20-Hydroxyecdysone activates the protective arm of the RAAS via the MAS receptor.

20-Hydroxyecdysone (20E) is a steroid hormone that plays a key role in insect development through nuclear ecdysteroid receptors (EcR/RXR complex) and at least one membrane GPCR receptor (DopEcR). It also displays numerous pharmacological effects in mammals, where its mechanism of action is still debated, involving either an unidentified GPCR or the estrogen ERß receptor. The goal of this study was to better understand 20E mechanism of action in mammals. A mouse myoblast cell line (C2C12) and the gene expression of myostatin (a negative regulator of muscle growth) were used as a reporter system of anabolic activity. Experiments using protein-bound 20E established the involvement of a membrane receptor. 20E-like effects were also observed with angiotensin(1-7), the endogenous ligand of MAS. Additionally, the effect on myostatin gene expression was abolished by Mas receptor knock-down using siRNA or pharmacological inhibitors. 17ß-Estradiol (E2) also inhibited myostatin gene expression, but protein-bound E2 was inactive, and E2 activity was not abolished by angiotensin(1-7) antagonists. A mechanism involving cooperation between the MAS receptor and a membrane-bound palmitoylated estrogen receptor is proposed. The possibility to activate the MAS receptor with a safe steroid molecule is consistent with the pleiotropic pharmacological effects of ecdysteroids in mammals and, indeed, the proposed mechanism may explain the close similarity between the effects of angiotensin(1-7) and 20E. Our findings open up many possible therapeutic developments involving stimulation of the protective arm of the renin-angiotensin-aldosterone system (RAAS) with 20E.

STL-seq reveals pause-release and termination kinetics for promoter-proximal paused RNA polymerase II transcripts.

Despite the critical regulatory function of promoter-proximal pausing, the influence of pausing kinetics on transcriptional control remains an active area of investigation. Here, we present Start-TimeLapse-seq (STL-seq), a method that captures the genome-wide kinetics of short, capped RNA turnover and reveals principles of regulation at the pause site. By measuring the rates of release into elongation and premature termination through the inhibition of pause release, we determine that pause-release rates are highly variable, and most promoter-proximal paused RNA polymerase II molecules prematurely terminate (∼80%). The preferred regulatory mechanism upon a hormonal stimulus (20-hydroxyecdysone) is to influence pause-release rather than termination rates. Transcriptional shutdown occurs concurrently with the induction of promoter-proximal termination under hyperosmotic stress, but paused transcripts from TATA box-containing promoters remain stable, demonstrating an important role for cis-acting DNA elements in pausing. STL-seq dissects the kinetics of pause release and termination, providing an opportunity to identify mechanisms of transcriptional regulation.

The homotetramerization of a GPCR transmits the 20-hydroxyecdysone signal and increases its entry into cells for insect metamorphosis.

Animal steroid hormones initiate signaling by passive diffusion into cells and binding to their nuclear receptors to regulate gene expression. Animal steroid hormones can initiate signaling via G protein-coupled receptors (GPCRs); however, the underlying mechanisms are unclear. Here, we show that a newly discovered ecdysone-responsive GPCR, ErGPCR-3, transmits the steroid hormone 20-hydroxyecdysone (20E) signal by binding 20E and promoting its entry into cells in the lepidopteran insect Helicoverpa armigera Knockdown of ErGPCR-3 in larvae caused delayed and abnormal pupation, inhibited remodeling of the larval midgut and fat body, and repressed 20E-induced gene expression. Also, 20E induced both the interaction of ErGPCR-3 with G proteins and rapid intracellular increase in calcium, cAMP and protein phosphorylation. ErGPCR-3 was endocytosed by GPCR kinase 2-mediated phosphorylation, and interacted with ß-arrestin-1 and clathrin, to terminate 20E signaling under 20E induction. We found that 20E bound to ErGPCR-3 and induced the ErGPCR-3 homodimer to form a homotetramer, which increased 20E entry into cells. Our study revealed that homotetrameric ErGPCR-3 functions as a cell membrane receptor and increases 20E diffusion into cells to transmit the 20E signal and promote metamorphosis.

In vitro adjuvant antitumor activity of various classes of semi-synthetic poststerone derivatives.

