Molecular Design for Specific Recognition of Functional DNA Sequences

项目总结： 编码和非编码的DNA基因组序列及其功能意义正在变得 越来越多的可用资源。用序列特异性、细胞通透性来调节这些序列的功能 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。描述的项目 这将消除这一障碍，并将通过潜在的新的 抗癌和抗寄生虫药物以及新的生物技术应用。这个项目建立在非常重要的基础上 在我们最初的资助期取得了成功的结果。我们发现，合理地设计和准备是可能的 用于识别目标DNA序列中的AT和GC碱基对(BP)的新模块。这是我们第一次 我设计了几个新的杂环，阳离子模块，它们选择性地和强烈地识别单个 带有侧翼AT序列的GC-BP。这是我们最初建议的主要目标，也是我们在这里提出的建议 从那次成功中建立起。我们的设计研究是从经典类型的细胞渗透性在特定的 基于包括临床有用化合物的分子平台的次要沟槽粘结剂。我们的 目标化合物在加入新的GC识别模块的同时保持了这些特征。的垮台 治疗中的大多数微小沟槽粘结剂都缺乏足够的细胞和核渗透性。我们的新产品 化合物逃过了这个障碍，但由于我们希望针对由癌症引起的疾病 微生物，在目标1中，我们将继续为细胞设计、准备和测试新的序列特定模块 渗透性和生物活性。提案的目标2描述了编制全新类型的 混合序列识别化合物与我们已建立的来自目标1的模块通过串联和连接 与广泛的混合DNA序列紧密和特定结合的并行方法。初步 我们第一个资助期的生物物理发现证明了我们的链接模块设计 方法是可行的，并可以在下一个资助期扩展到更复杂的序列。一系列广泛的 对新的化合物-DNA络合物进行了生物物理研究，包括高分辨核磁共振 方法和结晶学。目标3是全新的，不是我们最初提议的一部分。它建立在 令人振奋的初步结果，说明了我们设计的化合物的重要生物学功能。作为一种测试 系统，与PU.1转录因子(Tf)的合作结果表明，新的次要沟槽试剂， 我们的生物传感器在体外实验中确认，进入细胞和细胞核并变构抑制主要的沟槽结合 TFS。这一关于一系列转铁蛋白抑制剂未来开发的重要结果导致了对 我们的化合物对低PU.1急性髓系白血病(AML)患者细胞具有抑制作用。化合物进入细胞 对正常PU.1细胞无毒性的AML细胞死亡和选择性抑制PU1的细胞核。 这些结果是目标3的基础，对新药开发具有重大影响和重大意义。

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.