identification of amino acid residues involved in the binding of

Protein Science (1994), 3:1770-1778. Cambridge University Press. Printed in the USA Copyright 0 1994 The Protein Society

Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases

ASHIMA SAXENA,’ NAIFENG QIAN,2 ILDIKO M. KOVACH,2 A.P. KOZIKOWSKI,3 Y.P. PANG: DANIEL C. VELLOM? ZORAN RADIC;,’ DANIEL QUINN,4,6 PALMER TAYLOR,4 AND BHUPENDRA P. DOCTOR’ ’ Division of Biochemistry, Walter Reed Army Institute of Research, Washington, D.C. 20307 Department of Chemistry, The Catholic University of America, Washington, D.C. 20064 The Mayo Clinic, Jacksonville, Florida 32224 University of California San Diego, La Jolla, California 92093 Visiting Fogarty Fellow, Institute of Medical Research, University of Zagreb, Croatia

(RECEIVED January 31, 1994; ACCEPTED June 26, 1994)

Abstract

Huperzine A, a potential agent for therapy in Alzheimer’s disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with K , values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with K , values of 0.25 pM and 22 pM, respectively. Far larger K , values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337(330) Phe and Tyr 337(330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in K , value for the binding of Huperzine A. Mo- lecular mechanics energy minimization of the complexes formed between each of the 2 stereoisomers of Huper- zine A and fetal bovine serum acetylcholinesterase, Torpedo acetylcholinesterase, or human butyrylcholinesterase also revealed that (-)-Huperzine A gave a better fit than (+)-Huperzine A and implicated Tyr 337(330) in the stereoselectivity of Huperzine A.

Keywords: cholinesterases; Huperzine A; inhibitor; molecular modeling; site-directed mutagenesis

Huperzine A, an alkaloid constituent of Huperzia serrata has been a longstanding agent in Chinese herbal medicine for the treatment of dementia in the elderly population. It has been syn- thesized (Kozikowski et al., 1991) and characterized as a potent and selective inhibitor of acetylcholinesterase (EC 3.1.1.7) (McKinney et ai., 1991). The potentially superior inhibition char-

Reprint requests to: Bhupendra P. Doctor, Walter Reed Army Insti- tute of Research, Washington, D.C. 20307-5100.

Permanent address: Department of Chemistry, University of Iowa, Iowa City, Iowa.

Abbreviations: ChE, cholinesterase; AChE, acetylcholinesterase; BChE, butyrylcholinesterase; ATC, acetylthiocholine iodide; BTC, bu- tyrylthiocholine iodide; DTNB, 5,5‘-dithiobis(2-nitrobenzoic acid); FBS, fetal bovine serum; iso-OMPA, tetraisopropylpyrophosphoramide; BW284C5 I , di( p-allyl-N-methylaminophenyl)pentane-3-one; CMV, cytomegalovirus.

acteristics of Huperzine A, as compared to other cholinesterase inhibitors, have been attributed to the very slow rate of dissociation (to.s = 35 min) of AChE-Huperzine A complex in solution (Ashani et al., 1992). Also, the interaction of Huperzine A with AChE appears reversible and does not result in any detect- able chemical modification of the inhibitor (Ashani et at., 1992). These anti-AChE properties of Huperzine A, which are not shared by other commonly used anti-AChE drugs such as phy- sostigmine and tetrahydroaminoacridine, may prove to be use- ful pharmacological characteristics, not only for the treatment of Alzheimer’s disease and other nervous system-related demen- tias, but also for prophylaxis against organophosphate toxicity. Compared to AChE, Huperzine A has been reported to be 1,000-fold less potent as an inhibitor of butyrylcholinesterase (EC 3.1.1.8). The difference in the activity of Huperzine A toward AChE and BChE has been proposed to be due to differ-

1770

ChE-Huperzine A interactions

ences in the aromatic amino acid residues lining the pocket of the catalytic region of the enzyme molecule (Ashani et al., 1992).

The naturally occurring (-)-Huperzine A was a mixed competitive inhibitor of AChE (Wang et al., 1986). Inhibition studies with the 2 stereoisomers of Huperzine A revealed that (-)-Huperzine A inhibited crude preparations of rat cortical AChE 38-fold more potently than (+)-Huperzine A (McKinney et al., 1991). In the present study, this stereoselectivity of Hu- perzine A was examined in more detail with 3 distinct families of ChEs with known sequence differences. The basis for par- ticular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in a single template enzyme. These findings enabled us to model the ChE- Huperzine A interactions and propose an orientation for these bound stereoisomers in the complex.

