Ecdysteroid metabolism in mammals: The fate of ingested 20-hydroxyecdysone in mice and rats.

Phytoecdysteroids are molecules derived from sterol metabolism and found in many plants. They display a wide array of pharmacological effects on mammals (e.g. anabolic, anti-diabetic). Although these effects have been long established, the molecular targets involved remain to be identified. Like endogenous steroid hormones and bile acids, which are biochemically related, ingested or injected phytoecdysteroids undergo a set of reactions in mammals leading to the formation of numerous metabolites, only some of which have been so far identified, and it is presently unknown whether they represent active metabolites or inactivation products. In the large intestine, ecdysteroids undergo efficient 14-dehydroxylation. Other changes (reductions, epimerization, side-chain cleavage) are also observed, but whether these occur in the liver and/or large intestine is not known. The purpose of this study was to investigate the pharmacokinetics of 20-hydroxyecdysone (20E), the most common phytoecdysteroid, when administered to mice and rats, using, when required, tritium-labelled molecules to permit metabolic tracking. Bioavailability, the distribution of radioactivity and the kinetics of formation of metabolites were followed for 24-48 hours after ingestion and qualitative and quantitative analyses of circulating and excreted compounds were performed. In mice, the digestive tract always contains the majority of the ingested 20E. Within 30 min after ingestion, 20E reaches the large intestine, where microorganisms firstly remove the 14-hydroxyl group and reduce the 6-one. Then a very complex set of metabolites (not all of which have yet been identified) appears, which correspond to poststerone derivatives formed in the liver. We have observed that these compounds (like bile acids) undergo an entero-hepatic cycle, involving glucuronide conjugation in the liver and subsequent deconjugation in the intestine. Despite the very short half-life of ecdysteroids in mammals, this entero-hepatic cycle helps to maintain their plasma levels at values which, albeit low (≤0.2 µM), would be sufficient to evoke several pharmacological effects. Similar 20E metabolites were observed in mice and rats; they include in particular 14-deoxy-20E, poststerone and 14-deoxypoststerone and their diverse reduction products; the major products of this metabolism have been unambiguously identified. The major sites of metabolism of exogenous ecdysteroids in mammals are the large intestine and the liver. The entero-hepatic cycle contributes to the metabolism and to maintaining a low, but pharmacologically significant, concentration of ecdysteroids in the blood for ca. 24 h after ingestion. These data, together with parallel in vitro experiments provide a basis for the identification of 20E metabolite(s) possibly involved in the physiological effects associated with ecdysteroids in mammals.

Phytochemical analysis and bioactivity of the aerial parts of Abutilon theophrasti (Malvaceae), a medicinal weed.

Phytochemical investigations of aerial parts of Abutilon theophrasti yielded (6S,9R)-roseoside (1) and (6S,9S)-roseoside (2) which are new for the genus. The elucidation of the chemical structures was established by mass spectrometry, 1D and 2D NMR experiments. Although methanol extracts contained 48.5 ± 7.2 mg of caffeic acid equivalents and 15.87 ± 4.6 mg of quercetin equivalents, the antioxidant activity, as revealed by DPPH and ABTS assays, was of medium strength (EC50 of 306.2 ± 16.3 and 394.3 ± 14.8 µg/mL, respectively). A. theophrasti extract inhibits soybean 5-LOX with IC50 value 2.89 ± 0.2 mg/mL. The cytotoxicity of the methanol extract against MCF-7, CCRF-CEM and CEM/ADR5000 cancer cells resulted in IC50 values of 505.8 ± 34.7 µg/mL for MCF-7, 75.6 ± 7.1 µg/mL for CCRF-CEM, and 89.5 ± 13.4 µg/mL for CEM/ADR 5000 cells.

The metabolism of 20-hydroxyecdysone in mice: relevance to pharmacological effects and gene switch applications of ecdysteroids.

