Chem. Res. Toxicol. 1993,6, 724-730
724
Interaction of Acetylcholinesterase with the Enantiomers of Malaoxon and Isomalathion Clifford E. Berkman,+Debra A. Quinn, and Charles M. Thompson* Department of Chemistry, Loyola University of Chicago, 6525 North Sheridan Road, Chicago, Illinois 60626 Received May 10, 199P
The bimolecular reaction constants (hi), dissociation constants (&), and phosphorylation constants (k,) were determined for the enantiomers of malaoxon against rat brain acetylcholinesterase, and for the stereoisomers of isomalathion against rat brain acetylcholinesterase and electric eel acetylcholinesterase. (R)-Malaoxon was an 8.6-fold more potent anti-cholinesterase than (5')-malaoxon. Isomalathion stereoisomers with the R configuration a t carbon were 3-13fold stronger inhibitors than those with the S configuration. The isomalathion stereoisomers with the R configuration at phosphorus were 4.3-8.8-fold stronger inhibitors of rat brain acetylcholinesterase,yet 3.4-5.8-fold weaker inhibitors of electric eel acetylcholinesterase,than the isomalathion stereoisomers with the S configuration a t phosphorus. The rat brain acetylcholinesterase spontaneous (ko = approximately 13.0 X 103min-l) and oxime-mediated (hodme= 51.0 X min-') reactivation rate constants following inhibition by isomalathion stereoisomers with the R configuration a t phosphorus were comparable to spontaneous (11.3 X min-') and oxime-mediated (50.2 X min-l) reactivation rates obtained for (S)-isoparathion methyl. These data support a common phosphorylation mechanism, namely, the displacement of the thiosuccinyl moiety from isomalathion stereoisomers with the R configuration a t phosphorus, and displacement of the p-nitrophenoxy ligand from (S)-isoparathion methyl to form the same 0,s-dimethyl phosphorothiolated enzyme. Rat brain acetylcholinesterase inhibited by the isomalathion stereoisomers with the S configuration a t phosphorus were refractory to reactivation, suggesting an alternate mechanism of inhibition, i.e., the loss of the methylthio ligand. Several mechanisms are proposed to account for the subsequent nonreactivation. Similar reactivation kinetics are predicted for electric eel acetylcholinesterase inhibited by the isomalathion stereoisomers on the basis of lz, values.
Introduction Malathion is a commonly used organophosphate (OP) with a broad spectrum of insecticidal activity and a relatively low mammalian toxicity owing to rapid degradation by carboxylesterases (I,2). The selective toxicity of malathion toward insects is due to the deficit of esterasemediated hydrolysis in insects. The acute toxicity toward mammals and insects presumably results from first metabolic oxidation to malaoxon (eq l),apotent inhibitor
c oxldaion
s
co2c2n,
CH,O-/kS&CO&*HI CH,O
malathion
isomerizalion
0
CO*C*H,
CH,O-P\S 'I
&C02Cznt
(1)
CH,O
malaoxon
o
co1c2n5
CH,S-~.S&C02C*Hr
(2)
cn,o isomalathlon
(3, 4 ) of the neurotransmitter mediating enzyme acetylcholinesterase (AChE)l(5). Although purified malathion is relatively innocuous to mammals, thermal or photochemical isomerization (6, 7) leads to the more toxic f Present address: IGEN Research Institute, 130 5th Ave. N., Seattle, WA 98109. * Abstract published in Advance A C S Abstracts, September 1,1993. 1 Abbreviations: AChE, acetylcholinesterase;RBAChE,solubilizedrat brain AChE; EEAChE, electric eel AChE; m-CPBA, m-chloroperoxybenzoic acid; MMPP, monoperoxyphthalic acid, magnesium salt; PEX, potassium ethyl xanthate; DMS, dimethyl sulfa*, 2-PAM, 2-pyridinealdoxime methiodide; OP, organophosphate.
