Acetylcholinesterase Complexed with Bivalent Ligands Related to Huperzine A: Experimental Evidence for Species-Dependent Protein−Ligand Complementarity

Acetylcholinesterase (AChE) inhibitors improve the cognitive abilities of Alzheimer patients. (−)-Huperzine A [(−)-HupA], an alkaloid isolated from the club moss, Huperzia serrata, is one such inhibitor,
but the search for more potent and selective drugs continues. Recently, alkylene-linked dimers of 5-amino-5,6,7,8-tetrahydroquinolinone (hupyridone, 1a), a fragment of HupA, were shown to serve as more potent
inhibitors of AChE than (−)-HupA and monomeric 1a. We soaked two such dimers, (S,S)-(−)-bis(10)-hupyridone [(S,S)-(−)-2a] and (S,S)-(−)-bis(12)-hupyridone [(S,S)-(−)-2b] containing, respectively, 10 and
12 methylenes in the spacer, into trigonal TcAChE crystals, and solved the X-ray structures of the resulting
complexes using the difference Fourier technique, both to 2.15 Å resolution. The structures revealed one
HupA-like 1a unit bound to the “anionic” subsite of the active-site, near the bottom of the active-site gorge,
adjacent to Trp84, as seen for the TcAChE/(−)-HupA complex, and the second 1a unit near Trp279 in the
“peripheral” anionic site at the top of the gorge, both bivalent molecules thus spanning the active-site gorge.
The results confirm that the increased affinity of the dimeric HupA analogues for AChE is conferred by
binding to the two “anionic” sites of the enzyme. Inhibition data show that (−)-2a binds to TcAChE ∼6−7-
and > 170-fold more tightly than (−)-2b and (−)-HupA, respectively. In contrast, previous data for rat AChE
show that (−)-2b binds ∼3- and ∼2-fold more tightly than (−)-2a and (−)-HupA, respectively. Structural
comparison of TcAChE with rat AChE, as represented by the closely related mouse AChE structure
(1maa.pdb), reveals a narrower gorge for rat AChE, a perpendicular alignment of the Tyr337 ring to the
gorge axis, and its conformational rigidity, as a result of hydrogen bonding between its hydroxyl group and
that of Tyr341, relative to TcAChE Phe330. These structural differences in the active-site gorge explain
the switch in inhibitory potency of (−)-2a and 2b and the larger dimer/(−)-HupA potency ratios observed
for TcAChE relative to rat AChE. The results offer new insights into factors affecting protein−ligand
complementarity within the gorge and should assist the further development of improved AChE inhibitors.