Diversifying microbial natural products for drug discovery.

Historically, nature has provided the source for the majority of the drugs in use today. More than 20,000 microbial secondary metabolites have been described, but only a small percentage of these have been carried forward as natural product drugs. Natural products are in tough competition with large chemical libraries and with combinatorial chemistries. Hence, each step of a natural product program has to be more efficient than ever, starting from the collection of environmental samples and the selection of strains, to metabolic expression, genetic exploitation, sample preparation and chemical dereplication. This review will focus on approaches for diversifying microbial natural product strains and extract libraries, while decreasing genetic and chemical redundancy.

Sanglifehrin A, a novel cyclophilin-binding compound showing immunosuppressive activity with a new mechanism of action.

We report here on the characterization of the novel immunosuppressant Sanglifehrin A (SFA). SFA is a representative of a class of macrolides produced by actinomycetes that bind to cyclophilin A (CypA), the binding protein of the fungal cyclic peptide cyclosporin A (CsA). SFA interacts with high affinity with the CsA binding side of CypA and inhibits its peptidyl-prolyl isomerase activity. The mode of action of SFA is different from known immunosuppressive drugs. It has no effect on the phosphatase activity of calcineurin, the target of the immunosuppressants CsA and FK506 when complexed to their binding proteins CypA and FK binding protein, respectively. Moreover, its effects are independent of binding of cyclophilin. SFA inhibits alloantigen-stimulated T cell proliferation but acts at a later stage than CsA and FK506. In contrast to these drugs, SFA does not affect IL-2 transcription or secretion. However, it blocks IL-2-dependent proliferation and cytokine production of T cells, in this respect resembling rapamycin. SFA inhibits the proliferation of mitogen-activated B cells, but, unlike rapamycin, it has no effect on CD154/IL-4-induced Ab synthesis. The activity of SFA is also different from that of other known late-acting immunosuppressants, e.g., mycophenolate mofetil or brequinar, as it does not affect de novo purine and pyrimidine biosynthesis. In summary, we have identified a novel immunosuppressant, which represents, in addition to CsA, FK506 and rapamycin, a fourth class of immunophilin-binding metabolites with a new, yet undefined mechanism of action.

Sanglifehrins A, B, C and D, novel cyclophilin-binding compounds isolated from Streptomyces sp. A92-308110. I. Taxonomy, fermentation, isolation and biological activity.

A novel class of macrolides for which the name sanglifehrins is proposed, has been discovered from actinomycete strains based on their high affinity binding for cyclophilin A (CypA), an immunophilin originally identified as a cytosolic protein binding cyclosporin A (CsA). The sanglifehrins were produced by Streptomyces sp. A92-308110. They were isolated and purified by extraction and several chromatographic, activity-guided steps. Sanglifehrins A and B exhibit a 10 to approximately 20 fold higher affinity for CypA than CsA, whereas the affinity of sanglifehrins C and D for CypA is comparable to that of CsA. Sanglifehrins exhibit a lower immunosuppressive activity than CsA when tested in the mixed lymphocyte reaction. Their in vitro activity indicates that they belong to a novel class of immunosuppressants.

Sanglifehrins A, B, C and D, novel cyclophilin-binding compounds isolated from Streptomyces sp. A92-308110. II. Structure elucidation, stereochemistry and physico-chemical properties.

A novel class of macrolides, the sanglifehrins, was discovered by screening of actinomycete strains with a cyclophilin-binding assay. The chemical structures and absolute stereochemistries of the sanglifehrins A, B, C and D were determined unambiguously by NMR-techniques and by X-ray crystallography of the complex with cyclophilin A. Sanglifehrin A consists of a 22-membered macrocycle containing a tripeptide subunit and features in position 23 a chain of nine carbon atoms bearing a spirocyclic substituent. Sanglifehrins A and B are genuine metabolites whereas sanglifehrins C and D are artefacts.

Microbial conversion products of leptomycin B.

Leptomycin B (LMB), a secondary metabolite produced by Streptomyces sp. strain ATS 1287, with known antifungal and antitumor effects, inhibits the nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 regulatory protein Rev and exhibits significant antiproliferative activity. Since LMB itself turned out to be distinctly cytotoxic, a bioconversion screening with a selected set of 29 bacterial and 72 fungal strains was performed in order to obtain metabolites of LMB with reduced antiproliferative effects. Several derivatives of LMB, more polar than the parent compound and produced in yields of > 5%, were detected. Liquid chromatography-mass spectroscopy analysis indicated the type of bioconversion. Fermentations (1-liter scale) of those strains with high rates of transformation were suitable for isolation and characterization of the most prominent metabolites. Thus, bioconversion of LMB with Aspergillus flavus ATCC 9170 and Emericella unguis ATCC 13431 served for isolation of the novel derivatives 26-hydroxy-LMB (30% was the concentration of the metabolite [with respect to LMB] used for bioconversion) and LMB-24-glutaminamide (90%), respectively. Streptomyces rimosus ATCC 28893 converted LMB into 4,11-dihydroxy-LMB (13%) and 2,3-dihydro-LMB (55%). Although the antiproliferative effects of the LMB metabolites could be reduced through microbial conversion, none of these metabolites inhibited the nuclear export of Rev better than LMB itself.

