Chambers C. Hughes

The Marinopyrroles, Antibiotics of an Unprecedented Structure Class from a Marine Streptomyces sp.

Cultivation of an obligate marine Streptomyces strain has furnished the marinopyrroles A and B, densely halogenated, axially chiral metabolites that contain an uncommon bispyrrole structure. X-ray analysis of marinopyrrole B showed that the natural product exists as an atropo-enantiomer with the M-configuration. Though configurationally stable at room temperature, M-(-)-marinopyrrole A can be racemized at elevated temperatures to yield the non-natural P-(+)-atropo-enantiomer. The marinopyrroles possess potent antibiotic activities against methicillin-resistant Staphylococcus aureus.

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.

Antibacterials from the Sea

The ocean contains a host of macroscopic life in a great microbial soup. Unlike the terrestrial environment, an aqueous environment provides perpetual propinquity and blurs spatial distinctions. Marine organisms are under a persistent threat of infection by resident pathogenic microbes including bacteria, and in response they have engineered complex organic compounds with antibacterial activity from a diverse set of biological precursors. The diluting effect of the ocean drives the construction of potent molecules that are stable to harsh salty conditions. Members of each class of metabolite-ribosomal and non-ribosomal peptides, alkaloids, polyketides, and terpenes-have been shown to exhibit antibacterial activity. The sophistication and diversity of these metabolites points to the ingenuity and flexibility of biosynthetic processes in Nature. Compared with their terrestrial counterparts, antibacterial marine natural products have received much less attention. Thus, a concerted effort to discover new antibacterials from marine sources has the potential to contribute significantly to the treatment of the ever increasing drug-resistant infectious diseases.

Marinopyrrole A Target Elucidation by Acyl Dye Transfer

The targeting of marinopyrrole A to actin was identified using a fluorescent dye transfer strategy. The process began by appending a carboxylic acid terminal tag to a phenol in the natural product. The resulting probe was then studied in live cells to verify that it maintained activity comparable to marinopyrrole A. Two-color fluorescence microscopy confirmed that both unlabeled and labeled materials share comparable uptake and subcellular localization in HCT-116 cells. Subsequent immunoprecipitation studies identified actin as a putative target in HCT-116 cells, a result that was validated by mass spectral, affinity, and activity analyses on purified samples of actin. Further data analyses indicated that the dye in the marinopyrrole probe was selectively transferred to a single residue K(115), an event that did not occur with related acyl phenols and reactive labels. In this study, the combination of cell, protein, and amino acid analysis arose from a single sample of material, thereby, suggesting a means to streamline and reduce material requirements involved in mode of action studies.

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.

Bile salt–induced intermolecular disulfide bond formation activates Vibrio cholerae virulence

To be successful pathogens, bacteria must often restrict the expression of virulence genes to host environments. This requires a physical or chemical marker of the host environment as well as a cognate bacterial system for sensing the presence of a host to appropriately time the activation of virulence. However, there have been remarkably few such signal–sensor pairs identified, and the molecular mechanisms for host-sensing are virtually unknown. By directly applying a reporter strain of Vibrio cholerae, the causative agent of cholera, to a thin layer chromatography (TLC) plate containing mouse intestinal extracts, we found two host signals that activate virulence gene transcription. One of these was revealed to be the bile salt taurocholate. We then show that a set of bile salts cause dimerization of the transmembrane transcription factor TcpP by inducing intermolecular disulfide bonds between cysteine (C)-207 residues in its periplasmic domain. Various genetic and biochemical analyses led us to propose a model in which the other cysteine in the periplasmic domain, C218, forms an inhibitory intramolecular disulfide bond with C207 that must be isomerized to form the active C207–C207 intermolecular bond. We then found bile salt–dependent effects of these cysteine mutations on survival in vivo, correlating to our in vitro model. Our results are a demonstration of a mechanism for direct activation of the V. cholerae virulence cascade by a host signal molecule. They further provide a paradigm for recognition of the host environment in pathogenic bacteria through periplasmic cysteine oxidation.

Total Synthesis of the Ammosamides

The ammosamides A-C are chlorinated pyrrolo[4,3,2-de]quinoline metabolites isolated from the marine-derived Streptomyces strain CNR-698. The natural products, which possess a dense array of heteroatoms, were synthesized in 17-19 steps from 4-chloroisatin. That the five nitrogen atoms were introduced at the appropriate time and in a suitable oxidation state was key to the success of the total synthesis. Compared to synthetic deschloro ammosamide B, natural ammosamide B is much less susceptible to oxidative degradation.

Function-Oriented Biosynthesis of β-Lactone Proteasome Inhibitors in Salinispora tropica

The natural proteasome inhibitor salinosporamide A from the marine bacterium Salinispora tropica is a promising drug candidate for the treatment of multiple myeloma and mantle cell lymphoma. Using a comprehensive approach that combined chemical synthesis with metabolic engineering, we generated a series of salinosporamide analogues with altered proteasome binding affinity. One of the engineered compounds is equipotent to salinosporamide A in inhibition of the chymotrypsin-like activity of the proteasome yet exhibits superior activity in the cell-based HCT-116 assay.

Pharmacological Properties of the Marine Natural Product Marinopyrrole A against Methicillin-Resistant Staphylococcus aureus

ABSTRACT The ongoing spread of methicillin-resistant Staphylococcus aureus (MRSA) strains in hospital and community settings presents a great challenge to public health and illustrates the urgency of discovering new antibiotics. Marinopyrrole A is a member of a structurally novel class of compounds identified from a species of marine-derived streptomycetes with evidence of antistaphylococcal activity. We show that marinopyrrole A has potent concentration-dependent bactericidal activity against clinically relevant hospital- and community-acquired MRSA strains, a prolonged postantibiotic effect superior to that of the current first-line agents vancomycin and linezolid, and a favorable resistance profile. Marinopyrrole A showed limited toxicity to mammalian cell lines (at >20× MIC). However, its antibiotic activity against MRSA was effectively neutralized by 20% human serum. A variety of marinopyrrole analogs were isolated from culture or synthetically produced to try to overcome the inhibitory effect of serum. While many of these compounds retained potent bactericidal effect against MRSA, their activities were also inhibited by serum. Marinopyrrole A has significant affinity for plastic and may therefore have potential as a potent anti-MRSA agent in cutaneous, intracatheter, or antibiotic-lock applications.

Chlorizidine, a Cytotoxic 5H-Pyrrolo[2,1-a]isoindol-5-one-Containing Alkaloid from a Marine Streptomyces sp.

Cultivation of an obligate marine Streptomyces strain has provided the cytotoxic natural product chlorizidine A. X-ray crystallographic analysis revealed that the metabolite is composed of a chlorinated 2,3-dihydropyrrolizine ring attached to a chlorinated 5H-pyrrolo[2,1-a]isoindol-5-one. The carbon stereocenter in the dihydropyrrolizine is S-configured. Remarkably, the 5H-pyrrolo[2,1-a]isoindol-5-one moiety has no precedence in the field of natural products. The presence of this ring system, which was demonstrated to undergo facile nucleophilic substitution reactions at the activated carbonyl group, is essential to the molecules cytotoxicity against HCT-116 human colon cancer cells.