Enhanced Uptake of Anticancer C6-Pep Dimer in a Model Membrane Caused by Differential pK_a in Acidic Microenvironment

Abstract

Acidic tumor microenvironment (TME) presents a challenge for the action of antitumor drugs by acting as an additional barrier for the passive crossing of the cell membrane by chemotherapic agents playing a critical role in the proliferation of tumor cells. Anticancer lipopeptide C6-Pep dimer containing the leucine zipper motif shows an increased uptake into the model tumor membrane in TME, and application of external heat might lead to the uncoiling of the zipper, which could result in cell lysis. This work investigated the cause of this increased uptake of C6-Pep dimer into the bilayer model in TME. Accurate protonation states of all the titratable residues of the C6-Pep dimer in TME were determined using constant pH molecular dynamics. In TME, except for two terminal Glu5 residues, all other Glu residues in the C6-Pep dimer were permanently protonated. The remaining Glu5 residues had differential pK a values, leading to the construction of four possible dimers with different fixed protonation states, and molecular dynamics was used to study their interaction with the anionic bilayer. Except for the dimer at a physiological pH, the other dimers were positively charged and could readily adsorb on the membrane surface. The free energy of insertion of these dimers in the bilayer was lower for single and double protonated Glu5-containing dimers than for the others. After the insertion of the lipopeptides into the membrane, thinning of the bilayer in the vicinity of dimers and an increase in area per lipid of the bilayer were observed for all systems, indicating destabilization of the bilayer due to this intercalation. This study shows that the anticancer lipopeptide C6-Pep utilizes the TME around a tumor cell for insertion into the membrane.

A Rigorous Framework for Calculating Protein–Protein Binding Affinities in Membranes

“…The investigation by Balusek et al, on the other hand, supports the reliability of CHARMM36 for GpA dimerization. It should be noted that the accuracy and reliability of this force field in describing membrane protein interactions was demonstrated in various studies, − making it a robust choice for investigating protein dimerization in the membrane. Consequently, any need for a dispersion correction, as suggested, may be primarily attributed to the choice of simulation conditions and the specific collective variables (CVs) utilized in the calculations conducted in the study by Domański et al These choices can significantly influence the overall binding affinity calculation as they play a pivotal role in shaping the simulation outcomes.…”