Results

Inhibition of ChEs by the 2 stereoisomers of Huperzine A

Plots of percent residual activity versus time in the presence of (+)-Huperzine A, (-)-Huperzine A, and (+)-Huperzine A for purified preparations of FBS AChE and Torpedo AChE are shown in Figure 1. The results confirm the previous observation that (-)-Huperzine A was the active stereoisomer (Wang et al., 1986; McKinney et al., 1991). There was no inhibition of either

50

25

1

" J 0 10 20 30

TIME (MINUTES)

Fig. 1. Time course for the inhibition of various AChEs by Huperzine A. Stock solutions of (f)-Huperzine A (A), (-)-Huperzine A (B), or (+)-Huperzine A (C) were diluted into either FBS AChE (0) or Torpedo AChE (A), and the rate of inhibition was followed by measuring residual enzyme activity at various time intervals as described in the Mate- rials and methods. The final concentrations of (+)-Huperzine A and (-)-Huperzine A were 0.0115 pM, and that of (+)-Huperzine A was 0.026 pM.

1771

Table 1. Dissociation constants for the inhibition of ChEs by the 2 stereoisomers of Huperzine A

KI (PM)

Enzyme (+)-Huperzinea (-)-Huperzine (+)-Huperzine

FBS AChEb 0.02 k 0.005 0.006 f 0.004 0.21 k 0.005 Torpedo AChEb 0.22 + 0.005 0.25 k 0.01 22.0 k 2.0 Human BChEC 42.0 15.0 75.6 f 5.0 36.0 + 5.0 Horse BChEC 24.0 f 3.0 27.5 f 5.0 37.0 + 5.0

a K, values reported by Ashani et al. (1991).

A and measurement of residual enzyme activity.

at various Huperzine A concentrations.

K , values determined by equilibration of enzyme with Huperzine

K , values determined from analysis of slopes of l / u versus l/s plots

horse or human serum BChE at concentrations of up to 10 pM of the 2 stereoisomers of Huperzine A (data not shown). The K , values for the 2 stereoisomers of Huperzine A show that (-)-Huperzine A inhibited FBS AChE 35-fold more potently than (+)-Huperzine A (Table 1). On the other hand, (-)- Huperzine A was 88-fold more potent than (+)-Huperzine A in inhibiting Torpedo AChE. The preparation of (+)-Huperzine A had approximately 1% contamination of (-)-Huperzine A, which precluded us from observing more than 2 orders of magnitude difference between the K , values for the 2 stereoisomers. Therefore the stereoselectivity reflected by the K , values for the 2 stereoisomers of Huperzine A is minimal. Due to the high association and dissociation rates for the interaction of BChE and Huperzine A, K , values for horse and human serum BChE for the 2 stereoisomers of Huperzine A were determined by the anal- yses of steady-state kinetic data (Table 1). Unlike AChE, no significant differences in the K I values were observed for the 2 stereoisomers of Huperzine A with horse and human serum BChEs, suggesting a lack of stereoselectivity of this compound for BChE. Differences in the reactivity of (-)-Huperzine A toward FBS AChE, Torpedo AChE, and horse and human serum BChEs showed that Tyr 337(330)' in mammalian AChE may be an important amino acid residue in the binding of (-)- Huperzine A to ChEs. This prediction was tested by conduct- ing inhibition studies of (-)-Huperzine A with recombinant mouse AChE mutants, where Tyr 337(330) in the wild-type enzyme was modified to either Phe as in Torpedo AChE or Ala as in BChE. Plots of percent residual activity vs. time in the presence of (-)-Huperzine A for recombinant mouse AChE and the Tyr 337(330) Phe and Tyr 337(330) Ala mutants are shown in Figure 2. These results show that (-)-Huperzine A was a more potent inhibitor of wild-type mouse AChE as compared to the Tyr 337(330) Phe and Tyr 337(330) Ala mutants, implicating the amino acid residue Tyr 337(330) in the binding of Huperzine A to ChEs. As shown in Table 2, mutation of Tyr 337(330) in a mouse template to Phe or Ala yielded a difference in reactivity that closely mimicked the native enzymes (Table 1). Eliminat- ing the charge deep in the active-center gorge by mutation of

'The dual numbering system gives the residue number in the species designated followed by the corresponding residue in Torpedo AChE (Massoulie et al., 1992).

1772 A. Saxena et a/.

100

80

60

40

*

0 " ~ ' " " ' ~ ' ~ ' 0 5 10 15 20 25 30

TIME (MINUTES)

Fig. 2. Time course for the inhibition of various recombinant mouse AChEs by (-)-Huperzine A . Stock solution of (-)-Huperzine A was diluted into either wild-type recombinant mouse AChE (O), Tyr 337 Phe mutant AChE (A), or Tyr 337 Ala mutant AChE (0) such that the final concentration of (-)-Huperzine A was 0.01075 pM. The rate of inhibition was followed by measuring residual enzyme activity at various times as described in the Materials and methods.

Glu 199 in Torpedo AChE to Gln resulted in only a 3-fold increase in KI value of (-)-Huperzine A for this enzyme.

