Biology subjects

Pieraccini, S.

Publications and source records attributed to Pieraccini, S..

3 recordsLinked to original sources

bioRxiv2026

Ligand Binding Free Energy Landscapes at the Tubulin Colchicine Site from Coarse-Grained Metadynamics

Accessing deeply buried binding sites remains a major challenge in structure-based drug discovery, where accurate description of both protein dynamics and ligand binding pathways is required. Funnel metadynamics enables simulation of complete binding processes but is computationally demanding at the all-atom resolution. By adopting the Martini 3 force field, coarse-grained funnel metadynamics (CG-FMD) substantially reduces computational requirements while retaining enhanced sampling capabilities. In this work, we assess the capability of CG-FMD to model ligand recognition at the deeply buried colchicinoids site of the tubulin {beta}-heterodimer, a multisite protein of strategic importance. We investigated the binding of colchicine, podophyllotoxin and combretastatin-A4, recovering free energy profiles with improved statistical convergence compared to AA-FMD and comparable to experimental references. In particular CG-FMD binding free energies present mean absolute errors between 3 and 10 kJ mol-1. These results propose CG-FMD as an efficient, physics-based framework for probing ligand binding to challenging sites.

biophysics↗

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↗

bioRxiv2020

Substrate Recognition by γ-Glutamyltransferase: A Molecular Dynamics Simulations Study

{gamma}-glutamyltransferase (GGT) is an enzyme that uses {gamma}-glutamyl compounds as substrate and catalyzes their transfer into a water molecule or an acceptor substrate with varied physiological-function in bacteria, plants and animals. Crystal structures of GGT are known for different species and in different states of the chemical reaction; however, structural dynamics of the substrate binding to the catalytic site of GGT is unknown. Here, we modeled Escherichia Coli GGTs glutamine binding by using a swarm of accelerated molecular dynamics (aMD) simulations. Characterization of multiple binding events identified three structural binding motifs composed of polar residues in the binding pocket that govern glutamine binding into the active site. Simulated open and closed conformations of a lid-loop protecting the binding cavity suggests its role as a gating element by allowing or blocking substrates entry into the binding pocket. Partially open states of the lid-loop are accessible within thermal fluctuations, while the estimated free energy cost of a complete open state is 2.4 kcal/mol. Our results suggest that both specific electrostatic interactions and GGT conformational dynamics dictate the molecular recognition of substrate-GGT complexes.

biophysics↗