WO2006079792A1 - Procedes d'induction d'autophagie dans des cellules mammaliennes - Google Patents

Procedes d'induction d'autophagie dans des cellules mammaliennes Download PDF

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WO2006079792A1
WO2006079792A1 PCT/GB2006/000224 GB2006000224W WO2006079792A1 WO 2006079792 A1 WO2006079792 A1 WO 2006079792A1 GB 2006000224 W GB2006000224 W GB 2006000224W WO 2006079792 A1 WO2006079792 A1 WO 2006079792A1
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autophagy
impase
polypeptide
compound
cells
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David Rubinsztein
Sovan Sarkar
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Cambridge University Technical Services Ltd CUTS
Cambridge Enterprise Ltd
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Cambridge Enterprise Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • 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
    • G01N33/502—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 for testing non-proliferative effects
    • G01N33/5041—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 for testing non-proliferative effects involving analysis of members of signalling pathways

Definitions

  • This invention relates to the induction of autophagy, and in particular to autophagy inducing compounds which may be useful in the treatment of disease .
  • the ubiquitin-proteasome and autophagy-lysosomal pathways are the two major routes for protein and organelle clearance in eukaryotic cells .
  • Proteasomes predominantly degrade short-lived nuclear and cytosolic proteins .
  • the bulk degradation of cytoplasmic proteins or organelles is largely mediated by macroautophagy, generally referred to as autophagy 9 . It involves the formation of double membrane structures called autophagosomes , which fuse with lysosomes to form autolysosomes .
  • Autophagy substrates generally have long half-lives 9 .
  • Autophagy can also help cells clear the toxic , long-lived, aggregate-prone proteins causing many neurodegenerative disorders , like Huntington' s disease and forms of Parkinson' s disease 3 ' 4 . Induction of autophagy reduces the levels of mutant huntingtin and protected against its toxicity in cells and in transgenic Drosophila and mouse models 6 .
  • the only suitable pharmacological strategy for up regulating autophagy in mammals .. is to use rapamycin, or its analogues , that inhibit the mammalian target of rapamycin (mTOR) , a negative regulator of autophagy .
  • the present inventors have identified a novel pathway for the regulation of autophagy which involves inositol monophosphatase (IMPase) and levels of free inositol (IP 3 ) . Inhibition of IMPase and reductions in the level of free IP 3 are found to induce autophagy in cells , providing a new pharmacological strategy for modulating autophagy in mammals .
  • IMPase inositol monophosphatase
  • IP 3 free inositol
  • One aspect of the invention provides a method of screening for an autophagy inducer comprising; contacting an inositol monophosphatase (IMPase) polypeptide with a test compound; and, determining the interaction of the inositol monophosphatase polypeptide and the test compound .
  • IMPase inositol monophosphatase
  • test compound which interacts with the polypeptide is a candidate autophagy inducer .
  • Interaction may include the binding of the compound to the polypeptide and/or the activity of the polypeptide in the presence of the compound .
  • An aspect of the invention provides a method of screening for an autophagy inducer comprising; contacting an inositol monophosphatase (IMPase) polypeptide with a test compound; and, determining the activity of the IMPase polypeptide .
  • IMPase inositol monophosphatase
  • a decrease in monophosphatase activity in the presence of the compound relative to its absence may indicative that the compound is an autophagy inducer .
  • An IMPase polypeptide may comprise or consist of the sequence of a eukaryotic IMPase , in particular a mammalian IMPase .
  • a IMPase polypeptide may be a myo-inositol monophosphatase 1 (IMPAl) polypeptide comprising or consisting of the amino acid sequence of NP_005527.1 (GI : 5031789) or a myoinositol monophosphatase 2 (IMPA2) polypeptide comprising or consisting of the amino acid sequence of NP_055029.1 (GI : 7657236) or may be a fragment or variant of either of these sequences .
  • IMPAl myo-inositol monophosphatase 1
  • IMPA2 myoinositol monophosphatase 2
  • An IMPAl polypeptide may be encoded by a nucleotide sequence comprising or consisting of the nucleic acid sequence of NM_005536.2 (GI : 8393607) .
  • An IMPA2 polypeptide may be encoded by a nucleotide sequence comprising or consisting of the nucleic acid sequence of NM_014214.1 (GI : 7657235) .
  • a fragment or variant of a wild-type IMPase sequence as described herein may differ from the wild-type sequence by the addition, deletion, substitution and/or insertion of one or more amino acids , provided the function of hydrolysing inositol monophosphate to produce inositol is retained .
  • a polypeptide which is a variant of a wild-type IMPase sequence may comprise an amino acid sequence which shares greater than about 30% sequence identity with the wild-type IMPase sequence, greater than about 40% , greater than about 45% , greater than about 55% , greater than about 65% , greater than about 70% , greater than about 80% , greater than about 90% or greater than about 95% .
  • the sequence may share greater than about 30% similarity with the wild-type IMPase sequence, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity .
  • GAP Genetics Computer Group, Madison, WI
  • GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps .
  • Use of GAP may be preferred but other algorithms may be used, e . g . BLAST (which uses the method of
  • Similarity allows for "conservative variation” , i . e . substitution of one hydrophobic residue such as isoleucine , valine , leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine .
  • Determining the activity of a polypeptide may include detecting the presence of activity, detecting the presence of activity above a threshold value and/or measuring the level of activity.
  • polypeptide fragments which retain all or part of the IMPase activity of the full-length protein may be generated and used in the methods described herein, whether in vitro or in vivo.
  • Suitable ways of generating fragments include , but are not limited to, recombinant expression of a fragment from encoding DNA.
  • fragments may be generated by taking encoding DNA, identifying suitable restriction enzyme recognition sites either side of the portion to be expressed, and cutting out said portion from the DNA. The portion may then be operably linked to a suitable promoter in a standard commercially available expression system.
  • Another recombinant approach is to amplify the relevant portion of the DNA with suitable PCR primers . Small fragments (e . g .
