Oscar Salazar

Discovery of platencin, a dual FabF and FabH inhibitor with in vivo antibiotic properties.

Emergence of bacterial resistance is a major issue for all classes of antibiotics; therefore, the identification of new classes is critically needed. Recently we reported the discovery of platensimycin by screening natural product extracts using a target-based whole-cell strategy with antisense silencing technology in concert with cell free biochemical validations. Continued screening efforts led to the discovery of platencin, a novel natural product that is chemically and biologically related but different from platensimycin. Platencin exhibits a broad-spectrum Gram-positive antibacterial activity through inhibition of fatty acid biosynthesis. It does not exhibit cross-resistance to key antibiotic resistant strains tested, including methicillin-resistant Staphylococcus aureus, vancomycin-intermediate S. aureus, and vancomycin-resistant Enterococci. Platencin shows potent in vivo efficacy without any observed toxicity. It targets two essential proteins, β-ketoacyl-[acyl carrier protein (ACP)] synthase II (FabF) and III (FabH) with IC50 values of 1.95 and 3.91 μg/ml, respectively, whereas platensimycin targets only FabF (IC50 = 0.13 μg/ml) in S. aureus, emphasizing the fact that more antibiotics with novel structures and new modes of action can be discovered by using this antisense differential sensitivity whole-cell screening paradigm.

A novel actinomycete strain de-replication approach based on the diversity of polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways

The actinomycetes traditionally represent one of the most important sources for the discovery of new metabolites with biological activity; and many of these are described as being produced by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We present a strain characterization system based on the metabolic potential of microbial strains by targeting these biosynthetic genes. After an initial evaluation of the existing bias derived from the PCR detection in a well defined biosynthetic systems, we developed a new fingerprinting approach based on the restriction analysis of these PKS and NRPS amplified sequences. This method was applied to study the distribution of PKS and NRPS biosynthetic systems in a collection of wild-type actinomycetes isolated from tropical soil samples that were evaluated for the production of antimicrobial activities. We discuss the application of this tool as an alternative characterization approach for actinomycetes and we comment on the relationship observed between the presence of PKS-I, PKS-II and NRPS sequences and the antimicrobial activities observed in some of the microbial groups tested.

Isolation, structure, and antibacterial activity of philipimycin, a thiazolyl peptide discovered from Actinoplanes philippinensis MA7347

Bacterial resistance to antibiotics, particularly to multiple drug resistant antibiotics, is becoming cause for significant concern. The only really viable course of action is to discover new antibiotics with novel mode of actions. Thiazolyl peptides are a class of natural products that are architecturally complex potent antibiotics but generally suffer from poor solubility and pharmaceutical properties. To discover new thiazolyl peptides potentially with better desired properties, we designed a highly specific assay with a pair of thiazomycin sensitive and resistant strains of Staphylococcus aureus, which led to the discovery of philipimycin, a new thiazolyl peptide glycoside. It was isolated along with an acid-catalyzed degradation product by bioassay-guided fractionation. Structure of both compounds was elucidated by extensive application of 2D NMR, 1D TOCSY, and HRESIFT-MS/MS. Both compounds showed strong antibacterial activities against gram-positive bacteria including MRSA and exhibited MIC values ranging from 0.015 to 1 microg/mL. Philipimycin was significantly more potent than the degradation product. Both compounds showed selective inhibition of protein synthesis, indicating that they targeted the ribosome. Philipimycin was effective in vivo in a mouse model of S. aureus infection exhibiting an ED50 value of 8.4 mg/kg. The docking studies of philipimycin suggested that a part of the molecule interacts with the ribosome and another part with Pro23, Pro22, and Pro26 of L11 protein, which helped in explaining the differential of activities between the sensitive and resistant strains. The design and execution of the bioassay, the isolation, structure, in vitro and in vivo antibacterial activity, and docking studies of philipimycin and its degradation product are described.

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. Journal of Industrial Microbiology & Biotechnology (2000) 25, 315–327.

Molecular phylogenetic studies on the diatrypaceae based on rDNA-ITS sequences

The order Diatrypales (Ascomycota) contains one single family, the Diatrypaceae. To obtain insight in the phylogenetic relationships within this family, the complete sequences of the ITS region (ITS1, 5.8S rRNA gene and ITS2) of 53 isolates from the five main genera in the family (Diatrype, Diatrypella, Cryptosphaeria, Eutypa and Eutypella) were determined and aligned for phylogenetic reconstruction. Sequence analysis revealed the presence of tandem repeated motifs 11 nucleotides-long, placed in homologous positions along the ITS1 region. Parsimony analysis established the existence of nine monophyletic groups and one branch with a single isolate of Eutypella quaternata. The phylogenetic relationships established by parsimony analysis did not correlate well with classical taxonomic schemes. None of the five genera included in this study was found to be monophyletic. The genera Diatrype, Eutypa and Cryptosphaeria each were divided into two groups. Isolates of Diatrype flavovirens appeared in a clade separated from the one that grouped Diatrype disciformis and the rest of Diatrype species. The Eutypa strains appeared distributed into two clades, one grouping Eutypa lata and related species (Eutypa armeniacae, Eutypa laevata, Eutypa petrakii), and another with the remaining species of the genus. Eutypella (excluding Eutypella quaternaria) appeared as an unstable monophyletic group, which was lost when the sequence alignment was subjected to neighbor-joining analysis. The genus Diatrypella was not associated with any monophyletic group, suggesting that the multisporate asci character has appeared several times during the evolution of the group. Overall, our study suggests the need to revise many of the concepts usually applied to the classification of members of the family.

