Investigation of overlap between mitotic and ferroptotic pathways using novel hybrid small molecule inhibitors. - Project Summary/Abstract: Molecules that target microtubules have been essential tools to understand conserved mechanisms that regulate mitosis. For example, the initial yeast studies that identified Bub and Mad mitotic checkpoint proteins relied on mitotic arrest induced by small molecules that disrupt spindle microtubules. Human cells arrested in mitosis may undergo apoptosis. This response is at least part of the reason why tubulin targeted anti-mitotics including taxanes, epothilones, and vinca alkaloids, are effective anti-cancer agents. However, toxicity to normal tissues can limit the effectiveness of anti-mitotics in some contexts. Understanding how cells respond to anti- mitotics can provide fundamental knowledge about this phase of the cell cycle and may lead to the development of better therapeutic agents. In the process of characterizing new imidazole-based chalcones (IBCs) we identified a new class of small molecules capable of inducing lethal mitotic arrest. Additional structure-activity-relationship (SAR) studies have identified a minimal active region of these molecules. We also found that these molecules are amenable to molecular hybridization with other bioactive molecules. Hybrid small molecules, which include molecular glues, PROTACs, and protein dimerizers, have been used to investigate a variety of important biological questions. In other studies, we identified a small chemical moiety capable of inducing ferroptosis, a ROS-dependent form of programmed cell death. With this information we generated a novel series of hybrid molecules that combine an anti-mitotic functionality and a pro-ferroptotic moiety into a single molecule. Analysis of these hybrids, along with other molecules, has identified a nexus between mitotic arrest and ferroptosis. For example, some cells that are normally resistant to ferroptosis become sensitive when arrested in mitosis. We proposed to use cell and molecular biological methods as well as a traceable forward genetic screen to understand the molecular overlap between mitosis and ferroptosis. AIM 1. Targeting tubulin with imidazole chalcones and hybrid analogues. We hypothesize that IBCs disrupt chromosome alignment and cause mitotic arrest by binding to the colchicine binding site of tubulin to alter microtubule dynamics. These predictions will be tested by measuring interaction with tubulin as well as microtubule dynamics by live-cell confocal microscopy. AIM 2. Cross-talk between pathways regulating mitosis and ferroptosis. We hypothesize that prolonged mitotic arrest, in response to molecules that disrupt microtubules, sensitizes cells to ferroptosis. We will investigate the mechanisms involved and assess how this cross-talk impacts the biological effects of hybrid molecules we have designed. AIM 3. Genetic analysis of the interplay between ferroptosis and mitosis. We plan to use a lentiviral system designed for efficient mutagenesis of endogenous genes. Advances in RNA sequencing have vastly simplified the identification of integration sites making large scale screens accessible to undergraduate researchers. This system will be used to search for novel regulators of mitosis and ferroptosis and to investigate the interplay between these two processes.