  • a fragment of a full-length sequence may consist of fewer amino acids than the full-length sequence .
  • a fragment may- consist of at least 10 , at least 20 , at least 30 , at least 40 , at least 50 , at least 60 , at least 70 , at least 80 , at least 90 or at least 100 amino acids of the full length sequence but 800 or less , 700 or less , 600 or less , 500 or less , 250 or less , 200 or less , 150 or less , or 125 or less amino acids of the full length sequence .
  • Methods described herein may be in vivo cell-based methods , or in vitro non-cell-based methods .
  • the precise format for performing methods of the invention may be varied by those of skill in the art using routine skill and knowledge .
  • IMPase activity may be determined using standard biochemical techniques .
  • IMPase activity can be measured spectrophotometrically as inorganic phosphate liberated from inositol-1-phosphate (Shaltiel G et al World J Biol Psychiatry . 2001 Apr; 2 (2 ) : 95-8. )
  • IP 3 inositol-1 , 4 , 5-triphosphate
  • Another aspect of the invention provides a method of screening for an autophagy inducer comprising; contacting a cell with a test compound; and determining the level of IP 3 in the cell .
  • a decrease in the level of IP 3 in the presence of the compound is indicative that the compound is an autophagy inducer .
  • Suitable cells for use in the present methods include mammalian cells , preferably human cells .
  • the cell is an isolated and/or cultured cell .
  • Any suitable cultured mammalian cell may be used, for example Chinese hamster ovary cells , baby hamster kidney cells , COS cells, PC12 and many others .
  • the level of IP 3 may be determined by using conventional biochemical techniques .
  • levels of inositol-1 , 4 , 5- trisphosphate [IP 3 (1 , 4 , 5) ] can be measured in perchloric acid extracted samples (10 7 cells/aliquot) using the [ 3 H] Biotrak Assay System (Amersham Biosciences , UK) according to manufacturer' s instruction .
  • a method may further comprise; contacting the test compound with a mammalian cell; and, determining the autophagy activity of said cell .
  • An increase in autophagy activity in the presence of the compound is indicative that the compound is a candidate agent for use in the treatment of a protein conformational disorder .
  • Autophagy activity may be determined by any convenient method, for example monodansylcadaverine (MDC) staining (Ravikumar et al Hu . MoI . Gen . (2003 ) 12 9 1-10) , LC3 processing (Y . Kabeya et al EMBO J . 19 5720-5728 ) , or electron microscopy visulating autophagosome numbers .
  • MDC monodansylcadaverine
  • LC3 processing Y . Kabeya et al EMBO J . 19 5720-5728
  • Autophagy activity may be determined in the presence of proteosome inhibitors , such as epoximicin.
  • the ability of said compound to increase the clearance of cytoplasmic protein aggregates may be determined.
  • a cell for use in determining autophagy activity may comprise a heterologous nucleic acid encoding an aggregation-prone polypeptide , for example A53T or A3 OP mutant forms of ⁇ - synuclein, huntingtin or GFP-tagged with expanded polyalanine repeats .
  • An aggregation-prone polypeptide may comprise an aggregation-inducing mutation, for example a codon iteration mutation such as a polyQ or a polyA insertion, or may have the non-mutant , wild-type sequence . Clearance of the encoded aggregation-prone polypeptide , either in an aggregated or a soluble monomeric form, may be determined.
  • heterologous nucleic acid may be reversible i . e . expression may be induced and repressed as required, for example by adding or removing an inducer compound.
  • a method may comprise inducing and repressing the expression of said nucleic acid prior to contacting the mammalian cell with the test compound .
  • inducible and/or reversible expression systems and constructs are known in the art , including, for example the Tet- onTM expression (Clontech) , in particular in combination with the pTet-tTsTM vector (Clontech) .
  • An autophagy inducer identified by a method described herein may be useful in the treatment of a protein conformational disorder, in particular a neurodegenerative protein conformational disorder, or a bacterial infection, such as tuberculosis or streptococcus infection .
  • Protein conformational disorders which may be treated include codon reiteration mutation disorders , in particular polyQ expansion disorders such as Huntington' s disease, spinocerebellar ataxias types 1 , 2 , 3 , 6 , 7 , and 17 , Kennedy' s disease and dentatorubral-pallidoluysian atrophy. These disorders are characterised by the aggregation of mutant proteins that contain an expanded tract of repeated glutamine residues . For example , HD is characterised by an expanded polyQ stretch in exon 1 of the Huntington gene . Protein conformational disorders also include polyA expansion disorders . These disorders are characterised by the aggregation of mutant proteins which contain an expanded tract of repeated alanine residues .
  • codon reiteration mutation disorders in particular polyQ expansion disorders such as Huntington' s disease, spinocerebellar ataxias types 1 , 2 , 3 , 6 , 7 , and 17 , Kennedy' s disease and dentatorubral-pallid
  • oculapharyngeal muscular dystrophy is characterised by a polyadenine (polyA) expansion mutation in the polyadenine binding protein 2 gene .
  • Protein conformational disorders also include ⁇ -synucleiopathies such as Parkinson' s disease, LB variant Alzheimer' s disease and LB dementia . These are disorders characterised by the accumulation of cytoplasmic aggregates called Lewy bodies , which comprise ⁇ -synuclein.
  • Protein conformational disorders also include tauopathies such as Alzheimer' s disease, which are disorders characterised by the accumulation of cytosolic aggregates of tau protein within neurons .
  • Protein conformational disorders also include prion disorders such as Creutzfeldt-Jakob disease (CJD) , Kuru, Gerstmann-Straussler-Scheiker syndrome .
  • CJD may include sporadic, familial , Iatrogenic and variant CJD .
  • Test compounds may be natural or synthetic chemical compounds used in drug screening programmes . Extracts of plants which contain several characterised or uncharacterised components may also be used .
