof having 1, 2, 3, 4, 5, 4-10, 5-10, or 5 to 15 amino acid substitutions, or the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof.
  • vectors which contain one or more of the aforementioned nucleic acids.
  • the vector comprises at least one protein encoding nucleic acid, e.g. , nucleic acids encoding the mitochondrial fission inhibitor peptide sequences for Formulas disclosed herein.
  • a nucleotide sequence encoding a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) as described herein can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in the intended target cells (e.g., a cell that is genetically modified to synthesize the encoded mitochondrial fission inhibitor peptide or construct).
  • a subject nucleic acid is a recombinant expression vector.
  • the present disclosure provides isolated genetically modified host cells (e.g., transformed cells or cell-lines) that are genetically modified with a nucleic acid comprising a nucleic acid sequence which encodes a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) or which harbor an expression vector construct as described herein.
  • a subject isolated genetically modified host cell can produce a mitochondrial fission inhibitor construct or peptide.
  • Codon optimization for expression in a host cell e.g. , bacteria such as E. coli or insect Hi5 cells
  • CODONOPT Codon Optimization Tool
  • Embodiments disclosed herein further include antibodies or antigen-binding fragments thereof which bind specifically to one or more of the aforementioned peptides or fusion proteins thereof.
  • the antibodies or fragments thereof bind to polypeptides comprising the amino acid sequences identified as SEQ ID NOs: 1-8 or an immunogenic fragment thereof. In another embodiment, the antibodies bind to fragment of these polypeptides. Antigen-binding fragments of such antibodies, include, e.g. , F(ab) domain, F(ab) 2 domains, scFv domains, including synthetically generated antibodies (using, e.g. , phase display technology).
  • the antibodies bind to polypeptide sequences identified as SEQ ID NOs: 20-28, or an immunogenic fragment thereof.
  • biomolecules e.g., nucleic acids, proteins, peptides, and/or antibody molecules, including, conjugates thereof.
  • substantially purified refers to nucleic acids, amino acids or antibodies that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, most preferably about 90% and especially about 95% free from other components with which they are naturally associated.
  • the biomolecules may be altered by combining with various components of the constructs of Formula I or Formula II, e.g., fission inhibitor peptide or linker or carrier, such that their form and/or functionality is significantly changed compared to any natural counterparts.
  • a change in property may include physiochemical properties of the peptides, e.g. , molecular weight, isoelectric point, solubility, hydrophobicity and/or functionality of the peptides, e.g. , Drpl-Fisl inhibition, mitophagy, anti-apoptotic property, mitochondrial targeting, and the like.
  • the agents for use in accordance with the present disclosure are or comprise nucleic acids.
  • mitochondrial fission inhibitors comprise RNA and/or DNA molecules which target Drpl.
  • fission inhibitors are RNAi agents (for example, miRNAs, siRNAs, shRNAs, antisense oligonucleotides, ribozymes) and/or gene therapy vectors.
  • nucleic acid fission inhibitors for use in accordance with the present disclosure have a nucleotide sequence that corresponds to or hybridizes with a portion of a polynucleotide that encodes a Drpl, particularly, an mRNA sequence thereof.
  • nucleic acid agents for use in accordance with the present embodiment have a nucleotide sequence that includes a binding site for a Drpl binding partner, e.g. , Fisl, which mediates its cellular effects, e.g., mitochondrial localization, induction of oxidative and ER stress, activation of pro-apoptotic pathway, cell lysis and/or cell-death, etc.
  • the Drpl inhibitor is a complex comprising clustered regularly interspaced short palindromic repeat (CRISPR) and a CRISPR associated protein (CAS) or nucleic acid encoding the CRISPER/CAS complex, wherein the complex targets nucleic acid encoding Drpl.
  • CRISPR clustered regularly interspaced short palindromic repeat
  • CAS CRISPR associated protein
  • a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure can be incorporated into a variety of formulations for therapeutic use (e.g. , for treating a subject diagnosed with or suffering from a disease which is associated with abnormal mitochondrial fission).
  • a mitochondrial fission inhibitor peptide or construct can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols.
  • administration of a mitochondrial fission inhibitor peptide or construct can be achieved in various ways, including oral, vaginal, buccal, rectal, parenteral, intraperitoneal, intravenous, intramuscular, intradermal, transdermal, intratracheal, etc., administration.
  • a mitochondrial fission inhibitor peptide or construct can be systemic after administration or may be localized by the use of an implant or other formulation that acts to retain the active dose at the site of implantation.
  • a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be administered alone, in a combination of two or more mitochondrial fission inhibitor peptide or construct, or a mitochondrial fission inhibitor peptide or construct can be used in combination with known compounds (e.g., therapeutic agents suitable for treating a disease associated with abnormal mitochondrial fission, etc.)
  • a mitochondrial fission inhibitor peptide or construct may be administered in the form of its pharmaceutically acceptable salt.
  • a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, com starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
  • conventional additives such as lactose, mannitol, com starch or potato starch
  • binders such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins
  • disintegrators such as corn starch, potato starch or sodium carb
  • a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure can be formulated into preparations for injections by dissolving, suspending or emulsifying the peptide in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or nonaqueous solvent such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol
  • solubilizers isotonic agents
  • suspending agents emulsifying agents, stabilizers and preservatives.
  • a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure can be utilized in aerosol formulation to be administered via inhalation.
  • a mitochondrial fission inhibitor construct or peptide of the present disclosure can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
  • a mitochondrial fission inhibitor peptide or construct of the present disclosure can be used in topical formulations, by formulation with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases.
  • a mitochondrial fission inhibitor peptide (or a construct) of the present disclosure can be administered rectally via a suppository.
  • the suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
  • Unit dosage forms for oral, vaginal or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure.
  • unit dosage forms for injection or intravenous administration may comprise a mitochondrial fission inhibitor peptide (or a construct) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
  • Implants for sustained release formulations are well known in the art. Implants can be formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or gly colic acid form an erodible polymer that is well-tolerated by the host.
  • An implant containing a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be used, so that the local concentration of active agent (mitochondrial fission inhibitor peptide or construct) is increased relative to the rest of the body.
  • Liposomes can be used as a delivery vehicle.
  • the lipids can be any suitable combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine.
  • the remaining lipid can include neutral or acidic lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
  • compositions such as vehicles, adjuvants, carriers or diluents
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and non-human animal subjects, each unit containing a predetermined quantity of a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the unit dosage forms depend on the particular mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) employed and the effect to be achieved, and the pharmacodynamics associated with the mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) in the host.
  • Exemplary dosages for systemic administration range from 0.1 ⁇ g to 100 milligrams per kg weight of subject per administration.
  • An exemplary dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient.
  • the time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
  • an active agent e.g., a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) may be administered in dosages of, for example, 0.1 ⁇ g to 500 mg/kg body weight per day, e.g.
  • oral dosages may be about ten times the injection dose. Higher doses may be used for localized routes of delivery.
  • a specific mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be administered in an amount of from about 1 mg to about 1000 mg per dose, e.g. , from about 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, from about 250 mg to about 300 mg, from about 300 mg to about 350 mg, from about 350 mg to about 400 mg, from about 400 mg to about 450 mg, from about 450 mg to about 500 mg, from about 500 mg to about 750 mg, or from about 750 mg to about 1000 mg per dose.
  • dose levels can vary as a function of the specific mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II), the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific peptides may be more potent than others. Preferred dosages for a given peptide are readily determinable by those of skill in the art by a variety of means. One means is to measure the physiological potency of a given peptide.
  • An active agent e.g., a mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II
  • an individual uses any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration.
  • Administration can be acute (e.g., of short duration, e.g. , a single administration, administration for one day to one week), or chronic (e.g. , of long duration, e.g. , administration for longer than one week, e.g. , administration over a period of time ranging from about 2 weeks to about one month, from about 1 month to about 3 months, from about 3 months to about 6 months, or more).
  • routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, transdermal, sublingual, topical application, intravenous, ocular (e.g. , topically to the eye, intravitreal, etc.), rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect.
  • a mitochondrial fission inhibitor peptide or (or a construct of Formula I or Formula II) can be administered in a single dose or in multiple doses.
  • An active agent e.g., a mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II
  • routes of administration contemplated by the disclosure include, but are not necessarily limited to, enteral, parenteral, and inhalational routes.
  • Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, ocular, and intravenous routes, i.e., any route of administration other than through the alimentary canal.
  • Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration can involve invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
  • a mitochondrial fission inhibitor peptide or (or a construct of Formula I or Formula II) can also be delivered to the subject by enteral administration.
  • Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g. , using a suppository) delivery.
  • Methods of administration of a mitochondrial fission inhibitor peptide or (or a construct of Formula I or Formula II) through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration.
  • a suitable pharmaceutical preparation for transdermal transmission, absorption promoters or iontophoresis are suitable methods.
  • Iontophoretic transmission may be accomplished using commercially available "patches" which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
  • mitochondrial fission inhibitor or a construct of Formula I or Formula II
  • a peptide Drpl inhibitor in reducing mitochondrial dysfunction, as characterized by (a) increased mitochondrial interconnectivity or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death or cell lysis; (g) increased mitochondrial accumulation of mitophagy mediators selected from LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1); or (h) increased c-Jun N-terminal kinase (JNK) signaling.
  • MMP mitochondrial membrane potential
  • ROS reactive oxygen species
  • JNK c-Jun N-terminal kinase
  • mitochondrial fission inhibitor or a construct of Formula I or Formula II, i.e., a peptide Drpl inhibitor, for the manufacture of a medicament for treating a neurological disorder, e.g., ALS.
  • the present disclosure contemplates various assays for identifying agents that can increase neuron survival, as well as candidate therapeutic agents for treating neurodegenerative disorders (e.g., ALS).
  • neurodegenerative disorders e.g., ALS
  • the disclosure relates to methods for identifying a test compound that promotes neuron survival and/or is useful for the treatment of ALS comprising (a) contacting a cell system or a cell-free system comprising Drpl and Fisl with a test compound; (b) detecting a parameter associated with mitochondrial dysfunction in the absence and in the presence of the test agent; and (c) selecting a test compound if it modulates the parameter associated with mitochondrial dysfunction.
  • the method may comprise incubating the cell or cell-free system with one or more mitochondrial fission inhibitor peptides or a construct of Formula I or Formula II (disclosed hereinbefore) as a positive control.
  • test compound refers to agents and/or compositions that are to be screened for their ability to stimulate and/or increase and/or promote cell survival.
  • the test agents can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g. , small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g. , peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof.
  • the test agent is a small molecule or a peptide.
  • the modulation comprises a change of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or more compared to a control.
  • the screening method(s) may be carried out using any cells from a biological tissue and/or cell -containing bodily fluid that has been obtained directly from an individual, donor patient or animal.
  • the cells may be directly-derived from a subject, e.g. , without intermediate steps of subculture through a series of cultures and/or hosts.
  • a suspension of single cells is produced directly from the biological tissue and/or cell-containing bodily fluid. This is in contrast to established methods in which stable and highly passaged cell lines are used.
  • Such cell-lines are far removed from being directly derived from their progenitor tissue by several intermediate culture steps.
  • the cell is a fibroblast cell.
  • Methods of maintaining primary fibroblast cultures, as well as transformed fibroblast cell-lines, are known in the art. See, U.S. Pat. No. 9,029,148.
  • the cell is a fibroblast cell of ALS patients carrying pathogenic mutations in SOD1 (I113T), in FUS1 (fused in sarcoma; R521G) or in TDP43 (TAR DNA-binding protein 43; G289S).
  • the cell is a neuron or a neuronal cell.
  • Neurons include, without limitation, primary cultures such as primary cultures of embryonic dorsal root ganglion (DRG) neurons and primary cultures of fetal spinal cord neurons, for example, primary cultures of murine fetal spinal cord neurons (Elaine et al. , J. Cell Biol., 147, 1249-1260 (1999).
  • a neuron can be a neuron from, e.g. , a primary culture, an embryonic dorsal root ganglion primary culture or a fetal spinal cord primary culture.
  • cells useful according to a method disclosed in the present specification can include, a primary neuronal cell that contains an exogenous Drpl/Fisl, such as, e.g. , a rat embryonic dorsal root ganglion (DRG) neuron containing exogenous Drpl/Fisl or a murine fetal spinal cord neuron that contains an exogenous Drpl/Fisl.
  • a primary neuronal cell that contains an exogenous Drpl/Fisl
  • a primary neuronal cell that contains an exogenous Drpl/Fisl
  • an exogenous Drpl/Fisl such as, e.g. , a rat embryonic dorsal root ganglion (DRG) neuron containing exogenous Drpl/Fisl or a murine fetal spinal cord neuron that contains an exogenous Drpl/Fisl.
  • DDG rat embryonic dorsal root ganglion
  • Neuronal cell lines useful in aspects of the disclosure include, without limitation, neuroblastoma cell lines, neuronal hybrid cell lines, spinal cord cell lines, central nervous system cell lines, cerebral cortex cell lines, dorsal root ganglion cell lines, hippocampal cell lines and pheochromocytoma cell lines.
  • methods of the disclosure employ cells that are not neurons, wherein the cells can comprise a mutation in a gene associated with a neurodegenerative disorder.
  • some methods the present disclosure employ fibroblasts comprising a mutation in a gene associated with a neurodegenerative disorder.
  • methods of the disclosure employ fibroblasts comprising a mutation in a SODl gene, such as, without limitation, SOD1A4V, SOD1G8SR, and SOD1G93A.
  • SODl refers to either the gene encoding superoxide dismutase 1 or the enzyme encoded by this gene.
  • the SODl gene or gene product is known by other names in the art including, but not limited to, ALS1, Cu/Zn superoxide dismutase, indophenoloxidase A, IPOA, and SODC HUMAN. Those of ordinary skill in the art will be aware of other synonymous names that refer to the SODl gene or gene product.
  • the SODl enzyme neutralizes supercharged oxygen molecules (called superoxide radicals), which can damage cells if their levels are not controlled.
  • the human SODl gene maps to cytogenetic location 21q22.1.
  • Certain mutations in SODl are associated with ALS in humans including, but not limited to, Ala4Val, Gly37Arg. G85R and Gly93Ala, and more than one hundred others. Those of ordinary skill in the art will be aware of these and other human mutations associated with ALS. Certain compositions and methods disclosed herein employ cells comprising a SODl mutation.
  • SODl mutations refer to mutations in the SODl gene (NC 000021.8; NT_011512.11; AC_000064.1; NW_927384.1 ; AC_000153.1 ; NW_001838706.1 NM_000454.4; NP_000445.1 and NCBI Entrez Gene ID: 6647) including but are not limited to Ala4Val, Cys6Gly, Val7Glu, Leu8Val, GlylOVal, Glyl2Arg, Vall4Met, Glyl6Ala, Asnl9Ser, Phe20Cys, Glu21Lys, Gln22Leu, Gly37Arg. Leu38Arg. Gly41 Ser, His43Arg.
  • SODl is also known as ALS, SOD, ALS1, IPOA, homodimer SODl.
  • "SODl mutation” databases can be found at Dr. Andrew C. R. Martin website at the University College of London, the ALS/SOD1 consortium website and the human gene mutation database (HGMD®) at the Institute of Medical Genetics at Cambridge, UK.
  • the cell is a mouse motor neuron-like hybrid cell line (NSC-34), especially an NSC-34 cell-line harboring a SODl G93A mutation.
  • NSC-34 mouse motor neuron-like hybrid cell line
  • the cells or cell-lines comprise mutations in the fused-in-sarcoma (FUS 1 ) gene or trans-active response DNA binding protein 43 kDa (TDP-43) gene, or human homologs thereof.
  • the cells or cell-lines comprise mutations in the FLJSl gene (human homolog: ALS6).
  • the cells or cell-lines comprise mutations in the TDP-43 gene. Cell lines harboring the mutations can be purchased commercial! ⁇ ' .
  • the disclosure contemplates measuring the effect of the test compound on one or more parameters associated with mitochondrial dysfunction, including, but not limited to, (a) increased mitochondrial interconnectivity and/or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production and/or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death and/or cell lysis; (g) increased accumulation of mitophagy mediators selected from LC3- phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1), or a combination thereof in the mitochondria; (h) increased c-Jun N-terminal kinase (INK) signaling (e.g.
  • the parameter associated with mitochondrial dysfunction is selected from (1) increased mitochondrial Drpl recruitment, (2) increased Drpl phosphorylation and (3) increased mitochondrial accumulation of mitophagy mediators such as LC3-II and p62.
  • a combination of the parameters e.g. , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or more, may also be employed.
  • the parameter associated with mitochondrial dysfunction is mitochondrial interconnectivity and/or mitochondrial elongation score, which can be measured using routine immunofluorescence techniques coupled with analysis of images using software analysis. For instance, mean area' erimeter ratio can be employed as an index of mitochondrial interconnectivity and inverse circularity can be used as a measure of mitochondrial elongation. See, Wiemerslage et al. Journal of Neuroscience Methods 262, 56-65 (2016).
  • a reduction in the mitochondrial interconnectivity and/or elongation score of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more (compared to control) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
  • the parameter associated with mitochondrial dysfunction is mitochondrial membrane potential (MMP), ATP production, reactive oxygen species (ROS) production and mitochondrial superoxide generation.
  • MMP mitochondrial membrane potential
  • ROS reactive oxygen species
  • a reduction in MMP or mitochondrial ATP production is associated with mitochondrial dysfunction, the restoration of which by the test agent indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
  • mitochondrial membrane potential may be assessed via art known-methods, for example, as described in U.S. Pat. Pub. No. 2010/0209960.
  • measurement of MMP includes fluorescent detection, e.g., using fluorescent dyes comprising rhodamine 123, JC-1, tetrabromorhodamine 123, rhodamine 6G, TMRM, TMRE, tetramethylrosamine or rhodamine B (Guo et al, JCI 123, 5371-5388 (2013)).
  • fluorescent dyes comprising rhodamine 123, JC-1, tetrabromorhodamine 123, rhodamine 6G, TMRM, TMRE, tetramethylrosamine or rhodamine B (Guo et al, JCI 123, 5371-5388 (2013)).
  • ATP production may be measured using art known methods, for example, as described in U.S. Patent No. 6,261,796.
  • the mitochondrial ATP production is measured via colorimetric/fluorometric assay and reading in a SPETRAMAX M2 device.
  • the test compound restores the parameter to about 70%, about 80%, about 90% about 100% or a higher % of the normal level, e.g., baseline level of MMP or ATP production in cells obtained from healthy patients or cell- lines not harboring any mutations, then the test compound may be selected as promoting neuron survival and/or as being useful for the treatment of ALS.
  • test compound elevates MMP or ATP production in the cell (e.g., a fibroblast cell or a hybrid motor neuron cell) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline MMP or ATP production (e.g., measured in the absence of the test compound), then the test compound may be selected as promoting neuron survival and/or as being useful for the treatment of ALS.
  • the cell e.g., a fibroblast cell or a hybrid motor neuron cell
  • the parameter associated with mitochondrial dysfunction is an increase in ROS production and/or mitochondrial superoxide generation, the attenuation of which by the test agent indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
  • ROS may be measured using routine fluorescence detection techniques, for example, as taught in U.S. Pub. No. 2015/0219676.
  • mitochondrial superoxide production may be measured using specific fluorescent dyes, e.g. , MITOSOX RED, as detailed in the studies described herein.
  • a reduction in the total ROS production and mitochondrial superoxide generation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more (compared to control, e.g., an untreated sample) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
  • the parameter associated with mitochondrial dysfunction is Drpl or p62 recruitment (or both Drpl and p62 recruitment) from the cytosoi to the mitochondrial membrane, which can be quantitated using immuno-detection methods described above, e.g., immunoblotting or ELISA assays.
  • a reduction in Drpl and/or p62 recruitment by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline mitochondrial Drpl and/or p62 levels (e.g. , measured in the absence of the test compound) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
  • Drpl and/or p62 levels can be identified by routine methods, e.g., Western blot, dot blot, ELISA, flow cytometry, electrochemiluminescence, multiplex bead assay (e.g. , using Luminex or fluorescent microbeads), immunohistochemistry and the like. Representative methods are described in the Examples section.
  • the parameter associated with mitochondrial dysfunction is Drpl phosphorylation.
  • Phosphorylated Drpl can be detected using art known-methods, e.g. , immunodetection using anti-p-Drpl antibody S616 or anti-p-Drpl antibody S637 (see Example 1 and FIG. 2).
  • a reduction in Drpl phosphorylation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline phospho-Drpl levels (e.g., measured in the absence of the test compound) indicates that the test compound promotes neuron survival and/or is useful for ALS therapy.
  • the parameter associated with mitochondrial dysfunction is cell death and/or cell lysis.
  • Methods for detecting cell death and/or cell lysis are known in the art, e.g. , using a colorimetric CYTOTOX 96 non-radioactive assay to detect lactate dehydrogenase (LDH) levels (Promega Corporation), as disclosed in U.S. Pub. No. 2003/0049829.
  • cell death may be detected using routine apoptotic markers, e.g. , mitochondrial cytochrome c release and/or accumulation of Bax on the mitochondria. Under this embodiment, a reduction in cell death or cell lysis (e.g.
  • cytochrome c or Bax as assessed by measuring the levels or activity of LDH or levels of cytochrome c or Bax by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g., levels of LDH, cytochrome c or Bax in untreated cell or cell-free systems) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
  • baseline levels e.g., levels of LDH, cytochrome c or Bax in untreated cell or cell-free systems
  • embodiments described herein include specific devices or methods known in the art for the detection of fluorescence, e.g., from fluorophores or fluorescent proteins. Such include, but are not limited to, in vivo near-infrared fluorescence (see, Frangioni et al, Curr Opin. Chem.
  • the parameter associated with mitochondrial dysfunction is accumulation of mitophagy mediators in the mitochondria.
  • the mitophagy mediator is selected from LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1 ), or a combination thereof.
  • a reduction in the levels of mitophagy mediator in the mitochondria e.g., as assessed by measuring the levels of LC3-II and/or p62
  • at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
  • the parameter associated with mitochondrial dysfunction is increased c-Jun N -terminal kinase (JNK) signaling.
  • JNK c-Jun N -terminal kinase
  • increased INK signaling is signified by increased phosphoryiated JNK levels and/or increased levels of downstream effectors such as XBPi, ATF6a, phosphoryiated eIF2a, GRP78 and/or CHOP.
  • a reduction in JNK signaling e.g.
  • phosphoryiated JNK levels and/or a downstream effector selected from XBPI, ⁇ , phosphoryiated eIF2 , GRP78 and CHOP by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g. , levels of phosphoryiated JNK levels and/or a downstream effector thereof) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
  • cells may be allowed to grow for a period time, after which, the test compounds contacted with the cells, e.g., by supplementing the media. Period of cell growth can be optimized depending on the assay format, initial plating density of the cells. In some embodiments, a researcher can obtain cells that are already planted in the appropriate vessel and allowed to grow for a period of time. In other embodiments, the cells are plated in the appropriate vessel and allowed to grow for a period time, e.g., at least 1 day, at least 2 days, at least 3 days, at last 4 days, at least 5 days, at least 6 days, at least 7 days or more in presence of the test compound. In one embodiment, cells are grown for about 2-3 days in presence of the test compound prior to assaying for one or more of the aforementioned parameters.
  • a control can be a sample that is that is not contacted with a compound or a sham compound, e.g. , a random peptide or a carrier peptide alone (i.e. , a negative control).
  • a control can be a sample that is treated with a known promoter of cell survival (e.g., mitochondrial fission inhibitor peptide or construct of Formula I). This can serve as a positive control.
  • a known promoter of cell survival e.g., mitochondrial fission inhibitor peptide or construct of Formula I.
  • Other mediators of mitochondrial autophagy e.g. , Drp lK38A or Fis l RNAi, may also be used.
  • a control can be a sample that is treated with a known inhibitor of Drpl, e.g., Mdivi-1 or dynasore monohydrate.
  • a chemical library or compound library is a collection of stored chemicals usually used ultimately in high-throughput screening or industrial manufacture.
  • the chemical library can consist in simple terms of a series of stored chemicals.
  • Each chemical has associated information stored in some kind of database with information such the chemical structure, purity, quantity, and physiochemical characteristics of the compound.
  • test agents can be provided free in solution, or may be attached to a carrier, or a solid support, e.g., beads.
  • a carrier or a solid support, e.g., beads.
  • suitable solid supports include agarose, cellulose, carboxymethyl cellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resins, plastic films, polyaminomethylvinylether maleic acid copolymer, glass beads, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • test agents may be screened individually, or in groups. Group screening is particularly useful where hit rates for effective test agents are expected to be low such that one would not expect more than one positive result for a given group.
  • the assay can be performed any suitable container or apparatus available to one of skill in the art for cell culturing.
  • the assay can be performed in 24-, 96-, or 384-well plates.
  • the assay is performed in a 384-well plate.
  • cells can be plated at any density that provides an optimal signal-to-noise ratio.
  • neurons can be plated at a density of 1,000 to 20,000 cells/well in a 384-well plate.
  • motor neurons are plated at density of 1,000 cells/well, 2,000 cells/well, 4,000 cells/well, 8,000 cells/well, 12,000 cells/well, 16,000 cells/well, or 20,000 cells/well in a 384-well plate.
  • neurons are plated at a density of 8,000 cells/well in a 384-well plate.
  • one of ordinary skill can adjust the plating density for other cell culturing vessels. For example one can calculate the dimensions of a well in the 384-well plate and the vessels to be used and scale the number of cells to be plated based on volume or surface area ratio between a well from the 384-well plate and the vessel to be used.
  • the screening method is a high-throughput screening.
  • High-throughput screening is a method for scientific experimentation that uses robotics, data processing and control software, liquid handling devices, and sensitive detectors.
  • High-Throughput Screening allows a researcher to quickly conduct millions of biochemical, genetic or pharmacological tests.
  • HTS techniques are well known to one skilled in the art, for example, those described in U.S. Pat. Nos. 5,976,813; 6,472,144; 6,692,856; 6,824,982; and 7,091,048, and contents of each of which is herein incorporated by reference in its entirety. Diagnostic Tests
  • the disclosure also contemplates diagnostic tests and methods of diagnosing a neurological disorder (e.g., ALS) and/or disorders characterized by neuronal cell death.
  • a neurological disorder e.g., ALS
  • characterized by neuronal cell death e.g., ALS
  • the method of diagnosing a neurological disorder in a subject comprises: (a) obtaining a biological sample from the subject comprising a sample; (b) conducting at least one assay on the sample to detect mitochondrial dysfunction; and (c) diagnosing the subject as having the neurological disorder if the level of mitochondrial dysfunction in the patient sample is greater than the level of mitochondrial dysfunction in a control.
  • the level of mitochondrial dysfunction correlates with Drpl/Fisl activity.