Energy-minimized structures of Huperzine A bound to ChEs

Because AChE from Torpedo californica is the only ChE whose structure has been determined experimentally at atomic resolu- tion, docking of each of the 2 stereoisomers of Huperzine A into the active site and subsequent molecular mechanics energy minimization of the formed conjugate were carried out in Torpedo AChE. The procedure used for Torpedo AChE was then extended to both FBS AChE and human BChE.

Panel 8 2 in Figure 3 shows the interaction of (-)-Huperzine A with various amino acid residues in the active site of Torpedo AChE. There are 3 energetically favorable regions for the interaction of Huperzine A with the enzyme molecule: (1) the hy-

Table 2. Dissociation constants and free energy differences for the inhibition of mutant AChEs by (-)-Huperzine A

Enzyme K I (PM) AAG (kcal)a

Mouse AChE Wild typeb 0.0085 * 0.004 0.0 Tyr 337 PheC 0.273 * 0.01 2.1 Tyr 337 AlaC 8.2 L 0.1 4.9

Torpedo AChE Wild typeb 0.185 * 0.01 0.0 Glu 199 Glnb 0.56 * 0.07 0.7

a Calculated according to the formula AAG = RTln Kj/K, , where KI and K j are the dissociation constants for the mutant and wild-type AChE, respectively (Radic et al., 1993).

b K I values determined by equilibration of enzyme with (-)- Huperzine A and measurement of residual enzyme activity.

KI values determined from the slopes of I/v versus I / . plots at various (-)-Huperzine A concentrations.

drophobic core, represented in green, includes the ethylidene group and the bridge double bond of (-)-Huperzine A, inter- acting with Trp 84, Phe 330, Tyr 334, and Tyr 442 of the enzyme molecule; (2) the oxyanion region, represented by default color, includes the 3 partial hydrogen bonds formed between the carbonyl oxygen of Huperzine A and the amide backbone hydro- gens of Gly 118, Gly 119, Ala 201, the electrostatic interaction between the electrophilic carbonyl carbon of Huperzine A, and the y-oxygen of Ser 200 of the enzyme molecule; and (3) the electrostatic region, represented in red, indicates the possible interaction of the primary amine group of Huperzine A with the carboxylate of Glu 199, which is at a distance of 6-8 A.

Panel A2 in Figure 3 shows the complex of (-)-Huperzine A with FBS AChE. Like the Torpedo AChE-(-)-Huperzine A complex, this complex also has favorable interactions in the hydrophobic core involving the ethylidene group and the bridge double bond of (-)-Huperzine A, with Trp 86(84), Tyr 337(330), Tyr 341(334), and Tyr 449(442) of the enzyme. In the oxyanion hole region and electrostatic region, the interactions between the inhibitor and the enzyme molecule are as favorable as in Torpedo AChE.

Panel C2 in Figure 3 shows (-)-Huperzine A complexed with human BChE. According to the molecular mechanics calculations, there are numerous soft van der Waals interactions between Huperzine A and the active site residues of AChE. With both stereoisomers, the total number of these interactions is smaller for BChE as compared to AChE. The predominant interactions in the hydrophobic region are with Trp 86(84), which is within 3.4-4.0 A from a number of atoms in the inhibitor in either orientation. There are fewer contacts between Trp 86(84) of BChE and the 2 stereoisomers of Huperzine A. In BChE, Ala 330 provides a weaker interaction with both stereoisomers of Huperzine A as compared to Phe 330 in Torpedo AChE and Tyr 337 in FBSAChE, all of which are within optimal (3.4-4.0A) distance from some of the atoms of Huperzine A.

The complexes of (+)-Huperzine A with FBS AChE, Torpedo AChE, and human BChE are shown in panels AI, B1, and C1, respectively. The stereoselectivity of Huperzine A for FBS AChE (35-fold) and Torpedo AChE (88-fold) can be correlated to differences in interactions in the oxyanion hole region and weak electrostatic forces between Glu 199 and the primary amine group of Huperzine A. Although the former of these interactions are also soft van der Waals forces, there are weak hydrogen bonds between the carbonyl group of Huperzine A and amide backbone of Gly 1 18 and Gly 119, and the hydroxyl group of Ser 200, especially for FBS AChE, with the (-)-Huperzine A. The active site serine has the strongest tendency to hydrogen bond, which is not surprising because our model does not en- gage this serine in a covalent bond. The electrostatic interactions in this model are between Glu 199 and the primary amine of Hu- perzine A, but they are maximum at -2.0 kcal/mol or less because the distances between the nitrogen and one of the carboxyl oxygens are between 6 and 8 A (FBS AChE-(-)-Huperzine A, 7.12 A; FBS AChE-(+)-Huperzine A, 7.5 A ; Torpedo AChE- (-)-Huperzine A, 6.15 A ; Torpedo AChE-(+)-Huperzine A, 7.02 A; BChE-(-)-Huperzine A, 7.06 A ; and BChE-(+)- Huperzine A, 8.25 A).

