Understanding the Eribulin-Tubulin Interaction Using Sea Urchins
Microtubules are cellular polymers comprised of tubulin heterodimers and they play a crucial role in many cell functions, particularly mitotic cell division. As a result, many anti-cancer drugs are microtubule-targeting agents (MTAs) that aim to inhibit cell division. However, MTAs also target microtubules in normal cells, particularly neurons, resulting in potentially very serious side effects including peripheral neuropathy. However, the mechanism of the drug-induced neuropathy is not well understood. One of these MTAs, eribulin, is used for metastatic breast cancer treatment. Understanding how eribulin interacts with the tubulin protein at a molecular level may provide insights into how eribulin inhibits mitosis and causes neuropathy. In order to probe the drug-tubulin interaction, we sought to create and purify eribulin-tubulin co-crystals inside living cells that could then be used to investigate the molecular details of the interaction. Sea urchin eggs, available in large quantity and containing substantial amounts of the highly conserved tubulin protein, have been used successfully to form and isolate co-crystals of other MTAs and tubulin. First, we established that eribulin had the expected effect of disrupting sea urchin cell division. Dose dependency experiments indicated that sub-micromolar concentrations of eribulin delayed cell division while higher concentrations resulted in complete inhibition of cell division. Results from the dose response experiments were then used to select an eribulin concentration for in vivo crystallization experiments, which are ongoing. The eventual goal is to purify the co-crystals and determine the eribulin-tubulin binding affinity, stoichiometry and binding site(s).