Antibacterial and mechanism of action studies of boxazomycin A.

Boxazomycins A-C are potent broad-spectrum antibiotics isolated from Actinomycetes strain G495-1 in 1987. We now report that boxazomycin A inhibits bacterial growth by selectively inhibiting protein synthesis, its effect is bacteriostatic, and it is equally active against drug resistant bacterial strains. No cross-resistance to protein synthesis inhibitors was observed suggesting that its inhibition is distinct from clinical protein synthesis inhibitors. We also report in vivo efficacy in a Staphylococcus aureus murine infection model supported by corresponding pharmacokinetic studies.

Isolation, structure elucidation and antibacterial activity of a new tetramic acid, ascosetin.

The ever-increasing bacterial resistance to clinical antibiotics is making many drugs ineffective and creating significant treatment gaps. This can be only circumvented by the discovery of antibiotics with new mechanisms of action. We report here the identification of a new tetramic acid, ascosetin, from an Ascomycete using the Staphylococcus aureus fitness test screening method. The structure was elucidated by spectroscopic methods including 2D NMR and HRMS. Relative stereochemistry was determined by ROESY and absolute configuration was deduced by comparative CD spectroscopy. Ascosetin inhibited bacterial growth with 2-16 µg ml(-1) MIC values against Gram-positive strains including methicillin-resistant S. aureus. It also inhibited the growth of Haemophilus influenzae with a MIC value of 8 µg ml(-1). It inhibited DNA, RNA, protein and lipid synthesis with similar IC50 values, suggesting a lack of specificity; however, it produced neither bacterial membrane nor red blood cell lysis. It showed selectivity for bacterial growth inhibition compared with fungal but not mammalian cells. The isolation, structure and biological activity of ascosetin have been detailed here.

Isolation, structure elucidation, and biological activity of altersolanol P using Staphylococcus aureus fitness test based genome-wide screening.

Bacteria continue to evade existing antibiotics by acquiring resistance by various mechanisms, leading to loss of antibiotic effectiveness. To avoid an epidemic from infections of incurable drug-resistant bacteria, new antibiotics with new modes of action are desperately needed. Using a genome-wide mechanism of action-guided whole cell screening approach based on antisense Staphylococcus aureus fitness test technology, we report herein the discovery of altersolanol P (1), a new tetrahydroanthraquinone from an unknown fungus from the Hypocreales isolated from forest litter collected in Puerto Rico. The structure was elucidated by high-resolution mass spectrometry and 2D NMR spectroscopy. Relative stereochemistry was established by NOESY correlations, and absolute configuration was deduced by the application of MPA ester-based methodology. Observed (1)H and (13)C NMR shifts were well aligned with the corresponding chemical shifts predicted by DFT calculations. Altersolanol P exhibited Gram-positive antibacterial activity (MIC range 1-8 µg/mL) and inhibited the growth of Gram-negative Haemophilus influenzae (MIC 2 µg/mL). The isolation, structure elucidation, and antibacterial activity of altersolanol P are described.

Occurrence, distribution, dereplication and efficient discovery of thiazolyl peptides by sensitive-resistant pair screening.

Natural products have been major sources of antibacterial agents and remain very promising. Frequent rediscoveries of known compounds hampers progress of new discoveries and demands development and utilization of new methods for rapid biological and chemical dereplication. This paper describes an efficient approach for discovery of new thiazolyl peptides by sensitive-resistant pair screening and dereplication in a time and cost-effective manner at industrial scale. A highly effective library-based dereplication of thiazolyl peptides by high resolution fourier transform liquid chromatography mass spectrometry (HRFTLCMS) has been developed, which can detect and dereplicate very low levels of thiazolyl peptides particularly when combined with miniaturized high-throughput 96-well solid-phase extraction separation, and as well can be automated. Combination of sensitive (susceptible)-resistant pair screening, diversified screening collection and miniaturized high-throughput SPE and HRFTLCMS techniques were applied for discovery of new thiazolyl peptides. The combined approach allowed for identification of over 24 thiazolyl peptides represented by three of the five structural subgroups, including three novel compounds. In addition, it is possible for the first time to mechanistically group three structural subgroups of over 24 thiazolyl peptides. Furthermore, these studies helped to understand natural frequency of distribution of these compounds and helped in discovery of new producing strains of many thiazolyl compounds.

