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.     

Substituted thiotrifluoropropanones as potent selective inhibitors of juvenile hormone esterase

A series of 27 substituted thio-1,1,1-trifluoropropanones was synthesized by reacting the corresponding thiol with 1,1,1-trifluoro-3-bromopropanone. The resulting sulfides were screened as inhibitors of hemolymph juvenile hormone esterase and α-naphthyl acetate esterase activity of the cabbage looper, Trichoplusia ni, electric eel acetylcholinesterase, bovine trypsin, and bovine α-chymotrypsin. The presence of the sulfide bond increased the inhibitory potency on all of the enzymes tested when compared with compounds lacking the sulfide. In general, the compounds proved to be poor inhibitors of chymotrypsin and moderate inhibitors of trypsin. By varying the substituent on the sulfide, good inhibitory activity was obtained on α-naphthyl acetate esterase, acetylcholinesterase, while some of the compounds proved to be extremely powerful inhibitors of juvenile hormone esterase. The most powerful inhibitor tested was 3-octylthio-1,1,1-trifluoro-2-propanone, with an I₅₀ of 2.3 × 10^?9M on JH esterase. This compound showed a molar refractivity similar to that of the JH II backbone, was not toxic to T. ni, and was moderately toxic to mice, with a 48-hr LD₅₀ of >750 mg/kg. It effectively delayed pupation when applied to prewandering larvae of T. ni, as expected for a JH esterase inhibitor. Thus, some members of this series are promising for evaluating the role of JH esterase in insect development. The series also indicates that, by varying the substituent on the sulfide moiety, potent “transition-state” inhibitors can be developed for a wide variety of esterases and proteases.     

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.     

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.     

Binding of mercury compounds to the chloroplasts in relation to the inhibition of the Hill reaction

The binding behavior of mercuric chloride (HgCl₂), phenylmercuric acetate (PMA), and ethylmercuric chloride (EMC) to the spinach chloroplasts in relation to the inhibition of the Hill reaction was studied at pH 6.8 and 7.8 using ²⁰³Hg labeled compounds. The pH of the reaction medium did not influence the amount of mercury binding of the chloroplast at various mercurial concentrations, but it altered the inhibition curve of the Hill reaction. Between 0–1 × 10^?5M the binding of Hg²⁺ and EMC were similar and increased linearly with the concentration, while the binding of PMA was similar to the binding of Hg²⁺ only at a concentration below 4 × 10^?6M and was less when the concentration was above 4 × 10^?6M. However, the inhibition of the Hill reaction by these mercury compounds was quite different; at pH 7.8, the I₅₀ values for Hg²⁺, PMA, and EMC were 5 × 10^?6, 2.5 × 10^?6, and 2.5 × 10^?6M, respectively, while at pH 6.8, these values were 4 × 10^?6, 4 × 10^?5, and 2 × 10^?4M, respectively. The differential block of electron flow by the mercury compounds at pH 6.8 and 7.8 was further confirmed by electron spin resonance study.     

Effects of herbicidal carbamates on mitochondria and chloroplasts

At concentrations near 2 × 10^?4M, barban, chlorpropham, and phenmedipham are inhibitors of the electron transfer in potato and mung bean mitochondria. The inhibition seems to be localized in the flavoprotein region. It affects preferentially the exogenous NADH dehydrogenation, in potato mitochondria (I₅₀, 10^?4M). Succinate dehydrogenation is less inhibited. At noninhibiting concentrations, the studied carbamates cannot uncouple the oxidative phosphorylations. Photosynthesis is completely inhibited by 2.10^?7M phenmedipham, 5 × 10^?5M barban, and 2 × 10^?4M chlorpropham. The inhibition takes place at the PS II level. Moreover, barban and chlorpropham are uncouplers of the photophosphorylations for concentrations between 5 × 10^?5 and 5 × 10^?4M. The effects observed on mitochondrial respiration can also be found on respiration of Acer cultured cells. The effects on isolated chloroplast photosynthesis are also observed for slightly higher concentrations on cultured Chlorella and on pea and oat leaf fragments.     

