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.     

Selective inhibition of JH esterases from cockroach hemolymph

Juvenile hormone III was tritium labeled on the methyl ester and utilized with other substrates in an investigation of inhibition and substrate specificity of hemolymph esterases from the cockroach, Blaberus giganteus. The structure of labeled juvenile hormone III was supported both chemically and biochemically. Forty-two potential inhibitors were examined, and the best inhibitors included phosphoramidothiolates and S-phenylphosphates. One of these inhibitors was found useful in hormone biosynthesis studies dealing with the enzymatic conversion of methyl farnesoate to juvenile hormone in corpora allata homogenates. Several commonly used inhibitors of carboxyesterases caused only weak inhibition of JH esterases. Gel filtration elution patterns, inhibitor relationships, and specific activities of the hemolymph esterases indicate that juvenile hormones I and III are degraded by similar if not identical enzymes. In some cases, α-naphthyl acetate and juvenile hormone esterase activity could be differentially inhibited. Hemolymph esterases were not capable of degrading ethyl or isopropyl conjugated esters of two juvenoids or three model substrates.     

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.     

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.     

Oxidative activation of O,S-dimethyl phosphoramidothiolate

O,S-Dimethyl phosphoramidothiolate, the insecticide methamidophos, is converted oxidatively into a potent anti-cholinesterase agent by the action of m-chloroperbenzoic acid. The active intermediate is not stable enough to be isolated. From some chemical evidence, the sulfoxide, i.e., dimethyl phosphoramidothiolate S-oxide, is most probably the active intermediate. A similar activation may occur by biological oxidation.     

Separation and characterization of the pyrethroid-hydrolyzing esterases of the cattle tick, Boophilus microplus

The principal esterases present in homogenates of cattle tick larvae have been separated by gel filtration and preparative isoelectric focusing. Substrate specificities have been determined using trans-permethrin, trans-cypermethrin, p-nitrophenyl butyrate, and the pyrethroid analog, p-nitrophenyl-(1R,S)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate (t-NPDC). One of the esterases, with pI = 4.6, and molecular weight ～67,000, hydrolyzed the α-cyano-substituted pyrethroid, trans-cypermethrin, but not permethrin. The major esterase activity was found in the pI 5.6–5.8 region, and corresponded to a molecular weight of ～89,000. Small differences in substrate specificity and differences in the banding pattern after isoelectric focusing were detected between esterases of ticks of a pyrethroid-resistant strain (Malchi) and a pyrethroid-susceptible strain (Yeerongpilly). Rate constants were determined for the inhibition of the different esterases by the organophosphate coroxon and by naphthyl N-propylcarbamate, using p-nitrophenyl butyrate and t-NPDC as substrates.     

The biochemistry of insecticide resistance in Anopheles sacharovi: Comparative studies with a range of insecticide susceptible and resistant Anopheles and Culex species

Fourth instar larvae, the progeny from wild-caught Anopheles sacharovi females, were subjected to a number of biochemical tests and the results were compared to those from similar tests on laboratory insecticide resistant and susceptible strains of anopheline and culicine mosquitoes. DDT resistance in An. sacharovi is associated with the ability to rapidly metabolise DDT to DDE. The organophosphorus and carbamate resistance was not associated with quantitative changes in esterases, multifunction oxidases, or glutathione S-transferase. The acetylcholinesterase was less sensitive to malaoxon and propoxur than laboratory susceptible An. albimanus, and plots of inhibition suggest that the population was polymorphic for more than one form of acetylcholinesterase. Metabolism studies on malathion and pirimiphos methyl did not indicate resistance due to increased metabolism. There was no evidence of penetration barriers contributing to resistance to either DDT or malathion, and there was no indication of any resistance to pirimiphos methyl in our tests.     

Mechanisms of resistance to diflubenzuron in the house fly, Musca domestica (L.)

