Ecdysteroid Analogues Research Articles

A new binding mode for ecdysteroid analog tebufenozide as a multitargeted IGR with chitinase

Tebufenozide, an efficient insect growth regulator (IGR) against lepidopteran pests, presents a novel mode of action with minimal non-target impact. By competing with ecdysteroids for ecdysone receptor (EcR) binding, it regulates insect growth precisely. This study explores tebufenozide's potential as a multitarget IGR, targeting both EcR and Ostrinia furnacalis chitinase I (OfChtI). The inhibitory activity against OfChtI is comparable to that of substrates (GlcNAc)5, with an IC50 of 45.77 ​μM. Our computational findings indicate that tebufenozide binds at the subsite −1 to +1 of OfChtI through various interactions. Notably, tebufenozide establishes a pi-pi interaction with the flipped sidechain of Trp107, enabling tebufenozide to deeply penetrate into the S1 pocket, thereby obstructing substrate binding to OfChtI. These insights highlight the potency of multitarget strategies, laying the groundwork for innovative IGR designs that offer comprehensive pest management solutions.

Autophagy genes AMBRA1 and ATG8 play key roles in midgut remodeling of the yellow fever mosquito, Aedes aegypti.

The function of two autophagy genes, an activating molecule BECN1 regulated autophagy (AMBRA1) and autophagy-related gene 8 (ATG8) in the midgut remodeling of Aedes aegypti was investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of RNA samples collected from the last instar larvae and pupae showed that these two genes are predominantly expressed during the last 12h and first 24h of the last larval and pupal stages, respectively. Stable ecdysteroid analog induced and juvenile hormone (JH) analog suppressed these genes. RNA interference (RNAi) studies showed that the ecdysone-induced transcription factor E93 is required for the expression of these genes. JH-induced transcription factor krüppel homolog 1 (Kr-h1) suppressed the expression of these genes. RNAi-mediated silencing of AMBRA1 and ATG8 blocked midgut remodeling. Histological studies of midguts from insects at 48h after ecdysis to the final larval stage and 12h after ecdysis to the pupal stage showed that ATG gene knockdown blocked midgut remodeling. AMBRA1 and ATG8 double-stranded (dsRNA)-treated insects retained larval midgut cells and died during the pupal stage. Together, these results demonstrate that ecdysteroid induction of ATG genes initiates autophagy programmed cell death during midgut remodeling. JH inhibits midgut remodeling during metamorphosis by interfering with the expression of ATG genes.

Regulation of juvenile hormone and ecdysteroid analogues on the development of the predatory spider, Pardosa pseudoannulata, and its regulatory mechanisms

Insecticides harm the beneficial organisms, such as predatory spiders, through direct killing or regulation of the development and reproduction. In this study, the bioassay showed that the treatment of juvenile hormone (JH) analogue fenoxycarb delayed the moulting of Pardosa pseudoannulata, a dominant predatory spider in paddy fields. In order to figure out the regulatory mechanism of fenoxycarb on the spider development, we systematically analyzed JH biosynthesis in P. pseudoannulata. All genes involved in JH biosynthesis pathway were retrieved from the genome of P. pseudoannulata, except for CYP15A1. The absence of CYP15A1 was in agreement with the identification of methyl farnesoate (MF) rather than JH III in the spider. The delayed moulting and decreased expression of JH biosynthesis-related genes in the MF-applied spiderlings supported that MF was an active JH. Fenoxycarb treatment significantly upregulated the transcriptional level of JH biosynthesis-related genes and consequently delayed the spiderling moulting. In the spider development, ecdysteroid played the opposite role, in contrast to MF, to accelerate the development, as our previous study. Here we found that the treatment of ecdysteroid analogue tebufenozide accelerated P. pseudoannulata spiderling moulting, which resulted from the expressional suppression of ecdysteroid biosynthesis-related genes. In total, the JH and ecdysteroid analogues affected the development of P. pseudoannulata by the expressional regulation of biosynthesis-related genes, which would be helpful for the evaluation of hormone analogue insecticides in environmental safety, and useful for the protection and application of P. pseudoannulate and related spider species.

