Quantitative structure-activity analysis of acetylcholinesterase inhibition by oxono and thiono analogs of organophosphorus compounds

Download Quantitative structure-activity analysis of acetylcholinesterase inhibition by oxono and thiono analogs of organophosphorus compounds

Post on 24-Feb-2017

215 views

Category:

Documents

1 download

Embed Size (px)

TRANSCRIPT

<ul><li><p>66 Chem. Res. Toxicol. 1992,5, 66-71 </p><p>(35) Di Netta, J., and Egerton, J. R. (1967) Compositions and methods for treating helminthiasis, US. Patent 3,325,356. </p><p>(36) Di Netta, J., and Egerton, J. R. (1969) Compositions and methods for treating helminthiasis comprising combinations of organo-phophates and certain dibenzocycloheptenes, U.S. Patent 3,484,520. </p><p>(37) Egerton, J. R., and Di Netta, J. (1970) Anthelminthic compo- sitions and method of using same, U.S. Patent 3,524,000. </p><p>(38) Little, P. J., and Ryan, A. J. (1982) Inhibitors of hepatic mix- ed-function oxidases. 4. Effecta of benzimidazole and related compounds on aryl hydrocarbon hydroxylase activity from phe- nobarbitone and 3-methylcholanthrene induced rats. J. Med. Chem. 25,622426. </p><p>(39) Hansch, C., and Leo, A. J. (1979) Substituent Constants for Correlation Analysis in Chemistry and Biology, Wiley-Intersci- ence, New York. </p><p>(40) March, J. (1980) Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, pp 89-92, McGraw-Hill Kogakusha La., Tokyo. </p><p>(41) Ortiz de Montellano, P. R., Kunze, K. L., Yost, G. S., and Mico, B. A. (1979) Self-catalyzed destruction of cytochrome P-450 Covalent binding of ethynyl sterols to prosthetic heme. h o c . Natl. Acad. Sci. U.S.A. 76, 746-749. </p><p>(42) Ortiz de Montellano, P. R., and Kunze, K. L. (1980) Inactiva- tion of hepatic cytochrome P-450 by allenic substrates. Biochem. Biophys. Res. Commun. 94,443-449. </p><p>(43) Guengerich, F. P. (1990) Mechanism-based inactivation of hu- man liver microsomal cytochrome P-450 IIIA4 by gestodene. Chem. Res. Toxicol. 3, 363-371. </p><p>(44) Tephly, T. R., Black, K. A., Green, M. D., Coffman, B. L., Dannan, G. A., and Guengerich, F. P. (1986) Effect of the suicide </p><p>substrate 3,5-diethoxycarbonyl-2,6-dimethyl-4-ethyl-l,4-dihydro- pyridine on the metabolism of xenobiotics and on cytochrome P-450 apoproteins. Mol. Pharmacol. 29, 81-87. </p><p>(45) B&amp;ker, R. H., and Guengerich, F. P. (1986) Oxidation of 4aryl- and 4-alkyl-substituted 2,6-dimethyl-3,5-bis(alkoxycarbonyl)-1,4 dihydropyridines by human liver microsomes and immunochem- ical evidence for the involvement of a form of cytochrome P-450. J. Med. Chem. 29, 1596-1603. </p><p>(46) Halpert, J., Hammond, D., and Neal, R. A. (1980) Inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion (diethyl p-nitrophenyl phosphorothionate). J. Biol. Chem. 255,1080-1089. </p><p>(47) Guengerich, F. P. (1986) Covalent binding to apoprotein is a major fate of heme in a variety of reactions in which cytochrome P-450 is destroyed. Biochem. Biophys. Res. Commun. 138, </p><p>(48) Buening, M. K., and Franklin, M. R. (1976) SKF 525-A inhib- ition, induction, and 452-nm complex formation. Druz Metab. </p><p>193-198. </p><p>Dispos. 4, 244-255. (49) Pershing, L. K., and Franklin, M. R. (1982) Cytochrome P-450 </p><p>metabolic-intermediate complex formation and induction bv ma- </p><p>- </p><p>crolide antibiotics: A ned class of agents. Xenobiotiia 12, 687-699. </p><p>(50) Reidy, G. F., Mehta, I., and Murray, M. (1989) Inhibition of oxidative drug metabolism by orphenadrine: In vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics. Mol. Pharmacol. 35, 136-743. </p><p>(51) Murray, M., Zaluzny, L., and Farrell, G. C. (1986) Selective reactivation of steroid hydroxylases following dissociation of the isosafrole metabolite complex with rat hepatic cytochrome P-450. Arch. Biochem. Biophys. 