Isolation and Structure Elucidation of Thiazomycin —A Potent Thiazolyl Peptide Antibiotic from Amycolatopsis fastidiosa

Thiazolyl peptides are a class of rigid macrocyclic compounds richly populated with thiazole rings. They are highly potent antibiotics but none have been advanced to clinic due to poor aqueous solubility. Recent progress in this field prompted a reinvestigation leading to the isolation of a new thiazolyl peptide, thiazomycin, a congener of nocathiacins. Thiazomycin possesses an oxazolidine ring as part of the amino-sugar moiety in contrast to the dimethyl amino group present in nocathiacin I. The presence of the oxazolidine ring provides additional opportunities for chemical modifications that are not possible with other nocathiacins. Thiazomycin is extremely potent against Gram-positive bacteria both in vitro and in vivo. The titer of thiazomycin in the fermentation broth was very low compared to the nocathiacins I and III. The lower titer together with its sandwiched order of elution presented significant challenges in large scale purification of thiazomycin. This problem was resolved by the development of an innovative preferential protonation based one- and/or two-step chromatographic method, which was used for pilot plant scale purifications of thiazomycin. The isolation and structure elucidation of thiazomycin is herein described.

Evaluation of different PCR-based DNA fingerprinting techniques for assessing the genetic variability of isolates of the fungus Epicoccum nigrum

F. ARENAL, G. PLATAS, J. MARTÍN, O. SALAZAR and FERNANDO PELÁEZ.1999.Thirty‐six strains of the fungus Epicoccum nigrum, isolated from different substrata and ecosystems of Europe, America and Africa, were analysed using 14 molecular markers included in 5 different genetic fingerprinting techniques: AP‐PCR, tDNA‐PCR, microsatellite‐primed PCR, ARDRA and AFLP. All of the techniques used were able to differentiate the isolates, showing a high genetic diversity within the species. However, the different techniques detected different levels of similarity among the strains; ARDRA shows the most homogeneous results whilst AP‐PCR shows the most heterogeneous. The similarity indices achieved for each strain were compared for the different techniques. The distribution obtained by microsatellite‐primed PCR was similar to those shown by AP‐PCR techniques. tDNA‐PCR and AFLP rendered similar distributions, and ARDRA showed remarkably different results from the other techniques. The results also reveal the lack of an overall correlation between geographical or ecological origin of the isolates and their genotypes.

Discovery of okilactomycin and congeners from Streptomyces scabrisporus by antisense differential sensitivity assay targeting ribosomal protein S4.

Protein synthesis inhibition is a highly successful target for developing clinically effective and safe antibiotics. There are several targets within the ribosomal machinery, and small ribosomal protein S4 (RPSD) is one of the newer targets. Screening of microbial extracts using antisense-sensitized rpsD Staphylococcus aureus strain led to isolation of okilactomycin and four new congeners from Streptomyces scabrisporus. The major compound, okilactomycin, was the most active, with a minimum detection concentration of 3–12 μg ml−1 against antisense assay, and showed an MIC of 4–16 μg ml−1 against Gram-positive bacteria, including S. aureus. The congeners were significantly less active in all assays, and all compounds showed a slight preferential inhibition of RNA synthesis over DNA and protein synthesis. Antisense technology, due to increased sensitivity, continues to yield new, even though weakly active, antibiotics.

Isolation, Structure, and Antibacterial Activities of Lucensimycins D−G, Discovered from Streptomyces lucensis MA7349 Using an Antisense Strategy⊥

Bacterial resistance to existing antibiotics continues to grow, necessitating the discovery of new compounds of this type. Antisense-based whole-cell target-based screening is a new and highly sensitive antibiotic discovery approach that has led to a number of new natural product antibiotics. Screening with a rpsD-sensitized strain led to the discovery of a number of natural product polyketides from Streptomyces lucensis. Complete workup of the fermentation extract of this strain allowed for the isolation of seven new compounds, lucensimycins A-G (1-3, 4a, 5-7), with varying degrees of antibacterial activities. Lucensimycin E (5) exhibited the best activity and showed MIC values of 32 microg/mL against Staphylococcus aureus and 8 microg/mL against Streptococcus pneumoniae. The isolation, structure elucidation, and antibacterial activities of four new members, lucensimycins D-G, are described. Lucensimycins D (4a) and E (5) are N-acetyl-l-cysteine adducts of lucensimycin A (1). Semisynthesis of lucensimycins D and E from lucensimycin A has also been described. Lucensimycins F and G are myo-inositolyl-alpha-2-amino-2-deoxy-l-idosyl amide derivatives of lucensimycins D and E, respectively. The relative configuration of these compounds was determined, in part, by molecular dynamics simulations.

New genus-specific primers for the PCR identification of novel isolates of the genera Nocardiopsis and Saccharothrix

The taxonomic position of the genera Saccharothrix and Nocardiopsis has evolved in recent years to accommodate an increasing number of actinomycete strains that cannot be clearly distinguished by morphological characters. More recently, the taxonomic reorganization of the genus Saccharothrix has determined the creation of new, related genera, increasing the complexity of the identification of this taxon. Nevertheless, today these genera can still only be identified by applying chemotaxonomic and molecular criteria, and no other tools are available for the rapid distinction of members of the two genera. Phylogenetic analysis based on 16S rDNA nucleotide sequences of reference strains has shown that both genera represent complete distinct lineages within the order Actinomycetales. Differences in the nucleotide sequences of the 16S rDNAs of reference strains were used to design two pairs of genus-specific primers to identify novel members of the genera Nocardiopsis and Saccharothrix by PCR amplification. The genus specificity of these primers was validated with reference strains as well as with wild-type isolates that exhibited morphological characteristics common to both genera. The diversity and taxonomic position of the isolates identified with these tools is also discussed.