Targeting the Inverted CCAAT Box 2 in the Topoisomerase II Promoter by JH-37, an Imidazole-Pyrrole Polyamide Hairpin: Design,
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- 作者：James A. Henry ; N. Minh Le ; Binh Nguyen ; Cameron M. Howard ; Suzanna L. Bailey ; Sarah M. Horick ; Karen L. Buchmueller ; Minal Kotecha ; Daniel Hochhauser ; John A. Hartley ; W. David Wilson ; and Moses Lee
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：September 28, 2004
- 年：2004
- 卷：43
- 期：38
- 页码：12249 - 12257
- 全文大小：198K
- 年卷期：v.43,no.38(September 28, 2004)
- ISSN：1520-4995
文摘
The topoisomerase II
promoter is regulated through transcription factor interactions withfive inverted CCAAT boxes (ICBs). In confluent cancer cells, binding of nuclear factor Y to ICB2 repressesthe expression of this gene, contributing to resistance to topoisomerase II poisons. The ICB sites withinthe topoisomerase II promoter are, therefore, potential targets for the design of anticancer drugs andgene 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 footprintingstudies on the topoisomerase II promoter showed JH-37 to preferentially bind to ICB2,3 and ICB1sites. The larger 
TM values for ICB2,3 (8-9 
C) over ICB1,4,5 (4-5 C) indicated a preference ofJH-37 for ICB2,3. CD titration studies confirmed the binding of JH-37 to the minor groove, with a 1:1binding stoichiometry. Results from SPR studies showed JH-37 to bind most strongly to ICB2 (K = 3 ×107 M-1), followed by ICB1, the non-ICB sequence (TGCA), and finally the ICB mutant (ICB2m). Theimproved binding to ICB2 is largely due to a lower dissociation rate of the compound at the preferredsite. To our knowledge, this is the first example on the use of SPR for studying the interactions of hairpinpolyamides with DNA. Binding of JH-37 to ICB2 was corroborated by ITC studies, in which the Gof binding is driven by both enthalpy and entropy. With knowledge of the fundamental thermodynamicand kinetic properties that govern the molecular recognition of polyamides with DNA, we are poised tosystematically edit the structure of JH-37 in order to further enhance its binding affinity and selectivityfor ICB2,3. Our strategy for designing molecules that control gene expression is to target shorter, butmultiple, binding sites that are in close array within the promoter. Binding of JH-37 to multiple ICB sitesin the topoisomerase II promoter is an ideal test for this strategy. This approach is in contrast to thetraditional strategy of targeting 15-16 base pairs, which has not been successful in actual biologicalsystems due to poor cell uptake and distribution.
promoter is regulated through transcription factor interactions withfive inverted CCAAT boxes (ICBs). In confluent cancer cells, binding of nuclear factor Y to ICB2 repressesthe expression of this gene, contributing to resistance to topoisomerase II poisons. The ICB sites withinthe topoisomerase II promoter are, therefore, potential targets for the design of anticancer drugs andgene 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 footprintingstudies on the topoisomerase II promoter showed JH-37 to preferentially bind to ICB2,3 and ICB1sites. The larger TM values for ICB2,3 (8-9 C) over ICB1,4,5 (4-5 C) indicated a preference ofJH-37 for ICB2,3. CD titration studies confirmed the binding of JH-37 to the minor groove, with a 1:1binding stoichiometry. Results from SPR studies showed JH-37 to bind most strongly to ICB2 (K = 3 ×107 M-1), followed by ICB1, the non-ICB sequence (TGCA), and finally the ICB mutant (ICB2m). Theimproved binding to ICB2 is largely due to a lower dissociation rate of the compound at the preferredsite. To our knowledge, this is the first example on the use of SPR for studying the interactions of hairpinpolyamides with DNA. Binding of JH-37 to ICB2 was corroborated by ITC studies, in which the Gof binding is driven by both enthalpy and entropy. With knowledge of the fundamental thermodynamicand kinetic properties that govern the molecular recognition of polyamides with DNA, we are poised tosystematically edit the structure of JH-37 in order to further enhance its binding affinity and selectivityfor ICB2,3. Our strategy for designing molecules that control gene expression is to target shorter, butmultiple, binding sites that are in close array within the promoter. Binding of JH-37 to multiple ICB sitesin the topoisomerase II promoter is an ideal test for this strategy. This approach is in contrast to thetraditional strategy of targeting 15-16 base pairs, which has not been successful in actual biologicalsystems due to poor cell uptake and distribution.