Quantitative structure–activity relationships for the pre-steady-state inhibition of cholesterol esterase by 4-nitrophenyl-n-substituted carbamates

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  • Quantitative StructureActivity Relationships for thePre-Steady-State Inhibition of Cholesterol Esterase by

    4-Nitrophenyl-N-substituted Carbamates

    Gialih Lin,a,* Wei-Cheng Liaoa and Shyh-Ying Chioub

    aDepartment of Chemistry and Institute of Biochemistry, National Chung-Hsing University, Taichung, TaiwanbDepartment of Neurosurgery, China Medical College Hospital, Taichung, Taiwan

    Received 10 April 2000; accepted 7 July 2000

    Abstract4-Nitrophenyl-N-substituted carbamates (16) are the pseudo-substrate inhibitors of porcine pancreatic cholesterolesterase. Thus, the first step of the inhibition (Ki step) is the formation of the enzymeinhibitor tetrahedral adduct and the secondstep of the inhibition (kc) is the formation of the carbamyl enzyme. The formation of the enzymeinhibitor tetrahedral adduct isfurther divided into two steps, the formation of the enzymeinhibitor complex with the dissociation constant, KS, at the first stepand the formation of the enzymeinhibitor tetrahedral adduct from the complex at the second step. The two-step mechanism for theformation of the enzymeinhibitor tetrahedral adduct is confirmed by the pre-steady-state kinetics. The results of quantitativestructureactivity relationships for the pre-steady-state inhibitions of cholesterol esterase by carbamates 16 indicate that values oflogKS and logk2/k2 are correlated with the Taft substituent constant, , and the values from these correlations are 0.33 and0.1, respectively. The negative value for the logKSs*-correlation indicates that the first step of the two-step formation of theenzymeinhibitor tetrahedral adduct (KS step) is the formation of the positive enzymeinhibitor complex. The positive

    value forthe logk2/k2s*-correlation indicates that the enzymeinhibitor tetrahedral adduct is more negative than the enzymeinhibitorcomplex. Finally, the two-step mechanism for the formation of the enzymeinhibitor tetrahedral adduct is proposed according tothese results. Thus, the partially positive charge is developed at nitrogen of carbamates 16 in the enzymeinhibitor complexprobably due to the hydrogen bonding between the lone pair of nitrogen of carbamates 16 and the amide hydrogen of the oxyanionhole of the enzyme. The second step of the two-step formation of the enzymeinhibitor tetrahedral adduct is the nucleophilic attackof the serine of the enzyme to the carbonyl group of carbamates 16 in the enzymeinhibitor complex and develops the negative-charged oxygen in the adduct. # 2000 Elsevier Science Ltd. All rights reserved.

    Introduction

    Recently there has been increased interest in pancreaticcholesterol esterase (CEase, EC 3.1.1.13, also known asbile salt-activated lipase) due to the correlation betweenenzymatic activity in vivo and absorption of dietarycholesterol.1,2 Results of a recent study showed thatCEase is responsible for mediating intestinal absorptionof cholesteryl esters but does not play a primary role infree cholesterol absorption.3 Physiological substrates ofCEase include cholesteryl esters, retinyl esters, acylgly-cerols, vitamin esters, and phospholipids.47 CEaseplays a role in digestive lipid absorption in the upperintestinal tract, though its role in cholesterol absorptionin particular is controversial.1,2,8 A recent report indi-cates that CEase is directly involved in lipoprotein

    metabolism, in that the enzyme catalyzes the conversionof large low-density liproprotein (LDL) to smaller,denser, more cholesteryl ester-rich lipoproteins, and thatthe enzyme may regulate serum cholesterol levels.9 Amajor cause of atherosclerosis is an excess of plasma LDL,a phenomenon that is particularly evident in individualswith familial hypercholesterolemia, who lack functionalLDL receptors. The enzyme may function in these rolesby acting as a cholesterol transfer protein.10 CEaseshares the same catalytic machinery as serine proteasesin that they have an active site serine residue which, witha histidine and an aspartic or glutamic acid, forms acatalytic triad.11 The conservation of this catalytic triadsuggests that as well as sharing a common mechanismfor substrate hydrolysis, that is, formation of a discreteacyl enzyme intermediate via the serine hydroxyl group,serine proteases, CEase, serine phospholipase A2, andlipases may well be expected to be inhibited by the sameclasses of mechanism-based inhibitors. To date, this hasbeen demonstrated for diethyl-p-nitrophenol phosphate,12

    0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.PI I : S0968-0896(00 )00196-6

    Bioorganic & Medicinal Chemistry 8 (2000) 26012607

    *Corresponding author. Tel.: +886-930-383816; fax: +886-4286-2547; e-mail: gilin@dragon.nchu.edu.tw

