Design of a Hairpin Polyamide, ZT65B, for Targeting the Inverted CCAAT Box (ICB) Site in the Multidrug Resistant (MDR1) Gene

Abstract

A novel hairpin polyamide, ZT65B, containing a 3-methylpicolinate moiety was designed to target the inverted CCAAT box (ICB) of the human multidrug resistance 1 gene (MDR1) promoter. Binding of nuclear factor-Y (NF-Y) to the ICB site upregulates MDR1 gene expression and is, therefore, a good target for anticancer therapeutic agents. However, it is important to distinguish amongst different promoter ICB sites so that only specific genes will be affected. All ICB sites have the same sequence but they differ in the sequence of the flanking base pairs, which can be exploited in the design of sequence-specific polyamides. To test this hypothesis, ten ICB-containing DNA hairpins were designed with different flanking base pairs; the sequences ICBa and ICBb were similar to the 3'-ICB site of MDR1 (TGGCT). Thermal-denaturation studies showed that ZT65B effectively targeted ICBa and ICBb (DeltaTM=6.5 and 7.0 degrees C) in preference to the other DNA hairpins (<3.5 degrees C), with the exception of ICBc (5.0 degrees C). DNase I-footprinting assays were carried out with the topoisomerase IIalpha-promoter sequence, which contains five ICB sites; of these, ICB1 and ICB5 are similar to the ICB site of MDR1. ZT65B was found to selectively bind ICB1 and ICB5; footprints were not observed with ICB2, ICB3, or ICB4. A strong, positive induced ligand band at 325 nm in CD studies confirmed that ZT65B binds in the DNA minor groove. The selectivity of ZT65B binding to hairpins that contained the MDR1 ICB site compared to one that did not (ICBd) was confirmed by surface-plasmon studies, and equilibrium constants of 5x10(6)-1x10(7) and 4.6x10(5) M-1 were obtained with ICB1, ICB5,and ICB2 respectively. ZT65B and the previously published JH37 (J. A. Henry, et al. Biochemistry 2004, 43, 12 249-12 257) serve as prototypes for the design of novel polyamides. These can be used to specifically target the subset of ubiquitous gene elements known as ICBs, and thereby affect the expression of one or a few proteins.

Facile Dimer Synthesis for DNA-Binding Polyamide Ligands

“…Pyrrole-imidazole-containing polyamides are a versatile class of DNA-binding ligands. The syntheses of dimeric building blocks reported herein solve the difficulties associated with direct imidazole coupling instead of moving them to a different step, 3,11,12 reduce ligand synthesis times by half, may be broadly applicable for synthesis of other heterocyclic polyamides, 14,15 and result in overall ligand yields higher than previously reported, 3 thereby allowing facile synthesis of this useful class of compounds.…”

“…However, the imidazole amine 4 is a poor nucleophile, with reported yields as low as 5% for on-resin coupling. 10 Common synthetic approaches include varying the activation chemistry, 3,11,12 heating the resin, 13 or prolonging individual coupling steps to as long as 60 h. 11,13 However, none of these strategies directly address the inherent difficulty of coupling an electron-rich electrophile to an electron-poor nucleophile. To facilitate faster synthesis of polyamide ligands with reduced need for purification, as well as to obviate the difficulties traditionally encountered with on-resin coupling to Im monomers, 2,3,11,12 we report a simple and uniform synthesis of the eight terminal and Fmoc-protected Py and Im dimer combinations, as well as representative syntheses for two previously unpublished polyamide ligands, using these dimers.…”

“…However, the imidazole amine 4 is a poor nucleophile, with reported yields as low as 5% for on-resin coupling . Common synthetic approaches include varying the activation chemistry, ,, heating the resin, or prolonging individual coupling steps to as long as 60 h. , However, none of these strategies directly address the inherent difficulty of coupling an electron-rich electrophile to an electron-poor nucleophile.…”

An overview of recent advances in duplex DNA recognition by small molecules

“…In order to target the inverted CCAAT box (ICB) of the human multidrug resistance 1 gene (MDR1) promoter and to distinguish between different promoter ICB sites, several ICB-containing DNA hairpin polyamides were designed with different flanking base pairs. It was confirmed via thermal-denaturation studies and DNase I-footprinting assays that one of these conjugates containing a 3-methylpicolinate moiety (ZT65B, compound 18 ) binds in the minor groove and effectively targeted ICBa and ICBb, similar to the 3 ' -ICB site of MDR1 (TGGCT) [ 77 ].…”

DNA microstructure influences selective binding of small molecules designed to target mixed-site DNA sequences

“…Small molecules that bind in the minor groove of DNA have been validated for this approach from studies using synthetic polyamides (22–24). However, polyamides have limitations such as aggregation and cell uptake and a wider variety of agents is needed for diverse biological systems (25,26).…”