The mechanisms of resistance to the chitin synthesis inhibitor diflubenzuron were investigated in a diflubenzuron-selected strain of the house fly (Musca domestica L.) with > 1000 × resistance, and in an OMS-12-selected strain [O-ethyl O-(2,4-dichlorophenyl)phosphoramidothioate] with 380 × resistance to diflubenzuron. In agreement with the accepted mode of action of diflubenzuron, chitin synthesis was reduced less in larvae of the resistant (R) than of a susceptible (S) strain. Cuticular penetration of diflubenzuron into larvae of the R strains was about half that of the S. Both piperonyl butoxide and sesamex synergized diflubenzuron markedly in the R strains, indicating that mixed-function oxidase enzymes play a major role in resistance. Limited synergism by DEF (S,S,S-tributyl phosphorotrithioate) and diethylmaleate indicated that esterases and glutathione-dependent transferases play a relatively small role in resistance. Larvae of the S and R strains exhibited a similar pattern of in vivo cleavage of ³H- and ¹⁴C-labeled diflubenzuron at N₁C₂ and N₁C₁ bonds. However, there were marked differences in the amounts of major metabolites produced: R larvae metabolized diflubenzuron at considerably higher rates, resulting in 18-fold lower accumulation of unmetabolized diflubenzuron by comparison with S larvae. Polar metabolites were excreted at a 2-fold higher rate by R larvae. The high levels of resistance to diflubenzuron in R-Diflubenzuron and R-OMS-12 larvae are due to the combined effect of reduced cuticular penetration, increased metabolism, and rapid excretion of the chemical.     

The effect of O,S,S,-trimethyl phosphorodithioate on the in vivo metabolism of phenthoate in the rat

The in vivo metabolism of phenthoate (O,O-dimethyl S-[α-(carboethoxy)benzyl]phosphorodithioate) was followed in rats after oral administration of a nontoxic dose of 100 mg/kg. The same metabolic study was conducted following coadministration of 0.5% O,S,S-trimethyl phosphorodithioate (OSS-Me). When administered alone, phenthoate was metabolized principally by carboethoxy ester hydrolysis and cleavage of the PO and CS bonds, resulting in at least six metabolites. The primary urinary metabolite excreted was phenthoate acid. Coadministration of 0.5% OSS-Me did not alter the types of metabolites excreted. However, a reduction of the carboxylesterase-catalyzed product (phenthoate acid) was observed, indicating that the enzyme responsible for the major pathway of phenthoate detoxication was inhibited. Alternate detoxication processes did not compensate for the reduction in carboxylesterase-catalyzed detoxication. It was concluded that inhibition of the carboxylesterase enzymes is the major cause of the potentiation of phenthoate toxicity by OSS-Me.     

In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O-alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI₅₀ of O-(2,6-dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.     

Inhibition of juvenile hormone biosynthesis in the tobacco hornworm, Manduca sexta, by a bisthiolcarbamate juvenoid and related compounds

Biosynthesis of juvenile hormone in the tobacco hornworm, Manduca sexta, is inhibited by the bisthiolcarbamate juvenoid N-ethyl-1,2-bis(isobutylthiolcarbamoyl)ethane both in vitro and in vivo. In vitro an extremely steep dose-response curve was obtained with an ID₅₀ value of 6 × 10^?6M. However, in vivo topical treatment with the compound resulted in mild JH antagonistic symptoms, suggesting rapid metabolism of the compound. In agreement with results from metabolic studies performed on plants and in mammals, sulfoxidation of the thiocarbamate S-(4-chlorobenzyl)N,N-diethylthiocarbamate resulted in an enhanced inhibitory effect on JH biosynthesis in vitro. This suggests that the corresponding thiocarbamate sulfoxides may act as intermediates in carbomylating critical thiol sites important in the terpenoid biosynthesis pathway. Furthermore, this study shows that these prototype compounds are interesting tools for further investigation of chemical inhibition of JH biosynthesis in insects.     

Indoxacarb resistance in the house fly, Musca domestica

Indoxacarb (DPX-MP062) is a recently introduced oxadiazine insecticide with activity against a wide range of pests, including house flies. It is metabolically decarbomethoxylated to DCJW. Selection of field collected house flies with indoxacarb produced a New York indoxacarb-resistant (NYINDR) strain with >118-fold resistance after three generations. Resistance in NYINDR could be partially overcome with the P450 inhibitor piperonyl butoxide (PBO), but the synergists diethyl maleate and S,S,S-tributyl phosphorothioate did not alter expression of the resistance, suggesting P450 monooxygenases, but not esterases or glutathione S-transferases are involved in the indoxacarb resistance. Conversely, the NYINDR strain showed only 3.2-fold resistance to DCJW, and this resistance could be suppressed with PBO. Only limited levels of cross-resistance were detected to pyrethroid, organophosphate, carbamate or chlorinated hydrocarbon insecticides in NYINDR. Indoxacarb resistance in the NYINDR strain was inherited primarily as a completely recessive trait. Analysis of the phenotypes vs. mortality data revealed that the major factor for indoxacarb resistance is located on autosome 4 with a minor factor on autosome 3. It appears these genes have not previously been associated with insecticide resistance.     