  • Combinatorial library technology (Schultz, JS ( 1996) Biotechnol . Prog . 12 : 729-743 ) provides an efficient way of testing a potentially vast number of different substances for ability to modulate activity of a polypeptide .
  • Suitable test compounds include analogues and derivatives of IMPase inhibitors set out below .
  • test substance or compound that may be added to an assay will normally be determined by trial and error depending upon the type of compound used. Typically, from about 0.1 to 100 ⁇ M concentrations of putative inhibitor compound may be used, for example from 1 to 10 ⁇ M .
  • the test substance or compound is desirably membrane permeable in order to access intracellular targets .
  • the compound may be identified as an autophagy inducer using the methods described herein .
  • a method may further comprise modifying the compound to optimise the pharmaceutical properties thereof .
  • the modification of a ⁇ lead' compound identified as biologically active is a known approach to the development of pharmaceuticals and may be used to avoid randomly screening large number of molecules for a target property .
  • the optimisation commonly comprises several steps . Firstly, the particular parts of the compound that are critical and/or important in determining the target property are determined . These parts or residues constituting the active region of the compound are known as its "pharmacophore" .
  • the pharmacophore Once the pharmacophore has been found, its structure is modelled to according its physical properties , e .g . stereochemistry, bonding, size and/or charge, using data from a range of sources , e . g . spectroscopic techniques , X-ray diffraction data and NMR .
  • Computational analysis e.g . similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms) and other techniques can be used in this modelling process .
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted .
  • the template molecule and the chemical groups grafted on to it can conveniently be selected so that the modified compound is easy to synthesise , is likely to be pharmacologically acceptable , and does not degrade in vivo, while retaining the autophagy inducing activity of the lead compound .
  • the modified compounds found by this approach can then be screened to see whether they inhibit IMPase, reduce IP 3 levels and/or stimulate autophagy, or to what extent they exhibit one or more of these activities .
  • a method may comprise modifying a IMPase inhibitor, for example an IMPase inhibitor described below, or a IMPase substrate (such as IMP) or product (such as inositol or phosphate) to produce an analogue or derivative , and determining the ability of said derivative to inhibit autophagy .
  • a IMPase inhibitor for example an IMPase inhibitor described below
  • a IMPase substrate such as IMP
  • product such as inositol or phosphate
  • test compound may be manufactured and/or used in preparation, i . e . manufacture or formulation, of a composition such as a medicament , pharmaceutical composition or drug .
  • a method may comprise formulating the test compound into a pharmaceutical composition with a pharmaceutically acceptable excipient , vehicle or carrier as discussed further below.
  • Another aspect of the invention provides the use of an IMPase inhibitor in the manufacture of a medicament for increasing autophagy in an individual
  • the medicament may be useful in the treatment of a neurodegenerative protein conformational disorder, or a bacterial infection, such as tuberculosis or streptococcus infection.
  • IMPase inhibitors are well known in the art and include L- 690330 , L-690488 (Atack JR et al J Pharmacol Exp Ther . 1994 JuI ; 270 (1) : 70 -6) , lithium, valproate and carbemazapine .
  • Other examples of suitable IMPase inhibitors are described in Fauroux CM, Freeman S . J Enzyme Inhib . 1999 / 14 (2) : 97-108 , Miller DJ et al . Org Biomol Chem. 2004 Mar 7 ,- 2 (5) : 671-88. and Atack JR et al J Pharmacol Exp Ther . 1994 Jul ; 270 (1) : 70-6.
  • IMPase inhibitors may include inositol-1- monophosphate analogues or variants , for example with one or more cyclohexane hydroxyl groups deleted or substituted (Fauroux CM, Freeman S . J Enzyme Inhib . 1999 / 14 (2 ) : 97-108)
  • IMPase inhibitors may also include phosphonates such as ( - ) - (Ii?, 222, 422, 6i?) -2 , 4 , 6-trihydroxycyclohexyl cyclohexylammonium 1- methylphosphonate; ( - ) - (IR, 2R 1 AR, 6R) -6-Propyloxy-2 , 4- dihydroxycyclohexyl cyclohexylammonium 1-methylphosphonate ; ( - ) - (Ii?, 2R 1 AR, 6R) -6-Propyloxy-2 , 4 -dihydroxycyclohexyl cyclohexylammonium 1-ethylphosphonate ; ( - ) - (1S, 2R, AS, 6R) -6- Methylamino-2 , 4-dihydroxycyclohexyl cyclohexylammonium 1- methylphosphonate; ( -) - (IS, 2
  • IMPase inhibitors may also include inositol analogues such as ( ⁇ ) - (IS, 2R, AS, 6R) -6- (2-phenylethyl) aminocyclohexane-1, 2 , 4-triol ; ( - ) - ( 1S, 2R, AS, 6R) - ⁇ -Hexylaminocyclohexane-l ⁇ , 4-triol ; ( - ) - ( IS, 2R 1 AR 1 6R) -6-Propyloxycyc1ohexane-1 , 2 , 4-triol ; ( - ) - (IS, 2R, AS, 6R) -6-Hexyloxycyclohexane-l , 2 , 4-triol ; ( - ) - (IS, 2R 1 AS, 6R) ⁇ 6- [4- (2 -Hydroxyphenyloxy) butyloxy] cyclohexan
  • IMPase inhibitors may also include bisphosphonates , such as 1- hydroxyethylidene-1, 1 bisphosphonic acid and hydroxymethylene bisphosphonic acid, terpenoids such as sesquiterpene L-671776 from Memnoniella echinata and puberulonic acid from Penicillum spp and tropolones , in particular hydroxyl substituted tropolones such as 7-hydroxytropolone and 3 , 7- dihydroxytropolone and analogues , derivatives and salts thereof .