The inhibition of HEOM epoxide hydrase in mammalian liver microsomes and insect pupal homogenates

A range of compounds were tested as inhibitors of the enzyme epoxide hydrase, using a cyclodiene epoxide (HEOM) as substrate. Rat and rabbit liver microsomes and pupal homogenates of the blowfly (Calliphora erythrocephala) and the yellow mealworm (Tenebrio molitor) were compared as sources of the enzyme. Only minor differences were found between the four enzyme preparations, when considering I₅₀ values and percentage inhibition at standard concentration. The simple epoxide 1,1,1-trichloropropane-2,3-epoxide and two glycidyl ethers p-nitrophenyl glycidyl ether and p-ethylphenyl glycidyl ether tended to have lower I₅₀ values (1.8×10^?6 to 8.0×10^?5M) than triphenyl phosphate and SKF 525A (4.5×10^?5 to 1.4×10^?4M). Triphenyl phosphate and SKF 525A were competitive inhibitors for both the rat and Tenebrio enzymes. The only clear difference found between these two epoxide hydrase preparations was with respect to their inhibition by 1,1,1-trichloropropane-2,3-epoxide, which was an uncompetitive inhibitor with the rat enzyme, but showed kinetics of mixed inhibition with the insect preparation.     

Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5), 10^?3M acetylthiocholine (ASCh), and enzyme extract equivalent to 80 ± 3 μg protein incubated for 15 min at 30°C. The K_m value for ASCh (6.7 × 10^?5M) was much lower than that of butyrylthiocholine (BuSCh) (1.25 × 10^?2M). The enzyme activity was almost completely inhibited by 10^?6M paraoxon or 10^?5M eserine and was 84% inhibited by 10^?5M BW284C51 (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sulfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids.     

Metabolism of substituted diphenylether herbicides in plants. I. Enzymatic cleavage of fluorodifen in peas (Pisum sativum L.)

A “soluble” glutathione S-transferase that catalyzes the cleavage of the herbicide, 2,4′-dinitro-4-trifluoromethyl diphenylether (fluorodifen), was isolated and partially characterized from epicotyl tissues of pea seedlings. A 32-fold purification of the enzyme was achieved by differential centrifugation, ammonium sulfate precipitation, Sephadex gel filtration, and DEAE-cellulose ion exchange chromatography. The enzyme had a pH optimum of 9.3–9.5 and was specific for reduced glutathione, with an estimated apparent K_m value of 7.4 × 10^?4M. Limited specificity studies with four substituted ¹⁴C-labeled diphenylether compounds indicated that fluorodifen was the only effective substrate, with an estimated apparent K_m value of 1.2 × 10^?5M. Differences and similarities between the pea epicotyl enzyme and other plant and animal glutathione S-transferases were discussed from the standpoint of substrate specificity, pH optima, distribution, stability, and inhibitor studies.     

Structure-activity relationships of 1,2,3-benzothiadiazole insecticide synergists as inhibitors of microsomal oxidation

The activities of 47 substituted 1,2,3-benzothiadiazoles as inhibitors of microsomal epoxidation and/or hydroxylation in enzyme preparations from rat liver or armyworm (Spodoptera eridania) gut have been evaluated. Many were found to be effective inhibitors of microsomal oxidation, the most active being the 6-butyl and 6-propoxy derivatives with I₅₀ values of 4.9 × 10^?7 and 7.0 × 10^?7M, respectively, for the epoxidation reaction. Regression analyses have established that activity of the 5-, 6-, and 5,6-substituted compounds can be satisfactorily described in equations in terms of π², π, and σ whereas that of the 4-substituted derivatives depends on π and the steric parameter E₈.     

