Biology subjects

Passarella, D.

Publications and source records attributed to Passarella, D..

3 recordsLinked to original sources

bioRxiv2025

Dissecting structural and functional determinants of microtubule stabilization through guided chemical modulation

Paclitaxel (PTX) is a widely used chemotherapeutic, but its efficacy is limited by peripheral neuropathy, likely due to its structural impact on neuronal microtubules (MTs). To decouple MT stabilization from adverse structural changes, we designed, synthetized and characterized several PTX analogues. Our compound 1b retains PTXs stabilizing activity in vitro and in cells, and the 1b-bound MTs preserve a native-like structure. These facts allowed us to investigate the influence of specific MT structural features on motor proteins behavior and interaction with MT-associated protein Tau. PTX-induced lattice expansion disrupted dynein-mediated retrograde transport and altered kinesin-1 motility. Additionally, PTX reduced Taus initial non-cooperative binding and envelopes growth rate while increasing dwell time and suppressing dynamic binding. In contrast, 1b preserved more physiological Tau dynamics. These findings reveal that MT stabilization and structural modulation can be separated and highlight the functional importance of MT heterogeneity in maintaining neuronal transport and MAP interactions.

molecular biology↗

bioRxiv2025

Effective Tubulin Degradation by Rationally Designed Proteolysis Targeting Chimeras

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that induce the degradation of proteins of interest (POIs) via the ubiquitin-proteasome pathway by recruiting E3 ligases to form a ternary complex with the POI. In this study, we rationally designed and synthesized PROTACs targeting the {beta}-tubulin heterodimer, the building block of microtubules (MTs) that are essential for numerous cellular functions and represent important therapeutic targets in cancer and neurodegenerative diseases. Maytansinol, a known MT-destabilising agent, was selected as the POI ligand, functionalised and conjugated to linkers bearing cereblon or Von Hippel-Lindau ligands as E3 ligase recruiters. Four compounds were synthesized and characterized through structural, biophysical and cell biology studies to evaluate their ability to form degradation-prone tubulin-PROTAC-E3 ligase ternary complexes. We confirmed that the PROTACs effectively bind tubulin and recruit E3 ligases. Remarkably, two PROTACs exhibited cellular degradation activity, representing an important advancement in chemically inducing tubulin-E3-ligase interactions. This work integrates rational design, biophysical and structural validation, and cell-based studies to establish a robust framework for developing tubulin-targeting PROTACs, offering significant implications for basic research and therapeutic developments.

biochemistry↗

bioRxiv2022

Quantitative live cell imaging of a tauopathy model enables the identification of a polypharmacological drug candidate that restores physiological microtubule regulation

Tauopathies such as Alzheimers disease are characterized by the aggregation and increased phosphorylation of the microtubule-associated protein tau. The pathological changes in tau are closely linked to neurodegeneration, making tau a prime candidate for intervention. However, the multiple facets of tau function and the lack of cellular tauopathy models that could support mechanism-based drug development hampers progress. Here we report the development of a live-cell imaging approach to quantitatively monitor pathological changes of human tau as it interacts with axonal microtubules. We show that a full-length aggregation-prone tau construct exhibits reduced interaction with microtubules as it increasingly aggregates. Through chemoinformatic analyses, we identified 2-phenyloxazole (PHOX) derivatives as putative polypharmacological small molecules that inhibit tau aggregation and modulate tau phosphorylation. We found that PHOX15 restores the physiological microtubule interaction of aggregation-prone tau in neurons and inhibits the first phase of tau aggregation in vitro. Furthermore, we report that PHOX15 inhibits the tau kinases GSK3{beta} and Cdk5, alters the kinome activity of model neurons, and reduces tau phosphorylation at disease-relevant sites. Molecular dynamics simulations highlight cryptic channel-like pockets crossing tau protofilaments and indicate that the binding of PHOX15 in one of the channels reduces the protofilaments ability to adopt a PHF-like conformation. The data show that our imaging approach provides a useful tool for identifying compounds that modulate tau-microtubule interaction in axons. We demonstrate that a polypharmacological approach to simultaneously treat tau aggregation and tau phosphorylation is able to restore physiological microtubule regulation, identifying PHOX15 as a promising drug candidate to counteract tau-induced neurodegeneration.

neuroscience↗