Two new constituents from mangrove Bruguiera gymnorrhiza.

A lignan and two aromatic compounds were isolated from the branches of the mangrove plant, Bruguiera gymnorrhiza. They were brugunin A (1), bruguierol D (2) and 2,3-dimethoxy-5-propylphenol (3). Among them, 1 and 2 were new compounds; 3 was isolated from a natural source for the first time. The structures of these compounds were determined by NMR spectroscopic studies as well as chemical evidence.

Three new pimaren diterpenoids from marine mangrove plant, Bruguiera gymnorrhiza.

Three new pimaren diterpenoids, ent-8(14)-pimarene-15R, 16-diol (1), ent-8(14)-pimarene-1alpha,15R,16-triol (2), and (5R, 9S, 10R, 13S, 15S)-ent-8(14)-pimarene-1-oxo-15R,16-diol (3), along with three known diterpenoids (4-6) have been isolated from the stem of mangrove plant Bruguiera gymnorrhiza. The structures of compounds 1-3 were determined by extensive spectroscopic (2D NMR, MS, IR, and CD) analysis, and 3 and 5 showed moderate cytotoxic activities against L-929 and K562, respectively.

Reversible inhibition of calcineurin by the polyphenolic aldehyde gossypol.

The reversible inhibition of calcineurin (CaN), which is the only Ca(2+)/calmodulin-dependent protein Ser/Thr phosphatase, is thought to be a key functional event for most cyclosporin A (CsA)- and tacrolimus (FK506)-mediated biological effects. In addition to CaN inhibition, however, CsA and FK506 have multiple biochemical effects because of their action in a gain-of-function model that requires prior binding to immunophilic proteins. We screened a small molecule library for direct inhibitors of CaN using CaN-mediated dephosphorylation of (33)P-labeled 19-residue phosphopeptide substrate (RII phosphopeptide) as an assay and found the polyphenolic aldehyde gossypol to be a novel CaN inhibitor. Unlike CsA and FK506, gossypol does not require a matchmaker protein for reversible CaN inhibition with an IC(50) value of 15 microm. Gossypolone, a gossypol analog, showed improved inhibition of both RII phosphopeptide and p-nitrophenyl phosphate dephosphorylation with an IC(50) of 9 and 6 microm, respectively. In contrast, apogossypol hexaacetate was inactive. Gossypol acts noncompetitively, interfering with the binding site for the cyclophilin 18.CsA complex in CaN. In contrast to CsA and FK506, gossypol does not inactivate the peptidyl-prolyl-cis/trans-isomerase activity of immunophilins. Similar to CsA and FK506, T cell receptor signaling induced by phorbol 12-myristate 13-acetate/ionomycin is inhibited by gossypol in a dose-dependent manner, demonstrated by the inhibition of nuclear factor of activated T cell (NFAT) c1 translocation from the cytosol into the nucleus and suppression of NFAT-luciferase reporter gene activity.

New 1-O-acyl alpha-L-rhamnopyranosides and rhamnosylated lactones from Streptomyces sp., inhibitors of 3 alpha-hydroxysteroid-dehydrogenase (3alpha-HSD).

Chemical screening with extracts of Streptomyces sp. (strain GT 61150) resulted in the detection, isolation, and structure elucidation of two new acyl alpha-L-rhamnopyranosides (1 and 2) and three new rhamnosyllactones A, B1 and B2 (3 approximately 5). Rhamnosyllactones B1 and B2 were obtained as a 5:1 mixture. The structures were confirmed by spectroscopic analysis, especially 2D-NMR techniques. The rhamnosyltransferase of our strain is able to connect the sugar moiety to heteroaromatic carboxylic acids and enols. The metabolites 1 and 4/5 as well as previously reported acylrhamnosides 6 approximately 11 inhibit the enzyme 3alpha-hydroxysteroid-dehydrogenase (3alpha-HSD).

Feigrisolides A, B, C and D, new lactones with antibacterial activities from Streptomyces griseus.

Four new lactone compounds, named feigrisolides A to D (1 to 4), have been isolated from Streptomyces griseus. The chemical structures were determined by detail analysis of their spectroscopic data and chemical transformations. Structurally, the feigrisolides A (1) and B (2) are hepta-lactones, feigrisolide C (3) and D (4) are 16-membered macrodiolides. Biological studies showed that feigrisolide B (2) exhibited strong antibacterial, as well as medium cyctotoxic, and antiviral activities. Feigrisolides A (1), C (3) and D (4) are medium inhibitors of 3alpha-hydroxysteroid-dehydrogenase (3alpha-HSD) inhibiting activity.

Synthesis and cytotoxic evaluation of cycloheximide derivatives as potential inhibitors of FKBP12 with neuroregenerative properties.