  • control is a subject that does not have the neurological disorder (e.g. , ALS). In some embodiments, the control is a reference standard or level indicative of a subject that does not have the neurological disorder (e.g., ALS).
  • the disclosure contemplates using any assay for measuring mitochondrial dysfunction, including, but not limited to, (a) increased mitochondrial interconnectivity and/or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production and/or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death and/or cell lysis; (g) increased accumulation of mitophagy mediators selected from LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1), or a combination thereof in the mitochondria; (h) increased c-Jun N-terminal kinase (JNK) signaling (e.g., increased phosphorylated JNK levels and/or increased levels of downstream effectors such as XBP1, ATF6a, phosphorylated eIF2a, GRP78
  • the parameter associated with mitochondrial dysfunction and/or predisposition to ALS is (1) increased mitochondrial Drpl recruitment, (2) increased Drpl phosphorylation and (3) increased mitochondrial accumulation of mitophagy mediators such as LC3-II and p62.
  • a combination of the parameters may also be employed.
  • the at least one binding assay comprises an assay that measures one or more of the aforementioned parameters in a cell, e.g., fibroblast cell.
  • a cell e.g., fibroblast cell.
  • Representative assays, techniques and systems for measuring the parameters have been described previously. It should be appreciated by the skilled artisan that detection of one or more of the aforementioned parameters in a subject (e.g., one who is suspected to have the neurological disorder) compared to a control (e.g. , healthy subject) is indicative that the subject has or is at risk for developing the neurological disorder and/or a disorder characterized by neuronal cell death.
  • the diagnostic methods further comprise selecting a subject suspected of having a neurological disorder (e.g. , ALS). In some embodiments, the diagnostic methods further comprise selecting a subject suspected of having a disorder characterized by neuronal cell death.
  • the selected patients may be placed on appropriate therapy for the treatment of the disorder, e.g. , treatment with the mitochondrial fission peptide (or a construct of Formula I or Formula II), or riluzole, or any other agents useful for the treatment or management of ALS.
  • ALS amyotrophic lateral sclerosis
  • the parameter associated with mitochondrial dysfunction includes, but is not limited to, (a) increased mitochondrial interconnectivity and/or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production and/or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death and/or cell lysis; (g) increased accumulation of mitophagy mediators selected from LC3- phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1), or a combination thereof in the mitochondria; (h) increased c-Jun N-terminal kinase (JNK) signaling (e.g.
  • the parameter associated with mitochondrial dysfunction is selected from (1) increased mitochondrial Drpl recruitment, (2) increased Drpl phosphorylation and (3) increased mitochondrial accumulation of mitophagy mediators such as LC3-II and p62.
  • a combination of the parameters e.g. , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or more, may also be employed.
  • a positive diagnosis is made if the parameter being measured is modulated by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or more compared to a control.
  • a method for treating a neurological disorder comprises administering to a subject in need thereof, an effective amount of a composition that inhibits mitochondrial fission.
  • a method for slowing the progression of or delaying progression of a neurological disorder is also contemplated, the method comprising administering to a subject in need thereof, an effective amount of a composition that inhibits mitochondrial fission.
  • the disclosure relates to treatment of amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease or classical motor neuron disease, and to slowing or delaying progression of ALS.
  • ALS amyotrophic lateral sclerosis
  • Lou Gehrig's disease also called Lou Gehrig's disease or classical motor neuron disease
  • motor neuron disease is a progressive, ultimately fatal disorder that eventually disrupts signals to all voluntary muscles.
  • doctors use the terms motor neuron disease and ALS interchangeably. Both upper and lower motor neurons are affected.
  • Approximately 75% of people with classic ALS will also develop weakness and wasting of the bulbar muscles (muscles that control speech, swallowing, and chewing). Symptoms are usually noticed first in the arms and hands, legs, or swallowing muscles. Muscle weakness and atrophy occur disproportionately on both sides of the body.
  • ALS a progressive neurodegenerative disease
  • SOD superoxide dismutase gone, or SOD
  • a rare juvenile-onset form of ALS is genetic. Most individuals with ALS die from respiratory failure, usually within 3 to 5 years from the onset of symptoms. However, about 10 percent of affected individuals survive for 10 or more years.
  • embodiments disclosed herein relate to methods for treating ALS comprising administration of an effective amount of a compound disclosed herein (e.g. , mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) to a subject in need thereof.
  • the method of treating comprises the slowing or delaying of progression of the disorder.
  • the subjects are mammals, e.g., a rodent, a human, a livestock animal, a companion animal, or a non-domesticated or wild animal.
  • the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc.
  • the subject is a human.
  • the human can be a subject who has been diagnosed with ALS or as being predisposed to ALS.
  • methods of treatment of ALS described herein allow for early intervention upon detection of at least one sign or symptom associated with ALS. Accordingly, under this embodiment, treatment with the compounds disclosed herein (mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in improvement in at least one sign or symptom associated with ALS in the subject.
  • the treatment with the compounds disclosed herein results in an improvement in a histopathological trait, a behavioral trait, a physiological trait or a combination of traits associated with ALS in the subject.
  • Improvement of the trait can be determined by comparing the trait prior to treatment versus post-treatment. Alternately, the improvement can be monitored at various time-points during the course of the therapy, e.g. , at day 30 versus day 90.
  • the improvement is a quantifiable measurement (e.g., improvement in muscle strength). In other embodiments, the improvement is a qualitative measurement (e.g., improvement in health-related quality of life (HRQL), disease severity, cognition, depression, fatigue, coping capacity and burden of care).
  • HRQL health-related quality of life
  • a subject is described as having been treated if the therapeutic agent leads to an improvement of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 10-fold, at least 50-fold, or more, compared to a control (e.g., untreated or placebo-treated subject).
  • a control e.g., untreated or placebo-treated subject.
  • treatment with the compounds disclosed herein results in an improvement in a histopathological trait associated with ALS.
  • the improved histopathological trait comprises (a) reduced mitochondrial interconnectivity and/or mitochondrial elongation score; (b) increased mitochondrial membrane potential (MMP) and/or ATP production; (c) reduced reactive oxygen species (ROS) production and/or reduced mitochondrial superoxide generation; (d) reduced Drpl or p62 recruitment to the mitochondria; (e) reduced Drpl phosphorylation; (f) reduced cell death and/or cell lysis; (g) diminished mitochondrial accumulation of mitophagy mediators selected from LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1); (h) reduced c-Jun N-terminal kinase (INK) signaling (e.g., attenuation in
  • treatment with the compounds disclosed herein results in an improvement in a behavioral trait associated with ALS.
  • the improved behavioral trait includes improved general mobility, improved motor function, reduced phobia, reduced stereotypy (e.g., twitches/paralysis), improved engagement time, improved engagement score; or a combination thereof.
  • improvement in general mobility includes improvement in ambulation episodes, increase in moving time, increase in ambulatory time, an improvement in resting time or a combination thereof.
  • treatment with the compounds disclosed herein improves a pooled outcome reflecting general mobility behavior comprising enhanced ambulatory behaviors and reduced resting time.
  • treatment with the compounds disclosed herein results in an improvement in a motor trait associated with ALS.
  • the improved motor trait includes improved motor response to allodynia (e.g. , response to hot or cold objects).
  • treatment with the compounds disclosed herein results in an improvement in a physiological trait associated with ALS.
  • the improved physiological trait includes improved muscle strength, improved strength of grip, improved posture.
  • treatment with the compounds disclosed herein results in an improvement in a speech trait associated with ALS.
  • the improved speech trait includes improved tone and quality of voice and/or reduction in the frequency and duration of slurring.
  • the treatment with the compounds disclosed herein results in an improvement in an outcome associated with ALS, e.g. , improvement in an epidemiological outcome, a histopathological outcome, or a physiological outcome, or a combination thereof.
  • treatment with the compounds disclosed herein improves a combination of the aforementioned outcomes, e.g., at least 2, 3, 4, 5, 6, 7 or more outcomes.
  • treatment with the compounds or constructs disclosed herein improves at least one epidemiological outcome selected from the group consisting of overall survival (OS), survival at clinical score 1 (SCSI), survival at terminal endpoint (STE), age at terminal endpoint (ATE), survival at clinical score 3 (SCS3), age at clinical score 3 (ACS3), total disease duration (TDD), duration between clinical score 2 to terminal endpoint (CS2T), time to progression of disease (TTP), time-to-death (TTD), and disease-free survival period (DFS), or a combination thereof.
  • OS overall survival
  • SCSI survival at clinical score 1
  • STE survival at terminal endpoint
  • ATE age at terminal endpoint
  • SCS3 survival at clinical score 3
  • ACS3 age at clinical score 3
  • TDD total disease duration
  • TTP duration between clinical score 2 to terminal endpoint
  • TTP time to progression of disease
  • TTD time-to-death
  • DFS disease-free survival period
  • treatment with the compounds or constructs disclosed herein improves an epidemiological outcome selected from the group consisting of survival at clinical score 1 (SCSI), survival at terminal endpoint (STE), age at terminal endpoint (ATE), survival at clinical score 3 (SCS3), age at clinical score 3 (ACS3), total disease duration (TDD), and duration between clinical score 2 to terminal endpoint (CS2T) or a combination thereof.
  • stage 2 is characterized by development of weakness/limpness in hind limbs
  • stage 3 is characterized by development of paralysis of a hind limb
  • terminal endpoint is characterized by paralysis in both hind limbs plus a 20% or greater drop in the body weight or paralysis in both hind limbs plus a lack of righting reflex.
  • treatment with the compounds or constructs disclosed herein improves at least one in vivo histopathological outcome comprising attenuation in the association of Drpl and/or p62 with mitochondria of the subject's cell sample (e.g., spinal cord neurons).
  • in vivo therapy with the compounds of the disclosure results in a reduction of mitochondrial Drpl and/or p62 localization by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g. , mitochondrial Drpl or p62 localization in identical cells obtained from the same subject prior to treatment or from an untreated subject).
  • treatment with the compounds or constructs disclosed herein improves at least one in vivo physiological outcome comprising increased body weight (or body mass).
  • in vivo therapy with the compounds of the disclosure results in an increase in body weight by at least 20%>, at least 30%>, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g. , body weight or body mass in the same subject prior to treatment or from an untreated subject).
  • treatment with the compounds disclosed herein delayed onset of disease and/or slowed down the progression of the disease; improved motor function and/or muscle strength; reduced stereotypy and/or paralysis; and improved survival. Improvement in these various outcomes was corroborated via histopathological characterization of attenuation in mitochondrial Drpl/p62 localization following treatment with the compounds disclosed herein.
  • the mitochondrial fission inhibitor may be administered 2, 3, 4, 5 or more times a day, every 1 day, every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or 25 days, or every 1 month, 2 months, 3 months, 4 months, or 6 months, or annually.
  • the first dose and one or more subsequent doses the mitochondrial fission inhibitor may be administered in a dosing regimen that is a pulsed dosing regimen (e.g., the dosing schedule produces escalating inhibitor levels early in the dosing interval followed by a prolonged dose-free period).
  • the first dose and one or more subsequent doses of the fission inhibitor is administered in a dosing regimen that is not continuous (i.e., the intervals between doses are uneven).
  • the first dose and one or more subsequent doses of the fission inhibitor is administered in a dosing regimen that is a continuous dosing regimen.
  • the first dose is administered upon detection of one or more symptoms of ALS.
  • the one or more subsequent doses of the fission inhibitor are administered every day, every other day, every 2 days, 3 days, 4 days, 5 days, 6 days, once a week, every 2 or 3 weeks, once a month, every 6 weeks, every 2 months, 3 months, 4 months, 5 months, 6 months or any combination thereof.
  • the methods disclosed herein can further include administering one or more additional therapeutic agents to the subject in need thereof, such as an agent that is effective in the treatment of ALS.
  • additional agents include an anti-spasticity agent (e.g., baclofen or diazepam), an analgesic (e.g., gabapentin), an antiparkinsonian agent (e.g. , trihexyphenidyl or amitriptyline), a calcimimetic (e.g., cinacalcet), an anti- cramp agent (e.g., mexiletine) or an anti-ALS agent (e.g. , riluzole).
  • an anti-spasticity agent e.g., baclofen or diazepam
  • an analgesic e.g., gabapentin
  • an antiparkinsonian agent e.g. , trihexyphenidyl or amitriptyline
  • a calcimimetic
  • the combination comprises a mitochondrial fission inhibitor peptide (e.g. , a construct of Formula I or Formula II) with one or more additional active agents selected from: riluzole, baclofen, cinacalcet, acamprosate, mexiletin, torasemide, sulfisoxazole, and riluzole.
  • a mitochondrial fission inhibitor peptide e.g. , a construct of Formula I or Formula II
  • additional active agents selected from: riluzole, baclofen, cinacalcet, acamprosate, mexiletin, torasemide, sulfisoxazole, and riluzole.
  • the combination may comprise a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) together with one or more drug(s) that ameliorate(s) symptoms of ALS, one or more drug(s) that could be used for palliative treatment of ALS or one or more drug(s) currently evaluated in the frame of clinical trials for treating of ALS.
  • said one or more drug(s) is/are selected from AEOL 10150, arimoclomol, AVP-923, botulinum toxin type B (Myobloc), ceftriaxone, celastrol, celecoxib, cistanche total glycosides, coenzyme Q10, Copaxone, creatine, creatinine, dronabinol, erythropoietin, escitalopram (Lexapro), glatiramer acetate, granulocyte-colony stimulating factor (G-CSF), growth hormone (Somatropin), GSK1223249, indinavir, insulin-like growth factor- 1 (IGF-I), IGF-l-AAV, K S-760704, leteprinim, leuprolide, levetiracetam, MCI-186, mecobalamin, minocycline, modafinil, Naaladase inhibitor, N- Acetylcysteine, NBQX,
  • the method encompasses co-administering at least one of the aforementioned agents (or a combination thereof) and a mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II.
  • co-administering refers to the administration of a plurality of agents, wherein the agents can be administered simultaneously, or at different times, as long as they work together (e.g. , additively or synergistically) to achieve the desired effect (e.g. , inhibit cell death).
  • the mitochondrial fission inhibitor (or a construct of Formula I or Formula II) and the additional agent can be administered within any time of each other.
  • the mitochondrial fission inhibitor (or a construct of Formula I or Formula II) and the additional agent can be administered within 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hours, 45 minutes, 30 minute. 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes or less of each other.
  • co-administration does not require the mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) and the additional agent to be administered by the same route. As such, each can be administered independently or as a common dosage form.
  • fibroblasts derived from ALS patients the mitochondrial network was fragmented as compared with fibroblasts from healthy subjects (control), with prevalence of round-shaped mitochondria or sphere-like clusters (FIG. 1A).
  • control fibroblasts from healthy subjects
  • FIG. 1A To quantify the mitochondrial structure change, automated image analysis was performed and the effects of these ALS mutations on mitochondrial morphology were examined.
  • ALS patient-derived fibroblasts carrying any one of these three mutations showed a -50% decrease in mitochondrial interconnectivity (1.01 vs. 048 for Control and ALS, respectively; p ⁇ 0.00001) and elongation scores (1.54 vs. 0.77 for control and ALS, respectively; p ⁇ 0.00001) (FIG. IB, FIG. 1C).
  • construct comprised the peptide identified as SEQ ID NO: 1, a linker (GG), and a carrier (TAT 47 -57; SEQ ID NO: 8), with the carrier at the N-terminus of the construct and the antagonist peptide at the C-terminus of the construct.
  • the construct is identified herein as SEQ ID NO: 28 and is referred to as "PI 10".
  • PI 10 is a construct consisting of a heptapeptide conjugated to TAT 47- 57 (TAT, for intracellular delivery) that selectively inhibits the interaction between Drpl and Fisl, one of its adaptor proteins on the mitochondria (Guo et al, JCI, 123, 5371-5388 (2013)).
  • TAT for intracellular delivery
  • Drpl and Fisl one of its adaptor proteins on the mitochondria
  • MMP mitochondrial membrane potential
  • ATP ATP production
  • mitochondrial superoxide production was determined.
  • TMRM tetra-methyl-rhodamine methyl ester
  • ROS mitochondrial reactive oxygen species
  • Drpl recruitment from the cytosol to the mitochondrial outer membrane is a hallmark of activated mitochondrial fission (Frank et al., Developmental Cell 1, 515-525 (2001)).
  • a 3.4-fold increase in levels of Drpl recruitment to the mitochondria was observed in ALS patient-derived fibroblasts, which was significantly reduced by PI 10 treatment to 1.9 fold relative to control cells (p ⁇ 0.00001) (FIG. 1H, FIG. 5A). No significant changes in the total protein levels of both Drpl and Fisl proteins were observed in these cells (FIG. 5A).
  • SODl G93A cells showed an approximately two-fold increase in Mi to SOX, a specific and mitochondrial-targeted detection probe for superoxide radical (0 2 ) (FIG. 2A), which was reduced when SODl G93A cells were treated with PI 10 (FIG. 2A) (from 202% to 165% with PI 10; p ⁇ 0.03).
  • PI 10 PI 10
  • PI 10 treatment greatly blocked the release of cytochrome c from the mitochondria (p ⁇ 0.05), reduced the accumulation of active Bax on the mitochondria (p ⁇ 0.006), and improved decreased Bcl-2 levels on the mitochondria (p ⁇ 0.002) (FIG. 2B, FIG. 5B, FIG. 6C).
  • PI 10 treatment inhibited the initiation of apoptosis.
  • Drpl translocation to mitochondria (Otera et al , Biochimica et Biophysica Acta 1833, 1256-1268 (2013)).
  • Phosphorylation of Drpl at Ser-616 by cyclin-dependent kinase (CDK) 1/ Cyclin B or CDK5 promotes mitochondrial fission whereas de-phosphorylation of Drpl at Ser-637 by calcineurin facilitates its translocation to mitochondria and subsequently increases mitochondrial fission, which leads to an increased response to apoptotic stimuli (Liesa et al , Physiological Reviews 89, 799-845 (2009); Campello et al, EMBO Reports 11, 678-684 (2010)).
  • Drpl Ser-616/Ser-637 phosphorylation ratio reflects Drpl activity.
  • Western blot analysis of total protein lysates showed a significant increase in Drpl phosphorylation at Ser-616 combined with a decrease in phosphorylation at Ser-637 in NSC- 34 SOD1 G93A cells (FIG. 2C, FIG. 5B; p ⁇ 0.002).
  • FIG. 5D, FIG. 6E Western blot analysis showed that the levels of other ER stress markers, GRP78 and CHOP, increased under serum starvation and were also elevated in G93A expressing motor neurons as compared to the WT (p ⁇ 0.05; FIG. 2F, FIG. 5D, FIG. 6E), which were significantly normalized by PI 10 (p ⁇ 0.05; FIG. 2F, FIG. 5D, FIG. 6E).
  • SOD1G93A mouse model has been used since 1994 for preclinical testing in ALS (Gurney et al , Science 264, 1772-1775 (1994)). Despite recent genetic advances in the understanding of ALS, transgenic mice expressing mutant SOD1 remain the best available, and most widely used, vertebrate model of the disease.
  • SOD1G93A mice (on a mixed genetic background) were treated with either PI 10 or vehicle control, using osmotic mini-pumps (ALZET, delivering 3 mg/kg/day). Treatment began from the age of 90 days, at the onset of clinical/motor symptoms, to assess efficacy of PI 10 treatment in modifying disease progression (FIG. 3A, FIG. 7A).
  • the activity chamber is a simple assessment test used to determine general activity levels, gross locomotor activity, and exploration habits in rodents (Tatem et al , JoVE, 51785 (2014)) and all the following studies were carried out by an observer blinded to the experimental conditions. Changes in mouse locomotor behavior were determined 10 and 24 days after the initiation of PI 10 treatment (FIG. 3A). PI 10- treated mice spent significantly more time exploring the chamber as well as travelled further distances, as measured by ambulatory distance, ambulatory episodes and time spent exploring (p ⁇ 0.05, FIG. 3B- 3D, FIGS. 7C, 7D) indicating better motor functions at both time points. This exploratory behavior correlated with decreased inactive time (FIG. 7H).
  • FIG. 3E Improvement in the hind/front grip strength in the P110-treated mice was also observed (FIG. 3E). No difference was observed in body weight (FIG. 7B). Furthermore, the P110-treated group showed tendency towards increased jumping, a behavior associated with intense motor function (FIGS. 7D-7E). PI 10 treatment showed increased locomotion (measured by center zone entries); however, the spatial distribution of this activity (latency to center) did not suggest an anxiety-related phenotype (FIG. 3H, FIG. 7F). Stereotypic counts are the number of times the mouse breaks the same beam in succession without breaking an adjacent beam. It was observed that PI 10 increases stereotypy, which is often associated with worsened pathology, but this could be attributed to increased movement and locomotion, as the stereotypy in treated mice was comparable to that observed in WT littermates (FIG. 7G).
  • PCA principal component analysis
  • FIGS. 4A-4G show that treatment of ALS patients with PI 10 (SEQ ID NO: 28) had an increased survival even when treatment was initiated at the onset of the clinical symptoms, e.g, for ALS model mice, when the mice already showed dragging feet/knuckles (clinical score 1, CS1). Furthermore, treatment with PI 10 significantly delayed disease progression to terminal endpoint in these mice as assessed by the age at terminal endpoint, age at clinical score (CS3), time taken for the disease to progress from CS2 to terminal endpoint, as well as the overall increase in the duration of the disease after the onset of first symptoms and significantly improved the probability of survival post- paralysis. Moreover, sustained treatment with PI 10 treatment in naive mice showed neither toxicity nor any behavioral changes after 5 months on treatment at 3 mg/kg/day.
  • FIG. 8A shows that increased GFAP staining, which correlates with astrocytosis, and beta-actin staining, a marker of microglia, were blunted by treatment with PI 10 (SEQ ID NO: 28) in ALS mice relative to wild type mice or control (TAT, SEQ ID NO: 8) treated mice.
  • GFAP levels were quantified in spinal cord lysates, and the results are shown in FIG. 8B.
  • Treatment with PI 10 (SEQ ID NO: 28) in ALS mice reduced GFAP expression relative to wild type mice or control (TAT, SEQ ID NO: 8).
  • FIGS. 8C-8G are bar graphs showing the tissue levels of several cytokines, interleukin-lbeta (FIG. 8D), interleukin 1 -alpha (FIG. 8E), interleukin-6 (FIG. 8F) and tumor necrosis factor-alpha (FIG. 8G) are all reduced in ALS mice treated with PI 10.
  • FIGS. 9A-9D show the results, with FIG. 9A showing the mitochondrial aspect ratio, FIG. 9B the intracellular ATP, FIG. 9C the mitoSOX and FIG. 9D the total ROS in the transfected cells untreated (squares), control-treated (triangles) and PI 10-treated (inverted triangles).
  • NSC34 cells stably expressing WT or G93A hSODl were a gift from Dr. Manfredi (Weill Medical College of Cornell University, USA). NSC34 were maintained in DMEM supplemented with 10% v/v FBS and 1% (v/v) penicillin/streptomycin. When differentiation was required, cells were plated onto poly-d-lysine-coated plates and grown in differentiation medium, which contains 1 : 1 DMEM/Ham's F12 supplemented with 1% FBS, 1% P/S and 1% modified Eagle's medium non-essential amino acids.
  • ALS patient-derived fibroblasts (ALS 1 : ND29509; ALS 2: ND30327; ALS 3: ND32969) and fibroblasts of control healthy individuals (H1 :AG07123; H2:AG04146) 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% C0 2 -95% air. NSC34 cells were treated with PI 10 (SEQ ID NO: 28) or vehicle (TAT 47 - 57, SEQ ID NO: 8) at a final concentration of ⁇ ⁇ every 24-hour in serum free media.
  • PI 10 SEQ ID NO: 28
  • vehicle TAT 47 - 57, SEQ ID NO: 8
  • TAT SEQ ID NO: 8
  • PI 10 SEQ ID NO: 28
  • the coverslips were mounted with SLOWFADE antifade reagent (Invitrogen), and images were acquired using an ALL-IN-ONE Fluorescence Microscope BZ-X700 (Keyence) (Guo et al. , JCI, 123, 5371-5388 (2013); Qi et al, Journal of Cell Science 126, 789-802 (2013)).
  • Mitochondrial membrane potential Cells were incubated with tetra-methyl-rhodamine methyl ester (TMRM, Invitrogen) in HBSS (Hank's balanced salt solution) for 30 min at 37°C and the fluorescence was analyzed using SPECTRAMAX M2E (Molecular Devices) (excitation, 360 nm; emission, 460 nm). All data were normalized with respect to the fluorescence intensity of the control cells.
  • TMRM tetra-methyl-rhodamine methyl ester
  • ATP measurements were measured by the ATP colorimetric/fluorometric assay kit (Biovision, Milpitas, CA) using the manufacturer's protocols and reading in a SPECTRAMAX M2E (Molecular Devices). ATP concentration at each time point was calculated as a percentage of levels in the control group.
  • ROS production For cellular ROS detection, cells were incubated with 2,7 dichloro- fluorescin diacetate (DCFDA) (Abeam) 100 ⁇ for 30 minutes at 37°C in the dark, and fluorescence was analyzed with excitation/emission at 495/529 nm using SPECTRAMAX M2E. Fluorescence intensity was then normalized for cell number. To determine mitochondrial ROS production, cells were treated with 5 ⁇ MITOSOXTM RED, a mitochondrial superoxide indicator (Invitrogen) for 10 min at 37°C according to the manufacturer's protocol and fluorescence was analyzed with excitation/emission at 510/580 nm using SPECTRAMAX M2E.
  • DCFDA 2,7 dichloro- fluorescin diacetate
  • the homogenates were spun at 800 g for 10 min at 4 °C (nuclear pellet), and the resulting supernatants were aliquoted and used as total ly sates. A second aliquot was spun at 10,000 g for 20 min at 4 °C. The pellets were washed with lysis buffer and spun at 10,000 g again for 20 min at 4 °C. The final pellets were suspended in lysis buffer containing 1% Triton X-100 and were mitochondrial-rich lysate fractions (Guo et al, JCI, 123, 5371-5388, 2013; Qi et ctl , JCS, 126, 789- 802, 2013).
  • Proteasome activity Cells were homogenized in cold buffer (20 mM TrisHCl pH 7.5, 2 mM EDTA) and centrifuged at 15000 g for 10 min at 4°C. Protein concentration in supernatants was determined using the BCA protein assay (Thermo Fisher Scientific). All samples were assayed in triplicate using 10 ⁇ g of freshly protein extracts. Proteasome activity was measured using the CHEMICON Proteasome Activity Assay Kit (APT280, Millipore), as described by the manufacturer.
  • the extracts were incubated (2h at 37°C) with a labeled substrate, LLVY-7-amino-4-methyl-coumarin, and the cleavage activity was monitored by detection of the free fluorophore 7-amino-4-methyl- coumarin, using a SPECTRAMAX M2E at 380/460 nm.
