Inhibition of [(3)H]ponasterone a binding by ecdysone agonists in the intact Sf-9 cell line

Ecdysone agonists, including dibenzoylhydrazines, significantly inhibited the binding of [(3)H]ponasterone A ([(3)H]PoA) in intact Sf-9 cells (Spodoptera frugiperda). The amount of [(3)H]PoA binding varied in a concentration-dependent manner. According to the IC(50), concentration at which there is 50% inhibition, the order of potency of typical ecdysone agonists is tebufenozide (RH-5992) > methoxyfenozide (RH-2485) > PoA > 20-hydroxyecdysone > cyasterone > RH-5849, makisterone A > or = inokosterone > ecdysone. The ranking is consistent with that obtained from a cultured integument system of the rice stem borer Chilo suppressalis except for methoxyfenozide. Other compounds whose modes of action are different from that of ecdysteroids, for example respiration inhibitors, plant steroid hormones, and chitin synthesis inhibitors, did not inhibit the binding of [(3)H]PoA significantly. The mammalian hormones estradiol and diethylstilbestrol, and a secondary bile acid, lithocholic acid, significantly inhibited the binding of [(3)H]PoA at 25 microM. However, their binding activity in terms of pIC(50) was either very low or not evaluated.     

Significance of absorption, oxidation, and binding to toxicity of four ecdysone agonists in multi-resistant cotton leafworm

Treatment of last-instar larvae of multi-resistant cotton leafworm Spodoptera littoralis with four dibenzoylhydrazines, methoxyfenozide (RH-2485), tebufenozide (RH-5992), halofenozide (RH-0345), and RH-5849, resulted in premature molting leading to death. Methoxyfenozide was the most toxic followed by tebufenozide, halofenozide, and RH-5849. To explain differences in toxicity, especially between multi-resistant and laboratory strains, absorption in the body tissues and oxidative metabolism were tested with 14C-labeled ecdysone agonist and a Lineweaver-Burk assay, respectively. Then to address different compound potencies in multi-resistant strains, the potency of the four ecdysone agonists was measured based on their ability to mimic the natural insect molting hormone, 20-hydroxyecdysone (20E) by inducing evagination in isolated imaginal wing discs. Using monoclonal antibody 9B9, the presence of ecdysteroid receptors in imaginal discs in vitro was confirmed. In parallel, Scatchard plot analysis with whole imaginal wing discs cultured with different concentrations of 3H-labeled ponasterone A indicated no significant difference in affinity and in number of target sites for binding between multi-resistant and susceptible laboratory strains. The four compounds tested caused the effect as agonists of 20E in vitro, and typically the order of their toxicities (LC50s) corresponded with that for evagination-induction with whole imaginal discs.     

Relationships between structure and molting hormonal activity of tebufenozide, methoxyfenozide, and their analogs in cultured integument system of Chilo suppressalis Walker

The molting hormonal activity of methoxyfenozide (RH-2485), tebufenozide (RH-5992), five analogs with various alkyl groups, and 18 acyl analogs was measured by using cultured integument of rice stem borers, Chilo suppressalis Walker. The hormonal activity of methoxyfenozide was remarkably high (EC(50) = 1.1 x 10(-9) M), being equivalent to that of tebufenozide (RH-5992). The hormonal activity of several tebufenozide analogs with varying alkyl groups such as CH(3), n-C(3)H(7), i-C(3)H(7), n-C(4)H(9) and n-C(5)H(11) at the para-position of the benzene ring furthest from the tert-butyl group was lower than that of tebufenozide (alkyl group is C(2)H(5)). The activity decreased to varying degrees as a result of replacement of the 3,5-dimethylphenyl moiety of tebufenozide with either a phenyl, naphthyl, or cyclohexyl group. Both 1- and 2-naphthyl derivatives were very active (EC(50) = 4.3 x 10(-8) M and 3.2 x 10(-8) M, respectively) without any significant difference between them. The activity of the 1-cyclohexenyl analog (EC(50) = 1.0 x 10(-7) M) was about 40x that of the corresponding 3-cyclohexenyl analog (EC(50) = 4.4 x 10(-6) M), but 1/100 that of tebufenozide. The activity varied parabolically with respect to the molecular hydrophobicity, and decreased with longer acyl moieties.     

Comparative toxicity of three ecdysone agonist insecticides against the Mediterranean flour moth

The Mediterranean flour moth, Ephestia Kuehniella Zeller (Lepidoptera: Pyralidae), is an important pest in stored products worldwide, and is one of the major pests in flour mills in Algeria. Because environmental consideration, alternative approaches to neurotoxic insecticides, as well as safe, effective, and sound integrated pest management strategies are developed pest control agents such as the insect growth regulator (IGRs). Among these IGRs, the bisacylhydrazine derivatives are nonsteroidal ecdysterold agonists that mimic the action of moulting hormones and induce a precocious and incomplete moult in several insect orders. In topical bioassays using the pupae of E. kuehniella, three ecdysteroid agonists: RH-5849, the first bisaclhydrazine ecdysone agonist and two analogs, RH-5992 (tebufenozide) and RH-0345 (halofenozide), were evaluated on the reproduction under laboratory conditions. In a first series of experiments, the efficacy of these compounds was tested. These compounds exhibited insecticidal activity and the duration of pupal development was reduced with a dose-response relationship. Among the three tested compounds, tebufenozide (LD50 = 0.005 microg) appeared the most potent ecdysteroid agonist against E. kuehniella (RH-5849: LD50 = 0.05 microg and RH-0345: LD50 = 5.10 microg). In a second series of experiments, the effects of the ecdysone agonists (LD50) were investigated on the reproduction. Data showed that the three compounds affected growth of ovaries as evidenced by morphometric measurements of the ovaries from newly emerged adult females. In addition, the thickness of the chorion from basal oocytes was reduced only by RH-5992 and RH-0345. However, electron microscopic observations revealed that the three compounds had no significant effect on the fine structure of chorion. Finally, measurements of ovarian ecdysteroids' production by an enzyme immunoassay showed an increase in the hormonal amounts recorded in treated series compared to control series.     

LC/MS/MS identification of 20-hydroxyecdysone in a scorpion (Liocheles australasiae) and its binding affinity to in vitro-translated molting hormone receptors

Recent advances in mass spectrometry (MS) technology have facilitated the detection and quantification of minor components in organisms and the environment. In this study, we successfully identified 20-hydroxyecdysone (20E) in first instar nymphs (7 days after hatching) of the scorpion Liocheles australasiae, using tandem mass spectrometry combined with high-performance liquid chromatography (LC/MS/MS). This substance was not found in adults after the fifth stage. Other possible molting hormone candidates such as makisterone A (MaA) and ponasterone A (PoA), both of which are reported to be the molting hormones of a few arthropod species, were not detected in this scorpion. The ligand-receptor binding of 20E and its analogs was quantitatively evaluated against the in vitro-translated molting hormone receptor, the heterodimer of ecdysone receptor (EcR) and the retinoid X receptor (RXR) of L. australasiae (LaEcR/LaRXR). The concentrations of ecdysone (E), MaA, 20E, and PoA that are required to inhibit 50% of [(3)H]PoA binding to the LaEcR/LaRXR complex were determined to be 1.9, 0.69, 0.05, and 0.017 μM, respectively. The activity profiles of these 4 ecdysteroids are consistent with those obtained for the molting hormone receptors of several insects. The binding of a non-steroidal E agonist, tebufenozide, to EcR was not observed even at high concentrations, indicating that the structure of the ligand-binding pocket of LaEcR is not favorable for interaction with tebufenozide.     

