WO2009121176A1 - Compositions peptidiques de gène induit par l'insuline (insig) et procédés de cytoprotection - Google Patents

Compositions peptidiques de gène induit par l'insuline (insig) et procédés de cytoprotection Download PDF

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Publication number
WO2009121176A1
WO2009121176A1 PCT/CA2009/000413 CA2009000413W WO2009121176A1 WO 2009121176 A1 WO2009121176 A1 WO 2009121176A1 CA 2009000413 W CA2009000413 W CA 2009000413W WO 2009121176 A1 WO2009121176 A1 WO 2009121176A1
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seq
polypeptide
absent
srebp1
tat
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PCT/CA2009/000413
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English (en)
Inventor
Yu Tian Wang
Changiz Taghibiglou
Henry Giles Stratten Martin
Neil Cashman
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The University Of British Columbia
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

  • INSULIN-INDUCED GENE INSIG
  • the preseNt invention relates to lipid metabolism pathways, and more specifically to relationship between these pathways and cytotoxic cell death. Furthermore, therapeutic compositions and methods are presented.
  • SREBPs Sterol regulatory element binding proteins
  • ER endoplasmic reticulum
  • TFs membrane bound transcription factors
  • SCAP SREBP cleavage activating protein
  • INSIG Insulin-Induced Gene
  • INSIG-I protein is rapidly ubiquitinated at lysine- 156 and lysine-158 and degraded by proteasomes (Gong et al. 2006a; Gong et al. 2006b; Lee et al.2006a; Lee et al. 2006b).
  • INSIG-I is ubiquitinated and rapidly degraded and that ubiquitin ligase gp78 binds with much higher affinity to INSIG-I than INSIG-2 (Lee et al. 2006b).
  • Cellular stress such as hypotonic shock and ER stress also activates SREBP via rapid turnover of INSIG-I (Lee and Ye 2004).
  • the degradation of INSIG-I in the ER is a key determining step in the release of immature SREBPl and its subsequent proteolytic activation in Golgi.
  • NMDARs N-methyl-D-aspartate subtype of glutamate receptors
  • Embodiments of this invention are based in part on the surprising discovery that in primary cortical neuronal cultures, excitotoxic neuronal death produced by a brief NMDA stimulation or ischemic insult simulated with oxygen-glucose deprivation (OGD) is associated with a dose- and time-dependent activation and nuclear translocation of sterol regulatory element binding protein- 1 (SREBPl).
  • OGD oxygen-glucose deprivation
  • FIG. 1 For purposes of SREBPl activation and nuclear translocation by inhibiting INSIG-I degradation with a membrane-permeant INSIG-I derived interference peptide (Indip) significantly reduces either NMDA or OGD-induced neuronal death and that agents capable of reducing SREBPl activation such as Indip may represent a new class of NMDAR-based neuroprotective therapeutics against stroke and other neurologic diseases and conditions.
  • Indip membrane-permeant INSIG-I derived
  • MCAO medial cerebral artery occlusion
  • Indip peptide protects against glutamate induced excitotoxicity cell death in both wild type and AI-S (G93A) transgenic mouse embryonic spinal cord neurons.
  • Indip peptide protects against glutamate induced DNA fragmentation (apoptosis) in G93 A transgenic embryonic spinal cord neurons.
  • a pharmaceutical composition including a polypeptide and a carrier suitable for facilitating delivery of the polypeptide to a cell, wherein the polypeptide comprises one or more of the amino acid sequences set forth in SEQ ID NOS: 1 - 24 and 26-45.
  • the pharmaceutical composition may be for the treatment of stroke or amyotrophic lateral sclerosis (ALS).
  • ALS amyotrophic lateral sclerosis
  • a method of inhibiting cytotoxic cell death or reducing cytoxic stress including, treating a cell, a mammal including the cell, or a tissue including the cell, with an effective amount a polypeptide described herein.
  • a method of treating stroke or ALS including, treating a cell, a mammal including the cell, or a tissue including the cell, with an effective amount a polypeptide described herein.
  • polypeptide for treating stroke or ALS in a subject wherein the polypeptide is selected from any one or more of SEQ ID NOS: 1-24 and 26-45.
  • polypeptide to formulate a ' medicament treating stroke or ALS in a subject, wherein the polypeptide is selected from any one or more of SEQ ID NOS: 1-24 and 26-45.
  • a commercial package including a polypeptide, wherein the polypeptide is selected from any one or more of SEQ ID NOS : 1 - 24 and 26-45 or a pharmaceutical composition thereof.
  • a kit including a polypeptide, wherein the polypeptide is selected from any one or more of SEQ ID NOS: 1-24 and 26-45.
  • the polypeptide may be selected from one or mote of the following: GEPHKFOJEW (SEQ ID NO: 1); GGEPHKFKREW (SEQ ID NO; 3); GGGEPHKFKREW (SEQ ID NO: 4); GGGGEPHKFKREW (SEQ ID NO: 5); GGGGGEFHKFKREW (SEQ ID NO: 6); EPHKFKREW (SEQ ID NO: 7); GEPHKFKRE (SEQ ID NO: 8); EPHKFKRE (SEQ ID NO: 9); GEPHKFKRE (SEQ ID NO: 10); EPHKFKRE (SEQ ID NO: 11); PHKFKRE (SEQ ID NO: 12); PHKFKR (SEQ ID NO: 13); PHKFKK (SEQ ID NO: 14); DPHKFKREW (SEQ ID NO: 15); EPHKFKREF (SEQ ID NO: 16); DPHKFKREF (SEQ ID NO: 17); EPHKFKRDW (S
  • the peptide may also have one or more conserved amino acid changes.
  • the polypeptide may further include a delivery moiety.
  • the delivery moiety may allow for localization to the central nervous system.
  • the delivery moiety may be a TAT peptide.
  • the polypeptide may be selected from one or more of the following: YGRKKRRQRRRGEPHKFKREW (SEQ ID NO: 26); YGRKXRRQRRRGGEPHKFKREW (SEQ ID NO: 27); YGRKKRRQRRP-GGGEPHKFKREW (SEQ ID NO: 28); YGRKKRRQRRRGGGGEPHKFKREW (SEQ ID NO: 29); YGRKK RRQRRRGGGGGEPrIKFK-REW (SEQ ID NO: 30); YGRKKRRQRRREPHKFKREW (SEQ ID NO: 31); YGRKKRRQRRRGEPHKFKRE (SEQ ID NO: 32); YGRKKRRQRR
  • nucleic acid including a non-naturally occurring nucleic acid capable of encoding a polypeptide selected from any one or more of SEQ ID NOS: 1-24 and 26-45.
  • the nucleic acid maybe encoded by SEQ ID NO: 25,
  • the nucleic acid may be contained in an expression vector.
  • the nucleic acid may be contained in a cell.
  • FIGURE IA is a bar graph of protein/DNA microarray for transcription factors with characterized response elements revealed altered activity of a large number of transcription factors.
  • Nuclear extracts from cortical cultured neurons following NMDA- induced excitotoxicity (50 ⁇ M NMDA for Ih followed by 4h incubation) were compared to control cells, and up or down regulated transcription factors were identified. Note an about two-fold increase in SREBP1 -binding activity in response to NMDA stimulation (Black Bar).
  • FIGURE IB is a bar graph showing the appearance of cleaved n-terminal (active) SREBP1 (nt-SREBP1) at 6 hours following NMDA insult as compared to ⁇ -tubulin.
  • FIGURE 1C is a bar graph showing that NMDA-induced activation (NMDA) is prevented by treatment of cortical cultures with NMDAR antagonist AP5 (50 ⁇ M) or NR2B-subunit specific antagonist Ro25-6981 (Ro 0.5 ⁇ M), but not NR2A-preferential antagonist NVP-AAM077 (NVP 0.4 ⁇ M).
