Molecular Design for Specific Recognition of Functional DNA Sequences

项目概要： 编码和非编码DNA基因组序列以及它们的功能意义正变得越来越重要。 越来越可用。用序列特异性的、细胞可渗透的 合成化合物将是非常有价值的。缺乏令人满意的合成化合物是一个障碍 我们的基因组知识在生物技术和药物开发中的应用。述工程 这将消除障碍，并将通过潜在新的 抗癌和抗寄生虫药物以及新的生物技术应用。该项目建立在非常 在我们最初的融资阶段取得了成功。我们发现合理设计和准备 用于靶DNA序列中AT和GC碱基对（BP）识别的新模块。我们第一次 我设计了几种新的杂环阳离子模块，它们选择性地强烈识别单个 具有侧翼AT序列的GC bp。这是我们最初提案的主要目标，我们在这里提出的是 建立在这一成功之上。我们的设计研究开始于经典类型的细胞渗透性AT特异性 小沟结合剂，其基于包括临床上有用的化合物的分子平台。我们 目标化合物保持这些特征，同时引入新的GC识别模块。的倒台 治疗中的大多数小沟结合剂缺乏足够的细胞和核渗透性。我们的新 化合物逃脱了这一障碍，但由于我们希望靶向癌症诱导的疾病， 在目标1中，我们将继续设计、制备和测试用于细胞的新的序列特异性模块， 渗透性和生物活性。该提案的目标2描述了制备全新类型的 混合序列识别化合物与我们建立的模块从目标1连接的串行和 平行的方法紧密和特异性地结合到广泛的混合DNA序列。初步 我们第一个资助期的生物物理发现证明了我们的模块化设计 这一方法行之有效，并可在下一个供资期扩大到更复杂的序列。一系列广泛的 对新化合物-DNA复合物进行了生物物理研究，包括高分辨率NMR 方法和晶体学。目标3是全新的，不是我们最初提案的一部分。它建立在 令人兴奋的初步结果，说明了我们设计的化合物的重要生物功能。作为测试 系统，与PU.1转录因子（TF）的合作结果表明，新的小沟因子， 在我们的生物传感器体外测定中鉴定，进入细胞和细胞核并变构抑制大沟结合 TF。这一重要结果对于未来开发一系列TF抑制剂具有重要意义， 我们的化合物针对低PU.1急性髓性白血病（AML）患者细胞。化合物进入细胞 和具有选择性PU.1抑制和AML细胞死亡的细胞核，对正常PU.1细胞没有细胞毒性。 这些结果是目标3的基础，对新药开发具有重大影响和重要意义。

Project Summary: Both coding and non-coding DNA genomic sequences as well as their functional significance are becoming increasingly available. Modulating the functions of these sequences with sequence-specific, cell-permeable synthetic compounds would be extremely valuable. Deficiency of satisfactory synthetic compounds is a barrier to applications of our genomic knowledge in biotechnology and drug development. The project described herein will remove that barrier and will have an impact on human health through the potential for new anticancer and antiparasitic drugs as well as new biotechnology applications. The project builds on very successful results in our initial funding period. We found that it is possible to rationally design and prepare novel modules for both AT and GC base pair (BP) recognition in target DNA sequences. For the first time we have designed several new heterocyclic, cationic modules which selectively and strongly recognize a single GC bp with flanking AT sequences. This was a primary goal of our initial proposal and what we propose here builds from that success. Our design research was initiated with classical types of cell permeable AT specific minor groove binders that are based on a molecular platform that includes clinically useful compounds. Our target compounds maintain these features while incorporating new GC recognition modules. The downfall of most minor groove binders in therapeutics has been lack of sufficient cell and nuclear permeability. Our new compounds escaped this block but since we wish to target diseases from those induced by cancer to microorganisms, in Aim 1 we will continue to design, prepare and test new sequence-specific modules for cell permeability and biological activity. Aim 2 of the proposal describes the preparation of entirely new types of mixed sequence recognition compounds with our established modules from Aim 1 linked by both serial and parallel methods to bind tightly and specifically to a broad array of mixed DNA sequences. Preliminary biophysical findings from our first funding period are a proof of concept that our linked modular design approach works and can be expanded to more complex sequences in the next funding period. A broad array of biophysical studies are performed on the new compound-DNA complexes including high resolution NMR methods and crystallography. Aim 3 is entirely new and was not a part of our initial proposal. It builds on exciting preliminary results that illustrate important biological functions of our designed compounds. As a test system, collaborative results with the PU.1 transcription factor (TF) showed that new minor groove agents, identified in our biosensor in vitro assay, enter cells and nuclei and allosterically inhibit major groove binding TFs. This important result for future development of an array of TF inhibitors lead to collaborative evaluation of our compounds against low PU.1 acute myeloid leukemia (AML) patient cells. The compounds entered the cell and nuclei with selective PU.1 inhibition and AML cell death with no toxicity to cells to normal PU.1 cells. These results are the basis for Aim 3 with significant impact and major relevance for new drug development.