  • bisphosphonates such as 1- hydroxyethylidene-1, 1 bisphosphonic acid and hydroxymethylene bisphosphonic acid
  • terpenoids such as sesquiterpene L-671776 from Memnoniella echinata and puberulonic acid from Penicillum spp
  • tropolones in particular hydroxyl substituted tropolones such as 7-hydroxytropolone and 3 , 7- dihydroxytropol
  • IMPase inhibitors may be analogues or variants of L- 690330 , L-690488 , lithium, valproate and carbemazapine .
  • IMPase inhibitors other than lithium may be employed .
  • aspects of the invention provide a combination of an mTOR inhibitor and an IMPase inhibitor for use in increasing autophagy in an individual , a pharmaceutical composition comprising an mTOR inhibitor, an IMPase inhibitor and a pharmaceutically acceptable excipient and the use of an mTOR inhibitor and an IMPase inhibitor in the manufacture of a medicament for increasing autophagy in an individual .
  • An mTor inhibitor may include a rapamycin macrolide such as rapamycin or a salt , analogue or derivatives of rapamycin. Suitable rapamycin macrolides are described in more detail below.
  • An IMPase inhibitor may include a compound described above .
  • an mTOR inhibitor and an IMPase inhibitor may be useful in the treatment of a neurodegenerative disorder, or a bacterial infection, such as tuberculosis , or streptococcus infection, as described herein.
  • compositions are described in more detail below. Lithium and other IMPase inhibitors are commonly used in the treatment of mood disorders . The data presented herein indicates that other compounds which modulate autophagy may also be useful in the treatment of mood disorders .
  • Another aspect of the invention provides a method of screening for a compound useful in the treatment of a mood disorder comprising; contacting the test compound with a mammalian cell ; and, determining the autophagy activity of said cell .
  • a change in autophagy activity in the presence of the compound is indicative that the compound is a candidate agent for use in the treatment of a mood disorder .
  • Autophagy activity may be determined by measuring the ability of said compound to increase the clearance of cytoplasmic protein aggregates .
  • Aggregate clearance may be determined for example , in the presence of proteosome inhibitors such as epoximicin. The determination of autophagy activity and the clearance of cytoplasmic protein aggregates are described in more detail above .
  • Suitable cells and test compounds are described in more detail above .
  • Mood disorders include depression, bipolar disorder (manic- depression) , cyclothymia and dysthymia .
  • mTOR inhibitors such as rapamycin are known to stimulate autophagy and may be particularly useful in the treatment of disorders previously treated with IMPase inhibitors , such as mood disorders .
  • Another aspect of the invention provides the use of an mTOR inhibitor in the manufacture of a medicament for use in the treatment of a mood disorder .
  • mTOR inhibitors include rapamycin and other rapamycin macrolides .
  • a macrolide is a macrocyclic lactone, for example a compound having a 12-membered or larger lactone ring .
  • Lactam macrolides are macrocyclic compounds which have a lactam (amide) bond in the macrocycle in addition to a lactone (ester) bond.
  • Rapamycin is a lactam macrolide produced by Streptomyces hygroscopicus (McAlpine J . B . et al . J.Antibiotics ( 1991) 44 : 688 ; Schreiber, S . L . et al . J . Am. Chem. Soc . ( 1991) 113 : 7433 ; US3 , 929 , 992 ) .
  • a rapamycin macrolide as described herein may include rapamycin or a salt , analogue or derivative of rapamycin .
  • Suitable rapamycin analogues well known in the art (see for example WO 94/09010 and WO 96/41807) and include 40-O- (2 - hydroxy) ethyl-rapamycin, 32 -deoxo-rapamycin, 16-O-pent-2 -ynyl- 32 -deoxo-rapamycin, 16-0-pent-2 -ynyl-32 -deoxo-40-0- (2- hydroxyethyl) -rapamycin, 16-O ⁇ pent-2-ynyl-32- (S) -dihydro- rapamycin and 16-O-pent-2-ynyl-32- (S) -dihydro-40-O- (2 - hydroxyethyl) -rapamycin .
  • rapamycin analogues include carboxylic acid esters as set out in WO 92/05179 , amide esters as set out in US5 , 118 , 677 , carbamates as set out in US5 , 118 , 678 , fluorinated esters as set out in US5 , 100 , 883 , acetals as set out in US5 , 151 , 413 , silyl ethers as set out in US5 , 120 , 842 and arylsulfonates and sulfamates as set out in US5 , 177 , 203.
  • rapamycin analogues which may be used in accordance with the invention may have the methoxy group at the position 16 replaced with alkynyloxy as set out in WO 95/16691. Rapamycin analogues are also disclosed in WO 93/11130 , WO 94/02136 , WO 94/02385 and WO 95/14023.
  • Administration of a compound for the treatment of a disorder, as described herein, is preferably in a "prophylactically effective amount " or a "therapeutically effective amount" (as the case may be , although prophylaxis may be considered therapy) , this being sufficient to show benefit to the individual .
  • the actual amount administered, and rate and time-course of administration will depend on the nature and severity of what is being treated. Prescription of treatment , e . g . decisions on dosage etc , is within the responsibility of general practitioners and other medical doctors .
  • a composition may be administered alone or in combination with other treatments , either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions may include , in addition to active ingredient , a pharmaceutically acceptable excipient , carrier, buffer, stabiliser or other materials well known to those skilled in the art . Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient .
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral , or by inj ection, e . g . cutaneous , subcutaneous or intravenous .
  • compositions for oral administration may be in tablet , capsule , powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant .
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils , mineral oil or synthetic oil .
  • Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol , propylene glycol or polyethylene glycol may be included.
  • the active ingredient For intravenous , cutaneous or subcutaneous inj ection, or inj ection at the site of affliction, the active ingredient will IS
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • Those of relevant skill in the art are well able to prepare suitable solutions using, for example , isotonic vehicles such as Sodium Chloride Inj ection, Ringer ' s Inj ection, or Lactated Ringer ' s Inj ection.
  • Preservatives , stabilisers , buffers , antioxidants and/or other additives may be included, as required .
  • the invention encompasses each and every combination and sub- combination of the features that are described above .
