Susana P. Gaudêncio

The Ammosamides: Structures of Cell Cycle Modulators from a Marine‐Derived Streptomyces Species

Marine-derived actinomycete bacteria are emerging as a valuable resource for bioactive natural products encompassing a variety of unique structural classes.[1] In our hands, early detection of cell growth inhibitors using in vitro cytotoxicity assays against the colon carcinoma cancer cell line HCT-116, followed by extensive mechanism of action studies, has proven to be an effective approach. As such, the HCT-116 assay has been instrumental in the identification of potentially important anticancer agents.[2] In the course of our continued studies, Streptomyces strain CNR-698[3] was isolated from bottom sediments collected at a depth of 1618 meters in the Bahamas Islands in 2003. Cytotoxicity-guided (HCT-116) fractionation by C18 flash chromatography and RP-HPLC of crude extract led to the isolation of ammosamides A (1) and B (2) as blue and red solids, respectively (3 and 4 mg L−1). Structure assignments for 1 and 2 proved to be particularly difficult due to their inherent insolubility (soluble only in dimethyl sulfoxide (DMSO)) and a lack of descriptive NMR signals, ultimately requiring the integration of NMR spectral analysis, mass spectrometry data, and single crystal X-ray diffraction studies. High-resolution (ESI) mass spectrometric analysis of ammosamide A (1) indicated a molecular formula C12H1035ClN5OS (m/z [M+H]+: 308.0303]. The molecular weight of ammosamide B (2) was found to be 16 amu lower (m/z [M+H]+: 292.0604) consistent with the molecular formula C12H1035ClN5O2. The UV/Vis spectrum of 1 was indicative of an unusually highly conjugated structure with absorptions at λmax=580, 430, 350, and 290 nm. Inspection of the 1H NMR spectrum of 1 in [D6]DMSO revealed six singlets between δ=6.0 and 9.0 ppm and one methyl singlet at δ=4.03 ppm, while the 13C NMR spectra revealed the presence of eleven sp2 hybridized carbon atoms and a single sp3 hybridized carbon atom at δc=33.3 ppm (Table 1). The addition of D2O (20 μL) to the sample in [D6]DMSO resulted in the immediate disappearance of 1H NMR signals at δ=7.16 (1H), 6.63 (1H), 6.89 ppm (2H) and the slower disappearance of singlets at δ=8.92 (1H), and 7.68 ppm (1H) (less than 10 min). The exchangeable protons at δ=7.16, 6.63 and 6.89 ppm were assigned as aromatic amines at C-6 and C-8 (based on HMBC correlations), while the slowly exchanging protons at δ=8.92 and 7.68 ppm were assigned to a primary amide on the basis of COSY and HMBC correlations. The only non-exchangeable hydrogen atoms were the methyl singlet resonance at δ=4.03 ppm and a one-proton singlet at δ=8.47 ppm. The 13C NMR spectrum of 1 indicated the presence of two carbonyl groups (δc=177.2 and 166.0 ppm), as well as two upfield sp2 carbon atoms (δc=103.1 and 110.5 ppm). HMBC correlations between the downfield carbonyl (δc=177.2 ppm) and the proton methyl singlet at δ=4.03 ppm, we thought, defined an N-methyl amide, although a carbon chemical shift so far downfield would not be expected. In addition to correlations from the aromatic δ=8.47 ppm singlet, the only other HMBC correlations were from the exchangeable protons at δ=7.16/6.63 ppm to C-7 (δc=103.1 ppm) and from δ=6.89 ppm to C-7 and C-8a (δc=110.5 ppm). Table 1 NMR spectral data for 1 and 2 ([D6]DMSO). The spectral data for 1 suggested a highly unsaturated azaaromatic metabolite possessing three rings. However, the lack of definitive NMR assignments that could be used to link these features forced us to concentrate efforts toward obtaining an X-ray crystal structure. We were fortunate to obtain small crystals of 1 by the slow diffusion of H2O into a saturated solution in DMSO.