  • NMDAR antagonist AP5 50 ⁇ M
  • NR2B-subunit specific antagonist Ro25-6981 Ro 0.5 ⁇ M
  • NVP-AAM077 NVP-AAM077
  • FIGURE IE is a western blot showing NMDA-induced SREBP1 requires calpain activity, whereby neurons treated with calpain inhibitors calpeptin (Calpeptin; 25 ⁇ M) or MDL (MDL; 50 ⁇ M) prior to NMDA-insult show reduced NMDA-induced SREBP1 activation.
  • FIGURE 2A is a western blot showing purified nuclear fractions from cortical cultured neurons probed for the active form of SREBP1 (Nuclear SREBP1 or nt-SREBP1) and the membranes were then stripped and re-probed for TATA-binding protein (TBP) as nuclear fraction confirmation and loading control (bottom blots).
  • SREBP1 SREBP1
  • TBP TATA-binding protein
  • Mature SREBP1 is detected in the nucleus in response to NMDA stimulation in a time-dependent fashion and the SREBP1 nuclear translocation is blocked by AP5, demonstrating its dependence on specific activation of NR2B-contai ⁇ ing NMDARs.
  • FIGURE 2B is a bar graph showing purified nuclear fractions from cortical cultured neurons probed for the active form of SREBP1 (Nuclear SREBP1 or nt-SREBP1) as compared to Lamin A/C as nuclear fraction confirmation and loading control. Mature SREBP1 is detected in the nucleus in response to NMDA stimulation and the SREBP1 nuclear translocation is blocked by AP5 and Ro, but not NVP (at least 4 independent experiments in each groups; **p ⁇ 0.01), demonstrating its dependence on specific activation of NR2B-containing NMDARs.
  • SREBP1 Nuclear SREBP1 or nt-SREBP1
  • FIGURE 2C is a series of immuno fluorescent micrographs showing nuclear translocation of SREBP1 is dependent on NMDAR activation and associated with NMDA-induced apoptosis.
  • Neurons were treated without (Control) or with NMDA alone (NMDA; 20 ⁇ M for 1 hour) or in the presence 50 ⁇ M AP5 (NMDA+AP5) and were stained for SREBP1 four hours later.
  • SREBP1 translocates into the nucleus following NMDA stimulation (NMDA) as demonstrated by the increased colocalization with Hoechst 33358 and this translocation is prevented by co-application of AP5. scale bar 40 ⁇ m.
  • FIGURE 3B is a bar graph showing cortical cultures were assayed for LDH release after excitotoxicNMDA treatment at various cholesterol concentrations. The addition of lOO ⁇ M cholesterol significantly reduced excitoxicity-induced cell death. *p ⁇ 0.05 from 3 independent experiments.
  • FIGURE 3 C is a western blot showing endogenous SREBP1 expression is reduced by between 40 and 50% after 48 hour expression of a shRNA knockdown construct in HEK293 cells compared to empty vector or scrambled control shRNA.
  • FIGURE 3D is a series of immunofluorescent micrographs showing flag- SREBP1 expression is dramatically reduced in bippocampal cultures upon co-transfection with SREBP1 shRNA construct as compared to control shRNA and GFP alone neurons. 4 to 5 days after co-transfection, anti-flag immunofluoresence was used to detect flag- SREBP1 expression in green cells, nuclear concentrated flag-SREBP1 was absent in SREBP1 shRNA co-transfected neurons.
  • FIGURE 3 E is a series of immunofluorescent micrographs showing hippocampal neurons transfected with SREBP1 shRNA construct are resistant to NMDA induced excitotoxicity. 4 to 5 days after transfection, neurons were exposed to NMDA challenge (50 ⁇ M) and allowed 2 hours recovery before fixation. Apoptotic nuclear condensations were assessed by Hoechst 33358 (5 ⁇ g/ml) fluorescent stain (transfected nuclei indicated by arrow).
  • FIGURE 3F is a bar graph showing results of coverslips that were scored for the percentage of green apoptotic neurons (bar chart for >10 coverslips per condition from 3 independent experiments). **p ⁇ 0.01, scale bar lO ⁇ m.
  • FIGURE 4A is a proposed mechanism underlying SREBP1 activation and its inhibition by high level cholesterol or TAT-Indip peptide, showing that under basal conditions, immature SREBP1 forms a SREBP1-SCAP complex and is retained in the ER by the interaction between SCAP and ER anchoring protein Insig-1, with low cholesterol or excitotoxicity leads to the sequential ubiquitination of and degradation of the anchoring protein Insig-1, causing the release SREBP1 -SCAP from ER for SREBP1 cleavage/activation in the Golgi, and with high levels of cholesterol or lnsig-1 degradation interference peptide (Indip, that carries ubiquitination sites of Insig-1) is predicted to inhibit Insig-1 degradation and hence stabilize the Insig-1 SCAP interaction, thereby preventing the release of and activation of SREBP1.
  • a control peptide (TAT-Indip K-R ) lacking the Insig-1 ubiquitination lysine residues is predicted not to affect In
  • FIGURE 4B is an immunoblot of HEK 293 cell lysates, where the cells were treated with 2 ⁇ M peptide followed by 24 h stimulation with 10 ⁇ M simvastatin.
  • the simvastatin-induced degradation of Insig-1 was inhibited by TAT-Indip, but not TAT-Indip K-R .
  • FIGURE 4C is a bar graph showing Insig-1 protein as compared to ⁇ -tubulin in cortical culture lysates pretreated with TAT-Indip or TAT-Indip K-R peptide and probed for Insig- 1.
  • Insig-1 relative to ⁇ -tubulin control is unaffected by NMDA treatment in the presence of TAT-Indip, but not TAT-Indip K-R peptide, showing that TAT-Indip peptide can reduce NMDA-induced Insig-1 degradation. 4 experiments ⁇ 0.01
  • FIGURE 4D is a bar graph showing that TAT-Indip prevents NMDA-induced SREBP1 activation in neuronal cultures, where western blots of cortical cultures 6 hours after NMDA insult and the bar graph represents data from 4 independent experiments, p ⁇ 0.01.
  • FIGURE 5B is a bar graph that shows TAT-Indip reduces NMDA-induced LDH release. Cortical cultures were pretreated with 1 ⁇ M TAT-Indip peptide for 20 minutes before NMDA induced excitotoxicity assessed by LDH assay.
  • FIGURE 5C is a bar graph that shows TAT-Indip prevents NMDA-induced apoptosis.
  • Cortical cultures were treated without (Control) or with NMDA in the presence or absence of TAT-Indip (1 ⁇ M 20 min prior to NMDA application).
  • FIGURE 5D is a scatter plot quantification of Hoechst 33358 condensations showing that TAT-Indip peptide reduces apoptosis progression (data points for >60 neurons from 2 independent experiments). **p ⁇ 0.01
  • FIGURE 6A is a bar graph showing TAT-Indip reduces NMDA-induced cell death in cultured neurons. Cortical cultures were pretreated with 2 ⁇ M TAT-Indip or control peptide (Indip K-R ) for 20 minutes before NMDA induced excitotoxicity. Cell death assessed by LDH release 24 h later.
  • FIGURE 6B is a series of immunofluorescent micrographs that show TAT-Indip effectively reduces NMDA-induced neuronal death.
  • Individual neurons assessed for propidum iodide uptake 24 h after NMDA induced apoptosis in presence of TAT-Indip or TAT-Indip K-R - Neurons were identified by NeuN counterstain and nuclear propidiura iodide localized with Hoechst 33358.
  • TAT-Indip peptide (but not TAT-Indip K-R ) effectively blocks NMDA induced neuronal death (quantification for 6 coverslips from 2 independent experiments).
  • FIGURE 6C is a bar graph showing that Oxygen Glucose Deprivation (OGD) induces activation (nt-SREBP1) of SREBP1 as compared to ⁇ -Tubulin in cortical cultures exposed to 1 h OGD and immunobloted for nt-SREBP1 at indicated times post-OGD.
