De-shielding of activatable cell-penetrating peptides: recognizing and releasing in activation process

Abstract

Activatable cell-penetrating peptide (ACPP) would reduce the cytotoxicity of cell-penetrating peptide (CPP)-based drug delivery system by shielding the negative charge and improve the targeting of CPP when hydrolyzed by characteristic proteases. However, it is still not well understood how ACPP could be specifically recognized and how the shielding effect is removed. In this work, molecular dynamic simulations are used to study the interaction mechanism of the interacting domain of ACPP (the linking peptide) and its characteristic protein matrix metalloproteinase-9 (MMP-9). Results indicate the residue located in P1 site of the linking peptide is the key residue for recognition. The peptide bond skeleton rather than the side chain of residue determines the adsorption of residue in P1 site, which is also confirmed by mutation. The release process after hydrolysis is also found to be an important step to remove the shielding effect of ACPP. After enzyme hydrolysis, the binding of C-product (Ace-Pro-Leu-Gly) to MMP-9 is much stronger than that of N-product (Leu-Ala-Gly-Nme). Depending on the research for the departure mechanism of the hydrolysates, the dissociation constant (Kd) of C-product is much lower than N-product. Therefore, N-product (drug loaded CPP) prefers to leave MMP-9, while C-product (peptides providing shielding effects) tends to stay on MMP-9. This work highlights the recognizing and releasing process of substate-MMP-9 system, may provide a new perspective understanding for ACPP-based drug delivery system.