I t is difficult to assess the quantitative differences in binding energies that are the sum of a great number of small terms and are not isolated from the interactions with the surroundings. The

ChE-Huperzine A interactions 1773

35-fold stereoselectivity observed with FBS AChE, for example, would translate into a difference of 2.1 kcal/mol in binding energy. The weak hydrogen bonds observed for the (-)-Huperzine A complexes as well as more favorable interactions in the hydrophobic and electrostatic regions can support this difference.

Discussion

In the 3-dimensional structure of Torpedo AChE, the active site is in a gorge lined with the side chains of 14 aromatic amino acid residues (Sussman et al., 1991). Based on sequence alignments of ChEs, these residues are fully conserved in all known verte- brate AChEs (Gentry & Doctor, 1991). On the other hand, 6 of 14 aromatic amino acid residues at positions 70, 121,279,288, 290, and 330 in Torpedo AChE are replaced by aliphatic amino acid residues in BChE. These structural differences between AChE and BChE and recent investigations employing molecular modeling, site-directed mutagenesis, and studies of the catalytic and inhibitory properties of these mutants lend strong support to the involvement of these aromatic amino acid residues in the binding and selectivity of inhibitors to ChEs.

The role of Trp 86(84) in the orientation and stabilization of the quaternary ammonium group of the substrate has been dem- onstrated by chemical labeling studies (Weise et al., 1990; Kreienkamp et al., 19911, by crystallographic data (Sussman et al., 1991, 1992) and site-directed mutagenesis studies for Trp 86(84) in human AChE (Ordentlich et al., 1993). The 2 Phe residues at positions 295(288) and 297(290) define the di- mensions of the acyl pocket of mammalian AChEs, markedly reducing BTC hydrolysis and enhancing ATC hydrolysis by forming a clamp around the methyl moiety of the acetoxy group, and restricting its degrees of freedom (Vellom et al., 1993). Re- placement of either of these residues with nonaromatic amino acid residues, as in BChEs, results in an increased hydrolysis of BTC by the mutant enzyme and its increased sensitivity to the bulky BChE-specific organophosphate inhibitor iso-OMPA (Harel et al., 1992; Ordentlich et al., 1993; Vellom et al., 1993). This observation is also supported by a naturally occurring mutation in Drosophila AChE that contains a single Phe at position 368(290). Replacement of Phe by Tyr confers enzyme resistance to certain organophosphates (Fournier et al., 1992). The Trp residue at position 279 in Torpedo AChE is located near the lip of the gorge and has been designated as part of the “peripheral’’ anionic site. Mutation of this amino acid residue to a nonaromatic amino acid residue as in BChE results in a loss of sensitivity of the mutant enzyme to “peripheral” anionic site li- gands like propidium (Harel et al., 1992; Shafferman et al., 1992; RadiC et al., 1993). The 2 neighboring Tyr residues conserved in AChEs at position 72(70) and 124(121) also contrib- ute to the stabilization of “peripheral” site inhibitor complexes (RadiC et al., 1993). Another aromatic amino acid residue that is present near the choline binding site is Tyr 337(330) (Sussman et al., 1991). This residue appears to stabilize the binding of li- gands such as edrophonium, acridines, and 1 end of bisquater- nary compounds such as BW284C51 and decamethonium (Shafferman et al., 1992; Ordentlich et al., 1993; Radii et al., 1993). This residue destabilizes the binding of phenothiazines such as ethopropazine, which contains a bulky exocyclic substitution. Structure-activity relationships show that this is a con- sequence of steric hindrance between the diethylamino-2-isopropyl

moiety with the aromatic side chain of Tyr 337(330) in the mammalian enzyme (Radii et al., 1993).

The results presented here, using stereoisomers of Huperzine A also show the involvement of certain aromatic amino acids lining the gorge in the stabilization of AChE-Huperzine A complexes. The passage of Huperzine A through the gorge to reach the active site of AChE was tested in the Torpedo AChE model and found to be completely feasible. The inhibition of AChE by Huperzine A is probably because the lactam region has a resemblance to the staggered conformation of acetylcholine (Fig. 4). However, unlike esters, amides are not good substrates for AChEs. The 6-membered lactam ring is a thermodynami- cally favored internal amide that may resist hydrolysis because the catalytic residues for general acid/base catalysis like histi- dine are not at the proper distance and orientation. The lactam ring may open and close transiently in a reversible manner. The guiding principle in our modeling of the Huperzine A stereoisomers into the active site pockets of ChEs was to orient the carbonyl group of the lactam ring in the oxyanion hole. The interaction energies arising from the hydrogen bonding of C=O or P=O have been the most significant stabilizing forces in the active site region of serine hydrolase enzymes (Qian & Kovach, 1993). The optimal orientation for each of the 2 stereoisomers of Huperzine A in the active-site pockets of FBS AChE, Tor- pedo AChE, and human BChE was then determined. Our studies implicate Trp 86(84) and Tyr 337(330) as the critical amino acid residues that interact with Huperzine A and are supported by experimental data showing the stereoselectivity of AChE for Huperzine A and differences in the reactivity of (-)-Huperzine A toward AChE and BChE.

