Development of Ets2-Inhibitors

Ets2抑制剂的开发

• 批准号: 7458423
• 负责人: DEV PRIYA ARYA
• 金额: $ 22.24万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-10 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7458423
• 关键词: Abbreviations; Affinity; Aminoglycoside Antibiotics; Aminoglycosides; Antineoplastic Agents; Antisense DNA; Antisense Oligonucleotides; Area; Binding; Biological Assay; Biology; Breast; Breast Cancer Cell; Calorimetry; Cancer Etiology; Cause of Death; Cell Proliferation; Cells; Charge; Chemicals; Chemistry; Chromatin; Chromatin Structure; Circular Dichroism Spectroscopy; Classification; Clinic; Coffee; Collaborations; Commit; Complement; DNA; DNA-Directed RNA Polymerase; Development; Doctor of Medicine; Dominant-Negative Mutation; Electrophoretic Mobility Shift Assay; Face; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genomics; Goals; Growth; Health; Healthy People 2010; Human; Institutes; Interdisciplinary Study; Investigation; Joints; Kinetics; Knowledge; Laboratories; Life; Link; Luciferases; Malignant Neoplasms; Malignant neoplasm of prostate; Mammalian Cell; Measurement; Mediating; Methods; Molecular; Mutagenesis; Neomycin; Neoplastic Cell Transformation; Nuclear; Nucleic Acid Binding; Nucleic Acids; Numbers; Oligonucleotides; Organic Synthesis; Outcome; Pathogenesis; Permeability; Pharmaceutical Preparations; Phase; Plasmids; Play; Preparation; Principal Investigator; Property; Prostate; Proteins; Purpose; RNA; RNA analysis; Regulation; Reporter; Research; Role; Scanning; Site; Small Interfering RNA; Solid; Solutions; Structure; Students; Switzerland; Technology; Testing; Therapeutic; Thiourea; Titrations; Training; Transcription Initiation; Transfection; United States; United States Public Health Service; Work; anticancer activity; anticancer research; antigene; base; biophysical chemistry; c-myc Genes; cancer cell; cancer therapy; cost; design; disability; drug development; ds-DNA; gene function; gene repression; health application; improved; inhibitor/antagonist; innovation; interest; methyl sulfate; microcalorimetry; multidisciplinary; novel; novel strategies; novel therapeutics; oncology; phosphodiester; phosphorothioate; programs; promoter; research study; success; transcription factor; transmission process; triple helix; triplex DNA; tumor progression; ultraviolet; uptake

DESCRIPTION (provided by applicant): Nucleic acids are central molecules in the transmission, expression and conservation of genetic information. Recognition of duplex DNA by oligonucleotides, forming DNA triple helices, provides a promising approach to a chemical solution for DNA recognition. These triplex forming oligonucleotides (TFOs) have aroused significant interest as potential inhibitors of the expression of particular genes. The ets2 gene encodes a transcription factor that plays a critical role in controlling cell proliferation and differentiation and also has a key role in the pathogenesis and progression of breast and prostate cancer. In the Catapano (co- PI) lab, triplex formation has been shown to sequence-specifically inhibit transcription at specific sites in the ets2 promoter sequences and also directly inhibit transcription by blocking RNA polymerase. Triplex technology however remains to be improved before health applications to these specific areas can be made. The need for an improved target affinity in the therapeutic regulation of specific gene expression in cancer cells remains, as does the delivery of these TFOs and all oligonucleotides in cells. Our preliminary results show the ability of neomycin to stabilize key triplex forming targets in Ets2 gene and aid in delivery of oligonucleotides. The objective of this project is to develop neomycin-TFOs that directly target the ets2 gene, found amplified in breast and prostate cancer. The proposed work will overcome important challenges such as TFO affinity to the duplex and delivery to the cells, that traditional TFOs face. The hypothesis :} Neomycin-mediated delivery of TFO-neomycin conjugates can be used to develop sequence-specific anticancer agents with improved delivery properties and enhanced duplex affinity.