  • Figure 1 shows the activity of lithium in reducing mutant huntingtin aggregates in COS-7 cells transfected with pEGFP- HDQ74 and treated with or without 1OmM LiCl or 1OmM NaCl for 48h .
  • the effects of treatment on the percentage of EGFP-HDQ74- positive cells with aggregates or apoptotic morphology (cell death) are expressed as odds ratios 10 .
  • Figure 2 shows the activity of lithium in reducing mutant huntingtin aggregates in SK-N-SH cells transfected with pEGPP- HDQ74 and treated with or without 1OmM LiCl or 1OmM NaCl for 48h.
  • the effects of treatment on the percentage of EGFP-HDQ74- positive cells with aggregates or apoptotic morphology (cell death) are expressed as odds ratios 10 .
  • Figure 3 shows mutant huntingtin fragment (EGFP-HDQ74) clearance by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 12Oh in the presence (+) or absence ( - ) of 1OmM LiCl .
  • Figure 4 shows the percentage of GFP-positive cells with aggregates in stable PC12 cells expressing EGFP-HDQ74 after switching on EGFP74 expression then switching off for 12Oh in the presence (+) or absence ( - ) of 1OmM LiCl . Data are expressed as odds ratio compared to control condition (120h off) .
  • Figure 5 shows A53T ⁇ -synuclein mutant clearance in stable inducible PC12 cell lines by showing the amount of ⁇ -synuclein mutant remaining after switching on expression then switching off for 24h in the presence (+) or absence ( - ) of 1OmM LiCl or 1OmM NaCl .
  • Figure , 6 shows A3 OP ⁇ -synuclein mutant clearance in stable inducible PC12 cell lines by showing the amount of ⁇ -synuclein mutant remaining after switching on expression then switching off for 24h in the presence (+) or absence ( - ) of 1OmM LiCl or 1OmM NaCl .
  • Figure 7 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in COS-7 cells as in (Ia) , treated with or without lO ⁇ M SB216763 or lOO ⁇ M L-690330 for 48h, expressed as odds ratios .
  • Figure 8 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in SK-N-SH cells as in (figure 1) , treated with or without lO ⁇ M SB216763 or lOO ⁇ M L-690330 for 48h, expressed as odds ratios .
  • Figure 9 shows mutant huntingtin fragment (EGFP-HDQ74) clearance by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 17Oh in the presence (+) or absence ( - ) of lO ⁇ M SB216763.
  • Fig 10 shows mutant huntingtin fragment clearance by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 17Oh in the presence (+) or absence ( - ) of lOO ⁇ M L-690330.
  • Figure 11 shows levels of IPi -2 measured in COS-7 cells treated with 1OmM LiCl or lOO ⁇ M L-690 , 330 for 24h .
  • Figure 12 shows levels of IP 3 measured in COS-7 cells treated with 2 ⁇ M Bradykinin, 1OmM LiCl or lOO ⁇ M L-690 , 330 for 5min.
  • Figure 13 shows the percentage of EGFP-HDQ74-positive cells with aggregates and cell death in COS-7 cells treated with or without 50 ⁇ M CBZ or ImM VPA for 48h, expressed as odds ratios .
  • Figure 14 shows IP 3 levels measured in COS-7 cells treated for 5min with or without 2 ⁇ M bradykinin, 1OmM LiCl or 1OmM LiCl pre- treated for 5min with ImM myo-inositol (Ins) or 24 ⁇ M prolyl endopeptidase inhibitor (PEI) .
  • Figure 15 shows the percentage of EGFP-HDQ74-positive cells with aggregates in COS-7 cells , either left untreated or treated with 1OmM LiCl with (+) or without (-) ImM myo-inositol or 24 ⁇ M PEI for 48h, were expressed as odds ratios .
  • Figure 16 shows the percentage of EGFP-HDQ74-positive cell death in COS-7 cells , either left untreated or treated with 1OmM LiCl with (+) or without (-) ImM myo-inositol or 24 ⁇ M PEI for 48h, expressed as odds ratios .
  • Figure 17 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 12Oh in the presence (+) or absence (- ) of 1OmM LiCl and ImM myoinositol or 24 ⁇ M PEI .
  • Figure 18 shows IP 3 levels measured in COS-7 cells treated with or without ImM myo-inositol , 24 ⁇ M PEI or 0.2 ⁇ M rapamycin (Rap) for 5min.
  • Figure 19 shows the percentage of EGFP-HDQ74 -positive cells with aggregates and cell death in COS-7 cells treated with or without ImM myo-inositol or 24 ⁇ M PEI for 48h, expressed as odds ratios .
  • Figure 20 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 120h in the presence (+) or absence ( - ) of ImM myo-inositol or 24 ⁇ M PEI .
  • Figure 21 shows COS-7 cells transfected with pEGFP-HDQ74 along with empty vector (pCDNA3.1) or pRheb at 1 : 3 ratio .
  • the proportion of GFP-positive cells with aggregates or cell death were assessed after 48h and expressed as odds ratios .
  • Figure 22 shows COS-7 cells transfected with pEGFP-HDQ74 and pRheb and treated with or without 1OmM LiCl or IOOM L-690 , 330.
  • the control represents untreated rheb-transfected cells .
  • the proportion of GFP-positive cells with aggregates or cell death were assessed after 48h and expressed as odds ratios .
  • Figure 23 shows the percentage of EGFP-HDQ74-positive cells with aggregates in COS-7 cells as in (Ia) , either left untreated or treated with 0.2 ⁇ M rapamycin with (+) or without (-) ImM myoinositol (Ins) or 24 ⁇ M prolyl endopeptidase inhibitor (PEI) for 48h, expressed as odds ratios .
  • Figure 24 shows the percentage of EGFP-HDQ74-positive cells with cell death in COS-7 cells either left untreated or treated with 0.2 ⁇ M rapamycin with (+) or without (-) ImM myo-inositol (Ins) or 24 ⁇ M prolyl endopeptidase inhibitor (PEI) for 48h, expressed as odds ratios .