An analysis of the differences in free energy of binding for the 2 stereoisomers of Huperzine A to ChEs (Table 3) reveals 2 interesting facets of the ChE-Huperzine A interaction. First, the contributions of the functional moieties on the Tyr337(330) side chain appear additive rather than cooperative, as the effect of Tyr-to-Ala substitution appears to be a linear summation of the Phe-to-Tyr and Ala-to-Phe substitutions. Second, the con- tribution of the hydroxyl group is of comparable magnitude for the interaction of both stereoisomers of Huperzine A with ChEs, whereas the aromatic interactions contributed by the benzene ring prevail in the binding of (-)-Huperzine A.

Differences in the reactivity of Huperzine A toward FBS AChE, Torpedo AChE, and horse serum BChE implicate the involvement of Tyr 337(330) in the binding of (-)-Huperzine A to AChEs. The lone pair of electrons on 0 in the OH of Tyr 337 are H-bond acceptors from the NH of Trp 86(84). This increases the electron density in the indole side chain of Trp 86(84) and draws the 2 residues closer to provide a contin- uous ?r electron envelope around (-)-Huperzine A. Trp 86(84) provides the strongest overlap with the bridge methyl and primary amine segments of the frame, which pulls the ethylene bridge of the (-)-Huperzine A molecule in the vicinity of the aromatic ring of Tyr 337. This interaction is responsible for the low KI value of 6 nM for the mammalian enzyme. In Torpedo AChE, the corresponding Tyr residue has been replaced by Phe, resulting in the loss of this stabilizing hydrogen bond and a higher KI value of 0.25 p M for this enzyme. In human serum BChE, the Tyr residue has been replaced by Ala, which results in a loss of aromatic interactions and a further increase in KI value to 76 pM. In contrast, both Trp 86(84) and Tyr 337(330)

1774 A. Saxena et al.

AI

A2

Oly-118

Oly-119 3“ cilu-199 8er-200

Trp-84

Fig. 3. Molecular mechanics energy-minimized structures of Huperzine A bound to ChEs. The energy-minimized structures of each of the 2 stereoisomers of Huperzine A bound to FBS AChE (Al, A2), Torpedo AChE (Bl, B2), and human BChE (Cl, C2) are shown. The important amino acid residues of the enzyme molecule that interact with (-)-Huperzine A (A2, B2, C2) and (+)-Huperzine A (Al , Bl , C1) are Trp 84 and Phe 330 (green; hydrophobic residues); Gly 118, Gly 119, and Ser 200 (gray; oxyanion hole residues); and Glu 1 9 9 (red; weak electrostatic interaction). Weaker interactions (not shown) are with Gly 117, Asp 72, Tyr 121, and Tyr 432 (Al, A 2 , B1, B2). (Continues on facingpage.)

are near the NH3+ group of (+)-Huperzine A, which-appears to be a less favorable arrangement as indicated by the higher KI values of 0.21 pM, 22 pM, and 36 pM for FBS AChE, Torpedo AChE, and human serum BChE, respectively.

Inhibition studies of (-)-Huperzine A with mouse AChE mutants, where Tyr 337 (KI = 8.45 nM) was modified to either Phe, as in Torpedo AChE (KI = 0.273 pM), or Ala, as in BChE (K, = 8.2 pM), lend further support to our models. The corresponding KI values for (+)-Huperzine A were 0.36 pM for mouse wild-type AChE, 21 pM for Tyr 337 Phe mutant AChE, and 31.3 pM for Tyr 337 Ala mutant AChE. The results of our modeling studies also suggest that Glu 199 in Torpedo AChE may be involved in the binding of (-)-Huperzine A to the enzyme. Our experimental data using Torpedo Glu 199 Gln mutant AChE suggest that an energetically favorable electrostatic region generated by the interaction of the primary amine group of Huperzine A with the carboxylate of Glu 199, the carbonyl oxygen of Gly 441, and the hydroxyl group of Tyr 130 contrib- ute minimally to the stabilization of AChE-Huperzine A complex. This is consistent with the fact that the distance between the primary amine group of Huperzine A and Glu 1 9 9 is greater than 5 A in all cases.

