High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease.

African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.

New PCR primers for the screening of NRPS and PKS-I systems in actinomycetes: detection and distribution of these biosynthetic gene sequences in major taxonomic groups.

Nonribosomal peptide synthetases (NRPS) and type I polyketide synthases (PKS-I) are biosynthetic systems involved in the synthesis of a large number of important biologically active compounds produced by microorganisms, among others by actinomycetes. In order to assess the occurrence of these biosynthetic systems in this metabolically active bacterial group, we designed new PCR primers targeted to specifically amplify NRPS and PKS-I gene sequences from actinomycetes. The sequence analysis of amplified products cloned from two model systems and used to validate these molecular tools has shown the extreme richness of NRPS or PKS-I-like sequences in the actinomycete genome. When these PCR primers were tested on a large collection of 210 reference strains encompassing all major families and genera in actinomycetes, we observed that the wide distribution of these genes in the well-known productive Streptomyces species is also extended to other minor lineages where in some cases very few bioactive compounds have been identified to date.

Patterns of antimicrobial activities from soil actinomycetes isolated under different conditions of pH and salinity.

AIMS: To evaluate the patterns of the production of antimicrobial compounds by diverse collection of actinomycetes isolated from different geographies under alternative conditions of pH and salinity in the media. METHODS AND RESULTS: Actinomycetes were grouped based on their method of isolation and their phenotype diversity was determined by total fatty acid analysis. A total of 335 representative isolates, including 235 Streptomyces species and 100 actinomycetes from other taxa, were screened for the production of antimicrobial activities against a panel of bacteria, filamentous fungi and yeasts, including some of clinical relevance. Production of antimicrobial activities was detected in 230 strains. In the case of the genus Streptomyces, 181 antimicrobial activities (77% of the tested isolates) were recorded. The activities observed among the other actinomycetes taxa were lower (49% of the tested isolates). CONCLUSIONS: The results of this study support the idea that species of actinomycetes isolated in alternative selective conditions of pH and salinity present a significant capacity to produce compounds with antibacterial or antifungal activity. The best group of isolates in terms of production of active secondary metabolites was the one isolated in saline conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate that these actinomycetes strains isolated in alternative selective conditions of pH and salinity and collected from diverse geographical locations present a significant capacity to produce compounds with antibacterial or antifungal activity.

The complestatins as HIV-1 integrase inhibitors. Efficient isolation, structure elucidation, and inhibitory activities of isocomplestatin, chloropeptin I, new complestatins, A and B, and acid-hydrolysis products of chloropeptin I.

From the screening of a microbial extract library, isocomplestatin (1), a new axial-chiral isomer of complestatin (2) which is a known rigid bicyclic hexapeptide, was identified as a potent natural product inhibitor of HIV-1 integrase, a unique enzyme responsible for viral replication. Isocomplestatin showed inhibitory activities (IC(50)) in coupled 3'-end processing/strand transfer (200 nM), strand transfer (4 microM), and HIV-1 replication (200 nM) in virus-infected cells. Attempted large-scale isolation of 1 by the literature method, used for the isolation of complestatin, led to lower yield and limited availability. We have developed several new, two-step, high-yielding absorption/elution methods of isolation based on reverse-phase chromatography at pH 8 that are applicable to scales from one gram to potential industrial quantities. We have also discovered and determined the structure of two new congeners of 1, namely, complestatins A (4) and B (5), with almost equal HIV-1 integrase activity. They differ from 1 at C2' and C3' of the tryptophan moiety (residue F). Selective acid hydrolysis of chloropeptin I (3), itself a known acid-catalyzed rearranged isomer of 1 and 2 (8'- vs 7'-substitution in tryptophan residue F, respectively), an isomer of complestatin, and isocomplestatin resulted in a number of fragments (6-10) with retention of most of the HIV-1 integrase activity. The structure-activity relationship as revealed by these compounds could possibly lead to the design of better inhibitors or understanding of the HIV-1 integrase target.

New genus-specific primers for the PCR identification of members of the genus Saccharomonospora and evaluation of the microbial diversity of wild-type isolates of Saccharomonospora detected from soil DNAs.

