Studies on the chiral isomers of fonofos and fonofos oxon: I. Toxicity and antiesterase activities

The toxicity of the (R)_P and (S)_P chiral isomers and racemates of fonofos and fonofos oxon to insects and white mice were determined. (R)_P-Fonofos and (S)_P-fonofos oxon were 2- to 12-fold more toxic to house flies, mosquito larvae, and mice than were the corresponding enantiomers. The racemates were intermediate in toxicity. Stereoselectivity also was observed in the in vitro inhibition of house fly-head and bovine erythrocyte acetylcholinesterase, horse serum cholinesterase, chymotrypsin, trypsin, and a variety of esterases. In all cases the (S)_P-oxon was a more potent inhibitor than the (R)_P-oxon with k₁ ratios of $\text{(S)}{}_{\mathrm{<mtext>P</mtext>}}\text{(R)}{}_{\mathrm{<mtext>P</mtext>}}$ ranging from 4- to 60-fold. Further, differences in levels of house fly-head, mouse brain, and blood cholinesterase obtained from house flies and mice treated with the enantiomers and racemates of fonofos and fonofos oxon were observed. Differences in toxicity of the enantiomers and racemates to house flies and mice were more closely related to in vivo than to in vitro cholinesterase inhibition.     

In vitro inhibition of acetylcholinesterase by O,O-dimethyl S-aryl phosphorothioates

In vitro inhibition of house cricket head, house fly head, and bovine erythrocyte acetylcholinesterase by O,O-dimethyl S-aryl phosphorothioates was studied by Main's kinetic treatment. The potency of the compounds as reflected by the bimolecular reaction constants (k_i) indicated that house fly head acetylcholinesterase was the most sensitive to the inhibition followed by house cricket head and bovine erythrocyte acetylcholinesterase. There are no linear relationships between the phosphorylation rate constants and the total binding energies for the inhibition of three enzymes by this series of compounds, suggesting that the initial binding and the phosphorylation rate are not related. The structure and activity relationships were analyzed by multiple regression analyses with the use of Hammett's sigma, alkaline hydrolysis rates of the compounds, and p_i constants. The hydrophobic bonding of the compound on the enzyme surface as reflected by the p_i constant played a significant role in the determination of the potency of the inhibition of house cricket head and house fly head acetylcholinesterase by those compounds. However, the alkaline hydrolysis rates of the compounds, or the Hammett's sigma values seems to play a more important role in the determination of the inhibition of bovine erythrocyte acetylcholinesterase. Moderate insecticidal activity toward house crickets, house flies, and mosquito larvae were found.     

Selectivity, acetylcholinesterase inhibition kinetics, and quantitative structure-activity relationships of a series of N-(2-oxido-1,3,2-benzodioxa-phosphol-2-yl) amino acid ethyl or diethyl esters

The inhibitory effects of a recently introduced series of the titled compounds on insect and mammalian acetylcholinesterase (AChE) activity were examined, where the median inhibition concentration (I₅₀) and the inhibition kinetic parameters, bimolecular inhibition rate constant (k_i), affinity constant (K_a), and phosphorylation rate constant (k_p), were determined for each compound. Results indicated that all examined dioxaphospholenes had less inhibitory effects on mammalian AChE than fenitrothion, a commercial pesticide with moderate mammalian toxicity. The highest selectivity was obtained with compounds containing glutamic and leucine moieties (2.70 and 2.18, respectively) while selectivity of fenitrothion was 0.93. The low inhibitory effects of the examined dioxaphospholenes on mammalian AChE were attributed to their low phosphorylation rates (k_p < 2.2 min⁻¹) compared to that of fenitrothion (k_p = 4.84 min⁻¹). QSAR equations indicated that the inhibition process is controlled mainly by both the phosphorylation rate (direct effect) and the affinity of compounds toward the enzyme (inverse effect). Although the compounds’ hydrophobicity had no effects on the inhibition process, it affects the compounds’ toxicity since it affects the ability of compounds to penetrate insects to reach the enzyme active site.     