Ecdysteroids exert many pharmacological effects in mammals (including humans), most of which appear beneficial, but their mechanism of action is far from understood. Whether they act directly and/or after the formation of metabolites is still an open question. The need to investigate this question has gained extra impetus because of the recent development of ecdysteroid-based gene-therapy systems for mammals. In order to investigate the metabolic fate of ecdysteroids in mice, [1α,2α-(3)H]20-hydroxyecdysone was prepared and injected intraperitoneally to mice. Their excretory products (urine+faeces) were collected and the different tritiated metabolites were isolated and identified. The pattern of ecdysteroid metabolites is very complex, but no conjugates were found, in contrast to the classical fate of the (less polar) endogenous vertebrate steroid hormones. Primary reactions involve dehydroxylation at C-14 and side-chain cleavage between C-20 and C-22, thereby yielding 14-deoxy-20-hydroxyecdysone, poststerone and 14-deoxypoststerone. These metabolites then undergo several reactions of reduction involving, in particular, the 6-keto-group. A novel major metabolite has been identified as 2ß,3ß,6α,22R,25-pentahydroxy-5ß-cholest-8(14)-ene. The formation of this and the other major metabolites is discussed in relation to the various effects of ecdysteroids already demonstrated on vertebrates.

NMR applications for identifying ß-TrCP protein-ligand interactions.

In the absence of crystallographic data, NMR has emerged as the best way to define protein-ligand interactions. Using NMR experiments based on magnetization transfer, one can sort bound from unbound molecules, estimate the dissociation constant, identify contacts implied in the binding, characterize the structure of the bound ligand and conduct ligand competition assays.

Sheep prion protein synthetic peptide spanning helix 1 and beta-strand 2 (residues 142-166) shows beta-hairpin structure in solution.

According to the "protein only" hypothesis, a conformational conversion of the non-pathogenic "cellular" prion isoform into a pathogenic "scrapie" isoform is the fundamental event in the onset of prion diseases. During this pathogenic conversion, helix H1 and two adjacent surface loops L2 and L3 of the normal prion protein are thought to undergo a conformational transition into an extended beta-like structure, which is prompted by interactions with the pre-existing beta-sheet. To get more insight into the interaction between the helix and one of the beta-strands in the partially unfolded prion protein, the solution structure of a synthetic linear peptide spanning helix H1 and beta-strand S2 (residues 142-166 in human numbering) was studied by circular dichroism and nuclear magnetic resonance spectroscopies. We found that, in contrast to many prion fragments studied earlier, this peptide (i) is highly soluble and does not aggregate up to a millimolar concentration range in aqueous medium and (ii) exhibits an intrinsic propensity to a beta-hairpin like conformation at neutral pH. This beta-propensity can be one of the internal driving forces of the molecular rearrangement responsible for the pathogenic conversion of the prion protein.

Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system.

The first spectrophotometric study of the reaction of the myeloperoxidase/H2O2/Cl- system with NADPH and NMNH showed that the reaction products were not the corresponding oxidized nucleotides and that modifications would take place on the nicotinamide part of the molecule [Auchère, F. & Capeillère-Blandin, C. (1999) Biochem. J. 343, 603-613]. In this report, in order to obtain more precise information on the structural modifications and mechanism of the reaction, we focus on the purification and isolation of products derived from NADPH and NMNH by RP-HPLC. Electrospray ionization mass spectra indicated that the relative height of the peaks reflected that of the natural isotopic abundance of 35Cl and 37Cl, providing evidence that the products derived from NADPH and NMNH were monochlorinated. Moreover, calculated masses revealed the 1 : 1 addition of HOCl to the molecule. Various 1D and 2D NMR experiments provided data for the assignments of the chemical shifts of protons and carbons and the coupling constants of the protons of the chlorinated nucleotides. Further NOESY experiments allowed the characterization of the spatial structure of the chlorinated product and showed that trans HOCl addition occurred at the C5=C6 carbon double bond of the nicotinamide ring, leading to a chlorohydrin.

