Sequence and chemistry requirements for a novel aptameric oligonucleotide inhibitor of EGF receptor tyrosine kinase activity.
We have previously identified a phosphorothioate oligonucleotide (PS-ODN) that inhibited epidermal growth factor receptor tyrosine kinase (TK) activity both in cell fractions and in intact A431 cells. Since ODN-based TK inhibitors may have anti-cancer applications and may also help understand the non-antisense mediated effects of PS-ODNs, we have further studied the sequence and chemistry requirements of the parent PS-ODN (sequence: 5'-GGA GGG TCG CAT CGC-3') as a sequence-dependent TK inhibitor. Sequence deletion and substitution studies revealed that the 5'-terminal GGA GGG hexamer sequence in the parent compound was essential for anti-TK activity in A431 cells. Site-specific substitution of any G with a T in this 5'-terminal motif within the parent compound caused a significant loss in anti-TK activity. The fully PS-modified hexameric motif alone exhibited equipotent activity as the parent 15-mer whereas phosphodiester (PO) or 2'-O-methyl-modified versions of this motif had significantly reduced anti-TK activity. Further, T substitutions within the two 5'-terminal G residues of the hexameric PS-ODN to produce a sequence, TTA GGG, representing the telomeric repeats in human chromosomes, also did not exhibit a significant anti-TK activity. Multiple repeats of the active hexameric motif in PS-ODNs resulted in more potent inhibitors of TK activity than the parent ODN. These results suggested that PS-ODNs, but not PO or 2'-O-methyl modified ODNs, containing the GGA GGG motif can exert potent anti-TK activity which may be desirable in some anti-tumor applications. Additionally, the presence of this previously unidentified motif in antisense PS-ODN constructs may contribute to their biological effects in vitro and in vivo and should be accounted for in the design of the PS-modified antisense ODNs.
Pharmaceutical Sciences Research Institute, Aston University, Aston Triangle, Birmingham, UK. s.akhtar@cf.ac.uk
Saghir
S
Akhtar
Debbie
D
Dunnion
David
D
Poyner
John
J
Ackroyd
Mike
M
Bibby
John
J
Double
D000818:Animals; D001483:Base Sequence; D004195:Disease Models, Animal; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D009154:Mutation; D009368:Neoplasm Transplantation; D009374:Neoplasms, Experimental / Q000517:prevention & control; D020319:Oligodeoxyribonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D013873:Thionucleotides / Q000737:chemistry / Q000494:pharmacology / Q000627:therapeutic use; D023041:Xenograft Model Antitumor Assays
D020319:Oligodeoxyribonucleotides, Antisense; D013873:Thionucleotides; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases