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Summary Teredinibacter turnerae is an intracellular bacterial symbiont in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T. turnerae and their host shipworms around the world, implying that they encode defensive compounds. Here, we describe turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T. turnerae strains. Turnercyclamycins are bactericidal against challenging Gram-negative pathogens, including colistin-resistant Acinetobacter baumannii. Phenotypic screening identified the outer membrane as the likely target. Turnercyclamycins and colistin operate by similar cellular, although not necessarily molecular, mechanisms, but turnercyclamycins kill colistin-resistant A. baumannii, potentially filling an urgent clinical need. Thus, by exploring environments that select for the properties we require, we harvested the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of shipworms is a powerful example of this principle.

HIV-1 cDNA pre-integration complexes have been shown to persist for weeks in macrophages and to be transcriptionally active. Early and late gene transcripts are produced, along with some viral proteins, yet whole virus is not. While previous work has focused on the transcription and translation of HIV-1 genes; our understanding of cellular milieu that accompanies viral production is incomplete. We have used an in vitro system to model HIV-1 infection of macrophages, and single cell RNA sequencing (scRNA-seq) to compare the transcriptomes of uninfected cells, cells harboring pre-integration HIV-1 complexes (PIC) and those containing integrated provirus and actively making late HIV proteins. These are also compared to control cells, not exposed to virus.Several observations provide new perspective on the effects of HIV-1 transcription from pre-integrated cDNA versus from integrated provirus. First, HIV-1 transcript levels do not necessarily correlate with virus production, cells harboring PIC cDNA have transcript loads comparable to cells transcribing from provirus and making p24, mCherry, and vpu proteins. Second, all HIV-1 transcripts are easily detectable in abundance from PIC cDNA transcription, as is the case with cells transcribing from provirus, although the frequency of PIC cells with detectable gag-pol, tat, env, and nef transcripts is higher than the corresponding frequencies observed for “Provirus cells”. Third, the background transcriptomes of cells harboring pre- integrated HIV-1 cDNA are not otherwise detectably altered from cells not containing any HIV- 1 transcript. Fourth, integration and production of p24, mCherry, and Vpu proteins is accompanied by a switch from transcriptomes characterized by NFkB and AP-1 promoted transcription to a transcriptome characterized by E2F family transcription products. While some of these observations may seem heretical, single cell analysis provides a more nuanced understanding of PIC cDNA transcription and the transcriptomic changes that support HIV-1 protein production from integrated provirus.Author SummarySingle cell analysis is able to distinguish between HIV-1 infected macrophage cells that are transcribing pre-integrated HIV-1 cDNA and those transcribing HIV-1 provirus. Only cells transcribing HIV-1 provirus are making p24, marker mCherry and Vpu proteins, which corresponds with a change in the host cell’s background transcriptome from one expressing viral restriction and immunological response genes to one that is expressing genes associated with cell replication and oxidative phosphorylation.

Teredinibacter turnerae is an intracellular bacterial symbiont that lives in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T. turnerae and in their host shipworms around the world, implying that they encode the important defensive antibiotics. Here, we describe the turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T. turnerae strains. Turnercyclamycins A and B are bactericidal against challenging Gram-negative pathogens, including Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, at 1, 2, and 8 µg/mL, respectively. Additionally, these compounds kill colistin-resistant Acinetobacter strains, while lacking toxicity to mammalian cells. Phenotypic screening identified the outer membrane as the likely target. By exploring the inhabitants of environments that select for the properties we require, we can harvest the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of animals, and shipworms in particular, is a powerful example of this principle.

Teredinibacter turnerae is an intracellular bacterial symbiont that lives in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T. turnerae and in their host shipworms around the world, implying that they encode the important defensive antibiotics. Here, we describe the turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T. turnerae strains. Turnercyclamycins A and B are bactericidal against challenging Gram-negative pathogens, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii, at 1, 2, and 8 µg/mL, respectively, while lacking toxicity to mammalian cells. Phenotypic screening identified the outer membrane as the likely target. By exploring the inhabitants of environments that select for the properties we require, we can harvest the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of animals, and shipworms in particular, is a powerful example of this principle.

In search of new anti-tuberculars compatible with anti-retroviral therapy we re-identified amicetin as a lead compound. Amicetin's binding to the 70S ribosomal subunit of Thermus thermophilus (Tth) has been unambiguously determined by crystallography and reveals it to occupy the peptidyl transferase center P-site of the ribosome. The amicetin binding site overlaps significantly with that of the well-known protein synthesis inhibitor balsticidin S. Amicetin, however, is the first compound structurally characterized to bind to the P-site with demonstrated selectivity for the inhibition of prokaryotic translation. The natural product-ribosome structure enabled the synthesis of simplified analogues that retained both potency and selectivity for the inhibition of prokaryotic translation.

Antimicrobial resistance is a growing global health and economic concern. Current antimicrobial agents are becoming less effective against common bacterial infections. We previously identified pyrrolocins A and C, which showed activity against a variety of Gram-positive bacteria. Structurally similar compounds, known as pyrrolidinediones (e.g., TA-289, equisetin), also display antibacterial activity. However, the mechanism of action of these compounds against bacteria was undetermined. Here, we show that pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase (ACC), the first step in fatty acid synthesis. We used transcriptomic data, metabolomic analysis, fatty acid rescue and acetate incorporation experiments to show that a major mechanism of action of the pyrrolidinediones is inhibition of fatty acid biosynthesis, identifying ACC as the probable molecular target. This hypothesis was further supported using purified proteins, demonstrating that biotin carboxylase is the inhibited component of ACC. There are few known antibiotics that target this pathway and, therefore, we believe that these compounds may provide the basis for alternatives to current antimicrobial therapy.

IntroductionLatency reversal agents (LRAs), such as protein kinase C (PKC) agonists, constitute a promising strategy for exposing and eliminating the HIV-1 latent reservoir. PKC agonists activate NF-κB and, in turn, induce deleterious pro-inflammatory cytokine production. Adjuvant pharmacological agents, such as ruxolitinib, a JAK inhibitor, and rapamycin, an mTOR inhibitor, have previously been combined with LRAs to reduce deleterious pro-inflammatory cytokine secretion without inhibiting HIV-1 viral reactivation in vitro. Histone deacetylase inhibitors (HDACi) are known to dampen pro-inflammatory cytokine secretion in the context of other diseases and can synergize with other LRAs to bring dormant proviruses out of latency. In this study we investigated whether a broad panel of epigenetic modifiers, including HDACi, could effectively dampen PKC-induced pro-inflammatory cytokine secretion during latency reversal.MethodsWe screened an epigenetic modifier library to identify compounds that reduced intracellular IL-6 production induced by the PKC agonist Ingenol-3,20-dibenzoate. We further tested the most promising epigenetic inhibitor class, HDACi, for their ability to reduce a broad panel of pro-inflammatory cytokines and reactivate latent HIV-1 ex vivo.ResultsWe identified nine epigenetic modulators that reduced PKC-induced intracellular IL-6. In cells from aviremic individuals living with HIV-1, the HDAC1-3 inhibitor, suberohydroxamic acid (SBHA), reduced secretion of pro-inflammatory cytokines TNF-α, IL-5, IL-2r, and IL-17 but did not significantly reactivate latent HIV-1 when used in combination with Ingenol-3,20-dibenzoate.ConclusionThe addition of SBHA to Ingenol-3,20-dibenzoate reduces deleterious cytokine production during latency reversal but does not induce significant viral reactivation in aviremic donor PBMCs. The ability of SBHA to reduce PKC-induced pro-inflammatory cytokines when used in combination with Ingenol-3,20-dibenzoate suggests that SBHA can be used to reduced PKC induced pro-inflammatory cytokines but not to achieve latency reversal in the context of HIV-1.

Chemistry drives many biological interactions between the microbiota and host animals, yet it is often challenging to identify the chemicals involved. This poses a problem, as such small molecules are excellent sources of potential pharmaceuticals, pretested by nature for animal compatibility. We discovered anti-HIV compounds from small, marine tunicates from the Eastern Fields of Papua New Guinea. Tunicates are a reservoir for novel bioactive chemicals, yet their small size often impedes identification or even detection of the chemicals within. We solved this problem by combining chemistry, metagenomics, and synthetic biology to directly identify and synthesize the natural products. We show that these anti-HIV compounds, the divamides, are a novel family of lanthipeptides produced by symbiotic bacteria living in the tunicate. Neighboring animal colonies contain structurally related divamides that differ starkly in their biological properties, suggesting a role for biosynthetic plasticity in a native context where biological interactions take place.