Mechanisms of resistance to the juvenoid methoprene in the house fly Musca domestica L

The mechanisms of resistance and cross resistance to the juvenoids methoprene and R-20458 in the house fly, Musca domestica, were examined. Radiolabeled methoprene was found to be metabolized faster in resistant and cross-resistant house fly larvae than in susceptible larvae, and methoprene and R-20458 penetrated more slowly into larvae of the resistant strain. In vivo and in vitro metabolism of methoprene was largely by oxidative pathways followed by conjugation in all strains examined, and little or no ester change of methoprene was noted in vitro. In vitro oxidative metabolism of methoprene, R-20458, juvenile hormone I, and several model substrates was higher in resistant and cross-resistant larvae than in susceptible larvae. Juvenoid functionalities susceptible to metabolic attack by resistant strains are indicated.     

Metabolism of juvenile hormone I by microsomal oxidase,esterase, and epoxide hydrase of Musca domestica and some comparisons with Phormia regina and Sarcophaga bullata

House fly (Musca domestica L.) microsomes prepared from larvae, pupae, or adults contain three enzyme system which can metabolize juvenile hormone I: an esterase, an oxidase, and epoxide hydrase. The presence of the oxidase is indicated by the increased metabolism when microsomes are supplemented with NADPH and by the occurrence of additional metabolites tentatively identified as products arising from oxidation of the 6, 7 double bond. Additional evidence of the activity of the oxidase system is the increased metabolism of juvenile hormone I by the NADPH-dependent system from phenobarbital-induced insects, by inhibition of the oxidation by piperonyl butoxide and carbon monoxide, and by the greater metabolism of the hormone by microsomes from insecticide-resistant (high oxidase) strains. In vivo studies of house fly adults treated with ³H-labeled juvenile hormone I reveal a pattern of metabolism similar to that seen during NADPH-supplemented in vitro metabolism. The three enzymes have somewhat different patterns of activity during the larval stage of the house fly, juvenile hormone esterase and epoxide hydrase beginning at a high level of activity in the young larvae while the juvenile hormone oxidase is low at this stage. In the late larval stage all three enzymes show increased activity followed by declines during the pupal stage and further increases in the adult stage. Comparison of in vitro enzyme levels of the house fly, flesh fly (Sarcophaga bullata Parker), and blow fly [Phormia regina (Meigen)] showed that, although the enzymes were present in the latter two species, their activity on a per insect basis was considerably less than that of the house fly.     

The inhibition of insect juvenile hormone esterase by trifluoromethylketones: Steric parameters at the active site

A rationally designed structure-activity relationship study has been accomplished using trifluoromethylketone inhibitors of insect juvenile hormone esterase from the cabbage looper, Trichoplusia ni (Hubner) (Lepidoptera: Noctuidae). Several α- and α′-substituted derivatives of 3-octylthio-1,1,1-trifluoropropan-2-one have been prepared and assayed for inhibitory potency against juvenile hormone esterase. The results indicate that the sulfur/protein interaction does not occur in a sterically constrained environment. Substitution adjacent to sulfur did not dramatically effect activity. However, substitution adjacent to the carbonyl of the trifluoromethylketone moiety reduced inhibitory potency substantially, indicating that the active site region of juvenile hormone esterase which interacts with the carbonyl is restricted to rather small substrates. A small hydrophobic pocket near the active site has been identified and can serve to increase inhibitory potency by secondary binding of appropriate substituents. The present study has resulted in the preparation of two more effective in vitro inhibitors of juvenile hormone esterase than those previously reported. Evidence that there are two naturally occurring forms of juvenile hormone esterase has also been provided.     

Metabolism of fenitrothion by organophosphorus-resistant and -susceptible house flies, Musca domestica L

The metabolism of fenitrothion was investigated in highly resistant (Akita-f) and susceptible (SRS) strains of the house fly, Musca domestica L. The Akita-f strain was 3500 times more resistant to fenitrothion than the SRS strain. Fenitrothion, topically applied to the flies, was metabolized in vivo far faster in the Akita-f strain than in the SRS strain. In vitro studies revealed that fenitrothion was metabolized by a cytochrome P-450-dependent monooxygenase system and glutathione S-transferases. The former oxidase system metabolized fenitrothion in vitro into fenitrooxon and 3-methyl-4-nitrophenol as major metabolites, and into 3-hydroxymethyl-fenitrothion and 3-hydroxymethyl-fenitrooxon as minor metabolites. Glutathione S-transferases metabolized fenitrothion into desmethylfenitrothion. The cytochrome P-450-dependent monooxygenase system and glutathione S-transferases of the resistant Akita-f strain had 1.4 to 2.2 times and 9.7 times, respectively, as great activities as those of the susceptible SRS strain. These results suggest the importance of glutathione S-transferases in fenitrothion resistance in the Akita-f strain.     