Aryl N-methoxy-N-methylcarbamates: Potent competitive reversible inhibitors of cholinesterases

A series of 27 substituted aryl N-methoxy-N-methylcarbamates were synthesized and their ability to reversibly inhibit house fly-head and bovine-erythrocyte acetylcholinesterase and horse-serum cholinesterase was determined. These compounds were all competitive, reversible inhibitors of bovine erythrocyte acetylcholinesterase but some of them showed mixed competitive inhibition against the house fly-head and horse-serum enzymes. Dissociation constants (K_i) as small as 9.9 × 10^?9M and as large as 1.4 × 10^?4M were observed. A highly satisfactory correlation between log K_i for the inhibition of fly-head acetylcholinesterase by the N-methoxy-N-methylcarbamates and ?log I₅₀ for the inhibition of the same enzyme by the corresponding methylcarbamates was noted. Analysis of the anticholinesterase data by multiple regression showed -log K_i to be related to Hansch's π constant and ring position terms. The results indicate that reversible binding of these compounds to acetylcholinesterase occurs by hydrophobic bonding.     

Inhibition of two acetylcholinesterases by the 4-nitrophenyl esters of methyl-, ethyl-, and isopropyl(phenyl)phosphinic acid

The inhibition of eel acetylcholinesterase and bovine erythrocyte acetylcholinesterase by the 4-nitrophenyl esters of methyl-, ethyl-, and isopropyl(phenyl)phosphinic acid (MPP, EPP, and IPP, respectively) was investigated at pH 6.90 in 0.067 M phosphate buffer (25.0°C) using stopped-flow instrumentation and automated data processing. Our evaluation of the dissociation constant, K_d, the unimolecular bonding rate constant, k₂, and the bimolecular reaction constant, k_i, are the first reported values for these constants for a homologous series of this class of organophosphorus compounds. The largest k₁ value (29,428 M^?1 sec^?1) was observed for the reaction of eel acetylcholinesterase with 4-nitrophenyl methyl(phenyl)phosphinate. The smallest k_i value (9.6 M^?1 sec^?1) was observed for the reaction of bovine erythrocyte acetylcholinesterase with 4-nitrophenyl isopropyl(phenyl)phosphinate.     

Studies on the acetylcholinesterase of Anopheles albimanus resistant and susceptible to organophosphate and carbamate insecticides

Acetylcholinesterase from fourth instar Anopheles albimanus larvae was studied in vitro. The acetylcholinesterase from both the resistant and susceptible strains behaved as a single enzyme “type,” with straight pseudo first-order insecticide inhibition lines which intersected the Y axis at 100%. The enzyme from resistant larvae was more slowly inhibited than the susceptible enzyme; bimolecular rate constants (k_i) differed by approximately 1.2- to 6-fold for a range of organophosphorous compounds and 17- to 1570-fold for the carbamates. There was a good correlation between the levels of resistance and the acetylcholinesterase inhibition rates.     

Phosphinate inhibition studies of cholinesterases

The kinetic constants, K_d, k₂, and k_i, were determined for the inhibition by 4-nitro-phenyl methyl(phenyl)phosphinate of three cholinesterases: butyrylcholinesterase, bovine erythrocyte acetylcholinesterase and eel acetylcholinesterase. Stopped-flow kinetic evaluations and automated data acquisition and processing were employed. A broad range in affinity for the phosphinate inhibitor was observed as reflected by the binding constants, K_d. A similar wide range in the k₂ values for the unimolecular inhibition step was obtained. The net bimolecular rate constants, ki, indicate equal overall reactivity for butyrylcholinesterase and eel acetylcholinesterase with a smaller inhibition rate constant for bovine erythrocyte acetylcholinesterase.     

Phosphorylation rate constants and esteratic subsite normality employing the fluorescent organophosphate Maretin: A new approach to the study of the acylation mechanism of acetylcholinesterase and butyrylcholinesterase

The fluorescent organophosphate Maretin, O,O-diethyl O-(1–6 naphthaloximido)phosphate, is an optimum analytical reagent for measurement of acetylcholinesterase (AChE) and butyrylcholinesterase cholinesterase (BuChE) normality. It also permits direct measurement of the second-order kinetics of inhibition. Maretin is fluorescent in aqueous solution with excitation at 346 nm and emission at 398 nm. On phosphorylation, the N,N-naphtholoylhydroxylamine leaving group is nonfluorescent. Experimental advantages of this methodology include: readily correctable hydrolysis rate, commercial availability of pure analytical grade Maretin, ease of handling, and no significant side reactions. The bimolecular rate constant (K_i), equilibrium binding constant (K_eq), and phosphorylation rate constant (k₂) are measured as a function of pH and compared for the two enzymes. Phosphorylation rate data and enzyme activity studies indicate an independent pH functionality between acylation and deacylation for AChE.     

