Design and Synthesis of Gene-Specific Agents for HIR

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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.