WO2013102264A1 - Compositions et méthodes de modulation de l'excitoxicité médiée par les récepteurs ampa - Google Patents

Compositions et méthodes de modulation de l'excitoxicité médiée par les récepteurs ampa Download PDF

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Publication number
WO2013102264A1
WO2013102264A1 PCT/CA2013/000001 CA2013000001W WO2013102264A1 WO 2013102264 A1 WO2013102264 A1 WO 2013102264A1 CA 2013000001 W CA2013000001 W CA 2013000001W WO 2013102264 A1 WO2013102264 A1 WO 2013102264A1
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glur2
gapdh
polypeptide
protein
seq
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PCT/CA2013/000001
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English (en)
Inventor
Fang Liu
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Centre For Addiction And Mental Health
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Priority claimed from US13/343,555 external-priority patent/US8536115B2/en
Application filed by Centre For Addiction And Mental Health filed Critical Centre For Addiction And Mental Health
Publication of WO2013102264A1 publication Critical patent/WO2013102264A1/fr

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    • 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
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/61—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
    • 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
    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • the "calcium overload” hypothesis is the prominent theory explaining excitotoxicity (4).
  • the molecular mechanisms underlying NMDA-mediated excitotoxicity involve many Ca2+-regulated processes in the cell including activation of proteases (104), endonucleases (105), nitric oxide synthase (106), the production of free radicals (107) and mitochondrial membrane permeability (108).
  • the "calcium theory” can also apply to the Ca2+ permeable AMPA receptor-induced toxicity, however, there must be another explanation for the Ca2+-inpermeable AMPA receptor induced toxicity.
  • Ca2+-impermeable AMPA receptor induced toxicity is to induce membrane depolarization via Na+ influx.
  • GluR2-interacting proteins in excitotoxicity may be that the presence of GluR2 is required to maintain synaptic structure and organization. Accordingly, the toxicity observed in GluR2-deficient neurons may result from the effects on synaptic organization and function rather than due to AMPA receptor Ca2+ permeability.
  • An interesting candidate protein is the NSF, as it has been shown both to interact with GluR2 and to mediate membrane-fusion events (1 13-1 15). Interestingly, NSF expression increases following an ischemic insult (1 16). It is not yet clear whether an increase in NSF leads to an increase of surface expression of existing GluR2- containing AMPA receptors following ischemia.
  • polypeptides that not identical to any GluR2 subunit that occurs in nature.
  • GAPDH was specifically pulled down by GST- GluR2 N Ti (G) GST-G1UR2NTI-3 (H) and GST- GluR2 NT1 -3-2 (I) but not by the other GST fusion proteins or by GST alone.
  • J-L Using an in vitro binding assay, [ 35 S]- GAPDH probe bound with specific GST-GluR2 NT i (J) , GST-GluR2 NT i-3 ( ) and GST- GluR2 NT 1-3-2 (L) fragments, but not with other GST fusion proteins or GST alone.
  • FIGURE 21 shows results validating agonist regulation of the extracellular GAPDH: A PAR complex formation.
  • A Co-immunoprecipitation of GAPDH with the GluR2 subunit from solubilized rat hippocampus.
  • B-C Activation of AMPAR (HEK-293T: 100 ⁇ glutamate, 30 min; neurons: 100 ⁇ kainic acid [KA], 30 min), enhanced the association of GAPDH and GluR2 subunit, which was blocked by preincubation with the GluR2 NT1-3-2 peptide in both HEK-293T cells expressing GluRl/2 subunits (B) and primary cultures of rat hippocampal neurons (C) but did not affect directly immunoprecipitated GluR2 levels (B, C, bottom panels).
  • CM conditioned medium
  • Rabbit IgG was used as negative control.
  • E Conditioned media of nontransfected HEK-293T cells and HEK-293T cells transfected with GluRl/2 subunits, in the presence or absence of glutamate (Glut), was concentrated to examine the expression of GAPDH and a-tubulin. GAPDH was present in conditioned media and cell lysates, while a-tubulin was only present in cell lysates.
  • hybridization to filter- bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHP0 4 , 7% SDS, 1 mM EDTA at 65°C, and washing in 0.1 x SSC/0.1% SDS at 68° C for at least 1 hour (see Ausubel, et al. (eds), 1989, supra).
  • Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology ⁇ Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York).
  • stringent conditions are selected to be about 5°C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
  • a polypeptide of the invention can be synthesized in vitro or delivered to a cell in vivo by any conventional method.
  • the polypeptide may be chemically synthesized in vitro, or may be enzymatically synthesized in vitro in a suitable biological expression system.
  • a DNA, RNA, or DNA/RNA hybrid molecule comprising a nucleotide sequence encoding a polypeptide of the invention is introduced into an animal, and the nucleotide sequence is expressed within a cell of an animal.
  • the invention also provides a method of inhibiting GluR2 subunit association with GAPDH comprising: administering a nucleic acid capable of expressing a polypeptide comprising the GluR2 NT 1-3-2 (Y142-K172) amino acid sequence (SEQ ID NO: l), or SEQ ID NO:6 to a cell, cell culture, tissue or subject comprising GluR2 subunit and GAPDH.
  • a nucleic acid capable of expressing a polypeptide comprising the GluR2 NT 1-3-2 (Y142-K172) amino acid sequence (SEQ ID NO: l), or SEQ ID NO:6 to a cell, cell culture, tissue or subject comprising GluR2 subunit and GAPDH.
  • the method may be practiced in a human subject.
  • the human subject may have or be susceptible to stroke, epilepsy or other forms of brain injury, for example, but not limited to traumatic brain injury or injury from cardiac bypass surgery.
  • HA-GluR2 after pre-treatment with glutamate was presented as the ratio of colorimetric readings under non-permeabilized conditions to those under permeabilized conditions, and then normalized to their respective control groups (pretreated with ECS). Afterwards, cells were scrapped from the dishes, and the protein concentration of each dish was measured. The results of cell surface expression of receptors or proteins were calibrated by the protein concentration of each well. Analysis was done using at least 9 separate wells in each group. Cell EL1SA using primary hippocampal neurons was performed identically with assays using HEK-293T cells, with the exception that the anti-GluR2 antibody (MAB397; Chemicon) was used as primary antibody instead of anti-HA.
  • MAB397 anti-GluR2 antibody
  • EXAMPLE 5 Functional characterization of the GAPDH and GluR2 interaction - Modulation of GluR2 cell surface expression through the GAPDH and GluR2 interaction
  • GluR2 -mediated GAPDH nuclear translocation is responsible for GluR2- containing AMPAR-mediated cell death, which facilitates the interaction between GAPDH and p53 and activates p53 -dependent apoptosis pathway.
  • Cresyl violet was used to stain alive neurons in hippocampus region of each animal. Cresyl violet-stained nuclei wereobserved by microscope and total number of stained nuclei in CA1 region was summarized and normalized to the sham-operated group.
  • GRIP a synaptic PDZ domain-containing protein that interacts with AMPA receptors. Nature 386, 279-284.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
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  • Animal Behavior & Ethology (AREA)
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  • Genetics & Genomics (AREA)
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  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des polypeptides médiant l'excitoxicité AMPAR, comprenant la séquence d'acides aminés (SEQ ID N° 1) de la GluR2 NT 1-3-2 (Y142-K172), un fragment de celle-ci (SEQ ID N° 6) ou la séquence d'acides aminés (SEQ ID N° 2) de la GAPDH (2-2-1-1) (I221-E250). L'invention concerne également des séquences nucléotidiques codant pour lesdits polypeptides, des méthodes d'inhibition de l'association de la GAPDH à la sous-unité GluR2 ou p53. L'invention concerne également des méthodes d'inhibition d'excitoxicité médiée par les récepteurs AMPA à l'aide desdits polypeptides et acides nucléiques.
PCT/CA2013/000001 2012-01-04 2013-01-03 Compositions et méthodes de modulation de l'excitoxicité médiée par les récepteurs ampa WO2013102264A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13/343,555 2012-01-04
US13/343,555 US8536115B2 (en) 2006-08-31 2012-01-04 Compositions and methods for modulating AMPA receptor-mediated excitotoxicity