Biochemical characteristics of insecticide resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

A strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from corn in Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl parathion (354-fold). Biochemical studies revealed that various detoxification enzyme activities were higher in the field strain than in the susceptible strain. In larval midguts, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase) and hydrolases (general esterase, carboxylesterase, β-glucosidase) were 1.2- to 1.9-fold higher in the field strain than in the susceptible strain. In larval fat bodies, various activities of microsomal oxidases (epoxidases, hydroxylase, N-demethylase, O-demethylases, and S-demethylase), glutathione S-transferases (CDNB, DCNB, and p-nitrophenyl acetate conjugation), hydrolases (general esterase, carboxylesterase, β-glucosidase, and carboxylamidase) and reductases (juglone reductase and cytochrome c reductase) were 1.3- to 7.7-fold higher in the field strain than in the susceptible strain. Cytochrome P450 level was 2.5-fold higher in the field strain than in the susceptible strain. In adult abdomens, their detoxification enzyme activities were generally lower than those in larval midguts or fat bodies; this is especially true when microsomal oxidases are considered. However, activities of microsomal oxidases (S-demethylase), hydrolases (general esterase and permethrin esterase) and reductases (juglone reductase and cytochrome c reductase) were 1.5- to 3.0-fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b₅ were 2.1 and 1.9-fold higher, respectively, in the field strain than in the susceptible strain. In addition, acetylcholinesterase from the field strain was 2- to 85-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, propoxur, carbofuran, bendiocarb, thiodicarb) and organophosphates (methyl paraoxon, paraoxon, dichlorvos), insensitivity being highest toward carbaryl. Kinetics studies showed that the apparent K_m value for acetylcholinesterase from the field strain was 56% of that from the susceptible strain. The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S-transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase. Resistance appeared to be correlated better with detoxification enzyme activities in larval fat bodies than in larval midguts, suggesting that the larval fat body is an ideal tissue source for comparing detoxification capability between insecticide-susceptible and -resistant insects.     

Kinetic constants for the inhibition of acetylcholinesterase by phenyl carbamates

The kinetic constants of a variety of substituted phenyl N-methyl- and N,N-dimethylcarbamates, which inhibit bovine erythrocyte acetylcholinesterase, were determined by various experimental procedures. A procedure in the presence of a chromogenic substrate was developed, based on the suggestion of Hart and O'Brien, and was compared with the conventional Main method. The dissociation equilibrium constant, K_d, and the carbamylation rate constant, k₂, were shown to apparently depend on the inhibitor concentration range used for the determination in both procedures. Assuming that the binding of further inhibitor molecules to the reversible complex and the carbamylated enzyme is significant under conditions with high inhibitor concentrations, the concentration dependence of the kinetic constants was nicely delineated. It is indicated that reliable constants are obtainable with a rather low inhibitor concentration range, whose product by k_i is of the order of 0.2–1.0 min^?1.     

Comparative effects of two plant growth regulators; indole-3-acetic acid and chlorogenic acid on human and horse serum butyrylcholinesterase

Butyrylcholinesterase (BChE), a major detoxification enzyme found abundantly in many tissues and organisms, constitutes the first line defense in the serum of higher organisms and is a marker for toxic exposure. In this study, the interaction of two plant growth regulators, indole-3-acetic acid (IAA) and chlorogenic acid (CA) with purified human and horse serum BChE is investigated. The time dependent interaction of IAA with the two enzyme species was concentration dependent and rapid. Through kinetic studies, IAA was found to be linear-mixed type inhibitor for human serum BChE, and uncompetitive inhibitor for the horse serum enzyme. For the human BChE, α and the K_i value was calculated as 2.15 ± 1.09 and 3.09 ± 0.98 mM, respectively, and for the horse enzyme the K_i value was calculated as 1.05 ± 0.09 mM. The time dependent interaction of CA with the two enzyme species was biphasic. At low CA concentrations, CA stimulated the activities of both enzyme species whereas at high CA concentrations, inhibition was observed. At high concentrations, the inhibition kinetics for both enzymes fitted the non-competitive inhibition model. The K_i values calculated for human and horse BChE were 2.75 ± 0.14 and 0.96 ± 0.07 mM, respectively. The differences in the interaction of these two growth regulators with the two enzymes species arises from the structural differences between the human and horse serum BChE which can be considered as a triple human mutant BChE.     

