Determination by LC-MS of juvenile hormone titers in hemolymph of the silkworm, Bombyx mori.

Juvenile hormone (JH) I, II and III in the hemolymph of the silkworm, Bombyx mori were quantified by liquid chromatography-mass spectrometry (LC-MS). JHs were treated with methanol and trifluoroacetic acid to convert into JH methoxyhydrines (JH-MHs). The key to the analytical condition for JH-MHs was the addition of 5 µM sodium acetate to the eluting solution. Each JH-MH was observed as the sodium adduct ion with good sensitivity. This improved method enabled the titration of JH I, II and III in hemolymph of the silkworm to be monitored from the 3rd instar through to the early pupal stage. A peak of JH I was observed immediately after ecdysis in the 3rd and 4th instar stages. The JH I titer sharply decreased on day 1 and reached the lowest level before ecdysis, but there was no peak at the beginning of the 5th stadium, and no apparent increase was observed until pupation.

An anti-juvenile hormone agent, ethyl 4-(2-benzylhexyloxy)benzoate, inhibits juvenile hormone synthesis through the suppression of the transcription of juvenile hormone biosynthetic enzymes in the corpora allata in Bombyx mori.

Ethyl 4-[(S)-2-benzylhexyloxy)]benzoate (KF-13S), derived from ethyl 4-[2-(tert-butylcarbonyloxy)butoxy]benzoate (ETB), has strong anti-juvenile hormone (JH) activity which causes precocious metamorphosis in Bombyx mori, and the mode of action of this compound was studied. Application of KF-13S inhibited JH biosynthesis by the corpora allata (CA) in a reversible manner, and in vitro culture experiments showed that this inhibition was due to the direct action of this compound on the CA. When mRNA expression of the JH biosynthetic enzymes were studied, KF-13S strongly suppressed those of HMG Co-A synthase and HMG Co-A reductase. mRNA levels of other mevalonate enzymes and JH acid O-methyltransferase were also suppressed but were less sensitive to the compound. These studies showed that KF-13S prevents the transcription of many of the JH biosynthetic enzymes so that JH synthesis is suppressed.

A novel cytochrome P450 gene (CYP4G25) of the silkmoth Antheraea yamamai: cloning and expression pattern in pharate first instar larvae in relation to diapause.

A new cytochrome P450 gene, CYP4G25, was identified as a differentially expressed gene between the diapausing and post-diapausing pharate first instar larvae of the wild silkmoth Antheraea yamamai, using subtractive cDNA hybridization. The cDNA sequence of CYP4G25 has an open reading frame of 1674 nucleotides encoding 557 amino acid residues. Sequence analysis of the putative CYP4G25 protein disclosed the motif FXXGXRXCXG that is essential for heme binding in P450 cytochromes. Hybridization in situ demonstrated predominant expression of CYP4G25 in the integument of pharate first instar larvae. Northern blotting analysis showed an intensive signal after the initiation of diapause and no or weak expression throughout the periods of pre-diapause and post-diapause, including larval development. These results indicate that CYP4G25 is strongly associated with diapause in pharate first instar larvae.

Juvenile hormone activity of ethyl 4-(2-aryloxyhexyloxy)benzoates with precocious metamorphosis-inducing activity.

Ethyl 4-[2-(6-methyl-3-pyridyloxy)hexyloxy]benzoate (1) and ethyl 4-(2-phenoxyhexyloxy)benzoate (2), which induce precocious metamorphosis in larvae of Bombyx mori, a clear sign of juvenile hormone (JH) deficiency, showed JH activity when topically applied to allatectomized 4th instar larvae of B. mori. Compounds 1 and 2 induced precocious metamorphosis with doses at which they were effective as JH agonists.

Effect of tyramine and stress on sex-pheromone production in the pre- and post-mating silkworm moth, Bombyx mori.

