Abstract
Inhibitors of the enzyme acetylcholinesterase (AChE) slow and sometimes reverse the cognitive decline experienced by individuals with Alzheimer's disease. Huperzine A, a natural product used in traditional Chinese herbal medicine, and tacrine (Cognex) are among the potent AChE inhibitors used in this treatment, but the search for more selective inhibitors continues. We report herein the synthesis and characterization of (−)-12-amino-3-chloro-9-ethyl-6,7,10,11-tetrahydro-7,11-methanocycloocta[b]quinoline hydrochloride (huprine X), a hybrid that combines the carbobicyclic substructure of huperzine A with the 4-aminoquinoline substructure of tacrine. Huprine X inhibited human AChE with an inhibition constantK I of 26 pM, indicating that it binds to this enzyme with one of the highest affinities yet reported. Under equivalent assay conditions, this affinity was 180 times that of huperzine A, 1200 times that of tacrine, and 40 times that of E2020 (donepezil, Aricept), the most selective AChE inhibitor currently approved for therapeutic use. The association and dissociation rate constants for huprine X with AChE were determined, and the location of its binding site on the enzyme was probed in competition studies with the peripheral site inhibitor propidium and the acylation site inhibitor edrophonium. Huprine X showed no detectable affinity for the edrophonium-AChE complex. In contrast, huprine X did form a ternary complex with propidium and AChE, although its affinity for the free enzyme was found to be 17 times its affinity for the propidium-AChE complex. These data indicated that huprine X binds to the enzyme acylation site in the active site gorge but interferes slightly with the binding of peripheral site ligands.
Footnotes
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Send reprint requests to: Terrone L. Rosenberry, Department of Pharmacology, Mayo Clinic Jacksonville, 4500 San Pablo Rd., Jacksonville, FL 32224. E-mail:rosenb{at}mayo.edu
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↵1 Throughout this paper, we number residues according to the TcAChE sequence. For example, W84 and S200 in this sequence correspond to W86 and S203, respectively, in mammalian AChE.
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↵2 One unit of AChE activity corresponds to 1 μmol of acetylthiocholine hydrolyzed per minute under standard pH-stat assay conditions, and these conditions correspond to maximal AChE activity at pH 8 (Rosenberry and Scoggin, 1984). Our conventional spectrophotometric assay at 412 nm is conducted in pH 7 buffer with 0.5 mM acetylthiocholine, conditions that result in 4.5 ΔA412 nm/min with 1 nM AChE or ∼76% of the maximal activity.
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↵4 See the Physicians' Desk Reference (1999), Medical Economics Co., Montvale, New Jersey.
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↵3 The K I determined for TMTFA (50 pM; Szegletes et al., 1998) was based on the total concentration of TMTFA and its hydrate in solution. Because the hydrate is not an AChE inhibitor but is present in 105-fold excess over TMTFA (Nair et al., 1993), the intrinsic equilibrium dissociation constant K D for TMTFA itself with AChE is about 10−15 M.
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This work was supported by Grant NS-16577 from the National Institutes of Health, DAMD 17-98-2-8019 from the U.S. Army Medical Research Acquisition Activity, and by grants from the Muscular Dystrophy Association of America. Financial support from the Comisión Interministerial de Ciencia y Tecnologı́a (Programa Nacional de Tecnologı́a de los Procesos Quı́micos, Project QUI96–0828), Fundació “La Marató de TV3” (Project 3004/97), Comissionat per a Universitats i Recerca of the Generalitat de Catalunya (Project 1997-SGR-00140), and Medichem, S.A., and fellowships from Comissió Interdepartamental de Recerca i InnovacióTecnològica of the Generalitat de Catalunya to J. Morral and from Agencia Española de Cooperación Internacional (Instituto de Cooperación con el Mundo Arabe, Mediterráneo y Paı́ses en Desarrollo) to R.E.A. are gratefully acknowledged.
- Abbreviations:
- AChE
- acetylcholinesterase
- MPLC
- medium-pressure liquid chromatography
- DTNB
- 5,5′-dithiobis-(2-nitrobenzoic acid)
- TcAChE
- Torpedo californica acetylcholinesterase
- TMTFA
- m-(N, N,Ntrimethylammonio)trifluoroacetophenone
- COSY
- correlation spectroscopy
- HMQC
- heteronuclear multiple-quantum coherence
- Received August 23, 1999.
- Accepted November 3, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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Huprine X is a Novel High-Affinity Inhibitor of Acetylcholinesterase That Is of Interest for Treatment of Alzheimer's Disease
