title: Targeting the ets binding site of the HER2/neu promoter with pyrrole-imidazole polyamides.
abstract: Three DNA binding polyamides () were synthesized that bind with high affinity (K(a) = 8.7. 10(9) m(-1) to 1.4. 10(10) m(-1)) to two 7-base pair sequences overlapping the Ets DNA binding site (EBS; GAGGAA) within the regulatory region of the HER2/neu proximal promoter. As measured by electrophoretic mobility shift assay, polyamides binding to flanking elements upstream () or downstream (2 and 3) of the EBS were one to two orders of magnitude more effective than the natural product distamycin at inhibiting formation of complexes between the purified EBS protein, epithelial restricted with serine box (ESX), and the HER2/neu promoter probe. One polyamide, 2, completely blocked Ets-DNA complex formation at 10 nm ligand concentration, whereas formation of activator protein-2-DNA complexes was unaffected at the activator protein-2 binding site immediately upstream of the HER2/neu EBS, even at 100 nm ligand concentration. At equilibrium, polyamide 1 was equally effective at inhibiting Ets/DNA binding when added before or after in vitro formation of protein-promoter complexes, demonstrating its utility to disrupt endogenous Ets-mediated HER2/neu preinitiation complexes. Polyamide 2, the most potent inhibitor of Ets-DNA complex formation by electrophoretic mobility shift assay, was also the most effective inhibitor of HER2/neu promoter-driven transcription measured in a cell-free system using nuclear extract from an ESX- and HER2/neu-overexpressing human breast cancer cell line, SKBR-3.
authors.affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
authors.forename: S Y
authors.initials: SY
authors.lastname: Chiang
authors.forename: R W
authors.initials: RW
authors.lastname: Burli
authors.forename: C C
authors.initials: CC
authors.lastname: Benz
authors.forename: L
authors.initials: L
authors.lastname: Gawron
authors.forename: G K
authors.initials: GK
authors.lastname: Scott
authors.forename: P B
authors.initials: PB
authors.lastname: Dervan
authors.forename: T A
authors.initials: TA
authors.lastname: Beerman
mesh_terms: D000577:Amides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D018983:DNA Footprinting; D018734:Genes, erbB-2; D006801:Humans; D007093:Imidazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D011758:Pyrroles / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D000577:Amides; D007093:Imidazoles; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011758:Pyrroles; D018719:Erb-b2 Receptor Tyrosine Kinases; D014157:Transcription Factors