In this regard, it is relevant to mention that a model for the orientation of (-)-Huperzine A into the active site gorge of Torpedo AChE using systematic docking studies has been proposed (Pang & Kozikowski, 1993). These modeling studies implicate the involvement of Phe 330, Tyr 121, and Phe 290 in the binding of Huperzine A to AChEs and attribute the substitution of these aromatic amino acid residues by nonaromatic residues to the increase in the KI value of Huperzine A for BChE. Although the orientation of Huperzine A in the active site pocket of AChE proposed in our modeling studies, using molecular mechanics, is different from that proposed by systematic docking studies, both models implicate the involvement of Trp 84 and Phe 330 in the binding of Huperzine A to AChEs. It is likely that Huperzine A may also interact with other regions in the active site of the AChE molecule; we have not explored this possibil- ity either by molecular modeling studies or by testing the inhibition of site-specific AChE mutants of the other 5 aromatic amino acids located in the gorge and replaced by nonaromatic residues in mammalian BChE. The differences in KI values for the inhibition of FBS AChE, Torpedo AChE, and mammalian BChE by Huperzine A and increase in KI values caused by the mutation of Tyr 337 in mouse AChE to Phe and Ala are essen-

1775 ChE-Huperzine A interactions

B1 "334

82

c 1

c 2

Glu-199 Q

,330

-330

Fig. 3. Continued.

1776

Huperrine-A

Acetylcholine

Fig. 4. Structures of Huperzine A and acetylcholine. Structures were drawn using Isis Draw for Windows@, release 1 . 1 .

tially the same. This suggests that the other 5 aromatic amino acids may not affect the binding of Huperzine A to ChEs.

Materials and methods

Materials

ATC, BTC, and DTNB were obtained from Sigma Chemical Co., St. Louis, Missouri. The 2 stereoisomers of Huperzine A were isolated from synthetic (f)-Huperzine A as described (McKinney et al., 1991). Electrophoretically pure AChE from FBS was purified as described (De La Hoz et al., 1986), and AChE from 7: calijornica was provided by Professor I. Silman (Weizman Institute, Rehovot, Israel). BChE from horse serum was purified by affinity chromatography using the procedure similar to the one described for FBS AChE (De La Hoz et al.,

A . Saxena et al.

1986). One milligram of pure native AChE or BChE contained approximately 14 and 1 1 nmol of active sites, respectively.

Expression of Torpedo and mouse AChEs

Wild-type and mutant mouse AChE cDNAs were inserted into CMV-based expression vectors (Andersson et al., 1989; pRC/ CMV, Invitrogen). The presence of the neomycin resistance gene in the CMV vectors enabled the selection of stable transfectants (Radii et al., 1993; Vellom et al., 1993). Wild-type mouse AChE and the mutant AChEs were expressed from stable transfectants in HEK-293 cells. The cell culture media were concentrated to 1-2% of the original volume by ultrafiltration as described (Vel- lom et al., 1993). The cloning, mutagenesis, and expression of Torpedo AChE and its mutant forms in a baculovirus Spodop- tera system and subsequent purification of expressed enzymes were all carried out as described (Radii et al., 1992).

Measurement of ChEs activity and inhibition

AChE and BChE activities were measured in 50 mM sodium phosphate, pH 8.0, at 22 "C as described (Ellman et al., 1961) using ATC and BTC as substrates, respectively. Inhibition of various ChEs was carried out by diluting an appropriate volume of stock solutions (2-5 mM) of each of the 2 stereoisomers of Huperzine A (in 20% acetonitrile) into the enzyme solutions (15- 20 units/mL in 50 mM sodium phosphate, pH 8.0, containing 0.01% BSA) and measuring residual enzyme activity at various times. When the inhibition of various mutant AChEs by (-)- Huperzine A was determined, approximately 1 unit/mL of enzyme activity was used.

Determination of kinetic and inhibition parameters

The interaction of each of the 2 stereoisomers of Huperzine A with various ChEs can be described by the scheme shown in Fig- ure 5A. K , values for FBS AChE, Torpedo AChE, and wild- type mouse AChE were determined by equilibrating 0.2 units/mL of AChE with various concentrations of the inhibitor and measuring residual enzyme activity after equilibrium was reached. The data obtained were analyzed according to the following equa- tion, where [HUP-A] is the initial Huperzine A concentration:

[HUP-A] x 070 residual activity Vo inhibited activity

KI =

Table 3. Free energy differences for the inhibition of ChEs by the 2 stereoisomers of Huperzine A

AAG (kcal)a

Enzyme pair Mutation (+)-Huperzine A (-)-Huperzine A (+)-Huperzine A

Torpedo-FBS AChE Phe to Tyr 1.4 2.3 2.8 BChEh-Torpedo AChE Ala to Phe 3.0 3.2 0.3 BChEh-FBS AChE Ala to Tyr 4.5 5.4 3.1

a Calculated as described in Table 2. Calculated using average K, of human and horse serum BChEs.

ChE-Huperzine A interactions 1777

km

A E + HUP-A 8 E * HUP-A koff

B E + s * ESk% E + P

+ + HUP-A HUP-A

KI It lt UKI

E * HUP-A + S ES HUP-A

Fig. 5. A: Scheme describing the interaction of each of the 2 stereoisomers of Huperzine A with various ChEs. B: Scheme for analysis of kinetic data on inhibition of horse and human serum BChE and mouse mutant AChEs with either stereoisomer of Huperzine A. K , and a K I reflect the interaction of Huperzine A with the free enzyme and the enzyme-substrate complexes, respectively. K , is the competitive inhibition constant.

