Hopeahainol A binds reversibly at the acetylcholinesterase (AChE) peripheral site and inhibits enzyme activity with a novel higher order concentration dependence

Abstract

Natural product inhibitors of AChE are of interest both because they offer promise as inexpensive drugs for symptomatic relief in Alzheimer’s disease and because they may provide insights into the structural features of the AChE catalytic site. Hopeahainol A is an uncharged polyphenol AChE inhibitor from the stem bark of H. hainanensis with a constrained, partially dearomatized bicyclic core. Molecular modeling indicates that hopeahainol A binds at the entrance of the long but narrow AChE active site gorge because it is too bulky to be accommodated within the gorge without severe distortion of the gorge as depicted in AChE crystal structures. We conducted inhibitor competition experiments in which AChE inhibition was measured with hopeahainol A together with either edrophonium (which binds at the base of the gorge) or thioflavin T (which binds to the peripheral or P-site near the gorge mouth). The results agreed with the molecular modeling and indicated that hopeahainol A at lower concentrations (< 200 µM) bound only to the P-site, as hopeahainol A and thioflavin T were unable to form a ternary complex with AChE while hopeahainol A and edrophonium did form a ternary complex with essentially no competition between them. Inhibition increased to a striking extent at higher concentrations of hopeahainol A, with plots analogous to classic Dixon plots showing a dependence on hopeahainol A concentrations to the third- or fourth order. The inhibition at higher hopeahainol A concentrations was completely reversed on dilution and blocked by bound edrophonium. We hypothesize that bound hopeahainol A induces conformational changes in the AChE active site that allow binding of additional hopeahainol A molecules, a phenomenon that would be unprecedented for a reversible inhibitor that apparently forms no covalent bonds with AChE.

An Unusual Dimeric Inhibitor of Acetylcholinesterase: Cooperative Binding of Crystal Violet

“…For numerous reversible ligands, dose-response curves are also available. A recent study of the inhibitor hopeahainol shows an inhibition kinetics that is consistent with cooperative binding to AChE [ 29 ]. Still, cooperativity appears rare, and is linked to the specific chemistry and structure of the CV•AChE complex and is not a common, inherent property of cholinesterases.…”

Comparison of the Binding of Reversible Inhibitors to Human Butyrylcholinesterase and Acetylcholinesterase: A Crystallographic, Kinetic and Calorimetric Study

“…Such structures involving 4-ketoamyltrimethylammonium or choline with AChE [ 4 , 5 ] and acetylcholine with the S203A mutant of AChE [ 5 ] involve simultaneous binding of ligand to the A- and P-sites. Recently, AChE complexes with two or more bound ligands have been deduced from kinetic data for hopeahainol A [ 46 ] and from both kinetic data and the crystal structure with the dye crystal violet [ 47 ]. Neither of these complexes appear to include ligand binding to the A-site, but instead involve simultaneous ligand binding near the entrance of the active site, possibly including the P-site.…”