The hypothesis underlying this current work is that fresh juice expressed from Papua New Guinea (PNG) medicinal plants (succus) will inhibit human Cytochrome P450s (CYPs). The CYP inhibitory activity identified in fresh material was compared with inhibition in methanol extracts of dried material. Succus is the most common method of traditional medicine (TM) preparation for consumption in PNG. There is increasing concern that TMs might antagonize or complicate drug therapy. We have previously shown that methanol extracts of commonly consumed PNG medicinal plants are able to induce and/or inhibit human CYPsin vitro. In this current work plant succus was prepared from fresh plant leaves. Inhibition of three major CYPs was determined using human liver microsomes and enzyme-selective model substrates. Of 15 species tested, succus from 6/15 was found to inhibit CYP1A2, 7/15 inhibited CYP3A4, and 4/15 inhibited CYP2D6. Chi-squared tests determined differences in inhibitory activity between succus and methanol preparations. Over 80% agreement was found. Thus, fresh juice from PNG medicinal plants does exhibit the potential to complicate drug therapy in at risk populations. Further, the general reproducibility of these findings suggests that methanol extraction of dried material is a reasonable surrogate preparation method for fresh plant samples.

Microbial symbionts in humans, animals, and plants are unparalleled sources of compounds for drug discovery. However, methods are needed to access natural biodiversity and make it amenable to pharmaceutical and biotechnological development. Here, we employ a novel approach marrying traditional natural products chemistry with modern metagenomics and synthetic biology in Escherichia coli to discover, synthesize, and analogue divamides, anti‐HIV molecules from rare coral reef animals. We demonstrate that divamides are produced by Prochloron symbiotic bacteria, which have eluded cultivation for >40 years. This represents a new paradigm of direct compound discovery from a true symbiotic relationship. We show that the divamides arise from a diversitygenerating biosynthetic pathway, which was exploited to generate derivatives for mechanism of action studies. The synthesis of closely related, yet functionally distinct compounds by a single biosynthetic pathway provides the first direct evidence supporting the "speculative metabolism" hypothesis of chemical diversity.

International audience; The extract of a sample of the tunicate Didemnum molle (MAY13-117) collected in Mayotte afforded eight new metabolites, mollecarbamates A−D (1−4) and molleureas B−E (5−8), along with the two known natural products, N,N′-diphenylethyl urea (10) and molleurea A (11). Another sample of D. molle (MAD11-BA065) collected in Baie des Assassins, Madagascar, afforded molledihydroisoquinolone (9). Mollecarbamates 1−4 are a family of compounds that possess repeating o-carboxyphenethylamide units and a carbamate moiety, while the molleureas 5−8 contain tetra-and penta-repeating carboxyphenethylamide units and a urea bridge in different positions. Molledihydroisoquinolone (9) is a cyclic form of o-carboxyphenethylamide. We propose that these unique natural products are most probably produced by an unprecedented biosynthetic pathway that contains a yet unknown chorismate mutase variant. The structures of the compounds were elucidated by interpretation of the data from 1D and 2D NMR, HRESIMS, and MS/MS analyses of the positive ESIMS experiments. Compounds 1−8 were tested against pathogenic bacteria and in a cytoprotective HIV cell based assay but did not show any significant effects in these assays.

Strategies are needed for the robust production of cryptic, silenced, or engineered secondary metabolites in fungi. The filamentous fungus Fusarium heterosporum natively synthesizes the polyketide equisetin at >2 g L(-1) in a controllable manner. We hypothesized that this production level was achieved by regulatory elements in the equisetin pathway, leading to the prediction that the same regulatory elements would be useful in producing other secondary metabolites. This was tested by using the native eqxS promoter and eqxR regulator in F. heterosporum, synthesizing heterologous natural products in yields of ∼1 g L(-1). As proof of concept for the practical application, we resurrected an extinct pathway from an endophytic fungus with an initial yield of >800 mg L(-1), leading to the practical synthesis of a selective antituberculosis agent. Finally, the method enabled new insights into the function of polyketide synthases in filamentous fungi. These results demonstrate a strategy for optimally employing native regulators for the robust synthesis of secondary metabolites.

A racemic, prenylated polyketide dimer, oxazinin A (1), was isolated from a novel filamentous fungus in the class Eurotiomycetes, and its structure was elucidated spectroscopically. The pentacyclic structure of oxazinin A (1) is a unique combination of benzoxazine, isoquinoline, and a pyran ring. Oxazinin A (1) exhibited antimycobacterial activity and modestly antagonized transient receptor potential (TRP) channels.

The methanol extract of Melochia odorata yielded three 4-quinolone alkaloids including waltherione A (1) and two new alkaloids, waltherione C (2) and waltherione D (3). Waltheriones A and C showed significant activities in an in vitro anti-HIV cytoprotection assay at concentrations of 56.2 and 0.84 μM, and inhibition of HIV P24 formation of more than 50% at 1.7 and 0.95 μM, respectively. The structures of the alkaloids were established by spectroscopic data interpretation.