Various classes of semi-synthetic analogs of poststerone, the product of oxidative cleavage of the C20-C22 bond in the side chain of the phytoecdysteroid 20-hydroxyecdysone, were synthesized. The analogs were obtained by reductive transformations using L-Selectride and H2-Pd/C, by molecular abeo-rearrangements using the DAST reagent or ultrasonic treatment in the NaI-Zn-DMF system, and by acid-catalyzed reactions of poststerone derivatives with various aldehydes (o-FC6H4CHO, m-CF3C6H4CHO, CO2Me(CH2)8CHO). The products were tested on a mouse lymphoma cell line pair, L5178 and its ABCB1-transfected multi-drug resistant counterpart, L5178MDR, for their in vitro activity alone and in combination with doxorubicin, and for the ability to inhibit the ABCB1 transporter. Among the tested compounds, new 2,3-dioxolane derivatives of the pregnane ecdysteroid were found to have a pronounced chemosensitizing activity towards doxorubicin and could be considered as promising candidates for further structure optimization for the development of effective chemosensitizing agents.

Highly Efficient Extraction Procedures Based on Natural Deep Eutectic Solvents or Ionic Liquids for Determination of 20-Hydroxyecdysone in Spinach.

A novel, efficient extraction procedure based on natural deep eutectic solvents (NADES) and ionic liquids (ILs) for determination of 20-hydroxyecdysone (20-E) in spinach has been developed. NADES, the first green extraction agent, with different hydrogen bond donors and acceptors are screened in order to determine extraction efficiencies. NADES consisting of lactic acid and levulinic acid at a molar ratio of 1:1 exhibits the highest yields. ILs, the second green extraction agent, with various cations and anions are also investigated, where [TEA] [OAc]·AcOH, χAcOH = 0.75 displays the highest recovery. Moreover, NADES-SLE and IL-SLE (SLE, solid-liquid extraction) parameters are investigated. Using the obtained optimized method, the recoveries of the target compound in spinach are above 93% and 88% for NADES-SLE and IL-SLE procedure, respectively. The methods display good linearity within the range of 0.5-30 µg/g and LODs of 0.17 µg/g. The proposed NADES-SLE-UHPLC-UV and IL-SLE-UHPLC-UV procedures can be applied to the analysis of 20-E in real spinach samples, making it a potentially promising technique for food matrix. The main advantage of this study is the superior efficiency of the new, green extraction solvents, which results in a significant reduction of extraction time and solvents as compared to those in the literature.

In Silico Identification of Chemicals Capable of Binding to the Ecdysone Receptor.

The process of molting, known alternatively as ecdysis, is a feature integral in the life cycles of species across the arthropod phylum. Regulation occurs as a function of the interaction of ecdysteroid hormones with the arthropod nuclear ecdysone receptor-a process preceding the triggering of a series of downstream events constituting an endocrine signaling pathway highly conserved throughout environmentally prevalent insect, crustacean, and myriapod organisms. Inappropriate ecdysone receptor binding and activation forms the essential molecular initiating event within possible adverse outcome pathways relating abnormal molting to mortality in arthropods. Definition of the characteristics of chemicals liable to stimulate such activity has the potential to be of great utility in mitigation of hazards posed toward vulnerable species. Thus the aim of the present study was to develop a series of rule-sets, derived from the key structural and physicochemical features associated with identified ecdysone receptor ligands, enabling construction of Konstanz Information Miner (KNIME) workflows permitting the flagging of compounds predisposed to binding at the site. Data describing the activities of 555 distinct chemicals were recovered from a variety of assays across 10 insect species, allowing for formulation of KNIME screens for potential binding activity at the molecular initiating event and adverse outcome level of biological organization. Environ Toxicol Chem 2020;39:1438-1450. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Detection and quantitation of ecdysterone in human serum by liquid chromatography coupled to tandem mass spectrometry.