} We propose to develop neomycin conjugated TFOs that (1) enhance the binding affinity of the TFO to its target duplex, (2) improve the delivery and uptake of the TFO to the cancer cells. Using the recent findings from our labs, we will synthesize neomycin-TFO conjugates for triplex formation with sites of therapeutics interest in Ets-2 gene. We will then use neomycin- TFO conjugates to determine their efficacy in stabilizing the triplexes on Ets-2. Appropriate neomycin-TFO conjugates will be synthesized on a DNA synthesizer, and evaluated for anti- transcriptional and anticancer activity in binding to the Ets-2 targets of interest. The cellular and nuclear uptake of neomycin-conjugated TFOs is being done in collaboration with Carlo Catapano, M.D., Director, Laboratory of Experimental Oncology, Oncology Institute of Southern Switzerland (IOSI) Bellinzona, Switzerland. This testing will involve studies focused on the stability of the conjugate within cancer cells, examination of the sequence and target selectivity of the conjugate, systematic determination of the number of neomycin molecules per oligonucleotide needed for optimal delivery and anti-gene activity, the ability of the neomycin-TFO to discriminate for target sequences and test the effects of these high binding TFOs on endogeneous gene expression and growth of human breast cancer cells. The development of neomycin-TFO conjugates has the potential for bringing the much needed improvements in cancer therapy by vastly improving the permeability of TFOs and deregulating expression of ets2 gene in proliferation of breast and prostate cancer cells. The success of this approach will open up new avenues of TFO development and delivery not only as they relate to cancer, but in other oligonucleotide based therapies as well. Health Relevance Statement One of the challenges of research in oncology is to find ways to use the increasing knowledge of the mechanisms underlying neoplastic transformation and tumor progression to develop novel therapeutic strategies for cancer. Targeting specific genes, such as Ets2 or c-myc, which are involved in proliferation and survival of cancer cells is a promising approach. Our preliminary results show the ability of neomycin to stabilize key triplex forming targets in Ets2 gene and aid in delivery of oligonucleotides. This property of neomycin will now be extended to develop aminoglycoside-TFO conjugates to target Ets2 promoter sequences that bind with high affinity and can be delivered without external transfection agents. These conjugates can then be used to down regulate gene expression and inhibit proliferation of cancer cells. Proposed studies in our labs, using undergraduate and graduate students, and collaborative efforts with the Catapano lab will further help establish the efficacy of this approach. The work proposed here, a multidisciplinary effort encompassing organic synthesis, biophysical chemistry, and cancer research describes the development and neomycin mediated delivery/anticancer activity of novel positively charged TFOs (neomycin-TFOs). In particular, this work proposes to initiate a multidisciplinary team that leads to the creation and use of TFO conjugates that have novel properties and functions because of their conjugation with neomycin. The success of the proposed work would be a significant addition to currently available approaches in breast and prostate cancer drug development and TFO (oligonucleotide) delivery. We propose using 25 base Ets2 target sequences to design neomycin-TFO conjugates that can be employed to inhibit cancer growth, opening possibilities for the development of small sequence- designed anti-cancer agents. Using neomycin's ability to stabilize triplexes and deliver TFOs, neomycin-TFO (PS) conjugates will be developed as triplex-forming anticancer agents.