Members of the genus Saccharomonospora are isolated infrequently, probably due to the low occurrence of these actinomycetes in the environment. Although members of this genus can easily be identified by micromorphological criteria, the extensive chemotaxonomic characterization of each new isolate is a time-consuming task which cannot always be undertaken when handling large numbers of strains as is the case in natural products screening programmes. In this work, the design of one set of genus-specific oligonucleotides which allows rapid detection of members of the genus Saccharomonospora by means of PCR-specific amplification is presented. The genus specificity of these primers was validated on a wide range of collection and wild-type strains, and subsequently applied to evaluate the presence of representatives of this taxon directly from soil DNAs. Partial 16S rDNA sequencing of representative wild-type strains was used to validate their genus assignment. Further analyses of PCR fingerprinting patterns and 16S-23S internal transcribed spacer sequences were used to determine the diversity of wild-type isolates obtained from soils. This study shows the usefulness of the application of these primers for the direct identification of members of this genus and in assessment of its occurrence within natural microbial habitats.

Inhibitors of farnesylation of Ras from a microbial natural products screening program.

Mutant ras oncogenes are associated with various human tumors such as pancreas, colon, lung, thyroid, bladder and several types of leukemia. Prenylation of Ras proteins plays a major role in cell proliferation of both normal and cancerous cells. Normal and oncogenic Ras proteins are posttranslationally modified by a farnesyl group that promotes membrane binding. Inhibitors of farnesyl protein transferase (FPTase), the enzyme that catalyzes the prenylation of Ras proteins, inhibit growth of tumor cells. In an effort to identify structurally diverse and unique inhibitors of FPTase, a program devoted to screening of natural products was initiated. This effort led to the identification of 10 different families of compounds, all of which selectively inhibit FPTase with a variety of mechanisms that are reviewed in this manuscript. These compounds originated from the fermentations of a number of microorganisms, either actinomycetes or fungi, isolated from different substrates collected in tropical and temperate areas. A chemotaxonomic discussion on the distribution of each compound among single or different types of microorganisms, either phylogenetically related or unrelated species, is included.

New genus-specific primers for the PCR identification of members of the genera Pseudonocardia and Saccharopolyspora.

Members of the family Pseudonocardiaceae are difficult to identify on the basis of their micromorphology only. The biochemical characterization of each new isolate is a painstaking and time-consuming task which cannot always be undertaken when handling large numbers of strains as is the case in natural product screening programmes. In this study, two sets of genus-specific oligonucleotides were designed which allow rapid detection of members of the genera Pseudonocardia and Saccharopolyspora by means of PCR-specific amplification. The genus specificity of these primers was validated on a wide range of collection strains and the primers were subsequently used to study a group of 106 wild-type isolates that possessed morphological characteristics of the family. Out of this group, 51 strains could be identified as members of the genus Pseudonocardia and only nine isolates could be assigned to the genus Saccharopolyspora. The diversity indicated by whole-cell fatty acid profiles of both wild-type and reference strains was compared with that identified using the oligonucleotide primers. The partial 16S rDNA sequencing of representative wild-type strains was used to validate their genus assignment by PCR-specific amplification. This study shows the industrial usefulness of the application of these direct identification tools as well as the complementary use of two sources of data, PCR-specific amplification results and fatty acid composition, to assess the diversity of a microbial population.

Inhibition of fungal sphingolipid biosynthesis by rustmicin, galbonolide B and their new 21-hydroxy analogs.

The mode of action of the known antifungal macrolides rustmicin (1) and galbonolide B (2) has been determined to be the inhibition of sphingolipid biosynthesis. A large scale fermentation and isolation process was developed for production of large quantities of rustmicin. New 21-hydroxy derivatives of both compounds were isolated from pilot scale fermentations and were also produced by biotransformation of rustmicin and galbonolide B.

Quinoxapeptins: novel chromodepsipeptide inhibitors of HIV-1 and HIV-2 reverse transcriptase. I. The producing organism and biological activity.

Quinoxapeptin A and B are novel chromodepsipeptides which were isolated from a nocardioform actinomycete with indeterminant morphology. Quinoxapeptins A and B are potent inhibitors of HIV-1 and HIV-2 reverse transcriptase and almost equally active against two single mutants forms as well as a double mutant form of HIV-1 reverse transcriptase. Quinoxapeptin A and B are specific inhibitors of HIV-1 and HIV-2 reverse transcriptase because they did not inhibit human DNA polymerase alpha, beta, gamma and delta. Quinoxapeptin A and B are structurally similar to luzopeptin A which was also active against HIV-1 and HIV-2 reverse transcriptase.

Actinoplanic acids A and B as novel inhibitors of farnesyl-protein transferase.