  • OGD Oxygen Glucose Deprivation
  • FIGURE 6D is a bar graph showing OGD-induced activation of SREBP 1 is NMDA-receptor dependent OGD treated cortical cultures in the presence of NMDA- receptor antagonists were immunoblotted.
  • the pan NMDA-receptor antagonist (AP5, 50 ⁇ M) and NR2B specific antagonist (Ro25-698l, 0.5 ⁇ M) showed inhibition of OGD- dependent SREBP1 activation, but NR2A antagonist (NVP-AAM077, 0.4 ⁇ M) showed no effect.
  • FIGURE 6E is a bar graph showing TAT-Indip prevents QGD-raduced cell death.
  • TAT- Indip peptide (but not TAT-Indip K-R ) reduces OGD-induced cell death. **p ⁇ 0.01
  • FIGURE 7A is a schematic of pre-TAT-Indip peptide treatment.
  • FIGURE 7B is an immunoblot showing MCAO-induced Insig-1 degradation is reduced in i.v. injected TAT-Indip (3 nmol/g) treated animals. Cortical tissue collected from indicated areas and probed for Insig-1 on an immunoblot. MCAO causes Insig-1 loss in the occluded hemisphere (saline, occ), but TAT-Indip treated animals show no loss of Insig-1.
  • FIGURE 7C is an immunoblot showing SREBP1 activation is reduced in TAT- Indip treated animals.
  • the immunoblot is of cortical tissue collected at indicated time point in 7A.
  • nt-SREBP1 is increased with MCAO (saline, occ), but not in the presence of TAT-Indip.
  • FIGURE 7D is a bar graph showing TAT-Indip peptide protects from ischemia induced neuronal damage.
  • TTC stain was used to show healthy tissue (pink).
  • MCAO induces dramatic neuronal death 24 h after occlusion (saline).
  • TAT-Indip effectively reduces.
  • FIGURE 7F is a Schematic of post-TAT-Indip peptide treatment, where TAT- Indip and TAT-Indip K _ R injected i.v, 0.5 h after MCAO.
  • FIGURE 7G is a bar graph showing that post-MCAO TAT-Indip peptide treatment reduces ischemic cell death. FluoroJade immunohistochemisty for dead cells 7 days post-MCAO. Quantification of FuoroJade signal shows reduction of MCAO damage in the occluded hemisphere with TAT-Indip treatment, however saline injected or TAT-Indip K-R injected animais showed significantly more cell death. ⁇ 0.01, n>6.
  • FIGURE 8 is a plot showing that TAT-Indip peptide reduces tactile response deficit post-ischaemia.
  • Animals were treated with TAT-Indip or TAT-Indip K-R peptide 30 minutes after MCAO were assessed for limb response due to tactile stimuli on the indicated days post-ischaemia (O - no deficit, 1 - mild deficit, 2 - severe deficit).
  • O - no deficit, 1 - mild deficit, 2 - severe deficit Immediately after MCAO all animals showed severe deficits in tactile response, but 7 days post-ischaemia TAT-Indip treated animals show significantly improved responses. **p ⁇ 0.01.
  • FIGURE 9 is a an immunoblot from spinal cord lysates of wild type and ALS (G93A) transgenic mice that were subjected to SDS-PAGE and immunoblotting for Insig- 1 and mature SREBP1.
  • ALS tg mice showed increased SREBP1 activation and Insig-1 degradation.
  • Data from 4 individual experiments were summarized in the Bar graph.
  • FIGURE 10 is a bar graph showing TAT-Indip reduces glutamate-induced cell death in cultured mouse spinal coxd neurons.
  • Mouse embryonic spinal cord cultured neurons were pretreated with 2 ⁇ M TAT-Indip or TAT-Indip K-R peptide for 45 minutes before glutamate (100 ⁇ M for 30 min) induced excitotoxicity. Cell death assessed by LDH release after 6 hours.
  • FIGURE 11 is a bar graph showing G93A transgenic mouse embryonic spinal cord cultured neurons were pretreated with.2 ⁇ M TAT-Indip or TAT-Indip K-R peptide for 45 minutes before glutamate (100 ⁇ M for 30 rain) induced excitotoxicity. Cells were washed and then transferred to the regular culture media for additional 6 h. Neurons were then fixed and subjected to the Tunel assay to measure percentage of DNA fragmented cells (apoptotic cells). TAT-Indip effectively blocked glutamate induced apoptosis in G93A spinal cord neurons while TAT-Indip K-R had some neuronal toxic effect on its own and failed to block glutamate-induced toxicity. Data from 4 independent experiments.
  • antibody' refers to immune system proteins, also called immunoglobulins, produced in response to antigens.
  • Antibodies typically contain two heavy chains and two light chains, which are joined. Variability in the structure of these chains provides antigen specificity, thereby allowing individual antibodies to recognize specific antigens.
  • the term antibody may include polyclonal and monoclonal antibodies, chimeric, single chain, or humanized antibodies, as well as Fab or F(ab)2 fragments, including the products of an Fab or other immunoglobulin expression library. Methods of making such antibodies or fragments are known in the art and may be found in, for example Harlow E. and Lane D. Antibodies: A Laboratory Manual. (1988). Cold Spring Harbor Laboratory Press.
  • Antibodies may also include intracellular antibodies, sometimes referred to as intrabodies. Methods for designing, making and/or using such antibodies has been described in the art, for instance Lecerf et al. 2001 ; Hudson and Sourjau 2003. Selection or identification of specific peptides for use as epitopes for production of antibodies that differentiate between proteins, or isoforms of proteins may be made using sequence comparisons - one of skill in the art will be able to identify suitable peptide or protein sequences that may be useful for producing antibodies with the desired selectivities. Polyclonal antibodies axe antibodies that are derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope.
  • antibodies are typically produced by immunization of a suitable mammal, .such as a mouse, rabbit or goat. Larger mammals are often preferred as the amount of serum that can be collected is greater.
  • An antigen is injected into the mammal, which induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen. This IgG is purified from the mammal's serum.
  • monoclonal antibodies may be derived from a single cell line.
  • Adjuvants may be used to improve or enhance an immune response to antigens.
  • bioactive refers to one or more of the following: protection against glutamate induced DNA fragmentation; protection against glutamate induced excitotoxicity cell death; reduction of INSIG-I degradation; reduction of NMDA and/or OGD-induced neuronal death; neuroprotection; reduction of SREBP1 activation; prevention the SREBPI activation; mediation of excitotoxic neuronal injuries; reduction of nuclear translocation of SREBP1 ; reduction of infarct volume following stroke; reduction of SREBP1 activation in sporadic and familial amyotrophic lateral sclerosis; and reduction of ischemic injury.
  • a 'subject' refers to an animal, for example a bird or a mammal.
  • Animals may include a zebra fish, a rat, a mouse, a dog, a cat, a cow, a sheep, a horse, a pig or a primate (for example, a monkey or an ape).
  • a subject may further be a human, alternatively referred to as a patient.
  • a subject may be a transgenic animal.
  • NMDAR N-methyl-D-asparate
  • 'NMDAR' refers to NMDA receptor(s).
  • NMDAR is an ionotropic receptor for glutamate.
  • NMDA is a selective specific agonist of NMDAR, and the activation of NMDAR leads to ion channel opening, allowing for Na + and Ca 2+ ions into the cell and K + out of the cell.
  • the amount of Ca 2+ flux is small, it is thought that the movement of Ca 2+ plays a critical role in synaptic plasticity, which is implicated in learning and memory.
  • There are 8 NRl variants formed by alternative splicing of the GRINl gene or NMDARl i,e.
  • NRl-Ia, NRl-Ib, NRl -2a, NRl -2b, NRl -3 a, NRl -3b, NRl -4a, NRl -4b) and 4 isoforms of NR2 are generated by 4 genes (GRIN2A-D or NMDAR2A-D).
