3D Structure of Torpedo californica Acetylcholinesterase Complexed with Huprine X at 2.1 Å Resolution: Kinetic and Molecular Dynamic Correlates ²,‡ H. Dvir, $,§ D. M. Wong, $ M. Harel, $ X. Barril, | M. Orozco, ⊥ F. J. Luque, | D. Mun ˜oz-Torrero, # P. Camps, # T. L. Rosenberry, ∇ I. Silman, § and J. L. Sussman* ,$ Department of Structural Biology, Weizmann Institute of Science, RehoVot, Israel, 76100, Department of Neurobiology, Weizmann Institute of Science, RehoVot, Israel, 76100, Departament de Fisicoquı ´mica, Facultat de Farma ` cia, UniVersitat de Barcelona, AV. Diagonal 643, 08028 Barcelona, Spain, Departament de Bioquı ´mica i Biologia Molecular, Facultat de Quı ´mica, UniVersitat de Barcelona, c/. Martı ´ i Franque ´ s 1, 08028 Barcelona, Spain, Laboratori de Quı ´mica Farmace ` utica, Facultat de Farma ` cia, UniVersitat de Barcelona, AV. Diagonal 643, 08028 Barcelona, Spain, and Department of Pharmacology and Program in Neurosciences, Mayo Foundation for Medical Education and Research, Mayo Clinic, JacksonVille, Florida 32224, USA ReceiVed August 13, 2001; ReVised Manuscript ReceiVed NoVember 28, 2001 ABSTRACT: Huprine X is a novel acetylcholinesterase (AChE) inhibitor, with one of the highest affinities reported for a reversible inhibitor. It is a synthetic hybrid that contains the 4-aminoquinoline substructure of one anti-Alzheimer drug, tacrine, and a carbobicyclic moiety resembling that of another AChE inhibitor, (-)-huperzine A. Cocrystallization of huprine X with Torpedo californica AChE yielded crystals whose 3D structure was determined to 2.1 Å resolution. The inhibitor binds to the anionic site and also hinders access to the esteratic site. Its aromatic portion occupies the same binding site as tacrine, stacking between the aromatic rings of Trp84 and Phe330, whereas the carbobicyclic unit occupies the same binding pocket as (-)-huperzine A. Its chlorine substituent was found to lie in a hydrophobic pocket interacting with rings of the aromatic residues Trp432 and Phe330 and with the methyl groups of Met436 and Ile439. Steady-state inhibition data show that huprine X binds to human AChE and Torpedo AChE 28- and 54-fold, respectively, more tightly than tacrine. This difference stems from the fact that the aminoquinoline moiety of huprine X makes interactions similar to those made by tacrine, but additional bonds to the enzyme are made by the huperzine-like substructure and the chlorine atom. Furthermore, both tacrine and huprine X bind more tightly to Torpedo than to human AChE, suggesting that their quinoline substructures interact better with Phe330 than with Tyr337, the corresponding residue in the human AChE structure. Both (-)-huperzine A and huprine X display slow binding properties, but only binding of the former causes a peptide flip of Gly117. Anticholinergic drugs that block acetylcholine receptors in the brain have adverse effects on memory (1), and postmortem data from the brains of Alzheimer’s disease (AD) patients indicate selective loss of cholinergic neurons (2). Such observations gave rise to the “cholinergic hypothesis” that AD is associated with impairment in cholinergic transmission (3-5). This hypothesis led to the suggestion that cholinesterase (ChE) inhibitors might alleviate a putative deficit in acetylcholine (ACh) levels associated with AD, and thus might slow the process of cognitive impairment characteristic of the disease (5, 6). Consequently, a number of ChE inhibitors have been considered as candidates for the symptomatic treatment of AD, with some already approved by the United States FDA for general use. They include natural substances, such as physostigmine, (-)- huperzine A (7-9), and galanthamine, also known as Reminyl (10, 11), all of which are alkaloids, and synthetic compounds such as tacrine, also known as Cognex (12- ² This work was supported by the U. S. Army Medical and Materiel Command under Contract No. DAMD17-97-2-7022, the EC fifth Framework Program on the Quality of Life and Management of Living Resources, the Kimmelman Center for Biomolecular Structure and Assembly (Rehovot, Israel), the Benoziyo Center for Neurosciences, the Ministerio de Ciencia y Tecnologı ´a (PB98-1222 and PB99-0046), Grant NS-16577 from the National Institutes of Health, the Muscular Dystrophy Association of America, and the Fundacio ´ La Marato ´-TV3, project 3004/97. X.B. received a fellowship from the Ministerio de Ciencia y Tecnologı ´a. I.S. is the Bernstein-Mason Professor of Neurochemistry. ‡ Coordinates for the structure of the complex of TcAChE/huprine X and its structure factors have been deposited at the PDB with the accession code 1E66. * Corresponding author: Joel L. Sussman, E-mail: joel.sussman@ weizmann.ac.il, Tel: +972-8-9344531, Fax: +972-8-9344159. $ Department of Structural Biology, Weizmann Institute of Science. § Department of Neurobiology, Weizmann Institute of Science. | Departament de Fisicoquı ´mica, Facultat de Farma `cia, Universitat de Barcelona. ⊥ Facultat de Quı ´mica, Universitat de Barcelona. # Laboratori de Quı ´mica Farmace `utica, Facultat de Farma `cia, Uni- versitat de Barcelona. ∇ Mayo Foundation for Medical Education and Research. 1 Abbreviations: ACh, acetylcholine; ChE, cholinesterase; AChE, acetylcholinesterase (EC 3.1.1.7); Tc, Torpedo californica; h, human; AD, Alzheimer’s disease; MD, molecular dynamics; PDB, Protein Data Bank; NSLS, National Synchrotron Light Source; BNL, Brookhaven National Laboratory; CNS, Crystallography & NMR System; SA, simulated annealing; IB, individual B-factors; E int, interaction energy; STD, standard deviation; rmsd, root-mean-square deviation; PEG, poly- (ethylene glycol); TMTFA, m-(N,N,N-trimethylammonio)-2,2,2-tri- fluoroacetophenone; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); MES, 2-[N-morpholino]ethanesulfonic acid. 2970 Biochemistry 2002, 41, 2970-2981 10.1021/bi011652i CCC: $22.00 © 2002 American Chemical Society Published on Web 02/06/2002