Inhibitors of proteins from the rho-gef family

Abstract

A method for screening peptides with an aptamer library for determining inhibitors of any one of the proteins from the Rho-GEFs family.

1 . A method for determining inhibitors of proteins from the Rho-GEF family, comprising screening proteins with an aptamer library.  
     
     
         2 . The method according to  claim 1 , further comprising determining inhibitors of the protein Trio.  
     
     
         3 . The method according to  claim 2 , further comprising determining specific inhibitors of the GEFD2 domain of the protein Trio (TrioGEFD2).  
     
     
         4 . The method according to  claim 3 , further comprising determining inhibitors of the protein Trio which specifically bind to the GEFD1 domain of the protein Trio (TrioGEFD1).  
     
     
         5 . The method according to  claim 4 , further comprising determining inhibitors of the protein Trio which do not inhibit TrioGEFD1.  
     
     
         6 . Process for determining inhibitors of any one of the proteins from the Rho-GEF family, comprising: 
 a step of contacting said protein for which an inhibitor is sought with the peptides from an aptamer library,    a step of recovering a first group of peptides which specifically bind to said protein, particularly to the catalytic domain of the GEF region of said protein,    a step of recovering, among the first group of peptide as mentioned above, the peptide which specifically inhibits the in vitro exchange activity of said protein towards any one of the proteins from the Rho-GTPase family, as determined for example in the GEF assay, and    a step of testing the peptide as mentioned above for its capacity to inhibit in intact cells the activity of the Rho-GEF on the appropriate GTPase, using for example the GTPase activity assay.    
     
     
         7 . Process for determining inhibitors of any one of the proteins from the Rho-GEFs family, comprising: 
 a step of contacting said protein for which an inhibitor is sought with the peptides from an aptamer library,    a step of recovering a first group of peptides which specifically bind to said protein, particularly to the catalytic domain of the GEF region of said protein,    a step of recovering, among the first group of peptides as mentioned above, the peptide which specifically inhibits the in vitro exchange activity of said protein towards RhoA, as determined for example in the GEF assay, and    a step of testing the peptide as mentioned above for its capacity to inhibit in intact cells the activity of the Rho-GEF on RhoA, using for example the GTPase activity assay.    
     
     
         8 . A polypeptide, characterized in that said polypeptide comprises or consists of any one of the following amino acid sequences: 
 the sequence SEQ ID NO: 2,    or any sequence derived from the sequence SEQ ID NO: 2 as defined above, especially by substitution, deletion or addition of one or more amino acids, with the proviso that said derived sequence inhibits TrioGEFD2,    or any homologous sequence of the sequence SEQ ID NO: 2 as defined above, preferably having a homology of at least about 30% with the sequence SEQ ID NO: 2, with the proviso that said homologous sequence inhibits TrioGEFD2,    or any fragment of any one of the sequences as defined above, said fragment being preferably consisted of at least about 40 to about 140, preferably of about 137 amino acids of the sequence SEQ ID NO: 2, with the proviso that said fragment inhibits TrioGEFD2.    
     
     
         9 . A polypeptide according to  claim 8 , characterized in that said polypeptide comprises or consists of any one of the following amino acid sequences: 
 the sequence SEQ ID NO: 4,    or any sequence derived from the sequence SEQ ID NO: 4 as defined above, especially by substitution, deletion or addition of one or more amino acids, with the proviso that said derived sequence inhibits TrioGEFD2,    or any homologous sequence of the sequence SEQ ID NO: 4 as defined above, preferably having a homology of at least about 30% with the sequence SEQ ID NO: 4, with the proviso that said homologous sequence inhibits TrioGEFD2,    or any fragment of any one of the sequences as defined above, said fragment being preferably consisted of at least about 25 to about 40, preferably of about 30 amino acids of the sequence SEQ ID NO: 4, with the proviso that said fragment inhibits TrioGEFD2.    
     
     
         10 . A fragment of a polypeptide according to  claim 9 , characterized in that said polypeptide is chosen among the following sequences: SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16 and SEQ ID NO: 18.  
     
     
         11 . A variant of a polypeptide according to  claim 8 , characterized in that said polypeptide is chosen among the following sequences: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58 and SEQ ID NO: 60.  
     
     
         12 . A polypeptide according to  claim 8  further comprising flanking parts consisting of fragments of the thioredoxin.  
     