  • Multiple receptor isoforms with distinct brain distributions and functional properties arise by selective splicing of the NRl transcripts and differential expression of the NR2 subunits.
  • NMDAR forms a heterodimer between NRl and NR2 subunits.
  • SCAP' refers to SREBP cleavage activating protein (EntrezGene ID: 22937), which is required for proteolytic cleavage of SRBBP.
  • SCAP is an integral membrane protein located in the endoplasmic reticulum (ER), with a cytosolic region that contains a hexapeptide amino acid sequence (MELADL), which detects cellular cholesterol. When cholesterol is present, SCAP undergoes a conformational change, which prevents activation of SREBP and thereby inhibiting cholesterol synthesis. .
  • SREBP-I refers to sterol regulatory element binding protein 1, which may alternately be referred to as SREBF-I or SREBP1 (EntrezGene ID: 6720).
  • SREBPs belong to the basic-helix-loop-helix leucine zipper class of transcription factors and are attached to the endoplasmic reticulum membrane in an inactive form.
  • SREBPs In cells with low levels of sterols (i.e. cholesterol) SREBPs are cleaved to form a soluble N-terminal domain that translocates to the nucleus.
  • SREs specific sterol regulatory elements
  • ' ⁇ nsig-1 ' refers to the protein product of insulin-induced gene 1 (EntrezGene H): 3638), which produces a 277 amino acid polypeptide.
  • This protein binds to the sterol-sensing domains of SREBP cleavage-activating protein (SCAP) and HMG CoA reductase, and is involved in sterol-mediated trafficking of SCAP and HMG CoA reductase.
  • SCAP SREBP cleavage-activating protein
  • HMG CoA reductase HMG CoA reductase
  • Alternatively spliced transcript variants encoding distinct isoforms have been observed and degradation of Ins ⁇ g-1 by ubiquitin-mediated pathways is. important for SCAP/SREBP regulation, as described herein.
  • Provided herein are various compositions and methods for cytoprotection.
  • Provided herein are various compositions and methods for modulating ubiquitination of Insig-1.
  • Insig degradation peptide inhibitor referes to the lysines 156 and 158 and flanking sequences as set out herein and represented by human Ihsig-1 (i.e. GenBank Accession no. AYl 12745) as set out in Gong et al. (2006b). Based on a proposed membrane topology of human Insig-1, the region containing lysines 156 and 158 and flanking sequence is shown as loop outside of the ER membrane on the cytosolic surface (Gong et al. 2006b).
  • Indip peptides or polypeptides may be selected from one or more of the following: GEPHKPKREW (amino acids 152 to 162 of AYl 12745); EPHKFKREW (amino acids 153 to 162 of AYl 12745); HLGEPHKFKREW (amino acids 148 to 162 OfAYl 12745); GEPHKFKREW (amino acids 96 to 105 of AY527632).
  • 'peptide' or 'polypeptide' are used interchangeably herein, and refer to a compound comprised of at least two amino acid residues, but not a full length protein, covalently linked by peptide bonds or modified peptide bonds (for example peptide isosteres), whereby the modified peptide bonds may provide additional desired properties to the peptide or polypeptide, such as increased half-life.
  • a peptide or a polypeptide may comprise portions of one or more different proteins.
  • SEQ ID NO: 2 shows a TAT, derived HTV TAT protein transduction domain, linked to an Indip peptide, derived from human insig protein.
  • amino acids comprising a peptide or polypeptide as described herein may also be modified either by other processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Modifications can occur anywhere in a peptide or polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It is understood that the same type of modification may be present in the same or varying degrees at several sites in a given peptide. Furthermore, it is well known in the art how to select an appropriate expression system depending on the type of modification desired.
  • substitutions refers to the substitution of one or more amino acids for another at a given location or locations in the peptide or polypeptide, where the substitution can be made without substantial loss of the relevant function, hi making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like (described in more detail below), and such substitutions may be assayed for their effect on the function of the peptide or polypeptide by routine testing.
  • the term 'recombinant' means that something has been recombined, so that when made in reference to a nucleic acid construct the term refers to a molecule that is comprised of nucleic acid sequences that are joined together or produced by means of molecular biological techniques.
  • the term "recombinant" when made in reference to a protein or a polypeptide or a peptide refers to a protein or polypeptide or peptide molecule, which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques.
  • the term 'recombinant' when made in reference to genetic composition refers to a gamete or progeny with new combinations of alleles that did not occur in the parental genomes.
  • Recombinant nucleic acid constructs may include a nucleotide sequence, which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Recombinant nucleic acid constructs, therefore, indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e. by human intervention. Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species. Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of transformation of the host cells, or as the result of subsequent recombination events. . .
  • isolated compounds such as nucleic acids and amino acids (i.e. peptides or polypeptides).
  • 'Isolated' as used herein, is meant to convey that the isolated substance has been substantially separated or purified away from other components, such as biological components, with which it would otherwise be associated, for example in vivo, so that the isolated substance may be itself be manipulated or processed.
  • the term 'isolated' therefore includes substances purified by purification methods known in the art, as well as substances prepared by recombinant expression in a host, as well as chemically synthesized substances.
  • a compound is 'isolated' when it is separated from the components that naturally accompany it so that it is at least 60%, more generally 75% or over 90%, by weight, of the total relevant material in a sample.
  • a polypeptide that is chemically synthesised or produced by recombinant technology maybe generally substantially free from its naturally associated components.
  • a nucleic acid molecule is substantially pure when it is not immediately contiguous with (i.e., covalently linked to) the coding sequences with which it is normally contiguous in the naturally occurring genome of the organism from which the DNA of the invention is derived.
  • An isolated compound can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a polypeptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis or HPLC.
  • the term 'medicament' as used herein, refers to a composition that may be administered to a patient or test subject and is capable of producing an effect in the patient or test subject.
  • the medicament may be comprised of the effective chemical entity (i.e. one or more of the peptides or polypeptides described herein) alone or in combination with a pharmaceutically acceptable excipient.
  • excipient' may include any and all solvents, dispersion media, coatings, antibacterial, antimicrobial or antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • An excipient may be suitable for intravenous, intraperitoneal, intramuscular, subcutaneous, intrathecal, topical or oral administration.
  • An excipient may include sterile aqueous solutions or dispersions for extemporaneous preparation of sterile injectable solutions or dispersion- Use of such media for preparation of medicaments is known in the art.
  • RNA RNA, cDNA, genomic DNA, synthetic forms, and mixed polymers, both, sense and antisense strands, and may be chemically or biochemically modified or may contain non- natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • the term 'vector' refers to a polynucleotide compound used for introducing exogenous or endogenous polynucleotide into host cells.
  • a vector comprises a nucleotide sequence which may encode one or more polypeptide molecules. Plasmids, cosmids, viruses and bacteriophages, in a natural state or which have undergone recombinant engineering, are examples of commonly used vectors to provide an isolated polynucleotide molecule to a cell.
  • modifications to peptides or polypeptides may include, but are not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodjnation, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitin
  • polypeptides with N-terminal or C- terminal amino acids removed may be assayed for their effect on the function of the protein by routine testing.
  • Amino acids may be described as, for example, polar, non-polar, acidic, basic, aromatic or neutral
  • a polar amino acid is an amino acid that may interact with water by hydrogen bonding at biological or near-neutral pH.
  • the polarity of an amino acid is an indicator of the degree of hydrogen bonding at biological or near-neutral pH.
  • Examples of polar amino adds include serine, proline, threonine, cysteine, asparagine, glutarnine, lysine, histidine, arginine, aspartate, tyrosine and glutamate.
  • non-polar amino acids examples include glycine, alanine, valine leucine, isoleucrae, methionine, phenylalanine, and tryptophan.
  • Acidic amino acids have a net negative charge at a neutral pH. Examples of acidic amino acids include aspartate and glutamate.