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WO2013102264A1 true WO2013102264A1 (fr) 2013-07-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2800763A4 (fr) * 2012-01-05 2015-11-11 Camh Compositions et méthodes de traitement de la sclérose en plaques

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6312945B1 (en) * 1992-05-19 2001-11-06 Eli Lilly And Company Human glutamate receptor proteins and associated DNA compounds
WO2004022705A2 (fr) * 2002-09-04 2004-03-18 Incyte Corporation Proteines associees a la neurotransmission
WO2008025163A1 (fr) * 2006-08-31 2008-03-06 Centre For Addiction And Mental Health Compositions et procédés pour la modulation de l'excitotoxicité induite par le récepteur ampa
US7820398B2 (en) * 2003-11-06 2010-10-26 Grace Laboratories Inc. Immunosorbent blood tests for assessing paroxysmal cerebral discharges

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6312945B1 (en) * 1992-05-19 2001-11-06 Eli Lilly And Company Human glutamate receptor proteins and associated DNA compounds
WO2004022705A2 (fr) * 2002-09-04 2004-03-18 Incyte Corporation Proteines associees a la neurotransmission
US7820398B2 (en) * 2003-11-06 2010-10-26 Grace Laboratories Inc. Immunosorbent blood tests for assessing paroxysmal cerebral discharges
WO2008025163A1 (fr) * 2006-08-31 2008-03-06 Centre For Addiction And Mental Health Compositions et procédés pour la modulation de l'excitotoxicité induite par le récepteur ampa

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2800763A4 (fr) * 2012-01-05 2015-11-11 Camh Compositions et méthodes de traitement de la sclérose en plaques
US9518104B2 (en) 2012-01-05 2016-12-13 Centre For Addiction And Mental Health Compositions and methods for treating multiple sclerosis

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