Tanious Farial, Wilson W David, Wang Lei, Kumar Arvind, Boykin David W, Marty Carine, Baldeyrou Brigitte, Bailly Christian
Department of Chemistry and Laboratory for Chemical and Biological Sciences, Georgia State University, Atlanta, Georgia 30303, USA.
Biochemistry. 2003 Nov 25;42(46):13576-86. doi: 10.1021/bi034852y.
In the course of a program aimed at discovering novel DNA-targeted antiparasitic drugs, the phenylfuran-benzimidazole unfused aromatic dication DB293 was identified as the first diamidine capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Its preferred binding sequence encompasses the tetranucleotide 5'-ATGA.5'-TCAT to which DB293 binds tightly with a strong positive cooperativity. Here we have investigated the influence of the DNA sequence on drug binding using two complementary technical approaches: surface plasmon resonance and DNase I footprinting. The central dinucleotide of the primary ATGA motif was systematically varied to represent all of the eight possible combinations (AXGA and ATYA, where X or Y = A, T, G, or C). Binding affinities for each site were precisely measured by SPR, and the extent of cooperative drug binding was also determined. The sequence recognition process was found to be extremely dependent on the nature of the central dinucleotide pair. Modification of the central TG step decreases binding affinity by a factor varying from 2 to over 500 depending on the base substitution. However, the diminished binding affinity does not affect the unique binding mode. In nearly all cases, the SPR titrations revealed a positive cooperativity in complex formation which reflects the ease of the dication to form stacked dimeric motifs in the DNA minor groove. DNase I footprinting served to identify additional binding sites for DB293 in the context of long DNA sequences offering a large variety of randomly distributed or specifically designed sites. The ATGA motif provided the best receptor for the drug, but lower affinity sequences were also identified. The design of two DNA fragments composed of various targeted tetranucleotide binding sites separated by an "insulator" (nonbinding) sequence allowed us to delineate further the influence of DNA sequence on drug binding and to identify a novel high-affinity site: 5'-ACAA.5'-TTGT. Collectively, the SPR and footprinting results show that the consensus sequence 5'-(A/T)-TG-(A/T) represents the optimal site for cooperative dimerization of the heterocyclic diamidine DB293.
在一项旨在发现新型DNA靶向抗寄生虫药物的研究项目中，苯基呋喃 - 苯并咪唑非稠合芳香二价阳离子DB293被鉴定为首个能够在含GC序列的DNA小沟中形成堆叠二聚体的双脒。其偏好的结合序列包含四核苷酸5'-ATGA.5'-TCAT，DB293与之紧密结合且具有很强的正协同性。在此，我们使用两种互补的技术方法研究了DNA序列对药物结合的影响：表面等离子体共振和DNase I足迹法。对主要ATGA基序的中央二核苷酸进行系统变化，以代表所有八种可能的组合（AXGA和ATYA，其中X或Y = A、T、G或C）。通过表面等离子体共振精确测量每个位点的结合亲和力，并确定协同药物结合的程度。发现序列识别过程极其依赖于中央二核苷酸对的性质。中央TG步骤的修饰会使结合亲和力降低2至500倍以上，具体取决于碱基取代情况。然而，结合亲和力的降低并不影响独特的结合模式。在几乎所有情况下，表面等离子体共振滴定显示复合物形成过程中具有正协同性，这反映了二价阳离子在DNA小沟中形成堆叠二聚体基序的容易程度。DNase I足迹法用于在长DNA序列的背景下识别DB293的其他结合位点，这些序列提供了各种随机分布或专门设计的位点。ATGA基序为该药物提供了最佳受体，但也鉴定出了亲和力较低的序列。设计两个由“绝缘体”（非结合）序列隔开的各种靶向四核苷酸结合位点组成的DNA片段，使我们能够进一步描绘DNA序列对药物结合的影响，并鉴定出一个新的高亲和力位点：5'-ACAA.5'-TTGT。总体而言，表面等离子体共振和足迹法结果表明，共有序列5'-(A/T)-TG-(A/T)代表杂环双脒DB293协同二聚化的最佳位点。
