Ligand binding pocket peptide fragment of estrogen-related receptor 3 (err3) and uses thereof

Abstract

The present invention relates to the identification of novel biologically active compounds having an agonist or an antagonist effect on the transcriptional-activating activity of the Estrogen-Related Receptor 3 (ERR3).

1 . A peptide fragment that mimics, when fused to a polypeptide containing a DNA-binding domain, the ligand dependence of the transcriptional activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1, wherein said peptide fragment has an amino acid chain of up to 267 amino acids in length and wherein said peptide fragment comprises the amino acid sequence starting at the Prolyl amino acid residue in position 229 and ending at the Valyl amino acid residue in position 458 of the amino acid sequence SEQ ID N°1.  
     
     
         2 . A fusion polypeptide consisting of the peptide fragment of  claim 1  which is fused to a polypeptide containing a DNA-binding domain.  
     
     
         3 . The fusion polypeptide of  claim 2 , wherein the polypeptide containing a DNA-binding domain consists of the Gal4 protein.  
     
     
         4 . A protein complex between (i) a protein comprising the peptide fragment according to  claim 1  and (ii) the SRC-1 coactivator peptide of SEQ ID N°2 or a peptide fragment thereof comprising the Receptor Interaction Domain (RID) of SRC-1, wherein said protein complex, when the peptide fragment of  claim 1  is fused to a polypeptide containing a DNA-binding domain, mimicks the transcriptional-activating activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1.  
     
     
         5 . The protein complex of  claim 4 , wherein the protein (i) consists of a fusion polypeptide wherein the peptide fragment of  claim 1  is fused to a polypeptide containing a DNA-binding domain.  
     
     
         6 . The protein complex of  claim 5 , wherein the polypeptide containing a DNA-binding domain consists of the Gal4 protein.  
     
     
         7 . The protein complex of  claim 4 , wherein said SRC-1 coactivator peptide fragment comprises the amino acid sequence starting from the amino acid residue in position 614 and ending at the amino acid residue in position 764 of the amino acid sequence SEQ ID N°2.  
     
     
         8 . A nucleic acid encoding the peptide fragment according to  claim 1 .  
     
     
         9 . A nucleic acid encoding a fusion polypeptide according to  claim 2 .  
     
     
         10 . An expression cassette comprising a nucleic acid according to  claim 8  and a regulatory polynucleotide to which said nucleic acid is operably linked.  
     
     
         11 . The expression cassette of  claim 10 , wherein said regulatory polynucleotide is functionally-active in eukaryotic cells, preferably mammalian cells and most preferably in human cells.  
     
     
         12 . A recombinant vector comprising a nucleic acid according to  claim 8 .  
     
     
         13 . A cell host which is transformed with a nucleic acid encoding a peptide fragment that mimics, when fused to a polypeptide containing a DNA-binding domain, the ligand dependence of the transcriptional activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1, wherein said peptide fragment has an amino acid chain of up to 267 amino acids in length and wherein said peptide fragment comprises the amino acid sequence starting at the Prolyl amino acid residue in position 229 and ending at the Valyl amino acid residue in position 458 of the amino acid sequence SEQ ID N°1, wherein the cell host is transformed with an expression cassette according to  claim 10 .  
     
     
         14 . A method for the screening of compounds which are agonists or antagonists of the transcriptional-activating activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1, wherein said method comprises the steps of: 
 a) incubating a culture of transformed cell hosts, wherein said transformed cell hosts express a fusion polypeptide as defined in  claim 2 , and wherein said transformed cell hosts comprise a reporter polynucleotide comprising (i) at least one nucleic acid sequence which is recognised by the DNA-binding domain contained within said fusion polypeptide and (ii) an open reading frame encoding a detectable protein, with a candidate compound to be assayed;    b) measuring the amount of the detectable protein which is produced by said transformed cell hosts;    c) comparing the amount of the detectable protein measured at step b) with the amount of the detectable protein which is produced by a control culture of said transformed cell hosts wherein step a) is performed without said candidate substance;    d) selecting, respectively:    (i) candidate compounds which inhibit the detectable protein production, which consist of antagonist compounds of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1; and    (ii) candidate compounds which enhance the detectable protein production, which consist of agonist compounds of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1.    
     
     
         15 . The method of  claim 14 , wherein the detectable protein consists of the luciferase protein.  
     
     
         16 . A crystallised peptide fragment of  claim 1 .  
     
     
         17 . A crystallised protein complex of  claim 4 .  
     