As part of our screening for anti-HIV agents from marine invertebrates, the MeOH extract of Didemnum molle was tested and showed moderate in vitro anti-HIV activity. Bioassay-guided fractionation of a large-scale extract allowed the identification of two new cyclopeptides, mollamides E and F (1 and 2), and one new tris-phenethyl urea, molleurea A (3). The absolute configurations were established using the advanced Marfey's method. The three compounds were evaluated for anti-HIV activity in both an HIV integrase inhibition assay and a cytoprotective cell-based assay. Compound 2 was active in both assays with IC(50) values of 39 and 78 μM, respectively. Compound 3 was active only in the cytoprotective cell-based assay, with an IC(50) value of 60 μM.

Traditional knowledge of medicinal plant use in many regions of Papua New Guinea and the Autonomous Region of Bougainville is poorly described and rapidly disappearing. A program initiated by the University of Papua New Guinea to systematically document and preserve traditional knowledge of medicinal plant use was initiated with WHO help in 2001.To document and compare medicinal plant use in the Siwai and Buin districts of the Island of Bougainville. Siwai and Buin districts represent two adjacent geographic regions of differing language traditions.This report is a combination of two University of Papua New Guinea reports generated using a University of Papua New Guinea and Papua New Guinea Department of Health approved survey questionnaire "Information sheet on traditional herbal reparations and medicinal plants of Papua New Guinea".Although Siwai and Buin districts are adjacent in Southern Bougainville, there is considerable variation in the specific plants used medicinally and the specific uses of those plants that are used commonly in the two regions. In addition, many of the plants used in the region are widely distributed species that are used medicinally in other settings. Nevertheless, the high endemicity of plants and the extraordinary cultural diversity in the Autonomous Region of Bougainville has yielded description of the medicinal use of many plants that have not previously been reported in the wider scientific literature.Efforts to document and preserve traditional knowledge of plant use in Papua New Guinea have yielded important new records of plants with potential application in the provision of health care for a developing nation with an under developed Western style rural health care system. This report documents substantial commonality in the general modes of medicinal plant preparation and in the health care applications of plant use in the Siwai and Buin traditions, however, there was considerable difference noted in the particular uses of the specific plants used in one or another of the districts.

Exocarpic acid (13 E-octadecene-9,11-diynoic acid) from Exocarpos latifolius R.Br. (Santalaceae) was previously shown to have specific antimycobacterial activity. Microarray data suggested inhibition of fatty acid metabolism as a potential mode of action. Experiments designed to elucidate the mechanism of action showed that exocarpic acid was effective at inhibition of mycolic acid biosynthesis and did not act by dissipating the proton gradient in treated M. tuberculosis. Amide derivatives of exocarpic acid displayed similar properties to exocarpic acid, while other polyacetylenic fatty acids varied in their effects on mycolic acid biosynthesis.

An integrated and coordinated set of programs has been established to meet International Cooperative Biodiversity Group (ICBG) goals in Papua New Guinea (PNG). Here we give an overview of the PNG ICBG and focus on the key elements and major steps taken to establish a program necessary for the pharmacological assessment of botanicals and traditional medicines in PNG and, by extrapolation, in other developing countries.

Lipophilic fractions of stem extracts from Exocarpos latifolius, native to Papua New Guinea, showed significant activity against Mycobacterium tuberculosis H37Ra. Bioactivity-guided fractionation yielded exocarpic acid (E-octadeca-13-ene-9,11-diynoic-acid) as the major active component. Several new exocarpic acid analogs were also shown to be active. Exocarpic acid has previously been reported active against gram-positive, but not gram-negative bacteria. Work presented here demonstrates the selective activity of exocarpic acid against Mycobacterium tuberculosis H37Ra.

Marine pyridoacridines are a class of aromatic chemicals that share an 11H-pyrido[4,3,2-mn]acridine skeleton. Pyridoacridine alkaloids display diverse biological activities including cytotoxicity, fungicidal and bactericidal properties, production of reactive oxygen species (ROS) and topoisomerase inhibition. These activities are often dependent on slight modifications to the pyridoacridine skeleton. Here we demonstrate that while structurally similar to neoamphimedine and amphimedine, the biological activity of deoxyamphimedine differs greatly. Deoxyamphimedine damages DNA in vitro independent of topoisomerase enzymes through the generation of reactive oxygen species. Its activity was decreased in low oxygen, with the removal of a reducing agent and in the presence of anti-oxidants. Deoxyamphimedine also showed enhanced toxicity in cells sensitive to single or double strand DNA breaks, consistent with the in vitro activity.