[4] The X-ray assignment of ammosamide A (1) is shown in Figure 1. Once X-ray data became clear, the spectral data for 1 could be assigned. Figure 1 X-ray crystal structure of ammosamide A (1). Red O, blue N, yellow Cl, black C, white H. The structure assignment of ammosamide B (2) followed from analysis of spectral data and chemical interconversion. Comparison of the C-2 carbonyl chemical shifts in 1 (δC=177.2 ppm) and 2 (δC=164.0 ppm) revealed a difference of 13 ppm, consistent with the typical 13C chemical shift difference between a carbonyl and a thiocarbonyl (ca. 20 ppm).[5] In order to chemically confirm the presence of the thiolactam functionality, we used Lawessons reagent [2,4-bis(p-methoxyphenyl)-1,3-dithiadiphosphetane-2,4-disulfide] to convert lactam 2 into thiolactam 1.[6] The low yield of this reaction is likely attributable the nucleophilic amines in 2.[7] Exposed to air during storage, 1 was gradually converted to ammosamide B (2). Notably, the transformation could also be accomplished in 10 min, upon treatment of 1 with hydrogen peroxide in aqueous methanol.[8] This reactivity has been previously observed in other thioamide-containing compounds.[9] The structural similarities between the ammosamides and the microbial product lymphostin (3) are clear,[10] as is the relationship of the ammosamides to several sponge-derived pyrroloiminoquinone natural products, including batzelline A (4),[11a] isobatzelline D (5),[11b] and makaluvamine A (6)[11c] (Scheme 1). The sponge metabolites 4–6 possess different patterns of Cl and NH2 substitution and assume p-iminoquinone and o-quinone structures. The presence of an amino group at C-8 in the ammosamides results in a fundamentally different structure type in which the quinoline tautomer predominates. The pyrrole moiety in 3–6 is uniquely oxidized to the pyrrolidinone in ammosamide B (2). Finally, though methyl sulfides are present in 4 and 5, ammosamide A (1) is the first natural product to contain a thio-γ-lactam functionality.[12] Scheme 1 Related metabolites from bacteria and sponges. The fact that the ammosamides are highly colored (1: λmax=580 nm; 2: λmax=530 nm), yet lack quinone or iminoquinone functionalities, leads to speculation about the electronic character and reactivity of these metabolites. The intense colors of these compounds could reflect a strong charge separation between the two six-membered aromatic rings due to the effects of electron-donating groups on the chlorine-containing ring and electron-withdrawing substituents on the pyridine ring. It is, conceptually, also explained by the potential for ammosamide A to exist in an equilibrium with its bis-iminoquinone tautomer (Scheme 2). Furthermore, in 1 and 2 the chlorine atom at C-7 is poised to engage in nucleophilic aromatic substitution with a suitable nucleophile, particularly when the molecule exists as its bis-iminoquinone tautomer.[13] This reactivity may be relevant to the molecules interaction with its protein target.[14] Scheme 2 Possible tautomeric form of ammosamide A (1). Ammosamides A (1) and B (2) exhibited significant in vitro cytotoxicity against HCT-116 colon carcinoma, each with IC50=320 nM. These compounds also demonstrated pronounced selectivity in a diversity of cancer cell lines with values ranging from 20 nM to 1 μM, indicating a specific target mechanism of action. To explore the intracellular target of the ammosamides, ammosamide B (2) was converted to a highly fluorescent molecule by conjugation.[14] Treatment of HCT-116 colon carcinoma or HeLa cells with this fluorescent molecule produced immediate and irreversible labeling of a specific protein in the cellular cytosol. Using a cell and molecular biology approach, the target of the ammosamides was identified as a member of the myosin family, important cellular proteins that are involved in numerous cell processes, including cell cycle regulation, cytokinesis, and cell migration.

