The unbinding process of E2020 ((R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]-methylpiperidine) leaving
from the long active site gorge of Torpedo californica acetylcholinesterase (TcAChE) was studied by using
steered molecular dynamics (SMD) simulations on a nanosecond scale with different velocities, and unbinding
force profiles were obtained. Different from the unbinding of other AChE inhibitors, such as Huperzine A
that undergoes the greatest barrier located at the bottleneck of the gorge, the major resistance preventing
E2020 from leaving the gorge is from the peripheral anionic site where E2020 interacts intensively with
several aromatic residues (e.g., Tyr70, Tyr121, and Trp279) through its benzene ring and forms a strong
direct hydrogen bond and a water bridge with Ser286 via its O24. These interactions cause the largest rupture
force, 550 pN. It was found that the rotatable bonds of the piperidine ring to the benzene ring and
dimethoxyindanone facilitate E2020 to pass the bottleneck through continuous conformation change by rotating
those bonds to avoid serious conflict with Tyr121 and Phe330. The aromatic residues lining the gorge wall
are the major components contributing to hydrophobic interactions between E2020 and TcAChE. Remarkably,
these aromatic residues, acting in three groups as ”°sender”± and ”°receiver”±, compose a ”°conveyer belt”± for
E2020 entering and leaving the TcAChE gorge.