Due to the high association and dissociation rates for the interaction between Huperzine A and BChE (Ashani et al., 1992), the K I values for the inhibition of horse and human serum BChE and mouse Tyr 337(330) Phe and Tyr 337(330) Ala mutant AChEs with either stereoisomer of Huperzine A were determined by analysis of kinetic data described according to the scheme shown in Figure 5B, where K I and aKI reflect the interaction of Huperzine A with the free enzyme and the enzyme- substrate complexes, respectively. K , is the competitive inhibition constant. Data for this analysis were obtained by measuring inhibition of enzyme activity over a substrate concentration range of 0.025-0.4 mM and a series of Huperzine A concentrations of 0-66 pM depending on the enzyme. Plots of reciprocal ve- locities versus reciprocal substrate concentrations at a series of Huperzine A concentrations yielded a family of slopes. Replots of the slopes versus Huperzine A concentrations were used for the determination of the K I values of these enzymes.

Modeling and energy minimization by rnolecufar mechanics

Molecular modeling and all calculations were carried out on a Silicon Graphics Personal Iris W-4D35TG workstation. Prep- aration of ChE structures for molecular mechanics computation was done as follows: the X-ray diffraction-determined coordinates of AChE from T. californica (Sussman et al., 1991) were obtained from the Brookhaven Protein Data Bank, and a missing 5-amino acid segment (485-489) of the AChE structure was reconstructed. The 4 and $ dihedral angles of the main chain of the segment were adopted from the results of a statistical search of the database in molecular modeling package GEMM (V7.8) (B.K. Lee, National Institutes of Health, Bethesda, Mary- land), and the side chains were optimized with molecular mechanics program YETI (V5.3) (Vedani, 1988). Missing atoms from the side chains of 27 amino acid residues on the protein surface were also inserted and their positions optimized. YETI was used to generate the hydrogen positions at heteroatoms. Op- timization in YETI was carried out in an internal/Cartesian co-

ordinate space with a conjugate-gradient minimizer. All bond lengths, bond angles, and the positions of main chain atoms were kept constant during the calculations. The YETI force field consisted of 9.5/10.0 A for electrostatic interactions, 6.5/7.0 A for van der Waals interactions, and 4.5/5.0 A for hydrogen bonding interactions. Convergence criteria were set to 0.025 kcal mol" deg" for torsional RMS first derivative, to 0.050 kcal mol" deg" for rotational FMS first derivative, and to 0.750 kcal mol" A" for translational RMS first derivative. The energy convergence criterion was k0.05 kcal mol". The atomic structures of FBS AChE (B.P. Doctor & J.L. Sussman, unpubl. results) and human serum BChE (Hare1 et al., 1992) were reconstructed models based on the structure of AChE from T. californica.

X-ray fractional coordinates for Huperzine A (Geig et al., 1991) were converted to Cartesian coordinates. For molecular mechanics calculation, the partial atomic charges for Huperzine A were calculated with MNDO, as implemented in MOPAC (V6.3) (Dewar et al., 1985). Each of the 2 stereoisomers of Hu- perzine A was interactively docked into the active site of T. californica AChE with the GEMM 7.3 package, and the formed conjugates were individually subjected to energy minimization by molecular mechanics. Based on the energy calculated for all the conjugates, the orientation with the lowest energy was selected, and fine manual adjustments for the selected orientation were systematically performed. After each fine adjustment, energy minimization was carried out, and the interaction energy between the inhibitor and the enzyme was calculated and compared to obtain the best orientation for the (-)-Huperzine A molecule in the active site of Torpedo AChE. The procedure used for Torpedo AChE was then extended to both FBS AChE and human serum BChE.

Acknowledgments

We thank Regina Hur and Mary Kay Gentry for the preparation of the manuscript.

References

Anderson S , Davis D, Daheback H, Journvall M, Russell DW. 1989. Clon- ing, structure, and expression of the mitochondrial cytochrome P-450 sterol 26-hydroxylase, a bile acid biosynthetic enzyme. J Biol Chem 264~8222-8229.

Ashani Y, Peggins JO 111, Doctor BP. 1992. Mechanism of inhibition of cholinesterases by Huperzine A. Biochem Biophys Res Commun 184:719- 726.

De La Hoz D, Doctor BP, Ralston JS, Rush RS, Wolfe AD. 1986. A sim- plified procedure for the purification of large quantities of fetal bovine serum acetylcholinesterase. Life Sci 39:195-199.

Dewar S , Zoebisch EG, Healy EF, Stewart JJP. 1985. AMI: A new general purpose quantum mechanical molecular model. J A m Chem Soc 107:3902-3909.