phosphorodithiolate impurity isomalathion (eq 2). Isomalathion is a 1000-fold more potent anti-cholinesterase than malathion (6,8) and was implicated in the epidemic malathion poisoning of 2800 Pakistani spraymen in 1976 that resulted in 5 fatalities (9). Malathion, malaoxon, and isomalathion are structurally related through an asymmetric carbon center on the diethylsuccinate ligand Figure 1, which likely affects the interaction with AChE. For example, Hassan and Dauterman showed that the (R)-0,O-diethyl malaoxon was a 4.4-fold stronger inhibitor of bovine erythrocyte AChE than (SI-0,O-diethyl malaoxon (IO). To date, however, the stereoselectiveinhibition of AChE by the 0,O-dimethyl malaoxon enantiomers has not been reported. The isomerization of malathion to isomalathion (eq 2) is of special interest because it creates a second center of asymmetry at phosphorus (Figure 1).2 A priori, each of the four isomalathion stereoisomers is expected to interact uniquelywith AChE (11-13). Althoughapriorreport (14) alluded to possible differential anti-AChE potencies of the isomalathion stereoisomers, there has been little experimental support for this premise. We recently reported the synthesis of the stereoisomers of malathion, malaoxon, and isomalathion (15-1 7),and 2 The preceding paper ( 1 7 )designated the configurational identities of the isomalathion stereoisomersas follows: (1R,3R)-,(1R,3S)-,(1S,3R):, and (IS,3S)-isomalathion,where the phosphorus and carbon asymmetric atoms are the 1and 3 positions, respectively. Racemates are denoted as (RS)-malaoxon and (1RS,3RS)-isomalathion. Diastereomeric mixtures at phosphorus are denoted as (1RS,3S)- and (lRS,3R)-iaomalathion.
0893-228~/93/2706-0724$04.00/0 0 1993 American Chemical Society
Chem. Res. Toxicol., Vol.6,No. 5, 1993 725
Enantiomers of Malaoxon and Isomalathion S
C02C2H5
CH,O'IP\sI'
~ C 0 2 C 2 H 5
-
CH,O
II CH,O I CH,O
1
(Spmalathion
(R)-malathion
C02C2H5
0
CH,O/,!
C0ZCZH5
\ S -C02C2H5
\s ~ c 0 2 c 2 H 5
CH,O
(S)-malaoxon
(R)-malaoxon
CH,Sl"
[\s It
&c02c2H5
CH,O
(1R, 3R)-isomalathlon
C02C2H,
0
c02cfi5
CH,Ol"
I'
C0 ,2H ,5)
cH301
II Ap \
C0ZCZH5
II
$02C2H5 :
5
S &cO2c2HS
CH,S
CH,S
(lS, 3R)-isomalathion
(1S, 3s)-isomalathlon
CH,Sll 1\s&C02C2H5 CH,O
(1 R, 3S)-isomalathion
Figure 1. Stereoisomers of malathion, malaoxon, and isomalathicon.
established the isomer configurations through X-ray crystallographicanalysis of a phosphorothioic acid alkaloid salt precursor (18).Acquisition and structural assignment of these enantio-enriched molecules now permit an investigation of the dual role of phosphorus and carbon asymmetry upon the inhibition and reactivation of AChE. In this analysis, we show that the asymmetric carbon of malaoxon and isomalathion significantly influences the AChE inhibitory potency. The asymmetric phosphorus center of isomalathion also affects the anti-AChE potency, but it does not dominate the stereoselective action. An interesting postinhibitory fate linked to isomalathion phosphorus stereochemistry (19) and a species-dependent, stereoselective anti-AChE potency by the isomalathion isomers are identified.