Relation of acetylcholinesterase sensitivity to cross-resistance of a resistant house fly strain to organophosphates and carbamates

Acetylcholinesterase (AChE, E.C. 3.1.1.7) from an organophosphate-resistant strain of house fly, Musca domestica (L.) exhibited a decrease in sensitivity towards four organophosphates and two carbamates in comparison with enzyme from the parent susceptible strain. Sensitivity was less, as measured by the bimolecular reaction constant (k_i), by a factor of 117 for dichlorvos, 94 for paraoxon, 11 for diazoxon, 7 for Tetram, 62 for propoxur, and 50 for dimetilan. These differences in bimolecular reaction constants were attributed entirely to differences in their affinity for the enzyme, as measured by the dissociation constant, K_d. It is suggested that the cross resistance to these inhibitors is due at least in part to insensitive acetylcholinesterase.     

Eel acetylcholinesterase inhibition studies with heteroarylphosphinates

The inhibition of eel acetylcholinesterase by the 4-nitrophenyl esters of 2-furyl(methyl)-, methyl(2-thienyl)-, di-2-furyl-, and di-2-thienylphosphinic acid (I, II, III, and IV, respectively) was investigated at pH 6.90 in 0.067 M phosphate buffer (25.0°C) using stopped-flow instrumentation and automated data processing. Our evaluation of the dissociation constant, K_d, the unimolecular bonding rate constant, k₂, and the bimolecular reaction constant, k_i, are the first reported values for these constants for alkyl/heteroaryl and diheteroaryl esters of phosphinic acids. The largest k_i value (19,330 M^?1 sec^?1) was observed for the reaction of I with the enzyme. The order for the remaining three is II > IV > III. There is no direct relationship between the hydrolysis rates of the esters and their anticholinesterase activities on eel acetylcholinesterase. Likewise, there is no direct relationship between their anticholinesterase activities and the LD₅₀ values in rats.     

Esterase-mediated malathion resistance in the human head louse, Pediculus capitis (Anoplura: Pediculidae)

Resistance in a dual malathion- and permethrin-resistant head louse strain (BR-HL) was studied. BR-HL was 3.6- and 3.7-fold more resistant to malathion and permethrin, respectively, compared to insecticide-susceptible EC-HL. S,S,S-Tributylphosphorotrithioate synergized malathion toxicity by 2.1-fold but not permethrin toxicity in BR-HL. Piperonyl butoxide did not synergize malathion or permethrin toxicity. Malathion carboxylesterase (MCE) activity was 13.3-fold and general esterase activity was 3.9-fold higher in BR-HL versus EC-HL. There were no significant differences in phosphotriesterase, glutathione S-transferase, and acetylcholinesterase activities between strains. There was no differential sensitivity in acetylcholinesterase inhibition by malaoxon. Esterases from BR-HL had higher affinities and hydrolysis efficiencies versus EC-HL using various naphthyl-substituted esters. Protein content of BR-HL females and males was 1.6- and 1.3-fold higher, respectively, versus EC-HL adults. Electrophoresis revealed two esterases with increased intensity and a unique esterase associated with BR-HL. Thus, increased MCE activity and over-expressed esterases appear to be involved in malathion resistance in the head louse.     

Aryl N-hydroxy- and N-methoxy-N-methylcarbamates as potent reversible inhibitors of acetylcholinesterase

Several aryl N-hydroxy- and N-methoxy-N-methylcarbamates were examined as inhibitors of bovine erythrocyte acetylcholinesterase (AChE). These carbamate derivatives were generally strong inhibitors of AChE, but, unlike the typical N-methyl- and N,N-dimethylcarbamates which are carbamylating agents, they proved to be reversible, competitive inhibitors of the enzyme. The values for the dissociation constant (K_a) for the enzyme-inhibitor complex to enzyme and inhibitor were in the range of 2 × 10^?5?1 × 10^?7M.     

Kinetic properties of the inhibition of juvenile hormone esterase by two trifluoromethylketones and O-ethyl,S-phenyl phosphoramidothioate

Some inhibition kinetic properties and in vivo inhibition of the plasma juvenile hormone esterase from the cabbage looper (Trichoplusia ni Hübner) by one phosphoramidothioate and two trifluoromethylketones were examined. O-ethyl,S-phenyl phosphoramidothioate was shown to react irreversibly with the enzyme in a time-dependent manner, and the inhibition reaction can be factored into a reversible step with a dissociation constant, K_d, of 4.55 × 10^?5M followed by a phosphorylation step with a rate constant, k₂, of 1.98 min^?1. The phosphorylated enzyme did not show spontaneous recovery after 48 hr of dialysis. On the other hand, the two trifluoromethylketones were shown to act as reversible inhibitors, as their inhibited enzyme was regenerated completely after dialysis. However, 1,1,1,-trifluoro-3-thiooctylpropan-2-one, in contrast to 1,1,1-trifluorotetradecan-2-one, showed progressive time-dependent inhibition, and its reaction with the enzyme followed characteristic bimolecular second-order kinetics with a rate constant, k_i, of 3.37 × 10⁷M^?1 min^?1. The in vivo inhibition data of topically treated larvae at equimolar amounts of the tested compounds indicated rapid penetration, and the stability of the inhibition was higher for the phosphoramidothioate than for the trifluoromethylketones. The relationship of the mechanism of inhibition and the in vivo inhibition of these compounds to the understanding of the interactions between juvenile hormone and juvenile hormone esterase is discussed.     

