Rational Discovery of Microtubule-Stabilizing Peptides

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 ∼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 MT-binding 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 MT-stabilizing 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.