  • Table 1 provides a list of antibodies used for Western blot analysis:
  • the Activity Chamber was used to determine general activity levels, gross locomotor activity, and exploration habits in rodents. Assessment took place in an Open Field Activity Arena (Med Associates Inc., St. Albans, VT. Model ENV-515) mounted with three planes of infrared detectors, within a specially designed sound-attenuating chamber (Med Associates Inc., St. Albans, VT. MED-017M-027).
  • the arena is 43 cm (L) x 43 cm (W) x 30 cm (H) and the sound attenuating chamber is 74 cm (L) x 60 cm (W) x 60 cm (H).
  • the animal was placed in the corner of the testing arena and allowed to explore the arena for 10 minutes while being tracked by an automated tracking system. Parameters including distance moved, time immobile, and times spent in pre-defined zones of the arena were recorded.
  • PCA Principal component analysis
  • mice/group was used for behavioral tests and 5 mice/group were used for biochemical analysis from the same litter.
  • cell culture studies each study was performed with at least three independent experiments done in duplicates. An observer who was blind to the experimental groups conducted all the animal studies. From the age-matched mice, one of eight TAT-treated and two of sixteen P110-treated mice were excluded from the study due to death during the surgery to implant the second pump. The data from these three mice were not included in any of the behavioral analysis.
  • All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference.

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Abstract

Embodiments of the present disclosure relate generally to the use of compositions comprising Drp1 inhibitors, e.g., peptide compounds which inhibit interaction between Drp1 and mitochondrial fission protein (Fis1), in the prevention and treatment of amyotrophic lateral sclerosis (ALS), e.g., familial ALS1, including delaying the progression of ALS.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF AMYOTROPHIC LATERAL
SCLEROSIS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 62/488,575 filed April 21, 2017, incorporated by reference herein.
[0002] This invention was made with Government support under contract HL052141 awarded by the National Institutes of Health. The Government has certain rights in this invention.
REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM
[0003] This application contains a Sequence Listing which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on April 20, 2018, is named 091511-0621_8283_WO00_SL.txt and is 10,957 bytes in size.
TECHNICAL FIELD
[0004] Embodiments of the present disclosure relate generally to the use of compositions which inhibit Dynamin-related protein 1 (Drpl), e.g., peptide compounds which inhibit translocation of Drpl into effector organelles such as mitochondria, as agents for the treatment of amyotrophic lateral sclerosis.
BACKGROUND
[0005] Amyotrophic lateral sclerosis (ALS), which clinically manifests by progressive muscle atrophy and paralysis, is a fatal neurodegenerative disease characterized by the death of upper and lower motor neurons (MN) (Boillee et al, Neuron 52, 39-59 (2006)). Regardless of the region of onset, muscle weakness and atrophy invariably spread to other parts of the body as the disease progresses (Carri et al , BBRC, 483, 1187-1193 (2017)). Toward the end stages of the disease, as the diaphragm and intercostal muscles weaken, most patients require ventilator support. Individuals with ALS most commonly die of respiratory failure or pneumonia within 3-5 years from initial diagnosis (Pisa et al, BMC Pulmonary Medicine 16, 136 (2016)). There are no current treatments for ALS (Hervias et al , Muscle & Nerve 33, 598-608 (2006)).
[0006] For most patients, the underlying cause for ALS is not known (sporadic ALS), but over 100 different mutations in superoxide dismutase 1 (SODl) account for ~20% of familial ALS forms
(Cozzolino et al , Progress in Neurobiology 97, 54-66 (2012)). Although only accounting for about
2% of all ALS cases, SODl -associated ALS has been the most studied form of ALS, due to the early discovery of the disease-causing mutations and the availability of mouse models. Mutations in SOD gene are gain-of-function mutations that cause autosomal dominant inheritance of ALS. It is the toxicity of the mutant SODl protein, rather than a defect in the function of the normal SODl protein, that is thought to lead to the disease. See, US Pat. App. Pub. No. 2016/0082015. Previous studies have suggested that mutations in SODl cause various cellular events, including alteration of gene expression, abnormal protein interactions, dysfunction of mitochondria and cytoskeletal abnormalities (Sau et al, Human Molecular Genetics 16, 1604-1618 (2007)). However, the causal relationship between these events and the death of the motor neurons remains unclear.
[0007] Several recent studies suggested possible defects in mitochondrial dynamics in models of ALS, regardless of the causative mutation (Sharma et al, Neurochemical Research 41, 965-984 (2016); Tafuri et al , Frontiers in Cellular Neuroscience 9, 336 (2015)). SODl is found in mitochondria, and abnormal mitochondrial morphology and cristae ultrastructure have been observed in mutant SODl mice and ALS patient samples, predominantly in the spinal cord (De Vos et al , Human Molecular Genetics 16, 2720-2728 (2007)). Furthermore, mutant SODl binds preferentially to mitochondria, impairs respiration, decreases the Ca2+ buffering capacity, blocks mitochondrial protein import, and induces apoptosis through Bcl-2 inhibition (Song et al , Neurobiology of Disease 51, 72- 81 (2013)). Mutant SOD1G93A also affects mitochondrial dynamics; there is a significant decrease in mitochondrial length and an accumulation of round fragmented mitochondria (Tafuri et al, supra). The increase of fragmented mitochondria coincides with an arrest in both anterograde and retrograde axonal transport and increased cell death (Magrane et al , The Journal of Neuroscience, 32, 229-242 (2012)). Mutant SOD1G93A induces a reduction in neurite length and branching that is accompanied with an abnormal accumulation of rounded mitochondria in growth cones of motor neurons (Rosen et al, Nature 362, 59-62 (1993); Magrane et al , Human Molecular Genetics 18, 4552-4564 (2009)). Abnormal mitochondrial dynamics was also recently observed in skeletal muscle of the SODl G93A mice (Luo et al , PloS One 8, e82112 (2013), together indicating the importance of mitochondrial dynamics in ALS.
[0008] Mitochondria exist in the cells as highly dynamic entities, ranging from elaborate tubular networks to small organelles. These morphological changes are orchestrated through rapid and reversible fission and fusion processes. Mitochondrial fusion is mediated by mitochondrial large GTPase on the outer mitochondrial membrane; mitochondrial fission is mediated by the recruitment of dynamin-related protein 1 (Drpl or Dynamin-l-like protein DNM1L; UNIPROT Accession Nos. 000429 (human); Q8K1M6 (mouse)), a cytosolic large GTPase, to the outer mitochondrial membrane (OMM) by mitochondrial fission factor (Mff), fission 1 (Fisl ; UNIPROT Accession Nos. Q9Y3D6 (human); Q9CQ92 (mouse)) and mitochondrial dynamics proteins of 49 kDa and 51 kDa (Mid49/ Mid51). While Drpl-mediated mitochondrial fission is important for cell survival and brain development, excessive Drpl -mediated fission causes mitochondrial fragmentation, mitochondrial membrane depolarization, increase in reactive oxygen production (ROS) and oxidative stress, a decrease in ATP production and other mitochondrial physiological functions (Babbar et al , Molecular & Cellular Pharmacology 5, 109-133 (2013); Youle et al , Science 337, 1062-1065 (2012); Wu et al , FEBS Journal, 278, 941-954 (2011)). Although excessive Drpl activity has been linked to Huntington's disease, Parkinson's disease, multiple sclerosis and stroke (Filichia et al , Scientific Reports 6, 32656 (2016); Guo et al , Biochem J. 461, 137-146 (2014); Guo et al , JCI, 123, 5371-5388 (2013)), its role in the pathogenesis of ALS and neurodegeneration of motor neurons was hitherto unknown. Additionally, these previous studies are silent with regard to usefulness of new inhibitors of Drpl in the prevention and treatment of neurological disorders such as ALS.
[0009] It should be noted in this context that the only FDA-approved drug, Riluzole, does not prevent or stop the onset of ALS but is merely effective to slow the disease's progression in certain patients that have increased levels of glutamate in the brain. Thus, Riluzole only acts by reducing an excitotoxic component of the disease, and while it prolongs life by 2 to 3 months, it provides little functional improvement (Miller et al, "Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND)." Cochrane Database Systemic Reviews, CD001447, 2007).
[0010] Therefore, there is an unmet need for effective compositions for promoting motor neuron survival and compositions and methods for treatment of motor neuron diseases such as ALS.
SUMMARY
[0011] Embodiments disclosed herein provide for mitochondrial fission inhibitors (including constructs thereof) in inhibiting mitochondrial fragmentation and improving mitochondrial function. Embodiments disclosed herein provide for mitochondrial fission inhibitors (including constructs thereof) in reducing mitochondrial oxidative stress, triggering of apoptotic pathways and signaling cascades that lead to cell lysis and/or death. Particular embodiments relate to the use in vivo of mitochondrial fission inhibitors (including constructs thereof) in the treatment of ALS. For instance, it was surprisingly found that treatment of subjects with the mitochondrial fission inhibitors (including constructs thereof) delayed onset of disease and/or slowed down the progression of the disease; improved behavioral outcomes such as ambulatory time and/or duration; improved motor function and/or muscle strength; reduced stereotypy and/or paralysis; and improved survival. Improvement in these various traits and/or outcomes correlated strongly with attenuation in mitochondrial localization of the Drpl/p62 following treatment with the compounds disclosed herein.
[0012] In one aspect, a method for the treatment of amyotrophic lateral sclerosis (ALS) in a subject in need thereof is provided, wherein the method comprises administering to the subject a therapeutically effective amount of a composition comprising a mitochondrial fission inhibitor or a derivative thereof.
[0013] In some embodiments, the mitochondrial fission inhibitor inhibits the interaction of Drpl with mitochondrial fission 1 protein (Fisl). In other embodiments, the mitochondrial fission inhibitor is an inhibitor of Drpl/Fisl mediated fission of mitochondria. [0014] In some embodiments, the mitochondrial fission inhibitor is a mitochondrial fission inhibitor peptide. In other embodiments, the mitochondrial fusion inhibitor peptide has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, or 99% sequence identity to a peptide selected from the group consisting of DLLPRGS (SEQ ID NO: 1), DLLPRGT (SEQ ID NO: 2), STQELLRFPK (SEQ ID NO: 3), KLSAREQRD (SEQ ID NO: 4), CSVEDLLKFEK (SEQ ID NO: 5), KGSKEEQRD (SEQ ID NO: 6), and ELLPKGS (SEQ ID NO: 7). In still other embodiments, the mitochondrial fission inhibitor is a peptide selected from the group consisting of the peptides identified herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
[0015] In some embodiments, the mitochondrial fission inhibitor comprises a salt, a solvate, a hydrate, a polymorph or a prodrug of the mitochondrial fission inhibitor peptide.
[0016] In some embodiments, the mitochondrial fission inhibitor comprises a mitochondrial fission inhibitor peptide and a linker. In other embodiments, the linker is a poly-glycine linker comprising 2-6 glycine residues.
[0017] In some embodiments, the mitochondrial fission inhibitor is administered as a composition comprising the mitochondrial fission inhibitor peptide and a carrier. In other embodiments, the carrier is a cationic lipid. In still other embodiments, the carrier facilitates intracellular delivery of the mitochondrial fission inhibitor.
[0018] In some embodiments, the carrier is a carrier peptide selected from the group consisting of YGRKKRRQRRR (SEQ ID NO: 8), RRRQRRKKRGY (SEQ ID NO: 9), RKKRRQRR (SEQ ID NO: 10), YARAAARQARA (SEQ ID NO: 11), THRLPRRRRRR (SEQ ID NO: 12), GGRRARRRRRR (SEQ ID NO: 13) and a combination thereof.
[0019] In some embodiments, the mitochondrial fission inhibitor comprises a mitochondrial fission inhibitor peptide which is conjugated at its carboxyl-terminus or its amino-terminus, directly or via a linker, to the carrier.
[0020] In some embodiments, the carrier is a carrier peptide and the mitochondrial inhibitor peptide is conjugated at its amino-terminus or its carboxyl-terminus to the carrier peptide by a polyglycine linker. In other embodiments, the polyglycine linker comprises 1-2, 1-3, 2-4, 2-3, 1-4, 1-5 or 1-6 glycine residues.
[0021] In some embodiments, the method for treatment of ALS comprises administering to the subject a therapeutically effective amount of a composition comprising a compound comprising the structure PEP-L-CAR (Formula I), wherein, PEP is a peptide selected from the group consisting of peptides identified as SEQ ID NOs: 1-7, L is a linker selected from the group consisting of a peptide bond, GG, GGG, GGS, GGSG (SEQ ID NO: 14), GGSGG (SEQ ID NO: 15), GSGSG (SEQ ID NO: 16), GSGGG (SEQ ID NO: 17), GGGSG (SEQ ID NO: 18), and GSSSG (SEQ ID NO: 19), CAR is a carrier selected from the group consisting of sequences identified herein as SEQ ID NOs: 8-13, and where the position of PEP and CAR with respect linker L can be interchanged. That is, in another embodiment, a method for treating ALS or for delaying progression of ALS is contemplated, where a composition comprising a therapeutically effective amount of a compound having the structure CARL-PEP (Formula II) or PEP-L-CAR (Formula I), wherein, PEP is a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a peptide selected from the group consisting of peptides identified as SEQ ID NOs: 1-7, L is a linker having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a linker selected from the group consisting of a peptide bond, GG, GGG, GGS, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, and CAR is a carrier linker having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a carrier selected from the group consisting of sequences identified herein as SEQ ID NOs: 8-13.
[0022] In some embodiments, the structure of Formula I comprises a sequence selected from the group consisting of STQELLRFPKGGYGRKKRRQRRR (SEQ ID NO: 20),
KLSAREQRDGGYGRKKRRQRRR (SEQ ID NO: 21), DLLPRGSGGYGRKKRRQRRR (SEQ ID NO: 22), DLLPRGTGGYGRKKRRQRRR (SEQ ID NO: 23),
CSVEDLLKFEKGGYGRKKRRQRRR (SEQ ID NO: 24), KGSKEEQRDGGYGRKKRRQRRR (SEQ ID NO: 25), and ELLPKGSGGYGRKKRRQRRR (SEQ ID NO: 26).
[0023] In some embodiments, the construct of Formula I comprises, consists or consists essentially of the sequence YGRKKRRQRRRGGDLLPRGT (SEQ ID NO: 27). In other embodiments, the construct of Formula I comprises, consists or consists essentially of the sequence YGRKKRRQRRRGGDLLPRGS (SEQ ID NO: 28).
[0024] In some embodiments, the ALS is familial ALS.
[0025] In some embodiments, the subject has a mutation in the superoxide dismutase (SOD) gene, fused-in-sarcoma (FUS1) gene, or trans active response DNA binding protein 43 kDa (TDP-43) gene, or a human homolog thereof. In other embodiments, the subject has a mutation in the superoxide dismutase 1 (SOD1) gene. In still other embodiments, the subject is a human who has a G93A mutation in the human SOD1 gene.
[0026] In some embodiments, the treatment results in improvement in at least one sign or symptom associated with ALS.
[0027] In some embodiments, the treatment results in improvement in a histopathological trait, a behavioral trait, a physiological trait or a combination of traits associated with ALS. In other embodiments, the improvement in the histopathological trait associated with ALS comprises reduced oxidative stress, reduced cellular apoptosis, reduced death, or reduced degeneration of neuronal cells or muscle cells. [0028] In some embodiments, the improvement in the behavioral trait associated with ALS comprises improved general mobility, improved motor function, reduced phobia, reduced stereotypy, improved engagement time, improved engagement score or a combination thereof.
[0029] In some embodiments, the improvement in general mobility includes improvement in the number of ambulatory episodes, improvement in the number of total ambulatory episodes, increase in moving time, increase in ambulatory time, an improvement in resting time or a combination thereof. In other embodiments, the improvement in motor function includes improved motor response to allodynia.
[0030] In some embodiments, the improvement in the physiological trait associated with ALS comprises improved strength of grip.
[0031] In some embodiments, the treatment with the inhibitor results in improvement in an outcome of the ALS disease in the subject. In other embodiments, the outcome is an epidemiological outcome, a histopathological outcome, or a physiological outcome, or a combination thereof. In still other embodiments, the outcome is an epidemiological outcome selected from the group consisting of overall survival (OS), survival at clinical score 1 (SCS I), survival at terminal endpoint (STE), age at terminal endpoint (ATE), survival at clinical score 3 (SCS3), age at clinical score 3 (ACS3), total disease duration (TDD), duration between clinical score 2 to terminal endpoint (CS2T), time to progression of disease (TTP), time-to-death (TTD), and disease-free survival period (DFS), or a combination thereof. In yet other embodiments, the outcome is selected from the group consisting of survival at clinical score 1 (SCSI), survival at terminal endpoint (STE), age at terminal endpoint (ATE), survival at clinical score 3 (SCS3), age at clinical score 3 (ACS3), total disease duration (TDD), and duration between clinical score 2 to terminal endpoint (CS2T) or a combination thereof. In some embodiments, CS2 comprises development of weakness/limpness in hind limbs and CS3 comprises development of paralysis of a hind limb.
[0032] In some embodiments, the terminal endpoint includes paralysis in both hind limbs plus about a 20% or greater drop in the body weight or paralysis in both hind limbs plus a lack of righting reflex.
[0033] In some embodiments, the improved histopathological outcome comprises attenuation in the association of Drpl with mitochondria of spinal cord neurons in the subject treated with the inhibitor compared to an untreated subject. In other embodiments, the improved histopathological outcome comprises attenuation in the levels of p62 in the mitochondrial fraction.
[0034] In some embodiments, the improved physiological outcome comprises increase in body weight compared to untreated subjects.
[0035] In one aspect, a method for identifying a test compound that promotes neuron survival and/or is useful for the treatment of ALS is provided. The method comprises (a) contacting a cell system or a cell-free system comprising Drpl and Fisl with a test compound; (b) detecting a parameter associated with mitochondrial dysfunction in the absence and in the presence of the test agent; and (c) selecting a test compound if it modulates the parameter associated with mitochondrial dysfunction.
[0036] In some embodiments, the compound is selected if it modulates the parameter associated with mitochondrial dysfunction by at least 30% compared to a control.
[0037] In some embodiments, wherein the modulation is an increase or decrease in the parameter and the control comprises an untreated cell system or an untreated cell-free system.
[0038] In some embodiments, the parameter associated with mitochondrial dysfunction comprises (a) increased mitochondrial interconnectivity or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death or cell lysis; (g) increased mitochondrial accumulation of mitophagy mediators selected from LC3- phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1); (h) increased c-Jun N-terminal kinase (INK) signaling. In other embodiments, the increased c-Jun N-terminal kinase (JNK) signaling results in increased phosphorylated JNK levels and/or increased levels of downstream effectors XBP1, ATF6a, phosphorylated eIF2a, GRP78 and/or CHOP. In still other embodiments, the parameter associated with mitochondrial dysfunction comprises (1) increased mitochondrial Drpl recruitment; (2) increased Drpl phosphorylation; or (3) increased mitochondrial accumulation of mitophagy mediators selected from LC3-II and p62.
[0039] In one aspect, a method of diagnosing a neurological disorder in a subject is provided. The method comprises: (a) obtaining a biological sample from the subject comprising a sample; (b) conducting at least one assay on the sample to detect mitochondrial dysfunction; and (c) diagnosing the subject as having the neurological disorder if the level of mitochondrial dysfunction in the patient sample is greater than the level of mitochondrial dysfunction in a control.
[0040] In some embodiments, the mitochondrial dysfunction comprises (a) increased mitochondrial interconnectivity or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death or cell lysis; (g) increased mitochondrial accumulation of mitophagy mediators selected from LC3-phosphatidylethanolamine conjugate (LC3- II) and p62 (SQSTM1); or (h) increased c-Jun N-terminal kinase (JNK) signaling.
[0041] In some embodiments, the increased c-Jun N-terminal kinase (JNK) signaling results in increased phosphorylated JNK levels and/or increased levels of downstream effectors XBP1, ATF6a, phosphorylated eIF2a, GRP78 and/or CHOP. [0042] In some embodiments, the mitochondrial dysfunction comprises (1) increased mitochondrial Drpl recruitment; (2) increased Drpl phosphorylation; or (3) increased mitochondrial accumulation of mitophagy mediators selected from LC3-II and p62.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] The details of one or more embodiments of the disclosure are set forth in the accompanying drawings/tables and the description below. Other features, objects, and advantages of the disclosure will be apparent from the drawings/tables and detailed description, and from the claims.
[0044] FIGS. 1A-1I show mitochondrial structural and functional defects in ALS patient-derived fibroblasts are mediated by Drpl/ Fisl interaction.
[0045] FIG. 1A shows results of cell-staining experiments, wherein ALS-patient derived fibroblasts were treated with or without PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours) for 48 hours in defined medium and then stained with anti-TOM20 (a marker of mitochondria, 1 :500 dilution). Side panels show enlarged areas of the boxed region. Scale bar: 0.5 μιη.
[0046] FIG. IB shows mitochondrial elongation in healthy control fibroblasts and ALS-patient derived fibroblasts from the stained images was quantified using a macro in Image! At least 200 cells were quantified per condition, n = 3. *** indicates PO.001 ; **** indicates PO.0001, each compared to respective vehicle treated cells.
[0047] FIG. 1C shows mitochondrial interconnectivity in healthy control fibroblasts and ALS- patient derived fibroblasts from the stained images, as quantified using a macro in IMAJEJ software. At least 200 cells were quantified per condition. n = 3. *** indicates PO.001; **** indicates PO.0001, each compared to respective vehicle-treated cells.
[0048] FIG. ID shows mitochondrial membrane potential was determined using TMRM dye after 48 hours in control and ALS-patient derived fibroblasts in the presence or absence of the Drpl inhibitor, PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours). Results are presented as percent of control, n = 3. ** indicates PO.01 ; * indicates PO.05, each compared to respective vehicle treated cells.
[0049] FIG. IE shows mitochondrial ATP levels in control and ALS patient-derived fibroblasts in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours). Results are presented as percent of control, n = 3. ** indicates PO.01; * indicates PO.05, each compared to respective vehicle treated cells.
[0050] FIG. IF shows measurement of mitochondrial ROS production using MITOSOX in ALS patient-derived fibroblasts. Results are presented as percent of control, n = 3. *** indicates P .001 ; **** indicates PO.0001, each compared to respective vehicle-treated cells.
[0051] FIG. 1G shows measurement of total cellular ROS production in control and ALS-patient derived fibroblasts in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours). Results are presented as percent of control, n = 3. ** indicates PO.01; **** indicates PO.0001, each compared to respective vehicle-treated cells.
[0052] FIG. 1H shows levels of Drpl in mitochondrial fractions, as measured by immunoblotting (VDAC was used as a loading control). The protein levels were quantified and represented as fold- change of control, n = 3. ** indicates P<0.01; **** indicates PO.0001, each compared to respective vehicle-treated cells.
[0053] FIG. II shows levels of p62 in mitochondrial fractions, as measured by immunoblotting (VDAC was used as a loading control). The protein levels were quantified and represented as fold- change of control, n = 3. ** indicates PO.01; **** indicates PO.0001, each compared to respective vehicle-treated cells.
[0054] FIGS. 2A-2F show that the expression of SOD-1 G93A mutant in motor neurons induces cellular stress in a Drpl -dependent manner.
[0055] FIG. 2A shows measurement of mitochondrial membrane potential, mitochondrial specific ROS production, and cell death using LDH assay, in hSODl-WT and hSODl-G93A expressing NSC- 34 differentiated cells, as determined under serum starvation and H2O2 injury in the presence or absence of Drpl inhibitor PI 10 (1 μΜ/ 24 hours). Results are presented as percent or fold of MOCK (empty vector) (means ± SD; n = 3. *P<0.05; ***P<0.001; ****P<0.0001).
[0056] FIG. 2B shows levels of Drpl in mitochondrial fractions, as determined by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (1 μΜ/ 24 hours). VDAC was used as a loading control. The protein levels were quantified and represented as fold-change of hSODl-WT (means ± SD; n = 3-5.
***P<0.001; ****P<0.0001).
[0057] FIG. 2C shows levels of Drpl phosphorylation in mitochondrial fractions, as determined by immunoblotting using anti-p-Drpl S616 or anti-p-Drpl S637 antibodies in hSODl-WT and hSODl- G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (1 μΜ/ 24 hours), β-actin was used as a loading control. The protein levels were quantified and represented as fold-change of hSODl-WT (means ± SD). n = 6. **P<0.01; ****P<0.0001.
[0058] FIG. 2D shows activities of various enzymes in homogenates of hSODl-WT and hSODl- G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (1 μΜ/ 24 hours). Chymotrypsin-like activity was measured using fluorogenic substrate; Suc- LLVY-AMC was used to measure proteasome activity. The levels were quantified and represented as fold-change of MOCK (empty vector) (means ± SD; n = 3. *PO.05; **PO.01).
[0059] FIG. 2E shows levels of Parkin and LC3BII in mitochondrial fractions as determined via immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours). VDAC was used as loading control. The protein levels were quantified and represented as fold-change of MOCK (empty vector) (means ± SD; n = 3. *P<0.05; **P<0.01 ; ****P<0.0001).
[0060] FIG. 2F shows levels of phosphorylated- eIF2a, XBP1, and CHOP in total fractions, as measured by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours), β- actin was used as a loading control. The protein levels were quantified and represented as fold-change of hSOD-1 WT (means ± SD; n = 3. *P<0.05; ***P<0.001 ; ****P<0.0001).
[0061] FIGS. 3A-3H show inhibition of Drpl hyper-activation and its interaction with Fisl using PI 10 (SEQ ID NO: 28) improves behavioral outcomes in SOD1-G93A ALS mice. For these animal- model experiments, n = 7 for WT mice; n = 7 for G93A ALS mice + TAT; n = 14 for G93A ALS mice + PI 10 (SEQ ID NO: 28).
[0062] FIG. 3A shows a cartoon of the treatment regime starting at day 90 with pumps replaced at day 120 as well as open field test regime.
[0063] FIG. 3B shows tracks of median control mice, SOD1 G93A + vehicle and SOD1 G93A + PI 10 (SEQ ID NO: 28) as measured in a 10-min open field test.
[0064] FIG. 3C shows ambulatory episodes as analyzed using activity chamber after 10 days or 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice (*P<0.05; **P<0.01; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0065] FIG. 3D shows ambulatory distances as analyzed using activity chamber after 10 days or 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice (*P<0.05; **P<0.01; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0066] FIG. 3E shows grip strength test was carried out to assess the on muscular degeneration after 25 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P<0.05; ***p< 0.001 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post- hoc analysis).
[0067] FIG. 3F shows total resting time was measured using activity chamber after 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice (*P<0.05; **P<0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0068] FIG. 3G shows animal behavior outcomes associated in the context of fear; center zone entries were analyzed using activity chamber after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P<0.05; **P<0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis). [0069] FIG. 3H shows PCA of the entire behavioral data shows behavioral separation between the three groups after treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice.
