Unraveling the Numerous Biosynthetic Products of the Marine Sediment-Derived Fungus, Aspergillus insulicola.

A new tripeptide, pre-sclerotiotide F (3), was isolated from a marine sediment-derived fungus, Aspergillus insulicola, along with five known compounds, one of which was new at the time of isolation, scerotiotide F (4). The absolute configuration elucidation of the new compound was determined using a combination of NMR, HR-ESI-MS, and optical rotation analyses. Cytotoxicities were measured in vitro against selected cancer cells. The effects of pre-sclerotiotide F (3) and sclerotiotide F (4) on LPS-induced NF-κB and iNOS expression were also measured.

Probing sponge-derived terpenoids for human 15-lipoxygenase inhibitors.

A human 15-lipoxygenase (15-HLO) assay has been employed to discover new marine-sponge-derived bioactive compounds. Extracts from two different sponges, Jaspis splendens (order Choristida, family Jaspidae) and Suberea sp. (order Verongida, family Aplysinellidae), exhibited potent IC(50) values of 0.4 and 0.1 microg/mL, respectively. Both are sources of terpenoids, and the former is a known source of (+)-jasplakinolide (7), which is inactive as a 15-HLO inhibitor. The terpenoids included (+)-(5S,6S)-subersin (1, IC(50) > 100 microM), (-)-(5R,10R)-subersic acid (2, IC(50) = 15 microM), jaspaquinol (3, IC(50) = 0.3 microM), and (-)-jaspic acid (4, IC(50) = 1.4 microM). Structure elucidations and lipoxygenase activity studies of these compounds are reported.

Synthesis and antitumor activity of ester-modified analogues of bengamide B.

Bengamide B, a novel sponge-derived marine natural product with broad spectrum antitumor activity, was not suitable for further preclinical development because of its difficult synthesis and very poor water solubility. Bengamide B produced a 31% T/C at its solubility-limited maximum intravenous dose of 33 micromol/kg in MDA-MB-435 breast carcinoma implanted subcutaneously as a xenograft in nude mice. Compound 8a, a bengamide B analogue with three structural changes (t-Bu alkene substituent, unsubstituted lactam nitrogen, and inverted lactam 5'-myristoyloxy group), was as potent as bengamide B in vitro and more efficacious than bengamide B in vivo. A series of ester-modified analogues based on 8a were synthesized and tested in vitro and in vivo (MDA-MB-435). The cyclohexyl- and phenethyl-substituted esters, 8c and 8g, respectively, had in vitro and in vivo activities similar to that of 8a and enhanced water solubility (ca. 1 mg/mL). Consequently, 8c and 8g were tested in the MDA-MB-435 xenograft model at 100 micromol/kg and produced 29% and 57% tumor regression, respectively.

Bengamides revisited: new structures and antitumor studies.

The structural chemistry and biological activity of the bengamide class of compounds have been further characterized. Extracts prepared from recollected Jaspis cf. coriacea from five sites in Fiji were pooled. Six new bengamides, M (7b), N (8a), O (8b), P (9a), Q (9b), and R (10), were identified, accompanied by the known bengamides A (1a), B (1b), E (3a), F (3b), Y (5), Z (6), L (7a), G (11a), H (11b), and I (12). The structures of the new compounds were determined from spectroscopic data, and some were additionally confirmed by semisynthesis. Cytotoxicity screening data were obtained from the NCI-DTP 60 cell screen for bengamides A, B, and P. Bengamides A and B were more potent than bengamide P, with average IC(50) values of 0.046, 0.011, and 2.70 FM, respectively. The in vitro antitumor activity against MDA-MB-435 human mammary carcinoma was also determined for natural bengamides A, B, E, F, P, M, O, and Z and for synthetic samples of B and O. The best activity was observed for the natural bengamides A (IC(50) = 1 nM) and O (IC(50) = 0.3 nM).

Jasplakinolide induces apoptosis in various transformed cell lines by a caspase-3-like protease-dependent pathway.

To clarify the mechanisms underlying the antiproliferative effects of jasplakinolide, a cyclic depsipeptide from marine sponges, we examined whether jasplakinolide induces apoptosis in a variety of transformed and nontransformed cells. Jasplakinolide inhibited proliferation of human Jurkat T cells, resulting in cell death. This was accompanied by chromatin condensation and DNA cleavage at the linker regions between the nucleosomes. When caspase-3-like activity in the cytosolic extracts of Jurkat T cells was examined with a fluorescent substrate, DEVD-MAC (N-acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide), the activity in the cells treated with jasplakinolide was remarkably increased in a time-dependent manner. Pretreatment of Jurkat T cells with the caspase inhibitor zVAD [benzyloxycarbonyl(Cbz)-Val-Ala-beta-Asp(OMe)-fluoromethylketone] or DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-1-aldehyde) prevented the induction of apoptosis by jasplakinolide. Moreover, exposure of various murine transformed cell lines to jasplakinolide resulted in cell death, which was inhibited by zVAD. Although it has been well established that murine immature thymocytes are sensitive to apoptosis when exposed to various apoptotic stimuli, these cells as well as mature T lymphocytes were resistant to jasplakinolide-induced apoptosis. The results suggest that jasplakinolide induces apoptotic cell death through a caspase-3-like protease-dependent pathway. Another important outcome is that transformed cell lines were more susceptible to jasplakinolide-induced apoptosis than normal nontransformed cells.