Microsomal metabolism of juvenile hormone analogs in the house fly,Musca domestica L.

Of six juvenile hormone analogs of the alkyl 3,7,11-trimethyl-2,4-dodecadienate type, only the isopropyl ester was strongly morphogenic in the house fly, Musca domestica L. In vitro assays revealed that house fly microsomes contain B-esterases as well as oxidases which metabolize such analogs. However, these esterases did not hydrolyze the isopropyl ester, ZR-515. Enzymes prepared from larvae, pupae, and adults were all active and there was evidence that in the late larval stage the esterase activity was cyclic, showing a minimum in the early third instar and a maximum a few hours later. When microsomes from two susceptible and two resistant house fly strains were compared for metabolic activity against the juvenile hormone analogs, those from the resistant strains were 1.3 to 20 × higher in oxidase activity but there was no difference in esterase activity. The oxidative metabolism of two analogs ZR-515 and 512 was greatly enhanced when the flies were induced with phenobarbital but there was no enhancement in metabolism of three of the remaining analogs and only a slight enhancement of a fourth. It is concluded that the insecticidal action of ZR-515 is largely due to its stability in the presence of the house fly esterases.     

Developmental changes in trans-permethrin and α-naphthyl acetate ester hydrolysis during the last larval instar of Pseudoplusia includens

The hydrolysis of trans-permethrin and α-naphthyl acatate by midgut, fat body, and cuticle homogenates from Pseudoplusia includens (Walker) was monitored during the development of the last instar. The midgut homogenates appeared to have two pH optima (7.6 and 8.6) for the hydrolysis of trans-permethrin, the fat body homogenates had one optimum (7.4–7.8), and the cuticle homogenates had a major optimum at 6.6. Hydrolysis of both substrates peaked during the late feeding stages for midgut and cuticle homogenates, although relative changes were not the same. Hydrolysis of trans-permethrin peaked during the late feeding stage in fat body homogenates, while hydrolysis of α-naphthyl acetate continually increased through the prepupal stage. Thus, the hydrolysis of α-naphthyl acetate is not necessarily associated with the hydrolysis of trans-permethrin. The LD₅₀ values for trans-permethrin on the different stages appeared to reflect the influence of hydrolysis.     

Identification of mutations in the para sodium channel of Bemisia tabaci from Crete, associated with resistance to pyrethroids

We investigated the mechanisms of resistance to α-cypermethrin in a Q biotype, highly resistant Bemisia tabaci strain (GRMAL-RP) isolated from Crete. Cytochrome P450-dependent monoxygenase activity with the substrate ethoxycoumarin, and carboxylesterase activity with the substrates α-naphthyl-acetate, β-naphthyl-acetate, and para-nitrophenol acetate were substantially elevated in the GRMAL-RP, compared to the susceptible SUD-S strain, while glutathione-S-transferase activity with the substrate 1-chloro-2,4-dinitrobenzene was not different. The metabolic inhibitors piperonyl butoxide and S,S,S-tributyl phosphorotrithioate synergised cypermethrin toxicity in the GRMAL-RP strain, however, mortality was still lower than that of the susceptible strain, indicating the presence of an additional resistance mechanism. Analysis of the sequence of the IIS4-IIS6 region of the para sodium channel gene of the GRMAL-RP strain revealed two amino acid replacements compared to that of the SUD-S susceptible strain. One is the leucine to isoleucine substitution at position 925 (L925I) previously implicated in B. tabaci pyrethroid resistance and the other is a novel kdr resistant mutation for B. tabaci, a threonine to valine substitution at position 929 (T929V). Genotype analysis showed that the L925I and T929V were present in all GRMAL-RP males tested, at an approximately 1:1 frequency, but never in combination in the same haplotype.     

Fluorinated sterols,hormones and pheromones: Enzyme-targeted disruptants in insects

Substitution of fluorine for hydrogen in essential sterols, juvenile hormones, and pheromones can lead to materials that interfere with insect growth, development, or communication, in novel and potentially useful ways. Examples have been drawn from work in these areas to illustrate (a) fluorocitrate generation via dealkylation of 29-fluorophytosterols; (b) fluoroacetate release by β-oxidation of fatty alcohols and acids; (c) inhibition of juvenile hormone esterase by trifluoromethyl ketones; (d) inhibition of antennal esterases that hydrolyse acetate pheromones; and (e) sensory disruption by acyl fluorides that mimic aldehyde pheromones.