The polyhydroxylated phytosteroid ecdysterone is present in various plants (e.g. spinach). It is widely marketed as the active component of dietary supplements, due to its reported health and performance promoting effects. For evaluation of its actual bioavailability, a fast and sensitive method was developed, optimized and validated for human serum. Instrumental analysis was performed utilizing liquid chromatography-tandem mass spectrometry with positive electrospray ionization and acquisition in multiple reaction mode. Solid phase extraction and dilute-and-inject (following protein precipitation) were tested to isolate ecdysterone from human serum. Both methods were compared in the light of the preset analytical target profile. The limit of detection (LOD) and quantitation (LOQ) for ecdysterone in human serum after SPE extraction corresponded to 0.06 ng/mL and 0.14 ng/mL, respectively, meeting the requested sensitivity of the method. The assay was linear over the range of 0.10 ng/mL to 20.83 ng/mL. As expected, the sensitivity of the SPE method was better than that of the dilute-and-inject procedure, which did not allow for quantitation of all post administration serum samples. Accuracy (relative error; %) and precision (coefficient of variation; %), were both within acceptance criteria (<15%). The developed method was successfully applied to a ten week intervention study conducted in young men performing regular resistance training. Different doses of supplements containing ecdysterone from spinach extract have been administered during the study and the quantitation of ecdysterone in serum samples has been successfully conducted. Ecdysterone could be quantified in all post-administration samples using solid phase extraction (SPE) for sample pretreatment.

Ecdysteroids are present in the blood of wild passerine birds.

Ecdysteroids (arthropod molting hormones) play an important role in the development and sexual maturation of arthropods, and they have been shown to have anabolic and "energizing" effect in higher vertebrates. The aim of this study was to assess ecdysteroid diversity, levels according to bird species and months, as well as to observe the molting status of hard ticks (Acari: Ixodidae) infesting the birds. Therefore, blood samples and ticks were collected from 245 birds (244 songbirds and a quail). Mass spectrometric analyses showed that 15 ecdysteroids were regularly present in the blood samples. Molting hormones biologically most active in insects (including 20-hydroxyecdysone [20E], 2deoxy-20E, ajugasterone C and dacryhainansterone) reached different levels of concentration according to bird species and season. Similarly to ecdysteroids, the seasonal presence of affected, apolytic ticks peaked in July and August. In conclusion, this study demonstrates the presence of a broad range and high concentrations of ecdysteroids in the blood stream of wild-living passerine birds. These biologically active, anabolic compounds might possibly contribute to the known high metabolic rate of songbirds.

Two new phytoecdysteroids from Sphenocentrum jollyanum Pierre root.

The crude methanol extract of Sphenocentrum jollyanum root exhibited 98% and 80% antimicrobial activity against Aspergillus fumigatus Pinh and Vancomycin resistant enterococcus (VRE) at a concentration of 200 µg/mL, with IC50 11.45 and 12.95 µg/mL, respectively. The ethyl acetate fraction of methanol extract showed in-vitro antimicrobial activity against A. fumigatus Pinh at 83% with IC50 of <8 µg/mL. The phytochemical investigation of ethyl acetate fraction yielded six compounds, which were identified by their NMR, IR and MS spectral analyses as two new phytoecdysteroidal glycosides Sphenocentroside A (1), and Sphenocentroside B (2), and four known phytoecdysteroids: polypodoaurein (3), polypodine B (4), ecdysterone (5), and 20, 26-dihydroxyecdysone (6).

Molecular rearrangements of poststerone derivative steroid core with formation of unique D-homostructures of pregnane and androstane series.

20R-Hydroxy short-chain ecdysteroids were synthesized by chemo- and stereoselective reduction of poststerone acetonide with L-Selectride or LiAlH4. The same reaction with the excess of L- Selectride followed by the treatment of the reaction mixture with hydrochloric acid is accompanied by (8R)-13(14 → 8)abeo- rearrangements, which resulted in the contraction/expansion of C/D pregnane rings. The reaction of 20R-hydroxy poststerone analogs with (diethylamino)sulfur trifluoride (DAST) proceeds through intramolecular rearrangements and provides D-homo- or 13,14-seco- androstane structures.

Molecular dynamics and free energy studies on the Drosophila melanogaster and Leptinotarsa decemlineata ecdysone receptor complexed with agonists: Mechanism for binding and selectivity.