251,471-478. </p><p>Quantitative Structure-Activity Analysis of Acetylcholinesterase Inhibition by Oxono and Thiono Analogues of Organophosphorus </p><p>Compounds </p><p>Donald M. Maxwell* and Karen M. Brecht United States Army Medical Research, Institute of Chemical Defense, Aberdeen Proving </p><p>Ground, Maryland 21010-5425 Received June 27, 1991 </p><p>A comparison of the bimolecular rate constants (hi) for inhibition of electric eel acetyl- cholinesterase (AChE) by the oxono (i.e., P=O) and thiono (i.e., P=S) analogues of parathion, methylparathion, leptophos, fonofos, sarin, and soman revealed that the oxono/thiono ratios of ki values varied from 14 for soman to 1240 for parathion. Analysis of the relative importance of the dissociation equilibrium constant and the phosphorylation rate constant in producing this variation in ki values indicated that the oxono analogues had phosphorylation rate constant values that varied in a narrow range from 8- to 14-fold greater than their thiono counterparts, while the oxono/thiono ratios for dissociation constants varied widely from 1 for soman to 82 for fonofos. The lower affmities of thiono analogues for AChE probably resulted from differences in the hydrophobic binding of oxono and thiono analogues to the active site of AChE, inasmuch as the hydrophobicities (i.e., octanol/water partition coefficients) of thiono organophosphorus compounds were much greater than the hydrophobicities of their oxono analogues. Quantitative structure-activity analysis indicated that the hydrophobic effects of oxono and thiono moieties correlated with log ki for AChE inhibition to a greater extent (r2 = 0.79) than their electronic effects (r2 I 0.48). These observations suggest that the differences in hydrophobicity of oxono and thiono analogues of organophosphorus compounds may be as important as their electronic differences in determining their effectiveness as AChE inhibitors. </p><p>Introduction The low reactivity of thiono organophosphorus compounds for mammalian AChE provides a safety factor for agri- </p><p>The poor reactivity of phosphothioates and phospho- cultural of pesticides, such as or nothioaks for acetylchoheskrase (AChE) in while the rapid metabolic oxidation of these thiono (i-e., to their oxono analogues has been well documented (1-3). </p><p>Abbreviations: AChE, acetylcholinesterase; Pr, isopropyl; Pin, pi- * Author to whom correspondence should be addressed. nacolyl. </p><p>This article not subject to U.S. Copyright. Published 1992 by the American Chemical Society </p></li><li><p>AChE Inhibition by Oxono and Thiono Analogues </p><p>Table I. Structures of Oxono and Thiono Analogues of Organophosphorus Compounds </p><p>0,s II I A2 </p><p>A,-PP-X </p><p>oxono/thiono analogues A, A2 X" paraoxon/ parathion Et0 Et0 OPh-4-NO2 methylparaoxon/methylparathion Me0 Me0 OPh-4-N02 fonofos oxon/fonofos Et Et0 SPh leptophos oxon/leptophos Ph Me0 OPh-4-Br-2,5-C12 sarin/ thiosarin Me Pr'O F soman/ thiosoman Me Pin0 F </p><p>a Leaving group. </p><p>P=S) pesticides to their oxono (i.e., P=O) analogues in insects, but not mammals, produces a beneficial selective toxicity. Although oxono organophosphorus inhibitors of AChE are consistently better inhibitors than their thiono analogues, the ratios of their reactivities with AChE vary considerably. The bimolecular rate constant for inhibition of AChE by paraoxon was reported to be 10000-fold greater than that of its thiono analogue ( l ) , while the rate constant for inhibition of AChE by soman was only 3-fold greater than the rate constant for its thiono analogue (2). </p><p>The usual explanation for the greater inhibition of AChE by oxono organophosphorus inhibitors is that oxygen is more electronegative than sulfur, which results in a cor- respondingly greater reduction in the electron density around phosphorus (4). The greater reduction of the electron density around phosphorus produced by oxygen vs sulfur enhances the electrophilicity of phosphorus and thereby increases its reactivity toward nucleophiles, such as water or