     
         18 . A crystallised protein complex between (i) the peptide fragment of  claim 1  and (ii) a peptide fragment of the SRC-1 coactivator peptide of SEQ ID N°2 comprising the Receptor Interaction Domain (RID) thereof.  
     
     
         19 . The crystallised protein complex of  claim 18 , wherein peptide fragment (ii) consists of the Receptor Interaction Domain (RID) of the SRC-1 protein.  
     
     
         20 . The crystallised protein complex of  claim 19 , wherein two protein complexes form together a dimeric protein complex.  
     
     
         21 . The crystallised protein complex of  claim 20  wherein each of the protein complex of said dimeric protein complex comprises the Ligand Binding Pocket (LBP) of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1 and wherein each dimeric protein complex delineates a contact area of 1245 Å 3  and a cavity volume of 220 Å 3 .  
     
     
         22 . A method for selecting a compound that interacts with the Ligand Binding Pocket (LBP) of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1, wherein said method comprises the steps of: 
 a) generating a three-dimensional model of an ERR3 LBP dimer using the relative structural coordinates according to Table 1; and    b) employing said three-dimensional model to design or select a compound, from a serial of compounds, that interacts with said ERR3 LBP.    
     
     
         23 . The method of  claim 22 , further comprising the steps of: 
 c) obtaining the compound designed or selected at step b); and    d) contacting the compound obtained at step c) with a protein containing a peptide fragment in order to determine the effect the compound has on transcriptional-activating activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1;    wherein the peptide fragment mimics, when fused to a polypeptide containing a DNA-binding domain, the ligand dependence of the transcriptional activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1, wherein said peptide fragment has an amino acid chain of up to 267 amino acids in length and wherein said peptide fragment comprises the amino acid sequence starting at the Prolyl amino acid residue in position 229 and ending at the Valyl amino acid residue in position 458 of the amino acid sequence SEQ ID N°1.    
     
     
         24 . The method of  claim 23 , wherein step d) consists of the method according to  claim 14 , using said compound as the candidate agonist or antagonist compound.  
     
     
         25 . The method according to  claim 22 , wherein said compound is selected from a library of compounds.  
     
     
         26 . The method according to  claim 22 , wherein said compound is selected from a database.  
     
     
         27 . The method according to  claim 22 , wherein said compound is designed de novo.  
     
     
         28 . The method according to  claim 22 , wherein said compound is selected from the group consisting of estradiol (E2), diethylstilbestrol (DES) and 4-hydroxytamoxifen (4-OHT).  
     
     
         29 . A method for selecting a compound that interacts with the Ligand Binding Pocket (LBP) of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1, wherein said method comprises the step of: 
 a) selecting or designing a candidate agonist or antagonist compound for the estrogen-related receptor 3 (ERR3) of SEQ ID N°1 by performing computer fitting analysis of the candidate agonist or antagonist compound with the three-dimensional structure of the Ligand Binding Pocket (LBP) of said ERR3 receptor.    
     
     
         30 . The method of  claim 29 , further comprising steps of: 
 b) obtaining the compound selected or designed at step a); and    c) contacting the identified agent with a protein containing the peptide fragment of  claim 1  in order to determine the effect the compound has on the transcriptional-activating activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1.    
     
     
         31 . A method for selecting a compound that interacts with the Ligand Binding Pocket (LBP) of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1, wherein said method comprises the step of:  
       a) selecting or designing a candidate agonist or antagonist compound for the estrogen-related receptor 3 (ERR3) of SEQ ID N°1 by performing computer fitting analysis of the candidate agonist or antagonist compound with the three-dimensional structure of the Ligand Binding Pocket (LBP) of said ERR3 receptor,  
       wherein step c) consists of the method according to  claim 14 , using the identified compound as the candidate agonist or antagonist compound.  
     
     
         32 . The method according to  claim 29 , wherein said compound is selected from a library of compounds.  
     
     
         33 . The method according to  claim 29 , wherein said compound is selected from a database.  
     
     
         34 . The method according to  claim 29 , wherein said compound is designed de novo.  
     
     
         35 . The method according to  claim 29 , wherein said compound is selected from the group consisting of estradiol (E2), diethylstilbestrol (DES) and 4-hydroxytamoxifen (4-OHT).  
     