The ecdysone receptor is a nuclear hormone receptor that plays a pivotal role in the insect metamorphosis and development. To address the molecular mechanisms of binding and selectivity, the interactions of two typical agonists Ponasterone A and 20-Hydroxyecdysone with Drosophila melanogaster (DME) and Leptinotarsa decemlineata ecdysone (LDE) receptors were investigated by homology modeling, molecular docking, molecular dynamic simulation, and thermodynamic analysis. We discover that 1) the L5-loop, L11-loop, and H12 helix for DME, L7-loop, and L11-loop for LDE are more flexible, which affect the global dynamics of the ligand-binding pocket, thus facilitating the ligand recognition of ecdysone receptor; 2) several key residues (Thr55/Thr37, Phe109/Phe91, Arg95/Arg77, Arg99/Arg81, Phe108/Leu90, and Ala110/Val92) are responsible for the binding of the proteins; 3) the binding-free energy is mainly contributed by the van der Waals forces as well as the electrostatic interactions of ligand and receptor; 4) the computed binding-free energy difference between DME-C1 and LDE-C1 is -4.65 kcal/mol, explains that C1 can form many more interactions with the DME; 5) residues Phe108/Leu90 and Ala110/Val92 have relatively position and orientation difference in the two receptors, accounting most likely for the ligand selectivity of ecdysone receptor from different orders of insects. This study underscores the expectation that different insect pests should be able to discriminate among compounds from different as yet undiscovered compounds, and the results firstly show a structural and functional relay between the agonists and receptors (DME and LDE), which can provide an avenue for the development of target-specific insecticides. Communicated by Ramaswamy H. Sarma.

Bioavailability and structural study of 20-hydroxyecdysone complexes with cyclodextrins.

20-Hydroxyecdysterone - (2ß,3ß,5ß,22R)-2,3,14,20,22,25-hexahydroxycholest-7-en-6-one was isolated in satisfactory yield using ethanol extraction from the aerial part of Silene wolgensis (Hornem.) Otth; sometimes Silene wolgensis (Willd.) Bess. ex Spreng. The complexation of the phytoecdysteroid with ß-cyclodextrin was studied by NMR spectroscopy. By studying the changes in chemical shifts of protons of substrates and receptors it was found that ecdysterone interacts with cyclodextrins to form supramolecular inclusion complexes of stoichiometric composition of 1:1 or 1:2. Ecdysterone-ß-cyclodextrin complexes exhibit 100 times higher solubility in water than the parent compound.

Ponasterone A and F, Ecdysteroids from the Arctic Bryozoan Alcyonidium gelatinosum.

A new ecdysteroid, ponasterone F (1) and the previously reported compound ponasterone A (2) were isolated from specimens of the Arctic marine bryozoan Alcyonidium gelatinosum collected at Hopenbanken, off the coast of Edgeøya, Svalbard. The structure of 1 was elucidated, and the structure of 2 confirmed by spectroscopic methods including 1D and 2D NMR and analysis of HR-MS data. The compounds were evaluated for their ability to affect bacterial survival and cell viability, as well as their agonistic activities towards the estrogen receptors α and ß. The compounds were not active in these assays. Compound 2 is an arthropod hormone controlling molting and are known to act as an allelochemical when produced by plants. Even though its structure has been previously reported, this is the first time a ponasterone has been isolated from a bryozoan. A. gelatinosum produced 1 and 2 in concentrations surpassing those expected of hormonal molecules, indicating their function as defence molecules against molting predators. This work adds to the chemical diversity reported from marine bryozoans and expanded our knowledge of the chemical modifications of the ponasterones.

Comparative Transcriptome Analysis Identifies Genes Putatively Involved in 20-Hydroxyecdysone Biosynthesis in Cyanotis arachnoidea.

Cyanotis arachnoidea contains a rich array of phytoecdysteroids, including 20-hydroxyecdysone (20E), which displays important agrochemical, medicinal, and pharmacological effects. To date, the biosynthetic pathway of 20E, especially the downstream pathway, remains largely unknown. To identify candidate genes involved in 20E biosynthesis, the comparative transcriptome of C. arachnoidea leaf and root was constructed. In total, 86.5 million clean reads were obtained and assembled into 79,835 unigenes, of which 39,425 unigenes were successfully annotated. The expression levels of 2427 unigenes were up-regualted in roots with a higher accumulation of 20E. Further assignments with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways identified 49 unigenes referring to the phytoecdysteroid backbone biosynthesis (including 15 mevalonate pathway genes, 15 non-mevalonate pathway genes, and 19 genes for the biosynthesis from farnesyl pyrophosphate to cholesterol). Moreover, higher expression levels of mevalonate pathway genes in roots of C. arachniodea were confirmed by real-time quantitative PCR. Twenty unigenes encoding CYP450s were identified to be new candidate genes for the bioreaction from cholesterol to 20E. In addition, 90 transcription factors highly expressed in the roots and 15,315 unigenes containing 19,158 simple sequence repeats (SSRs) were identified. The transcriptome data of our study provides a valuable resource for the understanding of 20E biosynthesis in C. arachnoidea.