  • hexadecylsulfonyl fluoride,13 fluoroketones,14,15 boronicacid,16 chloroisocoumarin,17 b-lactones,18,19 b-lactams,20

    and carbamates.2128

    Two dierent X-ray crystal structures of bovine pan-creatic CEase have been reported recently.29,30 Althoughdierent bile salt-activation mechanisms for CEase areproposed, the active site of CEase is similar to those ofPseudomonas cepacia lipase31,32 and Candida rugosalipase (CRL).33 The active site of CEase may consist ofat least five major binding sites (Fig. 1): (a) an alkylchain binding site (ACS) that binds to the acyl chain ofcholesteryl ester and the first alkyl chain of triacylgly-cerol, (b) an oxyanion hole (OH), the H-bonding pep-tide NH functions of Gly107, Ala108, and Ala195, thatstabilizes the incipient carbonyl CO of the esterfunction during turnover, (c) an esteratic site (ES),comprised of the Ser194-His435-Asp320 active site triadthat is involved in nucleophilic and general acidbasecatalysis, (d) a leaving group hydrophobic binding site(LHOS) that binds to the hydrophobic part of the leavinggroup, (e) the second alkyl chain binding site (SACS)that binds to the second alkyl chain of triacylglyceroland is in a crevice above the catalytic site, and (f) a

    leaving group hydrophilic binding site (LHIS) thatbinds to the hydrophilic part of the leaving group and islocated at the opposite direction of ACS.

    In the presence of substrate, the mechanism of transientor pseudo-substrate inhibitions of CEase has been pro-posed in Scheme 1.2128

    Because the inhibition of CEase follows first-orderkinetics over the observed time period for the steady-state kinetics, the rate of hydrolysis of EI0 must be sig-nificantly slower than the rate of formation of EI0(kck3).34 Therefore, values of Ki and kc can be calcu-lated from eqn (1):2128

    kapp kc I Ki 1 S

    Km

    I

    1

    In eqn (1), kapp values are the first-order rate constantswhich are obtained according to Hosies method.21

    Bimolecular rate constant, ki kc=Ki, is related tooverall inhibitory potency.

    Figure 1. (A) Interactions between cholesteryl ester and the active sites of CEase. (B) Interactions between triacylglycerol and the active sites of CEase.

    2602 G. Lin et al. / Bioorg. Med. Chem. 8 (2000) 26012607

  • Quantitative structureactivity relationships (QSARs)for the serine protease- and acetylcholinesterase-cata-lyzed hydrolyses have been reported.3537 QSARs for theinhibitions of CEase by aryl carbamates have beenreported by us23,24,28 and Quinn et al.21,22 There exist theHammett type of QSAR (eqn (2))3841 for the inhibitionof CEase by substituted phenyl-N-butyl carbamates2123

    and the TaftIngold type of QSAR (eqn (3))3841 for theinhibition of CEase by 4-nitrophenyl-N-alkyl carba-mates.24 In eqn (2), values of k0,

    , and are the kvalue for the non-substituted phenyl parent compound,the reaction constant (the intensity factor of the induc-tive eect or the slope for the logk versus s plot), andthe Hammett substituent constant, respectively. In eqn(3), values of k0,

    , and are the k value for the non-substituted methyl parent compound, the reaction con-stant (the slope for the logk versus plot), and the Taftsubstituent constant, respectively. In this paper, we further

    draw attention to QSARs for the pre-steady-state inhi-bitions of CEase by carbamates 16 (Fig. 2).

    logk logk0 2

    logk logk0 3According to the results of the pre-steady-state kineticdata, more detailed reaction mechanism for the inhibi-tion of CEase by carbamates 16 may be proposed.

    Results

    The Ki values for the inhibitions of CEase by carba-mates 16 are obtained from non-linear least squarescurve fitting of eqn (1) (Table 1).2128 The results ofQSARs for the steady-state inhibition of CEase by car-bamates 16 (Table 2) indicate that values of logKiare only correlated with and the value for thecorrelation is 0.50, which is close to the valuesobtained previously.24 Since the negative value(should form a positive intermediate) for the logKi--correlation and the negative enzymeinhibitor tetra-hedral adduct are contradictory to each other, the Kistep is further divided into two steps, the formation ofthe enzymeinhibitor complex (E.I) with the dissociationconstant, KS k1=k1, and then the formation of EIfrom E.I (Scheme 2), where k1, k1; k2, and k2 are therate constants for the formation and redissociation ofE.I, the formation and redissociation of EI fromE.I.35,36,4246 Values of KS, k2, and k2 are obtainedfrom the non-linear least squares curve fitting of eqn (4)(Table 1).

    Scheme 1. Kinetic scheme for pseudo-substrate inhibition of CEase inthe presence of substrate.

    Figure 2. Structures of carbamates 16.

    Table 1. Pre-steady-state kinetic data for the inhibitions of CEase by carbamates 16a

    Inhibitor R= Es Ki(mM)b

    KS(mM)c

    k2(103 s1)c

    k2(104 s1)d

    1 n-Bu 0.13 0.39 2.60.2 242 0.820.06 1.20.12 n-Hex 0.15 0.40 3.20.4 233 0.820.07 1.120.053 n-Oct 0.13 0.33 3.60.4 232 0.730.04 1.10.14 C2H4Cl 0.39 0.43 5.80.7 353 2.20.2 2.90.15 CH2Ph 0.22 0.38 4.40.5 313 2.20.2 2.70.16 Allyl 0.1 0.39 3.80.5 275 1.