Tyramine (TA) increased significantly after mating, whereas there were no significant differences in octopamine (OA) and dopamine (DA) levels in the brain-suboesophageal ganglion (SOG) complexes between virgin and mated females. The effects of various biogenic amines were tested on pheromone production of virgin and mated females of the silkworm moth, Bombyx mori. After 8h a significant reduction by TA (46%) was observed. Meanwhile, when OA or DA was injected, a significant increase of pheromone titer was observed in both virgin and mated females. This study also presents evidence for an increase in levels of OA and DA in the brain-SOG complexes in response to mechanical stress in B. mori female. TA suppressed pheromone production in an in vitro pheromone gland (PG) homogenate preparation, thus suggesting that the target of TA is the PG. TA inhibited pheromone production in vitro in a dose-dependent manner and DA had a lower inhibitory activity than TA, whereas OA had no effect, suggesting that TA is a candidate for regulating pheromone production in the PG, although other factors could be responsible for the pheromonostatic function.

Synthesis and anti-juvenile hormone activity of ethyl 4-(2-aryloxyhexyloxy)benzoates.

A series of ethyl 4-(2-aryloxyhexyloxy)benzoates was prepared and tested for their activity to induce precocious metamorphosis in larvae of the silkworm. Phenyl analog 5 showed activity comparable to that of the 6-methyl-3-pyridyl analog reported as a novel anti-JH agent. The activity of 5 could be fully counteracted by methoprene, a JH agonist. The ethoxycarbonyl group of 5 was essential for its activity.

The pheromone production of female Plodia interpunctella is inhibited by tyraminergic antagonists.

Several compounds were found to suppress the calling behavior and in vitro pheromone biosynthesis of the Indian meal moth, Plodia interpunctella. The compounds were screened by means of a calling-behavior bioassay with female P. interpunctella. Five derivatives with activities in the nanomolar range were identified, in order of decreasing pheromonostatic activity: 4-hydroxybenzaldehyde semicarbazone (42) > 5-(4-methoxyphenyl)-1,3-oxazole (38) > 5-[4-(tert-butyl)phenyl]-1,3-oxazole (40) > 5-(3-methoxyphenyl)-1,3-oxazole (35) > 5-(4-cyanophenyl)-1,3-oxazole (36). These compounds also showed in vitro inhibitory activity in intracellular de novo pheromone biosynthesis, as determined with isolated pheromone-gland preparations that incorporated [1-(14)C]sodium acetate in the presence of the so-called pheromone-biosynthesis-activating neuropeptide (PBAN). The non-additive effect of the inhibitor with antagonist (yohimbine) for the tyramine (TA) receptor suggests that it could be a tyraminergic antagonist. Three-dimensional (3D) computer models were built from a set of compounds. Among the common-featured models generated by the program Catalyst/HipHop, aromatic-ring (AR) and H-bond-acceptor-lipophilic (HBAl) features were considered to be essential for inhibitory activity in the calling behavior and in vitro pheromone biosynthesis. Active compounds, including yohimbine, mapped well onto all the AR and HBAl features of the hypothesis. Less-active compounds were shown to be unable to achieve an energetically favorable conformation, consistent with our 3D common-feature pharmacophore models. The present hypothesis demonstrates that calling behavior and PBAN-stimulated incorporation of radioactivity are inhibited by tyraminergic antagonists.

Comparative receptor surface analysis of octopaminergic antagonists for the locust neuronal octopamine receptor.

In drug discovery, it is common to have measured activity data for a set of compounds acting upon a particular protein but not to have knowledge of the three-dimensional structure of the protein active site. In the absence of such three-dimensional information, one can attempt to build a hypothetical model of the receptor site that can provide insight about receptor site characteristics. Such a model is known as a comparative receptor surface analysis (CoRSA) model, which provides compact and quantitative descriptors which capture three-dimensional information about a putative receptor site. The quantitative structure-activity relationship (QSAR) of a set of 20 antagonists for octopamine (OA) receptor 3 in locust nervous tissue, was analyzed using CoRSA. Three-dimensional energetics descriptors were calculated from receptor surface model (RSM)-ligand interaction and these three-dimensional descriptors were used in QSAR analysis. The predictive character of the QSAR was further assessed using 24 agonists for OA receptor as test molecules. An RSM was generated using some subset of the most active structures and the results provided useful information in the characterization and differentiation of OA receptor.

Ethyl 4-[2-(6-Methyl-3-pyridyloxy)butyloxy]benzoate, a novel anti-juvenile hormone agent.