Leptomycin B is an inhibitor of nuclear export: inhibition of nucleo-cytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev is required for unspliced and incompletely spliced viral mRNAs to appear in the cytoplasm and thus for viral replication. Translocation of Rev from the nucleus to the cytoplasm is essential if Rev is to function. We wanted to identify inhibitors of this transport process because they would be potential antiviral agents. RESULTS: The Streptomyces metabolite, leptomycin B, and other antibiotics of the leptomycin/kazusamycin family were identified as inhibitors of the nucleo-cytoplasmic translocation of Rev at nanomolar concentrations. Rev-dependent export of mRNA into the cytoplasm is also blocked by leptomycin B, which inhibits Rev-dependent, but not Rev-independent gene expression in a short-term transfection assay. In primary human monocytes, leptomycin B suppresses HIV-1 replication. CONCLUSIONS: Leptomycin B is the first low molecular weight inhibitor of nuclear export to be identified. Although it cannot be used therapeutically, it should serve as a valuable tool for dissecting nuclear export pathways.

Evaluation of Streptomyces species-groups by pyrolysis mass spectrometry.

Curie-point pyrolysis mass spectrometry was used to evaluate the taxonomic integrity of three subclusters, provisionally labelled S. albidoflavus, S. anulatus and S. halstedii, which formed a species-group in an extensive numerical phenetic survey of the genus Streptomyces. Excellent agreement was found between the results of the triplicate analyses of each strain while the duplicated strains clustered adjacent to one another. Sequential principal component-canonical variates analysis of the experimental data collected on the 32 representative organisms showed that the S. albidoflavus strains formed a distinct group. This result taken together with earlier chemical, molecular and numerical taxonomic data indicates that the S. albidoflavus subcluster corresponds to a distinct species. In contrast, the subclusters equated with S. anulatus and S. halstedii were found to be heterogeneous and hence in need of further study. However, it is evident from the present investigation that Curie-point pyrolysis mass spectrometry provides a rapid and reproducible way of evaluating the taxonomic integrity of Streptomyces species-groups.

Cymbimicin A and B, two novel cyclophilin-binding structures isolated from actinomycetes.

Two novel metabolites, cymbimicins A and B, were isolated from the culture broth of a strain of Micromonospora sp. by screening for cyclophilin binding metabolites from actinomycete strains. Cymbimicin A binds to cyclophilin A with a high affinity six fold lower than to that of cyclosporin A. The binding affinity of cymbimicin B is about 100 times lower. The taxonomy of the producing strain, fermentation, isolation, physical and biological properties and structure elucidation are described.

Microbial biotransformation products of cyclosporin A.

In order to mimic the human metabolic pathway of cyclosporin A (CyA) a total of 28 bacterial and 72 fungal strains was screened for their ability to transform CyA. Among 3 bacteria and 11 fungi, which produced the main human metabolite OL-17 [eta HyMeBmt1]CyA, Actinoplanes sp. (ATCC 53771) achieved the best transformation rate (5.4%). Furthermore, the two N-demethylated minor products [Leu4]CyA (3.2%) and [Leu9]CyA (4.7%) were isolated, both known as minor natural metabolites and the first one also as a human biotransformation product. Microbial conversion of CyA using the actinomycete Sebekia benihana (NRRL 11111) yielded [gamma HyMeLeu4]CyA (35%), [gamma HyLeu4]CyA (4.5%) and [gamma HyMeLeu4, gamma HyMeLeu6]CyA (8.6%). The structures of these derivatives correspond with those of the human metabolic pathway. The related compounds [Nva2]CyA (CyG) and [D-MeSer3]CyA were similarly converted to the corresponding 4-gamma-hydroxylated analogues. None of the biotransformation products showed a better immunosuppressive effect than CyA, although in various cases the cyclophilin binding affinity was comparable to that of CyA.

Antascomicins A, B, C, D and E. Novel FKBP12 binding compounds from a Micromonospora strain.

5 novel ascomycin-like compounds, antascomicins A, B, C, D and E were isolated from a strain of Micromonospora. The antascomicins bind strongly to the FK506-binding protein FKBP12 and antagonize the immunosuppressive activity of FK506 and rapamycin. The strain description, fermentation, structure elucidation and biological activity of these compounds are described.

Novel bioactive compounds from Actinomycetes: a short review (1988-1992).