On the basis of the new finding that the protein synthesis inhibitor cycloheximide (1, 4-[2-(3, 5-dimethyl-2-oxocyclohexyl)-2-hydroxyethyl]-2,6-piperidinedione) is able to competitively inhibit hFKBP12 (K(i) = 3.4 microM) and homologous enzymes, a series of derivatives has been synthesized. The effect of the compounds on the activity of hFKBP12 and their cytotoxicity against eukaryotic cell lines (mouse L-929 fibroblasts, K-562 leukemic cells) were determined. As a result, several less toxic or nontoxic cycloheximide derivatives were identified by N-substitution of the glutarimide moiety and exhibit IC(50) values in the range of 22.0-4.4 microM for inhibition of hFKBP12. Among these compounds cycloheximide-N-(ethyl ethanoate) (10, K(i) = 4.1 microM), which exerted FKBP12 inhibition to an extent comparable to that of cycloheximide (1), was found to cause an approximately 1000-fold weaker inhibitory effect on eukaryotic protein synthesis (IC(50) = 115 microM). Cycloheximide-N-(ethyl ethanoate) (10) was able to significantly speed nerve regeneration in a rat sciatic nerve neurotomy model at dosages of 30 mg/kg.

Strep-tag II for one-step affinity purification of active bHLHzip domain of human c-Myc.

The c-Myc protein, the product of the c-myc protooncogene, is a nuclear phosphoprotein with DNA-binding properties when heterodimerized with the Max protein. It contains an amino-terminal transcriptional activation domain and a carboxy-terminal basic helix-loop-helix leucine zipper (bHLHzip) domain that directs heterodimerization and promotes DNA binding. Here, we describe the isolation of the bHLHzip domain of human c-Myc with a technique for efficient single-step purification. Using a C-terminal Strep-tag II affinity peptide and a novel Streptactin-Sepharose matrix, elution is performed under mild conditions by competition with the biotin analog desthiobiotin. No significant influence of the affinity tag on the activity of the bHLHzip domain was observed when the fusion protein was subjected to glutathione S-transferase (GST) pull-down assays for investigating its in vitro-binding properties with GST-Max. The use of the C-terminal Strep-tag II was shown to be more suitable for obtaining pure product fractions than use of the N-terminal GST affinity tag.

Dual reporter systems in yeast and mammalian cells for assessing progesterone receptor modulators.

In the present study we describe the set-up of a new one-hybrid reporter gene assay in Saccharomyces cerevisiae composed of the human progesterone receptor fused to the DNA-binding domain of the yeast transcriptional activator Gal4. This assay allows the convenient estimation of receptor mediated progestogenic as well as antiprogestogenic actions of compounds. The induction of the beta-galactosidase reporter gene expression correlated well with the progesterone receptor affinity and the concentration of the progestins tested. The results corresponded to those obtained from a reporter gene assay in the cancer cell line CV-1 and in vitro binding experiments using rabbit uterus cytosol. In both the yeast and CV-1 cells the activity of antiprogestins was detectable by inhibition of the progestin-induced reporter gene expression. Secondary reporter genes under the transcriptional control of receptor unrelated promoters have been introduced into yeast and mammalian test strains to distinguish between specific receptor mediated antihormone actions and nonspecific effects on cellular metabolism.

Bioactive agents from natural sources: trends in discovery and application.

About 30% of the worldwide sales of drugs are based on natural products. Though recombinant proteins and peptides account for increasing sales rates, the superiority of low-molecular mass compounds in human diseases therapy remains undisputed mainly due to more favorable compliance and bioavailability properties. In the past, new therapeutic approaches often derived from natural products. Numerous examples from medicine impressively demonstrate the innovative potential of natural compounds and their impact on progress in drug discovery and development. However, natural products are currently undergoing a phase of reduced attention in drug discovery because of the enormous effort which is necessary to isolate the active principles and to elucidate their structures. To meet the demand of several hundred thousands of test samples that have to be submitted to high-throughput screening (HTS) new strategies in natural product chemistry are necessary in order to compete successfully with combinatorial chemistry. Today, pharmaceutical companies have to spend approximately US $350 million to develop a new drug. Currently, approaches to improve and accelerate the joint drug discovery and development process are expected to arise mainly from innovation in drug target elucidation and lead finding. Breakthroughs in molecular biology, cell biology, and genetic engineering in the 1980 s gave access to understanding diseases on the molecular or on the gene level. Subsequently, constructing novel target directed screening assay systems of promising therapeutic significance, automation, and miniaturization resulted in HTS approaches changing the industrial drug discovery process drastically. Furthermore, elucidation of the human genome will provide access to a dramatically increased number of new potential drug targets that have to be evaluated for drug discovery. HTS enables the testing of an increasing number of samples. Therefore, new concepts to generate large compound collections with improved structural diversity are desirable.

Natural Products in High Throughput Screening: Automated High-Quality Sample Preparation.