Sequence-Based Analysis of Secondary-Metabolite Biosynthesis in Marine Actinobacteria

ABSTRACT A diverse collection of 60 marine-sediment-derived Actinobacteria representing 52 operational taxonomic units was screened by PCR for genes associated with secondary-metabolite biosynthesis. Three primer sets were employed to specifically target adenylation domains associated with nonribosomal peptide synthetases (NRPSs) and ketosynthase (KS) domains associated with type I modular, iterative, hybrid, and enediyne polyketide synthases (PKSs). In total, two-thirds of the strains yielded a sequence-verified PCR product for at least one of these biosynthetic types. Genes associated with enediyne biosynthesis were detected in only two genera, while 88% of the ketosynthase sequences shared greatest homology with modular PKSs. Positive strains included representatives of families not traditionally associated with secondary-metabolite production, including the Corynebacteriaceae, Gordoniaceae, Intrasporangiaceae, and Micrococcaceae. In four of five cases where phylogenetic analyses of KS sequences revealed close evolutionary relationships to genes associated with experimentally characterized biosynthetic pathways, secondary-metabolite production was accurately predicted. Sequence clustering patterns were used to provide an estimate of PKS pathway diversity and to assess the biosynthetic richness of individual strains. The detection of highly similar KS sequences in distantly related strains provided evidence of horizontal gene transfer, while control experiments designed to amplify KS sequences from Salinispora arenicola strain CNS-205, for which a genome sequence is available, led to the detection of 70% of the targeted PKS pathways. The results provide a bioinformatic assessment of secondary-metabolite biosynthetic potential that can be applied in the absence of fully assembled pathways or genome sequences. The rapid identification of strains that possess the greatest potential to produce new secondary metabolites along with those that produce known compounds can be used to improve the process of natural-product discovery by providing a method to prioritize strains for fermentation studies and chemical analysis.

Ammosamides A and B Target Myosin

Cytoskeletal proteins, including microfilaments, microtubules, and intermediate filaments, play a pivotal role in the treatment of cancer, as their regulation by small molecules arrests progression through the cell cycle.[1] Marine natural products contain a diversity of molecules that target the cytoskeleton. For instance, the cyclic peptide jasplakinolide induces assembly and stabilization of actin microfilaments.[2] The cytoskeleton is also accessed by other classes of natural products. Several polyketides, including halichondrin B and spongistatin, target microtubule stabilization,[3] while phor-boxazole B employs cytokeratin as a foundation to recruit critical cycle regulators.[4] Our interest focused on deep-sea actinomycetes[5] in an effort to identify metabolites that target other components of the cytoskeleton.

Fijiolides A and B, Inhibitors of TNF-α-Induced NFκB Activation, from a Marine-Derived Sediment Bacterium of the Genus Nocardiopsis

Fijiolide A, a potent inhibitor of TNF-alpha-induced NFkappaB activation, along with fijiolide B, were isolated from a marine-derived bacterium of the genus Nocardiopsis. The planar structures of fijiolides A (1) and B (2) were elucidated by interpretation of 2D NMR spectroscopic data, while the absolute configurations of these compounds were defined by interpretation of circular dichroism and 2D NMR data combined with application of the advanced Moshers method. Fijiolides A and B are related to several recently isolated chloroaromatic compounds, which appear to be the Bergman cyclization products of enediyne precursors. Fijiolide A reduced TNF-alpha-induced NFkappaB activation by 70.3%, with an IC(50) value of 0.57 micro-M. Fijiolide B demonstrated less inhibition, only 46.5%, without dose dependence. The same pattern was also observed with quinone reductase (QR) activity: fijiolide A was found to induce quinone reductase-1 (QR1) with an induction ratio of 3.5 at a concentration of 20 microg/mL (28.4 microM). The concentration required to double the activity was 1.8 microM. Fijiolide B did not affect QR1 activity, indicating the importance of the nitrogen substitution pattern for biological activity. On the basis of these data, fijiolide A is viewed as a promising lead for more advanced anticancer testing.

Multiplex de novo sequencing of peptide antibiotics

Proliferation of drug-resistant diseases raises the challenge of searching for new, more efficient antibiotics. Currently, some of the most effective antibiotics (i.e., Vancomycin and Daptomycin) are cyclic peptides produced by non-ribosomal biosynthetic pathways. The isolation and sequencing of cyclic peptide antibiotics, unlike the same activity with linear peptides, is time-consuming and error-prone. The dominant technique for sequencing cyclic peptides is nuclear magnetic resonance (NMR)-based and requires large amounts (milligrams) of purified materials that, for most compounds, are not possible to obtain. Given these facts, there is a need for new tools to sequence cyclic non-ribosomal peptides (NRPs) using picograms of material. Since nearly all cyclic NRPs are produced along with related analogs, we develop a mass spectrometry approach for sequencing all related peptides at once (in contrast to the existing approach that analyzes individual peptides). Our results suggest that instead of attempting to isolate and NMR-sequence the most abundant compound, one should acquire spectra of many related compounds and sequence all of them simultaneously using tandem mass spectrometry. We illustrate applications of this approach by sequencing new variants of cyclic peptide antibiotics from Bacillus brevis, as well as sequencing a previously unknown family of cyclic NRPs produced by marine bacteria. Supplementary Material is available online at www.liebertonline.com/cmb.