Identification and quantitative analysis of the phytoecdysteroids in Silene species (Caryophyllaceae) by high-performance liquid chromatography. Novel ecdysteroids from S. pseudotites.

Many species in the genus Silene (Caryophyllaceae) have previously been shown to contain ecdysteroids and this genus is recognised as a good source of novel ecdysteroid analogues. We have used ecdysteroid-specific radioimmunoassays and the microplate-based Drosophila melanogaster B(II) cell bioassay for ecdysteroid agonist and antagonist activities to identify further phytoecdysteroid-containing species in this genus. The main ecdysteroid components from 10 Silene species (S. antirrhina, S. chlorifolia, S. cretica, S. disticha, S. echinata, S. italica, S. portensis, S. pseudotites, S. radicosa, S. regia) were isolated and identified, mainly by normal-phase and reversed-phase high-performance liquid chromatography. The amount of each ecdysteroid was determined by comparing chromatogram peak areas with those for reference 20-hydroxyecdysone (20E) on reversed-phase HPLC. 20E is the most abundant ecdysteroid in each of the Silene extracts. Polypodine B, 2-deoxy-20-hydroxyecdysone and ecdysone are also common ecdysteroids in these Silene species, but the proportions of these ecdysteroids vary between the Silene species. HPLC proved to be a quick and effective way to screen Silene species, determine ecdysteroid profiles and, hence, identify extracts containing novel analogues. An extract of the aerial parts of S. pseudotites was found to contain several new ecdysteroids. These have been isolated and identified spectroscopically (by NMR and mass spectrometry) as 2-deoxyecdysone 22beta-D-glucoside, 2-deoxy-20,26-dihydroxyecdysone and 2-deoxypolypodine B 3beta-D-glucoside. Additionally, (5alpha-H)-2-deoxyintegristerone A (5alpha-2H 91%, 5alpha-1H 9%) was isolated as an artefact. This study contributes to the understanding of ecdysteroid distribution in Silene species and provides further information on the chemotaxonomic significance of ecdysteroids in Silene species.

[Structural diversity of ecdysteroids of Lychnis flos-cuculi]. / A Lychnis flos-cuculi ekdiszteroidjainak szerkezeti diverzitása.

Eleven ecdysteroids have been isolated from Lychnis floscuculi; we are the first who report eight ecdysteroids of the eleven compounds in this plant. Two of these ecdysteroids, dihydrorubrosterone and 20-hydroxyecdysone 3-acetate are newly discovered natural products. The success of isolation of these new ecdysteroids has been based on the use of separation methods in a proper order; these separation procedures were completing each others. At the beginning steps of isolation simple separation methods were used, such a solvent-solvent distribution and fractionated precipitation. Two third of the contaminants were removed thereby. High capacity low resolution methods were used then, such as classical adsorption column chromatography and preparative thin-layer chromatography. The major component (20-hydroxyecdyssone) and certain minor ecdysteroids (polypodine B and rubrosterone) were isolated in pure form here. Purification of the further minor components (poststerone, 2-deoxy-20-hydroxyecdysone, vitikosterone E, dihydrorubrosterone, makisterone A, taxisterone, 20-hydroxyecdysone 2-acetate, 20-hydroxyecdysone 3-acetate) required HPLC and other absorption chromatographic methods. Our recent separation scheme means a generally applicable guiding principle for isolation of any plant ecdysteroid, major and minor alike. Structural identification of the known ecdysteroids was based on their spectral data and that of their literature information. Structural elucidation of 20-hydroxyecdysone 3-acetate was done by the help of a standard component prepared by acetylation of 20-hydroxyecdysone. From the mixture of seven acetates the corresponding compound (20-hydroxyecdysone 3-acetate) was isolated, and used for identification. Structural diversity of ecdysteroids of Lychnis flos-cuculi is evaluated, and a tentative explanation is introduced for the formation and biosynthesis of the versatility of phytoecdysteroids.

