Biology subjects

Prota, A. E.

Publications and source records attributed to Prota, A. E..

3 recordsLinked to original sources

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↗

bioRxiv2021

Crystal structures of taxane-tubulin complexes: Implications for the mechanism of microtubule stabilization by Taxol

Paclitaxel (Taxol(R)) is a taxane and a first-line chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the current lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect the drugs mechanism of action. Here, we solved the crystal structure of the core baccatin III moiety of paclitaxel lacking the C13 side chain in complex with tubulin at 1.9 [A] resolution. Based on this information, we engineered two tailor-made taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects along with those of paclitaxel, docetaxel, and baccatin III on the microtubule lattice by X-ray fiber diffraction. We then compared high-resolution structures of ligand-bound tubulin and microtubule complexes with apo forms and used molecular dynamics simulations to understand the consequences of taxane binding to tubulin as well as to simplified protofilament and microtubule-lattice models. Our combined approach sheds light on three mechanistic questions. Firstly, taxanes bind better to microtubules as compared to unassembled tubulin due to a dual structural mechanism: Tubulin assembly is linked to a conformational reorganization of the {beta}M loop, which otherwise occludes ligand access to the taxane site, while the bulky C13 side chains preferentially recognize the microtubule-assembled over the unassembled conformational state of tubulin. Second, the occupancy of the taxane site by a ligand has no influence on the straightness of tubulin protofilaments. Finally, binding of the taxane core to the taxane site displaces the S9-S10 loop of {beta}-tubulin resulting in microtubule expansion. Our results provide detailed new insights into the mechanism of microtubule-stabilization by taxanes.

biophysics↗

bioRxiv2020

Pharmaceutical-grade Rigosertib is a Microtubule-destabilizing Agent

We recently used CRISPRi/a-based chemical-genetic screens and targeted cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. In a recent manuscript, Reddy and co-workers suggest that this microtubule-destabilizing activity of rigosertib is mediated not by rigosertib itself but by a contaminating degradation product of rigosertib, ON01500, present in formulations obtained from commercial vendors (Baker et al., 2019). Here, we demonstrate that treatment of cells with pharmaceutical-grade rigosertib (>99.9% purity) results in qualitatively indistinguishable phenotypes as treatment with commercially obtained rigosertib across multiple assays. The two compounds have indistinguishable chemical-genetic interactions with genes involved in modulating the microtubule network (KIF2C and TACC3), both destabilize microtubules in cells and in vitro, and both show substantially reduced toxicity in cell lines expressing a rationally-designed mutant of tubulin (L240F TUBB mutant), in which the rigosertib binding site in tubulin is mutated. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents, which is disputed by Reddy and co-workers, was recently confirmed by an independent research group (Patterson et al., 2019). We conclude that rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

cancer biology↗