A short synthesis of staurosporinone (K-252c)

A new, simple, high-yield synthesis of the indolo[2,3-a]carbazole alkaloid staurosporinone is described.

A chemoinformatics approach to the discovery of lead-like molecules from marine and microbial sources en route to antitumor and antibiotic drugs.

The comprehensive information of small molecules and their biological activities in the PubChem database allows chemoinformatic researchers to access and make use of large-scale biological activity data to improve the precision of drug profiling. A Quantitative Structure–Activity Relationship approach, for classification, was used for the prediction of active/inactive compounds relatively to overall biological activity, antitumor and antibiotic activities using a data set of 1804 compounds from PubChem. Using the best classification models for antibiotic and antitumor activities a data set of marine and microbial natural products from the AntiMarin database were screened—57 and 16 new lead compounds for antibiotic and antitumor drug design were proposed, respectively. All compounds proposed by our approach are classified as non-antibiotic and non-antitumor compounds in the AntiMarin database. Recently several of the lead-like compounds proposed by us were reported as being active in the literature.

Genomic Insights into Specialized Metabolism in the Marine Actinomycete Salinispora

Comparative genomics is providing new opportunities to address the diversity and distributions of genes encoding the biosynthesis of specialized metabolites. An analysis of 119 genome sequences representing three closely related species of the marine actinomycete genus Salinispora reveals extraordinary biosynthetic diversity in the form of 176 distinct biosynthetic gene clusters (BGCs) of which only 24 have been linked to their products. Remarkably, more than half of the BGCs were observed in only one or two strains, suggesting they were acquired relatively recently in the evolutionary history of the genus. These acquired gene clusters are concentrated in specific genomic islands, which represent hot spots for BGC acquisition. While most BGCs are stable in terms of their chromosomal position, others migrated to different locations or were exchanged with unrelated gene clusters suggesting a plug and play type model of evolution that provides a mechanism to test the relative fitness effects of specialized metabolites. Transcriptome analyses were used to address the relationships between BGC abundance, chromosomal position and product discovery. The results indicate that recently acquired BGCs can be functional and that complex evolutionary processes shape the micro-diversity of specialized metabolism observed in closely related environmental bacteria.

Exopolysaccharide production by a marine Pseudoalteromonas sp. strain isolated from Madeira Archipelago ocean sediments.

Exopolysaccharides (EPS) are polymers excreted by some microorganisms with interesting properties and used in many industrial applications. A new Pseudoalteromonas sp. strain, MD12-642, was isolated from marine sediments and cultivated in bioreactor in saline culture medium containing glucose as carbon source. Its ability to produce EPS under saline conditions was demonstrated reaching an EPS production of 4.4g/L within 17hours of cultivation, corresponding to a volumetric productivity of 0.25g/Lh, the highest value so far obtained for Pseudoalteromonas sp. strains. The compositional analysis of the EPS revealed the presence of galacturonic acid (41-42mol%), glucuronic acid (25-26mol%), rhamnose (16-22mol%) and glucosamine (12-16mol%) sugar residues. The polymer presents a high molecular weight (above 1000kDa). These results encourage the biotechnological exploitation of strain MD12-642 for the production of valuable EPS with unique composition, using saline by-products/wastes as feedstocks.

QSAR-Assisted Virtual Screening of Lead-Like Molecules from Marine and Microbial Natural Sources for Antitumor and Antibiotic Drug Discovery

A Quantitative Structure-Activity Relationship (QSAR) approach for classification was used for the prediction of compounds as active/inactive relatively to overall biological activity, antitumor and antibiotic activities using a data set of 1746 compounds from PubChem with empirical CDK descriptors and semi-empirical quantum-chemical descriptors. A data set of 183 active pharmaceutical ingredients was additionally used for the external validation of the best models. The best classification models for antibiotic and antitumor activities were used to screen a data set of marine and microbial natural products from the AntiMarin database—25 and four lead compounds for antibiotic and antitumor drug design were proposed, respectively. The present work enables the presentation of a new set of possible lead like bioactive compounds and corroborates the results of our previous investigations. By other side it is shown the usefulness of quantum-chemical descriptors in the discrimination of biologically active and inactive compounds. None of the compounds suggested by our approach have assigned non-antibiotic and non-antitumor activities in the AntiMarin database and almost all were lately reported as being active in the literature.