Actinoplanic acids A and B are macrocyclic polycarboxylic acids that are potent reversible inhibitors of farnesyl-protein transferase. Actinoplanic acids A and B were isolated from Actinoplanes sp. MA 7066 while actinoplanic acid B was isolated from both MA 7066 and Streptomyces sp. MA 7099. Actinoplanic acids A and B are competitive with respect to farnesyl diphosphate and are selective inhibitors of farnesyl-protein transferase because they do not inhibit geranylgeranyl-protein transferase type 1 or squalene synthase. MA 7066 is believed to be a novel species of actinomycetes while MA 7099 is believed to be a novel strain of Streptomyces violaceusniger on the basis of morphological, biochemical and chemotaxonomic characteristics as well as its production of actinoplanic acids.

Diversity of actinomycetes and fungi on seaweeds from the Iberian coasts.

As a part of a screening program for natural products from microorganisms, 465 actinomycetes strains and 278 fungal strains were isolated from 44 seaweed specimens (Rhodophyta and Phaeophyta), collected in the Iberian peninsula seashore (Atlantic and Mediterranean coasts). Six different isolation procedures were followed, and seven isolation media were used, some of which included seawater in their composition. The isolated microorganisms were identified to the level of species or genus by microscopic morphology. Determination of species of Streptomyces was performed through total fatty acid analysis by gas chromatography. Richness and diversity of the Streptomyces and actinoplanetes species isolated from the seaweeds are compared, taking into account the geographic location of the samples. Most of the fungi recovered from these samples were terrestrial, although several marine species (Dendryphiella arenaria, Dendryphiella salina, Varicosporina ramulosa, Corollospora intermedia and Asteromyces cruciatus) were also isolated.

Cochinmicins, novel and potent cyclodepsipeptide endothelin antagonists from a Microbispora sp. I. Production, isolation, and characterization.

Cochinmicins I, II, III are novel peptolides produced in submerged-fermentation cultures of Microbispora sp. ATCC 55140. These closely related compounds are separated by HPLC and are novel competitive endothelin antagonists. Cochinmicins II and III are stereoisomeric to each other. Cochinmicin I is the deschloro analog of cochinmicin III.

DNA sequence, products, and transcriptional pattern of the genes involved in production of the DNA replication inhibitor microcin B17.

The 3.8-kilobase segment of plasmid DNA that contains the genes required for production of the DNA replication inhibitor microcin B17 was sequenced. The sequence contains four open reading frames which were shown to be translated in vivo by the construction of fusions to lacZ. The location of these open reading frames fits well with the location of the four microcin B17 production genes, mcbABCD, identified previously through genetic complementation. The products of the four genes have been identified, and the observed molecular weights of the proteins agree with those predicted from the nucleotide sequence. The transcription of these genes was studied by using fusions to lacZ and physical mapping of mRNA start sites. Three promoters were identified in this region. The major promoter for all the genes is a growth phase-regulated OmpR-dependent promoter located upstream of mcbA. A second promoter is located within mcbC and is responsible for a low-level basal expression of mcbD. A third promoter, located within mcbD, promotes transcription in the reverse direction starting within mcbD and extending through mcbC. The resulting mRNA appears to be an untranslated antisense transcript that could play a regulatory role in the expression of these genes.

A clinical isolate of transposon Tn5 expressing streptomycin resistance in Escherichia coli.

The central region of transposon Tn5 carries three antibiotic resistance markers: neo, ble, and str. The str gene codes for a phosphotransferase that inactivates streptomycin. This activity is phenotypically expressed in several gram-negative bacteria but not in Escherichia coli. We identified a Tn5 variant in E. coli clinical isolates that express streptomycin resistance. This transposon carries a 6-base-pair deletion within the str gene, near the 3' end. The same kind of mutation had been previously obtained experimentally from Tn5.

Expression of Tn5-encoded streptomycin resistance in E. coli.

Four Tn5 mutations able to express streptomycin resistance in E. coli were obtained independently. These mutations (called Tn5) were localized and sequenced. All of them consist of a 6 bp deletion in the str gene near the 3' end. The mutation affects a region peculiar for its repetition of an identical 6 bp sequence. The mutation does not affect the level of transcription of the kan, ble, str operon of Tn5, neither does it increase the level of translation of str. The mutation seems to interfere with a post-translational event.

The transposon Tn5 carries a bleomycin-resistance determinant.

Transposon Tn5 carries a determinant for resistance to bleomycin (Bm). Deletion mapping and cloning experiments have shown that this determinant, gene ble, is located between the determinant for kanamycin (Km) and neomycin resistance (gene neo) and the determinant for streptomycin resistance (gene str). Genes neo, ble, and str belong to an operon controlled by the common promoter. The Mr of the ble product, as determined by polyacrylamide gel electrophoresis, is 12000 to 13000.