Abstract

Microtubule (MT) stabilization is an attractive pharmacological strategy to hamper the progress of neurodegenerative diseases. In this regard, seeking peptides with MT-stabilizing properties has awoken great interest. This work reports the rational discovery of two structurally related MT-stabilizing octapeptides using a combination of protein-peptide docking, conventional molecular dynamics, Gaussian accelerated molecular dynamics (GaMD), and tubulin polymerization assays. FASTA sequences for similar to 1000 peptides were crafted from single and double mutants of davunetide (NAP) and docked against the Taxol (TX) site on an octameric MT model representing a portion of the MT wall. Docked peptides were rescored after MM minimization and binding free energy refinement through single-point MM/GBSA calculations. The 60 best-ranked peptides were subjected to 50 ns MD simulations on peptide-MT complexes at the terminal TX site in the octameric Tau-MT model resulting in 11 complexes with occupancies greater than 99% and peptide-protein binding free energies less than -40 kcal/mol. Selected peptides were then examined through 300 ns GaMD simulations in complexes containing two identical ligands at the terminal and intermediate TX sites in the Tau-MT model to account for the differential association of MTbinding peptides to different regions of the MT structure. Six candidates showed a favorable MT-binding potential based on the analysis of interaction frequencies and relative mobilities of the complex components, suggesting a pivotal role of Arg278, Gln281, and Arg369 residues for peptides recognition. Four candidates were predicted to preserve an adequate balance of longitudinal and lateral interactions between tubulin dimers in peptide-MT complexes such that MT-stabilizing effects could be expected. MT polymerization experiments confirmed that four peptides (HAPVSIHQ, NYPVSIHQ, NWPVSIWQ, HAPVSIIQ) exhibit MTstabilizing activity in vitro with NWPVSIWQ (P43) and HAPVSIIQ (P52) being the most active. Tryptophan quenching assays verified that P43 and P52 bind to nonpolymeric tubulin, whereas viability experiments on HEK cells confirmed their safety to pursue future pharmacological studies. The results herein presented are valuable to making progress in the rational design of MT-stabilizing peptides.