Hydrolysis Mechanism of the Linkers by Matrix Metalloproteinase‑9 Using QM/MM Calculations

Activatable cell-penetrating peptides
(ACPPs) are known to be able
to decrease the cytotoxicity of cell-penetrating
peptide (CPP)-based drug delivery systems. Furthermore, they can improve
the targeting of CPPs when specifically recognized and hydrolyzed
by characteristic proteases. A comprehensive and profound understanding
of the recognition and hydrolysis process will provide a better design
of the ACPP-based drug delivery system. Previous studies have clearly
described how ACPPs are recognized and bound by MMPs. However, the
hydrolysis mechanism of ACPPs is still unsolved. This work focuses
on a proteinase-sensitive cleavable linker of ACPPs (PLGLAG), the
key structure for recognition and hydrolysis, trying to determine
the mechanism by which MMP-9 hydrolyzes its substrate PLGLAG. The
quantum mechanics/molecular mechanics (QM/MM) calculations herein
show that MMP-9 proteolysis is a water-mediated four-step reaction.
More specifically, it consists of (i) nucleophilic attack, (ii) hydrogen-bond
rearrangement, (iii) proton transfer, and finally (iv) amide bond
rupture. Considering the reversibility of multistep reaction, the
second step (i.e., hydrogen-bond rearrangement) has the highest barrier
and is the rate-limiting step in the hydrolysis of PLGLAG. The possible
design and improvement of the key P1 and P1′ sites are also
explored through mutations. The present results indicate that, while
the mutations affect the reaction energy barriers and the rate-limiting
steps, all mutants considered could be hydrolyzed by MMP-9. To provide
further insights, the hydrolysis mechanism of MMP-2, which has a similar
hydrolysis process to that of MMP-9 but with different reaction barriers,
is also studied and compared. As a result, this work provides detailed
insights into the hydrolysis mechanism of ACPPs by MMP-9 and, thus,
also possible insights for the development of new strategies for ACPP-based
delivery systems.