Methods of screening apoptosis modulating compounds, compounds identified by said methods and use of said compounds as therapeutic agents

Abstract

A method of screening cellular polypeptides for pro-apoptotic or anti-apoptotic activity in a cell of a particular cell-type, said method comprising: (a) culturing cells of said particular cell-type under non apoptotic conditions and culturing cells of said particular cell-type under apoptotic conditions, and (b) determining subcellular localisation of said cellular polypeptides in the cultured cells, wherein a localization of a cellular polypeptide in lipid rafts in cultured cells under non apoptotic conditions and a segregation of said cellular polypeptide from lipid rafts in cultured cells under apoptotic conditions is indicative that said cellular polypeptide has a pro-apoptotic or an anti-apoptotic activity in said particular cell-type.

1 - 8 . (canceled)  
   
   
       9 . A method of screening compounds for their capacity to modulate apoptosis in cells which produce pro- or anti-apoptotic polypeptides which are located in lipid rafts when said cells are cultured under non apoptotic conditions, said method comprising: 
 a. culturing said cells in a growth medium for maintaining non apoptotic conditions;    b. contacting said cultured cells with a candidate compound;    c. determining the level of one or several pro- or anti-apoptotic polypeptides associated to lipid rafts ; and,    d. selecting the compound which interferes with the association of one or several pro- or anti-apoptotic polypeptides with lipid rafts, said compound having the capacity to modulate apoptosis.    
   
   
       10 . A method of screening compounds for their capacity to promote apoptosis in cells, said method comprising 
 a. culturing mammalian cells in a growth medium for maintaining non apoptotic conditions; wherein said cells produce a pro-apoptotic protein which is located in lipid rafts under non apoptotic conditions of said cells;    b. contacting said cultured cells with a candidate compound; and,    c. determining the absence or the presence of lipid rafts in said cultured cells;    d. in case of presence of lipid rafts, optionally determining the level of pro-apoptotic protein located in the lipid rafts,    wherein the absence of lipid rafts in the plasma membrane of, cells incubated with said candidate compound or if determined, the reduced level of pro-apoptotic protein in the rafts is indicative that said compound promotes apoptosis.    
   
   
       11 . A method of screening compounds for their capacity to inhibit or prevent apoptosis of cells, said method comprising 
 a. culturing cells in a growth medium for maintaining non apoptotic conditions; wherein said cells produce a pro-apoptotic protein which is located in lipid rafts under, non apoptotic conditions;    b. contacting said cells with a candidate compound;    c. culturing cells under apoptotic conditions;    d. determining the absence or the presence of lipid rafts;    e. in the case of presence of lipid rafts, optionally determining the level of pro-apoptotic protein located in the lipid rafts; wherein the presence of lipid rafts in the plasma membranes of cells incubated with said candidate compound and optionally the maintained level of pro-apoptotic protein in the rafts is indicative that said candidate compound inhibits or prevents apoptosis.    
   
   
       12 . The method according to  claim 9 , wherein a pro-apoptotic protein located in lipid rafts under proliferative conditions is the Bad protein.  
   
   
       13 . The method of  claim 12 , wherein said cells which produce a Bad protein are cells characteristic of the immune system, preferably T cells.  
   
   
       14 . The method according to  claim 9 , wherein the presence or the absence of lipid rafts is visualized by confocal microscopy.  
   
   
       15 . The method according to  claim 9 , wherein the presence or the absence of lipid rafts is determined by the following steps 
 i) recovering the cultured cells incubated with said compound candidate and resuspending said cells in a buffer appropriate for subcellular fractionation, such as gradient sucrose buffer;    ii) ultracentrifugating the fractionated cells and;    iii) recovering the subcellular fraction which should contain lipid rafts;    iv) determining whether the recovered subcellular fraction contains ganglioside and/or lipid raft associated molecule(s).    
   