Establishment and characterization of an Ostrinia nubilalis cell line, and its response to ecdysone agonists

Summary A cell line derived from embryonic tissues of the European corn borer, Ostrinia nubilalis (UMC-OnE), was established in EX-CELL 401 medium containing 10% fetal bovine serum. The cells grew in suspension, and were mainly spherical in shape. The cell doubling times at the 17th and 79th passages were 56 and 36 h, respectively. DNA amplification fingerprinting showed that the DNA profile of the OnE cell line was different from that of the southwestern corn borer, Diatraea grandiosella (UMC-DgE), and that of the cotton bollworm, Helicoverpa zea (BCIRL-HZ-AM1). The OnE cell line was responsive to treatments of 20-hydroxyecdysone and the ecdysone agonists, methoxyfenozide (RH-2485) and tebufenozide (RH-5992). These compounds caused similar effects on the cells, which included cell clumping and decreased cell proliferation. The clumps were observed on the third day of incubation, and became larger after 7 d of incubation. After 168 h of incubation, methoxyfenozide and tebufenozide were 35 and 11 times more effective, respectively, in inhibiting proliferation of the OnE cells than was 20-hydroxyecdysone.     

Molecular cloning and expression analysis of ultraspiracle (USP) from the rice stem borer Chilo suppressalis

cDNA for ultraspiracle (USP) from the lepidopteran rice stem borer Chilo suppressalis was cloned using PCR techniques. The deduced amino acid sequence of C. suppressalis USP (CsUSP) was very similar to those of other lepidopteran USPs, especially to the Manduca sexta USP-2 isoform. Northern hybridization analysis detected a 6.5-kb message in the epidermis, fat body, and midgut of wandering larvae. CsUSP mRNA expression in the epidermis varied little during the last larval instar. Gel mobility shift assays showed that in vitro translated C. suppressalis ecdysone receptor (CsEcR) and CsUSP proteins bound to the Pal1 or Drosophila melanogaster hsp27 ecdysone response element as a heterodimer. In a ligand-receptor binding assay, [(3)H]ponasterone A ([(3)H]PoA) did not bind to individual CsEcR or CsUSP protein, but bound strongly to the CsEcR/CsUSP complex. [(3)H]PoA binding to CsEcR/CsUSP complex was competed by 20-hydroxyecdysone and a non-steroidal ecdysteroid agonist, RH-5992, but not by cholesterol, indicating that compounds with molting hormone activity against C. suppressalis can bind specifically to the CsEcR/CsUSP complex.     

Effects of larval exposure to sublethal concentrations of the ecdysteroid agonists RH-5849 and tebufenozide (RH-5992) on male reproductive physiology in Spodoptera litura

Sublethal concentrations of the bisacylhydrazine moulting hormone agonists, RH-5849, and tebufenozide (RH-5992) were fed to sixth (final) instar larvae of Spodoptera litura. Both RH-5849 and tebufenozide adversely affected the mating success of S. litura when the surviving treated males were crossed with normal females. The ecdysone agonists decreased the longevity of treated males and of untreated females when crossed with treated males. The number of eggs laid by untreated females mated to treated males was decreased, and the fertility (percentage of hatching success) of the resulting eggs was reduced. These effects on male reproductive success were at least in part explained by a reduction in the number of sperm transferred during mating. The adverse effects of tebufenozide on male reproductive function were qualitatively the same as those of RH-5849, but tebufenozide was active at lower concentrations. To understand the reason for these adverse effects on male reproduction, we investigated the effects of the insecticides on male reproductive physiology. Male reproductive tract development and testicular volume of resulting adult moths were adversely affected by sublethal larval exposure to the ecdysone agonists. Dose-dependent reductions occurred in the production of eupyrene and apyrene spermatozoa in the adult testes, and in the number of spermatozoa released from the testes into the male reproductive tract. The descent into the male tract of both eupyrene and apyrene sperm was found to start at the normal stage of development in both normal and treated insects, but the daily rhythm of sperm descent was subsequently disturbed in the insecticide-treated moths. This affected the numbers of sperm in the upper vas deferens (UVD), seminal vesicle (SV), and duplex (duplex). Injections of RH-5849 given to pharate adult or newly emerged adult S. litura also caused drastic reduction in the number of sperm in the upper regions of the male tract, when measured 24 h after injection. The possible importance of pest population reduction through the sublethal anti-reproductive effects of insecticides is discussed.     