Materials and Methods Chemicals. The preparation of the compounds used in this study is reported in the preceding paper (17). (R)- and (SIMalaoxon were prepared by oxidation of (R)- and @)-malathion, respectively, with m-chloroperoxybenzoic acid (m-CPBA) or monoperoxyphthalic acid, magnesium salt (MMPP) (16). Racemic (RS)-malaoxon was prepared similarly from racemic malathion. Racemic (IRS,3RS)-isomalathionwas prepared from racemic malathion via potassium ethyl xanthate (PEX) dealkylation and dimethyl sulfate (DMS) realkylation (8, 20). Diastereomericmixtures that were racemicat phosphorus { (1RS,3R)and (lRS,3S)-isomalathion)also were prepared by the dealkylation/realkylation sequence from (R)- or (&malathion, respectively. Enantioenriched isomalathion stereoisomers were prepared by dealkylationof (R)-or (@-malathionwith strychnine to furnish diastereomeric, alkaloid salts. Following repeated fractional recrystallization, each enriched diastereomer was reacted with DMS to afford the individual isomalathion stereoisomers (15, 17). Note: The diastereomer recrystallizations performed for this study gave higher enantio-enrichment than those in the prior report ( 1 7). The structural identity and chemical purity (greater than 99%) of these materials were confirmed by phosphorus and proton NMR, optical rotation, gas chromatography, high-performanceliquid chromatography,and combustion analysis. Chiral HPLC (ChiralpakAD; 1090isopropanol/hexane) established the isomalathion stereoisomeric purities as follows: 1S,3R, 93%; 1S,3S, 95%; 1R,3R, 94%;and lR,3S, 98%. An 86: 14 mixture of the isomalathion stereoisomer lS,3S/lR,3R was prepared by reaction of the same ratio of the alkaloid precursor salts with DMS (15) and the composition confirmed by chiral HPLC.
Reagents used for the kinetic assays and the isolation of rat brain acetylcholinesterase (RBAChE)were described previously (8, 20, 21). Electric eel acetylcholinesterase (EEAChE) (type VI-S)and 2-pyridinealdoxime methiodide (2-PAM)were obtained from Sigma Chemical Co. (St. Louis, MO). Caution: All the organophosphorus compounds used in this study are hazardousand should be handled ina well-ventilated hood by trained personnel. Reaction of Isomalathion a n d Malaoxon with AChE. Details for the determination of the bimolecular reaction constant (ki),dissociation constant (&), and phosphorylation constant (k,)were summarizedin prior reports (8,20,21). The reactivation rate constants ko (spontaneous)and k 0 h e(oxime-mediated)were determined following a 40-fold dilution of a RBAChE solution that was inhibited by the individual isomalathion stereoisomers as summarized previously (19, 20). All kinetic assays were dependent upon the cholinesterase-catalyzed hydrolysis of acetylthiocholineand the estimation of thiocholineproduced (22). All RBAChE enzyme analyseswere conducted in 0.1 M phosphate buffer (pH 7.6), whereas EEAChE analyses were conducted in 0.1 M phosphate buffer (pH 8.0). Concentrations of inhibitors used for the determination of the kinetic constants against RBAChE were 0.02-0.04 M, (R)-malaoxon; 0.26-1.30 M, (S)malaoxon; 0.1-0.8 M, (lR,3R)-isomalathion;0.39-6.2 M, (1R,3S)isomalathion; 0.31-2.4 M, (lS,3R)-isomalathion,and 2.0-10 M, (lS,3S)-isomalathion. Concentrations of inhibitors used for the determination of the kinetic constants against EEAChE were 0.65-10 M, (1R,3R)-isomalathion; 0.63-10 M, (lR,3S)-isomalathion; 0.08-0.4 M, (lS, 3R)-isomalathion; and 0.57-1.3 M, (1S,3S)-isomalathion.
Results and Discussion Prior studies showed that AChE may be inhibited stereospecifically by OP's (3,10-12,21,23-26),although most investigations examined the influence of a sole asymmetric phosphorus center. One notable study showed that the enantiomers of isopropyl methyIphosphonofluoridate differed in inhibitory potency by 4200-fold (11). The inhibition of AChE by OP's bearing both phosphorus and ligand stereocenters also has been examined (12,24), but few studies of AChE inhibition by OP's containing only a ligand stereocenter have been documented (10). Last, most studies of OP stereoselective action have been conducted with phosphonates (P-C linkage) (12,23-261, whereas we sought to define the contribution of asymmetric phosphorus and carbon centers upon the interaction of phosphorothiolates with AChE.