Ethyl 4-[2-(6-methyl-3-pyridyloxy)butyloxy]benzoate (2) was prepared as a novel anti-juvenile hormone (anti-JH) agent. Compound 2 induced precocious metamorphosis in larvae of the silkworm and black pigmentation of the larval cuticle, which are clearly recognized as JH-deficiency symptoms. The 4-ethoxycarbonyl group on the benzene ring was indispensable for activity. The activity of compound 2 could be fully counteracted by methoprene, a JH agonist, but not by the dietary administration of 20-hydroxyecdysone.

1,5-Disubstituted imidazoles inhibit juvenile hormone biosynthesis by the corpora allata of the mosquito Aedes aegypti.

We investigated the effect of fifteen 1,5-disubstituted imidazoles (1,5-dis) on juvenile hormone III (JH III) and methyl farnesoate (MF) biosynthesis by the corpora allata (CA) of the mosquito Aedes aegypti in vitro. Four compounds (TH-35, TH-83, TH-62 and TH-28) significantly decreased JH biosynthesis in the CA dissected from 3-day old sugar-fed females. The decrease of JH synthesis was not always associated with increased MF. TH-30 and TH-83 increased MF levels, while TH-85 and TH-61 significantly decreased MF levels. Five compounds (TH-26, TH-60, TH-83, TH-35 and TH-30) significantly inhibited JH biosynthesis in the CA dissected from females 15 h after a blood meal. Four 1,5-dis (TH-30, TH-26, TH-28 and TH-66) caused MF increases in CA from blood-fed females. 1,5-Disubstituted imidazoles had higher inhibitory activity on JH synthesis when substituted at position 5 by a 3-benzyloxyphenyl group and at position 1 by a benzyl group (such as TH-35). Inhibition of JH and MF biosynthesis by TH-35 was age-dependent and influenced by nutritional status; inhibition differed when evaluated in the CA dissected from sugar-fed females at different days after emergence and in the CA dissected from females at different hours after a blood meal. Inhibition was always higher when the CA was more active. The addition of TH-35 significantly reduced the stimulatory effect of Aedes-allatotropin and farnesoic acid on JH synthesis. This is the first report of an inhibitory effect of 1,5-disubstituted imidazoles on JH synthesis in Diptera.

Three-dimensional molecular-field analyses of octopaminergic agonists for the cockroach neuronal octopamine receptor.

The quantitative structure-activity relationship of a set of 40 octopaminergic agonists against receptor 2 in cockroach nervous tissue, was analyzed using molecular-field analysis (MFA). MFA on the study set of those compounds evaluated effectively the energy between a probe and a molecular model at a series of points defined by a rectangular grid. Contour surfaces for the molecular fields were presented and the results provided useful information in the characterization and differentiation of octopaminergic receptor.

Three-dimensional pharmacophore hypotheses of octopamine/tyramine agonists which inhibit [1-14C]acetate incorporation in Plodia interpunctella.

Three-dimensional pharmacophore hypotheses were built from a set of 36 octopamine (OA)/tyramine (TA) agonists responsible for the inhibition of sex-pheromone production in Plodia interpunctella. Among the ten chemical-featured models generated by a program Catalyst/Hypo, hypotheses including hydrogen-bond acceptor (HBA), hydrogen-bond acceptor aliphatic (HBAl), hydrophobic (Hp), hydrophobic aromatic (HpAr) and hydrophobic aliphatic (HpAl) features were considered to be important and predictive in evaluating OA/TA agonists. Active agonists mapped well onto all the features of the hypothesis such as HBA, HBAl, Hp, HpAr and HpAl features. On the other hand, inactive compounds were shown to be poorly capable of achieving an energetically favorable conformation shared by the active molecules in order to fit the 3-D chemical-feature pharmacophore models. Those hypotheses are considered to be used in designing new leads for hopefully more active compounds. Further research on the comparison of models from the agonists may help elucidate the mechanisms of OA/TA receptor-ligand interactions.

Inhibitors of calling behavior of Plodia interpunctella.