This work presents a novel tool, the Continuous Flow Microspotter (CFM) and its use in patterning cellular microarrays of multiple cell types into the bottom of a tissue culture well. The CFM uses a system of isolated microfluidic channels to make an array of localized microspots of adhesion dependent cells in the bottom of a conventional tissue culture well. With this device we have created micropatterns of multiple cell lines in a single tissue culture well and used this system to conduct simultaneous cytotoxicity tests and recover dose survival curves in a parallel study. This mechanism of parallel testing allows the researcher to employ the use of positive and negative controls, as well as compare the chemical response of phenotypes in a tightly controlled microenvironment. For the experiments presented in this paper we have fabricated a CFM with a set of ten microchannels (five inlet channels and five outlet channels) to pattern a row of five microspots consisting of four cellular microspots and one empty spot for background measurements. Micropatterns containing a set of four different Chinese hamster ovarian cell (CHO) mutant phenotypes were deposited into the bottom of commercially available tissue culture wells then interrogated with mitomycin C, a chemotherapeutic agent. This study shows statistically significant (P < 0.05) hypersensitivity of the UV20 CHO mutant to a DNA interstrand cross-linking agent (mitomycin C). Because the CFM is also capable of depositing proteins and other biomolecules to the individual microspots of the array we foresee capabilities of the 48 microspot CFM to multiplex 48 cell types with 48 chemical reagents all within the confines of a 60 mm2 area.

Papuamide A is representative of a class of marine derived cyclic depsipeptides, reported to have cytoprotective activity against HIV-1 in vitro. We show here that papuamide A acts as an entry inhibitor, preventing human immunodeficiency virus infection of host cells and that this inhibition is not specific to R5 or X4 tropic virus. This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding. Furthermore, papuamide A was able to inhibit HIV pseudotype viruses expressing envelope glycoproteins from vesicular stomatitis virus or amphotropic murine leukemia virus indicating the mechanism of viral entry inhibition is not HIV-1 envelope glycoprotein specific. Time delayed addition studies with the pseudotyped viruses show that papuamide A inhibits viral infection only at the initial stage of the viral life cycle. Additionally, pretreatment studies revealed that the virus, and not the cell, is the target of papuamide A’s action. Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A. We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity.

Human immunodeficiency virus (HIV) and tuberculosis (TB) infections constitute a large portion of the disease burden in Papua New Guinea (PNG). Treatment of HIV and TB require long-term administration of antiretroviral and anti-TB drugs, respectively. In addition to Western medicine, traditional medicine (TM) is widely practiced in PNG. There is increasing concern that traditional medicines may antagonize antiretroviral and anti-tuberculin drug efficacy. Plant-drug, or drug-drug, interactions can occur at the level of metabolism through two major mechanisms: enzyme induction or enzyme inhibition. Our previous study of commonly-used medicinal plants from PNG extracted in methanol found almost one third of TM extracts induced cytochrome P450 (CYP) subtype expression of CYP1A2 or CYP3A4, or both. In addition, almost two thirds inhibited CYP1A2, CYP3A4, or CYP2D6, or combinations thereof. In PNG, the most common route by which traditional medicines are consumed is ingestion of expressed juice, succus. We investigated CYP inhibition of independently recollected medicinal plants (n = 17) and compared CYP inhibition by succus to the previous methanolic study through pair-wise comparisons of like genus and species. Results for the recollected succus samples were not significantly different from results obtained with methanolic extracts (p < 0.05). Therefore, data obtained previously with methanolic extracts likely represents biological activities found in more traditional medicinal preparations. In addition, these results confirm previous in vitro data that indicate use of traditional medicines concomitantly with ART or anti-TB drugs could dramatically alter their efficacy.

The yield of the regioselective and scalable reaction sequence marginally depends on the substitution pattern of the substrates.