  • Figure 25 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 12Oh in the presence (+) or absence ( - ) of 0.2 ⁇ M rapamycin and ImM myo-inositol or 24 ⁇ M PEI .
  • Figure 26 shows the percentage of EGFP-HDQ74-positive cells with aggregates (upper) and cell death (lower) in COS-7 cells , treated with (+) or without (-) 1OmM LiCl , 0.2 ⁇ M rapamycin or both for 48h, expressed as odds ratios .
  • Figure 27 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 72h in the presence (+) or absence ( - ) of 1OmM LiCl , 0.2 ⁇ M rapamycin or both.
  • Figure 28 shows the percentage of EGFP-HDQ74-positive cells with aggregates (upper) and cell death (lower) in COS-7 cells treated with (+) or without (-) lOO ⁇ M L-690 , 330 , 0.2 ⁇ M rapamycin or both for 48h, expressed as odds ratios .
  • Figure 29 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 72h in the presence (+) or absence ( -) of lOO ⁇ M L-690 , 330 , 0.2 ⁇ M rapamycin or both .
  • Figure 30 shows mutant huntingtin fragment clearance in stable PC12 cells by showing the amount of soluble EGFP74 remaining after switching on EGFP74 expression then switching off for 12Oh and 17Oh in the presence (+) or absence ( - ) of 1OmM NaCl .
  • Figure 31 shows the percentage of GFP-positive PC12 cells expressing EGFP-HDQ74 with aggregates after treatment for 17Oh with or without 1OmM LiCl or 1OmM NaCl , expressed as odds ratio, compared to the control untreated cells (17Oh off) .
  • Figure 32 shows the percentage of EGFP-HDQ74 -positive cells with aggregates in COS-7 cells treated with (+) or without (-) 1OmM 3 -MA, 1OmM LiCl or both for 48h, expressed as odds ratio .
  • Figure 33 shows a schematic diagram of some molecular targets of lithium and the relevant pathways .
  • Lithium affects different cellular processes , the two main pathways being the Wingless (Wnt) pathway and the inositol cycle 2 ' 17 .
  • Lithium inhibits glycogen synthase kinase-3 ⁇ (GSK-3 ⁇ . of the Wnt pathway and activates ⁇ -catenin, which mediates Tcf-mediated transcription.
  • Lithium also inhibits inositol monophosphatase (IMPase) to reduce free inositol that decreases intracellular inositol- 1 , 4 , 5-triphosphate (IP 3 ) levels and a rundown of the inositol cycle 5 .
  • IMPase inositol monophosphatase
  • Figure 34 shows the percentage of EGFP-HDQ74-positive cells with aggregates in COS-7 cells treated with (+) or without (-) 1OmM 3 -MA, lOO ⁇ M L-690 , 330 or both for 48h, expressed as odds ratio .
  • Figure 35 shows the percentage of EGFP-HDQ74-positive cells with aggregates in COS-7 cells treated with (+) or without (-) lO ⁇ M lactacystin (Lact) , lOO ⁇ M L-690 , 330 or both for 48h, expressed as odds ratio .
  • Figure 36 shows a schematic diagram of the inositol cycle and its modulation to reverse the effects of lithium.
  • Lithium inhibits inositol monophosphatase (IMPase) to reduce free inositol that decreases intracellular inositol-1, 4 , 5 - triphosphate (IP 3 ) levels 2 ' 5 , leading to enhanced clearance of mutant proteins through autophagy by a rundown of the inositol cycle .
  • IMPase inositol monophosphatase
  • IP 3 intracellular inositol-1, 4 , 5 - triphosphate
  • Figure 37 shows inositol-1 , 4 , 5-triphosphate (IP 3 ) levels measured in COS-7 cells treated with 1OmM LiCl for 0 min, 1 min, 5 min, 1 h and 24 h. *** , p ⁇ 0.001 ; ** , p ⁇ 0.01 ; * , p ⁇ 0.05 ; NS , Non-significant .
  • Figure 38 shows a schematic diagram of the regulation of autophagy in mammalian cells , which is governed by the activity of mammalian target of rapamycin (mTOR) .
  • mTOR mammalian target of rapamycin
  • Huntington' s Disease (HD) gene exon 1 fragment with 74 polyglutamine repeats (Q74) in pEGFP-Cl (Clontech) was described and characterised previously 12 .
  • DMEM Dulbecco ' s Modified Eagle Medium
  • FBS Fetal Bovine Serum
  • FBS Fetal Bovine Serum
  • 2mM L-Glutamine 2mM L-Glutamine
  • Cells were plated in six-well dishes at a density of IxIO 5 cells per well for 24h and transfe ⁇ ted with pEGFP-HDQ74 using LipofectAMINE reagent for COS-7 cells and LipofectAMINE PLUS reagent for SK-N-SH using manufacturer' s protocol (Invitrogen) .
  • Transfected cells were fixed with 4% paraformaldehyde (Sigma) after 48h and mounted in 4' , 6- diamidino-2 -phenylindole (DAPI , 3 mg/ml , Sigma) over coverslips on glass slides and analysed for aggregation and cell death .
  • DAPI 6- diamidino-2 -phenylindole
  • COS-7 cells were plated at a density of 3xlO s cells per well and treated for 24h .
  • Stable inducible PC12 cell lines expressing EGFP-HDQ74 , or A53T or A30P ⁇ -synuclein mutants were plated at 3xlO 5 per well in S- well dishes and induced with l ⁇ g/ml doxycycline (Sigma) for 8h and 48h respectively .
  • Expression of transgenes was switched off by removing doxycycline from the medium 3 ' 4 and cells were treated with or without various compounds for 12Oh for EGFP-HDQ74 clearance and 24h for mutant ⁇ -synuclein clearance .