Some octopamine agonists were found to suppress the calling behavior of the stored product Indian meal moth, Plodia interpunctella. Compounds were screened using a calling behavior bioassay using female P. interpunctella. Four active derivatives, with inhibitory activity at the nanomolar range, were identified in order of decreasing activity: 2-(1-phenylethylamino)-2-oxazoline > 2-(2-ethyl,6-methylanilino)oxazolidine > 2-(2-methyl benzylamino)-2-thiazoline > 2-(2,6-diethylanilino)thiazolidine. Three-dimensional pharmacophore hypotheses were built from a set of 15 compounds. Among the ten common-featured models generated by the program Catalyst/HipHop, a hypothesis including a hydrogen-bond acceptor lipid, a hydrophobic aromatic and two hydrophobic aliphatic features was considered to be essential for inhibitory activity in the calling behavior. Active compounds mapped well onto all the hydrogen-bond acceptor lipid, hydrophobic aromatic and hydrophobic aliphatic features of the hypothesis. On the other hand, less active compounds were shown not to achieve the energetically favorable conformation that is found in the active molecules in order to fit the 3D common-feature pharmacophore models. The present studies demonstrate that inhibition of calling behavior is via an octopamine receptor.

Octopaminergic agonists for the cockroach neuronal octopamine receptor.

The compounds 1-(2,6-diethylphenyl)imidazolidine-2-thione and 2-(2,6-diethylphenyl)imidazolidine showed the almost same activity as octopamine in stimulating adenylate cyclase of cockroach thoracic nervous system among 70 octopamine agonists, suggesting that only these compounds are full octopamine agonists and other compounds are partial octopamine agonists. The quantitative structure-activity relationship of a set of 22 octopamine agonists against receptor 2 in cockroach nervous tissue, was analyzed using receptor surface modeling. Three-dimensional energetics descriptors were calculated from receptor surface model/ligand interaction and these three-dimensional descriptors were used in quantitative structure-activity relationship analysis. A receptor surface model was generated using some subset of the most active structures and the results provided useful information in the characterization and differentiation of octopaminergic receptor.

Three-dimensional common-feature hypotheses of inhibitors of calling behaviour and in vitro [14C]acetate incorporation by pheromone glands of Plodia interpunctella.

Some octopamine (OA) agonists were found to suppress the calling behaviour and pheromone biosynthesis in vitro of the Indian meal moth, Plodia interpunctella (Hübner), a stored-product pest. Compounds were screened using a calling behaviour bioassay of female P interpunctella. Three active derivatives, with activity at the nanomolar level, were identified. In order of decreasing pheromonostatic activity these were: 2-(2-ethyl-6-methylanilino)oxazolidine > 2-(2,6-diethylanilino)thiazolidine > 2-(2,6-diethylanilino)oxazolidine. These compounds showed also in vitro inhibitory activities in de novo pheromone biosynthesis. Three-dimensional pharmacophore hypotheses were built from a set of 19 compounds. Among the ten common-featured models generated by the program Catalyst/HipHop, a hypothesis including a ring aromatic group (RA), a positive ionizable group (PI) and two hydrophobic aliphatic (HpA1) features was considered to be essential for inhibitory activity in the calling behaviour and pheromone biosynthesis in vitro. Active compounds mapped well onto all the RA, PI and HpA1 features of the hypothesis. Less-active compounds were shown not to achieve the energetically favourable conformation which was found in the active molecules in order to fit the 3-D common-feature pharmacophore models. The present studies demonstrate that inhibition of calling behaviour and PBAN-stimulated incorporation of radioactivity is by OA-agonistic activity.

Three-dimensional common-feature hypotheses for octopamine agonist arylethanolamines.

Three-dimensional pharmacophore hypotheses were built from a set of 12 octopamine (OA) agonist arylethanolamines (AEAs). Among the 10 common-featured models generated by program catalyst/HipHop, a hypothesis including a hydrogen-bond donor (HBD) and a hydrogen-bond acceptor lipid (HBA1) features was considered to be important in evaluating the OA activity. OA mapped well onto all the HBD and HBA1 features of the hypothesis. On the other hand, for some inactive compounds, their lack of affinity is primarily due to their inability to achieve an energetically favorable conformation shared by the active compounds. Taken together, structures of a 4-OH-Ph, alpha-OH, and a primary amine are important for OA activities. The present studies on OA agonists demonstrate that an HBD and an HBA1 sites located on the molecule seem to be essential for OA activity.