726 Chem. Res. Toxicol., Vol. 6, No. 5, 1993
Berkman et al.
Table I. Kinetic Data. for RBAChE Inhibited by Malaoxon. Isomalathion, and Isoparathion
& [mM
k t [M-' min-I (x103)i
isomer R
731 (0.07) 141 (0.09) 85 (0.09)
RS
S
k, koC [ m i d % spontaneous (min-1) (X103)I reactivationd Malaoxon (Asymmetric Carbon)
(xlob)] 44 235 387
(X10-91
7% oxime reactivationd
42 (0.13)
51.0 (0.05)
81 (0.09)
0.99 except 1S,3R where cc = 0.49. f Reference 21. 8 Reference 20. a
Our first aim was to assess the individual contribution of each stereocenter upon the inhibition reaction. The specific role of the phosphorus atom was explored in prior studies that showed that (SI-isoparathion methyl was a 3-15-fold more potent anti-cholinesterase than (R)-isoparathion methyl (21). Moreover, the phosphorylated enzyme resulting from inhibition by (S)-isoparathion methyl reactivated 3.2-3.6-fold faster (20). The character of a ligand stereocenter was examined by Hassan and Dauterman, who showed that (R)-0,O-diethyl malaoxon was a 4.4-fold better anti-cholinesterase (bovine erythrocyte) and an 8-fold better anti-carboxylesterase(rat liver) than (S)-0,O-diethyl malaoxon (10). Since even modest changes in structure can influence the anti-cholinesterase potency, the inhibitory potency of the 0,O-dimethyl malaoxon stereoisomers was investigated first. A prior study showed that RBAChE best differentiated the isoparathion methyl enantiomers (21), and therefore, we first examined the inhibition of this cholinesterase with enantioenriched malaoxon. Inhibition of RBAChE by Malaoxon Stereoisomers. (R)-Malaoxon, (S)-malaoxon, and (RS)-malaoxon (racemic) were reacted with RBAChE, and the bimolecular reaction constants (ki) were determined (Table I) (eq 3).
ENZ
+
OP-X
e Kd
[ENZ'OP-X]
kP
reactivation (ko or kWim$
ENZ-OP
t
x'
(3)
non-reactivation (kNR) or aging (kNR)
ENZ-OP
(R)-Malaoxon was an 8.6-fold more potent inhibitor than (S)-malaoxon. (RS)-Malaoxon had inhibitory strength approximately between the malaoxon antipodes. The stereoselective inhibition of RBAChE by malaoxon is comparable to the 8.3-fold difference observed for isoparathion methyl enantiomers, indicating that asymmetry in the ligand was as important as the stereochemistry at phosphorus. To more precisely evaluate the stereochemical influence by the malaoxon enantiomers, values for the dissociation constant (Kd) and the phosphorylation constant (k,) were
determined (Table I). The difference in inhibitory potency between these stereoisomers was clearly linked to formation of the enzyme-inhibitor complex (&), where (R)malaoxon was 10-foldgreater than (S)-malaoxon. Owing to the enzyme active site asymmetry, it is not surprising that formation of the Michaelis complex occurs preferentially with one enantiomer since a specific orientation may be more readily accommodated. Virtually no difference was observed in the k, values between (R)-and (S)-malaoxon, suggestingthat the carbon stereocenter was relatively unimportant in the phosphorylation step. If inhibition of AChE by malaoxon proceeds through the loss of the succinylthio ligand, this result is not surprising. Moreover, if isomalathion proceeds via the same mechanism of inhibition as malaoxon, it follows that very little difference should be displayed in the k, values between isomalathion stereoisomers. Inhibition of RBAChE by Isomalathion Stereoisomers. Since the asymmetric carbon in malaoxon affected the inhibitory potency, RBAChE inhibition by mixtures of isomalathion stereoisomers with established carbon configurations (phosphorus diastereomers) was conducted. We found that (1RS,3R)-isomalathion was only a 