Systematic chemical genetics approaches to studying known and novel cell death pathways

Cell death is a complex process that plays a vital role in development, homeostasis, and disease. The study of cell death is impeded by the difficulty in characterizing cell death processes and an incomplete characterization of the full range of death pathways that occur in mammalian systems. This thesis presents two systematic approaches to address these problems using small molecules.;The first approach, called "modulatory profiling," involves the creation of profiles composed of the changes in potency and efficacy of lethal compounds produced by a second cell-death-modulating agent. We show that compounds with the same characterized mechanism of action have similar modulatory profiles. Furthermore, clustering of modulatory profiles grouped compounds more accurately than clustering lethal compounds based on gene expression profiles or based on chemical structure. Modulatory profiling of novel compounds correctly predicted three compounds to be microtubule-destabilizing agents, classified numerous compounds that act nonspecifically, and identified compounds that cause cell death through a mechanism that is distinct from previously established ones. One novel compound, NPC26, was further characterized based on information obtained from its profile. Thus modulatory profiling facilitates the characterization of bioactive small molecules, allowing for the parallel analysis of large numbers of such compounds.;The second approach is a forward chemical genetic screen for small molecules that induce cell death in the presence of a cocktail of cell death inhibitors. This assay was validated and used to identify three small molecule inducers of a death process independent of established cell death mediators. Synthetic modification of one of these compounds, NID3, allowed for incorporation of a tag for use in affinity purification of its protein target. These target identification experiments are ongoing and may help characterize a novel death pathway.