DNA Repair Inhibition and Cancer Therapy

DESCRIPTION (provided by applicant): The ability to repair DNA damage is critical to the survival of a cell, whether normal or cancerous. Upon damage, DNA repair genes and other factors are activated either to remove the damage, or, if the DNA damage is too extensive, to initiate programmed cell death. The majority of current anticancer therapies rely on their ability to create unrepairable DNA lesions, leading to apoptosis/cell death. While chemotherapy tends to be more toxic to rapidly dividing cancer cells, it is often insufficiently specific and thus also affects normally growing cells. As a result, patients frequently experience debilitating side effects that can limit the effectiveness of the therapy because the clinician must avoid exceeding the maximum tolerated dose (MTD). An added complication is that patients acquire resistance to the DNA-damaging agent, resistant cancer cells being able to repair the damage inflicted to their DNA and thus reducing the effect of treatment. The most widely used drugs in cancer therapy are those based on platinum compounds of which the most commonly used is Cisplatin. Cisplatin's mechanism of cytotoxicity is the formation of DNA adducts, the covalent 1,2 intrastrand cross-link between two adjacent guanines being the most abundant. There is a large body of evidence that Nucleotide Excision Repair (NER) is an important source of resistance to platinum compounds. Cisplatin-DNA adducts are removed by NER, the only mechanism known by which platinum-DNA intrastrand adducts are removed from DNA. Although complex (>20 proteins have so far been identified in the pathway), NER mechanisms can be summarized by five basic steps: recognition of the DNA lesion; cleavage of the damaged strand and of the lesion; excision of the damaged strand, creating a gap; synthesis of new DNA to fill the gap; and ligation of the final nick. Studies indicate that inhibition of NER would provide a rational approach to enhance cisplatin's cytotoxicity and to overcome cisplatin resistance. Such an inhibitor should produce synergistic cytotoxicities when administered in combination with cisplatin. We are interested in developing this area to provide an effective, sensitive, reliable yet cost-effective assay to use to screen large numbers of compounds to identify an inhibitor of NER. There are several ways to measure DNA repair, and although all have been used extensively to investigate the details of DNA repair pathways, precise and specific assays are cumbersome and expensive, and reliable readouts for Drug Discovery have been difficult to develop. We are interested in developing this area for High Throughput Screening where large numbers of compounds are tested. Phase I will be the design, development and validation of assays suitable (robust, sensitive, cost-effective and safe) for screening a compound library in medium or high throughput (96-well microtitre plates). The specific aims of Phase I will be met when the assays for NER inhibition, XPG binding and XPG nuclease activity are used to screen a compound collection and hits are identified and confirmed.