  • Primary antibodies used include anti-EGFP (8362 -1 , Clontech) , anti-HA (12CA5 , Covance) , anti-mTOR (2972) , anti-Phospho-mTOR (Ser2448) (2971) , anti-p70 S6 Kinase (9202 ) , anti Phospho-p70 S6 Kinase (Thr389) ( 9206) , anti-4E-BPl (9452 ) , anti-Phospho-4E-BPl (Thr37/46) (9459) , anti-S6 Ribosomal Protein (2212) and anti-
  • Phospho-S6 Ribosomal Protein (Ser235/236 ) (2211) , all from Cell Signaling technology, anti-LC3 (gift from T . Yoshimori , National Institute of Genetics , Japan) , anti-actin (A2066, Sigma) and anti-tubulin (Clone DM IA, Sigma) . Blots were probed with anti- mouse or anti-rabbit IgG-HRP (Amersham) and visualised using ECL or ECL Plus detection kit (Amersham) .
  • Cos-7 cells were fixed with 4% paraformaldehyde .
  • Primary antibodies included anti-LC3 (gift from T . Yoshimori , National Institute of Genetics , Japan) and anti-Phospho-S6 Ribosomal Protein (Ser235/236) (2211 , Cell Signalling technology) . Standard fluorescence method was used for detection and secondary antibodies used were goat anti-rabbit Alexa 488 Green and Alexa 594 Red (Cambridge Biosciences) .
  • Autophagic vacuoles were labelled with monodansylcadaverine (MDC, Sigma) by incubating Cos-7 cells on coverslips with 0.05mM MDC for 1 hour at 37°C 1S . Cells were then rinsed thrice with PBS r and analysed immediately using fluorescent microscope .
  • MDC monodansylcadaverine
  • IPi, 2 were assayed as previously described 26 . Briefly, COS-7 cells labelled with myo- [ 3 H] inositol (l ⁇ Ci/10 6 cells) for 24h, stimulated and then subj ected to chloroform/methanol (1 : 2) extraction followed by Bligh-Dyer phase separation. Levels of IPi, 2 were determined by liquid scintillation counting of fractions eluted following Dowex (formate form) ion exchange chromatography of aliquots of the aqueous phase . Results were calculated as a percentage of total incorporated radioactivity.
  • Lithium was confirmed to significantly reduced aggregation and cell death in COS-7 (non-neuronal) and SK-N-SH (neural precursor) cells (Figs . 1 and 2 ) , caused by truncated forms of mutant huntingtin, the aggregate-prone protein that causes
  • Huntington' s disease (HD) 11 Expression levels of EGFP-tagged huntingtin exon 1 with 74 polyglutamine repeats (EGFP-HDQ74) correlate with aggregate formation (the proportion of transfected cells with aggregates) in such cell models 3 ' 12 .
  • Clearance of an aggregation prone huntingtin construct was determined using a stable doxycycline-inducible PC12 cell line expressing EGFP-HDQ7.
  • the transgene expression was first induced by adding doxycycline and then switched off by removing doxycycline from the medium.
  • the clearance of the construct at various times after switching off expression after an initial induction period can be used to assess whether specific agents alter the clearance of the transgene product , as its expression decays when synthesis is stopped 3 .
  • Lithium was observed to significantly enhance clearance of soluble EGFP-HDQ74 (Fig . 3 ) and reduce aggregates at 12Oh and 17Oh (Figs 4 & 31) , whereas sodium chloride had no effect (Figs 30 & 31) . Furthermore, clearance of A53T and A3OP ⁇ -synuclein mutants , which cause autosomal dominant forms of Parkinson' s disease 13 ' 14 was assessed. After an induction of stable inducible PC12 cell lines and a subsequent removal of doxycycline , lithium treatment for 24h enhanced the clearance of A53T (Fig . 5) and A30P (Fig . 6) ⁇ -synuclein mutants whereas no effects were observed with sodium chloride .
  • microtubule-associated protein 1 light chain 3 (LC3 ) , a homologue of Apg8p essential for autophagy in yeast , is processed post-translationally into LC3 -I , which is cytosolic , and LC3 -II , which associates with autophagosome membranes 16 .
  • Lithium increased the number of LC3 -positive autophagic vesicles in COS-7 cells , like rapamycin, and increased the levels of LC3 - II .
  • the modest increase in LC3 -II is similar to what has been observed previously when autophagy is induced 16 .
  • lithium facilitates clearance of EGFP-HDQ74 and mutant ⁇ - synucleins through autophagy, then it should be blocked by the autophagy inhibitor, 3 -methyladenine (3 -MA) .
  • 3 -MA itself increased EGFP-HDQ74 aggregate formation in COS-7 cells 3 (Fig . 32) and delayed clearance of A53T ⁇ -synuclein 4 .
  • 3 - MA abolished the effect of lithium in reducing EGFP-HDQ74 aggregation (Fig . 32) , or promoting A53T ⁇ -synuclein clearance .
  • lithium induces autophagy and enhances the clearance of known autophagy substrates like EGFP-HDQ74 and mutant ⁇ - synucleins .
  • Lithium inhibits a number of enzymes , including glycogen synthase kinase-3 ⁇ (GSK-3 ⁇ ) and inositol monophosphatase (IMPase) 2 ' 17 .
  • GSK-3 ⁇ glycogen synthase kinase-3 ⁇
  • IMPase inositol monophosphatase 2 ' 17 .
  • SB216763 specific inhibitors of GSK-3 ⁇
  • IMPase L-690 , 330
  • L-690 , 330 reduced aggregation and cell death caused by EGFP-HDQ74 in both COS-7 and SK-N-SH cells
  • SB216763 increased EGFP-HDQ74 aggregates but reduced cell death (Fig . 7) .
  • Inhibitors of autophagy (3 -MA) and the proteasome (lactacystin were used to test whether the enhanced clearance of these proteins by L-690 , 330 was by autophagy or the proteasomal route . Both these inhibitors reduced clearance of A53T ⁇ -synuclein in the PC12 stable line 4 and increased EGFP-HDQ74 aggregates in COS-7 cells 3 (Figs 34 & 35) .
