J.Med.Chem.2009,52,2543-2549 The Crystal Structure of a Complex of Acetylcholinesterase with a Bis-()-nor-meptazinol Derivative Reveals Disruption of the Catalytic Triad Aviv Paz, .+ Qiong Xie, .Harry M. Greenblatt, t Wei Fu,' Yun Tang, Israel Silman, Zhuibai Qiu, and Joel L Sussman* T d Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel, Department of medicinai Chemistry, School of Pharmacy, Fudan University, Shanghai 200032, P. R. China, School of Pharmacy, East China University of Science and Technology, Shanghai 200237 P.R. China Received December 31. 2008 A bis-()-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and aB peptide aggregation The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 A resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the"anionic subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440N, which is hydrogen-bonded to Ser2000 in the native enzyme. The second nor-meptazinol binds at the peripheral anionic"site at the gorge entrance. A number of GOLD models of the complex, using both native TCAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors The enzyme acetylcholinesterase(AChE") terminates im transmission at central and peripheral cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine(ACh). It has served as a therapeutic target for the symptomatic treatment of Alzheimer's disease(AD) due to the cholinergic ypothesis, which argues that the cognitive decrements observed in AD patients are associated with impairment of cholinergic Figure 1. Chemical structure of 5h transmission. The hypothesis thus predicts that inhibition of AChE should prolong the effect of the neurotransmitter ACh, Table 1. Data Collection and Refinement Statistics resulting in partial restoration of the cognitive abilities of the P121 a=112.17 patients. As a consequence, the first generation of AD drugs cell parameters(A) were all acetylcholinesterase inhibitors(AChEIs), and four are c=137.57 currently used extensively, including the synthetic compound molecules/asymmetric unit 2020 and rivastigmine and the alkaloids galanthamine'and 40-2.7 al reflections 338424 AChE has been demonstrated to colocalize with the amyloid-P unique reflections 993%099.76) peptide(AB) in the brains of AD patients, and has been shown av lo(n) 17.5(3.55) to accelerate the assembly of AB to amyloid fibrils. Several (% ligands that bind at the peripheral anionic site(PAS)of AChE 18.5 have been shown to retard aggregation. Furthermore, a 23.5(5.2% of data) 0.019 monoclonal antibody directed against the PAs also reversed the rmsd, bond angles(deg effect of AChE on AB deposition. In the active-site gorge of PDB code 2W6C AChE the "anionic" subsite of the active site. also known as Values in parenthe the catalytic"anionic"site( CAS), is located at the bottom of A). Rmerge=2ll-( ) V2 I, where I is the observed intensity, and()is the gorge, and the PAS is near its entrance. 4. I This led to the the average intensity obtained from multiple observations of symmetry. development of the bivalent ligand approach in which identical related reflections after rejections.“R=∑F。-uFM∑ where F and Fc are the observed and calculated structure factors, respectively. As Fax: 972-8-934-4159. E-mail: joel. sussman(@ weizmann. acil or distinct pharmacophores are linked, via an appropriate spacer, Department of Structural Biology, Weizmann Institute of Science to produce a bifunctional drug with enhanced affinity. This Department of Neurobiology, Weizmann Institute of Science in turn, proved valuable for developing drugs with dual action s Department of Medicinal Chemistry, School of Pharmacy, Fudan both in inhibition of catalytic activity and in arresting the Ache- School of Pharmacy, East China University of Science and Technology catalyzed assembly of amyloid fibrils(for recent reviews see These authors made equal contribut refs 17 and 18) yloid-B peptide: ACh, acetylcholine: AChE, Xie and co-workers recently reported the synthesis and acetylcholinesterase: AChEL, acetylcholinesterase inhibitor, AD, Alzheimer's characterization of novel bis-(-)-nor-meptazinols that inhibit house: MEP,(-)-meptazinol; PAS, peripheral anionic site; Tc, Torpedo both AChE and butyrylcholinesterase (BChE) as well as californica: a, axial; e, equatorial tarding AB aggregation % The most potent of these compounds l0.102lm801657vCCC:S40 c 2009 American Chemical Society Web03/27/2009The Crystal Structure of a Complex of Acetylcholinesterase with a Bis-(-)-nor-meptazinol Derivative Reveals Disruption of the Catalytic Triad Aviv Paz,†,‡,⊥ Qiong Xie,§,⊥ Harry M. Greenblatt,† Wei Fu,§ Yun Tang,| Israel Silman,‡ Zhuibai Qiu,§ and Joel L. Sussman*,† Departments of Structural Biology and Neurobiology, Weizmann Institute of Science, RehoVot 76100, Israel, Department of Medicinal Chemistry, School of Pharmacy, Fudan UniVersity, Shanghai 200032, P.R. China, School of Pharmacy, East China UniVersity of Science and Technology, Shanghai 200237 P.R. China ReceiVed December 31, 2008 A bis-(-)-nor-meptazinol derivative in which the two meptazinol rings are linked by a nonamethylene spacer is a novel acetylcholinesterase inhibitor that inhibits both catalytic activity and A