     
         13 . A nucleotide sequence coding for a polypeptide according to  claim 8 .  
     
     
         14 . A nucleotide sequence according to  claim 13 , characterized in that said nucleotide comprises or consists of: 
 the nucleotide sequence represented by SEQ ID NO: 1 coding for the polypeptide represented by SEQ ID NO: 2,    or any nucleotide sequence derived from the sequence SEQ ID NO: 1 by degeneration of the genetic code, and coding for a polypeptide represented by SEQ ID NO: 2,    or any nucleotide sequence derived from the sequence SEQ ID NO: 1, especially by substitution, deletion or addition of one or more nucleotides, and coding for a derived polypeptide such as defined in  claim 4 ,    or any nucleotide sequence homologous of SEQ ID NO: 1, having preferably a homology of at least about 15%, in particular of at least about 20%, with the sequence SEQ ID NO: 1 coding for a polypeptide homologous of SEQ ID NO: 2, such as defined in  claim 4 ,    or any fragment of the nucleotide sequence SEQ ID NO: 1 or of any one of the above-defined nucleotide sequences, said fragment being preferably consisted of at least about 120 to about 420, preferably of about 411 nucleotides of the sequence SEQ ID NO: 1,    or any complementary nucleotide sequence of the above-mentioned sequences or fragments,    or any nucleotide sequence capable of hybridizing in stringent conditions with the complementary sequence of one of the above-mentioned sequences or fragments.    
     
     
         15 . A nucleotide sequence according to  claim 14 , characterized in that said nuceotide comprises or consists of: 
 the nucleotide sequence represented by SEQ ID NO: 3 coding for the polypeptide represented by SEQ ID NO: 4,    or any nucleotide sequence derived from the sequence SEQ ID NO: 3 by degeneration of the genetic code, and coding for a polypeptide represented by SEQ ID NO: 4,    or any nucleotide sequence derived from the sequence SEQ ID NO: 3, especially by substitution, deletion or addition of one or more nucleotides, and coding for a derived polypeptide such as defined in  claim 6 ,    or any nucleotide sequence homologous of SEQ ID NO: 3, having preferably a homology of at least about 15%, in particular of at least about 20%, with the sequence SEQ ID NO: 3 coding for a polypeptide homologous of SEQ ID NO: 4, such as defined in  claim 6 ,    or any fragment of the nucleotide sequence SEQ ID NO: 1 or of any one of the above-defined nucleotide sequences, said fragment being preferably consisted of at least about 75 to about 120, preferably of about 90 nucleotides of the sequence SEQ ID NO: 3,    or any complementary nucleotide sequence of the above-mentioned sequences or fragments,    or any nucleotide sequence capable of hybridizing in stringent conditions with the complementary sequence of one of the above-mentioned sequences or fragments.    
     
     
         16 . A fragment of a nucleotide sequence according to  claim 13 , characterized in that it is chosen among the following sequences: SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15 and SEQ ID NO: 17.  
     
     
         17 . A variant of a nucleotide sequence according to  claim 13 , characterized in that it is chosen among the following sequences: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57 and SEQ ID NO: 59.  
     
     
         18 . A recombinant vector, especially a plasmid, a cosmid, a phage or a DNA virus, containing a nucleotide sequence according to  claim 13 .  
     
     
         19 . A recombinant vector according to  claim 18 , containing the elements necessary for the expression in a host cell of the polypeptides coded by a nucleic acids according to inserted in said vector.  
     
     
         20 . A host cell, in particular chosen from bacteria, viruses, yeasts, fungi, plants or mammalian cells, the said host cell being transformed, in such a way that its genome contains a nucleotide sequence according to  claim 17 .  
     
     
         21 . A pharmaceutical composition characterized in that said composition comprises a polypeptide according to  claim 8 , in association with a pharmaceutically acceptable vehicle.  
     
     
         22 . A pharmaceutical composition according to  claim 21 , characterized in that said composition contains from about 700 μg to about 80 mg, preferably from about 7 to about 40 mg, as a unit dose, of the polypeptide according to  claim 8 .  
     
     
         23 . A method for the preparation of a drug for the treatment of pathologies that are related to the peripheral nervous system or to the central nervous system, in particular for the treatment of accidental paralysis related to the spinal marrow, comprising adminstering a polypeptide according to  claim 8.