  • Basic amino acids have a net positive charge at a neutral pH. Examples of basic amino acids include arginine, lysine and histidine.
  • Aromatic amino acids are generally nonpolar, and may participate in hydrophobic interactions. Examples of aromatic amino acids include phenylalanine, tyrosine and tryptophan. Tyrosine may also participate in hydrogen bonding through the hydroxyl group on the aromatic side chain.
  • Neutral, aliphatic amino acids are generally nonpolar and hydrophobic. Examples of neutral amino acids include alanine, valine, leucine, isoleucine and methionine, An amino acid may be described by more than one descriptive category. Amino acids sharing a common descriptive category may be substitutable for each other in a peptide.
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6)s are assigned to ammo acid residues (as detailed in United States Patent No.
  • the hydropathy index of an ammo add may be represented as a scale indicating the tendency of an amino acid to seek out an aqueous environment (negative value) or a hydrophobic environment (positive value).
  • Hydropathy indices of the standard amino acids include alanine (+1.8), arginine (-4.5), asparagine (-3.5), aspartic acid (-3.5), cysteine (+2.5), glutamine (-3.5), glutamic acid (-3.5), glycine (-0.4), histidine (-3.2), isoleucine (+4.5), leucine (+3.8), lysine (-3.9), methionine (+1.9), phenylalanine (+2.8), proline (-1.6), serine (-0.8), threonine (-0.7), tryptophan (-0.9), tyrosine (-1.3), and valine (+4.2).
  • Amino acids with similar hydropathy indices may be substitutable for each other in a peptide (Kyte & Doolittle 1982).
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0).
  • each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5); VaI (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3,5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).
  • conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, VaI, Leu, He, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr. . .
  • conservative amino acid changes include changes based on considerations, of hydrophilicity or hydrophobicity, size or volume, or charge.
  • Amino acids can be generally characterized as hydrophobic or hydropbilic, depending primarily on the properties of the amino acid side chain.
  • a hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eiseriberg et al. (1984).
  • Genetically encoded hydrophobic amino adds include Gly, Ala, Phe, VaI, Leu, lie, Pro, Met and Trp, and genetically encoded hydrophilic Eanrao acids include Thr, His, Glu, Gln, Asp, Arg, Ser, and Lys.
  • Non-genetically encoded hydrophobic amino acids include t-butylalanine, while non-genetically encoded hydxophilic amino acids include citrulhne and homocysteine.
  • Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains.
  • an aromatic amin,o acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substiruents such as -OH, -SH, -CN, -F, -Cl, -Br, -I, - NO2, -NO, -NH2, -NHR, -NRR, -C(O)R, -C(O)OH 5 -C(O)OR 1 -C(O)NH2, -C(O)NHR, - C(O)NRR, etc., where R is independently (C1-C6) alkyl, substituted (C1-C6) alkyl, (Cl- C6) allcenyl, substituted (C1-C6) allcenyl, (C1-C6) alkynyl, substituted (C1-C6) al
  • An apolax amino acid is a hydrophobic amino acid "with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar).
  • Genetically encoded apolar amino acids include Gly, Leu, VaI, Be, Ala, and Met.
  • Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain.
  • Genetically encoded aliphatic amino acids include Ala, Leu, VaI, and lie.
  • a polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, hut which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms.
  • Genetically encoded polar amino acids include Ser, Thr, Asn, and Gln.
  • An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His.
  • conserved amino acid substitutions may be made by substituting an amino acid for another amino acid within the group containing similar amino acids (if there are others) and provided the amino acid is not known to be responsible for the desired activity, as per the following groupings: GAVLI; FYW; CM; ST; KRH; DENQ; P (Stothard 2000).
  • the lysines (K) at positions 5 and 7 of the 10 amino acid sequence represented by SEQ ID NO: 1 are needed for ubiquitination and are preferably not both substituted with any other amino acids.
  • amino acids can be classified based on known behaviour and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids.
  • Amino acids contained within the peptides described herein may be understood to be in the L- or D- configuration. In peptides and peptidomimetics, D-amino acids may be substitutable for L-amino acids.
  • Nonstandard amino acids may occur in nature, and may or may not be genetically encoded.
  • Examples of genetically encoded nonstandard amino acids may include selenocysteine, sometimes incorporated into some peptides at a UGA codon, which may normally be a stop codon, or pyrrolysine, sometimes incorporated into some proteins at a UAG codon, which may normally be a stop codon.
  • Some nonstandard amino acids that are not genetically encoded may result from modification of standard amino acids already incorporated in a peptide, or may be for example metabolic intermediates or precursors.
  • nonstandard amino acids may include, but are not limited to 4- hydroxyproline, 5-hydroxylysine, 6-N-methyllysine, gamma-carboxyglutamate, desmosine, selenocysteine, ornithine, citrulline, lanthionine, 1-aminocyclopropane-1- carboxylic acid, gamma-aminobutyric add, carnitine, sarcosine, or N-formylmethionine.
  • Synthetic variants of $tanda ⁇ d and non-standard amino acids are also known and may include chemically derivatized amino acids, amino acids labeled for identification or tracking, or amino acids with a variety of side groups on the alpha carbon.
  • Example of such side groups are known in the art end may include aliphatic, single aromatic, polycyclic aromatic, heterocyclic, heteronuclear, amino, alkylamino, carboxyl, carboxamide,.carboxyl ester, guanidine, amidine, hydroxyl, alkoxy, mercapto-, alkylmercapto-, or other heteroatom-containing side chains.
  • Other synthetic amino adds may include alpha-imino adds, non-alpha amino acids such as beta-amino adds, des- carboxy or des-amino acids. Synthetic variants of amino acids may be synthesized using general methods known in the art, or may be. purchased from commercial suppliers, for example RSP Amino Adds LLCTM (Shirley, MA)..
  • the peptide or polypeptide may be represented by one or more of the . sequences represented in TABLE 1 or elsewhere herein.
  • a linker sequence may be used to join an Indip peptide to a targeting, delivery or localization moiety, referred to herein collectively as a 'delivery moiety', and may be advantageous.
  • a linker may be used to link, for example, a transduction domain like TAT to an tidip peptide.
  • 1-5 G residues may be used as a linker (see for example SEQ ID NOs: 1, and 3-6).
  • linker sequences maybe at the C- terminus of an Indip peptide.
  • Alternative linkers are known in the art.
  • a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or glutaraldehyde are well known..
  • Peptides or peptide analogues can be synthesised by chemical techniques known in the art, for example, by automated synthesis using solution or solid phase synthesis methodology. Automated peptide synthesisers are commercially available and use techniques well known in the art. Peptides and peptide analogues can also be prepared using recombinant DNA technology using methods such as those described in, for example, Sambrook J. and Russell D. (2000) Molecular Cloning: A Laboratory Manual (Third Edition) Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.) or Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Suns, 1994).
  • the peptides or polypeptides may be used in isolation, or may be linked to or in combination with another moiety, such as a targeting, delivery or localization moiety, referred to herein collectively as a 'delivery moiety', for example, tracer compounds, protein transduction domains or sequences, antibodies, liposomes, carbohydrate carriers, polymeric carriers or other agents or excipients as will be apparent to one of skill in the art.
  • a targeting, delivery or localization moiety referred to herein collectively as a 'delivery moiety', for example, tracer compounds, protein transduction domains or sequences, antibodies, liposomes, carbohydrate carriers, polymeric carriers or other agents or excipients as will be apparent to one of skill in the art.
  • bioactive molecules such as peptides
  • Delivery of bioactive molecules such as peptides, to a cell or cells in a reasonably efficient manner may require more than just the 'dumping' of the naked peptide onto the cell, or administering the naked peptide into the patient or test subject.
  • Agents that enable delivery of bioactive molecules into cells (or 'delivery moieties') in a suitable manner so as to provide an effective amount, such as a pharmacologically effective amount are known in the art, and are described in, for example, Dietz et al 2004. MoI Cell. Neurosci 27:85-131.