描述（由申请人提供）：核酸是遗传信息传递、表达和保存的中心分子。通过寡核苷酸识别双链 DNA，形成 DNA 三螺旋，为 DNA 识别化学溶液提供了一种有前途的方法。这些形成三链体的寡核苷酸（TFO）作为特定基因表达的潜在抑制剂引起了人们的极大兴趣。 ets2基因编码的转录因子在控制细胞增殖和分化中发挥关键作用，并且在乳腺癌和前列腺癌的发病机制和进展中也发挥关键作用。在 Catapano (co-PI) 实验室中，三链体形成已被证明可以序列特异性地抑制 ets2 启动子序列中特定位点的转录，并且还可以通过阻断 RNA 聚合酶来直接抑制转录。然而，三重技术在应用于这些特定领域的健康应用之前仍有待改进。在癌细胞中特定基因表达的治疗调节中仍然需要改进的靶标亲和力，这些 TFO 和细胞中所有寡核苷酸的递送也是如此。我们的初步结果表明新霉素能够稳定 Ets2 基因中关键的三链体形成靶标并有助于寡核苷酸的递送。该项目的目标是开发直接靶向 ets2 基因的新霉素-TFO，该基因在乳腺癌和前列腺癌中被扩增。拟议的工作将克服传统 TFO 面临的重要挑战，例如 TFO 对双链体的亲和力以及向细胞的递送。假设：} 新霉素介导的 TFO-新霉素缀合物的递送可用于开发具有改善的递送特性和增强的双链体亲和力的序列特异性抗癌药物。} 我们建议开发新霉素缀合的 TFO，其 (1) 增强 TFO-新霉素缀合物的结合亲和力。 TFO 与其靶双链体结合，(2) 改善 TFO 向癌细胞的递送和摄取。利用我们实验室的最新发现，我们将合成新霉素-TFO 缀合物，以与 Ets-2 基因中感兴趣的治疗位点形成三联体。然后我们将使用新霉素-TFO 缀合物来确定其稳定 Ets-2 上三链体的功效。合适的新霉素-TFO缀合物将在DNA合成仪上合成，并评估与感兴趣的Ets-2靶标结合的抗转录和抗癌活性。新霉素缀合 TFO 的细胞和核摄取是与瑞士贝林佐纳南部瑞士肿瘤研究所 (IOSI) 实验肿瘤学实验室主任 Carlo Catapano 医学博士合作完成的。该测试将涉及的研究重点是癌细胞内缀合物的稳定性、缀合物的序列和靶点选择性的检查、系统确定最佳递送和抗基因活性所需的每个寡核苷酸的新霉素分子数量、新霉素-TFO 来区分靶序列并测试这些高结合 TFO 对人乳腺癌细胞内源基因表达和生长的影响。新霉素-TFO 缀合物的开发有可能通过极大地改善 TFO 的渗透性并解除乳腺癌和前列腺癌细胞增殖中 ets2 基因的表达来为癌症治疗带来急需的改进。这种方法的成功将为 TFO 开发和递送开辟新途径，不仅因为它们与癌症有关，而且也适用于其他基于寡核苷酸的疗法。健康相关性声明 肿瘤学研究的挑战之一是找到方法，利用对肿瘤转化和肿瘤进展机制的不断了解来开发新的癌症治疗策略。针对参与癌细胞增殖和存活的特定基因，例如 Ets2 或 c-myc，是一种有前途的方法。我们的初步结果表明新霉素能够稳定 Ets2 基因中关键的三链体形成靶标并有助于寡核苷酸的递送。新霉素的这一特性现在将得到扩展，以开发氨基糖苷-TFO 缀合物，以高亲和力结合的靶向 Ets2 启动子序列，并且无需外部转染剂即可递送。然后，这些缀合物可用于下调基因表达并抑制癌细胞的增殖。我们实验室利用本科生和研究生进行的拟议研究以及与卡塔帕诺实验室的合作将进一步帮助确定这种方法的有效性。这里提出的工作是一项涵盖有机合成、生物物理化学和癌症研究的多学科努力，描述了新型带正电 TFO（新霉素-TFO）的开发和新霉素介导的递送/抗癌活性。特别是，这项工作建议组建一个多学科团队，致力于创建和使用 TFO 缀合物，这些缀合物由于与新霉素缀合而具有新颖的特性和功能。这项工作的成功将是对目前乳腺癌和前列腺癌药物开发和 TFO（寡核苷酸）递送可用方法的重大补充。我们建议使用 25 个碱基的 Ets2 靶序列来设计可用于抑制癌症生长的新霉素-TFO 缀合物，为开发小序列设计的抗癌药物开辟了可能性。利用新霉素稳定三链体和递送 TFO 的能力，新霉素-TFO (PS) 缀合物将被开发为三链体形成抗癌剂。