Bacterial toxins and uses thereof as ras specific proteases for treating cell proliferation diseases and disorders

Abstract

Disclosed are bacterial toxins and uses thereof as specific proteases for Ras sarcoma oncoproteins (Ras proteins). The bacterial toxins may be modified for use as pharmaceutical agents for treating Ras-dependent diseases and disorders including cell proliferation diseases and disorders such as cancer.

1 . A method of treating a cell proliferative disease or disorder in a subject, the method comprising administering to the subject a therapeutic polypeptide comprising the amino acid sequence of a DUF5 protease, a portion of a DUF5 protease comprising the C2A subdomain and/or the C2B subdomain, or a variant thereof. 
     
     
         2 . The method of  claim 1 , wherein the cell proliferative disease or disorder is associated with an activating mutation in a Ras protein. 
     
     
         3 . The method of  claim 1 , wherein the Ras protein is selected from the group consisting of KRAS, HRAS, or NRAS. 
     
     
         4 . The method of  claim 2 , wherein the activating mutation is present in codon 12, 13, or 61 of the Ras protein. 
     
     
         5 . The method of  claim 1 , wherein the cell proliferative disease or disorder is a tumor of a primary tissue selected from the group consisting of adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, prostate, skin, testis, thymus, and uterus. 
     
     
         6 . The method of  claim 1 , wherein the DUF5 protease or the portion thereof is selected from the group consisting of  Vibrio vulnificus  DUF5 protease or a portion thereof,  Vibrio  ordalii DUF5 protease or a portion thereof,  Vibrio cholerae  DUF5 protease or a portion thereof,  Vibrio spendidus  DUF5 protease or a portion thereof,  Moritella dasanensis  DUF5 protease or a portion thereof,  Aeromonas salmonicida  DUF5 protease or a portion thereof,  Aeromonas hydrophila  DUF5 protease or a portion thereof,  Photorhabdus temperata  DUF5 protease or a portion thereof,  Xenorhabdus nematophila  DUF5 protease or a portion thereof,  Photorhabdus luminescens  DUF5 protease or a portion thereof,  Photorhabdus asymbiotica  DUF5 protease or a portion thereof,  Yersinia kristensenii  DUF5 protease or a portion thereof, and  Pasteurella multocida  DUF5 protease or a portion thereof. 
     
     
         7 . The method of  claim 1 , wherein the therapeutic polypeptide comprises the amino acid sequence of any of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 or an amino acid sequence having at least 50% sequence identity with the amino acid sequence of any of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 and the therapeutic polypeptide cleaves a Ras protein. 
     
     
         8 . The method of  claim 1 , wherein the therapeutic polypeptide comprises the amino acid sequence of any of SEQ ID NOs:2, 4, 6, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 or an amino acid sequence having at least 50% sequence identity with the amino acid sequence of any of SEQ ID NOs:2, 4, 6, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 and the therapeutic polypeptide cleaves a Ras protein. 
     
     
         9 . The method of  claim 7 , wherein the therapeutic polypeptide cleaves the Ras protein between a tyrosine at amino acid position 32 and an aspartic acid at amino acid position 33. 
     
     
         10 . The method of  claim 8 , wherein the therapeutic polypeptide cleaves the Ras protein between a tyrosine at amino acid position 32 and an aspartic acid at amino acid position 33. 
     
     
         11 . The method of  claim 1 , wherein the therapeutic polypeptide is formulated as a pharmaceutical composition for delivering the therapeutic polypeptide to proliferating cells. 
     
     
         12 . The method of  claim 11 , wherein the therapeutic polypeptide comprises the DUF5 protease or the portion of the DUF5 protease fused or complexed with a carrier in the pharmaceutical composition that facilitates transport of the DUF5 protease or the portion of the DUF5 protease into the proliferating cells. 
     
     
         13 . The method of  claim 12 , wherein the therapeutic polypeptide comprises the DUF5 protease or the portion of the DUF5 protease fused to anthrax toxin lethal factor N-terminus (LF N ). 
     
     
         14 . The method of  claim 13 , wherein the therapeutic polypeptide is contacted with anthrax toxin protective antigen (PA) to form a complex that is delivered to the cytosol of proliferating cells. 
     
     
         15 . A pharmaceutical composition comprising a DUF5 protease or a portion of the DUF5 protease comprising the C2A subdomain and/or the C2B subdomain fused or complexed with a carrier that facilitates transport of the DUF5 protease or the portion of the DUF5 protease into proliferating cells. 
     
     
         16 . The pharmaceutical composition of  claim 15 , comprising a fusion protein comprising the DUF5 protease or the portion of the DUF5 protease fused at its N-terminus to anthrax toxin lethal factor N-terminus (LF N ). 
     
     
         17 . The pharmaceutical composition of  claim 16  further comprising anthrax toxin protective antigen (PA) which forms a complex with the fusion protein and the complex is delivered to the cytosol of proliferating cells. 
     
     
         18 . The pharmaceutical composition of  claim 15 , wherein the DUF5 protease or the portion thereof is selected from the group consisting of  Vibrio vulnificus  DUF5 protease or a portion thereof,  Vibrio  ordalii DUF5 protease or a portion thereof,  Vibrio cholerae  DUF5 protease or a portion thereof,  Vibrio spendidus  DUF5 protease or a portion thereof,  Moritella dasanensis  DUF5 protease or a portion thereof,  Aeromonas salmonicida  DUF5 protease or a portion thereof,  Aeromonas hydrophila  DUF5 protease or a portion thereof,  Photorhabdus temperata  DUF5 protease or a portion thereof,  Xenorhabdus nematophila  DUF5 protease or a portion thereof,  Photorhabdus luminescens  DUF5 protease or a portion thereof,  Photorhabdus asymbiotica  DUF5 protease or a portion thereof,  Yersinia kristensenii  DUF5 protease or a portion thereof, and  Pasteurella multocida  DUF5 protease or a portion thereof. 
     
     
         19 . The pharmaceutical composition of  claim 15 , wherein the DUF5 protease or the portion of the DUF5 protease comprises the amino acid sequence of any of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, and 27 or an amino acid sequence having at least 50% sequence identity with the amino acid sequence of SEQ ID NO:1 and the DUF5 protease cleaves a Ras protein. 
     
     
         20 . The pharmaceutical composition of  claim 15 , wherein the DUF5 protease or the portion of the DUF5 protease comprises the amino acid sequence of any of SEQ ID NOs:2, 4, 6, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 or an amino acid sequence having at least 50% sequence identity with the amino acid sequence of any of SEQ ID NOs:2, 4, 6, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, and 28 and the DUF5 protease or the portion of the DUF5 protease cleaves a Ras protein.