Design and Synthesis of Gene-Specific Agents for HIR

项目3的总体目标是开发新的试剂，干扰细胞对热疗和电离辐射(IR)联合治疗的保护性反应，以评估它们作为放射增敏药物的潜力。其基本前提是，热通过展开和沉淀蛋白质而增强辐射诱导的细胞死亡，从而减少修复或获得细胞损伤的途径。还推测热休克的同时诱导 蛋白质通过帮助未折叠蛋白质的重折叠和增溶来降低放射增敏作用，如果能够阻止它们的诱导，放射增敏作用将会增强。首先，我们将重点关注HSP70作为开发干扰基因表达的化学试剂的模型系统，然后将研究扩展到热休克蛋白家族的其他成员，如HSP25、HSP110或HSC70以及确定的潜在重要靶点 在其他项目中。这个项目有4个具体目标。第一个是设计和评价通过阻断热休克转录因子(HSTF)启动子结合来干扰热休克基因转录启动的药物。我们将在体外设计、合成和测定聚酰胺和肽核酸(PNA)与热休克元件(HSE)结合和抑制HTSF结合的能力。第二个具体目标是设计和评估在转录延伸过程中干扰热休克蛋白基因表达的试剂。我们将检测形成PNA和伪互补PNA的三链钳在转录前或转录期间(自杀转录)与转录的DNA结合的能力。我们还将设计和合成一类新的分子手铐结构，并测试它们通过拓扑连接到DNA来增强PNA有效性的能力。第三个目标将是确定PNA和聚酰胺基试剂在体内阻断的有效性。 基因在体外和体内的表达。不同的PNA或聚酰胺结构在阻断mRNA延长方面的有效性将通过核提取物中目标模板DNA的体外转录来初步评估。然后将筛选有前景的药物，以了解细胞摄取和阻断体内GFP标记的HSP基因的热诱导合成的能力。第四个目标是观察使用PNA和聚酰胺试剂减少热休克蛋白的合成是否会增加 高温诱导的放射增敏。与此同时，我们将与其他项目一起开发针对HILT中其他可能涉及的基因的siRNA试剂，以确定新的抗基因靶点。这些药物还将由Core C在动物身上进行人体细胞异种移植的毒性和有效性测试。

The overall goal of Project 3 is to develop new agents that interfere with protective aspects of the cellular response to combined treatment with hyperthermia and ionizing radiation (IR) to evaluate their potential as radiosensitizing drugs. The underlying premise is that heat enhances radiation induced cell death by unfolding and precipitating proteins resulting in decreased repair or access to cellular damage. It is also presumed that concurrent induction of heat shock proteins decreases radiosensitization by aiding the refolding and solubilization of unfolded proteins, and that if their induction could be prevented, radiosensitization would be enhanced. Initially we will focus on HSP70 as a model system for developing chemical agents that interfere with gene expression and then extend the study to other members of the heat shock protein families such as HSP25, HSP110 or HSC70 as well as potentially important targets identified in other projects. There are 4 specific aims for this project. The first is to design and evaluate agents that interfere with initiation of heat shock gene transcription by blocking promoter binding of heat shock transcription factor (HSTF). We will design, synthesize and determine the ability of both polyamides and peptide nucleic acids (PNA) to bind to the heat shock element (HSE) and inhibit the binding of HTSF in vitro. The second specific aim is to design and evaluate agents that interfere with heat shock protein gene expression during transcriptional elongation. We will examine the ability of triplex clamp forming PNAs and pseudo-complementary PNAs to bind to transcribed DNA either prior to transcription, or during transcription (suicide transcription). We will also design and synthesize a new class of molecular handcuff structures and test their ability to enhance the effectiveness of the PNAs by topological linking to the DNA. The third aim will be to determine the in vivo effectiveness of PNA and polyamide-based agents in blocking gene expression in vitro and in vivo. The effectiveness of different PNA or polyamide constructs in blocking mRNA elongation will be evaluated initially by in vitro transcription of target template DNA's in nuclear extracts. Promising agents will then be screened for cellular uptake and the ability to block heat-induced synthesis in vivo of a GFP-tagged HSP gene. The fourth aim will be to see whether decreasing HSP synthesis with PNA and polyamide agents increases hyperthermia-induced radiosensitization. At the same time we will be developing siRNA agents against other possible genes involved in Hilt in concert with the other projects in order to identify new anti-gene targets. These agents will also be tested in animals for toxicity and efficacy in human cell xenografts by Core C.