Development of Ets2-Inhibitors

描述（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识别的化学解决方案提供了一种有前途的方法。这些形成三联体的寡核苷酸（TFOs）作为特定基因表达的潜在抑制剂引起了极大的兴趣。ets2基因编码的转录因子在控制细胞增殖和分化中起关键作用，在乳腺癌和前列腺癌的发病和进展中也起关键作用。在Catapano （co- PI）实验室中，三重结构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，并通过阻断RNA聚合酶直接抑制转录。然而，在这些特定领域的卫生应用之前，Triplex技术仍有待改进。在癌细胞中特定基因表达的治疗性调节中，需要改进靶标亲和力，这些TFOs和所有寡核苷酸在细胞中的递送也是如此。我们的初步结果表明，新霉素能够稳定Ets2基因中关键的三联体形成靶点，并有助于寡核苷酸的传递。该项目的目标是开发直接靶向乳腺癌和前列腺癌中扩增的ets2基因的新霉素- tfos。所提出的工作将克服传统TFO面临的重要挑战，例如TFO对双链的亲和力和对细胞的递送。假设：新霉素介导的tfo -新霉素偶联物的递送可用于开发具有改善递送性能和增强双相亲和力的序列特异性抗癌药物。我们建议开发新霉素偶联的TFO，以(1)增强TFO与其靶双链的结合亲和力，(2)改善TFO向癌细胞的传递和摄取。利用我们实验室最近的发现，我们将在Ets-2基因上合成具有治疗作用的位点的新霉素- tfo偶联物，形成三联体。然后，我们将使用新霉素- TFO偶联物来确定它们在稳定Ets-2上的三联体中的功效。将在DNA合成器上合成合适的新霉素- tfo偶联物，并评估其与感兴趣的Ets-2靶点结合的抗转录和抗癌活性。与瑞士南部肿瘤研究所（IOSI） Bellinzona实验肿瘤学实验室主任Carlo Catapano医学博士合作，完成了新霉素缀合TFOs的细胞和核摄取。这项测试将包括集中研究癌细胞内偶联物的稳定性，检查偶联物的序列和靶标选择性，系统地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，新霉素- tfo区分靶标序列的能力，以及测试这些高结合tfo对内源性基因表达和人类乳腺癌细胞生长的影响。新霉素- tfo偶联物的开发有可能通过大大提高tfo的通透性和解除乳腺癌和前列腺癌细胞增殖中ets2基因的表达调控，带来癌症治疗急需的改进。这种方法的成功将为TFO的开发和交付开辟新的途径，不仅因为它们与癌症有关，而且在其他基于寡核苷酸的治疗中也是如此。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增长的知识来开发新的癌症治疗策略。靶向特定基因，如参与癌细胞增殖和存活的Ets2或c-myc是一种很有前途的方法。我们的初步结果表明，新霉素能够稳定Ets2基因中关键的三联体形成靶点，并有助于寡核苷酸的传递。新霉素的这一特性现在将被扩展到开发氨基糖苷- tfo偶联物，以高亲和力结合Ets2启动子序列，并且可以在没有外部转染剂的情况下传递。这些结合物可以用来下调基因表达，抑制癌细胞的增殖。在我们的实验室中，通过本科生和研究生，以及与Catapano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，包括有机合成、生物物理化学和癌症研究的多学科努力，描述了新霉素介导的新型带正电TFOs（新霉素-TFOs）的发展和传递/抗癌活性。特别地，这项工作建议建立一个多学科团队，导致TFO偶联物的创造和使用，这些偶联物由于与新霉素偶联而具有新的性质和功能。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）递送方法的重要补充。我们建议使用25个碱基Ets2靶序列来设计可用于抑制肿瘤生长的新霉素- tfo偶联物，为开发小序列设计的抗癌药物开辟可能性。利用新霉素稳定三聚体和传递tfo的能力，新霉素- tfo （PS）偶联物将被开发为三聚体形成的抗癌药物。

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.