WO2014059091A2 - Procédés de modulation de la fonction mitochondriale par l'intermédiaire d'ifn2 - Google Patents

Procédés de modulation de la fonction mitochondriale par l'intermédiaire d'ifn2 Download PDF

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Publication number
WO2014059091A2
WO2014059091A2 PCT/US2013/064255 US2013064255W WO2014059091A2 WO 2014059091 A2 WO2014059091 A2 WO 2014059091A2 US 2013064255 W US2013064255 W US 2013064255W WO 2014059091 A2 WO2014059091 A2 WO 2014059091A2
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inf2
caax
mitochondrial
activity
cells
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PCT/US2013/064255
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WO2014059091A3 (fr
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Henry N. HIGGS
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Trustees Of Dartmouth College
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Publication of WO2014059091A2 publication Critical patent/WO2014059091A2/fr
Publication of WO2014059091A3 publication Critical patent/WO2014059091A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria

Definitions

  • Drpl Dnml in yeast
  • Drpl oligomerization of Drpl into a helical ring around the outer mitochondrial membrane, followed by ring constriction.
  • One issue is that the diameter of the Drpl ring is significantly narrower (100-130 nm for Dnml) than an unconstricted mitochondrion (Mears, et al . (2011) Nature Struct. Mol. Bol . 18:20) , suggesting that prior constriction is necessary.
  • INF2 inverted formin-2, FH2 and H2 domain containing protein
  • INF2 accelerates both actin polymerization and depolymerization (Chhabra & Higgs (2006) J. Biol. Che . 281:26754)
  • INF2 exists as two isoforms differing in C-terminal sequence: the CAAX isoform, which is tightly bound to ER (Chhabra, et al . (2009) J " . Cell Sci.
  • KRPSRNQEEFVPDSDDIKAKRLCIVQ SEQ ID N0:1
  • the non-CAAX isoform which is cytoplasmic (Ramabhadran, et al . (2011) Molecular biology of the cell 22, 4822 (Dec, 2011) and has a C-terminal sequence of KRPSRNQEGLRSRPKAK (SEQ ID NO : 2 ) .
  • Suppression of INF2-nonCAAX in culture cells causes Golgi dispersal.
  • the cellular function of INF2-CAAX is not described in the art, since its suppression has no apparent effect on ER structure or dynamics (Chhabra, et al .
  • the determining step includes real-time imaging of the cell to detect a change in mitochondrial constriction, fission or motility.
  • the eukaryotic cell has been genetically modified with an expression vector that includes a nucleotide sequence encoding an INF2-CAAX polypeptide.
  • An agent identified by the screening method and a method of using such agents in the amelioration of a disease associated with mitochondrial dysfunction are also provided.
  • the present invention includes the use of IFN2 effector molecules to modulate the activity of IFN2-CAAX and to ameliorate a variety of neurodegenerative diseases such as Charcot - Marie-Tooth disease (CMTD) , Alzheimer's, Huntington's, Parkinson's, and amyotrophic lateral sclerosis (ALS) , as well as cardiac disease and cancer, by targeted therapies that either activate or inhibit INF2-CAAX.
  • CMTD Charcot - Marie-Tooth disease
  • ALS amyotrophic lateral sclerosis
  • the invention also includes the use of INF2-CAAX in screening assay to identify effector molecules that alter mitochondrial fission and ameliorate neurodegenerative diseases .
  • any suitable expression vector can be used including, but are not limited to, baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral vectors (e.g., viral vectors based on vaccinia virus, poliovirus, adenovirus, adeno-associated virus, SV40, herpes simplex virus, and the like), Pl-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest. Numerous suitable expression vectors are known to those of skill in the art, and many are commercially available.