[0070] FIGS. 4A-4G show inhibition of Drpl hyperactivation and its association with Fisl in the symptomatic phase improves survival and slows disease progression. For panels A-F, n = 7 for WT; n = 7 for G93A ALS mice + TAT (SEQ ID NO 8); n = 14 for G93A ALS mice + PI 10 (SEQ ID NO: 28).
[0071] FIGS. 4A-4B show Kaplan-Meier survival curve of G93A SOD1 (ALS) mice showing increased survival following PI 10 (SEQ ID NO: 28) treatment (dotted trace) as compared to the vehicle-treated control ALS mice (TAT; SEQ ID NO: 8) (solid trace) (FIG. 4A) and that the age at terminal endpoint was significantly improved in the P110-treated ALS mice (FIG. 4B). Treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day using 28-day pumps with pumps replaced at 120 days (Log-rank Mantel-cox test; p<0.03; Mann Whitney test; p<0.03).
[0072] FIGS. 4C-4D show the curve of time to clinical score 3, defined as a point wherein mice showed single leg paralysis. PI 10 treatment (dotted trace) delayed the onset as compared to the vehicle treated control -treatment (TAT; SEQ ID NO: 8) (solid trace) (FIG. 4C). FIG. 4D shows that the age at which mice reached the clinical score 3 was significantly improved in the PI 10 (SEQ ID NO: 28) treated ALS mice. Treatment with vehicle or PI 10 at 3mg/kg/ day using 28-day pumps (ALZET®), with pumps replaced at 120 days (Log-rank Mantel-cox test; p<0.01, Mann Whitney test; p<0.01).
[0073] FIG. 4E shows overall disease progression was slowed down as indicated by increased days from clinical score 1 to terminal endpoint (Mann Whitney test; p=0.057).
[0074] FIG. 4F is a Kaplan-Meier survival curve of G93A SOD1 (ALS) mice showing increased probability of survival post-paralysis following PI 10 (SEQ ID NO: 28) treatment (dotted trace) as compared to the vehicle-treated control ALS mice (TAT; SEQ ID NO: 8) (solid trace).
[0075] FIG. 4G shows spinal cord levels of Drpl in mitochondrial fractions, as determined by immunoblotting in WT and SOD1-G93A treated with either vehicle control or with PI 10 (SEQ ID NO: 28) at 3mg/kg/ day. VDAC was used as a loading control. Protein levels were quantified and represented as fold-change of WT (means ± SD) (n = 3). ***P<0.001 ; ****P<0.0001.
[0076] FIGS. 5A-5E show results of immunoblotting experiments.
[0077] FIG. 5A shows representative Western blots showing association of Drpl and p62 in mitochondria-enriched fractions and Drpl and Fisl total levels in different ALS patient-derived fibroblasts in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours); VDAC and β-actin was used as a loading control for mitochondrial fraction and total fraction respectively (n = 3).
[0078] FIG. 5B shows representative Western blots showing levels of Drpl, cytochrome c, BAX and Bcl-2 in mitochondrial fractions and levels of Drpl phosphorylation using anti-p-Drpl S616 or anti-p-Drpl S637 antibodies by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC- 34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours). VDAC and β-actin was used as a loading control for mitochondrial fraction and total fraction, respectively (n = 3).
[0079] FIG. 5C shows representative Western blots showing levels of autophagy associated proteins. Parkin and LC3BII examined in mitochondrial fractions. Representative Western blots showing phosphorylated-JNK, total INK, p62 and LC3BII total levels in hSODl-WT and hSODl- G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours). VDAC and β-actin was used as a loading control for mitochondrial fraction and total fraction respectively (n = 3).
[0080] FIG. 5D shows representative western blots showing levels of ER stress associated protein, GRP78, CHOP, p-eIF2a, total eIF2a, XBP-1 and ATF-6 in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours); β-actin was used as a loading control (n = 3).
[0081] FIG. 5E shows representative western blots showing the levels of Drpl in the mitochondria enriched fraction in the spinal cord of in WT and SOD1-G93A treated with either vehicle control or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day; VDAC was used as a loading control (n = 3).
[0082] FIG. 6A shows nitric oxide levels in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells were determined under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours). Results are presented as percent or fold of MOCK (empty vector) (means ± SD; n = 3. *P<0.05; ***P<0.001).
[0083] FIG. 6B shows cell death measured using LDH assay, mitochondrial membrane potential, mitochondrial specific ROS production, and nitric oxide levels in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells were determined under H202 injury in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours). Results are presented as percent or fold of MOCK (empty vector) (means ± SD; n = 3. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).
[0084] FIG.6C shows levels of cytochrome c, BAX and Bcl-2 were examined in mitochondrial fractions by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours); VDAC was used as a loading control. The protein levels were quantified and represented as fold-change of hSODl-WT (means ± SD; n = 3-5. **P<0.01; ***P<0.001; ****P<0.0001).
[0085] FIG. 6D shows levels of phosphorylated- JNK, p62 and LC3BII in total fractions by immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours); beta-actin was used as a loading control for total fraction. The protein levels were quantified and represented as fold-change of MOCK (empty vector) (means ± SD; n = 3. *P<0.05; ***P<0.001).
[0086] FIG. 6E shows levels of ATF6 and GRP78 in total fractions were measured by
immunoblotting in hSODl-WT and hSODl-G93A expressing NSC-34 differentiated cells under serum starvation in the presence or absence of PI 10 (SEQ ID NO: 28) (1 μΜ/ 24 hours); beta-actin was used as a loading control. The protein levels were quantified and represented as fold-change of hSOD-1 WT (means ± SD; n = 3. *P<0.05; **P<0.01 ; ***P<0.001 ; ****P<0.0001).
[0087] FIG. 7A shows no significant difference in the onset of clinical score 1 between the two groups when the treatment was started.
[0088] FIG. 7B shows significant loss in body weight between the WT and SOD1-G93A. There was no significant difference in body weight of SOD1-G93A mice between treated with either vehicle control or PI 10 (SEQ ID NO: 28) after 25-days treatment. All data are mean ± SD; n = 7 for G93A ALS mice + TAT (SEQ ID NO: 8 )and n = 14 for G93A ALS mice + PI 10 (SEQ ID NO: 28) (***P<0.001).
[0089] FIG. 7C shows ambulatory time analyzed using activity chamber after 10 days or 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice (*P<0.05; **P<0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0090] FIG. 7D shows animal behavior outcome associated with motor function; jump time, analyzed using activity chamber after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P<0.05; **P<0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0091] FIG. 7E shows animal behavior outcome associated with motor function; jump count, analyzed using activity chamber after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P<0.05; **P<0.01; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0092] FIG. 7F shows animal behavior outcome associated in the context of fear; latency towards center, analyzed using activity chamber after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P<0.05; **P<0.01 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0093] FIG. 7G shows animal behavior outcome associated in the stereotypy analyzed using activity monitor after 10 days/ 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P<0.05; **P<0.01 ; ***P<0.001 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0094] FIG. 7H shows animal behavior outcome associated in the total resting time analyzed using activity monitor after 24 days of treatment with vehicle or PI 10 (SEQ ID NO: 28) at 3mg/kg/ day in G93A SOD1 mice. (*P<0.05; **P<0.01 ; ***P<0.001 ; one-way ANOVA, repeated measure with Uncorrected Fischer's LSD post-hoc analysis).
[0095] FIG. 71 shows ALS disease symptoms from clinical score 2 (weakness/limpness in 2 hind limbs) to terminal endpoint (includes paralysis in both hind limbs plus either a 20% BW drop vs. previous max weight or lack of righting reflex within 20 seconds) significantly slowed down in the PI 10 (SEQ ID NO: 28)-treated group (Kolmogorov-Smirnov test; p=0.042).
[0096] FIGS. 8A-8B are a Western blot and analysis that show increased GFAP staining, which correlates with astrocytosis, and beta-actin staining, a marker of microglia, were blunted by treatment with PI 10 (SEQ ID NO: 28) in ALS mice relative to wild type mice or control (TAT, SEQ ID NO: 8) treated mice.
[0097] FIG. 8C shows the fold change in calcium-binding protein (SI 00b) in wild type mice and in ALS mice treated with vehicle control or with PI 10 (SEQ ID NO: 28).
[0098] FIGS. 8D-8G are bar graphs showing the tissue levels of several cytokines, interleukin- lbeta (FIG. 8D), interleukin 1 -alpha (FIG. 8E), interleukin-6 (FIG. 8F) and tumor necrosis factor- alpha (FIG.8G).
[0099] FIGS. 9A-9D shows results of treating a microglial cell line transfected with SOD1G93A mutant gene (WT) and treated with TAT (SEQ ID NO: 8) control or with PI 10 (SEQ ID NO: 28), FIG. 9A showing the mitochondrial aspect ratio, FIG. 9B the intracellular ATP, FIG. 9C the mitoSOX and FIG. 9D the total ROS in the transfected cells untreated (squares), control-treated (triangles) and PI 10-treated (inverted triangles).
DETAILED DESCRIPTION
[00100] Various aspects now will be described more fully hereinafter. Such aspects may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete.
[0100] Where a range of values is provided in this disclosure, it is intended that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. For example, if a range of 1 μΜ to 8 μΜ is stated, it is intended that 2 μΜ, 3 μΜ, 4 μΜ, 5 μΜ, 6 μΜ, and 7 μΜ are also explicitly disclosed, as well as the range of values greater than or equal to 1 μΜ and the range of values less than or equal to 8 μΜ.
[0101] The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "amino acid" includes a single amino acid as well as two or more of the same or different amino acids; reference to an "excipient" includes a single excipient as well as two or more of the same or different excipients, and the like. [0102] The word "about" means a range of plus or minus 10% of that value, e.g., "about 50" means 45 to 55, "about 25,000" means 22,500 to 27,500, etc., unless the context of the disclosure indicates otherwise, or is inconsistent with such an interpretation. For example in a list of numerical values such as "about 49, about 50, about 55, "about 50" means a range extending to less than half the interval(s) between the preceding and subsequent values, e.g. , more than 49.5 to less than 52.5. Furthermore, the phrases "less than about" a value or "greater than about" a value should be understood in view of the definition of the term "about" provided herein.
[0103] The phrase "consisting essentially of means that specific further components or steps can be present provided the further component or step do not materially affect the basic and essential characteristic(s) of the method or composition.
Overview
[0104] Previous studies have indicated that mitochondrial dysfunction has a role in the progression of ALS disease (Manfredi et al . Mitochondrion 5, 77-87 (2005)). Abnormal mitochondrial morphology has been reported in ALS patients, mice, and cell culture models (Cozzolino et al . Frontiers in Cellular Neuroscience 9, 31 (2015); Jiang et al. , Translational Neurodegeneration 4, 14 (2015)). One of the most studied ALS causal mutations is in SODl . Mutant SOD1 (mSODl) aggregation in the mitochondria leads to increased free radical formation, electron transport chain (ETC), disruption and loss of the mitochondrial membrane potential, mitochondrial morphologic defects, and decreased ATP production (Tafuri et al.. Frontiers in Cellular Neuroscience 9, 336 (2015); Luo et al , PloS One 8, e82112 (2013)). Furthermore, mitochondria-targeted antioxidants have repeatedly been shown to be efficient in slowing disease progression in ALS mouse models (Miquel et al, FRBM, 70, 204-213 (2014); Pehar et al, PloS One 9, el03438 (2014); Petri et al , Journal of Neurochemistry 98, 1141-1148 (2006)). However, not much is known about the molecular pathways that lead to the observed mitochondrial dysfunction in ALS.
[0105] Embodiments described herein reveal that mutations in SODl, FUS 1 or TDP-43 in fibroblasts derived from patients with ALS exhibited decreased mitochondrial function due to Drpl hyperactivation and thereby increasing mitochondrial fragmentation. The data provided herein show that fibroblasts harboring mutations in any one of these three different genes all have a common pre- apoptotic mitochondrial defect trigger - Drpl-Fisl interaction. In these patient-derived cells, Drpl association with the mitochondria was much greater relative to cells from healthy subjects, which was reduced by treatment with a selective peptide inhibitor of Drpl-Fisl interaction, exemplified by the peptide of SEQ ID NO: 1, and administered in the form of a peptide-carrier construct referred to as "PI 10" (SEQ ID NO: 28). It will be appreciated that SEQ ID NO: 1 is exemplary and that the other peptides identified herein are contemplated and suitable, alone or in the form of a peptide-carrier construct, optionally with a linker. Pl lO-mediated reduction in Drpl recruitment and activation at the mitochondrial axis was independent of the aforementioned mutations. These findings corroborate with the observations made with fibroblast cell culture models comprising mutations in the superoxide dismutase (SOD) gene.
[0106] Additionally, the instant disclosure establishes a link between SOD1 mutation and impairment in proteasome activity in the context of pathogenesis and progression of ALS. in this regard, the present disclosure reveals that Drpl inhibitors improve mitochondrial health, as evidenced by attenuated ROS production, reduced pro-apoptotic triggers, increased ATP production and reduced mitochondrial autophagy in two different in vitro model systems for ALS. Concomitantly, these findings point to a role of Drpl inhibitors in the diagnosis of mitochondrial dysfunction associated with many neurological disorders, e.g. , Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, etc.
[0107] Furthermore, embodiments described herein elucidate an in vivo role of Drpl inhibitors, such as the antagonist peptide in the PI 10 construct, in the therapy of ALS. The therapeutic utility of Drpl inhibitors was elucidated in an ALS mouse model Treating ALS-model mice with the inhibitor comprising a peptide of SEQ ID NO: 1 conjugated to a TAT deliver}' moiety drastically improves clinical symptoms, which appear around day 90, such as reduced dragging hind feet/knuckles (Stage 1 ). Furthermore, sustained treatment with the peptide-TAT conj ugate (e.g. , SEQ ID NO: 28) starting at the onset of the symptoms significantly improved behavioral outcomes, motor functioning and also increased survival. The improvement in animal behavior and increased survival were associated with lower levels of Drpl association with the mitochondrial as well as the restored phosphorylation states of Drpl. Thus, embodiments disclosed herein provide the first evidence for the potential clinical and therapeutic utility of the peptides disclosed herein for the treatment of AL S patients.
[0108] in summary, based on the results derived from in vitro fibroblast cell-culture model, ALS patient-derived cell samples and in vivo mouse model of ALS, the present disclosure evidences a causal link between Drpl hyperactivation and mitochondrial impairment and neurodegeneration in ALS (which is mediated via recruitment of Drpl to the mitochondria). Moreover, Drpl-mediated mitochondrial impairment and autophagy were inhibited by SEQ ID NO: 1 , a selective peptide inhibitor of Drpl /Fi s i interaction. SEQ ID NO: 1 selectively inhibited pathological, but not basal, mitochondrial fission and fragmentation. Furthermore, the peptide also reduced motor dysfunctions and increased survival in an ALS mouse model harboring a mutation in the superoxide dismutase gene (SOD I G93A).
Mitochondrial Fission Inhibitor Compositions
[0109] The present disclosure relates to inhibitors of mitochondrial fission, particularly compositions which inhibit interaction between dynamin-related protein 1 (Drpl) and mitochondrial fission 1 protein (Fisl). [0110] The mitochondrial fission inhibitor can be a small molecule (e.g., a hapten), a peptide, a protein, an antibody, an aptamer, a nucleic acid, or a combination thereof.
[0111] In one embodiment, the mitochondrial fission inhibitor is a peptide (PEP), which optionally comprises a linker (L) and a carrier moiety (CAR).
[0112] In one specific embodiment, the mitochondrial fission inhibitor comprises the inhibitor peptide (PEP) compound only.
[0113] In another embodiment, the mitochondrial fission inhibitor comprises the inhibitor peptide compound (PEP) and the carrier moiety (CAR), wherein the inhibitor peptide is linked, either covalently or non-covalently, to the carrier moiety.
[0114] In yet another specific embodiment, the mitochondrial fission inhibitor comprises the inhibitor peptide compound (PEP) and both the linker (L) and the carrier moiety (CAR), wherein the inhibitor peptide is linked, either covalently or non-covalently, to the linker and the linker is linked, either covalently or non-covalently, to the carrier.
[0115] Under one embodiment, the mitochondrial fission inhibitor is a construct comprising the structure PEP-L-CAR (Formula I), wherein, PEP is the inhibitor peptide, L is a linker which is either present or absent, and CAR is a carrier moiety. Alternately, the mitochondrial fission inhibitor comprises the structure CAR-L-PEP (Formula II), wherein CAR, L and PEP are as described above.
[0116] Specifically, in the construct of Formula I or Formula II, the peptide inhibitor is linked to the linker, which is linked to a carrier via a covalent bond.
[0117] In one embodiment, the PEP, L and CAR groups in the construct of Formula I or Formula II are linked together via a peptide bond. As used herein, a "peptide bond" is formed by the condensation reaction between two amino acids, wherein the acid moiety of one reacts with the amino moiety of the other to produce a peptide bond (-CO-NH-) between the two amino acids.
[0118] In one embodiment, a mitochondrial fission inhibitor peptide construct comprises, in order from NH2 (amino) terminus to COOH (carboxy) terminus: (a) inhibitor peptide (PEP); (b) optionally a linker (L); and (c) carrier (CAR). Alternately, a mitochondrial fission inhibitor peptide comprises, in order from NH2 (amino) terminus to COOH (carboxyl) terminus: (a) carrier (CAR); (b) optionally a linker (L); and (c) inhibitor peptide (PEP). Under this embodiment, the various components of the constructs of Formula I or Formula II are linked together via one or more peptide bonds.
Peptide (PEP)
[0119] In one embodiment, the disclosure relates to a mitochondrial fission inhibitor peptide (PEP), which inhibits mitochondrial fission in a cell under pathological conditions, but does not inhibit mitochondrial fission in normal control cells. Thus, a mitochondrial fission inhibitor peptide of the present disclosure is useful for inhibiting aberrant (pathological) mitochondrial fission. Representative mitochondrial fission inhibitor peptides are disclosed in U.S. Patent Nos. 9,243,232 and 9,243,232, the disclosures in which are incorporated by reference in their entirety.
[0120] The mitochondrial fission inhibitor peptide can have a length of from about 7 amino acids to about 50 amino acids, e.g. , from about 7 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, or from about 45 amino acids to about 50 amino acids, or longer than 50 amino acids.
[0121] A mitochondrial fission inhibitor peptide can have a length of from about 7 amino acid residues to about 20 amino acid residues, e.g. , a mitochondrial fission inhibiting peptide can have a length of 7 residues, 8 residues, 9 residues, 10 residues, 11 residues, 12 residues, 13 residues, 14 residues, 15 residues, 16 residues, 17 residues, 18 residues, 19 residues, or 20 residues, or more.
[0122] As used herein, the term "peptide" includes a natural peptide comprising a linear chain or branched amino acids, peptidomimetics, as well as pharmaceutically acceptable salts thereof. Typically, a peptide comprises a plurality of amino acid residues, e.g. , 2, 3, 4, 5, 6, 8, 10, or more amino acid residues which are bonded to each other via covalent bonds, e.g., a peptide bond. "Amino acid residue" means the individual amino acid units incorporated into the peptides of the disclosure. As used herein, the term "amino acid" means a naturally occurring or synthetic amino acid, as well as amino acid analogs, stereoisomers, and amino acid mimetics that function similarly to the naturally occurring amino acids. Included by this definition are natural amino acids such as: (1) histidine (His; H) (2) isoleucine (He; I) (3) leucine (Leu; L) (4) Lysine (Lys; K) (5) methionine (Met; M) (6) phenylalanine (Phe; F) (7) threonine (Thr; T) (8) tryptophan (Tip; W) (9) valine (Val; V) (10) arginine (Arg; R) (11) cysteine (Cys; C) (12) glutamine (Gin; Q) (13) glycine (Gly; G) (14) proline (Pro; P) (15) serine (Ser; S) (16) tyrosine (Tyr; Y) (17) alanine (Ala; A) (18) asparagine (Asn; N) (19) aspartic acid (Asp; D) (20) glutamic acid (Glu; E) (21) selenocysteine (Sec; U); including unnatural amino acids: (a) citrulline (Cit); (b) cystine; (c) gama-amino butyric acid (GABA); (d) ornithine (Orn); (f) theanine; (g) homocysteine (Hey); (h) thyroxine (Thx); and amino acid derivatives such as betaine; carnitine; carnosine creatine; hydroxytryptophan; hydroxyproline (Hyp); N-acetyl cysteine; S-Adenosyl methionine (SAM-e); taurine; tyramine.
[0123] In one embodiment, the mitochondrial fission inhibitor peptide comprises the amino acid sequence set forth in the sequences identified herein as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or a variant thereof or a derivative thereof. [0124] As used herein, the term "derivative" includes salts, amides, esters, enol ethers, enol esters, acetals, ketals, acids, bases, solvates, hydrates, polymorphs or prodrugs of the individual amino acids or the aforementioned inhibitor peptides. Such derivatives may be readily prepared by those of skill in this art using known methods for such derivatization. The derivatives suitable for use in the methods described herein may be administered to animals or humans without substantial toxic effects and either are biologically active or are prodrugs.
[0125] In one example, the derivatives comprise salts of the amino acids or the inhibitor peptide. The term "salt" includes salts derived from any suitable of organic and inorganic counter ions well known in the art and include, by way of example, hydrochloric acid salt or a hydrobromic acid salt or an alkaline or an acidic salt of the aforementioned amino acids.
[0126] If desired, the derivative can, in addition or alternatively, be a solvent addition forms, e.g., a solvate or alcoholate. Solvates contain either stoichiometric or non-stoichiometric amounts of a solvent, and may be formed during the process of crystallization with acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water; alcoholates are formed when the solvent is alcohol. Solvates of compounds described herein can be conveniently prepared or formed using routine techniques. Polymorphs refer to alternate crystal forms of the compounds described herein. Polymorphic purity of drug samples can be checked using techniques such as powder X-ray diffraction, IR/Raman spectroscopy, and utilizing the differences in their optical properties in some cases (Thomas et al , Chemical Communications, 48: 10559-10561 (2012)).
[0127] The derivative can further comprise amides or esters of the amino acids and/or isomers (e.g. , tautomers or stereoisomers) of the amino acids, as desired.
[0128] In another embodiment, the mitochondrial fission peptide is a variant of the peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7. The term "variant" as used herein refers to a biomolecule (e.g., polypeptide or nucleic acid) whose sequence that differs from that of a parent sequence by virtue of at least one modification or amino acid substitution. Accordingly, variant peptides comprise at least one modification or substitution of an amino acid residue.
[0129] Variant peptides can have at least 1 amino acid substitution compared to the parent polypeptide, e.g. , from about 1 to 10 ten amino acid substitutions, 1 to about 5 amino acid substitutions compared to the parent, or 1, 2, 3, 4 or 5 amino acid substitutions, e.g., differing in amino acid sequence by one, two, three, four, or five amino acids, compared to amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7.
[0130] Under an alternate embodiment, the variant mitochondrial fission inhibitor peptide may comprise a sequence which is at least about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, identical to, for example, one of the following polypeptide sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
[0131] In one embodiment, conservative amino acid substitutions in the context of a mitochondrial fission inhibitor peptide are selected so as to preserve activity of the peptide. Residues that are semi- conserved may tolerate changes that preserve charge, polarity, and/or size. For example, a mitochondrial fission inhibitor peptide comprising the amino acid sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 may have 1, 2 or 3 amino acid substitutions, at position 1, 2, 3, 4, 5, 6, and/or 7, wherein the substituted amino acid may be any one of the known 20 amino acids, wherein the inhibitor peptide maintains a mitochondrial fission inhibiting function.
Linkers (L)
[0132] The mitochondrial fission inhibitor peptide of Formula I or Formula II can include a linker which joins or links a carrier moiety or peptide to a mitochondrial fission inhibitor peptide. The linker may be a peptide having any of a variety of amino acid sequences. A linker which is a spacer peptide can be of a flexible nature, although other chemical linkages are not excluded. A linker peptide can have a length of about 1, 2, 3, 4, 5 amino acids or from about 1 to 2, 1 to 3, 2 to 4, 2 to 5, 1 to 5, 5 to 10, 10 to 20, 20 to 30, or 30 to 40 amino acids in length. Peptide linkers with a degree of flexibility can be used. The linking peptides may have virtually any amino acid sequence, where in some embodiments the linker peptide will have a sequence that results in a generally flexible peptide. The use of small amino acids, such as glycine and alanine, are of use in creating a flexible peptide. The creation of such sequences is routine to those of skill in the art. Various linkers are commercially available and are considered suitable for use.
[0133] Exemplary flexible linkers which can be used to join or link a carrier moiety to a mitochondrial fission inhibitor peptide, for example, via peptide bonds, include glycine polymers (G)n, (e.g., where n is an integer from 1 to about 20); glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 29) and (GGGS)n (SEQ ID NO: 30), where n is an integer of between 1 and 10, e.g. , 1, 2, 3, 4, 5, 6, 7, or more), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components. Glycine polymers are used in some embodiments. See Scheraga et al, in Reviews in Computational Chemistry, K. B. Lipkowitz and D. B. Boyd, Eds., VCH Publishers, New York, 1992, Vol. 3, pp. 73-142 (1992). Exemplary flexible linkers include, but are not limited to GG, GGG, GGS, GGSG (SEQ ID NO: 14), GGSGG (SEQ ID NO: 15), GSGSG (SEQ ID NO: 16), GSGGG (SEQ ID NO: 17), GGGSG (SEQ ID NO: 18), GSSSG (SEQ ID NO: 19), and the like. [0134] In another embodiment, the linker is non-peptide linker. Non-peptide linker moieties can also be used to join or link a carrier moiety to a mitochondrial fission inhibitor peptide. The linker molecules are generally about 6-50 atoms long. The linker molecules may also be, for example, aryl acetylene, ethylene glycol oligomers containing 2-10 monomer units, diamines, diacids, amino acids, or combinations thereof. Other linker molecules which can bind to polypeptides may be used in light of this disclosure.
[0135] In an alternative embodiment, the inhibitor peptide may be linked to the carrier peptide by a disulfide bond. In some embodiments, the disulfide bond is formed between two cysteines, two cysteine analogs or a cysteine and a cysteine analog. In this embodiment, both the modulatory peptide and the carrier peptide contain at least one cysteine or cysteine analog. The cysteine residue or analog may be present as the N-terminal or C-terminal residue of the peptide or as an internal residue of the inhibitor peptide and of the carrier peptide. The disulfide linkage is then formed between the sulfur residues on each of the cysteine residues or analogs. Thus, the disulfide linkage may form between, for example, the N-terminus of the inhibitor peptide and the N-terminus of the carrier peptide, the C- terminus of the inhibitor peptide and the C-terminus of the carrier peptide, the N-terminus of the inhibitor peptide and the C-terminus of the carrier peptide, the C-terminus of the inhibitor peptide and the N-terminus of the carrier peptide, or any other such combination including at any internal position within the inhibitor peptide and/or the carrier peptide.