The topoisomerase I (top1)–targeted camptothecin class of anticancer drugs is important in the treatment of several types of cancers. This class of drug inhibits the top1 enzyme during its catalytic DNA relaxation cycle, stabilizing the transient covalent top1-DNA complex by simultaneous noncovalent interactions with DNA and top1. We examined top1 using phage display because of the significance of this known top1-directed drug action. Several peptides that bind top1 were discovered and these were examined for top1 affinity, top1 catalytic and cleavage complex effects, and cytotoxic effects in cultured cell lines and in an in vivo tumor model. Although several peptides exhibited nanomolar and low-micromolar affinity for top1, none had cytotoxic effects when administered alone. However, in combination with 9-aminocamptothecin, one 15-mer peptide (SAYAATVRGPLSSAS) had synergistic cytotoxic effects with 9-aminocamptothecin both in the cytotoxicity assay and in nude mouse xenograft human tumor models. This report details the investigation of this peptide. [Mol Cancer Ther 2006;5(3):739–45]

By means of bioassay-guided fractionation, a new steroidal alkaloid, plakinamine M (1), and the known compound, plakinamine L (2), with a unique acyclic side chain, were isolated from the marine sponge Corticium sp. collected from New Britain, Papua New Guinea. The structures were determined on the basis of extensive 1D and 2D NMR and HRESIMS. The two compounds showed inhibition of Mycobacterium tuberculosis with MIC values of 15.8 and 3.6 μg/mL, respectively.

Aurantosides G-I (1-3) have been isolated from the lithistid sponge Theonella swinhoei from Papua New Guinea. Their structures were established by spectroscopic and chemical methods. Compounds 1-3 represent new monochloropentaenoyl tetramic acids with mono-, di-, and tri-N-saccharide substituents, respectively. Aurantosides G-I (1-3) failed to show any significant cytotoxicity against the human colon tumor cell line HCT-116.

Pyridoacridines are marine natural products that contain planar structures. Almost all are cytotoxic and capable of DNA intercalation. Several pyridoacridines have demonstrated anti-cancer activity, being able to generate reactive oxygen species or to inhibit topoisomerase (Topo) II. Synthetic pyridoacridines were characterized and compared to other pyridoacridines as well as the Topo-inhibiting drugs (etoposide, 9-aminocamptothecin and wakayin) in a series of in vitro enzyme systems. We found AK37 was able to stabilize a DNA-Topo I cleavable complex, but not a DNA-Topo II cleavable complex. To our knowledge, this is the first report of a DNA-Topo I cleavable complex stabilizing pyridoacridine. Structure comparison studies demonstrated that this activity was lost when an extra 'F' ring was added, but activity was not affected when the 'D' ring was removed. AK37 inhibited the catalytic activity of both human Topo I and II.

An examination of organic extracts of four new species of South African latrunculid sponges, Tsitsikamma pedunculata, T. favus, Latrunculia bellae, and Strongylodesma algoaensis, yielded 13 known and eight new pyrroloiminoquinone alkaloids, 3-dihydro-7,8-dehydrodiscorhabdin C (4), 14-bromo-3-dihydro-7,8-dehydrodiscorhabdin C (5), discorhabdin V (6), 14-bromo-1-hydroxydiscorhabdin V (7), tsitsikammamine A N-18 oxime (10), tsitsikammamine B N-18 oxime (11), 1-methoxydiscorhabdin D (12), and 1-aminodiscorhabdin D (13). Standard spectroscopic methods provided the structures of the pyrroloiminoquinone metabolites, while chiral GC-MS analysis of the acylated ozonolysis products of 21 confirmed the stereochemistry of the l-histidine residue in this compound. The anticancer activity of 20 pyrroloiminoquinone compounds was explored in the HCT-116 cancer cell line screen, and the DNA intercalation of the tsitsikammamines, together with their ability to cleave DNA through topoisomerase I inhibition, is discussed.

Topoisomerase IIalpha (top2) is a target of some of the most useful anticancer drugs. All clinically approved top2 drugs act to stabilize a drug-enzyme-DNA cleavable complex. Here we report the novel top2 activity of neoamphimedine, an isomer of the marine pyridoacridine amphimedine. Neoamphimedine was cytotoxic in yeast and mammalian cell lines. Neoamphimedine exhibited enhanced toxicity in top2 over-expressing yeast cells and was toxic in every mammalian cell line tested. However, neoamphimedine did not possess enhanced toxicity in a mammalian cell line sensitive to stabilized cleavable complexes. Therefore, we hypothesized that neoamphimedine is a top2-dependent drug, whose primary mechanism of action is not the stabilization of cleavable complexes. Top2-directed activity was determined in purified enzyme systems. Neoamphimedine-induced catenation of plasmid DNA only in the presence of active top2. This catenation correlated with the ability of neoamphimedine to aggregate DNA. Catenation was also observed using a filter-binding assay and transmission electron microscopy. Catenation was confirmed when only restriction enzyme digestion could resolve the catenated plasmid complex to monomer length plasmid DNA. Neoamphimedine also showed potent anti-neoplastic activity in human xenograft tumors in athymic mice. Neoamphimedine was as effective as etoposide in mice bearing KB tumors and as effective as 9-aminocamptothecin in mice bearing HCT-116 tumors. Amphimedine did not induce DNA aggregation or catenation in vitro, nor did it display any significant anti-neoplastic activity. These results suggest that neoamphimedine has a novel top2-mediated mechanism of cytotoxicity and anticancer potential.

