Specific inhibitors of DNA methyltransferase

Abstract

The invention provides novel mechanism-based inhibitors of DNA methyltransferase enzyme, and diagnostic and therapeutic uses for the same. The novel inhibitors according to the invention form stable, noncovalent complexes with DNA methyltransferase enzyme in a manner which is independent of S-adenosylmethionine.

What is claimed is:  
     
         1 . An inhibitor of DNA methytransferase enzyme having the general structure:  
       
         
           
           
               
               
           
         
       
       wherein each N is independently any nucleotide, n is a number from 0-20, C is 5-methylcytidine, G is guanidine, y is a number from 0-20, L is a linker, each D is a nucleotide that is complementary to an N such that Watson-Crick base pairing takes place between that D and the N such that the N n —C—G—N y  and the D n —G—B—D y  form a double helix, B is cytosine, inosine, uridine, 5-bromocytosine or 5-fluorocytosine, orabasic deoxyribose, the linkage between B and G is a phosphorothioate or phosphorodithioate linkage, dotted lines between nucleotides represent hydrogen bonding between the nucleotides, and the total number of nucleotides ranges from about 10 to about 50.  
     
     
         2 . The inhibitor of DNA methytransferase enzyme according to    claim 1   , wherein the inhibitor is labeled.  
     
     
         3 . The inhibitor of DNA methytransferase enzyme according to    claim 1   , wherein L is a nucleoside or an oligonucleotide region having from 2 to about 10 nucleotides.  
     
     
         4 . The inhibitor of DNA methytransferase enzyme according to    claim 3    comprising at least one internucleoside linkage selected from the group consisting of phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof.  
     
     
         5 . The inhibitor of DNA methytransferase enzyme according to    claim 3    comprising at least one phosphorothioate internucleoside linkage.  
     
     
         6 . The inhibitor of DNA methyltransferase enzyme according to    claim 1   , having the sequence selected from the group consisting of SEQ. ID. NO. 1, SEQ. ID. NO. 2, SEQ. ID. NO. 3, SEQ. ID. NO. 4, SEQ. ID. NO. 5, SEQ. ID. NO. 6, SEQ. ID. NO. 7, SEQ. ID. NO. 8, SEQ. ID. NO. 9, SEQ. ID. NO. 10, SEQ. ID. NO. 11, SEQ. ID. NO. 12, SEQ. ID. NO. 13, 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, SEQ. ID. NO. 21, SEQ. ID. NO. 22, SEQ. ID. NO. 23, SEQ. ID. NO. 24, SEQ. ID. NO. 25, SEQ. ID. NO. 26, SEQ. ID. NO. 27, SEQ. ID. NO. 28, SEQ. ID. NO. 29, SEQ. ID. NO. 30, SEQ. ID. NO. 31, SEQ. ID. NO. 32, SEQ. ID. NO. 33, SEQ. ID. NO. 34, SEQ. ID. NO. 35, SEQ. ID. NO. 36, SEQ. ID. NO. 37, SEQ. ID. NO. 38, SEQ. ID. NO. 39, SEQ. ID. NO. 40 and SEQ. ID. NO. 41, wherein C is cytidine, T is thymidine, A is adenosine, G is guanine, m is a methyl group at the 5-position of cytosine, B is cytosine, inosine, uridine, 5-bromocytidine, or 5-fluorouridine, X is any base and F is 5-fluorocytosine.  
     
     
         7 . An inhibitor of DNA methytransferase enzyme having the general structure  
       
         
           
           
               
               
           
         
       
       wherein each N is independently any nucleotide, n is a number from 0-20, C is 5-methylcytidine, G is guanidine, y is a number from 0-20, L is a linker, each D is a nucleotide that is complementary to an N such that Watson-Crick base pairing takes place between that D and the N such that the N n —C—G—N y  and the D n —G—B—D y  form a double helix, B is cytosine, inosine, uridine, 5-bromocytosine, abasic deoxyribose, or 5-fluorocytosine, dotted lines between nucleotides represent hydrogen bonding between the nucleotides, B and G are linked by a phosphorothioate or phosphorodithioate linkage and the total number of nucleotides ranges from about 10 to about 50, X is an antisense oligonucleotide of from about 10 to about 50 nucleotides in length, which is complementary to a portion of an RNA encoding DNA MTase enzyme, and L can optionally be X.  
     
     
         8 . A diagnostic method for determining whether a particular sample of cells is cancerous, the method comprising preparing an extract from the cells in the cell sample, adding a labeled inhibitor according to    claim 2   , measuring the extent of formation of a complex between the labeled inhibitor and DNA MTase enzyme, normalizing the level of such complex formation to the number of cells represented in the sample to obtain a normalized complex formation value, and comparing the normalized complex formation value to a normalized complex formation value for non-cancerous and/ or cancerous cell samples.  
     
     
         9 . The method according to    claim 8   , wherein the extract is a nuclear extract.  
     
     
         10 . A method for inhibiting tumorigenesis in an animal, including a human, comprising administering to the animal, which has cancer cells present in its body, a therapeutically effective amount of an inhibitor according to    claim 1    for a therapeutically effective period of time.