Coniothyrione: anatomy of a structure revision.

Coniothyrione is a xanthone-derived antibiotic reported several years ago by researchers at Merck & Co. Inc. Revision of the position of the chloro substitution was recently proposed on the basis of empirical reinterpretation of the carbon chemical shift data and a hypothetical biosynthetic argument without the acquisition of any new spectral data to support the postulated change in substituent location. The originally published HMBC data lead to an equivocal assignment of the structure and do not provide a solid basis of support for either structure. Neural network (13)C chemical shift calculations and density functional theory calculations also led to undifferentiated structures. Definitive confirmation of the structure of coniothyrione based on the acquisition and interpretation of 1,1-ADEQUATE and inverted (1)J(CC) 1,n-ADEQUATE data is now reported.

Isolation, structure, and biological activity of Phaeofungin, a cyclic lipodepsipeptide from a Phaeosphaeria sp. Using the Genome-Wide Candida albicans Fitness Test.

Phaeofungin (1), a new cyclic depsipeptide isolated from Phaeosphaeria sp., was discovered by application of reverse genetics technology, using the Candida albicans fitness test (CaFT). Phaeofungin is comprised of seven amino acids and a ß,γ-dihydroxy-γ-methylhexadecanoic acid arranged in a 25-membered cyclic depsipeptide. Five of the amino acids were assigned with d-configurations. The structure was elucidated by 2D-NMR and HRMS-MS analysis of the natural product and its hydrolyzed linear peptide. The absolute configuration of the amino acids was determined by Marfey's method by complete and partial hydrolysis of 1. The CaFT profile of the phaeofungin-containing extract overlapped with that of phomafungin (3), another structurally different cyclic lipodepsipeptide isolated from a Phoma sp. using the same approach. Comparative biological characterization further demonstrated that these two fungal lipodepsipeptides are functionally distinct. While phomafungin was potentiated by cyclosporin A (an inhibitor of the calcineurin pathway), phaeofungin was synergized with aureobasidin A (2) (an inhibitor of the sphingolipid biosynthesis) and to some extent caspofungin (an inhibitor of glucan synthase). Furthermore, phaeofungin caused ATP release in wild-type C. albicans strains but phomafungin did not. It showed modest antifungal activity against C. albicans (MIC 16-32 µg/mL) and better activity against Aspergillus fumigatus (MIC 8-16 µg/mL) and Trichophyton mentagrophytes (MIC 4 µg/mL). The linear peptide was inactive, suggesting that the macrocyclic depsipeptide ring is essential for target engagement and antifungal activity.

Isolation, structure, and biological activities of Fellutamides C and D from an undescribed Metulocladosporiella (Chaetothyriales) using the genome-wide Candida albicans fitness test.

In a whole-cell mechanism of action (MOA)-based screening strategy for discovery of antifungal agents, Candida albicans was used, followed by testing of active extracts in the C. albicans fitness test (CaFT), which provides insight into the mechanism of action. A fermentation extract of an undescribed species of Metulocladosporiella that inhibited proteasome activity in a C. albicans fitness test was identified. The chemical genomic profile of the extract contained hypersensitivity of heterozygous deletion strains (strains that had one of the genes of the diploid genes knocked down) of genes represented by multiple subunits of the 25S proteasome. Two structurally related peptide aldehydes, named fellutamides C and D, were isolated from the extract. Fellutamides were active against C. albicans and Aspergillus fumigatus with MICs ranging from 4 to 16 µg/mL and against fungal proteasome (IC50 0.2 µg/mL). Both compounds showed proteasome activity against human tumor cell lines, potently inhibiting the growth of PC-3 prostate carcinoma cells, but not A549 lung carcinoma cells. In PC-3 cells compound treatment produced a G2M cell cycle block and induced apoptosis. Preliminary SAR studies indicated that the aldehyde group is critical for the antifungal activity and that the two hydroxy groups are quantitatively important for potency.

Platensimycin and platencin congeners from Streptomyces platensis.