Studies on the optical isomers of EPN and EPNO

The optical isomers of EPN (O-ethyl O-p-nitrophenyl phenylphosphonothionate) and EPNO (O-ethyl O-p-nitrophenyl phenylphosphonate) have been synthesized. No significant difference in the rate of alkaline hydrolysis of the isomers at the two pH's evaluated was observed. The (+)-isomers of EPN and EPNO were more toxic to house flies than the corresponding (?)-isomers, while the (+)- and (?)-isomers, as well as the racemic mixture of EPN, were almost equally toxic to mice. The (+)-EPNO is more toxic to mice than the corresponding (?)-isomer. Cholinesterase inhibition studies demonstrated that (+)-EPNO has a higher bimolecular rate constant, (k_i) than the corresponding (?)-isomer. This higher inhibitory power was due to a higher affinity (K_a) of the (+)-isomer.     

Mechanism of inhibition reaction of acetylcholinesterase by phenyl N-methylcarbamates: Separation of hydrophobic,electronic, hydrogen bonding and proximity effects of aromatic substituents

The association equilibrium constant, $\text{1}\text{K}{}_{\mathrm{d}}$, and the carbamylation constant, k₂, of 53 o-, m-, and p-substituted phenyl N-methylcarbamates with bovine erythrocyte acetylcholinesterase were determined. The $\text{1}\text{K}{}_{\mathrm{d}}$ value varied 1000-fold, whereas the k₂ value did not depend upon the nature and position of substituents. The variation in $\text{log}\text{(}\text{1}\text{K}{}_{\mathrm{d}}\text{)}$ was analyzed using free energy related substituent parameters and regression analyses. The effect of substituents at o-, m-, and p-positions was nicely separated into hydrophobic, electronic, hydrogen bonding, and proximity (steric and field electronic for o-substituents) factors. The physicochemical significance of these factors was established by comparison with those for model organic reactivities. The mechanism of the whole reaction process was elucidated in terms of physical organic chemistry.     

Stereodependent bioactivation of R(+)-ethyl S-propyl methylphosphonothioate: Is the S-oxide the active metabolite?

R(+)-Ethyl S-propyl methylphosphonothioate is bioactivated both in vivo and when perfused through isolated liver to give a product which is much more active as an inhibitor of acetylcholinesterase than the parent compound. The bioactivation does not occur in hepatectomised animals. Acetylcholinesterase inhibited by the active metabolite is not reactivated by pyridine-2-aldoxime methanesulphonate (P2S), whereas enzyme inhibited by the parent compound and its S(?) enantiomer is reactivatable. Attempts to identify the active metabolite were unsuccessful and experiments to explore its stability were inconclusive. Extensive in vitro studies of the inhibition of acetylcholinesterase by the enantiomers of ethyl S-propyl methylphosphonothioate and ethyl S-diisopropylaminoethyl methylphosphonothioates and subsequent reactivation of the enzyme by P2S showed that (a) there are large differences between the rates of inhibition of the R and S enantiomers of both compounds, (b) reactivation profiles are critically dependent on reaction conditions, and (c) the reactivation profiles of the R and S enantiomers of the former compound are indistinguishable under all conditions whereas differences are observed under some conditions for the latter pair of enantiomers. The results are discussed in terms of the possibility that the S-oxide of R(+)-ethyl S-propyl methylphosphonothioate is the active metabolite and it is concluded that this is unlikely.     

Insensitivity of acetylcholinesterase as a factor in resistance of houseflies to the organophosphate Rabon

Acetylcholinesterase from an organophosphate-resistant strain of housefly exhibited a decrease in sensitivity to Rabon in vitro, to the extent of 206-fold for the soluble enzyme and 38-fold for the particulate. These effects upon the bimolecular reaction constant, k_i, were due to a 573-fold decrease in affinity of the inhibitor for the soluble and 103-fold for the particulate enzyme, coupled with a small increase in reactivity.