At present, compound libraries from combinatorial chemistry are the major source for high throughput screening (HTS) programs in drug discovery. On the other hand, nature has been proven to be an outstanding source for new and innovative drugs. Secondary metabolites from plants, animals, and microorganisms show a striking structural diversity that supplements chemically synthesized compounds or libraries in drug discovery programs. Unfortunately, extracts from natural sources are usually complex mixtures of compounds, often generated in time-consuming and, for the most part, manual processes. Because quality and quantity of the provided samples play a pivotal role in the success of HTS programs, this poses serious problems. In order to make samples of natural origin competitive with synthetic compound libraries, we devised a novel, automated sample preparation procedure based on solid-phase extraction (SPE). By making use of modified Zymark (Hopkinton, MA) RapidTrace® SPE workstations, we developed an easy-to-handle and effective fractionation method that generates high-quality samples from natural origin, fulfilling the requirements for an integration in high throughput drug discovery programs.

Biomolecular-chemical screening: a novel screening approach for the discovery of biologically active secondary metabolites. I. Screening strategy and validation.

Chemical screening using thin-layer chromatography and various staining reagents offers the opportunity to visualize an almost complete picture of a microbial secondary metabolite pattern (metabolic finger-print). A thorough application of this strategy resulted in a number of biologically active new secondary metabolites, although the screening strategy is per se not correlated to any biological activity. In the present paper we report on a novel approach called biomolecular-chemical screening which combines the chemical screening strategy with binding studies of biological relevance. Making use of thin-layer chromatography (TLC) and subsequent staining, biomolecular-chemical screening allows to examine binding properties of low molecular weight metabolites to certain bio-macromolecules. The screening strategy itself, as well as independent validation of the results using DNA as selected bio-macromolecule are presented. The biomolecular-chemical screening method is useful to screen binding behaviour towards DNA of both, pure metabolites by one-dimensional TLC, and crude extracts by two-dimensional TLC. Investigation of pure secondary metabolites as well as screening of crude microbial extracts and new secondary metabolites obtained with this screening strategy are presented in accompanying papers.

Biomolecular-chemical screening: a novel screening approach for the discovery of biologically active secondary metabolites. II. Application studies with pure metabolites.

The novel screening strategy called "biomolecular-chemical screening" combines the advantages of the chemical screening approach--the analysis of the chromatographic and chemical behaviour of secondary metabolites on TLC plates--with binding studies of these molecules with bio-macromolecules like DNA. This approach was advantageously used to detect the interaction of pure compounds with DNA. In order to prove the reliability of the biomolecular-chemical screening and to examine DNA-binding properties, 470 pure secondary metabolites were analysed by this method. Besides the confirmation of already known binders with the TLC-based method, for a number of natural products DNA-binding properties were discovered for the first time. In consequence, binding of pure compounds can be measured by 1D TLC in a reliable and easy manner, in which DNA is applied together with the test compound at the starting spot. Analysis is performed via differences in Rf-values in comparison to a reference chromatogram without DNA.

Biomolecular-chemical screening: a novel screening approach for the discovery of biologically active secondary metabolites. III. New DNA-binding metabolites.

Based on the chemical screening technique, biomolecular-chemical screening has been developed which makes use of two-dimensional TLC analysis of microbial extracts and combines thin-layer chromatography (RP-18) with binding studies towards DNA. In the first dimension the metabolites of the crude microbial extract are separated, and in the second dimension binding properties towards DNA are analysed. An initial screening program with 500 microbial extracts prepared by solid-phase extraction with XAD-16 resin resulted in 17 samples which contained metabolites with significant DNA-binding behavior. Fermentation, isolation and structural characterization led to already known metabolites [phenazine-1,6-dicarboxylate (1), phencomycin (2), 11-carboxy-menoxymycin B (3), soyasaponine I (4), and (8S)-3-(2-hydroxypropyl)-cyclohexanone (5)], as well as to new secondary metabolites. Fermentation of the producing organisms of the new DNA-binding metabolites, ent-8,8adihydro-ramulosin (6). (2R,4R)-4-hydroxy-2-(1,3-pentadienyl)-piperidine (7), (5R)-dihydro-5-pentyl-4'-methyl-4'-hydroxy-2(3H)-furanone (8), and seco-4,23-hydroxyoleane-12-en-22-one-3-carboxylic acid (9), as well as isolation, structural characterization, and physico-chemical properties are reported.

Selective inactivation of parvulin-like peptidyl-prolyl cis/trans isomerases by juglone.

