Chimeric peptides for the regulation of GTPases

Abstract

Chimeric peptides or fusion proteins are disclosed that include a RhoGAP activity domain and at least one specificity domain that targets a specific Rho protein. The fusion proteins can be used to inhibit any GTPase activity within a cell. The fusion proteins are particularly advantageous for the treatment of cancer. The present invention generally relates to The present invention generally relates to chimeric peptides capable of regulating GTPases, and more particularly, to methods of targeting individual GTPases by using GTPase-activating proteins. Such proteins may be used for the treatment of cancers and other GTPase-related diseases. This invention relates to nucleic acid molecules and the encoded GTPase activating proteins, and variants thereof, and to the use of these molecules in the characterization, diagnosis, prevention, and treatment of cell signaling, immune, and cell proliferative disorders, particularly cancer. Disclosed herein are compounds and methods for regulating transcription of a selected gene. Methods are provided for constructing and for using such compounds to regulate transcription of a selected gene.

1 . A chimeric polypeptide, comprising: 
 a GAP activity domain; and    at least one targeting domain, wherein said targeting domain targets at least one specific GTPase protein.    
     
     
         2 . The chimeric polypeptide of  claim 1 , wherein said targeting domain is a C-terminal sequence from a GTPase. (is derived from a C-terminal sequence of GTPase.  
     
     
         3 . The chimeric polypeptide of  claim 1 , further comprising a membrane translocating protein domain.  
     
     
         4 . The chimeric polypeptide of  claim 1 , wherein said GTPase is a Rho GTPase.  
     
     
         5 . The chimeric polypeptide of  claim 1 , further comprising 6-12 amino acids at either end of the chimeric polypeptide.  
     
     
         6 . The chimeric polypeptide of  claim 2 , wherein said Rho C-terminal sequence is C-terminal to the GAP activity domain.  
     
     
         7 . The chimeric polypeptide of  claim 2  wherein said Rho C-terminal sequence is N-terminal to the GAP activity domain.  
     
     
         8 . The chimeric polypeptide of  claim 1 , wherein the GAP activity domain is a peptide sequence containing the minimum structural domain necessary for GTPase activating protein activity.  
     
     
         9 . The chimeric polypeptide of  claim 1 , wherein the GAP activity domain is a Rho family GAP selected from the group consisting of p50RhoGAP, p190, p122-RhoGAP, p190-A, p190-B, Oligophrenin-1, GRAF, MacGAP, Cdc42GAP (p50RhoGAP), SrGAP1-SrGAP3, n-chimaerin, CdGAP, Bcr, Abr, MgcRacGAP, ARAP1, ARAP2, RICH-1, NADRIN, 3BP-1, RhoGAP8, and their variants.  
     
     
         10 . The chimeric polypeptide of  claim 1 , wherein the GAP domain is a polypeptide fragment of a GAP GTPase domain from a GAP protein, wherein the fragment comprises at least 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 170, 200, 250 or more contiguous amino acids of the amino acid sequence of a GAP protein.  
     
     
         11 . The chimeric polypeptide of  claim 1 , wherein the specificity domain is derived from the C-terminus amino acid sequence of a GTPase selected from the group consisting of RhoA, RhoB, RhoC, Cdc42, Rac1, Rac2, Rac3, TC10, RhoG, RhoD, Chp, WRCH1, TCL, and RIF.  
     
     
         12 . The chimeric polypeptide of  claim 1 , wherein the specificity domain is a GTPase fragment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, or more contiguous amino acids of the C-terminus amino acid sequence of a GTPase.  
     
     
         13 . The chimeric polypeptide of  claim 2 , wherein the C-terminal sequenceis from one of the members of the RhoGTPase family.  
     
     
         14 . The chimeric polypeptide polypeptide of  claim 13 , wherein the RhoGTPase is selected from the group consisting of RhoA, RhoB, RhoC, Cdc42, Rac1, Rac2, Rac3, TC10, RhoG, RhoD, Chp, WRCH1, TCL, and RIF.  
     
     
         15 . The chimeric polypeptide of  claim 1 , wherein the GAP domain peptide is a polypeptide fragment of a GAP GTPase domain from a GAP protein, wherein the fragment comprises an amino acid sequence of the formula:  
         X N —GAP—X C    
       wherein “GAP” is the polypeptide fragment of a GAP GTPase domain from a GAP protein; 
 wherein X N  is an amino acid fragment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or more contiguous amino acids of the N-terminus amino acid sequence of a GAP protein; and  
 wherein X C  is an amino acid fragment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more contiguous amino acids of the C-terminus amino acid sequence of a GAP protein.  
 