Oxo-analogues of 20-hydroxyecdysone in the synthesis of novel fluorinated ecdysteroid derivatives

Novel side chain ω-fluorinated ecdysteroid analogues were obtained starting from ω-functionalized ecdysteroids. Thus, the reaction of diacetonides of 25RS-hydroxy- and 24-hydroxy 27-nor- and 25,26,27-tris-nor-20-hydroxyecdysone derivatives with diethylaminosulfur trifluoride (DAST) involves replacement of the terminal hydroxyl group and dehydration at the 14-position to give 25RS-fluoro- and 24-fluoro-14-anhydro analogues of ecdysteroids.

Characterization of synthetic ecdysteroid analogues as functional mimics of brassinosteroids in plant growth

Brassinosteroids (BRs) are plant steroidal hormones that play important roles in many stages of plant growth. Several plant species produce ecdysteroids, which are known as insect molting steroid hormones. In this study, we evaluated the biological activities of three hydroxysteroidal compounds, 20-hydroxyecdysone (ECD), 7,8-dihydro-8α-20-hydroxyecdysone (DHECD), and 7,8-dihydro-5α,8α-20-hydroxyecdysone (α-DHECD), and compared their activities with that of brassinolide (BL), the most potent BR. In rice, DHECD and α-DHECD enhanced the degree of lamina inclination, as do BRs. In Arabidopsis thaliana, DHECD and α-DHECD increased hypocotyl length in the wild-type, and also partially overcame the hypocotyl shortening in the wild-type caused by 0.3μM brassinazole, a specific BR biosynthesis inhibitor. DHECD and α-DHECD partially reduced dwarfism in the BR-biosynthesis-deficient mutant det2. Treatment with DHECD or α-DHECD downregulated the expression of the BR biosynthesis genes DWF4 and CPD, which are generally, suppressed by BR, and upregulated the expression of TCH4 and SAUR-AC1, which are generally promoted by BR. However, their regulated activities were less effective than BL. Moreover, the 10−4M DHECD and α-DHECD induced the accumulation of dephosphorylated BIL1/BZR1 that enhanced BR signaling as a master transcription factor. In contrast, ECD did not affect rice lamina bending, Arabidopsis hypocotyl elongation, the expression levels of BR-related genes and BIL1/BZR1 phosphorylation status. Based on these results, we hypothesize that both DHECD and α-DHECD have functional activities similar to those of BR.

Semi-synthetic preparation, structural elucidation and pharmacological study of nitrogen-containing ecdysteroid analogs

Ecdysteroids are specific hydroxysteroids that exert a wide a range of biological functions in the flora and the fauna. Our group has recently described that less polar ecdysteroid derivatives can strongly sensitize various cancer cell lines to certain chemotherapeutics, which can be observed both in multi-drug resistant (MDR) and drug susceptible cancer cells [1 – 2]. Recent studies also revealed that oxime groups can increase the antitumor activity of certain steroid derivatives [3], but ecdysteroid oximes have previously not been studied from this point of view. In our current work, we aimed to synthesize C6 ecdysteroid oximes of 20-hydroxyecdysone 2,3;20,22-diacetonide, optimizing the previously available methods [4]. Rotational planar chromatographic, analytical and preparative RP-HPLC and centrifugal partition chromatographic methods were developed for the purification. The diacetonides of two 14,15-anhydro ecdysteroid oxime isomers (E/Z-oximes) were obtained, and Beckmann rearrangement of the new 14,15-anhydro E-oxime yielded another new derivative containing a seven-membered lactam B-ring [5]. The ecdysteroid oximes were tested for their activity against L5178 mouse T-cell lymphoma cells (non-MDR) and their sub-cell line transfected with pHa MDR1/A retrovirus overexpressing the human ABCB1 efflux pump (MDR cell line). Each oxime was found to be a promising modulator of resistance on the MDR cell line, and the E-oxime could also sensitize the non-MDR cells to doxorubicin. Our preliminary SAR study also revealed, that the antitumor activity of ecdysteroid diacetonides follow the ecdysteroid < Z-oxime < E-oxime order. Acknowledgement: Szeged Foundation for Cancer Research.