   
       16 . The method according to  claim 15 , wherein the presence or the absence of lipid rafts is determined by the use of a marker which specifically recognizes ganglioside or a raft-associated molecule.  
   
   
       17 . The method according to  claim 16 , wherein said marker is selected among cholera toxin subunit B (CTx), anti-Bad antibody or anti-Lck antibody.  
   
   
       18 . The method of  claim 9 , wherein said cells are mammalian cells.  
   
   
       19 . The method of  claim 9 , wherein said non apoptotic conditions are proliferative conditions.  
   
   
       20 . The method of  claim 9 , wherein said growth medium comprises at least a cytokine or a growth factor necessary for maintaining proliferative growth conditions.  
   
   
       21 . The method of  claim 11 , wherein the apoptotic conditions of step c) are obtained by depriving the cells with said cytokine or growth factor.  
   
   
       22 . The method of  claim 20 , wherein a cytokine or growth factor necessary for maintaining proliferative growth conditions is an interleukin, preferably selected among IL-4, IL-2 or IL-9, or a mixture thereof.  
   
   
       23 . A use of a compound capable of modulating lipid rafts formation, in the preparation of a medicine for the treatment of disorders induced by or associated with a defective regulation of cell death or of any specific pathology in which cell death may be at least a part of the therapy.  
   
   
       24 . The use of  claim 23 , wherein said defective regulation affects cells which produce Bad protein.  
   
   
       25 . The use of  claim 24 , wherein said defective regulation affects cells of the immune system.  
   
   
       26 . The use of any of  claim 23 , wherein said compound is capable of disrupting lipid rafts and wherein said defective regulation of cell death results in an abnormal decrease of cell death.  
   
   
       27 . The use of  claim 26 , wherein said abnormal decrease of cell death is related to cancer disease and especially to lymphoproliferative cancers, infectious disease and especially viral disease, inflammatory disease or auto-immune disease.  
   
   
       28 . The use of any of  claim 23 , wherein a compound capable of disrupting lipid rafts is methyl-β-cyclodextrin or filipin.  
   
   
       29 . The use of  claim 23 , wherein said compound is capable of reconstituting lipid rafts in the plasma membrane of cells and wherein said defective regulation of apoptosis results in an abnormal increase of cell death.  
   
   
       30 . The use of  claim 29 , wherein a pathology resulting in an abnormal increase of cell death is a disease associated to senescence, neurodegenerative disease Alzheimer, AIDS, ischemic cell death or wound-healing.  
   
   
       31 . The use of  claim 29 , wherein a compound capable of reconstituting lipid rafts is edelfosine.  
   
   
       32 . An in vitro method for the detection of a defective regulation of apoptosis, in a sample of cells of an individual, said method comprising determining the presence or the absence of lipid rafts in said cells, wherein the absence of said lipid rafts is indicative of a defective regulation of apoptosis.  
   
   
       33 . The method according to  claim 32 , wherein said cells are cells of the immune system of an individual affected by a lymphoproliferative disease.  
   
   
       34 . The method of  claim 32 , wherein the presence or the absence of lipid rafts is determined by detecting the presence or absence of a pro-apoptotic or an anti-apoptotic protein which is known to be located in lipid rafts under non apoptotic conditions of cells.  
   
   
       35 . The method of  claim 34 , wherein a pro-apoptotic protein known to be located in lipid rafts under non apoptotic conditions is a Bad protein.  
   
   
       36 . A use of a compound appropriate for detecting the presence of lipid rafts, in the in vitro detection method according to  claim 32 .  
   
   
       37 . The use of  claim 36 , wherein a compound appropriate for detecting the presence of lipid rafts is a compound which specifically recognizes Bad protein, Lck protein or ganglioside GM1.  
   
   
       38 . The use of  claim 37 , wherein said compound is selected among cholera toxin subunit B (CTx), anti-Bad antibody and anti-Lck antibody.  
   