Inhibition of fatty acid biosynthesis in isolated bean and maize chloroplasts by herbicidal phenoxy-phenoxypropionic acid derivatives and structurally related compounds

The effects of nine phenoxy-phenoxypropionic acid derivatives and structurally related compounds on the incorporation of [¹⁴C]-acetate into free fatty acids in isolated bean and maize chloroplasts were studied. The compounds tested were esters and the corresponding free acids, OH-diclofop, a nonherbicidal metabolite of diclofop in plants, and d and l enantiomers of diclofop. Fatty acid biosynthesis in bean chloroplasts was not affected by all compounds. OH-Diclofop had a weak inhibitory effect on fatty acid synthesis in maize chloroplasts, while free acids were stronger inhibitors than the corresponding esters in the same system. Uptake studies with diclofop-methyl and diclofop indicated that the esters showed higher uptake rates in chloroplasts suspension. d-Diclofop (I₅₀, 9 × 10^?8M) was a more potent inhibitor than l-diclofop (I₅₀, 4 × 10^?6M). This agrees with the low herbicidal activity of the l enantiomer in vivo. The results suggest that the mode of action in this type of herbicide may be closely linked with the inhibition of fatty acid biosynthesis. The tolerance of beans could be based on an insensitivity of the target site.     

The action of insecticides on neurosecretory neurons in the stick insect, Carausius morosus

The action of insecticides on the spontaneous electrical activity of neurohemal tissue in the stick insect, Carausius morosus, has been studied using extracellular electrodes. The pyrethroid, permethrin, causes a massive increase in the frequency of the spontaneously generated action potentials at concentrations between 5 × 10^?5 and 5 × 10^?8M. Concentrations as low as 5 × 10^?11M are still effective in producing bursting activity.DDT, at concentrations between 5 × 10^?5M and 5 × 10^?6M, produces an overall increase in activity although the bursting activity is less violent than that shown with permethrin. DDT, 5 × 10^?7M, is ineffective at altering the resting pattern.Carbaryl and coroxon cause a transitory increase in electrical activity at 1 × 10^?4M, but are ineffective at 1 × 10^?5M.It is concluded that insecticides could have a direct effect upon the neurohormonal balance in insects.     

Hydrolysis of paraoxon and malaoxon in three strains of Myzus persicae with different degrees of parathion resistance

Homogenates of three strains of Myzus persicae, A, R, and E, with an LD₅₀ for topically applied parathion of 9, 93, and 263 ng per aphid, showed an in vitro hydrolytic degradation of paraoxon of 2.3, 4.7, and 8.6 pmol/mg aphid/h, respectively. These values represent V_max; K_m was <10^?7M. The three strains showed a malaoxon degradation of 2.4, 11.9, and 18.8 pmol/mg/h at 10^?6M substrate concentration. V_max for R and E was 21 and 27 pmol, respectively and K_m 7 and 4 × 10^?7M. Activity in strain A was too low to estimate these entities. The breakdown product of paraoxon was mainly diethyl phosphoric acid, that of malaoxon mainly dimethyl phosphoric acid. No hydrolysis of the carboxylester groups of malaoxon was found. Hydrolysis of paraoxon and malaoxon was inhibited by isopropyl and n-propyl paraoxon and by the salioxon-analog K₂. The two latter compounds were shown to act as synergists with parathion when added in amounts that caused little mortality when given alone. The hydrolytic enzyme is soluble and retains its activity during incubations of several hours. It is likely that it is responsible for at least part of the resistance. Resistance was maintained without selection over a period of three years. There was no correlation between degree of resistance and carboxylesterase activity of the strains.     