Platensimycin (1a) and platencin (2) are inhibitors of FabF and FabF/H bacterial fatty acid synthase. The discovery of natural congeners is an approach that can render a better understanding of the structure-function relationships of complex natural products. The isolation and structure elucidation of nine new congeners (11-20) of platensimycin and platencin are described from a fermentation broth of Streptomyces platensis. These hydroxylated congeners are likely derived by cytochrome P450 oxidation of the terpenoid units post-cyclization. Polar groups in the terpenoid portion of the molecule produce negative interactions with the hydrophobic pocket of FabF, resulting in poor activities. However, the discovery of these compounds serves an important purpose, not only to understand structure-function relationships, which cannot be easily accessed by chemical modification, but also to provide access to compounds that could be used for structural identification/confirmation of the oxidative trace metabolites produced in vivo during animal experiments.

Isolation, structure and biological activities of platencin A(2)-A(4) from Streptomyces platensis.

Natural products serve as a great reservoir for chemical diversity and are the greatest source for antibacterial agents. Recent discoveries of platensimycin and platencin as inhibitors of bacterial fatty acid biosynthesis enzymes supplied new chemical scaffolds for potential antibacterial agents to overcome resistant pathogens. Discovery of natural congeners augment chemical modification in understanding of structure-activity relationship (SAR). Chemical and biological screening of the extracts led to isolation of three hydroxylated analogs of platencin. The C-12, C-14 and C-15 hydroxylated analogs showed attenuated activities which provided significant understanding of functional tolerance in the diterpenoid portion of the molecule. A truncated and oxidized C-13 natural congener was isolated which suggested direct intermediacy of ent-copalyl diphosphate for the biosynthesis of platensimycins and platencins.

Isolation, structure and biological activities of platensimycin B4 from Streptomyces platensis.

Platensimycin and platencin are inhibitors of FabF and FabF/H bacterial fatty acid synthesis enzymes, respectively. Discovery of natural congeners provides one of the ways to understand the relationship of chemical structure and biological function. Efforts to discover the natural analogs of platensimycin by chemical screening led to the isolation of platensimycin B(4), a glucoside congener of platensimycin. This analog showed significantly attenuated activity and critically defined the limited binding space around the aromatic ring and established the importance of the free phenolic and carboxyl group for the activity.

Isolation, enzyme-bound structure and antibacterial activity of platencin A1 from Streptomyces platensis.

Natural products continue to serve as one of the best sources for discovery of antibacterial agents as exemplified by the recent discoveries of platensimycin and platencin. Chemical modifications as well as discovery of congeners are the main sources for gaining knowledge of structure-activity relationship of natural products. Screening for congeners in the extracts of the fermentation broths of Streptomyces platensis led to the isolation of platencin A(1), a hydroxy congener of platencin. The hydroxylation of the tricyclic enone moiety negatively affected the antibacterial activity and appears to be consistent with the hydrophobic binding pocket of the FabF. Isolation, structure, enzyme-bound structure and activity of platencin A(1) and two other congeners have been described.

Thiazomycins, thiazolyl peptide antibiotics from Amycolatopsis fastidiosa.

Thiazolyl peptides are a class of highly rigid trimacrocyclic compounds consisting of varying but large numbers of thiazole rings. The need for new antibacterial agents to treat infections caused by resistant bacteria prompted a reinvestigation of this class, leading to the previous isolation of thiazolyl peptides, namely, thiazomycin (5) and thiazomycin A (6), congeners of nocathiacins (1-4). Continued chemical screening led to the isolation of six new thiazolyl peptide congeners (8-13), of which three had truncated structures lacking an indole residue. From these, compound 8 showed activity similar to thiazomycin. Two compounds (9 and 10) showed intermediate activities, and the three truncated compounds (11-13) were essentially inactive. The discovery of the truncated compounds revealed the minimal structural requirements for activity and suggested probable biosynthetic pathways for more advanced compounds. The isolation, structure elucidation, antibacterial activity, and proposed biogenesis of thiazomycins are herein described.

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 microg ml(-1) against antisense assay, and showed an MIC of 4-16 microg 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 elucidation, and biological activity of virgineone from Lachnum Wirgineum using the genome-wide Candida albicans fitness test.