  • examples of such agents include liposomes, antibodies or receptor ligands that may be coupled to the bioactive molecule, viral vectors, and protein transduction domains (PTD).
  • PTDs include Antennapedia homeodomain (Perez et al 1992 J. Cell Sci 102:717-722), transportan (Pooga et al 1998 FASEB J 12: 67-77), the translocation domains of diphtheria toxin (Stenmark et al 1991 J Cell Biol 113:1025-1032; Wiedlocha et al 1994 Cell 76:1039-1051), anthrax toxin (Ballard et al 1998 Infect.
  • the TAT peptide has been used to transduce ghal-derived neurotrophic factor (GDNF) into the central nervous system (CNS) ofmice (Kilic E. et aL 2005).
  • CNS central nervous system
  • Schwarze et al. (1999) introduced TAT tagged proteins itito the brains of mice.
  • intravenous administration of brain- . derived neurotrophic factor conjugated to the OX26 monoclonal antibody (MAb) specific for the transferring recptor to cross the blood-brain barrier (Pardridge (2002); and Zhang & Partridge (2001)).
  • charged liposomes Haas et al; (2009)
  • • rhenacarborane Hawkins et al. (2009)
  • hyaluronan Horvat et al. (2008)
  • compositions or compounds according to some embodiments may be administered in any of a variety of known routes.
  • methods that may be suitable for the administration of a compound include orally, intravenous, inhalation, intramuscular, subcutaneous, topical, intraperitoneal, intra-rectal or intra-vaginai suppository, sublingual, and the like.
  • the compounds of the present invention may be administered as a sterile aqueous solution, or may be administered in a fat-soluble excipient, or in another solution, suspension, patch, tablet or paste format as is appropriate.
  • a composition comprising the compounds of the invention may be formulated for administration by inhalation. For instance, a compound may be combined with an excipient to allow dispersion in an aerosol.
  • inhalation formulations will be known to those skilled in the art.
  • Other agents may be included in combination with the compounds of the present invention to aid "uptake or metabolism, or delay dispersion within the host, such as in a controlled- release formulation.
  • controlled release formulations will be known to those of skill in the art, and may include microencapsulation, embolism within a carbohydrate or polymer matrix, and the like.
  • Other methods known in the art for making formulations are found in, for example, "Remington's Pharmaceutical Sciences", (19th edition), ed. A. Gennaro, 1995, Mack Publishing Company, Easton, Pa.
  • compositions or compounds of some embodiments of the invention may vary depending on the route of administration (oral, intravenous, inhalation, or the like) and the form in which the composition or compound is administered (solution, controlled release or the like). Determination of appropriate dosages is within the ability of one of skill in the art.
  • an 'effective amount', a 'therapeutically effective amount', or a 'pharmacologically effective amount' of a medicament refers to an amount of a medicament present in such a concentration to result in a therapeutic level of drug delivered over the term that the drug is used. This may be dependent on mode of delivery, time period of the dosage, age, weight, general health, sex and diet of the subject receiving the medicament. Methods of determining effective amounts are known in the art.
  • Suitable vectors may include, viral or bacterial vectors.
  • Suitable viral vectors may include, retroviral vectors, alphaviral, vaccinial, adenoviral, adenoassociated viral, herpes viral, and fowl pox viral vectors.
  • the vectors preferably have a native or engineered capacity to transform eukaryotic cells, e.g., CHO-Kl cells.
  • the vectors useful in the context of the invention can be 'naked' nucleic acid vectors (i.e., vectors having Httle or no proteins, sugars, and/o ⁇ lipids encapsulating them) such as plasmids or episomes, or the vectors can be complexed with other molecules.
  • nucleic acid vectors i.e., vectors having Httle or no proteins, sugars, and/o ⁇ lipids encapsulating them
  • Other molecules that may be combined with the present nucleic acids are viral coats, cationic lipids, liposomes, polyamines, gold particles, and targeting moieties such as ligands, receptors, or antibodies that target cellular molecules.
  • the nucleic acid molecules described herein may comprise a region coding for one or more of the ⁇ ndip peptides described herein, may be operatively linked to a suitable promoter, which promoter is preferably functional in eukaryotic cells.
  • a suitable promoter such as, the RSV promoter and the adenovirus major late promoter.
  • Suitable non-viral promoters may include, the phosphoglycerokinase (PGK) promoter and the elongation factor 1 ⁇ promoter.
  • Non- viral promoters may be human promoters.
  • Additional suitable genetic elements, known in the art may also be ligated to, attached to, or inserted into the nucleic acid and constructs to provide additional functions, such as level of expression, or pattern of expression.
  • the native promoters for expression of the Insig-1 nucleotides may also be used, in which event they are preferably not -used in the chromosome naturally encoding them unless modified by a process that substantially changes that chromosome.
  • Such substantially changed chromosomes can include chromosomes transfected and altered by a retroviral vector or similar process.
  • such substantially changed chromosomes can comprise an artificial chromosome such as a HAC, YAC, or BAC.
  • nucleic acid molecules described herein maybe operatively linked to enhancers to facilitate transcription.
  • Enhancers are cis-acting elements of DNA that stimulate the transcription of adjacent genes. Examples of enhancers which confer a high level of transcription on linked genes in a number of different cell types from many species include, without liinitation, the enhancers from SV40 and the RSV-LTR. Such enhancers can be combined with other enhancers which have cell type-specific effects, or any enhancer may be used alone. .
  • the inventive nucleic acid molecule can further comprise a polyadenylation site following the coding region of the nucleic acid molecule. Also, preferably all the proper transcription signals (and translation signals, where appropriate) will be correctly arranged such that the exogenous nucleic acid will be properly expressed in the cells into which it is introduced. If desired, the exogenous nucleic acid also can incorporate, splice sites (i.e., splice acceptor and splice donor sites) to facilitate mRNA production while maintaining an inframe, full length transcript Moreover, the inventive nucleic acid molecules can further comprise the appropriate sequences for processing, . secretion, intracellular localization, and the like.
  • the 9 amino acid sequence "EPHKFKREW”, maybe reverse translated to a 27 base sequence based on the most likely codons as follows: gaaccgcataaatttaaacgcgaatgg (SEQ ID NO : 25) .
  • Rabbit anti-SREBP1 was obtained from Santa Cruz BiotechnologyTM (Santa Cruz, CA).
  • Mouse mAb MAP2 was obtained from BD PharmagenTM (San Diego, CA).
  • ProLong GoldTM mounting medium and secondary fluorophore-bound IgGs (Alexa FluorTM series) for immunocytochemistry were obtained from Invitrogen-Molecular ProbesTM (Portland, OR).
  • Rabbit anti-Lamin A/C was obtained from Cell Signaling TechnologyTM (Danvers, MA).
  • Mouse anti ⁇ -tubulin was from Sigma AldrichTM (St. Louis, MO).
  • Mouse anti- neuronal nuclei (NeuN) was purchased from Chemicon, Inc.TM (Temecula, CA).
  • Mouse anti-Myc tag was from.
  • TATA binding protein (TBP) was purchased from Abeam Inc.TM (Cambridge, MA).
  • Leupeptin protease inhibitor was obtained from Peptides InternationalTM (Louisville, Kentucky) and all other lysate buffer components, including phosphatase and protease inhibitors, were obtained from Sigma AldrichTM (St Louis, MO).
  • BAPTA-AM was obtained from Invitrogen-Molecular ProbesTM (Portland, OR).
  • Calpeptin and MDL were obtained from CalbiocheraTM. (San Diego, CA).
  • NMDA was purchased from Ascent ScientificTM (Weston, UK).
  • Mouse anti Insig-1 antibody was a generous gift from Prof. Stephen A.
