Mechanism-Based Rational Discovery and In Vitro Evaluation of Novel Microtubule Stabilizing Agents with Non-Taxol-Competitive Activity

Abstract

Microtubules (MT) are cytoskeletal polymers of αβ-tubulin dimers that play a critical role in many cellular functions. Diverse antimitotic drugs bind to MT and disrupt their dynamics acting as MT stabilizing or destabilizing agents. The occurrence of undesired side effects and drug resistance encourages the search for novel MT binding agents with chemically diverse structures and different interaction profiles compared to known active compounds. This work reports the rational discovery of seven novel MT stabilizers using a combination of molecular modeling methods and in vitro experimental assays. Virtual screening, similarity filtering, and molecular mechanics generalized Born surface area (MM/GBSA) binding free energy refinement were employed to select seven potential candidates with high predicted affinity toward the non-taxoid site for MT stabilizers on β-tubulin. MD simulations of 150 ns on reduced MT models suggest that candidate compounds strengthen the longitudinal interactions between tubulin dimers across protofilaments, which is a primary molecular mechanism of action for known MT stabilizers. In vitro MT polymerization assays confirmed that all candidates promote MT assembly at concentrations of >50 mM and exhibit noncompetitive MT polymerization profiles when cotreating with Taxol. Preliminary HeLa cell viability assays revealed a moderate cytotoxic effect for the compounds under study at 100 μM concentration. These results support the validity of our rational discovery strategy and the use of molecular modeling methods to pursue the search and optimization of new MT targeting agents.

On the Microtubule-Stabilizing Properties of a Tau Oligopeptide

“…The energetics of the MT lattice was examined through MM/GBSA calculations on GaMD trajectories. MM/GBSA has been used in past studies to obtain reliable descriptors accounting for the strength of lateral and longitudinal interactions between tubulin dimers. ,, Thereby, this method is expected to provide insight into the MT-stabilizing properties of the Tau peptides based on the energetics of protein–protein contacts within the reduced MT models in ligand-free and Tau-complexed states. To this aim, two ligand–receptor schemes were applied to estimate the strength of lateral and longitudinal interactions between tubulin units within the octameric models (Figure ).…”

“…The Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method was applied using 600 frames retrieved from the 300 ns GaMD trajectories and a single trajectory approach. The modified GB model (igb = 5) with mbondi2 with α, β, and γ values of 1.0, 0.8, and 4.85 was used to treat the polar component of solvation free energy with dielectric constants of 80 and 1 for the solvent and the protein, respectively, based on our recently published reports. ,,, Kolmogorov–Smirnov (KS) and unpaired two-sample Wilcoxon (W) tests were applied to compare the distribution and mean values for backbone MT rmsd data in ligand-free and complexed states using the R software.…”

“… , This MT model is the smallest system with biochemical significance to deal with the binding of Tau-like peptides as the Tau binding domains span three consecutive monomeric units across protofilaments centered in α-tubulin . These systems are expected to account for the lateral and longitudinal protein–protein contacts involved in MT assembly and stabilization as observed in prior molecular modeling studies dealing with the binding effects of diversity of MT-stabilizing compounds. − Our simulation approach considers the association of two identical Tau oligopeptides to equivalent binding regions on adjacent protofilament ridges. This strategy was conceived as a means to enhance the exploration of the conformational space for the peptide by studying two independent systems from a single simulation run.…”

Interdimeric Curvature in Tubulin–Tubulin Complexes Delineates the Microtubule-Destabilizing Properties of Plocabulin

“…MM/GBSA binding free-energy calculations were applied to estimate the strength of plocabulin–tubulin contacts in model 1 and model 2 complexes (Table ), using previously reported protocols for the study of ligand–tubulin and tubulin–tubulin interactions. − Analogous results for rhizoxin complexes built from the 4TUV crystallographic model were also obtained for comparative purposes. Our aim in this regard was to discriminate between these two ligands in terms of their affinity toward the maytansine site in dimeric and tetrameric tubulin complexes using MM/GBSA data. − According to MM/GBSA calculations, plocabulin is predicted to have a high affinity toward the maytansine pocket in the tubulin dimer (model 1 ) with an average Δ G bind of −58.1 ± 0.2 kcal mol –1 .…”

“…The modified GB model (igb = 5) with mbondi2 with α, β, and γ values of 1.0, 0.8, and 4.85, respectively, was employed to treat the polar component of solvation free energy with dielectric constants of 80 and 1 for the solvent and the protein, respectively. This method was chosen because of its proven reliability to examine the energetic aspects of ligand–tubulin and tubulin–tubulin interactions in recent published reports. − The curvature of the tubulin–tubulin arrangement in plocabulin complexes with two tubulin dimers (model 2 ) was evaluated from the analysis of the interdimeric θ angle (°) formed by the vectors that connect the centers of mass of intradimeric α and β subunits. Average θ (°) values were used to estimate the number of tubulin units to complete an oligomeric ring and the curvature (κ) of this cyclic arrangement as the reciprocal of the ring radius ( R ).…”