The ABC transporter Pdr5p mediates the efflux of nonsteroidal ecdysone agonists in Saccharomyces cerevisiae.

We have previously shown that the synthetic nonsteroidal ecdysone agonist tebufenozide (RH-5992) is actively excluded by resistant cells of insects. To identify the transporter that could be involved in the efflux of RH-5992, the role of three ATP binding cassette transporters, Pdr5p, Snq2p and Ycf1p, has been studied using transporter-deletion mutants of yeast Saccharomyces cerevisiae. PDR5 (pleiotropic drug resistance 5) deletion mutants (Deltapdr5 and Deltapdr5Deltasnq2) retained significantly higher levels of 14C-radiolabeled RH-5992 within the cells when compared to wild-type strain or single deletion mutants of SNQ2 (Deltasnq2) and YCF1 (Deltaycf1). Introduction of an expression vector containing the PDR5 gene into the PDR5 single deletion mutant reversed the effect, resulting in the active exclusion of [14C]RH-5992 from these cells as efficiently as the wild-type cells. These results demonstrated that the ABC transporter Pdr5p but not Snq2p or Ycf1p was responsible for the active exclusion of [14C]RH-5992 in yeast. This exclusion was temperature-dependent and was blocked by the ATPase inhibitors oligomycin and vanadate, indicating that the efflux was an active process. The mutants with the PDR5 deletion can also selectively accumulate [14C]RH-0345 and [14C]RH-2485, but not [14C]RH-5849, indicating that these three compounds share the same transporter Pdr5p for efflux.     

Biological activity of two juvenoids and two ecdysteroids against three stored product insects

The insecticidal activity of juvenile hormone agonists methoprene and pyriproxyfen, and the ecdysone agonists RH-5849 and tebufenozide was evaluated against susceptible and actellic-resistant strains of Tribolium castaneum and susceptible strains of Rhyzopertha dominica and Sitophilus oryzae. Concentrations ranging from 0.1 to 20 ppm of the analogues were mixed in the food medium to which the tested insects were exposed. The results showed that all these compounds could affect the development of the tested species to differing extents but had no effect on the mortality of parental adults. The two JH analogues did not prolong the life span of R. dominica and S. oryzae, but very greatly extended that of T. castaneum. The extension led to the production of giant larvae and failure to pupate. Actellic-resistant strain of T. castaneum showed some cross-resistance to methoprene and pyriproxyfen, but not to RH-5849 and tebufenozide. Pyriproxyfen was the most effective compound among the four IGRs; a concentration of 0.1 ppm could completely inhibit the F(1) adult occurrence of both S- and R-strains of T. castaneum and its LC(90)s for controlling R. dominica and S. oryzae were 0.1 and 1.2 ppm, respectively. Methoprene was highly effective against R. dominica, but less active on S. oryzae. RH-5849 could achieve almost complete control of F(1) adults of T. castaneum and R. dominica at 10 ppm, but was less potent on S. oryzae. Tebufenozide appeared to be much less active on these three species compared with the other three compounds. The percentage reductions of F(1) adults for S- and R-strains of T. castaneum at a concentration of 20 ppm were 80 and 99%, respectively.     

Fecundity and fertility reductions in adult leafrollers exposed to surfaces treated with the ecdysteroid agonists tebufenozide and methoxyfenozide

The effects on the fecundity and fertility of redbanded leafroller, Argyrotaenia velutinana (Walker), and obliquebanded leafroller,Choristoneura rosaceana (Harris), exposed as adults to surfaces treated with the ecdysone agonists tebufenozide (RH-5992) and methoxyfenozide (RH-2485) were examined. The first part of the study consisted of recently emerged moths being exposed to treated surfaces continuously throughout their lives (including mating and oviposition). Continuous exposure to tebufenozide- or methoxyfenozide-treated surfaces significantly reduced the mean number of eggs laid and the percent of eggs that hatched in both species. The second part of the study involved exposure of recently emerged virgin moths (by sex) to treated surfaces for 24 h, after which, the exposed moths were paired with a nontreated partner to mate and oviposit on nontreated surfaces. In this experiment, for A. velutinana, significant reductions in fecundity occurred only when the female was exposed to methoxyfenozide-treated surfaces. Significant reductions in A. velutinana egg fertility occurred with both male and female exposure in the methoxyfenozide treatments and only female exposure in the tebufenozide treatments. For C. rosaceana, significant reductions in fecundity occurred with both male and female exposure in the tebufenozide and methoxyfenozide treatments. Significant reductions in C. rosaceana egg fertility occurred with both male and female exposure in the tebufenozide treatments and only with female exposure in the methoxyfenozide treatments.     