the active site serine of AChE. This expla- nation appears to adequately explain the 10-fold greater hydrolysis rate of paraoxon vs parathion and methyl- paraoxon vs methylparathion (5), but it was inadequate to explain the 10000-fold difference in AChE reactivities of paraoxon vs parathion (1,6) or to account for the tre- mendous variation in the effect of oxono vs thiono ana- logues for AChE inhibition by alkyl methylphosphono- fluoridates (2). </p><p>To address these unresolved questions concerning the differences in AChE inhibition by oxono and thiono or- ganophosphorus compounds, we performed a quantitative structure-activity analysis of the oxono and thiono ana- logues of a heterogeneous group of organophosphate and organophosphonate inhibitors (see Table I). The in vitro activity of these inhibitors was evaluated by measurement of their bimolecular rate constants for AChE inhibition, as well as their dissociation and phosphorylation constants, which had not been previously measured for any thiono organophosphorus compounds. A variety of electronic, hydrophobic, and steric properties for the substituents of these organophosphorus compounds were used as inde- pendent variables for quantitative structure-activity analysis. </p><p>Materials and Methods Chemicals. Parathion, paraoxon, methylparathion, methyl- </p><p>paraoxon, leptophos, leptophos oxon, fonofos, and fonofos oxon were obtained from the Pesticides and Chemicals Repository, U.S. Environmental Protection Agency (Research Triangle Park, NC). Soman, thiosoman, sarin, and thiosarin were obtained from the Chemical Research, Development and Engineering Center (Ab- erdeen Proving Ground, MD). The purity of all organophosphorus compounds was &gt;98.9% as determined by 31P nuclear magnetic resonance spectroscopy. Caution: All of the aboue-mentioned organophosphorus compounds are hazardous and should be </p><p>Chem. Res. Toxicol., Vol. 5, No. 1, 1992 61 </p><p>handled carefully (7). Acetylthiocholine iodide, 5,5'-dithiobis- (Znitrobenzoic acid), and electric eel AChE (type V-S) were purchased from Sigma Chemical Co. (St. Louis, MO). </p><p>Determination of Kinetic Constants for AChE Inhibition. The bimolecular rate constants (ki), dissociation equilibrium constant (&amp;), and phosphorylation rate constants (k ) for in- hibition of AChE by organophosphorus compounds, wiere ki = k /Kd, were determined by the method of Wang and Murphy (8). dectric eel AChE dissolved in 0.1 M phosphate buffer (pH 8.0) containing 0.1% bovine serum albumin was incubated a t 5 OC with a small volume of organophosphorus inhibitor dissolved in ethanol. The concentration of AChE used for inhibition studies was 0.3 pM except for organophosphorus compounds with ki &gt; 10' M-l min-l, where a 10-fold reduction in enzyme concentration was necessary. In order to satisfy the experimental requirements for pseudo-firsborder inhibition kinetics, inhibitor concentrations were a t least 10-fold greater than enzyme concentrations. For inhibitors with adequate aqueous solubility, k, and Kd were de- termined from first-order inhibition kinetics using inhibitor concentrations from </p></li><li><p>68 Chem. Res. Toxicol., Vol. 5, No. 1, 1992 Maxwell and Brecht </p><p>Table 11. Kinetic Constants for Inhibition of Eel Acetylcholinesterase" phosphorylation dissociation bimolecular </p><p>rate constant O P , ki, M-' min-' ratio paraoxon 32.7 (5.5) 3.5 x 10-4 (0.7) 9.3 x 104 1240 </p><p>methylparaoxon 22.7 (7.1) 9.1 6.5 X lo-' (1.4) 70 3.5 x 104 640 </p><p>fonofos oxon 27.4 (4.3) 8.2 2.3 X lo4 (0.3) 82 1.2 x 105 680 </p><p>leptophos oxon 28.3 (4.5) 1.3 X (0.4) 2.1 x 105 960 </p><p>sarin 30.2 (9.1) 13.5 7.0 X 10" (1.4) 3.8 4.3 x 106 51 </p><p>soman 45.1 (9.8) 12.9 3.5 X 10" (1.5) 1.1 1.3 x 107 14 </p><p>constant 0 ! S b inhibitor k,, min-' ratio Kd, M ratio O P , rate constant </p><p>7.5 x 10' parathion -C -C </p><p>methylparathion 2.5 (0.9) 4.6 X (0.7) 5.5 x 10' </p><p>fonofos 3.3 (1.4) 1.8 x 10-2 (0.2) 1.8 x 102 </p><p>-C 2.2 x 102 leptophos -C </p><p>thiosarin 2.2 (0.8) 2.6 X (0.4) 8.5 x 104 </p><p>thiosoman 3.5 (0.3) 3.8 X 10" (0.9) 9.2 x 105 a Inhibition constants determined at 5 "C in pH 8.0 phosphate buffer. Standard deviations are in parentheses. Oxono/thiono ratio of </p><p>kinetic constants for analogue pairs. Inadequate inhibitor solubility in water to perform measurement. </p><p>Table 111. Quantitative Structure- Activity Relationships for AChE Inhibition by Oxono and Thiono Analogues of </p><p>Organophosphorus Compounds independent explained variation (r2) </p><p>variables log k; log k, log (I/&amp;) 0.36 0.18 0.05 0.01 0.33 0.02 0.01 0.01 0.34 0.40 0.79 0.50 0.23 0.01 0.12 0.01 0.48 0.87 0.17 0.01 0.44 0.01 </p><p>0.12 0.15 0.54 0.01 0.24 0.67 0.39 0.21 0.11 0.29 0.60 </p><p>for all inhibitors except parathion and leptophos where the aqueous solubilities were too low to achieve inhibitor concentrations LKd, which is required for determination of these kinetic constants (Table 11). For the oxono ana- logues the k, values varied only 2-fold from 22.7 min-' for methylparaoxon to 45.1 min-' for soman, while the Kd values varied 186-fold from 6.5 X 10" M for methylpara- oxon to 3.5 X lo* M for soman. Similarly, the thiono analogues had a 2-fold range of k, values (2.2 min-' for thiosarin to 3.5 min-' for thiosoman) and a 12000-fold range of Kd values (4.6 X loT2 M for methylparathion to 3.8 X lo4 M for thiosoman). </p><p>The relative importance of phosphorylation and affinity in producing the differences in ki values between oxono and thiono analogues was examined by comparing the oxono/thiono ratios of k, and Kd values (Table 11). The oxono analogues had k, values that were consistently 8- to 14-fold greater than those of their thiono analogues. However, the Kd values of oxono analogues were from 1- to 82-fold greater than those of their thiono counterparts. Therefore, the greater ki values for AChE inhibition by the oxono analogues of fonofos and methylparathion in com- parison to the ki values of their thiono analogues were primarily due to the 70- to 82-fold differences in Kd and not to the 8- to 9-fold differences in k,. In contrast, the 51-fold difference in ki values between sarin and thiosarin was more the result of their 14-fold difference in k, than their 4-fold difference in Kd, and the 14-fold difference in the ki values for soman vs thiosoman was entirely due to their oxonof thiono difference in k,. </p><p>Quantitative structureactivity regression analysis (see Table 111) indicated that the independent variables that were most closely correlated with the variation of log ki were the pKa of the leaving group (r2 = 0.44), the oxo- nolthiono indicator variable D1 (r2 = 0.48), and the hy- </p><p>Table IV. Best Structure-Activity Regression Equations for AChE Inhibition Constants </p><p>kinetic constant regression equations ? </p><p>ki log ki = 1.36(*2 - ~ 0 , s ) + 5.89 0.79" O M b </p><p>Kd log (I/&amp;) = 0.86(~2 - Tois) + 4.62 0.67" </p><p>k, log k, = 28.2D1 + 2.88 0.87" </p><p>log ki = 2.880, - O.83pKa + 7.57 </p><p>log (I/Kd) = 0.58(~2 - ~ 0 1 s ) - 0.38pKa + 6.45 0.82b9C </p><p>log k, 29.301 + 4.32~2 - 5.00 0.94b*c log k, 23.701 + 3.51(~2 - T O / S ) + 9.28 0.94b*c </p><p>" Best one-variable regression equation for kinetic constant. * Best multiple-variable regression equation for kinetic constant. Significant difference between one-variable and multiple-variable </p><p>regression equations for kinetic constant. </p><p>drophobicity difference r2 - r o / s (r2 = 0.79). The best multiple-variable regression equation for explaining the variation in log ki was achieved with two variables, pKa and D1, which resulted in r2 = 0.85 (Table IV). Including additional independent variables in the regreasion equation did not increase ? values. When the 1.2 values for the best multiple-variable equation (i.e., p...</p></li></ul>

Recommended

View more >