Some marine animals are rich sources of unique polycyclic aromatic alkaloids that are cytotoxic against tumor cell lines and effective in mouse tumor xenograft models. Ascididemin is a pyridoacridine alkaloid originally derived from a Didemnum sp. tunicate. It has potent cytotoxicity against tumor cells in vitro and in vivo. Preclinical screening at NCI revealed the antineoplastic activities of ascididemin and a synthetic analogue 48. Ascididemin has been reported to inhibit topoisomerase II and induce topoisomerase II-mediated DNA cleavage. This study, however, focuses on the unique ability of ascididemin and two synthetic analogues (48 and 109) to cleave DNA in the absence of topoisomerase I or II. An in vitro assay revealed their concentration-dependent ability to cleave DNA and identified dithiothreitol as the sole requirement for maximal activity. On the basis of shared structural features of the three analogues, a double N-bay region and iminoquinone heterocyclic ring, two possible mechanisms of action were hypothesized: (1) generation of reactive oxygen species facilitated by metal binding to the common phenanthroline bay region, and (2) production of reactive oxygen species by direct reduction of the iminoquinone moiety. Experimental results supported direct iminoquinone reduction and ROS generation as the mechanism of ascididemin cytotoxicity. Antioxidants protected against DNA cleavage in vitro and protected cultured Chinese hamster ovary cells from toxicity. Additionally, it was shown that cells deficient in the ability to repair reactive oxygen species damage to their DNA were more susceptible to ascididemin and analogues than repair competent cells. Ascididemin-treated cells were also shown to induce oxygen-stress related proteins, further implicating the production of reactive oxygen species as the mechanism of cytotoxicity for these molecules.

Following proviral integration into the host cell genome and establishment of a latent state, the human immunodeficiency virus type 1 (HIV-1) can reenter a productive life cycle in response to various stimuli. HIV-1 reactivation occurs when transcription factors, such as nuclear factor-κB (NF-κB), nuclear factor of activated T cells (NFAT), and activator protein -1 (AP-1), bind cognate sites within the long terminal repeat (LTR) region of the HIV-1 provirus to promote transcription. Interestingly, pattern recognition receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) can reactivate latent HIV-1 through activation of the transcription factor NF-κB. Some PRRs are expressed on central memory CD4+ T cells (TCM), which in HIV-1 patients constitute the main reservoir of latent HIV-1. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), interacts with PRRs through membrane components. However, the ability of Mtb to reactivate latent HIV-1 has not been extensively studied. Here we show that phosphatidylinositol mannoside 6 (PIM6), a component of the Mtb membrane, in addition to whole bacteria in co-culture, can reactivate HIV-1 in a primary TCM cell model of latency. Using a JLAT model of HIV-1 latency, we found this interaction to be mediated through Toll-like receptor-2 (TLR-2). Thus, we describe a mechanism by which Mtb can exacerbate HIV-1 infection. We hypothesize that chronic Mtb infection can drive HIV-1 reactivation. The phenomenon described here could explain, in part, the poor prognosis that characterizes HIV-1/Mtb co-infection.

A cascade silver(I)-catalyzed hydroamination/Michael addition sequence has been developed to deliver highly substituted bicyclic guanidines. This transformation gives rise to geometrically and constitutionally stable ene–guanidines and generates a remote stereocenter with moderate to high diastereoselectivity.

Three new decalin-type tetramic acid analogues, pyrrolocins A (1), B (2), and C (3), were defined as products of a metabolic pathway from a fern endophyte, NRRL 50135, from Papua New Guinea. NRRL 50135 initially produced 1 but ceased its production before chemical or biological evaluation could be completed. Upon transfer of the biosynthetic pathway to a model host, 1–3 were produced. All three compounds are structurally related to equisetin-type compounds, with 1 and 3 having a trans-decalin ring system, while 2 has a cis-fused decalin. All were active against Mycobacterium tuberculosis, with the trans-decalin analogues 1 and 3 exhibiting lower MICs than the cis-decalin analogue 2. Here we report the isolation, structure elucidation, and antimycobacterial activities of 1–3 from the recombinant expression as well as the isolation of 1 from the wild-type fungus NRRL 50135.

Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these ‘chemical symbioses’. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=−0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and biotechnological studies.

Ethnopharmacological relevance A substantial proportion of the population in Papua New Guinea (PNG) lives with human immunodeficiency virus (HIV). Treatment requires lifelong use of antiretroviral therapy (ART). The majority of people in PNG use traditional medicines (TM) derived from plants for all types of health promotions. Consequently, there is a concern that herb–drug interactions may impact the efficacy of ART. Herb–drug, or drug–drug, interactions occur at the level of metabolism through two major mechanisms: enzyme induction or enzyme inhibition. In this study, extracts of commonly-used medicinal plants from PNG were screened for herb–drug interactions related to cytochrome P450s (CYPs). Materials and methods Sixty nine methanol extracts of TM plants were screened for their ability to induce CYPs by human aryl hydrocarbon receptor- (hAhR-) and human pregnane X receptor- (hPXR-) dependent mechanisms, utilizing a commercially available cell-based luciferase reporter system. Inhibition of three major CYPs, CYP1A2, CYP3A4, and CYP2D6, was determined using human liver microsomes and enzyme-selective model substrates. Results Almost one third of the TM plant extracts induced the hAhR-dependent expression of CYP1A2, the hPXR-dependent expression of CYP3A4, or both. Almost two thirds inhibited CYP1A2, CYP3A4, or CYP2D6, or combinations thereof. Many plant extracts exhibited both induction and inhibition properties. Conclusions We demonstrated that the potent and selective ability of extracts from PNG medicinal plants to affect drug metabolizing enzymes through induction and/or inhibition is a common phenomenon. Use of traditional medicines concomitantly with ART could dramatically alter the concentrations of antiretroviral drugs in the body; and their efficacy. PNG healthcare providers should counsel HIV patients because of this consequence.

An antimalarial screen for plants collected from Papua New Guinea identified an extract of Horsfieldia spicata as having activity. Isolation of the active constituents led to the identification of two new compounds: myristicyclins A (1) and B (2). Both compounds are procyanidin-like congeners of myristinins lacking a pendant aromatic ring. Myristicyclin A was found to inhibit the ring, trophozoite, and schizont stages of Plasmodium falciparum at similar concentrations in the mid-μM range.

The cytotoxic marine alkaloid ascididemin has been shown to be a thiol-dependent DNA cleaving agent. Previous mechanisms of action studies have concluded that DNA and/or the DNA processing enzyme topoisomerase II were the cellular targets for the alkaloid—this is the first direct evidence that a pyridoacridone alkaloid can cause DNA cleavage under physiological conditions.

The makaluvamines are marine natural products that were originally isolated because of their cytotoxicity in a cell-based mechanism screen. They have significant anti-cancer activity in animal models. There is, however, disagreement in the literature as to whether these compounds target topoisomerase II via a clinically relevant mechanism. This work shows that the makaluvamines can induce dose-dependent DNA cleavage via topoisomerase II. For most of the makaluvamines the levels of cleavage are significantly below those achieved by equimolar concentrations of etoposide. To some extent these results might explain the discrepancies present in the literature.

An ethyl acetate extract of bark from Evodia elleryana produced significant growth inhibition of Mycobacterium tuberculosis at concentrations only minimally inhibitory to human T cells. The crude extract yielded 95% inhibition of TB at 50 microg/ml. The crude extract yielded 29% growth inhibition of human T-cells in culture at that concentration.

A novel lumun-lumun sampling methodology was used to obtain a large diversity of micromollusks, including the new species Lienardia totopotens. In turn, from L. totopotens we cultivated a Streptomyces sp. strain that contained new and known spirotetronate polyketides, lobophorins (1–5). The structures were elucidated using spectroscopy, and the compounds were evaluated for cytotoxicity to human cells and activity against Mycobacterium tuberculosis. A structure-activity relationship was discerned, wherein the lack of digitoxose in 1 led to lack of both cytotoxic and antibacterial activity. For compounds 2–5 both activities were in the low μM to mid nM range. Although this likely precludes their direct application in tuberculosis therapy due to possible poor therapeutic index, very slight changes in structure led to widely varying antibacterial:cytotoxicity ratios, providing a possible basis to synthesize more selective derivatives.