Diastereospecific conjugation of 24-oxo-25,26,27-trinor analogs of ecdysteroids with L-ascorbic acid

Acid-catalyzed acetalization of 24-oxo-25,26,27-trinor analogs of ecdysteroids in the reaction with 2,3-di-O-benzylascorbic acid diastereospecifically gives the corresponding S-diastereoisomer with respect to the acetal chiral center.

Effects of an Ecdysteroid Analog ( RH-0345 ) on the Ovarian and Testicular Components of Eupolybothrus Nudicornis ( Myriapoda : Chilopoda )

We studied the effects of one ecdysteroid analog (RH-0345) on the ovarian and testicular components of the centipede Eupolybothrus nudicornis used as a model of predators of soil fauna. The injection of 10 µg of RH-0345 significantly reduced protein, lipid and carbohydrate concentrations in both the ovarian tissue of females and testicular tissue of males collected in spring or autumn. In consequence, this study shows that ecdysteroid analog pesticides can affect the reproduction of other arthropods than insects by modifying the level of metabolites in male and female gonads.

Transformations of ecdysteroids in the synthesis of their structural analogs and minor phytoecdysteroids

The review considers transformations of ecdysteroids (mostly of 20-hydroxyecdysone and its derivatives) involving the A, B, C, and D rings of the steroid skeleton and the side chain, which are used in the synthesis of minor phyto- and zooecdysteroids and structural analogs of ecdysteroids.

Detection of phytoecdysteroids by gustatory sensilla on chelicerae of the brown dog tick Rhipicephalus sanguineus

Ecdysteroids are polyhydroxylated steroids that act as moulting hormones in arthropods and regulate several important life‐cycle processes. Phytoecdysteroids are ecdysteroid analogues produced by some plants that disrupt the growth and development of insects feeding on them, and can be perceived by the taste receptors of insects. The present study tested the hypothesis that the blood‐feeding tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae) is capable of detecting phytoecdysteroids. By recording from the chelicerae, six phytoecdysteroids are tested: α‐ecdysone, 20‐hydroxyecdysone, ponasterone A, makisterone A, inokosterone and pterosterone. In unfed ticks, makisterone A and pterosterone elicit frequencies of neural impulses higher than in a negative control (a KCl solution at 10−3m), with detection thresholds of 10−6m and 10−12m, respectively. The spike amplitudes of the responses to these compounds, and also for 20‐hydroxyecdysone and ponasterone A, are higher than in the control, indicating that a different neurone may be involved: perhaps a deterrent cell, as observed in insects. In fed ticks, only pterosterone at 10−4m remains active. In behavioural attachment assays, no difference is observed between electrophysiologically active compounds and the negative control. These results show the capability of R. sanguineus ticks to detect phytoecdysteroids, although they do not clarify the role of ecdysteroids in tick biology, for which further studies are required.

Molecular characterization of an ecdysteroid inducible carboxylesterase with GQSCG motif in the corn borer, Sesamia nonagrioides

We obtained a full-length cDNA encoding a carboxylesterase in Sesamia nonagrioides. The complete cDNA sequence is comprised of 1838bp with an open reading frame encoding 576 amino acid residues with predicted molecular mass of 64.24kDa. The deduced amino acid sequence showed high identity to JHE-Related of Trichoplusia ni (65% amino acid identity) and 49–46% amino acid identity to JHEs of other lepidopterans and contained all five functional motifs of insect JHEs. The gene has been termed as SnJHE-Related (SnJHER) to denote its similarity to other insect JHE genes and the occurrence of an unusual cysteine residue immediately adjacent to the catalytic serine, instead of the conventional alanine residue. Phylogenetic analyses localised SnJHER together with TnJHER in a branch of the lepidopteran's JHEs group, with other carboxylesterases (COEs) occuring in separated groups. The JH analog methoprene did not affect the expression of SnJHER in contrast to other insect JHEs. Additionally, ecdysteroid analogs induced SnJHER gene expression. The SnJHER mRNA levels were higher in long-day non-diapausing larvae than in short-day diapausing ones. In the fifth instar of non-diapausing and ninth instar of diapausing larvae, the SnJHER mRNAs reached higher expression levels on the days close to each larval molt. In the last (sixth) non-diapausing larval instar, SnJHER mRNA levels peaked in the intermolt period but were lower than during the fifth instar.