peptide aggregation. The crystal structure of its complex with Torpedo californica acetylcholinesterase was determined to 2.7 Å resolution. The ligand spans the active-site gorge, with one nor-meptazinol moiety bound at the “anionic” subsite of the active site, disrupting the catalytic triad by forming a hydrogen bond with His440Nε2 , which is hydrogen-bonded to Ser200Oγ in the native enzyme. The second nor-meptazinol binds at the peripheral “anionic” site at the gorge entrance. A number of GOLD models of the complex, using both native TcAChE and the protein template from the crystal structure of the bis-(-)-nor-meptazinol/TcAChE complex, bear higher similarity to the X-ray structure than a previous model obtained using the mouse enzyme structure. These findings may facilitate rational design of new meptazinol-based acetylcholinesterase inhibitors. Introduction The enzyme acetylcholinesterase (AChEa ) terminates impulse transmission at central and peripheral cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (ACh).1 It has served as a therapeutic target for the symptomatic treatment of Alzheimer’s disease (AD) due to the cholinergic hypothesis, which argues that the cognitive decrements observed in AD patients are associated with impairment of cholinergic transmission.2,3 The hypothesis thus predicts that inhibition of AChE should prolong the effect of the neurotransmitter ACh, resulting in partial restoration of the cognitive abilities of the patients. As a consequence, the first generation of AD drugs were all acetylcholinesterase inhibitors (AChEIs),4 and four are currently used extensively, including the synthetic compounds E20205 and rivastigmine6 and the alkaloids galanthamine7 and (-)-huperzine A.8 AChE has been demonstrated to colocalize with the amyloid-
peptide (A
) in the brains of AD patients,9 and has been shown to accelerate the assembly of A
to amyloid fibrils.10 Several ligands that bind at the peripheral anionic site (PAS) of AChE have been shown to retard aggregation.11,12 Furthermore, a monoclonal antibody directed against the PAS also reversed the effect of AChE on A
deposition.13 In the active-site gorge of AChE, the “anionic” subsite of the active site, also known as the catalytic “anionic” site (CAS), is located at the bottom of the gorge, and the PAS is near its entrance.14,15 This led to the development of the bivalent ligand approach in which identical or distinct pharmacophores are linked, via an appropriate spacer, to produce a bifunctional drug with enhanced affinity.16 This, in turn, proved valuable for developing drugs with dual action both in inhibition of catalytic activity and in arresting the AChE￾catalyzed assembly of amyloid fibrils (for recent reviews see refs 17 and 18). Xie and co-workers recently reported the synthesis and characterization of novel bis-(-)-nor-meptazinols that inhibit both AChE and butyrylcholinesterase (BChE) as well as retarding A
aggregation.19 The most potent of these compounds * To whom correspondence should be addressed. Phone: 972-8-934-4531. Fax: 972-8-934-4159. E-mail:joel.sussman@weizmann.ac.il. † Department of Structural Biology, Weizmann Institute of Science. ‡ Department of Neurobiology, Weizmann Institute of Science. § Department of Medicinal Chemistry, School of Pharmacy, Fudan University. | School of Pharmacy, East China University of Science and Technology. ⊥ These authors made equal contributions. a Abbreviations: A
, amyloid-
peptide; ACh, acetylcholine; AChE, acetylcholinesterase; AChEI, acetylcholinesterase inhibitor; AD, Alzheimer’s disease; BuChE, butyrylcholinesterase; CAS, catalytic anionic site; m, mouse; MEP, (-)-meptazinol; PAS, peripheral anionic site; Tc, Torpedo californica; a, axial; e, equatorial. Figure 1. Chemical structure of 5h. Table 1. Data Collection and Refinement Statistics space group P3121 cell parameters (Å) a ) 112.17 b ) 112.17 c ) 137.57 molecules/asymmetric unit 1 resolution (Å) 40-2.7 total reflections 338424 unique reflections 26746 completeness (%)a 99.93% (99.76) av I/σ(I) 17.5 (3.55) Rmerge (%)b 11.7 R (%)c 18.5 Rfree (%)d 23.5 (5.2% of data) rmsd, bond lengths (Å) 0.019 rmsd, bond angles (deg) 2.1 PDB code 2W6C a Values in parentheses relate to the highest resolution shell (2.8-2.7 Å). b Rmerge ) ∑ |I - 〈I〉|/∑ I, where I is the observed intensity, and 〈I〉 is the average intensity obtained from multiple observations of symmetry￾related reflections after rejections. c R ) ∑ ||Fo| - ||Fc||/∑ |Fo|, where Fo and Fc are the observed and calculated structure factors, respectively. d As defined by ref 38. J. Med. Chem. 2009, 52, 2543–2549 2543 10.1021/jm801657v CCC: $40.75  2009 American Chemical Society Published on Web 03/27/2009