Differential inhibition of potassium ion absorption by difenzoquat in wild oat and cereals

Over a concentration range of 5.0 × 10^?6?7.5 × 10^?4M, the selective herbicide difenzoquat (1,2-dimethyl-3,5-diphenyl-1H-pyrazolium) caused more pronounced inhibition of potassium ion (K⁺) absorption by excised seedling roots of susceptible wild oat (Avena fatua L.) compared to those of tolerant barley (Hordeum vulgare L. cv. Bonanza) or wheat (Triticum aestivum L. cv. Neepawa). At 2.5 × 10^?5M difenzoquat, the relative inhibition of K⁺ (⁸⁶Rb) absorption by wild oat root segments inceased from 30% with a 10-min uptake period to 75% with an uptake period of 90 min, whereas no inhibition at all was evident for wheat root segments even after a 90-min exposure to the herbicide. An ion efflux compartmental analysis procedure demonstrated that difenzoquat did not affect the passive permeability properties of the plasma membrane of wild oat root cells. The experimental findings indicated that difenzoquat interfered directly with the process of active ion transport across the plasma membrane of root cells.     

Pyrethroid insecticides: Potent,stereospecific enhancers of mouse brain sodium channel activation

Deltamethrin and NRDC 157, pyrethroid insecticides that produce different poisoning syndromes in mammals, enhanced veratridine-dependent, sodium channel-mediated ²²Na⁺ uptake in mouse brain synaptosomes. Concentrations producing half-maximal enhancement were 2.5 × 10^?8M (deltamethrin) and 2.2 × 10^?7M (NRDC 157). This effect was stereospecific: The nontoxic 1S enantiomers had no significant effect on veratridine-dependent activation. At high deltamethrin concentrations, enhancement was maximal at 5 × 10^?5?1 × 10^?4M veratridine. Pyrethroid enhancement was completely blocked by 5 × 10^?6M tetrodotoxin, and neither pyrethroid affected ²²Na⁺ uptake in the absence of veratridine at concentrations up to 1 × 10^?5M. The relative potencies of deltamethrin and NRDC 157 in the synaptosomal sodium channel assay agree well with their relative acute toxicities to mice when administered by intracerebral injection. These findings demonstrate that pyrethroids exemplifying both characteristic poisoning syndromes are potent, stereospecific modifiers of sodium channel function in mammalian brain.     

Potassium uptake in atrazine-treated roots of sensitive and resistan plants

The action of atrazine and its biodegradation products on the membrane transport of potassium in roots was evaluated in both sensitive and resistant plants. Excised roots of maize and oat showed inhibition of potassium uptake efficiency in the presence of 1.4 × 10^?4M atrazine and 1.4 × 10^?4M deethylated atrazine. Other biodegradation products such as 2-chloro-4-amino-6-ethylamino-1,3,5-triazine,2-chloro-4,6-,bisamino-1,3,5-triazine, and 2-chloro-4-amino-1,3,5-triazine showed no inhibitory effect on the K⁺ uptake capacity. Two maize hybrids showing different uptake efficiency were inhibited differently by atrazine. We suggest that atrazine and deethylated atrazine inhibited the K⁺ transport interacting directly with the plant cell membranes without discerning between resistant and sensitive plants.     

The effects of pyrethroids on the electrical activity of neurosecretory cells from the brain of Rhodnius prolixus

The effects of pyrethroids on the on-going electrical activity of the axons of neurosecretory cells from the brain of fifth instar Rhodnius prolixus have been studied using extracellular electrodes. Low concentrations of the pyrethroids decamethrin, bioresmethrin, permethrin, and bioallethrin all produce dramatic increases in the overall frequency and dramatic changes in the pattern of electrical activity when applied directly to the exposed brain and corpora cardiaca in an otherwise intact insect. This change in activity was brought about by a recruitment in active units and the production of phasic acivity. A doubling of frequency over that of controls was brought about by low doses of the pyrethroids, namely decamethrin, 1 × 10^?10M; bioresmethrin, 2 × 10^?10M; permethrin, 1 × 10^?9M; and bioallethrin, 2 × 10^?7M. Similar hyperactivity of this system occurred during intoxication of intact insects following topical application of LD₉₅ bioresmethrin. The enhanced sensitivity shown by neurosecretory cells over that of other cell types is discussed, as is the possibility that the increases in electrical activity of neurosecretory axons may result in massive neurohormonal release and thereby contribute to the eventual poisoning of the insect.     