[0136] In yet another embodiment, the peptide inhibitor containing an additional amino acid comprising a reactive side chain, e.g., SH group of cysteine may be coupled to the carrier via click chemistry. See Liang et al. , J. Angew. Chem., Int. Ed., 48, 965 (2009).
Carrier (CAR)
[0137] As noted above, the construct of Formula I or Formula II may include a carrier moiety. "Carrier moiety" refers to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates traversing a lipid bilayer, micelle, cell membrane, organelle membrane, or vesicle membrane. A carrier moiety attached to another molecule facilitates the molecule traversing a membrane, for example going from extracellular space to intracellular space, or cytosol to within an organelle. In some cases, a carrier moiety facilitates crossing the blood-brain barrier. In some embodiments, a carrier moiety is covalently linked to the amino terminus of a mitochondrial fission inhibiting peptide. In some embodiments, a carrier moiety is covalently linked to the carboxyl terminus of a mitochondrial fission inhibiting peptide.
[0138] In some cases, the carrier moiety is a carrier peptide and is covalently linked to a fission inhibiting peptide, e.g. , via a peptide bond. For example, the carrier peptide can be a peptide having a length of from about 5 to 50 amino acids, e.g., from about 5 to 10, 5 to 15, 10 to 15, 10 to 20, 15 to 20, 10 to 25, 20 to 25, 20 to 30, or 30 to 40 amino acids. [0139] Exemplary carriers which may be linked to the mitochondria fission inhibitor peptide include but are not limited to a minimal undecapeptide protein transduction domain corresponding to residues 47-57 of human immunodeficiency virus-1 (HIV-1) TAT (GENBANK Acc. No. AEB53027;
including YGRKKRRQRRR (SEQ ID NO: 8) or RRRQRRKKRGY (SEQ ID NO: 9), a polyarginine sequence comprising a number of arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al , Cancer Gene Ther. 9(6):489-96 (2002)); a Drosophila Antennapedia protein transduction domain (Noguchi et al. Diabetes 52,1732-1737 (2003)); a truncated human calcitonin peptide (Trehin et al. Pharm. Research 21,1248-1256 (2004)); polylysine (Wender et al. , PNAS USA 97,13003-13008 (2000)); RRQRRTSKLMKR (SEQ ID NO: 31); transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 32);
KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 33); and
RQIKIWFQNRRMKWKK (SEQ ID NO: 34). Exemplary carriers include but are not limited to, YGRKKRRQRRR (SEQ ID NO: 8); RRRQRRKKRGY (SEQ ID NO: 9); RKKRRQRRR (SEQ ID NO: 10); an arginine homopolymer of from 3 arginine residues to 50 arginine residues; a TAT (48-60) polypeptide comprising the sequence GRKKRRQRRRPPQ (SEQ ID NO: 35); R9 peptide comprising the sequence RRRRRRRRR (SEQ ID NO: 36); a penetratin peptide comprising the sequence
RQIKIWFQNRRMKWKK (SEQ ID NO: 34); a pentratin-arginine fusion peptide (Pen-Arg) comprising the sequence RQIRIWFQNRRMRWRR (SEQ ID NO: 37); a pVEC peptide comprising the sequence LLIILRRRIRKQAHAHSK (SEQ ID NO: 38); an M918 peptide comprising the sequence MVTVLFRRLRIRRACGPPRVRV (SEQ ID NO: 39); a TP10 peptide comprising the sequence AGYLLGKINLKALAALAKKIL (SEQ ID NO: 40). Preferred carrier sequences include, but are not limited to, any of the following: YGRKKRRQRRR (SEQ ID NO: 8); RRRQRRKKRGY (SEQ ID NO: 9); RKKRRQRR (SEQ ID NO: 10); YARAAARQARA (SEQ ID NO: 11); THRLPRRRRRR (SEQ ID NO: 12); and GGRRARRRRRR (SEQ ID NO: 13).
[0140] In another embodiment, the carrier is a short amphipathic peptide carrier, Pep-1, which can facilitate rapid cellular uptake of various peptides, proteins, and even full-length antibodies with high efficiency and less toxicity. See Morris et al, Nat. Biotechnol., 19, 1173-1176, 2001.
[0141] It should be noted that in the aforementioned amino acid annotations, the left-most amino acid residue is normally the N-terminal end of the carrier peptide, which is conjugated to the C-terminal end of the mitochondrial fission inhibitor peptide (PEP) or the C-terminal end of the linker (L), e.g., via a covalent bond, particularly a peptide bond. However, the orientation of the carrier may be interchanged as long as the resulting construct possesses the desired activity, e.g. , inhibit the interaction between Drpl and Fisl, prevent mitochondrial fission, and/or attenuate cellular apoptosis or death due to mitochondrial injury. [0142] In embodiments wherein the carrier peptide (CAR) is conjugated to the N-terminal end of the mitochondrial fission inhibitor peptide (PEP) or the linker (L), e.g., to form a structure CAR-PEP or CAR-L-PEP, a reverse orientation of the conjugation, i.e. , C-terminal end of the carrier peptide (rightmost amino acid) is conjugated to the N-terminal end of the linker (L) or the N-terminal end of the mitochondrial fission inhibitor peptide (PEP), is particularly preferred.
[0143] In another embodiment, the carrier is a non-peptide molecule, e.g. , cationic lipids as described in Zuris et al., Nat Biotechnol. 33(l):73-80 (2015)).
[0144] Non-limiting examples of a mitochondrial fission inhibitor peptide construct of Formula I include the following fusion peptides:
[0145] STQELLRFPK-GG-YGRKKRRQRRR (SEQ ID NO: 20);
[0146] KLSAREQRD-GG-YGRKKRRQRRR (SEQ ID NO: 21);
[0147] DLLPRGS -GG- YGRKKRRQRRR (SEQ ID NO: 22);
[0148] DLLPRGT-GG-YGRKKRRQRRR (SEQ ID NO: 23);
[0149] CSVEDLLKFEK-GG-YGRKKRRQRRR (SEQ ID NO: 24);
[0150] KGSKEEQRD-GG-YGRKKRRQRRR (SEQ ID NO: 25); or
[0151] ELLPKGS-GG-YGRKKRRQRRR (SEQ ID NO: 26).
[0152] In one embodiment, the construct is a linear construct. In another embodiment, the construct is cyclic. In one embodiment, the construct is no more than 25, 30, 35, 40, 45, 50, 55, 60, 61, 62, 63, 64, or 65 amino acids in length.
[0153] With regard to the construct of Formula I or Formula II, embodiments further include variants which comprise an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, or 99% amino acid sequence identity to the constructs identified herein. Under this embodiment, the mitochondrial fission inhibitor peptide can comprise an amino acid sequence differing in amino acid sequence by 1, 2, 3, 4 or 5 amino acids, compared to the core structure of Formula I or Formula II. Purely as a representative embodiment, the variant peptide can comprise additional amino acids at the C-terminal end (e.g. , the arginine R residue in the aforementioned compounds), which allow functionalization or derivatization. Under a second representative embodiment, the variant peptide can comprise additional amino acids at the linker region (e.g. , the glycine G residues in the middle portion of aforementioned compounds), which confer desired length, hydrophilicity and other physiochemical properties to the variant peptides.
Mutants
[0154] In another embodiment, included herein are variant mitochondrial fission inhibitor peptides (or a construct of Formula I or Formula II) comprising a mutation in the core peptide sequence set forth in STQELLRFPK (SEQ ID NO: 3), KLSAREQRD (SEQ ID NO: 4), DLLPRGS (SEQ ID NO: 1), DLLPRGT (SEQ ID NO: 2), CSVEDLLKFEK (SEQ ID NO: 5), KGSKEEQRD (SEQ ID NO: 6) or ELLPKGS (SEQ ID NO: 7).
[0155] In one embodiment, the mutation is a substitution, deletion, addition of 1, 2, 3, 1-2, or 1-3 amino acids, wherein the mutation(s) does (do) not change the in vivo or in vitro activity of the inhibitor peptide construct as described herein.
Modifications
[0156] In some cases, a subject peptide comprises one or more modifications. For example, a mitochondrial fission inhibitor construct or peptide can be cyclized. As another example, a subject peptide can have one or more amino acid modifications. A subject mitochondrial fission inhibitor construct or peptide can include one or more D-amino acids.
[0157] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g. , acetylation, or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are peptides that have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
[0158] Also provided in the subject disclosure are mitochondrial fission inhibitor peptides (or a construct of Formula I or Formula II) that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. Analogs of such peptides include those containing residues other than naturally occurring L-amino acids, e.g. , D-amino acids or non-naturally occurring synthetic amino acids.
[0159] A subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) may be joined to a wide variety of other oligopeptides or proteins for a variety of purposes. By providing for expression of the subject peptides, various post-translational modifications may be achieved. For example, by employing the appropriate coding sequences, one may provide famesylation or prenylation. For example, a mitochondrial fission inhibitor construct or peptide can be bound to a lipid group at a terminus, so as to be able to be bound to a lipid membrane, such as a liposome.
[0160] Other suitable modifications on the mitochondrial fission inhibitor peptide (or a construct of
Formula I or Formula II) include, e.g. , (1) end-cappings of the terminal of the peptides, such as amidation of the C-terminus and/or acetylation or deamination of the N-terminus; (2) introducing peptidomimetic elements in the structure; and (3) cyclization, in which the cyclization of the peptide can occur through natural amino acids or non-naturally-occurring building blocks. [0161] A modified mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be a peptoid (N-substituted oligoglycines), e.g. , in which an amino acid side chain is connected to the nitrogen of the peptide backbone, instead of the a-carbon. See, e.g., Zuckermann et al , J. Am. Chem. Soc. 114, 10646 (1992).
[0162] A subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can include naturally-occurring and non-naturally occurring amino acids. A subject mitochondrial fission inhibitor construct or peptide can comprise D-amino acids, a combination of D- and L-amino acids, and various "designer" amino acids (e.g., β-methyl amino acids, Ca-methyl amino acids, and Na-methyl amino acids, etc.) to convey special properties to peptides.
[0163] Additionally, a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be a cyclic peptide. A subject mitochondrial fission inhibitor construct or peptide can include non-classical amino acids in order to introduce particular conformational motifs. Any known non-classical amino acid can be used. Non-classical amino acids include, but are not limited to, l,2,3,4-tetrahydroisoquinoline-3-carboxylate; (2S,3S)-methylphenylalanine, (2S,3R)-methyl- phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine; 2- aminotetrahy dronaphthalene-2-carboxylic acid; hydroxy- 1 ,2,3,4-tetrahy droisoquinoline-3-carboxylate; β-carboline (D and L); HIC (histidine isoquinoline carboxylic acid); and HIC (histidine cyclic urea). Amino acid analogs and peptidomimetics can be incorporated into a subject mitochondrial fission inhibitor construct or peptide to induce or favor specific secondary structures, including, but not limited to, LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), a β-turn inducing dipeptide analog; β-sheet inducing analogs; β-turn inducing analogs; a-helix inducing analogs; γ-turn inducing analogs; Gly-Ala turn analog; amide bond isostere; tretrazol; and the like.
[0164] Inclusion of a non-naturally occurring amino acid can provide for linkage to a polymer, a second polypeptide, a scaffold, etc. For example, a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) linked to a water-soluble polymer can be made by reacting a water-soluble polymer (e.g. , poly(ethylene glycol) (PEG)) that comprises a carbonyl group to an the subject mitochondrial fission inhibitor construct or peptide that comprises a non-naturally encoded amino acid that comprises an aminooxy, hydrazine, hydrazide or semicarbazide group.
[0165] In some embodiments, a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) is linked (e.g. , covalently linked) to a polymer (e.g. , a polymer other than a polypeptide). As another example, a subject mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be linked to a water-soluble polymer (e.g., PEG). Suitable polymers can have an average molecular weight in a range of from 500 Da to 50,000 Da, e.g. , from 5000 Da to 40,000 Da, from 25,000 to 40,000 Da, or from 40,000 to 60,000 Da. Methods of Making the Inhibitor Peptide and/or Constructs
[0166] A mitochondrial fission inhibitor peptide can be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified. For the most part, the compositions which are used will comprise at least 80% by weight of the desired product, at least about 85% by weight, at least about 95% by weight, or at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. The percentages can be based upon total protein.
[0167] A mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) may be prepared by in vitro (e.g. , cell-free) synthesis, using conventional methods as known in the art. Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, Calif, Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
[0168] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface, or provide some other desired property such as increased solubility, increased resistance to proteolysis, increased in vivo half-life, and the like. One or more cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.
Compositions
[0169] A mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) as described herein may be in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include acid addition salts, such as hydrochloride, hydrobromide, sulfurate, nitrate, phosphorate, acetate, propionate, glycolate, pyruvate, oxalate, malate, malonate, succinate, maleate, fumarate, tartarate, citrate, benzoate, cinnamate, mandelate, methanesulfonate, ethanesulfonate, p- toluene-sulfonate, salicylate and the like, and base addition salts, such as sodium, potassium, calcium, magnesium, lithium, aluminum, zinc, ammonium, ethylenediamine, arginine, piperazine, etc.
[0170] The present disclosure provides compositions comprising a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II). The composition can comprise, in addition to a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II), one or more of: a salt, e.g., NaCl, MgCl, KC1, MgS04, etc.; a buffering agent, e.g., a Tris buffer, N-(2- Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(Ν-Μο ηο1ίηο)ρΓορ3η68υ1ίοηίΰ acid (MOPS), N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.; a solubilizing agent; a detergent, e.g. , a non-ionic detergent such as TWEEN-20, etc.; a protease inhibitor; glycerol; and the like.
[0171] Compositions comprising a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) may include a buffer, which is selected according to the desired use of the peptide, and may also include other substances appropriate to the intended use. Those skilled in the art can readily select an appropriate buffer, a wide variety of which are known in the art, suitable for an intended use.
Pharmaceutical compositions
[0172] In some cases, a composition comprising the mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) is a pharmaceutical composition containing a pharmaceutically acceptable carrier. A pharmaceutical composition can be administered to a subject in need thereof (e.g., a subject in need of inhibition of abnormal (e.g. , pathological) mitochondrial fission). A subject pharmaceutical composition comprises: a) a mitochondrial fission inhibitor construct or peptide; and b) a pharmaceutically acceptable excipient, a variety of which are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, "Remington: The Science and Practice of Pharmacy", 19th Ed. (1995), or latest edition, Mack Publishing Co; A. Gennaro (2000) "Remington: The Science and Practice of Pharmacy", 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al , eds ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al , eds., 3rd ed. Amer. Pharmaceutical Assoc.
Kits
[0173] In some embodiments, disclosed herein are kits or other articles of manufacture which contains one or more of the mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) or a composition comprising the same, together with instructions for formulating and/or using the composition, e.g. , in the treatment of ALS. Kits or other articles of manufacture may include a container, a syringe, vial and any other articles, devices or equipment useful in administration (e.g. , intravenous, subcutaneous, or inhalation). Suitable containers include, for example, bottles, vials, syringes (e.g., pre-filled syringes), ampules, cartridges, reservoirs, pumps, or lyo-jects. The container may be formed from a variety of materials such as glass or plastic. In some embodiments, a container is an osmotic pump, e.g. , ALZET pump, DUROS pump (see, Rohloff et al , J Diabetes Sci Technol., 2, 461-467 (2008)) or Intarcia Pump 650 (Intarcia Therapeutics, Boston, MA, USA). Suitable pre- filled syringes include, but are not limited to, borosilicate glass syringes with baked silicone coating, borosilicate glass syringes with sprayed silicone, or plastic resin syringes without silicone. [0174] Typically, the container may hold one or more formulations and a label on, or associated with, the container that may indicate directions for reconstitution and/or use. For example, the label may indicate that the formulation is reconstituted to concentrations as described above. The label may further indicate that the formulation is useful or intended for, for example, subcutaneous administration. In some embodiments, a container may contain a single dose of a stable formulation containing the peptide inhibitor or the construct of Formula I or Formula II. In various embodiments, a single dose of the stable formulation is present in a volume of less than about 15 ml, 10 ml, 5.0 ml, 4.0 ml, 3.5 ml, 3.0 ml, 2.5 ml, 2.0 ml, 1.5 ml, 1.0 ml, or 0.5 ml, or less. Alternatively, a container holding the formulation may be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the formulation. Kits or other articles of manufacture may further include a second container comprising a suitable diluent (e.g., BWFI, saline, buffered saline). Upon mixing of the diluent and the formulation, the final peptide concentration in the reconstituted formulation will generally be at least 1 μg/ml (e.g. , at least 5 μg/ml, at least 10 μg/ml, at least 20 μg/ml, at least 50 μg/ml, at least 100 μg/ml, at least 300 μg/ml, at least 500 μg/ml, at least 1 mg/ml, at least 3 mg/ml, at least 10 mg/ml or more). Kits or other articles of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, kits or other articles of manufacture may include an instruction for self-administration.
Nucleic Acids Encoding the Peptides or Constructs
The present disclosure provides synthetic nucleic acids, where a subject synthetic nucleic acid comprises a nucleotide sequence encoding a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II). Included herein are nucleic acids encoding the mitochondrial fission inhibitor peptides identified herein by SEQ ID NOs: 1-7, or a variant thereof having 1, 2, 3, 4, or 5 amino substitutions, the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof.
[0175] Also included herein are nucleic acids encoding the following mitochondrial fission inhibitor peptide constructs of Formula I or Formula II, including the constructs identified herein by SEQ ID NOs: 21-28, or a variant thereof having 1, 2, 3, 4, 5, 4-10, 5-10, or 5 to 15 amino acid substitutions, or the complementary strand thereto, or the RNA equivalent thereof, or a complementary RNA equivalent thereof.
Vectors
[0176] Also included herein are vectors which contain one or more of the aforementioned nucleic acids. In one embodiment, the vector comprises at least one protein encoding nucleic acid, e.g. , nucleic acids encoding the mitochondrial fission inhibitor peptide sequences for Formulas disclosed herein. [0177] A nucleotide sequence encoding a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) as described herein can be operably linked to one or more regulatory elements, such as a promoter and enhancer, that allow expression of the nucleotide sequence in the intended target cells (e.g., a cell that is genetically modified to synthesize the encoded mitochondrial fission inhibitor peptide or construct). In some embodiments, a subject nucleic acid is a recombinant expression vector.
[0178] Accordingly, the present disclosure provides isolated genetically modified host cells (e.g., transformed cells or cell-lines) that are genetically modified with a nucleic acid comprising a nucleic acid sequence which encodes a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) or which harbor an expression vector construct as described herein. In some embodiments, a subject isolated genetically modified host cell can produce a mitochondrial fission inhibitor construct or peptide.
Codon optimized sequences
[0179] Included herein are codon-optimized sequences of the aforementioned nucleic acid sequences and vectors. Codon optimization for expression in a host cell, e.g. , bacteria such as E. coli or insect Hi5 cells, may be routinely performed using Codon Optimization Tool (CODONOPT), available freely from Integrated DNA Technologies, Inc., Coralville, Iowa.
Antibodies
[0180] Embodiments disclosed herein further include antibodies or antigen-binding fragments thereof which bind specifically to one or more of the aforementioned peptides or fusion proteins thereof.
[0181] In one embodiment, the antibodies or fragments thereof bind to polypeptides comprising the amino acid sequences identified as SEQ ID NOs: 1-8 or an immunogenic fragment thereof. In another embodiment, the antibodies bind to fragment of these polypeptides. Antigen-binding fragments of such antibodies, include, e.g. , F(ab) domain, F(ab)2 domains, scFv domains, including synthetically generated antibodies (using, e.g. , phase display technology).
[0182] In one embodiment, the antibodies bind to polypeptide sequences identified as SEQ ID NOs: 20-28, or an immunogenic fragment thereof.
Purified molecules
[0183] Included herein are purified biomolecules, e.g., nucleic acids, proteins, peptides, and/or antibody molecules, including, conjugates thereof. The term "substantially purified," as used herein, refers to nucleic acids, amino acids or antibodies that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, most preferably about 90% and especially about 95% free from other components with which they are naturally associated. [0184] In embodiments described herein, the biomolecules may be altered by combining with various components of the constructs of Formula I or Formula II, e.g., fission inhibitor peptide or linker or carrier, such that their form and/or functionality is significantly changed compared to any natural counterparts. A change in property may include physiochemical properties of the peptides, e.g. , molecular weight, isoelectric point, solubility, hydrophobicity and/or functionality of the peptides, e.g. , Drpl-Fisl inhibition, mitophagy, anti-apoptotic property, mitochondrial targeting, and the like.
Nucleic Acid Agents as Mitochondrial Fission Inhibitors
[0185] In some embodiments, the agents for use in accordance with the present disclosure are or comprise nucleic acids. In some such embodiments, mitochondrial fission inhibitors comprise RNA and/or DNA molecules which target Drpl. In some such embodiments, fission inhibitors are RNAi agents (for example, miRNAs, siRNAs, shRNAs, antisense oligonucleotides, ribozymes) and/or gene therapy vectors.
[0186] In some embodiments, nucleic acid fission inhibitors for use in accordance with the present disclosure have a nucleotide sequence that corresponds to or hybridizes with a portion of a polynucleotide that encodes a Drpl, particularly, an mRNA sequence thereof. In some embodiments, nucleic acid agents for use in accordance with the present embodiment have a nucleotide sequence that includes a binding site for a Drpl binding partner, e.g. , Fisl, which mediates its cellular effects, e.g., mitochondrial localization, induction of oxidative and ER stress, activation of pro-apoptotic pathway, cell lysis and/or cell-death, etc.
[0187] In certain embodiments, the Drpl inhibitor is a complex comprising clustered regularly interspaced short palindromic repeat (CRISPR) and a CRISPR associated protein (CAS) or nucleic acid encoding the CRISPER/CAS complex, wherein the complex targets nucleic acid encoding Drpl. See Kunin et al, Genome Biology 8 (4): R61, 2007; Carte et al , Genes & Development 22 (24): 3489-96; Wang et al, Structure 19 (2): 257-64, 2011; Niewoehner et al , Nucleic Acids Research 42 (2): 1341-53, 2014; Semenova et al , PNAS USA 108 (25): 10098-103, 2011; Gudbergsdottir et al, Molecular Microbiology 79 (1): 35-49, 2011; Mmica et al, Molecular Microbiology, 80 (2): 481-91, 2011.
Formulations. Dosages, and Routes of Administration
[0188] A mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure (also referred to below as "active agent") can be incorporated into a variety of formulations for therapeutic use (e.g. , for treating a subject diagnosed with or suffering from a disease which is associated with abnormal mitochondrial fission). More particularly, a mitochondrial fission inhibitor peptide or construct can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, lotions, and aerosols. As such, administration of a mitochondrial fission inhibitor peptide or construct can be achieved in various ways, including oral, vaginal, buccal, rectal, parenteral, intraperitoneal, intravenous, intramuscular, intradermal, transdermal, intratracheal, etc., administration. A mitochondrial fission inhibitor peptide or construct can be systemic after administration or may be localized by the use of an implant or other formulation that acts to retain the active dose at the site of implantation.
[0189] A mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be administered alone, in a combination of two or more mitochondrial fission inhibitor peptide or construct, or a mitochondrial fission inhibitor peptide or construct can be used in combination with known compounds (e.g., therapeutic agents suitable for treating a disease associated with abnormal mitochondrial fission, etc.) In pharmaceutical dosage forms, a mitochondrial fission inhibitor peptide or construct may be administered in the form of its pharmaceutically acceptable salt. The following methods and excipients are merely exemplary and are in no way limiting.
[0190] For oral preparations, a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be used alone or in combination with appropriate additives to make tablets, powders, granules or capsules, for example, with conventional additives, such as lactose, mannitol, com starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, com starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents.
[0191] A mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure can be formulated into preparations for injections by dissolving, suspending or emulsifying the peptide in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
[0192] A mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure can be utilized in aerosol formulation to be administered via inhalation. A mitochondrial fission inhibitor construct or peptide of the present disclosure can be formulated into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like.
[0193] A mitochondrial fission inhibitor peptide or construct of the present disclosure can be used in topical formulations, by formulation with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
[0194] Furthermore, a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure can be made into suppositories by mixing with a variety of bases such as emulsifying bases or water-soluble bases. A mitochondrial fission inhibitor peptide (or a construct) of the present disclosure can be administered rectally via a suppository. The suppository can include vehicles such as cocoa butter, carbowaxes and polyethylene glycols, which melt at body temperature, yet are solidified at room temperature.
[0195] Unit dosage forms for oral, vaginal or rectal administration such as syrups, elixirs, and suspensions may be provided wherein each dosage unit, for example, teaspoonful, tablespoonful, tablet or suppository, contains a predetermined amount of a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) of the present disclosure. Similarly, unit dosage forms for injection or intravenous administration may comprise a mitochondrial fission inhibitor peptide (or a construct) in a composition as a solution in sterile water, normal saline or another pharmaceutically acceptable carrier.
[0196] Implants for sustained release formulations are well known in the art. Implants can be formulated as microspheres, slabs, etc. with biodegradable or non-biodegradable polymers. For example, polymers of lactic acid and/or gly colic acid form an erodible polymer that is well-tolerated by the host. An implant containing a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be used, so that the local concentration of active agent (mitochondrial fission inhibitor peptide or construct) is increased relative to the rest of the body.
[0197] Liposomes can be used as a delivery vehicle. The lipids can be any suitable combination of known liposome forming lipids, including cationic or zwitterionic lipids, such as phosphatidylcholine. The remaining lipid can include neutral or acidic lipids, such as cholesterol, phosphatidyl serine, phosphatidyl glycerol, and the like.
[0198] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are readily available to the public. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
Dosages
[0199] The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for human and non-human animal subjects, each unit containing a predetermined quantity of a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms depend on the particular mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) employed and the effect to be achieved, and the pharmacodynamics associated with the mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) in the host. [0200] Exemplary dosages for systemic administration range from 0.1 μg to 100 milligrams per kg weight of subject per administration. An exemplary dosage may be one tablet taken from two to six times daily, or one time-release capsule or tablet taken once a day and containing a proportionally higher content of active ingredient. The time-release effect may be obtained by capsule materials that dissolve at different pH values, by capsules that release slowly by osmotic pressure, or by any other known means of controlled release.