  • neuronal cell- specific regulatory elements include those from a neuron- specific enolase (NSE) gene (Hannas-Dj ebarra , et al . (1997) Brain Res. Mol . Brain Res. 46:91-99; a PDGF gene; a Thl gene ⁇ e.g., mouse Thyl .2 (Caroni, et al . (1997) J " . Neurosci.
  • NSE neuron-specific enolase
  • a neurofilament gene e.g., NF- L, NF-M, and NF-L
  • a neurofilament gene e.g., NF- L, NF-M, and NF-L
  • GFAP glial filament acidic protein
  • myelin basic protein gene e.g., a PDGF promoter
  • microtubule-associated protein gene e.g., a synaptophysin gene
  • a tyrosine hydroxylase gene e.g., a suitable neuronal cell-specific regulator region includes, e.g., an NSE promoter; a PDGF promoter; an aromatic amino acid decarboxylase (7AADC) promoter; a neurofilament promoter (see, e.g., GENB/A K Accession No.
  • L0 147) a synapsin promoter (see, e.g., GENBANK Accession No. M55301) ; a thy-1 promoter (see, e.g., Chen, et al . (1987) Cell 51:7-19); a serotonin receptor promoter (see, e.g., GENBANK Accession No. S62283); a tyrosine hydroxylase promoter (TH) ; a GnRH promoter (see, e.g., Radovick, et al . (1991) Proc . Natl. Acad. Sci .
  • a synapsin promoter see, e.g., GENBANK Accession No. M55301
  • a thy-1 promoter see, e.g., Chen, et al . (1987) Cell 51:7-19
  • a serotonin receptor promoter see, e.g., GENBANK Accession No. S
  • NIH 3T3 cells e.g., ATCC No. CRL-1658
  • Huh-7 cells Huh-7 cells
  • BHK cells e.g., ATCC No. CCL10
  • PC12 cells ATCC No. CRL1721
  • COS cells COS-7 cells
  • RATI cells mouse L cells (ATCC No. CCLI.3)
  • HEK human embryonic kidney
  • a human medulloblastoma-derived cell line e.g., D342 Med (ATCC HTB-187), Daoy (ATCC HTB-186), D283 Med (ATCC HTB-185) ; a human tumor-derived neuronal - like cell, e.g., PFSK-1 (ATCC CRL-2060) , SK-N-DZ (ATCC CRL- 2149), SK-N-AS (ATCC CRL-2137), SK-N-FI (ATCC CRL-2142), IMR-32 (ATCC CCL-127) , etc.; a mouse neuronal cell line, e.g., BC3H1 (ATCC CRL-1443), EOCi (ATCC CRL-2467) , C8-D30
  • Agents that have an effect in an assay method of the invention may be further tested for cytotoxicity, bioavailability, and the like, using well-known assays.
  • Agents that have an effect in an assay method of the invention may be subjected to directed or random and/or directed chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • Such structural analogs include those that increase bioavailability, and/or reduced cytotoxicity.
  • Those skilled in the art can readily envision and generate a wide variety of structural analogs, and test them for desired properties such as increased bioavailability and/or reduced cytotoxicity and/or ability to cross the blood- brain barrier.
  • a test agent that INF2-CAAX activity and/or mitochondrial function is a candidate agent for treating a disease associated with dysfunctional mitochondria, including, e.g., a neurodegenerative disease or cancer.
  • a candidate agent identified can be further evaluated, in a secondary screen, for efficacy in vivo, using an appropriate animal model of the disease or condition.
  • the disease is a neurodegenerative disease
  • secondary screens can employ any phenomena associated learning impairment, dementia or cognitive disorders that can be readily assessed in an animal model .
  • the screening can include assessment of phenomena including, but not limited to: 1) assessment behavioral symptoms associated with memory and learning; and 2) detection of neurodegeneration characterized by progressive and irreversible differentiation of the limbic system, association neocortex, and basal forebrain
  • neurodegeneration can be measured by, for example, detection of synaptophysin expression in brain tissue
  • memory and learning deficits can be studied using a 3 runway panel for working memory impairment (attempts to pass through two incorrect panels of the three panel-gates at four choice points) (Ohno, et al . (1997) Pharmacol. Biochem. Behav. 57:257- 261) .
  • oligonucleotides of the siRNA molecule include 5 ' -ACA AAG AAA CTG TGT GTG A-3' (SEQ ID NO:5) or 5'-CCC TGA TTC TGA TGA TAA T-3' (SEQ ID NO:6), which inhibit the expression of INF2-CAAX thereby inhibiting INF2-CAAX activity and increasing mitochondrial length.