Toxicity of two ecdysone agonists, halofenozide and methoxyfenozide, against the multicoloured Asian lady beetle Harmonia axyridis (Col., Coccinellidae)

Abstract: The insecticidal activity of two ecdysone agonists, methoxyfenozide (RH-2485) and halofenozide (RH-0345), was tested against last-instar larvae of the natural predator Harmonia axyridis (Col., Coccinellidae). In addition, the relative weight gain of the larvae after application was followed. Both products proved to be equally toxic at concentrations of 25, 50 and 100 mg/l. The ecdysteroidal activity of the compounds caused premature induction of larval moulting, cessation of feeding and incomplete pupation in affected larvae. Although, compared with previous results with methoxyfenozide and halofenozide in target pests such as the Colorado potato beetle, Leptinotarsa decemlineata , these compounds caused mortality only after application at relatively high concentrations.     

Cyclic dibenzoylhydrazines reproducing the conformation of ecdysone agonists, RH-5849

We have investigated the biologically active conformation of the non-steroidal ecdysone agonist, 1-tert-butyl-1,2-dibenzoylhydrazine (RH-5849) by means of design, synthesis and conformational analysis of cyclic derivatives of RH-5849. Among the synthesized compounds, a 6-membered cyclic hydrazine bearing two benzoyl groups (5) exists in three conformational states in solution, and the major unsymmetrical conformer of 5 is similar to that of RH-5849 on the basis of 1H NMR and X-ray analyses. The 3,3-dimethyl derivative of 5 (10) exists as a single unsymmetrical conformer. Although there is conformational similarity of the cyclic derivatives with RH-5849, these compounds did not show any hormonal or insecticidal activity. The hydrogen bonding character of the amide N-H group of the dibenzoylhydrazine seems to play a critical role in the appearance of the biological activity.     

Synthesis of ponasterone A derivatives with various steroid skeleton moieties and evaluation of their binding to the ecdysone receptor of Kc cells

A series of ponasterone A (PNA) derivatives with various steroid moieties were synthesized to measure their binding activity to the ecdysone receptors of Drosophila Kc cells. The activity of compounds was evaluated by determining the concentration required to give the 50% inhibition (IC(50) in M) of the incorporation of [(3)H]PNA to Drosophila Kc cells. Compounds with no functional groups such as OH and CO group in the steroid skeleton moiety were inactive. By the introduction of functional groups such as the OH and the CO group in the steroidal structure, these compounds became active. Some compounds containing the A/B-trans ring fusion, which is different from that (A/B-cis) of ecdysteroids were also active. The oxidation of CH(2) at 6-position to CO, enhanced the activity 19 times, but the activity was erased by the reduction of oxo to OH group at 6-position. The activity was enhanced about 250 times by the conversion of A/B ring configuration from trans [(20R,22R)-2beta,3beta,20,22-tetrahydroxy-5alpha-cholestan-6-one: pIC(50)=4.84] to cis [(20R,22R)-2beta,3beta,20,22-tetrahydroxy-5beta-cholestan-6-one: pIC(50)=7.23]. The latter cis-type compound which is the most potent among compounds synthesized in this study was equipotent to the natural molting hormone, 20-hydroxyecdysone, even though it is 1/50 of PNA.     

Comparative toxicity and ecdysone receptor affinity of non-steroidal ecdysone agonists and 20-hydroxyecdysone in Chironomus tentans.

