Date of Award

January 2016

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Ranjit S. Bindra

Second Advisor

Roy Decker

Abstract

Mammalian cells mainly utilize two DNA repair pathways to repair double stranded breaks (DSB): homologous recombination (HR) and non-homologous end joining (NHEJ). While HR utilizes homologous DNA sequences as a template for repair, NHEJ processes and re-ligates the ends of the breaks. NHEJ is further sub-divided into a DNA-PK-dependent canonical pathway, and alternative pathways, which are more mutagenic. These pathways are essential for cell survival after ionizing radiation, and thus have become important therapeutic targets for radiosensitization. Our group recently performed multiple high-throughput small molecule screens for novel DSB repair inhibitors. These studies identified numerous selective NHEJ and HR inhibitors, many of which are unknown compounds. Our group now seeks to evaluate the underlying mechanisms by which these compounds regulate DSB repair. To this end, we built an automated, high-throughput secondary assay platform to interrogate hits identified from primary screens. This includes an adherent cell, imaging cytometry platform to rapidly assay GFP- and RFP-based reporter cell lines in 96- and 384-well microplates, to assess DNA content and S-phase proliferation, as well as to perform miniaturized clonogenic survival assays. We then applied this platform to the hits from our primary screens. We identified two groups of compounds from our initial screens: A) novel HSP90 inhibitors and B) cardiac glycosides. Compounds from group A resembled known HSP90 inhibitors, but demonstrated different effects on key DSB repair pathways. One agent from group A demonstrated radiosensitivity in vitro. Compounds from both groups inhibited NHEJ and HR and were remarkably non-toxic at the majority of doses tested. This is the first report to demonstrate that cardiac glycosides inhibit both major DNA repair pathways. These data suggest a novel collection of DSB repair inhibitors which can potently suppress key DSB repair pathways with little toxicity, and in a manner unique to previously described agents. Our work also reveals a cutting-edge secondary assay platform that can be used for mechanism of action studies for other small molecules and genes identified from high-throughput screens in the future.

Comments

This thesis is restricted to Yale network users only. It will be made publicly available on 12/31/2019

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