A glycosylated tetramic acid, virgineone (1), was isolated from saprotrophic Lachnum virgineum. The antifungal activity of the fermentation extract of L. virgineum was characterized in the Candida albicans fitness test as distinguishable from other natural products tested. Bioassay-guided fractionation yielded 1, a tyrosine-derived tetramic acid with a C-22 oxygenated chain and a beta-mannose. It displayed broad-spectrum antifungal activity against Candida spp. and Aspergillus fumigatus with a MIC of 4 and 16 microg/mL, respectively. Virgineone was also identified in a number of Lachnum strains collected from diverse geographies and habitats.

Isolation, structure and antibacterial activity of pleosporone from a pleosporalean ascomycete discovered by using antisense strategy.

Protein synthesis is one of the best antibacterial targets that have led to the development of a number of highly successful clinical drugs. Protein synthesis is catalyzed by ribosome, which is comprised of a number of ribosomal proteins that help the catalysis process. Ribosomal protein S4 (RPSD) is one of the proteins that is a part of the ribosomal machinery and is a potential new target for the discovery of antibacterial agents. Screening of microbial extracts using antisense-sensitized rpsD Staphylococcus aureus strain led to the isolation of pleosporone, a new compound, with modest antibacterial activities with MIC ranging from 1 to 64 microg/mL. This compound showed the highest sensitivity for Streptococcus pneumoniae and Haemophilus influenzae, and exhibited MIC's of 4 and 1 microg/mL, respectively. Pleosporone showed modest selectivity for the inhibition of RNA synthesis compared to DNA and protein synthesis, and showed activity against HeLa cells. Isolation, structure elucidation, and biological activity of pleosporone have been described.

Isolation, structure and fatty acid synthesis inhibitory activities of platensimycin B1-B3 from Streptomyces platensis.

Platensimycins B(1)-B(3) are natural congeners of platensimycin with modest to significant changes in the benzoic acid portion of the molecule, leading to attenuation in the biological activities and thus confirming the significance of the free carboxylate for the potent activity.

Isolation, structure, and antibacterial activity of phaeosphenone from a Phaeosphaeria sp. discovered by antisense strategy.

Ribosomal protein S4 (RPSD), a part of the ribosomal small subunit, is one of the proteins that is a part of the ribosomal machinery and is a potential new target for the discovery of antibacterial agents. Continued screening of microbial extracts using antisense-sensitized rpsD Staphylococcus aureus strain led to the isolation of a new dimeric compound, phaeosphenone (2). Compound 2 showed broad-spectrum antibacterial activity against Gram-positive bacteria, exhibiting MIC values ranging from 8 to 64 microg/mL. Phaeosphenone showed the highest sensitivity for Streptococcus pneumoniae (8 microg/mL) and inhibited the growth of Candida albicans with an MIC of 8 microg/mL. Phaeosphenone showed a modest selectivity for the inhibition of RNA synthesis over DNA and protein synthesis in S. aureus.

Structure and semisynthesis of platensimide A, produced by Streptomyces platensis.

Platensimycin and platencin are novel natural product antibiotics that inhibit bacterial growth by inhibiting condensing enzymes FabF and FabF/FabH of fatty acid biosynthesis pathways, respectively. Continued search for the natural congeners of these compounds led to the isolation of platensic acid, the free C-17 tetracyclic enoic acid, and platensimide A, a 2,4-diaminobutyric acid amide derivative. Isolation, structure, semisynthesis, and activity of these compounds are described.

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.

Coniothyrione, a chlorocyclopentandienylbenzopyrone as a bacterial protein synthesis inhibitor discovered by antisense technology.

Bacterial protein synthesis inhibitors interact mainly with rRNA and to some extent ribosomal proteins, which are potential targets for developing new antibacterial agents. Specifically, the ribosomal protein S4 of the 30s ribosomal subunit known as ribosomal protein small-subunit D (rpsD) may be useful as a target. The antisense-rpsD gene-sensitized two-plate assay led to the discovery of a novel chlorinated cyclopentandienylbenzopyrone antibiotic, coniothyrione, C14H9ClO6, isolated from Coniothyrium cerealis MF7209. It exhibited liquid MICs of 16-32 microg/mL against Staphylococcus aureus, Bacillus subtilis, Haemophilus influenzae, Streptococcus pneumoniae, and Enterococcus faecalis and >64 microg/mL against Escherichia coli. Isolation, structure elucidation, and antibacterial activity of coniothyrione are described.