  • L-690 , 330 could not facilitate clearance of A53T ⁇ - synuclein or reduce EGFP-HDQ74 aggregates (Fig 34) .
  • L- 690 , 330 enhanced clearance of A53T ⁇ -synuclein and significantly reduced EGFP-HDQ74 aggregates in cells treated with the proteasome inhibitor lactacystin (Fig 35) .
  • L-MA autophagy
  • lactacystin lactacystin
  • Lithium and L-690 , 330 were confirmed as inhibiting IMPase in our experimental conditions , as they increased levels of inositol mono- and bis-phosphate (IP 1-2 ) (Fig . 11) and decreased myo- inositol-l , 4 , 5-triphosphate (IP 3 ) levels (Fig . 12 ) , a consequence of reduced free inositol s .
  • Lithium significantly reduced IP 3 levels even at time-points from 1 min to 24h (Fig . 37) .
  • Inositol depletion is a common mechanism for mood stabilizing drugs like lithium, carbamazepine (CBZ) and valproic acid (VPA) 7 .
  • CBZ and VPA significantly reduced EGFP- HDQ74 aggregates and attenuated polyglutamine toxicity in COS-7 cells (Fig . 13 ) , and also enhanced clearance of A3OP ⁇ - synuclein .
  • these drugs may be of therapeutic value in HD and related neurodegenerative diseases .
  • the activity of mTOR can be inferred by the levels of phosphorylation of its substrates , S6K1 (p70S6K) and 4E-BP1 at Thr389 and Thr37/46 , respectively (Fig 38 ) , and the phosphorylation of the S6 ribosomal protein (S6P) , a substrate of S6K1. While rapamycin reduced phosphorylation of S6K1 , S6P and 4E-BP1 in COS-7 cells as expected, lithium or L-690 , 330 did not have any effect on their phosphorylation, which provides indication that their effects are independent of mTOR inhibition .
  • Lithium and rapamycin were observed to have additive effects in reducing EGFP-HDQ74 aggregates and cell death in COS-7 cells (Fig . 26) , compared to the single treatments of lithium or rapamycin. Furthermore, lithium and rapamycin together facilitated greater clearance of soluble EGFP-HDQ74 at 72h (Fig . 27) and A53T ⁇ -synuclein at 8h, compared to single either compound alone . In order to clearly demonstrate this effect , early time-points were chosen at which obvious reductions of the levels of these proteins are not yet observed when the cells are treated with either of the compounds alone .
  • rapamycin concentration used in these experiments was close to saturating, as a further increase in the rapamycin dose did not lead to more clearance of soluble EGFP-HDQ74. Consistent with the above observations , the combination of L-690 , 330 and rapamycin had an enhanced protective effect on EGFP-HDQ74 mediated toxicity in COS-7 cells (Fig . 28) and also facilitated greater clearance of soluble EGFP-HDQ74 and A53T ⁇ -synuclein at early time-points in stable PC12 cell lines , compared to the individual treatments (Fig . 29) .
  • Lithium induces autophagy by inhibiting inositol monophosphatase , which in turn decreases free inositol leading to enhanced clearance of mutant proteins . These effects are reversed by free inositol (myo-inositol) , which itself inhibits clearance of autophagy substrates , providing further support for the role of IMPase in autophagy regulation.
  • the abrogation of the effect of lithium on autophagy by PEI provides indication that these effects are mediated via IP 3 .

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Abstract

La présente invention se rapporte à des procédés d'induction d'autophagie dans des cellules mammaliennes. Cette invention se rapporte à l'induction de l'autophagie dans des cellules par l'intermédiaire de l'inhibition de l'inositol monophosphatase (IMPase) et de la réduction du taux d'inositol libre (IP3). Ceci peut s'avérer utile, par exemple, pour le traitement d'un ensemble de troubles, notamment les troubles neurodégénératifs et les infections bactériennes telles que la tuberculose. L'invention se rapporte également à des composés thérapeutiques, à leurs utilisations et à des méthodes de traitement ainsi qu'à des procédés de criblage d'inducteurs d'autophagie.
PCT/GB2006/000224 2005-01-25 2006-01-23 Procedes d'induction d'autophagie dans des cellules mammaliennes Ceased WO2006079792A1 (fr)

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WO2008059014A3 (fr) * 2006-11-17 2008-09-18 Univ Salamanca Agents antituberculeux
WO2010045270A3 (fr) * 2008-10-13 2010-08-19 San Diego State University Research Foundation Compositions de marquage et d'identification d'autophagosomes et leurs procédés de fabrication et d'utilisation
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WO2012061907A2 (fr) 2010-11-10 2012-05-18 Katholieke Universiteit Leuven Activité des ostéoclastes
EP2954896A1 (fr) * 2014-06-13 2015-12-16 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Inhibiteurs à petite molécule de la ligase de Skp2 E3 dans la dépression et d'autres maladies

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Cited By (8)

* Cited by examiner, † Cited by third party
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WO2008059014A3 (fr) * 2006-11-17 2008-09-18 Univ Salamanca Agents antituberculeux
ES2315133A1 (es) * 2006-11-17 2009-03-16 Universidad De Salamanca Agentes antituberculosis.
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WO2010045270A3 (fr) * 2008-10-13 2010-08-19 San Diego State University Research Foundation Compositions de marquage et d'identification d'autophagosomes et leurs procédés de fabrication et d'utilisation
WO2012029722A1 (fr) * 2010-08-30 2012-03-08 武田薬品工業株式会社 Procédé de criblage
WO2012061907A2 (fr) 2010-11-10 2012-05-18 Katholieke Universiteit Leuven Activité des ostéoclastes
EP2954896A1 (fr) * 2014-06-13 2015-12-16 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Inhibiteurs à petite molécule de la ligase de Skp2 E3 dans la dépression et d'autres maladies

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