3.0-fold stronger inhibitor of RBAChE than (1RS,3S)-isomalathion (Table I), suggesting that either the RBAChE could not distinguish the carbon configurational stereoisomersof isomalathion as well as malaoxon, or the inhibitory potency may be adjusted by the asymmetric phosphorus center. To address this issue, values for kiwere determined for the four individual stereoisomers of isomalathion as well as an 86:14 mixture of (lS,3S)- to (1R,3R)-isomalathion. A 29-fold difference in anti-AChE potency was found between the strongest (1R,3R) and weakest (1S,3S) isomalathion stereoisomers. While the inhibitory potencies (ki) of the diastereomeric mixtures (lRS,3R)- and (1RS,3S)-isomalathionwere midway in value between the enantio-enriched isomers, and the fully racemic material gave an average ki value relative to the four stereoisomers, the 86:14 mixture of (1S,3S)- to (1R,3R)-isomalathion reacted with RBAChE to give a ki value that closely reflected the molar ratio of the individual stereoisomers according to the following equation: [( % stereoisomer A)(ki of stereoisomer A)] + [(% stereoisomer B)(ki of
Chem. Res. Toxicol., Vol. 6, No. 5, 1993 727
Enantiomers of Malaoxon and Isomalathion
Scheme I. Comparative Mechanisms of Phosphorylation and Reactivation by the Stereoisomers of Isomalathion and Isoparathion Methyl
(1S, 3RS)-lsomalathlon
AChE
0 , S-dlmethylphosphorothiolated enzyme
(R)-isoparathion methyl
I
reactivation
AChE
T
reactivation
(1 R, 3RS)-lsomalathion
A
A
C
h
(S)-isoparathion methyl
E
0, Sdimethylphosphorothlolated enzyme
stereoisomer B)1 = ki of the AB mixture (calculated, 59 X 103; found, 56 X 103 M-1 min-I). Both (IR,3R)- and (IR,3S)-isomalathion were more potent inhibitors of RBAChE than the corresponding (IS,3R)- and (IS,SS)-isomalathion stereoisomers, a preference for a configurational arrangement that also was reported for nerve gas agents (12) and methyl phosphoand ascribed to specific complementary nates (24, W), active site features (11,12,24,25). In addition, (IS,3R)and (IR,3R)-isomalathionstereoisomers displayed greater inhibitory potency against RBAChE than (IS,3S)- and (IR,3S)-isomalathion. Yet, the impact on the inhibition reaction by the carbon stereocenter was attenuated by the configuration at phosphorus. For example, the difference in inhibitory strength associated with a change in the carbon configuration from R to S was 6.8-fold when the phosphorus configuration was S,while the difference was only 3.3-fold when the phosphorus configuration was R. Likewise, the phosphorus stereocenter was leveraged by the carbon stereocenter. This effect is evidenced by a comparison of the phosphorus S and R configurational isomers, which show a 4.3-fold ki difference when the configuration was R at carbon, while the difference was 8.8-fold when the carbon configuration was S. Therefore, the carbon and phosphorus asymmetric centers of isomalathion act interdependently during the inhibition of RBAChE. Values for Kd and k, were determined for the isomalathion stereoisomers (Table I). As with the malaoxon stereoisomers, the ki values paralleled the dissociation constants, Kd. For example, (IR,3R)-isomalathion displayed a 3.4-fold higher Kd and a 3.3-fold higher ki than (IR,3S)-isomalathion. The Kd values also parallel the inhibition potency for (IR,3S)-isomalathion and (1S,3S)isomalathion, the former an 8.8-fold stronger inhibitor. The (lS,3S)-stereoisomer yields estimates of 2.51 for k, and 20 OOO for Kd, which necessitate higher [I]for adequate kinetic analysis (27). To accommodate this aspect, the inhibition of RBAChE by the 1S,3Sisomer was conducted with 10-fold greater inhibitor concentrations. However,
