Conformation Driven Enhancement of Neurolysin Activity in Presence of a Small Molecule Activator

Neurolysin (Nln) is an M3 metallopeptidase that regulates neuropeptide concentration in the central nervous system. It has emerged as a therapeutic target for mitigating post-ischemic injury by hydrolyzing and inactivating several neuropeptides. It has been recently shown that small molecule activators, such as pyridine piperazine (Py-Pip) derivatives, can enhance Nln catalytic activity, facilitating hydrolysis of Nln substrate peptides. However, binding sites of these molecules and the mechanism of action remain unclear due to the dynamic nature of Nln. Here, we use molecular dynamics (MD) simulation of the apo and activator-bound Nln systems along with Markov state modeling to identify three binding sites of Py-Pip, and quantify their effects on the Nln conformational landscape and kinetics. Two of these sites have opposing functional outcomes: Binding inside the interhelical channel favors <i>closed</i> conformations by slowing down channel opening, thus allowing the substrates to orient favorably for the catalytic reaction, while binding at the exterior region of the channel stabilizes <i>open</i> states, potentially facilitating both substrate entry and cleaved product release. These findings suggest a possibility of a multi-site allosteric activation model in which distinct binding locations selectively modulate different steps within a catalytic cycle. This framework provides a structural and kinetic model for the action of small molecule Nln activators, offering insight into the design of cerebro-protective therapeutics that reduce neuropeptide accumulation after ischemic injury.