Ellman GL, Courtney D, Andres V, Featherstone RM. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Phar-

Fournier D, Bride JM, Hoffman E Karch F. 1992. Acetvlcholinesterase: Two macol I :88-95.

types of modifications confer resistance to inseckide. J Biol Chem 267:14270-14274.

Geig SJ, Tuckmantel W, Kozikowski AP. 1991. Huperzine A potent acetylcholinesterase inhibitor of use in the treatment of Alzheimer's disease. Acta Crystallogr C 472324821.

Gentry MK, Doctor BP. 1991. Alignment of amino acid sequences of ace- tylcholinesterases and butyrylcholinesterases. In: Massoulie J, Bacou F, Barnard EA, Chatonnet A, Doctor BP, Quinn DM, eds. Cholinesterases: Structure, function, mechanism, genetics and cell biology. Washington, D.C.: American Chemical Society. pp 394-398.

1778 A . Saxena et al.

Harel M, Sussman JL, Krejci E, Bon S, Chanal P, Massoulie J, Silman I. 1992. Conversion of acetylcholinesterase to butyrylcholinesterase, modeling and mutagenesis. Proc Nut1 Acad Sci USA 89:10827-10831.

Kozikowski AP, Xia Y, Reddy ER, Tuckmantel W, Hanin I , Tang XC. 1991. Synthesis of Huperzine A and its analogues and their anticholinesterase activity. J Org Chem 56:4636-4645.

Kreienkamp HJ, Weise C, Raba R, Aaviksaar A, Hucho F. 1991. Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom. Proc Natl Acad Sci USA 88:6117-6121.

Massoulie J, Sussman JL, Doctor BP, Soreq H , Velan B, Cygler M, Rotundo R, Shafferman A, Silman I , Taylor P. 1992. Recommendations for no- menclature in cholinesterases. In: Shafferman A, Velan B, eds. Multi- disciplinary approaches to cholinesterase functions. New York: Plenum Press. pp 285-288.

McKinney M, Miller JH, Yamada F, Tuckmantel W, Kozikowski AP. 1991. Potencies and stereoselectivities of enantiomers of Huperzine A for inhibition of rat cortical acetylcholinesterase. Eur JPharmaco1203:303- 305.

Ordentlich A, Barak D, Kronman C, Flashner Y, Leitner M, Segall Y, Ariel N, Cohen S, Velan B, Shafferman A. 1993. Dissection of the human acetylcholinesterase active center determinants of substrate specificity. J Biol Chem 268: 17083- 17095.

Pang YP, Kozikowski AP. 1993. Topography of the Huperzine A binding site in AChE: Ab initio docking of reversible inhibitors of AChE into the dynamic enzyme. In: Wermuth CG, ed. Trends in QSAR and molecular modeling '92. Leiden: ESCOM. pp 422-424.

Qian N, Kovach 1. 1993. Key active site residues in the inhibition of acetyl- cholinesterases by soman. FEBS Lett 336:263-266.

Radic Z, Gibney G, MacPhee-Quigley K, Kawamoto S, Bongiorno C, Tay- lor P. 1992. Expression of recombinant acetylcholinesterase in a Baculo-

31 :9760-9767. virus system: Kinetic properties of glutamate 199 mutants. Biochemistry

Radic Z, Pickering N, Vellom DC, Camp S, Taylor P. 1993. Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry 32:12074-12084.

Shafferman A, Velan B, Ordentlich A, Kronman C, Grosfeld H, Leitner M, Flashner Y, Cohen S, Barak D, Ariel N. 1992. Substrate inhibition of acetylcholinesterase: Residues involved in signal transduction from the surface to the catalytic center. EMBO J 11:3561-3568.

Sussman JL, Harel M, Frolow F, Oefner C, Goldman A, Toker L, Silman 1 . 1991. Atomic structure of acetylcholinesterase from Torpedo californica: A prototypic acetylcholine binding protein. Science253:872-879.

Sussman JL, Harel M, Silman I . 1992. Three dimensional structure of acetylcholinesterase. In: Shafferman A, Velan B, eds. Multidisciplinary approaches to cholinesterase functions. New York: Plenum Press. pp 95-108.

Vedani A. 1988. YETI: An interactive molecular mechanics program for small-molecule protein complexes. J Comp Chem 9:268-275.

Vellom DC, RadiC Z, Ying L, Pickering NA, Camp S, Taylor P. 1993. Amino acid residues controlling acetylcholinesterase and butyrylcholinesterase specificity. Biochemistry 32:12-17.

Wang Y, Yie D, Tang X. 1986. Anti-cholinesterase activity of huperzine A.

Weise C, Kreienkamp HJ, Raba R, Pedak A, Aaviksaar A, Hucho F. 1990. Acta Pharmacol Sin 7: 1 10- 1 13.

Anionic subsites of the acetylcholinesterase from Torpedo californica. EMBO J 9:3385-3388.

identification of amino acid residues involved in the binding of

Documents