  • TAT-Indip YGRKKRRQRRRGEPHKFKREW
  • YGRKKRRQRRRGEPHKFKREW was custom synthesized by Peptide Synthesizing Facility at UBC (Vancouver, BC, Canada)
  • Nuclear extraction kit was purchased from PanomicsTM (Redwood City,. CA)
  • NR2A-specific antagonist NVP-AAM077 was a generous gift from YP Auberson, Novartis Phanna AGTM (Basel, Switzerland).
  • NR2B-specific antagonist Ro 25-6981 was from Sigma AldrichTM (St. Louis, MO).
  • AP5 was purchased from TocrisTM (Cookson Bristol, UK).
  • Dissociated cultures of rat cortical neurons were prepared from 18-d-old Sprague Dawley rat embryos as described previously (Meilke & Wang 2005). To obtain mixed cortical cultures enriched with neurons, uridine (10 ⁇ M) and 5-Fluor-2'-deoxyuridine (10 ⁇ M) were added to the culture medium at 3 d in vitro (DIV) and maintained. for 48 h to inhibit non- neuronal cell proliferation before the cultures were returned to the normal culture medium. Mature neurons (12-14 DIV) were used for experiments. Dissociated spinal cord neurons were prepared from 16-d-old mice embryos hSOD-G93A or wt controls. Cultures were maintained for 10 d in vitro before excitotoxic challenge.
  • Oxygen-glucose deprivation was achieved by transferring cortical cultures to an anaerobic chamber (Thermo ECTM) containing a 5% CO 2 , 10% H 2 , and 85% N 2 ( ⁇ 0.01% O 2 ) atmosphere (Aarts et al. 2002; Goldberg & Choi 1.993; and Meilke & Wang 2005). Cultured neurons were then washed three times with glucose-free bicarbonate-buffered solution (deoxygenated in the anaerobic chamber for 30 min before use) and maintained anoxic for 1 h at 37°C. OGD was terminated by removing the cultures from the chamber, washing them twice with normal ECS, and returning them to the original growth conditions until additional assay.
  • Nuclear extracts were isolated from control and NMDA-treated cell cultures (10 8 cells) using the PanomicsTM nuclear extraction kit (PanomicsTM; catalog number AY2002) as recommended by the manufacturer. Biorin-labeled DNA binding oligonucleotides (TranSignalTM probe mix; PanomicsTM) were incubated with 10 ⁇ g of nuclear extract for 30 min. at 15°C to allow the formation of protein/DNA (or transcription factor/DNA) complexes. The protein/DNA complexes were then separated from the free probes by column purification as described by the manufacture's recommendations (PanomicsTM). Probes were then hybridized to the TranSignal Protein/DNA Combo ArraysTM overnight at 42°C followed by post-hybridization washes.
  • Each TranSignal Protein/DNA Combo ArrayTM contained 345 putative transcription factor nucleotide sequences.
  • hybridized arrays were incubated with horseradish peroxidase-labeled streptavidin and bound signals were detected using chemiluminescence imaging system
  • Necrotic neuronal death was quantified by measuring lactate dehydrogenase (LDH) release 20 h after treatments using a Cyto Tox 96TM assay kit (PromegaTM, Madison, WI); Apoptotic neuronal death was determined either by visualizing neurons stained with Hoechst-33342 or using a cell ELISA assay. For visualizing apoptotic neurons, H ⁇ echest- 33342TM ( ⁇ ⁇ g/ml) was added to the culture medium after treatments and incubated for 30 min. Images were taken with a Leica DMIRE2TM fluorescence microscope. Cells with condensed or fragmented chromatin were considered apoptotic.
  • LDH lactate dehydrogenase
  • MCAo Middle cerebral arterial occlusion
  • the operation wound was sutured, and the animal was allowed to recover from anaesthesia. After 90 minutes of occlusion, the animal was re-anaesthetized to facilitate removal of the occlusion. Sham-operated animals received the same surgical operation, except without the final occlusion. Body temperature was monitored and maintained between 36.5 and 37.5 °C throughout the surgical procedure with a heating pad. .
  • the behaviour tests used were described previously, and included a postural reflex test and five forelimb placement tests (Liu et al. 2007; and Aarts et al. 2002).. Each test gave a score between 0 (no deficit) and 2 (complete deficit) for a total score between 0 and 12. Only rats with a neurological score of 11 one hour following stroke were included in this study. The rats were scored again the next day to determine functional neurological outcome.
  • NMDARs results Activation of NMDARs has been linked to the modulation of a number of transcription factors (TF), with either pro-neuronal survival or pro-death activity (Camandola & Mattson 2007; Hardingham & Bading 2001; Hetman & Kharebava 2006; Rao & Finkbeiner 2007; West et al. 2002; Zhang et al. 2007; and zou & Crews 2006), suggesting that' alteration of TF activity may critically contribute to excitotoxic neuronal injuries following stroke.
  • TF transcription factors
  • SREBP1 SREBP1 activation has recently been linked to glucolipotbxic cell death in pancreatic ⁇ -cells (Sandberg et al; 2005; Takahashi et al.
  • Activation of SREBP1 requires the cleavage of the inactive membrane integral precursor protein of 130 kDa by twp dedicated proteases in the Golgi, leading to the release and translocation of the soluble mature N-terminal SREBP1 (nt-SREBP1) of 68kDa for transcriptional activity.
  • nt-SREBP1 soluble mature N-terminal SREBP1
  • NMDA stimulation changes in the amount of activated nt-SREBP1 in cortical neuronal cultures treated with and without NMDA (50 ⁇ M; 20 min) were determined, As shown in FIGURE IB, NMDA stimulation produced an about 4.5 fold increase in expression of mature nt-SREBP1 after 6 hours (FIGURE IB).
  • NMDAR Activation is specifically mediated by NMDARs as it was fully prevented when NMDA stimulation was performed in the presence of NMDAR.
  • antagonist AP5 50 ⁇ M
  • NR2B-subunit specific antagonist RO25-6981 Ros 0.5 ⁇ M
  • NVP-AAM077 NR2A- ⁇ referential agonist NVP-AAM077
  • Emerging evidence has suggested that activation of NMDAR could lead to pro-survival or pro-death of neuronal cells depending on the subunit composition and/or subcellular location of the receptors.
  • NMDA-induced SREBP1 activation is resistant to NVP-AAMQ77, a NR2A-containing NMDAR preferable antagonist, but blocked by Ro25- 6981, a NR2B subunit specific antagonist.
  • NVP-AAMQ77 a NR2A-containing NMDAR preferable antagonist
  • Ro25- 6981 a NR2B subunit specific antagonist.
  • NMDA-induced SREBP1. activation is primarily mediated by NR2B-subu ⁇ it-containing NMDARs, and hence may specifically contribute to the mediation of excitotoxic neuronal injuries.
  • calpain rnhibitor calpeptin 25 ⁇ M or MDL (50 ⁇ M) (FIGURE IE) inhibited NMDAR-mediated SREBP1 activation, further supporting a potential role of SREBP1 activation in NMDAR-mediated excitotoxicity.
  • nt-SREBP1 nuclear translocation of SREBP1 . (nt-SREBP1) following NMDA-induced excitotoxicity was examined. As shown in FIGURE 2A, western blotting of nuclear extract fractions, of control or NMDA-treated cortical neuronal cultures revealed a time dependent and NMDAR-dependent appearance of active form of SREBP1 in the nucleus (nt-SREBP1).
  • SREBP1 nuclear translocation was also primarily mediated by NR2B-containing NMDARs as it was resistant to NR2A antagonist NVP, but prevented by NR2B antagonist Ro (FIGURE 2B).
  • the NMDAR-dependent activation and nuclear translocation of SREBP1 was also further confirmed with co-immunolocalization of endogenous SREBP1 with nuclei stained using Hoechst 33342.
  • An increase nuclear SREBP1 was detected after NMDA-induced excitotoxicity and which was sensitive to AP5 (FIGURE 2C).