     
     
         16 . The chimeric polypeptide of  claim 15 , wherein the XN is a polypeptide fragment wherein the fragment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more contiguous amino acids of an amino acid sequence selected from the group consisting of amino acids 1-1260 of SEQ ID NO: 2, amino acids 1-256 of SEQ ID NO: 4, amino acids 1-1013 of SEQ ID NO: 6, or fragments thereof;  
     
     
         17 . The chimeric polypeptide of  claim 15 , wherein XN is selected from the group consisting of amino acids 1249-1260 of SEQ ID NO: 2, amino acids 237-256 of SEQ ID NO: 4, amino acids 996-1013 of SEQ ID NO: 6, or fragments thereof.  
     
     
         18 . The chimeric polypeptide of  claim 15 , wherein X C  is an amino acid fragment comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more contiguous amino acids of the amino acids 1432-1513 of SEQ ID NO:2, amino acids 427-439 of SEQ ID NO:4, amino acids 1206-1227 of SEQ ID NO:6, or fragments thereof.  
     
     
         19 . The chimeric polypeptide of  claim 15 , wherein X C  is selected from the group consisting of amino acids 1432-1513 of SEQ ID NO:2, amino acids 427-439 of SEQ ID NO:4, amino acids 1206-1227 of SEQ ID NO:6, and fragments thereof.  
     
     
         20 . The chimeric polypeptide of  claim 1 , wherein the targeting polypeptide specificity domain is a peptide fragment wherein the fragment comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or more contiguous amino acids of a sequence selected from the group consisting of the amino acid sequences: SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and fragments thereof.  
     
     
         21 . The chimeric polypeptide of  claim 1 , wherein the targeting polypeptide fragment comprises an amino acid sequence selected from the group consisting of amino acids: SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and fragments thereof.  
     
     
         22 . The chimeric polypeptide of  claim 1 , wherein the chimeric polypeptide further comprising a membrane-translocating peptide.  
     
     
         23 . The chimeric polypeptide of  claim 22 , wherein the membrane-translocating peptide comprises a membrane-associating isoprenylation modification.  
     
     
         24 . The chimeric polypeptide of  claim 22  wherein said membrane translocating protein comprises a lipid modified peptide.  
     
     
         25 . The chimeric peptide of claim. 22 wherein said membrane translocating protein is a peptide of about 8 to about 50 residues.  
     
     
         26 . The chimeric polypeptide of  claim 22 , wherein the membrane-translocating peptide is a peptide of about 8 to about 50 residues comprising at least eight consecutive residues of an amino acid sequence selected from the group consisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and fragments thereof.  
     
     
         27 . The chimeric polypeptide of  claim 22 , wherein the membrane-translocating peptide is an amino acid sequence having about 8 to about 24 residues comprising at least eight consecutive residues of an amino acid sequence selected from the group consisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, and fragments thereof.  
     
     
         28 . The chimeric polypeptide of  claim 22 , wherein the membrane-translocating peptide is a sequence of amino acids generally of a length of about 8 to about 50 or more amino acid residues, wherein at least 55% of the residues are hydrophobic such that they have a hydrophobic, lipid-soluble portion.  
     
     
         29 . The chimeric polypeptide of  claim 22 , wherein the membrane translocation sequence the amino acid sequence is selected from the group consisting of Pro-Thr-Arg-Gln-Gln-Lys-Arg-Pro-Cys-Ser-Leu-Leu (SEQ ID NO:21), Pro-Gln-Pro-Thr-Arg-Gln-Gln-Lys-Arg-Pro-Cys-Ser-Leu-Leu, (SEQ ID NO:22), Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Thr-Arg-Gln-Gln-Lys-Arg-Pro, (SEQ ID NO:23), Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Arg-Pro-Arg-Gln-Thr-Gln-Lys, (SEQ ID NO:24), and Gly-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Gly-Thr-Arg-Gln-Gln-Lys-Arg-Pro-Cys-Leu-Leu (SEQ ID NO:25).  
     
     
         30 . The chimeric polypeptide of  claim 1 , wherein the chimeric polypeptide comprises at least 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250 amino acids (e.g., contiguous amino acids) of an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12, and SEQ ID NO: 13.  
     
     
         31 . The chimeric polypeptide of  claim 1 , wherein the chimeric polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, or SEQ ID NO:13.  
     
     
         32 . A composition comprising a polypeptide according to  claim 1  in a pharmaceutically acceptable carrier.  
     
     
         33 . A method of treating a disease related to up-regulation of GTPase activity in a mammal, comprising administering to the mammal a composition of  claim 29  in an amount effective to reduce the GTPase activity.  
     
     
         34 . The method of  claim 30 , wherein the disease is a GTP hydrolysis-related disorder.  
     
     
         35 . A method for modulating GTPase activity in a cell, comprising: 
 contacting said cell with an active compound that modulates GTPase activity, wherein the active compound is a chimeric polypeptide comprising: a) a GAP activity domain and b) at least one specificity domain.    
     
     
         36 . A method for the treatment of cancer in a patient, comprising Administering the composition of  claim 29  to said patient in an amount effective to inhibit GTPase activity.  
     
     
         37 . The method of  claim 33 , wherein said cancer is characterized by increased activity of a GTPase.