(6-Benzyloxy-2,5,7,8-tetramethylchroman-2-yl)acetaldehyde conjugates with 20-hydroxyecdysone, stachysterone B, and their 20,22-acetonides

Conjugates consisting of ecdysteroid and α-tocopherol analog fragments were synthesized for the first time from 20-hydroxyecdysone and (6-benzyloxy-2,5,7,8-tetramethylchroman-2-yl)acetaldehyde.

Transient expression of specific Cotesia plutellae bracoviral segments induces prolonged larval development of the diamondback moth, Plutella xylostella

A polydnavirus, Cotesia plutellae bracovirus (CpBV), possesses a segmented and dispersed genome that is located on chromosome(s) of its symbiotic endoparasitic wasp, C. plutellae. When the host wasp parasitizes larvae of the diamondback moth, Plutella xylostella, at least 27 viral genome segments are delivered to the parasitized host along with the wasp egg. The parasitized P. xylostella exhibits significant immunosuppression and a prolonged larval development. Parasitized larvae take about 2 days longer than nonparasitized larvae to develop until the wandering stage of the final larval instar, and die after egress of the full grown wasp larvae. Developmental analysis using juvenile hormone and ecdysteroid analogs suggests that altering endocrine signals could induce the retardation of larval developmental rate in P. xylostella. In this study we used a transient expression technique to micro-inject individual CpBV genome segments, and tested their ability to induce delayed larval development of P. xylostella. We demonstrated that a CpBV segment was able to express its own encoded genes when it was injected into nonparasitized larvae, in which the expression patterns of the segment genes were similar to those in the larvae parasitized by C. plutellae. Twenty three CpBV genome segments were individually cloned and injected into the second instar larvae of P. xylostella and their effects assessed by measuring the time taken for host development to the cocooning stage. Three CpBV genome segments markedly interfered with the host larval development. When the putative genes of these segments were analyzed, it was found that they did not share any common genes. Among these segments able to delay host development, segment S27 was predicted to encode seven protein tyrosine phosphatases (CpBV-PTPs), some of which were mutated by insertional inactivation with transposons, while other encoded gene expressions were unaffected. The mutant segments were unable to induce prolonged larval development of P. xylostella. These results suggest that CpBV can induce prolonged larval development of P. xylostella, and that at least some CpBV-PTPs may contribute to the parasitic role probably by altering titers of developmental hormones.

Juvenile hormone regulation of male accessory gland activity in the red flour beetle, Tribolium castaneum

Male accessory gland proteins (Acps) act as key modulators of reproductive success in insects by influencing the female reproductive physiology and behavior. We used custom microarrays and identified 112 genes that were highly expressed in male accessory glands (MAG) in the red flour beetle, Tribolium castaneum. Out of these 112 identified genes, 59 of them contained sequences coding for signal peptide and cleavage site and the remaining 53 contained transmembrane domains. The expression of 14 of these genes in the MAG but not in other tissues of male or female was confirmed by quantitative real-time PCR. In virgin males, juvenile hormone (JH) levels increased from second day post adult emergence (PAE), remained high on third day PAE and declined on fourth day PAE. The ecdysteroid titers were high soon after adult emergence but declined to minimal levels from 1 to 5 days PAE. Feeding of juvenile hormone analog, hydroprene, but not the ecdysteroid analog, RH-2485, showed an increase in size of MAGs, as well as an increase in total RNA and protein content of MAG. Hydroprene treatment also increased the expression of Acp genes in the MAG. RNAi-mediated knock-down in the expression of JHAMT gene decreased the size of MAGs and expression of Acps. JH deficiency influenced male reproductive fitness as evidenced by a less vigor in mating behavior, poor sperm transfer, low egg and the progeny production by females mated with the JH deficient males. These data suggest a critical role for JH in the regulation of male reproduction especially through MAG secretions.