the unusual kinetic behavior of this isomer will require further investigation. The k, values obtained for (1R,3R)- and (lR,3S)isomalathion were virtually identical, consistent with the results obtained with malaoxon. However, (IS,3R)- and (lS,3S)-isomalathion k, values differed by 4.7-fold, and were significantly higher than (IR,3R)- and (1R,3S)isomalathion. Thus,the carbon stereochemistry influences the phosphorylation only when (lS,3R)- or (lS,3S)isomalathion is the inhibitor, and suggests that the succinylthio ligand remains attached. Conversely, since there was no difference in the k, for (IR,3R)- and (1R,3S)isomalathion, it is likely that the succinylthio ligand is displaced and has little stereochemical influence over this step. Alternatively, if RBAChE inhibition by the four isomalathion stereoisomers occurs via a common mechanism (i.e., displacement of the succinylthio ligand with inversion or retention of the phosphorus configuration; 241, then the putative S-or R-configured 0,s-dimethyl phosphorothiolated AChE would result. Thus, the two phosphorylated enzymes resulting from inhibition by the isomalathionstereoisomersshould be identical to RBAChE inhibited by (SI-or (R)-isoparathion methyl (loss of p-nitrophenoxy;Scheme I), and reactivation rates should be comparable. In an effort to distinguish the influence of stereochemistry upon the recovery processes and to clarify any mechanistic ambiguity, the rates of spontaneous and oxime-mediated reactivation (koand k0-e; eq 3) were desired. Reactivation of RBAChE Following Inhibition by Isomalathion Stereoisomers. In separate experiments, RBAChE was inhibited by each isomalathion stereoisomer and the lS,3SIlR,3R (86:14) diastereomer mixture, the enzyme was diluted 40-fold (to halt further inhibition), and the spontaneous reactivation rate constants, ko (eq 3), were calculated from the slope of the initial portion (0-30 min) of the graph (Figure 2, panel A; Table I). Rate constants for oxime-mediatedreactivation ( k 0 h e ) were determined similarly. Following inhibition of RBAChE by each of the isomalathion stereoisomers and
-
728 Chem. Res. Toxicol., Vol. 6, No. 5, 1993 2,0
Spontaneous Reactivation
Oxime-mediated Reactivation
1
1.5
bp
,
1.0
B
.
:: aa
*
0
0
.
II time (min)
time (min)
Figure 2. Spontaneous and oxime-mediated (2-PAM) reactivation of RBAChE inhibited by isomalathion stereoisomers: (0) 1S,3R; (e) 1S,3& (0) 1R,3R; ( 0 )1R,3& (A)lS,3S/lR,3R
the 1S,3SIl R,3R (86:14) diastereomer mixture, the enzyme was diluted 40-fold (to halt further inhibition) and reacted with 2-PAM, and the oxime-mediated rate constant, koime (eq 3), was calculated from the initial slope of the graph (Figure 2, panel B). By 60 min, the reactivation (spontaneous and oxime-mediated) reached a plateau and was assigned the total percent reactivation relative to uninhibited enzyme (Table I). No additional enzyme activity returned after 120 min. The koxkevalues for RBAChE inhibited by (lR,3R)- or (lR,SS)-isomalathion were 4-fold greater than the ko values, and the total percent returned enzyme activity was twice that of the spontaneous process. Both the spontaneous (approximately 13 X 10-3 min-') and oximemediated reactivation (approximately 51 X le3min-l) rates for these two diastereomers were nearly identical, suggesting that RBAChE's phosphorylated by (1R,3R)or (1R,SS)-isomalathion were chemically and stereochemically equivalent. In contrast, RBAChEs inhibited by (1S,3R)-or (lS,SS)-isomalathionwere refractory to spontaneous and oxime-mediated reactivation, and showed only a modest amount of total returned activity (
