Targeting the Inverted CCAAT Box 2 in the Topoisomerase IIα Promoter by JH-37, an Imidazole−Pyrrole Polyamide Hairpin:  Design, Synthesis, Molecular Biology, and Biophysical Studies

Abstract

The topoisomerase IIalpha promoter is regulated through transcription factor interactions with five inverted CCAAT boxes (ICBs). In confluent cancer cells, binding of nuclear factor Y to ICB2 represses the expression of this gene, contributing to resistance to topoisomerase II poisons. The ICB sites within the topoisomerase IIalpha promoter are, therefore, potential targets for the design of anticancer drugs and gene control agents. The synthesis and DNA binding properties of a hairpin polyamide molecule (JH-37) that targets 5'-TTGGT-3' found in ICB2 and ICB3 sites are described. Gel shift and DNase I footprinting studies on the topoisomerase IIalpha promoter showed JH-37 to preferentially bind to ICB2,3 and ICB1 sites. The larger DeltaT(M) values for ICB2,3 (8-9 degrees C) over ICB1,4,5 (4-5 degrees C) indicated a preference of JH-37 for ICB2,3. CD titration studies confirmed the binding of JH-37 to the minor groove, with a 1:1 binding stoichiometry. Results from SPR studies showed JH-37 to bind most strongly to ICB2 (K = 3 x 10(7) M(-1)), followed by ICB1, the non-ICB sequence (TGCA), and finally the ICB mutant (ICB2m). The improved binding to ICB2 is largely due to a lower dissociation rate of the compound at the preferred site. To our knowledge, this is the first example on the use of SPR for studying the interactions of hairpin polyamides with DNA. Binding of JH-37 to ICB2 was corroborated by ITC studies, in which the DeltaG degrees of binding is driven by both enthalpy and entropy. With knowledge of the fundamental thermodynamic and kinetic properties that govern the molecular recognition of polyamides with DNA, we are poised to systematically edit the structure of JH-37 in order to further enhance its binding affinity and selectivity for ICB2,3. Our strategy for designing molecules that control gene expression is to target shorter, but multiple, binding sites that are in close array within the promoter. Binding of JH-37 to multiple ICB sites in the topoisomerase IIalpha promoter is an ideal test for this strategy. This approach is in contrast to the traditional strategy of targeting 15-16 base pairs, which has not been successful in actual biological systems due to poor cell uptake and distribution.

Modulation of topoisomerase IIα expression by a DNA sequence-specific polyamide

“…Reagents JH-37 was synthesized as described previously (18) and dissolved in DMSO to a final concentration of 10 mmol/L. Etoposide was obtained from Sigma-Aldrich (Poole, Dorset, United Kingdom) and prepared in DMSO at a concentration of 100 mmol/L.…”

“…Double-stranded oligonucleotides were 5 ¶-end labeled with T4 kinase (Invitrogen, Paisley, United Kingdom) using [g- 32 P]ATP and subsequently purified on Bio-Gel P-6 columns (Bio-Rad, Hemel Hempstead, United Kingdom). EMSAs were essentially done as described (18). Briefly, f0.1 ng of radiolabeled probe was incubated for 2 h in a buffer containing 20 mmol/L K-HEPES (pH 7.9), 1 mmol/L MgCl 2 , 0.5 mmol/L K-EDTA, 10% glycerol, 50 mmol/L KCl, 0.5 mmol/L DTT, and 0.5 Ag poly(dI-dC).poly(dI-dC) (Pfizer, Tadworth, United Kingdom) and 1Â protease inhibitor mix (Complete) at 30jC with the competitor JH-37 in this case.…”

“…The concentration of purified RNA was determined by measuring the absorbance at 260 nm. Subsequently, the reverse transcription reaction was carried out at 48jC for 45 min using 5 Ag RNA, 4 AL of avian myeloblastosis virus reverse transcriptase enzyme (Promega), 2 AL RNasin RNase inhibitor (Promega), 8 AL reverse transcriptase buffer, 4 AL of 10 mmol/L deoxynucleotide triphosphate, 8 AL oligo(dT) [12][13][14][15][16][17][18] (Invitrogen), and nuclease-free water in a final volume of 40 AL. Avian myeloblastosis virus reverse transcriptase enzyme was inactivated by heating the reaction mix at 94jC for 2 min.…”

“…A hairpin polyamide molecule (JH-37) designed to recognize sequences encompassing ICB2/ICB3 within the promoter of the topo IIa gene was synthesized (18). Surface plasmon resonance studies confirmed that JH-37 binds more strongly to ICB2 than to ICB1.…”

Survey of the year 2004 commercial optical biosensor literature

“…And lastly, show a panel of sensorgrams, as well as the fit of a simple binding isotherm to the equilibrium data points. (If this model fits the data poorly and complementary biophysical analyses provide evidence of a more complicated interaction, more complex models, such as multiple bind sites of varying affinity or cooperativity, can be implemented [391, 438,613,845]). To illustrate peratures.…”