Cell‐Penetrating Peptides: Correlation between Peptide‐Lipid Interaction and Penetration Efficiency

Abstract

Cell‐penetrating peptides are used in the delivery of peptides and biologics, with some cell‐penetrating peptides found to be more efficient than others. The exact mechanism of how they interact with the cell membrane and penetrate it, however, remains unclear. This study attempts to investigate the difference in free energy profiles of three cell‐penetrating peptides (TAT, CPP1 and CPP9) with a model lipid bilayer (DOPC) using molecular dynamics pulling simulations with umbrella sampling. Potential mean force (PMF) and free energy barrier between the peptides and DOPC are determined using WHAM analysis and MM‐PBSA analysis, respectively. CPP9 is found to have the smallest PMF value, followed by CPP1 and TAT, consistent with the experimental data. YDEGE peptide, however, does not give the highest PMF value, although it is a non‐cell‐permeable peptide. YDEGE is also found to form water pores, alongside with TAT and CPP9, suggesting that it is difficult to distinguish true water pore formation from artefacts arising from pulling simulations. On the contrary, free energy analysis of the peptide‐DOPC complex at the lipid‐water interface with MM‐PBSA provides results consistent with experimental data with CPP9 having the least interaction with DOPC and lowest free energy barrier, followed by CPP1, TAT and YDEGE. These findings suggest that peptide‐lipid interaction at the lipid‐water interface has a direct correlation with the penetration efficiency of peptides across the lipid bilayer.

Free Energy Analyses of Cell-Penetrating Peptides Using the Weighted Ensemble Method

“…Please note that the total simulation time for each CPP simulation is about 5∼10 µs, which includes all the walkers (child simulations) in each bin and all the iterations. This time scale of a single trajectory might be too short to observe a water pore mentioned in the previous results [8,13,27,30]. Second, our PMF profiles are incomplete because we do not find any translocation of CPPs and PMF profiles spanned only from the top to the middle of the model membranes.…”

“…is very similar to a previous result [27], which used umbrella samplings. On the other hand, Tat's order of magnitude of free energy is much higher than those of previous simulations [8,13]. Note that most of the previous free energy analyses were done with a single lipid molecule (e.g., DOPC or DPPC), while we use a mixed composition of…”

“…Due to the short time scale achieved in current MD simulations, people have been using biased simulations (e.g., umbrella sampling [11][12][13], steered MD simulations [14]) to study CPPs and their interactions with membranes. The umbrella sampling is very popular to obtain free energy barrier between CPPs and membranes.…”

Free energy analyses of cell-penetrating peptides using the weighted ensemble method

“…The order of magnitude of free energy for Arg 9 (∼ 100 kT) is very similar to a previous result [27], which used umbrella samplings. On the other hand, Tat's order of magnitude of free energy is much higher than those of previous simulations [8,13]. Note that most of the previous free energy analyses were done with a single lipid molecule (e.g., DOPC or DPPC), while we use a mixed composition of lipid molecules (DOPC/DOPG(4:1)) in our simulations.…”

Recent Developments in Free Energy Calculations for Drug Discovery

“…Peptide sequences are often optimized to achieve a desired conformation capable of binding and inhibiting target proteins similarly to small molecule inhibitors, with the advantage that peptides can bind larger surface areas for greater specificity than small molecules. Simulation and in silico design of peptides has been performed to generate cyclic lead compounds against the hormone resistin through the alchemical method to optimize selectivity ( Chi and Vargas, 2020 ), construct peptide inhibitors derived from the LEDGF/P75 protein against HIV1 integrase receptor ( Kilburg and Gallicchio, 2018 ), investigate differences in free energy profiles of cell penetrating peptides on DOPC model lipid bilayers to characterize penetration efficiency ( Her Choong and Keat Yap, 2020 ), and identify determinants for peptide binding to the PSM9 PDZ domain found in synaptic junctions ( Harish et al, 2019 ).…”