   
       39 . A use of a compound capable of modulating pro- or anti-apoptotic protein rafts localization for the preparation of a medicine for the treatment of disorders induced by or associated with a defective regulation of cell death or of any specific pathology in which cell death may be at least a part of the therapy.  
   
   
       40 . The use of  claim 39 , wherein said compound promote pro- or anti-apoptotic protein segregation from lipid rafts.  
   
   
       41 . The use of  claim 39 , wherein said compound promotes pro- or anti-apoptotic protein localization in lipid rafts.  
   
   
       42 . An in vitro method for the detection of a defective regulation of apoptosis, in a sample of cells of an individual, said method comprising determining the presence or the absence of lipid rafts in said cells, wherein the presence of a great number of lipid rafts compared to normal cells is indicative of a defect regulation of apoptosis.  
   
   
       43 . The method according to  claim 10 , wherein a pro-apoptotic protein located in lipid rafts under proliferative conditions is the Bad protein.  
   
   
       44 . The method of  claim 43 , wherein said cells which produce a Bad protein are cells characteristic of the immune system, preferably T cells.  
   
   
       45 . The method according to  claim 10 , wherein the presence or the absence of lipid rafts is visualized by confocal microscopy.  
   
   
       46 . The method according to  claim 10 , wherein the presence or the absence of lipid rafts is determined by the following steps 
 i) recovering the cultured cells incubated with said compound candidate and resuspending said cells in a buffer appropriate for subcellular fractionation, such as gradient sucrose buffer;    ii) ultracentrifugating the fractionated cells and;    iii) recovering the subcellular fraction which should contain lipid rafts;    iv) determining whether the recovered subcellular fraction contains ganglioside and/or lipid raft associated molecule(s).    
   
   
       47 . The method according to  claim 45 , wherein the presence or the absence of lipid rafts is determined by the use of a marker which specifically recognizes ganglioside or a raft-associated molecule.  
   
   
       48 . The method according to  claim 47 , wherein said marker is selected among cholera toxin subunit B (CTx), anti-Bad antibody or anti-Lck antibody.  
   
   
       49 . The method of  claim 10 , wherein said cells are mammalian cells.  
   
   
       50 . The method of  claim 10 , wherein said non apoptotic conditions are proliferative conditions.  
   
   
       51 . The method of  claim 10 , wherein said growth medium comprises at least a cytokine or a growth factor necessary for maintaining proliferative growth conditions.  
   
   
       52 . The method according to  claim 11 , wherein a pro-apoptotic protein located in lipid rafts under proliferative conditions is the Bad protein.  
   
   
       53 . The method of  claim 52 , wherein said cells which produce a Bad protein are cells characteristic of the immune system, preferably T cells.  
   
   
       54 . The method according to  claim 11 , wherein the presence or the absence of lipid rafts is visualized by confocal microscopy.  
   
   
       55 . The method according to  claim 11 , wherein the presence or the absence of lipid rafts is determined by the following steps 
 i) recovering the cultured cells incubated with said compound candidate and resuspending said cells in a buffer appropriate for subcellular fractionation, such as gradient sucrose buffer;    ii) ultracentrifugating the fractionated cells and;    iii) recovering the subcellular fraction which should contain lipid rafts;    iv) determining whether the recovered subcellular fraction contains ganglioside and/or lipid raft associated molecule(s).    
   
   
       56 . The method according to  claim 54 , wherein the presence or the absence of lipid rafts is determined by the use of a marker which specifically recognizes ganglioside or a raft-associated molecule.  
   
   
       57 . The method according to  claim 56 , wherein said marker is selected among cholera toxin subunit B (CTx), anti-Bad antibody or anti-Lck antibody.  
   
   
       58 . The method of  claim 11 , wherein said cells are mammalian cells.  
   
   
       59 . The method of  claim 11 , wherein said non apoptotic conditions are proliferative conditions.  
   
   
       60 . The method of  claim 11 , wherein said growth medium comprises at least a cytokine or a growth factor necessary for maintaining proliferative growth conditions.