Isolation of 5 alpha- and 5 beta-dihydrorubrosterone from Silene otites L. (Wib).

5 alpha-Dihydrorubrosterone (2 beta, 3 beta, 14 alpha, 17 beta-tetrahydroxy-5 alpha-androst-7-ene-6-one), a new 19-carbon 5 alpha-ecdysteroid, was isolated together with its 5 beta counterpart from the aerial parts of Silene otites L. (Wib.) (Caryophyllaceae) by a combination of solvent partition, low-pressure column chromatography, thin-layer chromatography (normal-phase and reversed-phase) and finally HPLC. Mass spectrometry and nuclear magnetic resonance spectroscopic procedures were used for compound characterization.

Conformations in solution and bound to bacterial ribosomes of ketolides, HMR 3647 (telithromycin) and RU 72366: a new class of highly potent antibacterials.

The new class of antibiotics called ketolides is endowed with remarkable antibacterial activity against macrolide-resistant strains. Further modifications of the 3 keto-macrolactone backbone led to 11,12-hydrazonocarbamate ketolides with an imidazolyl pyridine chain: the file-leader of ketolide class, HMR 3647 (telithromycin), and its N-bis-demethyl-derivative, RU 72366. The potency of HMR 3647 is higher than that of RU 72366. Stereospecific 1H and 13C resonance assignments of HMR 3647 and RU 72366 have been determined and have allowed a detailed quantitative conformational analysis of the uncomplexed form of the molecules. The comparative conformation of HMR 3647 in solution and its N-bis-demethyl-derivative in D2O has been carried out using different heteronuclear correlation experiments in conjunction with nuclear Overhauser effect experiments and in particular long-range 3J(CH) coupling constants and using molecular dynamics (MD) methods. The study of ketolide ribosome interaction has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). The database of ribosome-bound ketolide structures has been used to compare the structure(s) of ketolide in ribosome-ketolide complexes with the conformational preferences of free ketolides and to highlight the significant differences between HMR 3647 and RU 72366. A comparison of the conformations bound to ribosome was made with those of other previously studied ketolide (RU 004) and macrolides and would explain the remarkable potencies of HMR 3647 in inhibiting protein synthesis.

Sileneoside H, a new phytoecdysteroid from Silene brahuica.

Sileneoside H (1), a new phytoecdysteroid, has been isolated from the roots of Silene brahuica and identified as 22-O-alpha-D-galactosylintegristerone A 25-acetate by MS and NMR analysis.

Lincomycin and clindamycin conformations. A fragment shared by macrolides, ketolides and lincosamides determined from TRNOE ribosome-bound conformations.

Two important lincosamide antibiotics, lincomycin and clindamycin were studied in the complex state with the bacterial ribosome after a conformational analysis by 1H and 13C NMR spectroscopy and molecular modelling of the unbound molecules. Lincosamide-ribosome interactions were investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY), resulting in a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrates in solution indicate that specific conformations are preferred in the bound state. Clindamycin, the more bioactive antibiotic studied, displayed a stronger NMR response than lincomycin showing that in lincosamide-ribosome interactions, a low affinity binding level is associated to the tight binding one and is related to biological activity. This study shows that conformation plays an essential role for the low affinity binding site. Superimposition of lincosamide, macrolide and ketolide bound structures exhibited conformational similarities in a particular fragment which is in agreement with a hypothesis of partial overlapping lincosamide and macrolide binding sites.

Conformational analysis of ketolide, conformations of RU 004 in solution and bound to bacterial ribosomes.

A new structurally distinct class of 14-membered-ring macrolides is characterized by a keto-function instead of the cladinose sugar, well-known for its fragility even in weakly acidic media. This new class called ketolides is endowed with remarkable antibacterial activity against macrolide-resistant strains. A complete assignment of the 1H and 13C NMR spectra of RU 004 in deuteriochloroform, methanol-d4 and D2O has been made using different two-dimensional (2D) chemical-shift correlation methods. The study of ketolide-ribosome interaction has been investigated using 2D transferred nuclear Overhauser effect spectroscopy (TRNOESY). A comparison of the conformations in solution and bound to ribosomes was made with those of previous macrolides. This study can highlight some of the significant differences between RU 004 and other antibiotics.