In contrast to FK506 binding proteins and cyclophilins, the parvulin family of peptidyl-prolyl cis/trans isomerases (PPIases; E.C. 5.2.1.8) cannot be inhibited by either FK506 or cyclosporin A. We have found that juglone, 5-hydroxy-1,4-naphthoquinone, irreversibly inhibits the enzymatic activity of several parvulins, like the E. coli parvulin, the yeast Ess1/Ptf1, and human Pin1, in a specific manner, thus allowing selective inactivation of these enzymes in the presence of other PPIases. The mode of action was studied by analyzing the inactivation kinetics and the nature of products of the reaction of E. coli parvulin and its Cys69Ala variant with juglone. For all parvulins investigated, complete inactivation was obtained by a slow process that is characterized by pseudo-first-order rate constants in the range of 5.3 x 10(-)4 to 4. 5 x 10(-)3 s-1. The inactivated parvulin contains two juglone molecules that are covalently bound to the side chains of Cys41 and Cys69 because of a Michael addition of the thiol groups to juglone. Redox reactions did not contribute to the inactivation process. Because thiol group modification was shown to proceed 5-fold faster than the rate of enzyme inactivation, it was considered as a necessary but not sufficient condition for inactivation. When measured by far-UV circular dichroism (CD), the rate of structural alterations following thiol group modification parallels exactly the rate of inactivation. Thus, partial unfolding of the active site of the parvulins was thought to be the cause of the deterioration of PPIase activity.

Label-free monitoring of DNA-ligand interactions.

We report on the label- and isotope-free monitoring of DNA interactions with low-molecular-weight ligands. An optical technique based on interference at thin layers was used to monitor in real time binding of ligands at DNA which was immobilized by Coulomb interactions at a positively charged surface. Approximately 2 ng DNA/m2 was irreversibly bound to the surface, which remained stable over several days. This result was confirmed by characterization of the layer using spectroscopic ellipsometry. During incubation of immobilized DNA with a variety of intercalators and other DNA-binding compounds in a flow system, interactions were monitored by reflectometric interference spectroscopy. Binding effects between 10 and 400 pg/ mm2 were detected unambiguously. Nonspecific binding effects were excluded by using a negatively charged reference surface. Variation of intercalator concentration allowed the characterization of interaction with respect to kinetics and thermodynamics by the evaluation of binding rate and equilibrium coverage. The affinity constants were determined in the range between 10(5) and 10(6) M-1, in good agreement to those obtained by homogeneous phase assays. Association rate constants between 10(3) and 10(5) M-1 s-1 and dissociation rate constants between 10(-1) and 10(-2) s-1 were determined by evaluation of the binding curves. Both the fast and simple test format and a universal applicability make the new technique described attractive for detecting and characterizing interaction of low-molecular-weight molecules with DNA.

Specific binding of low molecular weight ligands with direct optical detection.

The characterization of low molecular weight ligand interaction with receptor molecules is of importance for the investigation of biological processes and for drug research. We report on the investigation of the binding of low molecular weight ligands to immobilized receptors by label-free detection. Reflectometric interference spectroscopy, an optical transducer which allows the monitoring of a few picograms per square millimetre changes in surface coverage, was used to study two model systems. In both cases detection of the binding event was successful. High affinity binding of biotin to immobilized streptavidin was clearly detectable at receptor surface concentrations as low as 1-2 x 10(10) binding sites/mm2. Linear correlation between the receptor surface concentration and the response to biotin binding was observed. Using immobilized DNA, we investigated the binding of common intercalators with respect to kinetics and thermodynamics by evaluation of the association and the dissociation part of the binding curve. Bi-exponential increase and decrease of intercalator loading was observed, indicating complex interaction kinetics. The four structurally different intercalators showed significant distinction in binding kinetics and equilibrium signals. Improvement of experimental parameters is required to obtain more reliable kinetic data.

New cineromycins and musacins obtained by metabolite pattern analysis of Streptomyces griseoviridis (FH-S 1832). I. Taxonomy, fermentation, isolation and biological activity.

Chemical screening using thin layer chromatography and various staining reagents offers the opportunity to visualize a nearly complete picture of a microbial secondary metabolite pattern (metabolic finger-print). This approach can be used advantageously for both, the detection of so-called "talented" strains, and for qualifying microbial strain collections, especially as a fundamental step of efficiently applied biological high-throughput assays. Based on their metabolic finger-print, microbial isolates can be classified in: (i) non-producing organisms, which gave no indication of the formation of secondary metabolites up to a defined detection limit, (ii) organisms of narrow productivity, which produce one or two secondary metabolites as main products with a restricted dependence to alteration of the culture conditions, and (iii) talented organisms, which are able to synthesize an array of structurally different secondary metabolites. As an example, the talented strain, Streptomyces griseoviridis (FH-S 1832), was studied in detail. Investigations in its taxonomical characterization, fermentation, as well as the isolation and purification procedures leading to 14-membered macrocyclic lactones of the cineromycin-type (cineromycin B and three new congeners) and to the musacins A to F are reported. Musacin C exhibits anthelminthic and weak antiviral activities.