Ecdysone agonists belonging to the bisacylhydrazine class of compounds are a new generation of insecticidal compounds that cause premature lethal molts in susceptible intoxicated insects. While two of the bisacylhydrazines (coded as RH-5992 and RH-2485) are predominantly toxic to lepidopteran pests, RH-5849, which has not been commercialized, has a broader spectrum of toxicity. We have carried out toxicity bioassays with last (4th) instar Chironomus tentans L. larvae, radioligand binding assays using bacterial fusion proteins of C. tentans ecdysone receptor and ultraspiracle (CtEcR, CtUSP), and C. tentans imaginal disc development assays to compare the relative potencies of the three bisacylhydrazine compounds as well as of 20-hydroxyecdysone (20E). In all three assays, the potency of the three bisacylhydrazines was in the order RH-2485>RH-5992>RH-5849. While in toxicity assays 20E was ineffective, most likely due to rapid metabolism, it was more potent than RH-5849 but less so than RH-5992 and RH-2485 in imaginal disc assays. In summary, we compared the potencies of the ecdysone agonists for C. tentans at three levels: whole organism, imaginal discs and the receptor level, and our results indicate that the increased toxicity of the non-steroidal ecdysone agonists for C. tentans has a high correlation to the affinity of these compounds for CtEcR/CtUSP bacterially expressed proteins. Our results, though, do not exclude reasons of metabolic stability of the compounds in C. tentans, which we have not investigated in this report.     

Toxicity and residual activity of methoxyfenozide and tebufenozide to codling moth (Lepidoptera: Tortricidae) and oriental fruit moth (Lepidoptera: Tortricidae)

A series of studies were conducted to examine the residual activity and toxicity of the ecdysone agonists tebufenozide and methoxyfenozide to codling moth, Cydia pomonella (L.), and oriental fruit moth, Grapholita molesta (Busck), in North Carolina apple systems. Methoxyfenozide exhibited greater activity than tebufenozide against codling moth eggs in dose-response bioassays, with a 4.5- and 5.3-fold lower LC50 value to eggs laid on fruit treated before or after oviposition, respectively. Oriental fruit moth eggs were 57- and 12-fold less sensitive to methoxyfenozide than were codling moth eggs on fruit treated before and after oviposition, respectively. Methoxyfenozide was effective in reducing larval entries of both codling moth and oriental fruit moth in field residual activity bioassays, exhibiting activity for at least 28 d after application. Residue breakdown on fruit was approximately 80% at 28 d after treatment for both methoxyfenozide and tebufenozide, with the most rapid residue decline (60%) occurring during the first 14 d after application. Two applications of methoxyfenozide applied at 14-d intervals provided better canopy coverage and higher residue levels than one application. Spray volume (683 versus 2,057 liters/ha) did not affect the efficacy of methoxyfenozide. Leaf and fruit expansion during the season was measured to determine potential plant-growth dilution effects on residual activity. There was very little increase in leaf area after mid May, but increase in fruit surface area over the season was described by a second order polynomial regression. Implications for codling moth and oriental fruit moth management programs are discussed.     

A photoaffinity, non-steroidal, ecdysone agonist, bisacylhydrazine compound, RH-131039: characterization of binding and functional activity

In this paper we describe the synthesis, ligand-binding and functional activity characteristics of the photoaffinity, non-steroidal, ecdysone agonist, bisacylhydrazine compound, 3-benzoyl-benzoic acid N-tert-butyl-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide (RH-131039). Tritiated RH-131039 is the first non-steroidal photoaffinity compound that was shown to bind specifically to ecdysone receptors (EcRs) from insects belonging to the orders Diptera and Lepidoptera. The spruce budworm (Choristoneura fumiferana) ecdysone receptor (CfEcR) bound with high affinity (K(d)=2.23+/-0.27 nM) to this compound. When irradiated with UV light (lambda=350 nm) under equilibrium ligand-binding conditions, RH-131039 attached specifically and covalently to the CfEcR ligand-binding domain (LBD). RH-131039 also bound to cloned ecdysone receptor proteins from three dipteran insects, Drosophila melanogaster, Aedes aegypti and Chironomous tentans. This paper also describes and invokes caution in interpretation of ligand-binding results obtained using crude cellular extracts containing target receptors, as illustrated with the use of Drosophila Kc cells that have functional EcR and L57 cells (derivatives of Kc cells in which EcR-B isoforms have been knocked out by "parahomologous" recombination). Tritiated RH-131039 is a useful tool to dissect ligand-binding and functional differences for EcRs from different arthropod species.     