  • the agent is a constitutively activate INF2-CAAX protein, e.g., harboring a A149D mutation, which decreases mitochondrial length and increased ER/mitochondrial association.
  • the agents can be formulated into preparations for injection by dissolving, suspending or emulsifying them 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 such as solubilizers , isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of this invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or yehicle.
  • the specifications for the novel unit dosage forms of the invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host .
  • compositions such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • 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 .
  • An agent of this invention can be 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.
  • patient are treatable according to the subject methods.
  • hosts are “mammals” or “mammalian,” where these terms are used broadly to describe organisms that are within the class Mammalia, including the orders Carnivore
  • Oligonucleotides for human total INF2 siRNA were synthesized by IDT Oligo against target sequence 5'-GGA UCA ACC UGG AGA UCA UCC GC- 3 ' (siRNA#l; SEQ ID NO : 3 ) , and 5 ' - GCA GUA CCG CUU CAG CAU UGU CA- 3 ' (siRNA#2 ; SEQ ID NO: 4).
  • Live Imaging and Confocal Microscopy Imaging of live and fixed cells was performed using spinning disk or laser scanning confocal systems. For live imaging, cells grown on 18 mm coverslips were mounted into Rose chambers, then onto a Wave FX spinning disk confocal microscope (Quorum Technologies, Inc., Guelph, Canada, on a NIKON ECLIPSE Ti microscope) with Bionomic Controller (20/20 Technology, Inc.) temperature-controlled stage set to 37°C.
  • Proteins were separated by 7.5% SDS-PAGE and transferred to a PVDF membrane (polyvinylidine difluoride membrane, Millipore) .
  • the membrane was blocked with TBS-T (20 mM Tris-HCl, pH 7.6, 136 mM NaCl, and 0.1% TWEEN-20) containing 3% BSA
  • Additional molecules might be required to convert actin polymerization force into mitochondrial membrane deformation, including: actin bundling molecules, actin filament pointed (minus) end binding molecules on the mitochondrion, and molecules mediating ER/mitochondria interaction.
  • actin bundling molecules actin filament pointed (minus) end binding molecules on the mitochondrion
  • molecules mediating ER/mitochondria interaction This model supports findings suggesting that Drpl oligomeric rings are narrower than unconstricted mitochondria (Mears, et al . (2011) Nature Struct. Mol. Biol. 18:20), and that mitochondria can constrict in a Drpl - independent manner (Friedman, et al . (2011) supra; Labrousse, et al . (1999) Mol. Cell 4:815; Legesse-Miller, et al . (2003) Mol. Biol. Cell 14:1953).

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

L'invention concerne des dosages de criblage pour l'identification d'agents qui modulent la protéine contenant le domaine formine 2, FH2 et WH2 inversé, l'activité d'isoforme 2 (INF2-CAAX) ou la fonction mitochondriale à médiation par INF2-CAAX, et l'utilisation de tels agents dans l'amélioration d'une maladie associée au dysfonctionnement mitochondrial.
PCT/US2013/064255 2012-10-12 2013-10-10 Procédés de modulation de la fonction mitochondriale par l'intermédiaire d'ifn2 WO2014059091A2 (fr)

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US201261712951P 2012-10-12 2012-10-12
US61/712,951 2012-10-12

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WO2014059091A2 true WO2014059091A2 (fr) 2014-04-17
WO2014059091A3 WO2014059091A3 (fr) 2015-08-20

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WO2009114700A2 (fr) * 2008-03-12 2009-09-17 The Trustees Of Columbia University In The City Of New York Modèle cellulaire de maladie neurodégénérative et procédés d’utilisation de celui-ci pour identifier des inhibiteurs de fusion mitochondriale

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