[0201] Depending on the subject and condition being treated and on the administration route, an active agent (e.g., a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) may be administered in dosages of, for example, 0.1 μg to 500 mg/kg body weight per day, e.g. , from about 0.1 μg/kg body weight per day to about 1 μg/kg body weight per day, from about 1 μg/kg body weight per day to about 25 μg/kg body weight per day, from about 25 μg/kg body weight per day to about 50 μg/kg body weight per day, from about 50 μg/kg body weight per day to about 100 μg/kg body weight per day, from about 100 μg/kg body weight per day to about 500 μg/kg body weight per day, from about 500 μg/kg body weight per day to about 1 mg/kg body weight per day, from about 1 mg/kg body weight per day to about 25 mg/kg body weight per day, from about 25 mg/kg body weight per day to about 50 mg/kg body weight per day, from about 50 mg/kg body weight per day to about 100 mg/kg body weight per day, from about 100 mg/kg body weight per day to about 250 mg/kg body weight per day, or from about 250 mg/kg body weight per day to about 500 mg/kg body weight per day. The range is broad, since in general the efficacy of a therapeutic effect for different mammals varies widely with doses generally being 20, 30 or even 40 times smaller (per unit body weight) in man than in the rat. Similarly the mode of administration can have an effect on dosage. Thus, for example, oral dosages may be about ten times the injection dose. Higher doses may be used for localized routes of delivery.
[0202] A specific mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) can be administered in an amount of from about 1 mg to about 1000 mg per dose, e.g. , from about 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 20 mg, from about 20 mg to about 25 mg, from about 25 mg to about 50 mg, from about 50 mg to about 75 mg, from about 75 mg to about 100 mg, from about 100 mg to about 125 mg, from about 125 mg to about 150 mg, from about 150 mg to about 175 mg, from about 175 mg to about 200 mg, from about 200 mg to about 225 mg, from about 225 mg to about 250 mg, from about 250 mg to about 300 mg, from about 300 mg to about 350 mg, from about 350 mg to about 400 mg, from about 400 mg to about 450 mg, from about 450 mg to about 500 mg, from about 500 mg to about 750 mg, or from about 750 mg to about 1000 mg per dose.
[0203] Those of skill will readily appreciate that dose levels can vary as a function of the specific mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II), the severity of the symptoms and the susceptibility of the subject to side effects. Some of the specific peptides may be more potent than others. Preferred dosages for a given peptide are readily determinable by those of skill in the art by a variety of means. One means is to measure the physiological potency of a given peptide.
Routes of Administration
[0204] An active agent (e.g., a mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) is administered to an individual using any available method and route suitable for drug delivery, including in vivo and ex vivo methods, as well as systemic and localized routes of administration. Administration can be acute (e.g., of short duration, e.g. , a single administration, administration for one day to one week), or chronic (e.g. , of long duration, e.g. , administration for longer than one week, e.g. , administration over a period of time ranging from about 2 weeks to about one month, from about 1 month to about 3 months, from about 3 months to about 6 months, or more).
[0205] Conventional and pharmaceutically acceptable routes of administration include intranasal, intramuscular, intratracheal, subcutaneous, intradermal, transdermal, sublingual, topical application, intravenous, ocular (e.g. , topically to the eye, intravitreal, etc.), rectal, nasal, oral, and other enteral and parenteral routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the agent and/or the desired effect. A mitochondrial fission inhibitor peptide or (or a construct of Formula I or Formula II) can be administered in a single dose or in multiple doses.
[0206] An active agent (e.g., a mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) can be administered to a host using any available conventional methods and routes suitable for delivery of conventional drugs, including systemic or localized routes. In general, routes of administration contemplated by the disclosure include, but are not necessarily limited to, enteral, parenteral, and inhalational routes.
[0207] Parenteral routes of administration other than inhalation administration include, but are not necessarily limited to, topical, transdermal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intrasternal, ocular, and intravenous routes, i.e., any route of administration other than through the alimentary canal. Parenteral administration can be carried to effect systemic or local delivery of the agent. Where systemic delivery is desired, administration can involve invasive or systemically absorbed topical or mucosal administration of pharmaceutical preparations.
[0208] A mitochondrial fission inhibitor peptide or (or a construct of Formula I or Formula II) can also be delivered to the subject by enteral administration. Enteral routes of administration include, but are not necessarily limited to, oral and rectal (e.g. , using a suppository) delivery.
[0209] Methods of administration of a mitochondrial fission inhibitor peptide or (or a construct of Formula I or Formula II) through the skin or mucosa include, but are not necessarily limited to, topical application of a suitable pharmaceutical preparation, transdermal transmission, injection and epidermal administration. For transdermal transmission, absorption promoters or iontophoresis are suitable methods. Iontophoretic transmission may be accomplished using commercially available "patches" which deliver their product continuously via electric pulses through unbroken skin for periods of several days or more.
[0210] In another embodiment, disclosed herein is a use of the mitochondrial fission inhibitor (or a construct of Formula I or Formula II), i.e. , a peptide Drpl inhibitor, in reducing mitochondrial dysfunction, as characterized by (a) increased mitochondrial interconnectivity or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death or cell lysis; (g) increased mitochondrial accumulation of mitophagy mediators selected from LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1); or (h) increased c-Jun N-terminal kinase (JNK) signaling.
[0211] In another embodiment, disclosed herein is use of mitochondrial fission inhibitor (or a construct of Formula I or Formula II), i.e., a peptide Drpl inhibitor, for the manufacture of a medicament for treating a neurological disorder, e.g., ALS.
Methods of use
Use in screening compounds
[0212] The present disclosure contemplates various assays for identifying agents that can increase neuron survival, as well as candidate therapeutic agents for treating neurodegenerative disorders (e.g., ALS).
[0213] In one embodiment, the disclosure relates to methods for identifying a test compound that promotes neuron survival and/or is useful for the treatment of ALS comprising (a) contacting a cell system or a cell-free system comprising Drpl and Fisl with a test compound; (b) detecting a parameter associated with mitochondrial dysfunction in the absence and in the presence of the test agent; and (c) selecting a test compound if it modulates the parameter associated with mitochondrial dysfunction.
[0214] Additionally, the method may comprise incubating the cell or cell-free system with one or more mitochondrial fission inhibitor peptides or a construct of Formula I or Formula II (disclosed hereinbefore) as a positive control.
[0215] As used herein, the term "test compound" refers to agents and/or compositions that are to be screened for their ability to stimulate and/or increase and/or promote cell survival. The test agents can include a wide variety of different compounds, including chemical compounds and mixtures of chemical compounds, e.g. , small organic or inorganic molecules; saccharines; oligosaccharides; polysaccharides; biological macromolecules, e.g. , peptides, proteins, and peptide analogs and derivatives; peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; an extract made from biological materials such as bacteria, plants, fungi, or animal cells; animal tissues; naturally occurring or synthetic compositions; and any combinations thereof. In some embodiments, the test agent is a small molecule or a peptide.
[0216] In some embodiments, the modulation comprises a change of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or more compared to a control.
[0217] The screening method(s) may be carried out using any cells from a biological tissue and/or cell -containing bodily fluid that has been obtained directly from an individual, donor patient or animal. In some embodiments, the cells may be directly-derived from a subject, e.g. , without intermediate steps of subculture through a series of cultures and/or hosts. Thus, a suspension of single cells is produced directly from the biological tissue and/or cell-containing bodily fluid. This is in contrast to established methods in which stable and highly passaged cell lines are used. Such cell-lines are far removed from being directly derived from their progenitor tissue by several intermediate culture steps.
[0218] Cell-free systems, including ceil-free extracts, are well-known in the art, and have been described in the literature. See for example igawa et ai , FEBS Lett., 442, 15-19 (1999); Shimizu et al. , Nat Bioiechnol, 19, 751-55 (2001) and US Pat. Pub. No. 2006/0166306.
[0219] In one embodiment, the cell is a fibroblast cell. Methods of maintaining primary fibroblast cultures, as well as transformed fibroblast cell-lines, are known in the art. See, U.S. Pat. No. 9,029,148. Preferably, the cell is a fibroblast cell of ALS patients carrying pathogenic mutations in SOD1 (I113T), in FUS1 (fused in sarcoma; R521G) or in TDP43 (TAR DNA-binding protein 43; G289S).
[0220] In another embodiment, the cell is a neuron or a neuronal cell. Neurons include, without limitation, primary cultures such as primary cultures of embryonic dorsal root ganglion (DRG) neurons and primary cultures of fetal spinal cord neurons, for example, primary cultures of murine fetal spinal cord neurons (Elaine et al. , J. Cell Biol., 147, 1249-1260 (1999). In another embodiment, a neuron can be a neuron from, e.g. , a primary culture, an embryonic dorsal root ganglion primary culture or a fetal spinal cord primary culture. As non-limiting examples, cells useful according to a method disclosed in the present specification can include, a primary neuronal cell that contains an exogenous Drpl/Fisl, such as, e.g. , a rat embryonic dorsal root ganglion (DRG) neuron containing exogenous Drpl/Fisl or a murine fetal spinal cord neuron that contains an exogenous Drpl/Fisl.
[0221] Neuronal cell lines useful in aspects of the disclosure include, without limitation, neuroblastoma cell lines, neuronal hybrid cell lines, spinal cord cell lines, central nervous system cell lines, cerebral cortex cell lines, dorsal root ganglion cell lines, hippocampal cell lines and pheochromocytoma cell lines. [0222] In some embodiments, methods of the disclosure employ cells that are not neurons, wherein the cells can comprise a mutation in a gene associated with a neurodegenerative disorder. In one non- limiting example, some methods the present disclosure employ fibroblasts comprising a mutation in a gene associated with a neurodegenerative disorder. In some embodiments, methods of the disclosure employ fibroblasts comprising a mutation in a SODl gene, such as, without limitation, SOD1A4V, SOD1G8SR, and SOD1G93A.
[0223] As used herein, the term "SODl" refers to either the gene encoding superoxide dismutase 1 or the enzyme encoded by this gene. The SODl gene or gene product is known by other names in the art including, but not limited to, ALS1, Cu/Zn superoxide dismutase, indophenoloxidase A, IPOA, and SODC HUMAN. Those of ordinary skill in the art will be aware of other synonymous names that refer to the SODl gene or gene product. The SODl enzyme neutralizes supercharged oxygen molecules (called superoxide radicals), which can damage cells if their levels are not controlled. The human SODl gene maps to cytogenetic location 21q22.1. Certain mutations in SODl are associated with ALS in humans including, but not limited to, Ala4Val, Gly37Arg. G85R and Gly93Ala, and more than one hundred others. Those of ordinary skill in the art will be aware of these and other human mutations associated with ALS. Certain compositions and methods disclosed herein employ cells comprising a SODl mutation.
[0224] "SODl mutations" refer to mutations in the SODl gene (NC 000021.8; NT_011512.11; AC_000064.1; NW_927384.1 ; AC_000153.1 ; NW_001838706.1 NM_000454.4; NP_000445.1 and NCBI Entrez Gene ID: 6647) including but are not limited to Ala4Val, Cys6Gly, Val7Glu, Leu8Val, GlylOVal, Glyl2Arg, Vall4Met, Glyl6Ala, Asnl9Ser, Phe20Cys, Glu21Lys, Gln22Leu, Gly37Arg. Leu38Arg. Gly41 Ser, His43Arg. Phe45Cys, His46Arg, Val47Phe, His48Gln, Glu49Lys, Thr54Arg, Ser59Ile, Asn65Ser, Leu67Arg, Gly72Ser, Asp76 Val, His80Arg, Leu84Phe, Gly85Arg. Asn86Asp, Val87Ala, Ala89Val, Asp90Ala, Gly93Ala, Ala95Thr, Asp96Asn, Val97Met, GlulOOGly, AsplOAsn, Ilel04Phe, Serl05Leu, Leul06Val, Glyl08Val, Ilel2Thr, Ilel3Phe, Glyl l4Ala, Argl l5Gly, Vall l 8Leu, Alal40Gly, Alal45Gly, Aspl24Val, Aspl24Gly, Aspl25His, Leul26Ser, Serl34Asn, Asn 139His, Asnl39Lys, Glyl41Glu, Leul44Phe, Leul44Ser, Cysl46Arg, Alal45Thr, Glyl47Arg, Vall48Gly, Val l48Ile, Ilel49Thr, Ilel51Thr, and Ilel51 Ser. SODl is also known as ALS, SOD, ALS1, IPOA, homodimer SODl. "SODl mutation" databases can be found at Dr. Andrew C. R. Martin website at the University College of London, the ALS/SOD1 consortium website and the human gene mutation database (HGMD®) at the Institute of Medical Genetics at Cardiff, UK.
[0225] Preferably, the cell is a mouse motor neuron-like hybrid cell line (NSC-34), especially an NSC-34 cell-line harboring a SODl G93A mutation.
[0226] In another embodiment, the cells or cell-lines comprise mutations in the fused-in-sarcoma (FUS 1 ) gene or trans-active response DNA binding protein 43 kDa (TDP-43) gene, or human homologs thereof. In one embodiment, the cells or cell-lines comprise mutations in the FLJSl gene (human homolog: ALS6). in another embodiment, the cells or cell-lines comprise mutations in the TDP-43 gene. Cell lines harboring the mutations can be purchased commercial!}' .
[0227] The disclosure contemplates measuring the effect of the test compound on one or more parameters associated with mitochondrial dysfunction, including, but not limited to, (a) increased mitochondrial interconnectivity and/or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production and/or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death and/or cell lysis; (g) increased accumulation of mitophagy mediators selected from LC3- phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1), or a combination thereof in the mitochondria; (h) increased c-Jun N-terminal kinase (INK) signaling (e.g. , increased phosphorylated JNK levels and/or increased levels of downstream effectors such as XBP1, ATF6a, phosphorylated eIF2a, GRP78 and/or CHOP). Particularly, the parameter associated with mitochondrial dysfunction is selected from (1) increased mitochondrial Drpl recruitment, (2) increased Drpl phosphorylation and (3) increased mitochondrial accumulation of mitophagy mediators such as LC3-II and p62. A combination of the parameters, e.g. , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or more, may also be employed.
[0228] In one embodiment, the parameter associated with mitochondrial dysfunction is mitochondrial interconnectivity and/or mitochondrial elongation score, which can be measured using routine immunofluorescence techniques coupled with analysis of images using software analysis. For instance, mean area' erimeter ratio can be employed as an index of mitochondrial interconnectivity and inverse circularity can be used as a measure of mitochondrial elongation. See, Wiemerslage et al. Journal of Neuroscience Methods 262, 56-65 (2016). Under this embodiment, a reduction in the mitochondrial interconnectivity and/or elongation score of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more (compared to control) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
[0229] In another embodiment, the parameter associated with mitochondrial dysfunction is mitochondrial membrane potential (MMP), ATP production, reactive oxygen species (ROS) production and mitochondrial superoxide generation.
[0230] In one embodiment, a reduction in MMP or mitochondrial ATP production is associated with mitochondrial dysfunction, the restoration of which by the test agent indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS. Under this embodiment, mitochondrial membrane potential may be assessed via art known-methods, for example, as described in U.S. Pat. Pub. No. 2010/0209960. In one embodiment, measurement of MMP includes fluorescent detection, e.g., using fluorescent dyes comprising rhodamine 123, JC-1, tetrabromorhodamine 123, rhodamine 6G, TMRM, TMRE, tetramethylrosamine or rhodamine B (Guo et al, JCI 123, 5371-5388 (2013)). Alternately or additionally, under this embodiment, ATP production may be measured using art known methods, for example, as described in U.S. Patent No. 6,261,796. In one embodiment, the mitochondrial ATP production is measured via colorimetric/fluorometric assay and reading in a SPETRAMAX M2 device. Wherein the parameter is MMP or ATP production, if the test compound restores the parameter to about 70%, about 80%, about 90% about 100% or a higher % of the normal level, e.g., baseline level of MMP or ATP production in cells obtained from healthy patients or cell- lines not harboring any mutations, then the test compound may be selected as promoting neuron survival and/or as being useful for the treatment of ALS. In another embodiment, if the test compound elevates MMP or ATP production in the cell (e.g., a fibroblast cell or a hybrid motor neuron cell) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline MMP or ATP production (e.g., measured in the absence of the test compound), then the test compound may be selected as promoting neuron survival and/or as being useful for the treatment of ALS.
[0231] Alternately and/or additionally, the parameter associated with mitochondrial dysfunction is an increase in ROS production and/or mitochondrial superoxide generation, the attenuation of which by the test agent indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS. Under this embodiment, ROS may be measured using routine fluorescence detection techniques, for example, as taught in U.S. Pub. No. 2015/0219676. Likewise, mitochondrial superoxide production may be measured using specific fluorescent dyes, e.g. , MITOSOX RED, as detailed in the studies described herein. Under this embodiment, a reduction in the total ROS production and mitochondrial superoxide generation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more (compared to control, e.g., an untreated sample) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
[0232] In another embodiment, the parameter associated with mitochondrial dysfunction is Drpl or p62 recruitment (or both Drpl and p62 recruitment) from the cytosoi to the mitochondrial membrane, which can be quantitated using immuno-detection methods described above, e.g., immunoblotting or ELISA assays. Under this embodiment, a reduction in Drpl and/or p62 recruitment by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline mitochondrial Drpl and/or p62 levels (e.g. , measured in the absence of the test compound) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS. Drpl and/or p62 levels can be identified by routine methods, e.g., Western blot, dot blot, ELISA, flow cytometry, electrochemiluminescence, multiplex bead assay (e.g. , using Luminex or fluorescent microbeads), immunohistochemistry and the like. Representative methods are described in the Examples section.
[0233] In yet another embodiment, the parameter associated with mitochondrial dysfunction is Drpl phosphorylation. Phosphorylated Drpl can be detected using art known-methods, e.g. , immunodetection using anti-p-Drpl antibody S616 or anti-p-Drpl antibody S637 (see Example 1 and FIG. 2).
Under this embodiment, a reduction in Drpl phosphorylation by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline phospho-Drpl levels (e.g., measured in the absence of the test compound) indicates that the test compound promotes neuron survival and/or is useful for ALS therapy.
[0234] In yet another embodiment, the parameter associated with mitochondrial dysfunction is cell death and/or cell lysis. Methods for detecting cell death and/or cell lysis are known in the art, e.g. , using a colorimetric CYTOTOX 96 non-radioactive assay to detect lactate dehydrogenase (LDH) levels (Promega Corporation), as disclosed in U.S. Pub. No. 2003/0049829. In another embodiment, cell death may be detected using routine apoptotic markers, e.g. , mitochondrial cytochrome c release and/or accumulation of Bax on the mitochondria. Under this embodiment, a reduction in cell death or cell lysis (e.g. , as assessed by measuring the levels or activity of LDH or levels of cytochrome c or Bax) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g., levels of LDH, cytochrome c or Bax in untreated cell or cell-free systems) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
[0235] Wherein the assaying for mitochondrial dysfunction involves colorimetric or fluorometric devices, embodiments described herein include specific devices or methods known in the art for the detection of fluorescence, e.g., from fluorophores or fluorescent proteins. Such include, but are not limited to, in vivo near-infrared fluorescence (see, Frangioni et al, Curr Opin. Chem. Biol, 7:626-634 (2003)), the MAESTRO™ in vivo fluorescence imaging system (Cambridge Inc., Woburn, MA, USA), in vivo fluorescence imaging using a flying-spot scanner (see, Ramantiam et al, IEEE Transactions on Biomedical Engineering, 48: 1034-1041 (2001)), and the like. Other methods or devices for detecting an optical response include, without limitation, visual inspection, CCD cameras, video cameras, photographic film, laser-scanning devices, fluorimeters, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or signal amplification using photomultiplier tubes.
[0236] In another embodiment, the parameter associated with mitochondrial dysfunction is accumulation of mitophagy mediators in the mitochondria. In one embodiment, the mitophagy mediator is selected from LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1 ), or a combination thereof. Under this embodiment, a reduction in the levels of mitophagy mediator in the mitochondria (e.g., as assessed by measuring the levels of LC3-II and/or p62) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g., levels of LC3-1I and/or p62 in mitochondria obtained from untreated cells or ceil- free systems) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
[0237] In another embodiment, the parameter associated with mitochondrial dysfunction is increased c-Jun N -terminal kinase (JNK) signaling. In one embodiment, increased INK signaling is signified by increased phosphoryiated JNK levels and/or increased levels of downstream effectors such as XBPi, ATF6a, phosphoryiated eIF2a, GRP78 and/or CHOP. Under this embodiment, a reduction in JNK signaling (e.g. , as assessed by measuring the levels of phosphoryiated JNK levels and/or a downstream effector selected from XBPI, ΑΊΤόα, phosphoryiated eIF2 , GRP78 and CHOP) by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g. , levels of phosphoryiated JNK levels and/or a downstream effector thereof) indicates that the test compound promotes neuron survival and/or is useful for the treatment of ALS.
[0238] In the aforementioned screening assays wherein cells or cell-lines are used, cells may be allowed to grow for a period time, after which, the test compounds contacted with the cells, e.g., by supplementing the media. Period of cell growth can be optimized depending on the assay format, initial plating density of the cells. In some embodiments, a researcher can obtain cells that are already planted in the appropriate vessel and allowed to grow for a period of time. In other embodiments, the cells are plated in the appropriate vessel and allowed to grow for a period time, e.g., at least 1 day, at least 2 days, at least 3 days, at last 4 days, at least 5 days, at least 6 days, at least 7 days or more in presence of the test compound. In one embodiment, cells are grown for about 2-3 days in presence of the test compound prior to assaying for one or more of the aforementioned parameters.
[0239] The aforementioned parameters can be assessed using one or more controls. A control can be a sample that is that is not contacted with a compound or a sham compound, e.g. , a random peptide or a carrier peptide alone (i.e. , a negative control). A control can be a sample that is treated with a known promoter of cell survival (e.g., mitochondrial fission inhibitor peptide or construct of Formula I). This can serve as a positive control. Other mediators of mitochondrial autophagy, e.g. , Drp lK38A or Fis l RNAi, may also be used. A control can be a sample that is treated with a known inhibitor of Drpl, e.g., Mdivi-1 or dynasore monohydrate.
[0240] The number of possible test agents runs into millions. Methods for developing small molecule, polymeric and genome based libraries are known in the art. See Ding et al , J Am. Chem. Soc. 124: 1594-1596 (2002) and Lynn et ctl, J. Am. Chem. Soc. 123: 8155-8156 (2001). Commercially available compound libraries can be obtained from, e.g. , ARQULE, Pharmacopia, BIOMOL International and Oxford. These libraries can be screened using the screening devices and methods described herein. Chemical compound libraries such as those from NIH Roadmap, Molecular Libraries Screening Centers Network (MLSCN) can also be used. A comprehensive list of compound libraries can be found at the Harvard University Chemical Biology Platform. A chemical library or compound library is a collection of stored chemicals usually used ultimately in high-throughput screening or industrial manufacture. The chemical library can consist in simple terms of a series of stored chemicals. Each chemical has associated information stored in some kind of database with information such the chemical structure, purity, quantity, and physiochemical characteristics of the compound.
[0241] Depending upon the particular embodiment being practiced, the test agents can be provided free in solution, or may be attached to a carrier, or a solid support, e.g., beads. A number of suitable solid supports may be employed for Immobilization of the test agents. Examples of suitable solid supports include agarose, cellulose, carboxymethyl cellulose, polystyrene, polyethylene glycol (PEG), filter paper, nitrocellulose, ion exchange resins, plastic films, polyaminomethylvinylether maleic acid copolymer, glass beads, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. Additionally, for the methods described herein, test agents may be screened individually, or in groups. Group screening is particularly useful where hit rates for effective test agents are expected to be low such that one would not expect more than one positive result for a given group.
[0242] The assay can be performed any suitable container or apparatus available to one of skill in the art for cell culturing. For example, the assay can be performed in 24-, 96-, or 384-well plates. In one embodiment, the assay is performed in a 384-well plate.
[0243] Without limitations, cells can be plated at any density that provides an optimal signal-to-noise ratio. For example, neurons can be plated at a density of 1,000 to 20,000 cells/well in a 384-well plate. In some embodiments, motor neurons are plated at density of 1,000 cells/well, 2,000 cells/well, 4,000 cells/well, 8,000 cells/well, 12,000 cells/well, 16,000 cells/well, or 20,000 cells/well in a 384-well plate. In one embodiment, neurons are plated at a density of 8,000 cells/well in a 384-well plate. Based foregoing, one of ordinary skill can adjust the plating density for other cell culturing vessels. For example one can calculate the dimensions of a well in the 384-well plate and the vessels to be used and scale the number of cells to be plated based on volume or surface area ratio between a well from the 384-well plate and the vessel to be used.
[0244] In some embodiments, the screening method is a high-throughput screening. High-throughput screening (HTS) is a method for scientific experimentation that uses robotics, data processing and control software, liquid handling devices, and sensitive detectors. High-Throughput Screening (HTS) allows a researcher to quickly conduct millions of biochemical, genetic or pharmacological tests. HTS techniques are well known to one skilled in the art, for example, those described in U.S. Pat. Nos. 5,976,813; 6,472,144; 6,692,856; 6,824,982; and 7,091,048, and contents of each of which is herein incorporated by reference in its entirety. Diagnostic Tests
[0245] The disclosure also contemplates diagnostic tests and methods of diagnosing a neurological disorder (e.g., ALS) and/or disorders characterized by neuronal cell death.
[0246] In one aspect, the method of diagnosing a neurological disorder in a subject comprises: (a) obtaining a biological sample from the subject comprising a sample; (b) conducting at least one assay on the sample to detect mitochondrial dysfunction; and (c) diagnosing the subject as having the neurological disorder if the level of mitochondrial dysfunction in the patient sample is greater than the level of mitochondrial dysfunction in a control. In certain embodiments, the level of mitochondrial dysfunction correlates with Drpl/Fisl activity.
[0247] Any suitable control can be used. In some embodiments, the control is a subject that does not have the neurological disorder (e.g. , ALS). In some embodiments, the control is a reference standard or level indicative of a subject that does not have the neurological disorder (e.g., ALS).
[0248] The disclosure contemplates using any assay for measuring mitochondrial dysfunction, including, but not limited to, (a) increased mitochondrial interconnectivity and/or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production and/or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death and/or cell lysis; (g) increased accumulation of mitophagy mediators selected from LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1), or a combination thereof in the mitochondria; (h) increased c-Jun N-terminal kinase (JNK) signaling (e.g., increased phosphorylated JNK levels and/or increased levels of downstream effectors such as XBP1, ATF6a, phosphorylated eIF2a, GRP78 and/or CHOP). Particularly, the parameter associated with mitochondrial dysfunction and/or predisposition to ALS is (1) increased mitochondrial Drpl recruitment, (2) increased Drpl phosphorylation and (3) increased mitochondrial accumulation of mitophagy mediators such as LC3-II and p62. A combination of the parameters may also be employed.