Block of voltage-dependent K+ channels in insect muscle by the diacylhydrazine insecticide RH-5849, 4-aminopyridine,and quinidine

In calcium-free saline, voltage-clamped ventral longitudinal muscles of housefly larvae have maintained (I_K) and transient (I_A) voltage-dependent K⁺ currents. With 500 ms conditioning pulses, inactivation of I_A had a midpoint at ?53 mV and changed e-fold in 3.46 mV. I_A inactivated completely at ?40 mV, with a time constant of 71 ms, allowing the effects of various K⁺ channel blockers to be studied on I_K in isolation. RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), a novel insect growth regulator, induces a lethal premature molt in insect larvae by mimicking the action of the molting hormone at ecdysone receptors. RH-5849 also causes acute neurotoxicity in some insects by selectively blocking of I_K in nerve and muscle. While most channel blockers have a Hill coefficient near 1, consistent with a simple one molecule per channel block mechanism, RH-5849 and the analog RH-1266 were found in the present study to block I_K channels in insect muscle with a Hill coefficient of 1.5. The lC₅₀ (concentration that caused 50% block) for block of I_K was 59 μM for RH-5849 and 40 μM for RH-1266. While tetraethylammonium blocked I_K by only 20% at 100 mM, 4-aminopyridine blocked the current with an lC₅₀ of 1.2 mM and a Hill coefficient of 0.97. Quinidine was the most potent blocker of I_K in this study, with an lC₅₀ of 20 μM. Block of I_K by either RH-5849 or 4-aminopyridine was independent of test pulse potential, but block by quinidine increased with depolarization. Block of I_K by RH-5849 and quinidine was time dependent, suggesting an open channel block mechanism, but the time course was too fast relative to channel activation for kinetic analysis. The lC₅₀ for block of I_K by RH-5849 decreased with temperature, with a Q₁₀ of 0.52. I_A was also blocked by RH-5849, but was less sensitive than I_K. The lC₅₀ for block of I_A by RH-5849 was 775 μM, 13-fold higher than the lC₅₀ for block of I_K. © 1992 Wiley-Liss, Inc.     

Classical and three-dimensional QSAR for the inhibition of [3H]ponasterone A binding by diacylhydrazine-type ecdysone agonists to insect Sf-9 cells

The activity of 52 diacylhydrazine congeners was evaluated by measuring the inhibition of the incorporation of [3H]ponasterone A into intact Sf-9 cells. Eleven compounds were newly synthesized in this study. Results showed that the substitution of the 2-CH3 or 3-OCH3 moiety of methoxyfenozide with other groups or the removal of either group was unfavorable to the activity. The activity was quantitatively analyzed using both classical QSAR (Hansch-Fujita) and three-dimensional QSAR methods (comparative molecular field analysis, CoMFA). Sterically favorable fields were observed at the 3- and 4-positions of the benzene ring opposite from the t-butyl group (B-ring), and a sterically unfavorable field was evidenced at the 2-position. Another sterically unfavorable field developed surrounding the favorable field observed at the 4-position of the B-ring. Electrostatically negative fields were observed near the CO moiety, above the benzene ring, and at the 4-position of the B-ring. The optimum hydrophobicity of compounds in terms of their logP values was calculated to be approximately 4.1. Results of the three dimensional structure-activity relationship analyses were consistent with those obtained from the previously reported classical QSAR for 2-chlorobenzoyl analogs containing various para-substituents. The high activity of potent insecticides such as tebufenozide and chromafenozide were rationalized by CoMFA. Thus, this CoMFA result will be useful in the design of new compounds and in understanding the molecular mechanism of the ligand-receptor interactions.     

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.