The mechanism of fungistatic action of sec-butylamine: II. The effect of sec-butylamine on pyruvate oxidation by mitochondria of Penicillium digitatum and on the pyruvate dehydrogenase complex

sec-Butylamine at 5 mM inhibited the oxidation of pyruvate by mitochondria isolated from hyphae of Penicillium digitalum, but had little effect on the oxidation of citrate, isocitrate, succinate, malate, acetyl-coenzyme A, or reduced nicotinamide adenine dinucleotide. sec-Butylamine did not interfere with oxidative phosphorylation, as evidenced by similar $\text{P}\text{O}$ ratios in treated and control mitochondria. The pyruvate dehydrogenase complex (EC 1.2.4.1) isolated from young hyphae of P. digitatum was inhibited strongly by 20 mM sec-butylamine, whereas other tricarboxylic acid cycle enzymes were only slightly affected at most. Inhibition of the pyruvate dehydrogenase complex by sec-butylamine was competitive with respect to pyruvate. The K_i for sec-butylamine in the reaction was 1.38 × 10^?2M, and the K_m for pyruvate was 2.28 × 10^?4M. These observations and other evidence derived from studies with intact hyphae support the hypothesis that the pyruvate dehydrogenase complex is the primary site of the fungistatic action of sec-butylamine.     

The effect of DDT and its analogs on the fragility of human erythrocytes

The antihemolytic actions of DDT and eight analogs were examined with human erythrocytes. Apparent aqueous concentrations to produce 60% of control hemolysis ranged from 3.7 × 10^?4 to 2.4 × 10^?6M, with DDT being one of the least active. No correlation was found between antihemolytic potency and neurotoxicity, and it was concluded that the findings did not illuminate the toxic or neural actions of these compounds.     

Interaction of some unsubstituted phosphoramidate analogs of methamidophos (O,S-dimethyl phosphorothioamidate) with acetylcholinesterase and neuropathy target esterase of hen brain

At 37°C and pH 7.4–8.0, five higher O-alkyl analogs of methamidophos and four O-alkyl O-2,5-dichlorophenyl phosphoramidates all were more potent progressive inhibitors of hen brain AChE and neuropathy target esterase (NTE) than was methamidophos itself. For AChE, k_a increased from 7.2 × 10² to 1.0 × 10⁵ M⁻¹ min⁻¹ between methyl and n-hexyl S-methyl esters and from 9.3 × 10³ to 8.9 × 10⁵ M⁻¹ min⁻¹ between ethyl and n-hexyl dichlorophenyl analogs. For NTE, the ranges were from 16 to 7.9 × 10⁴ for S-methyl esters, and were 9.7 × 10⁴ to 7.8 × 10⁶ M⁻¹ min⁻¹ for dichlorophenyl. S-methyl esters were more active against AChE than against NTE and all the dichlorophenyl esters were more active against NTE than against AChE. Spontaneous reactivation of 75–100% activity without aging of AChE was found after 19 hr incubation at 37°C after inhibition by all nine straight-chain alkyl analogs. After inhibition by O-isopropyl S-methyl phosphorothioamidate, some spontaneous reactivation with complete aging of all remaining inhibited AChE occurred during 19 hr. No spontaneous reactivation or aging of inhibited NTE was detected. It was concluded that the molecular structures of the inhibited enzymes obtained from equivalent compounds in the two series of inhibitors were identical and that the leaving groups were, therefore, S-methyl and O-2,5-dichlorophenyl, respectively. Although hen brain NTE inhibited by methamidophos in vitro did not age, cases of delayed neuropathy in man have been reported and, presumably, require aging as well as inhibition of NTE. Possible explanations of this apparent discrepancy include (i) the fact that methamidophos consists of two chiral forms and that the form seen to be active in vitro may be disposed of preferentially in vivo, (ii) the possibility of activation in vivo to a different inhibitor, (iii) differences between conformation and ease of aging of inhibited NTE in vitro and in vivo, and (iv) species differences.