[0249] In some embodiments, the at least one binding assay comprises an assay that measures one or more of the aforementioned parameters in a cell, e.g., fibroblast cell. Representative assays, techniques and systems for measuring the parameters have been described previously. It should be appreciated by the skilled artisan that detection of one or more of the aforementioned parameters in a subject (e.g., one who is suspected to have the neurological disorder) compared to a control (e.g. , healthy subject) is indicative that the subject has or is at risk for developing the neurological disorder and/or a disorder characterized by neuronal cell death.
[0250] In some embodiments, the diagnostic methods further comprise selecting a subject suspected of having a neurological disorder (e.g. , ALS). In some embodiments, the diagnostic methods further comprise selecting a subject suspected of having a disorder characterized by neuronal cell death. The selected patients may be placed on appropriate therapy for the treatment of the disorder, e.g. , treatment with the mitochondrial fission peptide (or a construct of Formula I or Formula II), or riluzole, or any other agents useful for the treatment or management of ALS.
[0251] In one particular embodiment, the disclosure relates to a method of diagnosing amyotrophic lateral sclerosis (ALS) in a subject comprises: (a) obtaining a biological sample from the subject comprising neuronal cells; (b) detecting at least one parameter associated with mitochondrial dysfunction; and (c) diagnosing the subject as having ALS based on the detection of the parameter. Under this embodiment, the parameter associated with mitochondrial dysfunction includes, but is not limited to, (a) increased mitochondrial interconnectivity and/or mitochondrial elongation score; (b) reduced mitochondrial membrane potential (MMP) and/or ATP production; (c) increased reactive oxygen species (ROS) production and/or increased mitochondrial superoxide generation; (d) increased Drpl or p62 recruitment to the mitochondria; (e) increased Drpl phosphorylation; (f) increased cell death and/or cell lysis; (g) increased accumulation of mitophagy mediators selected from LC3- phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1), or a combination thereof in the mitochondria; (h) increased c-Jun N-terminal kinase (JNK) signaling (e.g. , increased phosphorylated JNK levels and/or increased levels of downstream effectors such as XBP1, ATF6a, phosphorylated eIF2a, GRP78 and/or CHOP). Particularly, the parameter associated with mitochondrial dysfunction is selected from (1) increased mitochondrial Drpl recruitment, (2) increased Drpl phosphorylation and (3) increased mitochondrial accumulation of mitophagy mediators such as LC3-II and p62. A combination of the parameters, e.g. , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or more, may also be employed.
[0252] In some embodiments, a positive diagnosis is made if the parameter being measured is modulated by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or more compared to a control.
Methods of Treatment
[0253] A method for treating a neurological disorder is contemplated that comprises administering to a subject in need thereof, an effective amount of a composition that inhibits mitochondrial fission. A method for slowing the progression of or delaying progression of a neurological disorder is also contemplated, the method comprising administering to a subject in need thereof, an effective amount of a composition that inhibits mitochondrial fission.
[0254] In one embodiment, the disclosure relates to treatment of amyotrophic lateral sclerosis (ALS), also called Lou Gehrig's disease or classical motor neuron disease, and to slowing or delaying progression of ALS. ALS is a progressive, ultimately fatal disorder that eventually disrupts signals to all voluntary muscles. In the United States, doctors use the terms motor neuron disease and ALS interchangeably. Both upper and lower motor neurons are affected. Approximately 75% of people with classic ALS will also develop weakness and wasting of the bulbar muscles (muscles that control speech, swallowing, and chewing). Symptoms are usually noticed first in the arms and hands, legs, or swallowing muscles. Muscle weakness and atrophy occur disproportionately on both sides of the body. Affected individuals lose strength and the ability to move their arms, legs, and body. Other symptoms include spasticity, exaggerated reflexes, muscle cramps, fasciculations, and increased problems with swallowing and forming words. Speech can become slurred or nasal. When muscles of the diaphragm and chest wall fail to function properly, individuals lose the ability to breathe without mechanical support. Although the disease does not usually impair a person's mind or personality, several recent studies suggest that some people with ALS may have alterations in cognitive functions such as problems with decision-making and memory. ALS most commonly strikes people between 40 and 60 years of age, but younger and older people also can develop the disease. Men are affected more often than women. Most cases of ALS occur sporadically, and family members of those individuals are not considered to be at increased risk for developing the disease. However, there is a familial form of ALS in adults, which often results from mutation of the superoxide dismutase gone, or SOD, located on chromosome 21. In addition, a rare juvenile-onset form of ALS is genetic. Most individuals with ALS die from respiratory failure, usually within 3 to 5 years from the onset of symptoms. However, about 10 percent of affected individuals survive for 10 or more years.
[0255] Accordingly, embodiments disclosed herein relate to methods for treating ALS comprising administration of an effective amount of a compound disclosed herein (e.g. , mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) to a subject in need thereof. In one embodiment, the method of treating comprises the slowing or delaying of progression of the disorder.
[0256] In certain embodiments, the subjects are mammals, e.g., a rodent, a human, a livestock animal, a companion animal, or a non-domesticated or wild animal. In one embodiment, the subject may be a rodent, e.g. a mouse, a rat, a guinea pig, etc. In an exemplary embodiment, the subject is a human. The human can be a subject who has been diagnosed with ALS or as being predisposed to ALS.
[0257] In some embodiments, methods of treatment of ALS described herein allow for early intervention upon detection of at least one sign or symptom associated with ALS. Accordingly, under this embodiment, treatment with the compounds disclosed herein (mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in improvement in at least one sign or symptom associated with ALS in the subject.
[0258] In one embodiment, the treatment with the compounds disclosed herein (e.g. , mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in an improvement in a histopathological trait, a behavioral trait, a physiological trait or a combination of traits associated with ALS in the subject. Improvement of the trait can be determined by comparing the trait prior to treatment versus post-treatment. Alternately, the improvement can be monitored at various time-points during the course of the therapy, e.g. , at day 30 versus day 90.
[0259] In some embodiments, the improvement is a quantifiable measurement (e.g., improvement in muscle strength). In other embodiments, the improvement is a qualitative measurement (e.g., improvement in health-related quality of life (HRQL), disease severity, cognition, depression, fatigue, coping capacity and burden of care).
[0260] In some embodiments, wherein the improvement is a quantifiable measurement, a subject is described as having been treated if the therapeutic agent leads to an improvement of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1-fold, at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 10-fold, at least 50-fold, or more, compared to a control (e.g., untreated or placebo-treated subject).
[0261] In one embodiment, treatment with the compounds disclosed herein (e.g., mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in an improvement in a histopathological trait associated with ALS. Particularly, the improved histopathological trait comprises (a) reduced mitochondrial interconnectivity and/or mitochondrial elongation score; (b) increased mitochondrial membrane potential (MMP) and/or ATP production; (c) reduced reactive oxygen species (ROS) production and/or reduced mitochondrial superoxide generation; (d) reduced Drpl or p62 recruitment to the mitochondria; (e) reduced Drpl phosphorylation; (f) reduced cell death and/or cell lysis; (g) diminished mitochondrial accumulation of mitophagy mediators selected from LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 (SQSTM1); (h) reduced c-Jun N-terminal kinase (INK) signaling (e.g., attenuation in phosphorylated JNK levels and/or reduction in the levels of downstream effectors such as XBP1, ATF6a, phosphorylated eIF2a, GRP78 and/or CHOP). Particularly, therapeutic improvement in a histopathological trait associated with ALS includes (1) reduced mitochondrial Drpl recruitment, (2) reduced Drpl phosphorylation and (3) diminished mitochondrial accumulation of mitophagy mediators such as LC3-II and p62.
[0262] In another embodiment, treatment with the compounds disclosed herein (e.g., mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in an improvement in a behavioral trait associated with ALS. Particularly, the improved behavioral trait includes improved general mobility, improved motor function, reduced phobia, reduced stereotypy (e.g., twitches/paralysis), improved engagement time, improved engagement score; or a combination thereof. Especially under this embodiment, improvement in general mobility includes improvement in ambulation episodes, increase in moving time, increase in ambulatory time, an improvement in resting time or a combination thereof. Particularly under this embodiment, treatment with the compounds disclosed herein improves a pooled outcome reflecting general mobility behavior comprising enhanced ambulatory behaviors and reduced resting time.
[0263] In another embodiment, treatment with the compounds disclosed herein (e.g., mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in an improvement in a motor trait associated with ALS. Particularly, the improved motor trait includes improved motor response to allodynia (e.g. , response to hot or cold objects).
[0264] In another embodiment, treatment with the compounds disclosed herein (e.g., mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in an improvement in a physiological trait associated with ALS. Particularly, the improved physiological trait includes improved muscle strength, improved strength of grip, improved posture.
[0265] In yet another embodiment, treatment with the compounds disclosed herein (e.g., mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in an improvement in a speech trait associated with ALS. Particularly, the improved speech trait includes improved tone and quality of voice and/or reduction in the frequency and duration of slurring.
[0266] In one embodiment, the treatment with the compounds disclosed herein (e.g. , mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) results in an improvement in an outcome associated with ALS, e.g. , improvement in an epidemiological outcome, a histopathological outcome, or a physiological outcome, or a combination thereof. In certain embodiments, treatment with the compounds disclosed herein improves a combination of the aforementioned outcomes, e.g., at least 2, 3, 4, 5, 6, 7 or more outcomes.
[0267] In one embodiment, treatment with the compounds or constructs disclosed herein improves at least one epidemiological outcome selected from the group consisting of overall survival (OS), survival at clinical score 1 (SCSI), survival at terminal endpoint (STE), age at terminal endpoint (ATE), survival at clinical score 3 (SCS3), age at clinical score 3 (ACS3), total disease duration (TDD), duration between clinical score 2 to terminal endpoint (CS2T), time to progression of disease (TTP), time-to-death (TTD), and disease-free survival period (DFS), or a combination thereof.
[0268] Particularly, treatment with the compounds or constructs disclosed herein improves an epidemiological outcome selected from the group consisting of survival at clinical score 1 (SCSI), survival at terminal endpoint (STE), age at terminal endpoint (ATE), survival at clinical score 3 (SCS3), age at clinical score 3 (ACS3), total disease duration (TDD), and duration between clinical score 2 to terminal endpoint (CS2T) or a combination thereof. Under this embodiment, stage 2 is characterized by development of weakness/limpness in hind limbs; stage 3 is characterized by development of paralysis of a hind limb; and terminal endpoint is characterized by paralysis in both hind limbs plus a 20% or greater drop in the body weight or paralysis in both hind limbs plus a lack of righting reflex. [0269] In one embodiment, treatment with the compounds or constructs disclosed herein improves at least one in vivo histopathological outcome comprising attenuation in the association of Drpl and/or p62 with mitochondria of the subject's cell sample (e.g., spinal cord neurons). Herein, in vivo therapy with the compounds of the disclosure results in a reduction of mitochondrial Drpl and/or p62 localization by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g. , mitochondrial Drpl or p62 localization in identical cells obtained from the same subject prior to treatment or from an untreated subject).
[0270] In yet another embodiment, treatment with the compounds or constructs disclosed herein improves at least one in vivo physiological outcome comprising increased body weight (or body mass). Herein, in vivo therapy with the compounds of the disclosure results in an increase in body weight by at least 20%>, at least 30%>, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more over baseline levels (e.g. , body weight or body mass in the same subject prior to treatment or from an untreated subject).
[0271] Especially, as evidenced in the data provided in FIG. 4 and FIG. 6, treatment with the compounds disclosed herein (e.g. , mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II) delayed onset of disease and/or slowed down the progression of the disease; improved motor function and/or muscle strength; reduced stereotypy and/or paralysis; and improved survival. Improvement in these various outcomes was corroborated via histopathological characterization of attenuation in mitochondrial Drpl/p62 localization following treatment with the compounds disclosed herein.
Dosing Regimen
[0272] In some embodiments of the use or methods described above or below, the mitochondrial fission inhibitor may be administered 2, 3, 4, 5 or more times a day, every 1 day, every 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 14 days, 16 days, 18 days, 20 days, or 25 days, or every 1 month, 2 months, 3 months, 4 months, or 6 months, or annually.
[0273] Wherein multiple doses are administered, the first dose and one or more subsequent doses the mitochondrial fission inhibitor (or a construct of Formula I or Formula II) may be administered in a dosing regimen that is a pulsed dosing regimen (e.g., the dosing schedule produces escalating inhibitor levels early in the dosing interval followed by a prolonged dose-free period). In some embodiments of the methods of treatment of ALS described above, the first dose and one or more subsequent doses of the fission inhibitor is administered in a dosing regimen that is not continuous (i.e., the intervals between doses are uneven). In some embodiments of the methods of treatment of ALS described above, the first dose and one or more subsequent doses of the fission inhibitor is administered in a dosing regimen that is a continuous dosing regimen. [0274] In some embodiments, the first dose is administered upon detection of one or more symptoms of ALS. In some embodiments, the one or more subsequent doses of the fission inhibitor are administered every day, every other day, every 2 days, 3 days, 4 days, 5 days, 6 days, once a week, every 2 or 3 weeks, once a month, every 6 weeks, every 2 months, 3 months, 4 months, 5 months, 6 months or any combination thereof.
Combination Therapies
[0275] If desired, the methods disclosed herein can further include administering one or more additional therapeutic agents to the subject in need thereof, such as an agent that is effective in the treatment of ALS. Examples of additional agents that can be used in the methods described herein include an anti-spasticity agent (e.g., baclofen or diazepam), an analgesic (e.g., gabapentin), an antiparkinsonian agent (e.g. , trihexyphenidyl or amitriptyline), a calcimimetic (e.g., cinacalcet), an anti- cramp agent (e.g., mexiletine) or an anti-ALS agent (e.g. , riluzole).
[0276] In one embodiment, the combination comprises a mitochondrial fission inhibitor peptide (e.g. , a construct of Formula I or Formula II) with one or more additional active agents selected from: riluzole, baclofen, cinacalcet, acamprosate, mexiletin, torasemide, sulfisoxazole, and riluzole.
[0277] In another embodiment, the combination may comprise a mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) together with one or more drug(s) that ameliorate(s) symptoms of ALS, one or more drug(s) that could be used for palliative treatment of ALS or one or more drug(s) currently evaluated in the frame of clinical trials for treating of ALS. Preferably, said one or more drug(s) is/are selected from AEOL 10150, arimoclomol, AVP-923, botulinum toxin type B (Myobloc), ceftriaxone, celastrol, celecoxib, cistanche total glycosides, coenzyme Q10, Copaxone, creatine, creatinine, dronabinol, erythropoietin, escitalopram (Lexapro), glatiramer acetate, granulocyte-colony stimulating factor (G-CSF), growth hormone (Somatropin), GSK1223249, indinavir, insulin-like growth factor- 1 (IGF-I), IGF-l-AAV, K S-760704, leteprinim, leuprolide, levetiracetam, MCI-186, mecobalamin, minocycline, modafinil, Naaladase inhibitor, N- Acetylcysteine, NBQX, nimesulide, nimodipine, olanzapine, olesoxime (TR019622), ONO-2506, oxepa, pioglitazone, R(+) pramipexole dihydrochloride monohydrate, olesoxime, oxandrolone, quinidine, phenyl butyrate, SB-509, Scriptaid, sN 0029, somatropine, talampanel, tamoxifen, tauroursodeoxycholic acid, TCH346, testosterone, thalidomide, trehalose, tretinoin, vitamin E, YAM80 or from 17-beta-estradiol, 2-MPPA (2-(3-mercaptopropyl)pentanedioic acid), 3,4- diaminopyridine, 5-hydroxytryptophan, 7-nitroindazole, alpha-lipoic acid, AM1241, aminophylline, angiogenin, anti -human SOD1 antibody, antisense peptide nucleic acid directed against p75(NTR), AP7, apocynin, BAPTA-AM, BDNF, BN82451, cannabinol, cardiotrophin-1, CD4 antibodies, CNTF, colivelin, dietary copper, corticotrophin, cyclophosphamide, Delta(9)-tetrahydrocannabinol, DHEA, diazepam, dietary zinc, diltiazem, DMPO, DP- 109, DP-460, edaravone, EGCG, epigallocatechin gallate, etidronate, FeTCPP, fluvoxamine, folic acid, gabapentin, galectin-1, GDNF, ginseng, GPI- 1046, guanidine, HGF, humanin, IFN-alpha, interleukin-3, ivermectin, L-745,870, L-carnitine, L- DOPA, lecithinized SOD, lenalidomide, leupeptin, LIF, L-NAME, lysine acetylsalicylate, melatonin, mepivacaine, methamphetamine, methylcobalamin, MK-801, MnTBAP, modafinil, morphine, Neu2000, NGF, nordihydroguaiaretic acid, nortriptyline, NT3, olmesartan, penicillamine, pentoxifylline, pimozide, polyamine-modified catalase, pramipexole, prednisone, progesterone, promethazine, putrescine-modified catalase, pyruvate, rasagiline, RK35, Ro 28-2653, rofecoxib, RPR 119990, RX77368, SB203580, selegiline, semapimod, sertraline, SS-31, SSR180575, stabilized siRNA against human Cu,Zn-superoxide dismutase (SOD1), tacrolimus, tamsulosin hydrochloride, TAT-modified Bcl-X(L), TGF-beta2, tianeptine, trientine, TRO 19622, U-74389F, VEGF, vincristine, WHI-P131, WIN55, 212-2, WX-340, xaliproden, ZK 187638 and zVAD-fmk.
[0278] In some embodiments, the method encompasses co-administering at least one of the aforementioned agents (or a combination thereof) and a mitochondrial fission inhibitor peptide or a construct of Formula I or Formula II.
[0279] The term "co-administering," "co-administration," or "co-administer" refers to the administration of a plurality of agents, wherein the agents can be administered simultaneously, or at different times, as long as they work together (e.g. , additively or synergistically) to achieve the desired effect (e.g. , inhibit cell death).
[0280] Without limitations, when administered in separate formulations, the mitochondrial fission inhibitor (or a construct of Formula I or Formula II) and the additional agent can be administered within any time of each other. For example, the mitochondrial fission inhibitor (or a construct of Formula I or Formula II) and the additional agent can be administered within 24 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hours, 45 minutes, 30 minute. 25 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes or less of each other.
[0281] Additionally, co-administration does not require the mitochondrial fission inhibitor peptide (or a construct of Formula I or Formula II) and the additional agent to be administered by the same route. As such, each can be administered independently or as a common dosage form.
[0282] Evidence of mitochondrial dysfunction in fibroblasts of ALS patients carrying pathogenic mutations in SOD1 (I113T), in FUS1 (fused in sarcoma; R521G) or in TDP43 (TAR DNA-binding protein 43; G289S) genes was first studied. As fibroblasts have a mainly glycolytic metabolism, they were cultured in oxidative conditions in galactose-containing medium for 48 h, to induce dependence on oxidative phosphorylation (OX-PHOS) for ATP production (Aguer et al , PloS One 6, e28536 (2011)). In fibroblasts derived from ALS patients, the mitochondrial network was fragmented as compared with fibroblasts from healthy subjects (control), with prevalence of round-shaped mitochondria or sphere-like clusters (FIG. 1A). To quantify the mitochondrial structure change, automated image analysis was performed and the effects of these ALS mutations on mitochondrial morphology were examined. ALS patient-derived fibroblasts carrying any one of these three mutations showed a -50% decrease in mitochondrial interconnectivity (1.01 vs. 048 for Control and ALS, respectively; p<0.00001) and elongation scores (1.54 vs. 0.77 for control and ALS, respectively; p <0.00001) (FIG. IB, FIG. 1C). Since similar observations of mitochondrial phenotypes were made in cells derived from PD and HD patients, experiments were conducted to determine if mitochondrial fragmentation was mediated by Drpl-hyperactivation. In these studies, construct comprised the peptide identified as SEQ ID NO: 1, a linker (GG), and a carrier (TAT47-57; SEQ ID NO: 8), with the carrier at the N-terminus of the construct and the antagonist peptide at the C-terminus of the construct. The construct is identified herein as SEQ ID NO: 28 and is referred to as "PI 10". That is, PI 10 is a construct consisting of a heptapeptide conjugated to TAT47-57 (TAT, for intracellular delivery) that selectively inhibits the interaction between Drpl and Fisl, one of its adaptor proteins on the mitochondria (Guo et al, JCI, 123, 5371-5388 (2013)). Treatment with PI 10 (1 μΜ/day for 2 days) significantly improved mitochondrial structure and improved mitochondrial interconnectivity (from 0.48 to 1.32 with PI 10 treatment; pO.00001) (FIGS.1A-1C).
[0283] To assess the functional benefit of PI 10 on mitochondrial structure observed in fibroblasts, parameters such as mitochondrial membrane potential (MMP), ATP production, and mitochondrial superoxide production were determined. The fluorescent probe tetra-methyl-rhodamine methyl ester (TMRM) was used to measure mitochondrial membrane potential, as previously published (Guo et al. , JCI, 123, 5371-5388 (2013)). SOD1 mutation caused a -50% decrease in MMP and in ATP production compared to controls cells (p<0.00001) (FIGS. ID-IE). To determine if the mitochondria bioenergetic dysfunctions were associated with oxidative stress, specific mitochondrial reactive oxygen species (ROS) were also measured using MITOSOX and total ROS levels in mutant cells were determined by DCFH2 reagent. Increased levels of mitochondria-specific ROS (186%, p<0.00001) and total cellular ROS (250%, p<0.00001) were observed in these ALS patient-derived cells (FIGS. 1F-1G). All these mitochondrial defects associated with mitochondrial damage were significantly reduced by treatment with PI 10 (1 μΜ/day for 2 days) (FIGS. 1D-1G).
[0284] Drpl recruitment from the cytosol to the mitochondrial outer membrane is a hallmark of activated mitochondrial fission (Frank et al., Developmental Cell 1, 515-525 (2001)). A 3.4-fold increase in levels of Drpl recruitment to the mitochondria was observed in ALS patient-derived fibroblasts, which was significantly reduced by PI 10 treatment to 1.9 fold relative to control cells (p<0.00001) (FIG. 1H, FIG. 5A). No significant changes in the total protein levels of both Drpl and Fisl proteins were observed in these cells (FIG. 5A).
[0285] Previously, p62, a protein implicated in protein aggregate formation, was shown to accumulate progressively in the G93A mouse spinal cord (Gal et al , JBC, 282, 11068-11077 (2007)). Since p62 recruitment and accumulation at mitochondria has been associated with increased ROS production as well as mitochondrial membrane depolarization (Narendra et al, Autophagy 6, 1090- 1106 (2010)), experiments were conducted to assess the levels of p62 in the mitochondrial fraction in these patient-derived cells. Indicative of aberrant autophagy stall, a 3.3-fold increased recruitment and association with mitochondria of p62 was observed, that was rectified by PI 10 treatment (reduced to 1.9-fold relative to controls; p<0.00001) (FIG. II, FIG. 5A).
[0286] Next, to further dissect the pathways involved in P 110-i.nduced benefit observed in ALS- patient-denved fibroblasts, we focused on motor neurons expressing G93A SOD1 mutation using two stressors; serum starvation for 72 hours or H202 injury. It has been previously shown that NSC34 motor neuron cells expressing human G93A SODl, compared to cells expressing the human WTSODL have a significant increase in cytosolic oxidative stress. The ability to concentrate the TMRM probe in mitochondria was also decreased in SODl G93A, which was improved by PI 10 treatment (from 66% in hSODl G93A without to 83% with PI 10; p<0.003) (FIG. 2A). SODl G93A cells showed an approximately two-fold increase in Mi to SOX, a specific and mitochondrial-targeted detection probe for superoxide radical (02 ) (FIG. 2A), which was reduced when SODl G93A cells were treated with PI 10 (FIG. 2A) (from 202% to 165% with PI 10; p<0.03). A causal role for endogenous nitric oxide (NO) produced by motor neurons and apoptosis under mutant SODl expression has been reported (Lee et al , BBRC, 387, 202-206 (2009)). As expected, we also observed a significant increase in NO levels in SODl G93A cells under both chronic oxidative stress conditions as measured by nitrite levels, and these were significantly reduced by PI 10 treatment, indicating that limiting mitochondrial dysfunction was sufficient in limiting NO levels (from 198% in hSODl G93A without to 145% with PI 10: p<0.001) (FIG. 6A). Using presence of lactate dehydrogenase (LDH) in the culture medium as a measurement of cell lysis, we assessed cell death. A two-fold increase in LDH release was observed in mutant SODl cells under serum starved conditions, which was significantly reduced by PI 10 treatment (from 198% to 145% with Pi 10; pO.001) (FIG. 2A). A similar effect was observed under oxidative stress conditions (FIG. 6B).
[0287] In NSC-34 cells expressing SODl G93A mutant that are cultured without serum for 72 hours, Drpl association with the mitochondria increased by greater than 2.4-fold (p<0.0002) (FIG. 2B, FIG. 5B) as compared to vehicle-treated cells, and treatment with PI 10 significantly reduced the Drpl recruitment to the mitochondria (to 1.7-fold of control levels: p<0.001). Impairment of mitochondrial fission is closely linked with increased apoptosis and autophagic cell death in response to various stimuli by increasing mitochondrial depolarization and ROS production. In NSC-34 SODl G93A cells, PI 10 treatment greatly blocked the release of cytochrome c from the mitochondria (p<0.05), reduced the accumulation of active Bax on the mitochondria (p<0.006), and improved decreased Bcl-2 levels on the mitochondria (p<0.002) (FIG. 2B, FIG. 5B, FIG. 6C). Thus, PI 10 treatment inhibited the initiation of apoptosis.
[0288] Regulation of Drpl by post-translational modifications is important for Drpl translocation to mitochondria (Otera et al , Biochimica et Biophysica Acta 1833, 1256-1268 (2013)). Phosphorylation of Drpl at Ser-616 by cyclin-dependent kinase (CDK) 1/ Cyclin B or CDK5 promotes mitochondrial fission whereas de-phosphorylation of Drpl at Ser-637 by calcineurin facilitates its translocation to mitochondria and subsequently increases mitochondrial fission, which leads to an increased response to apoptotic stimuli (Liesa et al , Physiological Reviews 89, 799-845 (2009); Campello et al, EMBO Reports 11, 678-684 (2010)). Therefore, a balance between Drpl Ser-616/Ser-637 phosphorylation ratio reflects Drpl activity. Western blot analysis of total protein lysates showed a significant increase in Drpl phosphorylation at Ser-616 combined with a decrease in phosphorylation at Ser-637 in NSC- 34 SOD1 G93A cells (FIG. 2C, FIG. 5B; p<0.002). These results indicate that Drpl hyperactivation and phosphorylation occur due to the expression of SOD1 G93A mutation in motor neurons and treatment by PI 10 inhibits this hyperactivation.
