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Ortho Effects for Inhibition Mechanisms ofButyrylcholinesterase by o-Substituted Phenyl N-ButylCarbamates and Comparison with Acetylcholinesterase,
Cholesterol Esterase, and LipaseGialih Lin,* Yu-Ru Lee, Yu-Chen Liu, and Yon-Gi Wu
Department of Chemistry, National Chung-Hsing University, Taichung 402, Taiwan
Received January 20, 2005
Phenyl carbamates are used to treat Alzheimers disease. These compounds inhibitacetylcholinesterase and butyrylcholinesterase. The goal of this work was to determine thechemical characteristics of ortho substituents that make some carbamates better inhibitors ofbutyrylcholinesterase than of acetylcholinesterase, cholesterol esterase, and lipase. Theinhibition constants, Ki, Ki, kc, and ki were measured for nine different carbamates. The valueswere plotted according to Hammett, Taft-Kutter-Hansch, and Swan-Lupton to obtainconstants that correlated the chemical nature of the substituents with inhibition potency. Itwas found that the negative charges of tetrahedral intermediates were more stabilized by orthoelectron-withdrawing substituents of the inhibitors in butyrylcholinesterase than in acetyl-cholinesterase. This result confirmed formation of 3-pronged hydrogen bonds for the oxyanionhole of butyrylcholinesterase and 2-pronged hydrogen bonds for the oxyanion hole ofacetylcholinesterase. Furthermore, it was found that ortho electron-donating substituents ofthe inhibitors accelerated inhibition of butyrylcholinesterase by ortho polar effects. Conforma-tions of enzyme-inhibitor tetrahedral intermediates for butyrylcholinesterase were differentfrom those for acetylcholinesterase and cholesterol esterase; ortho substituents in thetetrahedral intermediates were located far from the negatively charged carbonyl oxygens inbutyrylcholinesterase, but close to the negatively charged carbonyl oxygens in acetylcholines-terase and cholesterol esterase. In conclusion, electron-donating substituents in the orthoposition were better inhibitors of butyrylcholinesterase than acetylcholinesterase, whileelectron-withdrawing substituents were better inhibitors of acetylcholinesterase.
Butyrylcholinesterase (BChE1, EC 18.104.22.168) is a serinehydrolase related to acetylcholinesterase (AChE, EC22.214.171.124). Unlike AChE, which plays a vital role in thecentral and peripheral nervous systems, the physiologicalfunction of BChE remains unclear (1, 2). Despite havingno identified endogenous substrate, BChE plays a keyrole in detoxification, by degrading esters such as succi-nylcholine and cocaine (3). The X-ray structures of BChEand BChE-inhibitor complex have been recently reported(4, 5). Similar to AChE (6-9), the active site of BChE(Figure 1) contains (a) an esteratic site (ES) comprisedof the catalytic triad Ser198-His438-Glu325, which islocated at the bottom of the gorge (4, 5), (b) an oxyanion
hole (OAH) composed of Gly116, Gly117, and Ala199, thatstabilizes the tetrahedral intermediate, (c) an anionic
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1 Abbreviation: ABS, acyl group binding site; ACh, acetylcholine;AChE, acetylcholinesterase; AD, Alzheimers disease; AS, anionicbinding site; ATCh, acetylthiocholine; , intensity factor for ortho stericconstant; DTNB, 5,5-dithio-bis-2-nitrobenzoate; BCh, butyrylcholine;BChE, butyrylcholinesterase; BTCh, butyrylthiocholine; CEase, cho-lesterol esterase; CRL, Candida rugosa lipase; ES, esteratic site orcatalytic triad; ESo, Taft-Kutter-Hansch ortho steric constant; F,Swain-Lupton-Hansch ortho polar constant or polar constant throughspace; f, intensity factor to the ortho polar constant; kc, carbamylationconstant; kd, decarbamylation constant; Ki, inhibition constant; Ki,virtual inhibition constant; ki, bimolecular inhibition constant; OAH,oxyanion hole; PAS, peripheral anionic binding sites; PCL, Pseudomo-nas cepacia lipase; PSL, Pseudomonas species lipase; QSAR, quantita-tive-structure activity relationship; F, Hammett reaction constant; p,Hammett para-substituent constant or polar constant through bonds.
Figure 1. The acyl group binding site (ABS), oxyanion hole(OAH), esteratic site or catalytic triad (ES), anionic binding site(AS), and peripheral anionic binding site (PAS) of BChE.
1124 Chem. Res. Toxicol. 2005, 18, 1124-1131
10.1021/tx050014o CCC: $30.25 2005 American Chemical SocietyPublished on Web 06/24/2005
substrate-binding site (AS) composed of Trp82, where thequaternary ammonium pole of butyrylcholine (BCh) andof various active site ligands binds through a preferentialinteraction of quaternary nitrogens with the electronsof aromatic groups, (d) an acyl group-binding site (ABS)that binds the acyl or carbamyl group of substrate orinhibitor, and (e) a peripheral anionic binding site (PAS)composed of Phe278 (10), Tyr332 (11), and Asp70, whichis located at the entrance (mouth) of the active site gorgethat may bind to the tacrine-based heterobivalent ligands(10) and cage amines (12).
