Inducible phosphofructokinase and the warburg effect

Abstract

There is disclosed a cancer malignancy diagnostic assay comprising obtaining a sample of a body fluid or tissue, performing a sequence identity assay to look for the presence of iPFK-2 specific sequences; an anticancer pharmaceutical composition comprising a specific antisense oligonucleotide to the inventive isolated iPFK-2 sequence and a pharmaceutically acceptable oligonucleotide carrier; and a method for finding therapeutically active anti-cancer compounds comprising screening compounds for activity to inhibit iPFK-2, preferably kinase activity.

We claim:  
     
         1 . A cancer malignancy diagnostic assay comprising obtaining a sample of a body or tumor fluid or tissue, performing a sequence identity assay to look for the presence of iPFK-2 specific sequences.  
     
     
         2 . The cancer malignancy diagnostic assay of  claim 1  wherein the assay is selected from the group consisting of PCR (polymerase chain reaction) assays, ELISA immunologic assays, hybridization assays, and combinations thereof.  
     
     
         3 . An anticancer pharmaceutical composition comprising a specific antisense oligonucleotide of at least 10 bases complimentary to the iPFK-2 cDNA sequence [SEQ ID NO. 11] and a pharmaceutically acceptable oligonucleotide carrier.  
     
     
         4 . The anticancer pharmaceutical composition of  claim 3  wherein the antisense oligonucleotide is a 15-50 base oligonucleotide incorporating an oligonucleotide sequence selected from the group consisting of: 5′-CCAACGGCATCTTCGCGGCT-3′ [SEQ ID NO: 2], 5′-GTCAGTTCCAACGGCATCTT-3′ [SEQ ID NO: 4], and combinations thereof.  
     
     
         5 . A method for screening for anticancer therapeutic agents that inhibit kinase enzymatic activity of iPFK-2, comprising: 
 (a) expressing an iPFK-2 enzyme or kinase domain thereof from the cDNA sequence of SEQ ID NO. 11;    (b) purifying and isolating the expressed iPFK-2 enzyme or kinase domain thereof;    (c) providing a candidate therapeutic agent and fructose 6-phosphate substrate; and    (d) measuring formation of fructose 2,6-bisphosphate product as a measure of kinase enzymatic activity.    
     
     
         6 . The method of  claim 5  further comprising the step of determining that the therapeutic agent does not inhibit the kinase enzymatic activity of liver of PFK-2.  
     
     
         7 . An antibody that binds to an epitope of iPFK-2, wherein the epitope comprises a sequence of amino acids corresponding to amino acids 505-515 of iPFK-2.  
     
     
         8 . An isolated DNA sequence encoding an iPFK-2 polypeptide and comprising a coding region of the sequence described in SEQ ID NO.: 11.  
     
     
         9 . An isolated iPFK-2 polypeptide expressed by the cDNA sequence described in SEQ ID NO.: 11.  
     
     
         10 . An isolated DNA sequence encoding a kinase domain of an iPFK-2 polpeptide comprising base pairs 47-797 of SEQ ID NO. 11.  
     
     
         11 . An isolated iPFK-2 kinase domain polypeptide expressed by base pairs 47-797 of SEQ ID NO. 11.  
     
     
         12 . An anti-inflammatory pharmaceutical composition comprising a specific antisense oligonucleotide of at least 10 bases complimentary to an iPFK-2 cDNA sequence [SEQ ID NO. 11] and a pharmaceutically acceptable oligonucleotide carrier.  
     
     
         13 . The anti-inflammatory pharmaceutical composition of  claim 12  wherein the antisense oligonucleotide is a 15-50 base oligonucleotide incorporating an oligonucleotide sequence selected from the group consisting of: 5′-CCAACGGCATCTTCGCGGCT-3′[SEQ ID NO: 2], 5′-GTCAGTTCCAACGGCATCTT-3′[SEQ ID NO: 4], and combinations thereof.  
     
     
         14 . A method for treating inflammatory diseases, comprising administering an effective amount of an iPFK-2 antagonist.  
     
     
         15 . The method of  claim 14  wherein the iPFK-2 antagonist is an inhibitor of iPFK-2 enzymatic activity, an anti-iPFK-2 antibody, or an iPFK-2 antisense molecule.  
     
     
         16 . A method for treating cancers, comprising administering an effective amount of an iPFK-2 antagonist.  
     
     
         17 . The method of  claim 15  wherein the iPFK-2 antagonist is an inhibitor of iPFK-2 enzymatic activity, an anti-iPFK-2 antibody, or an iPFK-2 antisense molecule.