Acetylcholinesterase complexed with bivalent ligands related to Huperzine A

Experimental evidence for species-dependent protein-ligand complementarity

Dawn M. Wong, Harry M. Greenblatt, Hay Dvir, Paul R. Carlier, Yi Fan Han, Yuan-Ping Pang, Israel Silman, Joel L. Sussman

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)363-373
Number of pages11
JournalJournal of the American Chemical Society
Volume125
Issue number2
DOIs
StatePublished - Jan 15 2003

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Acetylcholinesterase
Rats
Cholinesterase Inhibitors
Catalytic Domain
Ligands
Dimers
Proteins
Lycopodiaceae
Huperzia
Enzyme inhibition
Alkaloids
Hydrogen Bonding
Rigidity
Hydroxyl Radical
Hydrogen bonds
Enzymes
Switches
X-Rays
X rays
Crystals

ASJC Scopus subject areas

  • Chemistry(all)

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Acetylcholinesterase complexed with bivalent ligands related to Huperzine A : Experimental evidence for species-dependent protein-ligand complementarity. / Wong, Dawn M.; Greenblatt, Harry M.; Dvir, Hay; Carlier, Paul R.; Han, Yi Fan; Pang, Yuan-Ping; Silman, Israel; Sussman, Joel L.

In: Journal of the American Chemical Society, Vol. 125, No. 2, 15.01.2003, p. 363-373.

Research output: Contribution to journalArticle

Wong, Dawn M. ; Greenblatt, Harry M. ; Dvir, Hay ; Carlier, Paul R. ; Han, Yi Fan ; Pang, Yuan-Ping ; Silman, Israel ; Sussman, Joel L. / Acetylcholinesterase complexed with bivalent ligands related to Huperzine A : Experimental evidence for species-dependent protein-ligand complementarity. In: Journal of the American Chemical Society. 2003 ; Vol. 125, No. 2. pp. 363-373.
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abstract = "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 {\AA} 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.",
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T1 - Acetylcholinesterase complexed with bivalent ligands related to Huperzine A

T2 - Experimental evidence for species-dependent protein-ligand complementarity

AU - Wong, Dawn M.

AU - Greenblatt, Harry M.

AU - Dvir, Hay

AU - Carlier, Paul R.

AU - Han, Yi Fan

AU - Pang, Yuan-Ping

AU - Silman, Israel

AU - Sussman, Joel L.

PY - 2003/1/15

Y1 - 2003/1/15

N2 - 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.

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