Novel secondary metabolites continue to be isolated from Actinomycetes. Their biological activities and chemical structures show a wide range of diversity. This short review provides information on the compounds isolated between 1988 and 1992, and highlights interesting substances discovered during screening.

Novel bioactive compounds from actinomycetes.

Actinomycetes form an enormous reservoir of secondary metabolites and enzymes. The potential for exploiting rare actinomycetes is highlighted by the discovery of novel compounds from strains of Spirillospora and Nocardioides. Novel compounds of well known classes of antibiotics, such as polyenes, continue to be discovered. For compounds containing a chromophore, the analysis by high-performance liquid chromatography coupled with a diode-array detector enables the elimination of producers of known compounds and facilitates the discovery of novel compounds or derivatives. The complexity of the regulatory mechanisms is illustrated by glutamine synthetase. The characterization of thermostable amylolytic, lignolytic, peroxidase and neuramidase activities, and the isolation of novel cellulolytic actinomycetes clearly demonstrate the potential of Actinomycetes as producers of enzymes.

Numerical classification and identification of Streptomyces species--a review.

Evidence is presented to show that numerical taxonomy is of proven value both for the circumscription and identification of Streptomyces species. In addition, 252 representatives of numerically defined species and species-groups of this taxon were examined for 273 unit characters and the resultant data analysed using conventional statistics. Clustering was only marginally affected by the proximity coefficients used or by test error, estimated at 3.37%. The numerical classification obtained confirmed and extended the results of previous taxometric surveys, notably by showing that the Streptomyces albidoflavus species-group encompassed taxospecies corresponding to S. albidoflavus, Streptomyces anulatus and Streptomyces halstedii. Rapid enzyme tests based upon the fluorophores, 7-amino-4-methylcoumarin and 4-methylumbelliferone, provide useful data for streptomycete systematics. It can be concluded that the genus Streptomyces is currently well circumscribed and underspeciated.

Pyrolysis mass spectrometry as a method for the classification, identification and selection of actinomycetes.

Pilot experiments were designed to determine the potential of Curie-point pyrolysis mass spectrometry (PyMS) in the classification, identification and typing of industrially significant actinomycetes, and for the detection of target and novel actinomycetes needed for pharmacological screening programmes. The results indicate that the method is of value for the separation of actinomycetes at and below the species level, in the detection and circumscription of novel actinomycetes, and for the detection of identical and duplicated strains. There is also evidence that the pyrolysis system will permit the identification of target actinomycetes directly from selective isolation plates. PyMS is one of the methods that should be used to generate polyphasic taxonomies of actinomycete genera.

Isolation of (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine, the characteristic structural element of cyclosporins, from a blocked mutant of Tolypocladium inflatum.

By mutagenic treatment of a strain of Tolypocladium inflatum, a cyclosporin non-producing mutant was obtained which accumulated the characteristic building unit of cyclosporins, (4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine (abbreviation Bmt; systematic name: (2S,3R,4R,6E)-2-amino-3-hydroxy-4-methyl-6-octenoic acid) in free form. The isolation from a culture filtrate was performed by extraction, chromatographic separation and final crystallization from methanol - water. The structure and stereochemistry of this amino acid was determined by chemical transformation and correlation to dihydro-MeBmt, with known chirality [(2S,3R,4R)-3-hydroxy-4-methyl-2-methylamino-octanoic acid], obtained by hydrolysis of dihydrocyclosporin A.

Protein kinase C activators of the teleocidin family decrease the IgE-binding capacity of rat basophilic leukemia cells.

The tumor promoters 12-O-tetradecanoylphorbol-13-acetate (TPA) and the teleocidins (TCDs) had similar inhibitory effects on IgE binding onto the membrane of rat basophilic leukemia (RBL)-2H3 cells. The level of expression of the functional IgE Fc receptor (Fc epsilon R), as measured by CELISA, was decreased up to a maximum of 60% within 5 min-1 h of treatment. This inhibition was obtained at concentrations of 0.1 microgram/ml for most TCDs, of 1 microgram/ml for TPA and of 20 micrograms/ml for one TCD (olivoretin A). These molecules also decreased the amount of cell-bound IgE detectable by CELISA on cells that had been coated with IgE prior to TCD treatment. When incubated with RBL-2H3 cells for 30 min-2 h, the TCDs and TPA stimulated serotonin release. Depending on their concentration, they had various effects on IgE-plus antigen-induced serotonin release. It is suggested that the down-regulation of IgE receptor expression by these tumor promoters is mediated through protein kinase C activation and phosphorylation of the Fc epsilon R.

Clavamycins, new clavam antibiotics from two variants of Streptomyces hygroscopicus. I. Taxonomy of the producing organisms, fermentation, and biological activities.

In a selective screening for antifungal metabolites, six new clavam antibiotics, clavamycins A, B, C, D, E and F, have been detected from two variants of Streptomyces hygroscopicus NRRL 15846 and NRRL 15879.