Interaction of proteins involved in ecdysone and juvenile hormone signal transduction

Ecdysteroids and juvenile hormones (JH) regulate a variety of developmental, physiological, behavioral, and metabolic processes. Ecdysteroids function through a heterodimeric complex of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP). An 85 kDa protein identified in Drosophila melanogaster methoprene-tolerant (Met) mutant binds to JH III with high affinity, and the mutant flies are resistant to juvenile hormone analog (JHA), methoprene. Reporter assays using the yeast two-hybrid system were performed in order to study the molecular interactions between EcR, USP and Met. As expected, EcR fused to the B42 activation domain and USP fused to the LexA DNA binding domain interacted with each other and supported induction of the reporter gene in the presence of stable ecdysteroid analog, RG-102240 or steroids, muristerone A and ponasterone A. The USP:USP homodimers supported expression of the reporter gene in the absence of ligand, and there was no significant increase in the reporter activity after addition of a JHA, methoprene. Similarly, Met:Met homodimers as well as Met:EcR and Met:USP heterodimers induced reporter activity in the absence of ligand and addition of ecdysteroid or JH analogs did not increase the reporter activity regulated by either homodimers or heterodimers of Met protein. Two-hybrid assays in insect cells and in vitro pull-down assays confirmed the interaction of Met with EcR and USP. These data suggest that the proteins that are involved in signal transduction of ecdysteroids (EcR and USP) and juvenile hormones (Met) interact to mediate cross-talk between these two important hormones. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.

Synthesis of analogs of ecdysteroids containing an isoxaline ring in the side chain

Starting with poststerone a synthesis was performed of ecdysteroid analogs containing an isoxaline ring in the side chain. The key stages of the route to the target products were Normant reaction and the subsequent 1,3-dipolar cycloaddition of nitrile oxides to 20-hydroxy-20-vinylsteroids.

Novel ecdysteroid analogs with oxygen-containing heterocycles in the steroid skeleton

The reaction of ecdysteroids (20-hydroxyecdysone and its acetonides) with lithium in liquid ammonia gave novel analogs with an oxetane 9α,14α-oxacycle in the steroid skeleton. In aqueous alcohol solution the 9,14-oxa analogs rearrange to the more stable 9α,13α-oxa analogs through a 1,2-migration of the 18-Me group from the C-13 to the C-14 atom.

Lack of interference of common phytoecdysteroids with production of nitric oxide by immune-activated mammalian macrophages

Effects of selected common phytoecdysteroids on immunobiological responses triggered by lipopolysaccharide and interferon-γ (IFN-γ) were tested under in vitro conditions using murine resident peritoneal macrophages. Namely, production of nitric oxide was investigated. The series of test agents encompassed ecdysteroids occurring often as major components of the ecdysteroid fraction in numerous plant extracts: 20-hydroxyecdysone (20E), polypodine B, ajugasterone C, ponasterone A and inokosterone. Their structural variability concerns only variation in the number and position of hydroxyls. Two additional side-chain modified ecdysteroids: makisterone A (with a methyl substituent at position 24) and carthamosterone (with a cyclic side-chain lactone), and three ecdysteroid analogs: poststerone, rubrosterone and dihydrorubrosterone (devoid of side chains) were included into the test series. All test compounds, except of ponasterone A, represent natural substances isolated from the medicinal plant Leuzea carthamoides and are supposed to be significant for the often reported pharmacological activities of preparations derived from this species. However, the tested ecdysteroids did not interfere with the immunobiological activity of the immunocompetent cells. Our results thus differ from the so far reported information.

7,8-dihydro analogs of ecdysteroids

Reactions of 20-hydroxyecdysone, its diacetonide, and 24,25(25,26)-anhydro derivative with lithium tetrahydridoaluminate gave the corresponding 6α- and 6β-epimeric alcohols and 7,8-dihydro analogs.

Effects and applications of arthropod steroid hormones (ecdysteroids) in mammals

Zooecdysteroids (arthropod steroid hormones) regulate the development of arthropods and probably many other invertebrates. Phytoecdysteroids are analogues occurring in a wide range of plant species, where they contribute to the deterrence of phytophagous invertebrates. The purpose of this short review is to summarise findings on the occurrence, metabolism and pharmacological effects of ecdysteroids in mammalian systems and to draw attention to their potential applications, particularly in gene-switch technology, where ecdysteroid analogues (steroidal and non-steroidal) can be used as effective and potent elicitors.