     
         36 . A method for selecting an antagonist compound for the transcriptional-activating activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1, wherein said method comprises the steps of: 
 a) generating a three-dimensional model of an ERR3 LBP dimer using the relative structural coordinates of amino acid residues LEU268, ALA272, GLU275, LEU309, ILE310, ARG316, VAL313, LEU345, HIS434, PHE435, LEU454 and PHE450 according to Table 1 for chain A± a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å; and    b) performing, for each candidate compound, a computer fitting analysis of said candidate antagonist compound with three-dimensional model generated at step a); and    c) selecting, as an antagonist compound, every candidate compound having a chemical structure inducing a steric constraint with PHE435 or LEU345 amino acid residues of the the estrogen-related receptor 3 (ERR3) Ligand Binding Pocket (LBP).    
     
     
         37 . The method of  claim 36 , further comprising steps of: 
 c) obtaining the compound selected or designed at step b); and    d) contacting the identified agent with a protein containing the peptide fragment of  claim 1  in order to determine the effect the compound has on the, the transcriptional-activating activity of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1.    
     
     
         38 . The method of  claim 37 , wherein step d) consists of the method according to  claim 14 , using the identified compound as the candidate antagonist compound.  
     
     
         39 . The method according to  claim 36 , wherein said compound is selected from a library of compounds.  
     
     
         40 . The method according to  claim 36 , wherein said compound is selected from a database.  
     
     
         41 . The method according to  claim 36 , wherein said compound is designed de novo.  
     
     
         42 . The method according to  claim 36 , wherein said compound is selected from the group consisting of estradiol (E2), diethylstilbestrol (DES) and 4-hydroxytamoxifen (4-OHT).  
     
     
         43 . A crystallised complex between: 
 (i) a complex comprising (i1) the peptide fragment of  claim 1  and (i2) a peptide fragment of the SRC-1 coactivator peptide of SEQ ID N°2 comprising the Receptor Interaction Domain (RID) thereof; and    (ii) a ligand of the the estrogen-related receptor 3 (ERR3) Ligand Binding Pocket (LBP).    
     
     
         44 . The crystallised complex of  claim 43 , wherein said ligand consists of an antagonist compound of the estrogen-related receptor 3 (ERR3) of SEQ ID N°1.  
     
     
         45 . The crystallised complex of  claim 44 , wherein said ligand is selected from the group consisting of estradiol (E2), diethylstilbestrol (DES) and 4-hydroxytamoxifen (4-OHT).  
     
     
         46 . A molecular complex model comprising: 
 (i) the estrogen-related receptor 3 (ERR3) Ligand binding Pocket (LBP) defined by the structural coordinates of amino acid residues LEU268, ALA272, GLU275, LEU309, ILE310, ARG316, VAL313, LEU345, HIS434, PHE435, LEU454 and PHE450 according to Table 1 for chain A± a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 Å; and    (ii) a ligand for said estrogen-related receptor 3 (ERR3) Ligand binding Pocket (LBP).    
     
     
         47 . The molecular complex of  claim 46 , wherein said ligand consists of an antagonist compound for the estrogen-related receptor 3 (ERR3) of SEQ ID N°1.  
     
     
         48 . The molecular complex of  claim 47 , wherein said ligand is selected from the group consisting of estradiol (E2), diethylstilbestrol (DES) and 4-hydroxytamoxifen (4-OHT).  
     
     
         49 . A mutant of the peptide fragment of  claim 1 , wherein, based on the amino acid residue numbering of SEQ ID N°1, said mutant is selected from the group of mutants consisting of: 
 1) the mutant “PHE435LEU”;    2) the mutant “ALA272PHE”;    3) the mutant “ALA272LEU”; and    4) the mutant “LEU345ILE; PHE435LEU; PHE450LEU”.    
     
     
         50 . A nucleic acid encoding a mutant according to  claim 49   
     
     
         51 . The nucleic acid of  claim 50 , wherein the open reading frame encoding the mutant is operably linked to a regulatory polynucleotide.  
     
     
         52 . A recombinant vector comprising, inserted therein, the nucleic acid of  claim 50 .  
     
     
         53 . A cell host transformed with a nucleic acid according to  claim 50 .  
     
     
         54 . A machine-readable data storage medium, comprising a data storage material encoded with machine-readable data, wherein said machine-readable data consist of the X-ray structural coordinate data of the Ligand Binding Pocket (LBP) of the estrogen-related receptor 3 (ERR3) according to Table 1.  
     
     
         55 . A machine-readable data storage medium, comprising a data storage material encoded with machine-readable data which, when using a machine programmed with instructions for using said data, displays a graphical three-dimensional representation of a crystal of the Ligand Binding Pocket (LBP) of the estrogen-related receptor 3 (ERR3).