Cholesterol esterase accelerates intestinal cholesterol absorption.

Mechanisms of acceleration of cholesterol absorption by cholesterol esterase were investigated in various experimental conditions. Lymphatic recovery of cholesterol intubated as a micellar solution containing phosphatidylcholine (PC) into the duodenum was enhanced by the co-administration of cholesterol esterase in rats drained of bile and pancreatic juice. However, no accelerated incorporation was observed when cholesterol was solubilized in PC-depleted micelles. Cholesterol esterase dose-dependently accelerated the incorporation of cholesterol into differentiated Caco-2 cells, only when cholesterol was solubilized in PC-containing micelles. The accelerated incorporation of cholesterol into Caco-2 cells by cholesterol esterase disappeared when the enzyme was preincubated with a suicide inhibitor of cholesterol esterase. Cholesterol esterase has an activity as phospholipase A(2). When 10% of PC in bile salt micelles was replaced by lysophosphatidylcholine (lysoPC), the incorporation of cholesterol into Caco-2 cells was significantly accelerated. Cholesterol esterase enhanced the incorporation of micellar cholesterol into brush border membranes prepared from the rat jejunum. The addition of cholesterol esterase to bile salt micelles accelerated the release of micellar cholesterol in a dose-dependent manner, only when the micelles contained PC. These observations strongly suggest that cholesterol esterase hydrolyzes PC in bile salt micelles and thereby, accelerating the release of cholesterol from bile salt micelles. This may be a major cause of the acceleration of cholesterol absorption by cholesterol esterase.

Three-dimensional common-feature hypotheses for octopamine agonist 2-(arylimino)imidazolidines.

Three-dimensional pharmacophore hypotheses were built from a set of 10 octopamine (OA) agonist 2-(Arylimino)imidazolidines (AIIs), 2-(Arylimino)thiazolidines (AITs) and 2-(Arylimino)oxazolidines (AIOs). Among the 10 common-featured models generated by program Catalyst/HipHop, a hypothesis including a ring aromatic (RA), a positive ionizable (PI) and three hydrophobic aliphatic (HpAl) features was considered to be important in evaluating the OA-agonist activity. Active OA agonist 2,6-Et2 AII mapped well onto all the RA, PI and HpAl features of the hypothesis. On the other hand, less active compounds were shown to be difficult to achieve the energetically favorable conformation which is found in the active molecules in order to fit the 3-D common-feature pharmacophore models. Taken together, 2,6-Et2-Ph and foramidine structures are important as OA agonists. The present studies on OA agonists demonstrate that a RA, a PI and three HpAl sites located on the molecule seem to be essential for OA-agonist activity.

Inhibitory Action of an Imidazole Compound on Ecdysone Synthesis in Prothoracic Glands of the Silkworm, Bombyx mori: (imidazole compound/ecdysteroids/prothoracic gland/precocious pupation/Bombyx mori).

The precocious pupation was induced either by allatectomy at the time of third ecdysis or by topical application of an imidazole compound (KK-42; 1-benzyl-5-[(E)-2, 6-dimethyl-1, 5-heptadienyl] imidazole) to the fourth (penultimate) instar larvae of the silkworm, Bombyx mori. However, the critical period for KK-42 treatment in induction of precocious pupation was longer than that for allatectomy. The effects of KK-42 depended on the doses applied and a half-maximum dose was estimated to be approx. 10 µg/larva. KK-42 suppressed the increase in hemolymph ecdysteroid titres leading to larval ecdysis in controls. Ecdysteroid levels remained at low levels for about 6 days after the treatment, followed by an increase toward precocious pupation. When the prothoracic glands from the mature fifth instar larvac were incubated in vitro in Grace's medium containing various concentrations of KK-42, secretion of ecdysone into the medium was suppressed depending upon the doses of KK-42 added and a half-inhibition concentration was estimated to be approx. 1 nM. Thus, KK-42 was shown to be an inhibitory agent to ecdysteroid secretion in silkworm larvae.