In Alzheimers disease (AD), a neurological disorder,cholinergic deficiency in the brain has been reported (13,14). Four drugs for treatment of AD, tacrine (Cognex),donepezil (Aricept), rivastigmine (Exelon) (Figure 2), andgalantamine (Reminyl), are dual inhibitors of AChE andBChE (14). The additional demonstration that centralBChE rather than AChE inhibition is the best correlationof cognitive improvement in AD clinical studies with thedual cholinesterase inhibitor rivastigmine (Figure 2)further suggests that BChE represents an intriguingtarget to develop drugs for the treatment of neurodegen-erative disease (15-17). The derivatives of physostigmine(Figure 2) are also potential drugs for the treatment ofAD (18). Since rivastigmine (19) and physostigmine arecarbamates, both inhibition mechanisms of AChE (20-26) and BChE (5, 27) by carbamates may play importantroles for treatment of AD.
Carbaryl (1-naphthyl N-methylcarbamate, Sevin) (Fig-ure 2), carbofuran (Furadan), propoxur (Baygon), andaldicarb (Temik) are carbamate pesticides that haveactivities against a broad range of insects and lowmammalian toxicity (28). These carbamate pesticides arealso dual inhibitors of AChE and BChE. Therefore, bothinhibition mechanisms of AChE and BChE by carbam-ates may also play important roles in understanding themechanism of pesticide toxicology.
The mechanism for BChE-catalyzed hydrolysis ofsubstrate is formation of the first tetrahedral intermedi-ate via nucleophilic attack of the active site Ser198(Figure 1) to substrate, then formation of the acyl enzyme
intermediate from the intermediate. In the presence ofsubstrate, the pseudosubstrates aryl carbamates serveas inhibitors (Scheme 1) (5, 27). The carbamylation stageis rapid compared to subsequent decarbamylation (kc .kd)2, thus the two stages are easily resolved kinetically(20-27, 29-37). In the presence of a carbamate inhibitor,time courses for hydrolysis of butyrylthiocholine (BTCh)are biphasic, and kapp values can be calculated from eq 1(31, 32). In eq 1, A0, kapp, vo, and vss are the absorbance
at t ) 0, observed first-order inhibition rate constant,initial velocity, and steady-state velocity, respectively.Reactions must be followed for at least six half-lives toobtain reliable estimates of the parameters, especiallyvss and kapp (33). Once kapp values have been determinedat various inhibitor concentrations, the resulting data arefit to eq 2 to obtain Ki and kc values. In other words, Kiand kc values are obtained from each nonlinear least-squares curve fittings of kapp values against [I] accordingto eq 2 (22-27, 31-38). Determination of the Ki and kc
values by this method is called the continuous assaymethod and is much more rapid than a traditionalstopped-time (or dilution) assay method (31). The bimo-lecular rate constant, ki ) kc/Ki, is related to overallinhibitory potency. Moreover, aryl carbamates meet thethird criterion for the pseudosubstrate inhibitors, asproposed by Abeles and Maycock (39), in that enzyme isprotected from the inhibitions by carbamates in thepresence of a reversible inhibitor, edrophonium (Figure2). Therefore, carbamates are characterized as pseu-dosubstrate inhibitors of BChE (5, 27).
Quantitative structure-activity relationships (QSARs)represent an attempt to correlate structural propertiesof compounds with biological activities or chemical reac-tivities (40, 41). These chemical descriptors, which in-clude parameters to account for hydrophobicity, elec-tronic, inductive, or polar properties, and steric effects,are determined empirically or by calculations. Littleadditional development of QSAR has occurred until thework of Louis Hammett, who has correlated electronicproperties of substituted benzoic acids with their equi-librium constants and reactivities by the Hammettequation (eq 3).
In eq 3, the h value is the log k0 value for the standardreaction (unsubstituted benzoic acid) and the parametersF and are the Hammett reaction constant and the
2 The kd value of (9 ( 2) 10-4 s-1 for BChE inhibition bycarbamates 1-9 is calculated from the progress curves (33).
Figure 2. Structures of carbamates 1-9, rivastigmine, phys-ostigmine, edrophonium, and carbaryl.
Scheme 1: Kinetic Scheme for PseudosubstrateInhibitions of BChE in the Presence of Substrate
A ) A0 + (vo - vss)(1 - exp(- kappt ))/kapp + vsst (1)
kapp ) kc[I]/(Ki(1 + [S]/Km) + [I]) (2)
log k ) h + F (3)
Effects for BChE, AChE, CEase, and Lipase Inhibitions Chem. Res. Toxicol., Vol. 18, No. 7, 2005 1125
substituents constant, respectively. The investigation alsoreveals that meta- and para-substituted compoundsgenerally correlate well, but ortho-substituted ones do not(40) due to complications from direct steric and polareffects (42). According to Fujita and Nishiokas sugges-tion, the total ortho effect is composed of the