Predictive study by molecular modeling to promote specific probes of glutamate receptors, using methylated cyclic glutamic acid derivatives (trans- and cis-ACPD). Comparison with specific agonists.

Two classes of glutamate receptors (metabotropic and ionotropic) and their subclasses (groups I-III and N-methyl-D-aspartic acid (NMDA), kainic acid (KA)), respectively, are characterized by the binding of a L-glutamate moiety in a specific conformation. The conformations may be grouped by the two backbone torsion angles, chi1 [alpha-CO2-C(2)-C(3)-C4)] and chi2 [+NC(2)-C(3)-C(4)-gamma-CO2] and by the two characteristic distances between the potentially active functional groups, alpha-N+-gamma-CO2 (d1) and alpha-CO2-gamma-CO2 (d2). The conformational preferences of 2,3,4-methyl(a and b)-cis and trans-1-aminocyclopentane-1,3-dicarboxylate are discussed in the light of the physical features known for specific metabotropic (groups I-II) and specific ionotropic (NMDA, KA) agonists, respectively. The spatial orientation of the perceived functional groups was elucidated in cyclic derivatives which contain an embedded L-glutamate moiety in a particularly restricted conformation (relative to the C(2)-C(3)-C(4) bond) using a combination of NMR experimental results and mechanics and dynamics calculations. One important conclusion of the study is that a single glutamate receptor is privileged for each theoretical model considered by molecular dynamics. This study showed clearly what would be conformational preferences of cyclic glutamate derivatives following the geometrical isomerism of the methyl group.

Solution conformation of methylated macrolide antibiotics roxithromycin and erythromycin using NMR and molecular modelling. Ribosome-bound conformation determined by TRNOE and formation of cytochrome P450-metabolite complex.

Conformational study of methylated derivatives of macrolide antibiotics roxithromycin (6-OMe-roxithromycin and 6,11-OMe-roxithromycin) has been achieved by NMR in solution and molecular dynamics (MD) simulations and compared to 6-OMe-erythromycin (clarithromycin). A complete conformational study by NMR has been led by determination of homonuclear coupling constants and NOEs. Heteronuclear 1H-13C coupling constants were also measured to investigate the orientation of the sugar moieties with respect to the erythronolide. MD simulations were performed using the crystallographic coordinates as the starting conformation. For each compound, experimental results were compared to calculated conformations in order to identify eventual conformational equilibrium in solution. It is shown that the effect of the methylation is opposite for roxithromycin compared to erythromycin especially on motional properties as the roxithromycin derivatives gain in mobility while the erythromycin derivatives behaves as a more restrained molecule. The study of macrolide-ribosome interactions has been investigated using transferred NOESY 1H NMR experiments and the conformations weakly bound to bacterial ribosomes were determined. Biological interactions of these compounds with membranar liver protein cytochrome P450 was also discussed with regard to their structural properties.

Transferred nuclear Overhauser effect study of macrolide-ribosome interactions: correlation between antibiotic activities and bound conformations.

The study of macrolide-ribosome interactions has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). A new medically important macrolide antibiotic, roxithromycin, with the replacement of the 9-keto group in erythromycin by a 9-oxime chain, was studied in the complex state with the bacterial ribosome. Analysis of transferred nuclear Overhauser effect (TRNOE) experiment resulted in a set of constraints for all protons pairs. These constraints were used in structure determination procedures based on molecular modelling to obtain a bound structure compatible with the experimental NMR data. The results compared with the conformational analysis of the substrate in solution indicate that only one specific conformation is preferred in the bound state while in the free state the sugar ring moities were relatively disordered. The bioactive macrolide antibiotics studied roxithromycin and erythromycin which displayed a strong NMR response, are metabolized in RU39001 and erythralosamine respectively which do not retain antimicrobial activity. The inactive major metabolites were used to define if TRNOEs observation may be characteristic of a biological activity. These control experiments gave essentially blank TRNOESY spectra. This study shows that Mg2+ does not play a direct role for the low affinity binding site studied by TRNOE what is in agreement with an hypothesis of two distinct binding levels, with a low affinity binding level necessary for the tight binding one.