[0289] Mutant SOD1 is degraded by the proteasome and partial inhibition of proteasome activity leads to the formation of large SOD 1 -containing aggregates, which is thought to contribute to neuropathology (Hyun et al , Journal of Neurochemistry 86, 363-373 (2003)). Recently, it has been shown that the levels of proteasomal 20S constitutive catalytic subunits were significantly reduced in the spinal cord of SOD1G93A mice at an advanced stage of the disease (Kabashi et al. , ALS- Official Publication of World Federation of Neurology Research Group, 13, 367-371 (2012)). A decrease in chymotrypsin-like proteasome activity in SOD1G93A motor neurons was observed (FIG. 2D) and PI 10 treatment prevented this decline in proteasomal activity (p<0.012) (FIG.2D), thereby indicating that improving mitochondrial health by blocking excessive fission is sufficient to improve overall cellular health and clearance systems.
[0290] The ubiquitin-proteasomal system, important for maintaining protein quality control, is also compromised in experimental models of familial ALS (Dantuma et al, Frontiers in Molecular Neuroscience 7, 70 (2014); Scotter et al, Journal of Cell Science 127, 1263-1278 (2014)). Inhibition of the fission machinery through DrplK38A or Fisl RNAi decreased mitochondrial autophagy and resulted in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion (Twig et al, EMBO J, 27, 433-446 (2008)). Further, increased levels of LC3BII and p62 have been previously reported in ALS models (Soo et al , Cell Death Discovery 1, 15030 (2015); Goode et al , Autophagy 12, 1094-1104 (2016); Oakes et al, Molecular Brain 10, 5 (2017)). To determine the effect of the SOD1 mutant on mitophagy, experiments were conducted to measure mitochondrial enrichment of autophagy mediators (including the LC3-phosphatidylethanolamine conjugate LC3-II and p62 (also known as SQSTM1)). Isolated mitochondria from SOD1 G93A motor neurons showed higher levels of LC3BII and p62 (p<0.05) and PI 10 treatment significantly reduced the mitophagy defects (p<0.05; FIG. 2E, FIG. 5C), indicating a direct role for Drpl/Fisl mediated fission in mitochondrial mitophagy stall. Furthermore, the increase in c-Jun N-terminal kinase (JNK) phosphorylation indicates increased cellular stress (p<0.05), whereas LC3BII conversion and p62- enhanced accumulation in total lysates demonstrate altered autophagy were significantly reduced by PI 10 treatments in SOD 1 G93A motor neurons (p < 0.05) (FIG. 2E, FIG. 5C, FIG. 6D).
[0291] Since activated JNK is one of the mediators of ER stress-induced apoptosis(52), experiments were conducted to investigate the expression of XBPl and ATF6a and phosphorylation of eIF2a (markers of ER stress; Szegezdi et al , EMBO Reports 7, 880-885 (2006)) and further determine whether improving mitochondrial function by PI 10 significantly reduces ER stress in SOD1G93A NSC34 cells. Increased levels of XBPl and ATF6a and phosphorylation of eIF2a were observed in the mutant cells, which levels were reduced by PI 10, thus indicating a functional crossover between Drpl hyperactivation and ER stress (p<0.05; FIG. 2F, FIG. 5D, FIG. 6E). Western blot analysis showed that the levels of other ER stress markers, GRP78 and CHOP, increased under serum starvation and were also elevated in G93A expressing motor neurons as compared to the WT (p<0.05; FIG. 2F, FIG. 5D, FIG. 6E), which were significantly normalized by PI 10 (p<0.05; FIG. 2F, FIG. 5D, FIG. 6E).
[0292] The SOD1G93A mouse model has been used since 1994 for preclinical testing in ALS (Gurney et al , Science 264, 1772-1775 (1994)). Despite recent genetic advances in the understanding of ALS, transgenic mice expressing mutant SOD1 remain the best available, and most widely used, vertebrate model of the disease. SOD1G93A mice (on a mixed genetic background) were treated with either PI 10 or vehicle control, using osmotic mini-pumps (ALZET, delivering 3 mg/kg/day). Treatment began from the age of 90 days, at the onset of clinical/motor symptoms, to assess efficacy of PI 10 treatment in modifying disease progression (FIG. 3A, FIG. 7A). The activity chamber is a simple assessment test used to determine general activity levels, gross locomotor activity, and exploration habits in rodents (Tatem et al , JoVE, 51785 (2014)) and all the following studies were carried out by an observer blinded to the experimental conditions. Changes in mouse locomotor behavior were determined 10 and 24 days after the initiation of PI 10 treatment (FIG. 3A). PI 10- treated mice spent significantly more time exploring the chamber as well as travelled further distances, as measured by ambulatory distance, ambulatory episodes and time spent exploring (p<0.05, FIG. 3B- 3D, FIGS. 7C, 7D) indicating better motor functions at both time points. This exploratory behavior correlated with decreased inactive time (FIG. 7H). Improvement in the hind/front grip strength in the P110-treated mice was also observed (FIG. 3E). No difference was observed in body weight (FIG. 7B). Furthermore, the P110-treated group showed tendency towards increased jumping, a behavior associated with intense motor function (FIGS. 7D-7E). PI 10 treatment showed increased locomotion (measured by center zone entries); however, the spatial distribution of this activity (latency to center) did not suggest an anxiety-related phenotype (FIG. 3H, FIG. 7F). Stereotypic counts are the number of times the mouse breaks the same beam in succession without breaking an adjacent beam. It was observed that PI 10 increases stereotypy, which is often associated with worsened pathology, but this could be attributed to increased movement and locomotion, as the stereotypy in treated mice was comparable to that observed in WT littermates (FIG. 7G).
[0293] Dimensional reduction analyses have previously used principal component analysis (PCA) to extract independent components from rodent behavioral data (Tanaka et al, Behavioral Brain Research 233, 55-61 (2012)). To determine if PI 10 treatment led to different behavioral outcomes, principal component analysis (PCA) was used to stratify the mice based on their behaviors. For each mouse, a vector containing normalized values for each behavior studied was assembled, and the top two principal components describing the variation in mouse behavior (accounting for 35% and 15% of the variance in the data; FIG. 3H) were identified. When the results were plotted using the 28 mice included in the study on these principal components, it was found that the WT mice segregated distinctly from the control -treated SOD1G93A mice by silhouette score (FIG. 3H), and the PI 10- treated mice fell between these two groups. Further analysis revealed that the first principal component (PCI) reflected general mobility of the mice: positive values for ambulatory behaviors, and negative values for resting time (FIG. 3H). The P110-treated mutant group, as a whole, showed higher values in PCI as compared to the control -treatment mutant group, indicating improved movement and suggesting treatment benefit. The improved overall neurological phenotype of SOD1G93A mice in response to the PI 10 treatment translated to an improvement in overall survival to phenotypic end- point. Notably, in this study, treatment was initiated at the onset of the clinical symptoms, when the mice already showed dragging feet/knuckles (clinical score, CS 1) (FIG. 7A). Median survival of vehicle-treated SOD1G93A mice was 125.3 ± 1.9 days, n=7, whereas treatment with PI 10 increased the lifespan of SOD1G93A mice to 132.6 ± 1.6 days, n=14 (p=0.0312, Mantel-Cox test, p=0.0319, Mann Whitney test) (FIG. 4A). Furthermore, treatment with PI 10 significantly delayed disease progression to terminal endpoint in these mice as assessed by the age at terminal endpoint, age at clinical score (CS) 3, time taken for the disease to progress from CS 2 to terminal endpoint, as well as the overall increase in the duration of the disease after the onset of first symptoms (p =0.0240 Mantel- Cox test) (FIGS. 4B-4E, FIG. 71). When the spinal cord mitochondrial fractions harvested after 24 days of treatment (age 114 days) were analyzed, increased Drpl mitochondrial association was observed in G93A mice as compared with WT mice, which was corrected by sustained PI 10 treatment (FIG. 4D, FIG. 5E) (relative values increased from 0.27 in WT mice to 2.08 in the ALS mice and down to 0.77 in ALS mice treated with PI 10 pO.001, n=3). These results indicate that inhibiting Drpl hyperactivation after disease onset ameliorates disease in the ALS mouse model. [0294] With reference to FIG. 4F, treatment of ALS mice with the P110 construct (SEQ ID NO: 28) provided an increased probability of survival post-paralysis, as evident from the animals treated with PI 10 (dotted trace) compared to the vehicle-treated control ALS mice (TAT; SEQ ID NO: 8) (solid trace).
[0295] The data in FIGS. 4A-4G show that treatment of ALS patients with PI 10 (SEQ ID NO: 28) had an increased survival even when treatment was initiated at the onset of the clinical symptoms, e.g, for ALS model mice, when the mice already showed dragging feet/knuckles (clinical score 1, CS1). Furthermore, treatment with PI 10 significantly delayed disease progression to terminal endpoint in these mice as assessed by the age at terminal endpoint, age at clinical score (CS3), time taken for the disease to progress from CS2 to terminal endpoint, as well as the overall increase in the duration of the disease after the onset of first symptoms and significantly improved the probability of survival post- paralysis. Moreover, sustained treatment with PI 10 treatment in naive mice showed neither toxicity nor any behavioral changes after 5 months on treatment at 3 mg/kg/day.
[0296] In another study, the effect of PI 10 treatment on astrogliosis and microglial activation in ALS mice was evaluated. FIG. 8A shows that increased GFAP staining, which correlates with astrocytosis, and beta-actin staining, a marker of microglia, were blunted by treatment with PI 10 (SEQ ID NO: 28) in ALS mice relative to wild type mice or control (TAT, SEQ ID NO: 8) treated mice. GFAP levels were quantified in spinal cord lysates, and the results are shown in FIG. 8B. Treatment with PI 10 (SEQ ID NO: 28) in ALS mice reduced GFAP expression relative to wild type mice or control (TAT, SEQ ID NO: 8). FIG. 8C shows the fold change in calcium-binding protein (SI 00b), a potential marker for glial dysfunction and blood-brain barrier disruption and which increases in serum of patients with neurodegenerative diseases such as ALS. As seen in FIG. 8C, SI 00b plasma levels in ALS mice treated with PI 10 was reduced. FIGS. 8D-8G are bar graphs showing the tissue levels of several cytokines, interleukin-lbeta (FIG. 8D), interleukin 1 -alpha (FIG. 8E), interleukin-6 (FIG. 8F) and tumor necrosis factor-alpha (FIG. 8G) are all reduced in ALS mice treated with PI 10.
[0297] In another study, a BV2 microglial cell line was transiently transfected with SOD1G93A mutant gene to mimic ALS. The transfected cells were left untreated, or were treated with vehicle control (TAT, SEQ ID NO: 8) or the PI 10 (SEQ ID NO: 28). FIGS. 9A-9D show the results, with FIG. 9A showing the mitochondrial aspect ratio, FIG. 9B the intracellular ATP, FIG. 9C the mitoSOX and FIG. 9D the total ROS in the transfected cells untreated (squares), control-treated (triangles) and PI 10-treated (inverted triangles).
EXAMPLES
[0298] The structures, materials, compositions, and methods described herein are intended to be representative examples of the disclosure, and it will be understood that the scope of the disclosure is not limited by the scope of the examples. Those skilled in the art will recognize that the disclosure may be practiced with variations on the disclosed structures, materials, compositions and methods, and such variations are regarded as within the ambit of the disclosure.
EXAMPLE 1
Materials & Methods:
[0299] All chemicals were purchased from Sigma- Aldrich (St Louis, MO) unless stated otherwise.
[0300] Peptide Synthesis. Peptides were synthesized using microwave chemistry, as previously described by Qi et al, Journal of Cell Science 126, 789-802 (2013).
[0301] Cell culture and peptide treatments. NSC34 cells stably expressing WT or G93A hSODl were a gift from Dr. Manfredi (Weill Medical College of Cornell University, USA). NSC34 were maintained in DMEM supplemented with 10% v/v FBS and 1% (v/v) penicillin/streptomycin. When differentiation was required, cells were plated onto poly-d-lysine-coated plates and grown in differentiation medium, which contains 1 : 1 DMEM/Ham's F12 supplemented with 1% FBS, 1% P/S and 1% modified Eagle's medium non-essential amino acids. ALS patient-derived fibroblasts (ALS 1 : ND29509; ALS 2: ND30327; ALS 3: ND32969) and fibroblasts of control healthy individuals (H1 :AG07123; H2:AG04146) 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% C02-95% air. NSC34 cells were treated with PI 10 (SEQ ID NO: 28) or vehicle (TAT47- 57, SEQ ID NO: 8) at a final concentration of Ι μΜ every 24-hour in serum free media. Similarly, for patient-derived fibroblasts, TAT (SEQ ID NO: 8) or PI 10 (SEQ ID NO: 28) peptides were added once daily for the duration of the experiment at 1 μΜ final concentration. All experiments were carried out in defined serum free media. Cells with fewer than 18 passages were used in all experiments.
[0302] Immunofluorescence. Cells cultured on 8-well chamber slides were washed with cold PBS, fixed in 4% formaldehyde, and permeabilized with 0.1% Triton X-100. After incubation with 2% normal goat serum (to block nonspecific staining), fixed cells were incubated overnight at 4°C with TOM20 (1 :500) (Santa Cruz, USA). Cells were washed with PBS and incubated for 60 minutes with FITC-conjugated goat anti-rabbit IgG (1 :500 dilution). The cells were then washed gently with PBS and counterstained with HOECHST 33342 (1 : 10,000 dilution, Molecular Probes) to visualize nuclei. The coverslips were mounted with SLOWFADE antifade reagent (Invitrogen), and images were acquired using an ALL-IN-ONE Fluorescence Microscope BZ-X700 (Keyence) (Guo et al. , JCI, 123, 5371-5388 (2013); Qi et al, Journal of Cell Science 126, 789-802 (2013)).
[0303] Analysis of mitochondrial morphology. Mitochondrial interconnectivity and elongation from epifluorescence micrographs of cells immunostained for mitochondria was measured and analyzed using IMAGEJ (an open source image processing program designed for scientific multidimensional images). Analysis of mitochondrial structure in patient-derived fibroblasts was conducted using art- known methods (Dagda et al , JBC, 284, 13843-13855 (2009)). Mean area/perimeter ratio was employed as an index of mitochondrial inter connectivity, with inverse circularity used as a measure of mitochondrial elongation Wiemerslage et al. , J. Neuroscience Methods , 262, 56-65 (2016)).
Cell and mitochondrial health assays
[0304] Mitochondrial membrane potential Cells were incubated with tetra-methyl-rhodamine methyl ester (TMRM, Invitrogen) in HBSS (Hank's balanced salt solution) for 30 min at 37°C and the fluorescence was analyzed using SPECTRAMAX M2E (Molecular Devices) (excitation, 360 nm; emission, 460 nm). All data were normalized with respect to the fluorescence intensity of the control cells.
[0305] ATP measurements. ATP levels were measured by the ATP colorimetric/fluorometric assay kit (Biovision, Milpitas, CA) using the manufacturer's protocols and reading in a SPECTRAMAX M2E (Molecular Devices). ATP concentration at each time point was calculated as a percentage of levels in the control group.
[0306] ROS production. For cellular ROS detection, cells were incubated with 2,7 dichloro- fluorescin diacetate (DCFDA) (Abeam) 100 μΜ for 30 minutes at 37°C in the dark, and fluorescence was analyzed with excitation/emission at 495/529 nm using SPECTRAMAX M2E. Fluorescence intensity was then normalized for cell number. To determine mitochondrial ROS production, cells were treated with 5 μΜ MITOSOX™ RED, a mitochondrial superoxide indicator (Invitrogen) for 10 min at 37°C according to the manufacturer's protocol and fluorescence was analyzed with excitation/emission at 510/580 nm using SPECTRAMAX M2E.
[0307] Cell death. Cytotoxicity was determined using Cytotoxicity Detection Kit (Goode et al, Autophagy 12, 1094-1104 (2016); Roche). In brief, media was collected at endpoints (in phenol red- free DMEM) to measure the percentage of released lactate dehydrogenase activity (LDH). To quantify total LDH, cells were lysed with Triton X (1% in serum free cell culture media) overnight at 4°C. 50 μΐ media or lysate were transferred with 50 μΐ of reaction mix in a 96 well-plate and incubated at RT for 30 minutes in the dark. Absorbance was measured at 490 nm using SPECTRAMAX M2E.
[0308] Isolation of mitochondria-enriched fraction and lysate preparation. Cells were washed with cold phosphate-buffered saline (PBS) at pH 7.4 and scraped off using mannitol-sucrose (MS) buffer, containing 210 mM mannitol, 70mM sucrose, 5 mM MOPS (3-(N-morpholino) propane-sulfonic acid), lmM EDTA, and protease inhibitor cocktail, pH 7.4. Spinal cords were minced and homogenized in the lysis buffer and then placed on ice for 30 minutes. Collected cells or tissue were disrupted 10 times by repeated aspiration through a 25-gauge needle, followed by a 30-gauge needle (10 times). The homogenates were spun at 800 g for 10 min at 4 °C (nuclear pellet), and the resulting supernatants were aliquoted and used as total ly sates. A second aliquot was spun at 10,000 g for 20 min at 4 °C. The pellets were washed with lysis buffer and spun at 10,000 g again for 20 min at 4 °C. The final pellets were suspended in lysis buffer containing 1% Triton X-100 and were mitochondrial-rich lysate fractions (Guo et al, JCI, 123, 5371-5388, 2013; Qi et ctl , JCS, 126, 789- 802, 2013).
[0309] Proteasome activity. Cells were homogenized in cold buffer (20 mM TrisHCl pH 7.5, 2 mM EDTA) and centrifuged at 15000 g for 10 min at 4°C. Protein concentration in supernatants was determined using the BCA protein assay (Thermo Fisher Scientific). All samples were assayed in triplicate using 10μg of freshly protein extracts. Proteasome activity was measured using the CHEMICON Proteasome Activity Assay Kit (APT280, Millipore), as described by the manufacturer. The extracts were incubated (2h at 37°C) with a labeled substrate, LLVY-7-amino-4-methyl-coumarin, and the cleavage activity was monitored by detection of the free fluorophore 7-amino-4-methyl- coumarin, using a SPECTRAMAX M2E at 380/460 nm.
[0310] Western blot analysis. Protein concentrations were determined using the Bradford assay (Pierce/Thermo Scientific). Proteins were resuspended in Laemmli buffer containing 2- mercaptoethanol, loaded on SDS/PAGE and transferred on to nitrocellulose membrane, 0.45 μιτι (Bio- Rad), as before (32, 75). Membranes were probed with the indicated antibody and then visualized by ECL (0.225mM p-Coumaric acid; Sigma), 1.25mM 3-amino-phtal-hydrazide (Luminol; Fluka) in 1M Tris pH 8.5). Scanned images of the exposed X-ray film were analyzed with ImageJ to determine relative band intensity. Quantification was performed on samples from independent cultures for each condition. The antibodies used in this study are in Table 1.
[0311] Table 1 provides a list of antibodies used for Western blot analysis:
Table 1
Figure imgf000061_0001
Antibody name Company Catalog No Dilution
Anti-JNK Cell Signaling Technology 9252 1 :500
Anti-LC3BII Cell Signaling Technology 3868 1 :500
Anti-p62 Abeam 56416 1 :500
Anti-Parkin Abeam 77924 1 :500
Anti-phospho-JNK Cell Signaling Technology 9251 1 :200
Anti-phospho-Drp 1
(Ser616) Cell Signaling Technology 3455 1 :200
Anti-phospho-Drp 1
(Ser637) Cell Signaling Technology 4867 1 :200
Anti-phospho-eIF2a Cell Signaling Technology 9721 1 :500
Anti-VDACl Abeam 14734 1 :2000
Anti-XBPl Abeam 37152 1 :500
Anti- -actin Cell Signaling Technology 3700 1 : 1000
[0312] Peptide treatment in mouse model All the experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committee of Stanford University and were performed based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals. G93A expressing SODl mice and their WT littermates were purchased from The Jackson Laboratory. The animals used in the PI 10 treatment study were implanted with a 28-days osmotic pump (ALZET) containing TAT47-57 carrier control peptide (SEQ ID NO: 8) or PI 10 (SEQ ID NO: 28) which delivered to the mice at a rate of 3 mg/Kg/day, as described previously (Disatnik et al , JEM, 213, 2655-2669 (2016)). The first pump was implanted at an average age of 90 days, when the animals showed clinical symptoms, with the subsequent pump implantation 30 days later, at 120 days.
[0313] Animal survival and behavior study. The overall survival during the study period was recorded, and the remaining mice were sacrificed when they reached terminal endpoint. An experimenter who was blind to genotypes and drug groups conducted all the behavior and survival studies. The following clinical scores were used in the study; 0 = Normal Gait; 0.5 = slight dragging of knuckles (at least 2x during circling of arena); 1 = dragging feet/knuckles; 1.5 = single leg extremely weak/ limp (little to no use for walking); 2 = weakness/limpness in 2 hind limbs; 3 = single leg paralysis; 4 = 2 legs paralysis; 4+= advanced paralysis or cannot right in 20 seconds.
[0314] Grip Strength Testing. The grip strength test was used to assess motor function and control of the fore and hind paws. Mice were allowed to grab the bar(s) on the Chatillon (Largo, Florida USA) DFIS-10 digital force gauge while being gently pulled parallel away from the bar by the tail. The maximum force prior to release of the mouse's paw from the bar was recorded. Five trials of front paw strength and subsequent, three trials of all paw strength were conducted.
[0315] Activity Chamber. The Activity Chamber was used to determine general activity levels, gross locomotor activity, and exploration habits in rodents. Assessment took place in an Open Field Activity Arena (Med Associates Inc., St. Albans, VT. Model ENV-515) mounted with three planes of infrared detectors, within a specially designed sound-attenuating chamber (Med Associates Inc., St. Albans, VT. MED-017M-027). The arena is 43 cm (L) x 43 cm (W) x 30 cm (H) and the sound attenuating chamber is 74 cm (L) x 60 cm (W) x 60 cm (H). The animal was placed in the corner of the testing arena and allowed to explore the arena for 10 minutes while being tracked by an automated tracking system. Parameters including distance moved, time immobile, and times spent in pre-defined zones of the arena were recorded.
[0316] Principal component analysis (PCA). For each mouse, the behavioral and bodyweight analyses from days 100, 114, and 115 of treatment were assembled into a vector, with a total of 31 values/vector/mouse. To avoid bias from behavioral measurements with a high range, all values were normalized (by subtracting the mean of each behavior and dividing by the standard deviation). PCA was performed using the Python scikit-learn package. Clustering of mouse treatment groups was quantified using a silhouette score, as described previously (Cunningham et al. , Cell Reports 18, 2592- 2599 (2017)). Briefly, for each point in a treatment group, this silhouette score (s) was defined as s = (b-a)/max(a,b), where a is the mean distance between the point and other points in the same group, and b is the mean distance between the point and other points in the next nearest group.
[0317] Statistics. Data are expressed as means ± SD. Statistical analysis was assessed by unpaired Student's t test and 1-way or 2-way ANOVA. Whenever significant F values were obtained, Tukey adjustment was used for multiple comparison purposes. The standard Mantel Cox log-rank test was used to assess survival. Significance of changes in neurological symptoms was analyzed with the Fisher's exact test. Statistical significance was considered achieved when the P value was less than 0.05. All analyses were conducted with GRAPHPAD PRISM software.
[0318] In animal studies, as indicated, 7-14 mice/group was used for behavioral tests and 5 mice/group were used for biochemical analysis from the same litter. In cell culture studies, each study was performed with at least three independent experiments done in duplicates. An observer who was blind to the experimental groups conducted all the animal studies. From the age-matched mice, one of eight TAT-treated and two of sixteen P110-treated mice were excluded from the study due to death during the surgery to implant the second pump. The data from these three mice were not included in any of the behavioral analysis. [0319] All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All published references, documents, manuscripts, scientific literature cited herein are hereby incorporated by reference. All identifier and accession numbers pertaining to scientific databases referenced herein (e.g., NCBI, GENBANK, EBI) are hereby incorporated by reference.

Claims

WE CLAIM:
1. A composition for use in delaying progression of amyotrophic lateral sclerosis (ALS) in a subject in need thereof, the composition comprising a therapeutically effective amount of a mitochondrial fission inhibitor or a derivative thereof.
2. A composition for use in delaying progression of neurodegeneration in a subject with a neurodegenerative disease, the composition comprising a therapeutically effective amount of a mitochondrial fission inhibitor or a derivative thereof.
3. The composition of claim 1 or 2, wherein the mitochondrial fission inhibitor inhibits the interaction of Drpl with mitochondrial fission 1 protein (Fisl).
4. The composition of any one of claims 1-3, wherein the mitochondrial fission inhibitor inhibits Drpl /Fisl mediated fission of mitochondria.
5. The composition of any one of claims 3-4, wherein the inhibitor of Drpl is a peptide.
6. The composition of any one of claims 1-5, wherein the mitochondrial fission inhibitor is a peptide having at least 90% sequence identity to a peptide having a sequence identified as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.
7. The composition of any one of claims 5-6, wherein the mitochondrial fission inhibitor peptide consists of 7, 8 or 9 amino acids and has at least 80% sequence identity to SEQ ID NO: 1.
8. The composition of any one of claims 5-7, wherein peptide is a salt of the peptide.
9. The composition of any one of claims 5-8, wherein the peptide is covalently attached to a linker.
10. The composition of claim 9, wherein the linker is a poly -glycine linker comprising between about 2-6 glycine residues.
11. The composition of any one of claims 1-10, wherein the composition or the mitochondrial fission inhibitor comprises a carrier.
12. The composition of claim 11, wherein the carrier is a cationic lipid.
13. The composition of any one of claims 1-10, wherein the mitochondrial fission inhibitor is a peptide conjugated to a carrier moiety that facilitates intracellular delivery.
14. The composition of claim 13, wherein the carrier is selected from the group consisting of sequences identified as SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
15. The composition of claim 13 or 14, wherein the peptide is conjugated at its carboxyl-terminus, directly or via a linker, to the carrier.
16. The composition of any one of claims 13-15, wherein the peptide is conjugated at its carboxyl- terminus, via a polyglycine linker comprising 2-6 glycine residues, to the carrier.
17. A method for the treatment of amyotrophic lateral sclerosis (ALS) or for delaying progression of ALS in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of a construct having a structure PEP-L-CAR (Formula I) or CAR-L- PEP (Formula II), wherein,
PEP is a peptide selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7;
L is a linker selected from the group consisting of a peptide bond, GG, GGG, GGS, GGSG (SEQ ID NO: 14), GGSGG (SEQ ID NO: 15), GSGSG (SEQ ID NO: 16), GSGGG (SEQ ID NO: 17), GGGSG (SEQ ID NO: 18), and GSSSG (SEQ ID NO: 19); and
CAR is a carrier selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13.
18. The method of claim 17, wherein the construct has a sequence identified as SEQ ID NO: 28.
19. The method of any one of claims 17-18, wherein the ALS is familial ALS.
PCT/US2018/028665 2017-04-21 2018-04-20 Compositions and methods for the treatment of amyotrophic lateral sclerosis WO2018195491A1 (en)

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