EFFECT OF MICROTUBULE-TARGETING AGENTS ON STRUCTURAL SIGNALING AND CHEMICAL TRANSPORT IN CELLS

Microtubules, along with actin and intermediate filaments, are key components of the cellular cytoskeleton and play pivotal roles in several biological functions, including intracellular trafficking, positioning of cellular components in interphase, mitotic spindle formation during cell division, maintaining of cell morphology and cell motility. Structurally, microtubules are formed by αβ-tubulin heterodimers that polymerize in a head-to-tail fashion to create protofilaments, which subsequently assemble into hollow cylindrical polar filaments. Microtubules’ behavior and function depend on their polymerization dynamics, with the first layer of regulation being the GTPase cycle of tubulin and its addition to the growing microtubule end. This process causes tubulin to transition from a curved conformation in solution to a straight conformation in the microtubule lattice. In cells, secondary layers of regulation (associated proteins, post-translational modifications, and tubulin isotypes levels) allow for a more precise modulation and differentiation of microtubule activities. Due to their involvement in key cellular activities, compounds that interfere with the microtubule cytoskeleton have been developed as cytotoxic agents, demonstrating remarkable efficacy against a wide spectrum of cancer types. Paclitaxel, a natural product extracted from yew tree bark, was the first microtubule-targeting agent (MTA) to be approved for clinical use in 1992, followed by two synthetic derivatives, docetaxel and cabazitaxel. These molecules bind to the taxane binding site in the microtubule, inducing its structural stabilization and preventing depolymerization events. Although the ability of taxanes to disrupt microtubule dynamics is closely linked to their chemotherapeutic effects, the exact cellular mechanism behind their anticancer activity remains under debate. At present, taxanes remain among the first-line treatments for several solid tumors, including breast cancer, ovarian carcinoma, pancreatic adenocarcinoma, non-small lung cancer, and prostate cancer. However, the clinical use of these molecules is limited by taxane-induced peripheral neurotoxicity, which severely affects up to 30% of patients and frequently leads to therapy discontinuation. To protect neurons from damage during taxane chemotherapy, it is essential to gain an improved mechanistic understanding of these drugs’ pathological effects on neurons. Due to their complex and elongated structures, neurons rely on intracellular active transport for cargo movement along the axon, making them especially susceptible to deficits in this process caused by taxane treatment. A recent hypothesis links axonal transport impairment to lattice expansion, a structural alteration induced by taxanes that appears to be unrelated to the stabilization effect. Locally expanded dimers may act as lattice point defects and cause premature detachment or stalling of the microtubule-associated motor proteins. Moreover, since current models of the microtubule structure describe the expanded lattice as a distinct structural feature localized near the tip, the spread of this structural signal along the whole lattice could impact the recognition patterns of motor proteins. Interestingly, unpublished results from our laboratory show that flutax-2, a C7-modified fluorescent taxane, does not induce microtubule lattice expansion. The present work, titled “Effects of microtubule-targeting agents on structural signaling and chemical transport in cells” focuses on two primary objectives. First, it aims to investigate the influence of C7-modifications of the taxane core on the cellular, biochemical, and structural properties of microtubules, in particular on lattice axial expansion. Second, it seeks to describe the relationship between the microtubule structural modifications induced by taxanes and the impairment of intracellular transport. These objectives were pursued through a multidisciplinary approach that included the in-silico design and chemical synthesis of new C7-decorated taxanes engineered to emulate the binding mode of flutax-2. Subsequently, the in vitro biochemical and structural characterization of these compounds was carried out, and their effect on cellular systems was extensively investigated. The findings presented in this thesis highlight the potential of C7-modification for generating taxanes able to override common cellular resistance mechanisms and/or to exert reduced impact on microtubule structure, thereby mitigating their interference over intracellular transport. Furthermore, the results obtained suggest that microtubule lattice expansion alone cannot account for the altered transport within taxanes-treated cells.