New minor ecdysteroids from Silene otites (L.) Wib.

Six minor new ecdysteroid components have been isolated from Silene otites (L.) Wib. by a combination of chromatographic methods. Three of them (2-deoxy-20-hydroxyecdysone 3,22-diacetate, 5 alpha-2-deoxy-20-hydroxyecdysone 3-acetate, and 2-deoxy-20-hydroxyecdysone 3-crotonate) are new natural products.

Structure of kainic acid totally elucidated by NMR and molecular modelling.

One class of glutamate receptors is characterized by the binding of the neuroexcitant and toxin kainic acid (KA), which contains an embedded L-glutamate moiety in a partially restricted (about the 2,3-bond) conformation. While there are a number of compounds that exhibit high specificity and selectivity at the ionotropic N-methyl-D-aspartate receptor, there has been a lack of selective and high-affinity ligands for the ionotropic KA subclass of excitatory amino acid receptors. This substance has received some attention recently being the least understood of the ionotropic type of glutamate receptor. The spatial orientation of the perceived functional groups of KA has been elucidated by a conformational analysis of an aqueous solution of KA using a combination of nuclear magnetic resonance (NMR) experimental results, mechanics and dynamics calculations, and theoretical simulation of NMR spectra. The weak pH-dependent effects on overall conformation and the structure of the principal '4E-envelope' KA conformer are established in aqueous solution. This study clearly shows the structural 'down' position of the double bond and the preferred 'g(-)-c' conformation of the C(3) carboxymethyl side-chain. The complex structure of this compound is thus definitively resolved. The conformation of the envelope ring such as C(3) carboxymethyl and C(4)-isopropenyl groups may strongly influence the potencies of KA interactions with the KA receptor.

24(24(1))[Z]-dehydroamarasterone B, a phytoecdysteroid from seeds of Leuzea carthamoides.

A new phytoecdysteroid, 24(24(1))[Z]-dehydroamarasterone B, has been isolated from seeds of Leuzea (Rhaponticum) carthamoides. It has been unambiguously identified by CIMS, 13C NMR and 1H NMR spectroscopy. The biological activity of the ecdysteroid has been determined in the Drosophila melanogaster BII bioassay. The ED50 (5.2 x 10(-7) M) is 70-fold higher than that for 20-hydroxyecdysone (7.5 x 10(-9) M).

Solution conformation of alpha, beta or gamma-methylglutamyl-containing derivatives as probes of vitamin K-dependent carboxylase using molecular modelling and nuclear magnetic resonance.

In the present study, the conformational behaviour of methyl substituted N-BOC glutamic acid methyl esters (2M, 3T, 3E, 4T, 4E) has been completely characterized through combined NMR and molecular modeling studies. Hetero- and homonuclear coupling constants were measured in order to assign the remaining diastereotopic methylene protons at C(3) and/or C(4), and used for comparison with theoretical data. In parallel, the complete conformational analysis of these analogues has been achieved using molecular mechanics and molecular dynamics (MD) methods. The conformation of the glutamyl residue is established by the excellent agreement between the experimental and calculated side chain scalar coupling constants. The theoretical NMR data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for internal motions. There is a significant influence of the methyl group on the conformational behaviour and on the biological relevance of these structures. Steric effect or electrostatic interaction may also have a considerable influence in stabilizing a conformational population in D2O solution. The conformational preferences of those different analogues in aqueous and methanol solution are discussed in the light of biological results obtained on the vitamin K-dependent carboxylase system.