Extraction optimization and UHPLC method development for determination of the 20-hydroxyecdysone in Sida tuberculata leaves.

Sida tuberculata (ST) is a Malvaceae species widely distributed in Southern Brazil. In traditional medicine, ST has been employed as hypoglycemic, hypocholesterolemic, anti-inflammatory and antimicrobial. Additionally, this species is chemically characterized by flavonoids, alkaloids and phytoecdysteroids mainly. The present work aimed to optimize the extractive technique and to validate an UHPLC method for the determination of 20-hydroxyecdsone (20HE) in the ST leaves. Box-Behnken Design (BBD) was used in method optimization. The extractive methods tested were: static and dynamic maceration, ultrasound, ultra-turrax and reflux. In the Box-Behnken three parameters were evaluated in three levels (-1, 0, +1), particle size, time and plant:solvent ratio. In validation method, the parameters of selectivity, specificity, linearity, limits of detection and quantification (LOD, LOQ), precision, accuracy and robustness were evaluated. The results indicate static maceration as better technique to obtain 20HE peak area in ST extract. The optimal extraction from surface response methodology was achieved with the parameters granulometry of 710 nm, 9 days of maceration and plant:solvent ratio 1:54 (w/v). The UHPLC-PDA analytical developed method showed full viability of performance, proving to be selective, linear, precise, accurate and robust for 20HE detection in ST leaves. The average content of 20HE was 0.56% per dry extract. Thus, the optimization of extractive method in ST leaves increased the concentration of 20HE in crude extract, and a reliable method was successfully developed according to validation requirements and in agreement with current legislation.

Evolutionarily Conserved Roles for Blood-Brain Barrier Xenobiotic Transporters in Endogenous Steroid Partitioning and Behavior.

Central nervous system (CNS) chemical protection depends upon discrete control of small-molecule access by the blood-brain barrier (BBB). Curiously, some drugs cause CNS side-effects despite negligible transit past the BBB. To investigate this phenomenon, we asked whether the highly BBB-enriched drug efflux transporter MDR1 has dual functions in controlling drug and endogenous molecule CNS homeostasis. If this is true, then brain-impermeable drugs could induce behavioral changes by affecting brain levels of endogenous molecules. Using computational, genetic, and pharmacologic approaches across diverse organisms, we demonstrate that BBB-localized efflux transporters are critical for regulating brain levels of endogenous steroids and steroid-regulated behaviors (sleep in Drosophila and anxiety in mice). Furthermore, we show that MDR1-interacting drugs are associated with anxiety-related behaviors in humans. We propose a general mechanism for common behavioral side effects of prescription drugs: pharmacologically challenging BBB efflux transporters disrupts brain levels of endogenous substrates and implicates the BBB in behavioral regulation.

Capillary electrophoresis study on the base-catalyzed formation of bioactive oxidized metabolites of 20-hydroxyecdysone.

A novel capillary electrophoretic method was developed for the analysis and monitoring of the base-catalyzed autoxidation of 20-hydroxyecdysone, a worldwide used non-hormonal anabolic food supplement. An effective separation of the starting material and its bioactive oxidized derivatives was achieved by using sulfobutyl-ß-cyclodextrin as selector at pH 11 and by fixing the separation voltage at +30kV. Only a dilution step was inserted before injecting the sample, taken from the crude reaction mixture, to the capillary electrophoresis instrument. The same alkaline pH was used for the analysis as for the reaction, unlike the previously reported HPLC study where sample neutralization was required prior to the measurement. Due to the very short analysis time (6min) in capillary electrophoresis, more frequent sampling and more detailed time scale analysis could be carried out. Furthermore, in contrast with the preceding HPLC results, the previously unobserved calonysterone could also be detected by capillary electrophoresis as a minor primary product. Our novel method demonstrated higher resolution than the one before. Baseline separation could be achieved and the resolution values were in the range of 1.9-7.0. The limit of detection was below 71µg/ml, the relative standard deviation values of the migration time and peak area for intra- and inter-day precision were less than 10%. The more precise, direct monitoring of the time dependency of the oxidation process is expected to have a significant impact on yield optimization initiatives to allow related pharmacological studies in the near future.