  • SREBP1 may causatjvely relate to NMDAR-mediated excitotoxic neuronal injuries.
  • SCAP SREBP cleavage activating protein
  • neurons Four to five days after transfection, neurons were exposed to NMDA challenge (50 ⁇ M) and allowed 2 hours recovery before fixation and apoptotic nuclear condensations were assessed by Hoechst 33358 (5 ⁇ g/ml) fluorescent stain (transfected nuclei indicated by arrow) and coverslips were scored for the percentage of apoptotic neurons (FIGURE 3F). .
  • Insig-1 degradation interference peptide (Indip) was designed (GEPHKFKREW (SEQ ID NO: I)) and which contains the lysine-156 and lysine-158 ubiquitination sites (positions 5 and 7 in bold of the 10 AA peptide) based on the prediction that the peptide would competitively block Insig-1 ubiquitination and hence reduce Insig- 1 degradation in the proteas ⁇ me (FIGURE 4A) .
  • the peptide was rendered membrane permeable by fusing the cell-membrane transduction domain of the human immunodeficiency virus-type 1 Tat protein (YGRXKRRQKRR) as per Schwarze et al. 1999 to the N-terminal of the Indip to generate TAT-Indip peptide (FIGURE 4A).
  • the delivery moiety such as TAT
  • Simvastatin-induced degradation of Insig-1 was inhibited by TAT-Indip in HEK 293 cells treated with 2 ⁇ M of Insig-1 peptide followed by 24 h stimulation with 10 ⁇ M simvastatin.
  • Insig-1 showed reduced Insig-1 in control cells as compared to TAT-Indip treated cells, but not TAT-Indip ⁇ -R treated cells (FIGURE 4B). Furthermore, rapid tn$ig-l ubiquitination in response to NMDA-induced excitotoxicity was inhibited by TAT- ⁇ dip, but not control peptide. Cortical cell cultures were pretreated with TAT-Indrp or TAT-Indip K .
  • TAT- Indip The neuroprotective effect of TAT- Indip was time-dependent as quantifying Hoechst signal revealed that although the protection could be noticeable as early as 30 min, it only became statistically significant 4 hours after NMDA treatments (FIGURE 5D). This delayed protective action of TAT- Indip corresponds well to the time course required for SREBP1 activation, consistent with TAT-Indip acting via the suppression of SREBP1.
  • TAT-Indip peptide was shown to protect against NMDA induced excitotoxicity and Oxygen Glucose Deprivation neuronal death in cultured neurons.
  • Cortical cultures were pretreated with TAT-Indip or TAT- Indip K-R before NMDA induced excitotoxicity and cell death was assessed by LDH release or propidum iodide uptake after NMDA induced apoptosis and showed that TAT- Indip effectively reduces NMDA-induced neuronal death (FIGURES 6A & 6B).
  • OGD-induced activation of SREBP1 is NMDA-receptor dependent.
  • OGD treated cortical cultures in the presence of NMDA-receptor antagonists AP5 and Ro25- 69Sl inhibit OGD-dependent SREBP1 activation, but NVP-AAM077 has no effect (FIGURE 6D).
  • TAT-Indip is also shown to prevent OGD-induced cell death in cortical cultures pretreated with TAT-Indip or TAT-mdip ⁇ - R before 1 h OGD. Cell death was assayed by LDH release and TAT-Indip, but not TAT-Indip ⁇ _ R reduces OGD-induced cell death (FIGURE 6E). .
  • the TAT- ⁇ ndip peptide was successful in reaching inside of the stroke affected area and effectively interfering with the degradation of Instg-1 and subsequently blocking SREBP1 activation.
  • the TAT-Indip peptide infusion significantly reduced the infarct areas.
  • MCAo produced an averaged infarct area represented 45 ⁇ 7.6 % of the total hemisphere volume.
  • MCAo-induced infarct was significantly reduced to 22 ⁇ 4.2 % of the hemisphere (FIGURE 7D).
  • TAT-Indip peptide treatment also reduced the impact of MCAo on. animals' behavioural performance on forelimb placing and postural reflex tests.
  • TAT-Indip treated animals (7 ⁇ 1.2) performed significantly better than control animals (ll ⁇ 0.3) 24 hours after MCAo surgery (FIGURE 7E).
  • SREBP1 is activated by MCAo challenge and that . ; inhibiting this activation with TAT-Indip not only reduces ischemic neuronal damage, but also improves neurological performance.
  • FIGURE 8 shows TAT-Indip peptide reduces tactile response deficit following an ischaemic insult
  • Animals that were treated with TAT-Indip or TATMndip ⁇ - R peptide after MCAo were assessed for limb response due to tactile stimuli for several days post-ischaemia and although all animals showed severe deficits in tactile response initially, TAT-Indip treated animals show significantly improved responses after 7 days.
  • the present results provide evidence supporting a causative role of SREBP1 in mediating neuronal injuries.. Specifically,.
  • the present disclosure identifies activation of SREBP1 as a potential target in the cascade leading to ischemic neuronal damage, upon which new compounds, such as ⁇ ndip, TAT-Indip and conserved peptides thereof may be developed as potentially a new : class of neuroprotective thereapeutics to reduce, neuronal damage following ischemic insults such as stroke and brain trauma!
  • lnsig-1 degradation and SREBP1 activation are shown in spinal cord lysate of ALS (G93A) transgenic mice-immunoblots as compared to wild type mouse spinal cord lysates. SREBP1 activation is increased in ALS tg mice, while Insig-1 shows degradation (FIGURE 9). Also, TAT-Indip peptide appears to protect against glutamate induced excitotoxicity cell death in both wild type and G93A transgenic embryonic spinal cord neurons.
  • TAT-Indip or TAT-Indip K _ R peptide were pretreated with TAT-Indip or TAT-Indip K _ R peptide before glutamate induced excitotoxicity, then assayed for cell death by LDH release and showed significant inhibition of cell death with TAT-Indip (FIGURE 10).
  • TAT-Indip peptide appears to protect against glutamate induced DNA fragmentation (apoptosis) in G93A transgenic embryonic spinal cord neurons.
  • FIG. 11 shows that SREBP1 is activated in post-mortem spinal cord samples from both sporadic and familial ALS patients, but not normal controls as compared to the ⁇ -Tubulin.
  • SREBPs are the major transcription factors that regulate the expression of a large number genes involved in cellular cholesterol and lipid biogenesis and metabolic alterations in some of these lipid products may be implicated in mediating neuronal damage following ischaemic injury and activation of SREBP1 may exert its pro- neuronal death action by altering one or more of these lipid products
  • SREBPl may regulate the expression of SRE-containing genes that are not directly involved in lipid metabolism, including genes encoding G proteins (Park et al. 2002) and voltage-gated ion channels (Park et al. 2008).
  • SREBP1 contributes to neuronal damage by a mechanism independent of lipid metabolism.
  • excitotoxicity is thought to be a common neuropathology associated with a large number of neurological disorders ranging from acute brain insults such as stroke to chronic neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), the present disclosure may have broad implications for new therapeutics for ' clinical treatment of these neurological disorders. . . .
  • INSIG a broadly conserved transmembrane chaperone for sterol-sensing domain proteins.
  • Step 2 a principal regulator of anaerobic gene expression in fission yeast.

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Abstract

L'invention porte sur des compositions peptidiques de gène induit par l'insuline (INSIG) et sur des procédés de cryoprotection. Les peptides sont issus d'une région du INSIG qui est ubiquitinée au niveau de deux résidus lysine et peut être couplée à une transduction cellulaire ou autre fraction de distribution ou ciblage ou localisation. Les procédés de cytoprotection peuvent être dirigés sur un accident vasculaire cérébral ou une sclérose latérale amyotrophique.
PCT/CA2009/000413 2008-03-31 2009-03-31 Compositions peptidiques de gène induit par l'insuline (insig) et procédés de cytoprotection WO2009121176A1 (fr)

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