Identification of modulators of serine protease inhibitor kazal and their use as anti-cancer and anti-viral agents

Abstract

This disclosure describes a relevant etiology of cancer and a novel anti-cancer therapeutic strategy, based on the discovery that a protein named serine protease inhibitor (SPIK/SPINK/PSTI) was up-regulated by hepatitis B and C virus infections consequently suppressing the cell apoptosis. Accordingly, the present disclosure provides, inter alia, an inhibitor of SPIK and/or a technology of suppression of over-expression of SPIK in cells. The inhibitors include: 1) chemical compounds, which can inhibit SPIK transcripts, protein activity, and gene expression, 2) SPIK siRNA (RNAi gene silence or dsRNA of SPIK, 3) DNA anti-sense and anti-SPIK antibody. Further, this disclosure provides methods of using the inhibitor as an anti-cancer agent to re-instate cancer cell apoptosis (e.g., serine protease dependent cell apoptosis).

1 . A method for identifying a Serine Protease Inhibitor-Kazal (SPIK) modulatory compound, wherein the modulatory compound modulates SPIK serine protease inhibitory activity, the method comprising:
 (a) contacting a serine protease with SPIK protein in the presence of a labeled serine protease substrate and measuring a first amount of serine protease activity;   (b) contacting a serine protease with SPIK protein in the presence of a labeled serine protease substrate and a test agent and measuring a second amount of serine protease activity;   (c) comparing the first amount and second amounts of serine protease activity to identify a compound which modulates SPIK serine protease inhibitory activity, wherein the test agent is selected from the group consisting of small molecules, proteins, nucleic acids, and antibodies.   
     
     
         2 . The method of  claim 1  wherein the label is fluorescent. 
     
     
         3 . The method of  claim 1  wherein the label can be detected by FRET. 
     
     
         4 . The method of  claim 1  wherein the modulation is selected from the group consisting of inhibition and activation. 
     
     
         5 . The method of  claim 1 , wherein a plurality of test agents are screened simultaneously. 
     
     
         6 . The method of  claim 1 , wherein the SPIK polypeptide consists essentially of a polypeptide encoded by a sequence of nucleotides selected from the group consisting of a sequence of nucleotides that:
 (a) is set forth in SEQ ID NO. 3;   (b) hybridizes under conditions of high stringency to a nucleic acid of SEQ ID NO: 3;   (c) hybridizes under conditions of high stringency to a nucleic acid complementary to the nucleic acid of SEQ ID NO: 3;   (d) encodes a biologically active variant of the polypeptide of SEQ ID NO: 4; and   (e) comprises degenerate codons of the sequences of nucleotides of (a), (b), (c) or (d).   
     
     
         7 . A compound which modulates SPIK serine protease inhibitory activity identified by the method of  claim 1 . 
     
     
         8 . A kit comprising the compound of  claim 7  for use in treating a disease selected from the group consisting of HBV infection, HCV infection, hepatitis, cancer, and hepatic cancer, in an animal and a [pharmaceutically acceptable carrier. 
     
     
         9 . An siRNA which is a member selected from the group consisting of L71 siRNA comprising sense and anti-sense oligonucleoside with the SPIK sequence of SEQ ID NO: 1, as shown in  FIGS. 9 , and L183 comprising sense and anti-sense oligonucleoside with the SPIK sequence SEQ ID NO: 2, as shown in  FIG. 9 . 
     
     
         10 . A diagnostic kit comprising an antibody to SEQ ID NO:4 or SEQ ID NO:5 to diagnose patients exhibiting hepatitis C virus infection symptoms or at risk for developing a disease, wherein the disease is hepatitis B virus infection, hepatitis C virus infection, hepatitis, cancer, or hepatic cancer. 
     
     
         11 . An inhibitor of SPIK expression or function used for treatment of a disease which is hepatitis B virus infection, hepatitis C virus infection, hepatitis, cancer, or hepatic cancer. 
     
     
         12 . A method of identifying therapeutically effective compounds comprising determining the ability of test agents to modulate SPIK serine protease inhibitory activity in apoptosis sensitive cells expressing SPIK exposed to apoptotic agents, wherein the test agent is a compound determined to have potential therapeutic efficacy if the apoptosis of the cells in response to the agents changes compared to control cells not exposed to the test agent. 
     
     
         13 . The method of  claim 12  wherein the cells are selected from the group consisting of hepatic cells, cancer cells, and hepatic cancer cells. 
     
     
         14 . A method for identifying a compound which is an inhibitor of SPIK expression comprising: (a) contacting a test agent in vitro with a cell that expresses SPIK protein; (b) determining the expression level of the SPIK protein in the cell; and (c) determining whether the expression level determined in step (b) is lower than the SPIK protein expression level determined in the absence of the test agent, such lower expression level indicating that the compound is an inhibitor of SPIK expression. 
     
     
         15 . The method of  claim 14 , wherein the SPIK protein comprises the amino acid sequence as set forth in SEQ ID NO: 4. 
     
     
         16 . The method of  claim 14 , wherein the cell is selected from the group consisting of a liver cell, a cancer cell, and a hepatic cancer cell. 
     
     
         17 . A method of screening for a compound that inhibits, diminishes, or modulates anti-apoptotic activity in an eukaryotic cell, said method comprising: (a) introducing into eukaryotic cells an expression vector comprising a polynucleotide encoding a SPIK polypeptide, or fragment thereof, having serine protease inhibitory activity, (b) treating one fraction of said cells with a test agent and leaving a second fraction of said cells untreated as a control, (c) treating both fractions of cells with an agent that induces cell death, and (d) detecting an inhibition, diminution or modulation in anti-apoptotic activity in the fraction of cells treated with the test agent in comparison to the untreated control, thereby screening for a test agent which is a compound that inhibits, diminishes, or modulates anti-apoptotic activity in an eukaryotic cell. 
     
     
         18 . The method of  claim 17 , wherein said test agent is selected from the group consisting of small molecules, proteins, nucleic acids, and antibodies. 
     
     
         19 . The method of  claim 17 , wherein the SPIK polypeptide consists essentially of a polypeptide encoded by a sequence of nucleotides selected from the group consisting of a sequence of nucleotides that:
 (a) is set forth in SEQ ID NO. 3;   (b) hybridizes under conditions of high stringency to a nucleic acid of SEQ ID NO: 3;   (c) hybridizes under conditions of high stringency to a nucleic acid complementary to the nucleic acid of SEQ ID NO: 3;   (d) encodes a biologically active variant of the polypeptide of SEQ ID NO: 4; and   (e) comprises degenerate codons of the sequences of nucleotides of (a), (b), (c) or (d).