CYP450s analysis across spiny lobster metamorphosis identifies a long sought missing link in crustacean development.

Cytochrome P450s (CYP450s) are a rapidly evolving family of enzymes, making it difficult to identify bona fide orthologs with notable lineage-specific exceptions. In ecdysozoans, a small number of the most conserved orthologs include enzymes which metabolize ecdysteroids. Ecdysone pathway components were recently shown in a decapod crustacean but with a notable absence of shade, which is important for converting ecdysone to its active form, 20-hydroxyecdysone (20HE), suggesting that another CYP450 performs a similar function in crustaceans. A CYPome temporal expression analysis throughout metamorphosis performed in this research highlights several un-annotated CYP450s displaying differential expression and provides information into expression patterns of annotated CYP450s. Using the expression patterns in the Eastern spiny lobster Sagmariasus verreauxi, followed by 3D modelling and finally activity assays in vitro, we were able to conclude that a group of CYP450s, conserved across decapod crustaceans, function as the insect shade. To emphasize the fact that these genes share the function with shade but are phylogenetically distinct, we name this enzyme system Shed.

Dual inhibition of BACE1 and Aß aggregation by ß-ecdysone: Application of a phytoecdysteroid scaffold in Alzheimer's disease therapeutics.

Current medications for the complex neurological disorder, Alzheimer's disease (AD), can neither stop disease progression nor revert back disease pathogenesis. The present study demonstrates the applicability of a phytoecdysteroid, ß-ecdysone, as a multi-potent agent in AD therapeutics. ß-ecdysone strongly binds to the active site cavity of BACE1 with calculated dissociation constant of 1.75±0.1µM. Steady-state and time-resolved fluorescence spectroscopy reveal that binding of ß-ecdysone induces conformational transition of the protein from open to closed form thereby blocking substrate binding. Even 500nM of the compound completely blocks the enzyme activity. Furthermore, ß-ecdysone strongly inhibits Aß aggregation, evident from ANS and ThT binding assay. Co-incubation of equimolar peptide and ß-ecdysone completely inhibits Aß fibril formation which is further complemented by the AFM study. Low systemic toxicity of ß-ecdysone further extends the applicability of the compound as functional food and dietary supplement for disease management.

Nitric Oxide and ERK mediates regulation of cellular processes by Ecdysterone.

The complex process of wound healing is a major problem associated with diabetes, venous or arterial disease, old age and infection. A wide range of pharmacological effects including anabolic, anti-diabetic and hepato-protective activities have been attributed to Ecdysterone. In earlier studies, Ecdysterone has been shown to modulate eNOS and iNOS expression in diabetic animals and activate osteogenic differentiation through the Extracellular-signal-Regulated Kinase (ERK) pathway in periodontal ligament stem cells. However, in the wound healing process, Ecdysterone has only been shown to enhance granulation tissue formation in rabbits. There have been no studies to date, which elucidate the molecular mechanism underlying the complex cellular process involved in wound healing. The present study, demonstrates a novel interaction between the phytosteroid Ecdysterone and Nitric Oxide Synthase (NOS), in an Epidermal Growth Factor Receptor (EGFR)-dependent manner, thereby promoting cell proliferation, cell spreading and cell migration. These observations were further supported by the 4-amino-5-methylamino- 2' ,7' -difluorofluorescein diacetate (DAF FM) fluorescence assay which indicated that Ecdysterone activates NOS resulting in increased Nitric Oxide (NO) production. Additionally, studies with inhibitors of both the EGFR and ERK, demonstrated that Ecdysterone activates NOS through modulation of EGFR and ERK. These results clearly demonstrate, for the first time, that Ecdysterone enhances Nitric Oxide production and modulates complex cellular processes by activating ERK1/2 through the EGF pathway.