Buzonamine, a new alkaloid from the defensive secretion of the millipede, Buzonium crassipes.

GC-MS analysis of the defensive secretion from the millipede Buzonium crassipes showed three volatile components, beta-pinene (35%), limonene (6%) and a new alkaloid, buzonamine (59%). Buzonamine had an HRMS molecular ion at m/z=221.1785 (calculated for C(14)H(23)NO, 221.1781), 14 carbons in the 13C-NMR and 23 hydrogens by SFORD, DEPT and APT experiments. All distinct 1H nuclei were assigned to 13C resonances with 2D 1H-13C COSY data, and the final structure was determined by 1H-1H COSY, 1H-13C HMBC and nOe experiments. Buzonamine contains four rings including an epoxy group and a tertiary nitrogen. beta-Pinene, limonene or buzonamine, kept the mound nesting ant, Formica obscuripes, from eating mealworms (Tenebrio molitor) treated with 1mg of the chemical. A 150mg millipede expels 4mg of secretion.

Biosynthetically diverse compounds from a saltwater culture of sponge-derived Aspergillus niger.

The new compound, asperic acid (1), and the known compounds hexylitaconic acid (2), malformin C (3), pyrophen (4), and asperazine (5) were isolated from the saltwater culture of Aspergillus niger derived from a Caribbean sponge, Hyrtios proteus. The structure elucidation of asperic acid is presented.

Deoxynortrichoharzin, a new polyketide from the saltwater culture of a sponge-derived Paecilomyces fungus.

The saltwater culture of a Paecilomyces cf. javanica isolated from the marine sponge Jaspis cf. coriacea has yielded a new polyketide, deoxynortrichoharzin (1), and two known diketopiperazines (2 and 3).

Cyclic polyketide peroxides and acyclic diol analogues from the sponge Plakortis lita.

The sponge Plakortis lita from Papua New Guinea is a source of three cyclic peroxides-ethyl plakortide Z (3), ethyl didehydroplakortide Z (4), and methyl didehydroplakortide Z (5)-and three acyclic diol analogues-ethyl seco-plakortide Z (6), epi-ethyl seco-plakortide Z (7), and ethyl didehydro-seco-plakortide Z (8). The absolute stereochemistry at the three chiral sites of 3 was assigned by preparing 6, which was investigated using the refined Mosher's method. Compounds 4, 5, and 6 were also concluded to have the same absolute stereochemistry as 3. The cyclic peroxides were generally cytotoxic, while the acyclic analogues were devoid of activity. Compound 3 was equally active in vitro against solid tumor and L-1210 leukemia cell lines. Alternatively, 4 was observed in vitro to be moderately solid-tumor selective but did not exhibit in vivo activity against solid tumors in mice.

Dihydrotubastrines: phenethylguanidine analogues from the Indo-Pacific marine sponge Petrosia cf. contignata.

Two phenethylguanidine derivatives, 7,8-dihydrotubastrine (1) and 4-deoxy-7,8-dihydrotubastrine (2), along with the sterol xestobergsterol A (3), were isolated from the marine sponge Petrosia cf. contignata. The structures of the new natural products 1 and 2 were based on spectroscopic data and comparison to the literature properties for semisynthetic 1. This is the first example from this compound class with a saturated acyclic C2 unit.

Isolation and cytotoxic evaluation of marine sponge-derived norterpene peroxides.

The marine sponge Diacarnus cf. spinopoculum has provided a series of norterpenes, including five new compounds (7-11), two new ent-compounds [(-)-1a and (+)-1b], and three known compounds (2a, 2b, and 12). Eight of these compounds represent additional examples of the muqubilin/sigmosceptrellin classes (norsesterterpene peroxides) or the nuapapuin class (norditerpene peroxides). Also isolated were dinorditerpenones 11 and 12, which are biosynthetically related to the muqubilin/sigmosceptrellin structure classes. In all, 11 compounds were evaluated for their cytotoxic properties using a soft agar assay system and the NCI's 60 cell-line screen. Compounds without peroxide functionality were inactive. Overall, the norsesterterpene peroxides were less selective as cytotoxins than norditerpene peroxide analogues. Two compounds, nuapapuin A methyl ester (3) and nuapapuin B (7), which were somewhat selective in their cytotoxic behavior, were selected for further in vivo evaluation.

Using scalarane sesterterpenes to examine a sponge taxonomic anomaly.

A parallel study was conducted on two Indo-Pacific foliose sponges. The first specimen contains 3-hydroxy-20,22-dimethyl-20-deoxoscalarin (2), while the second contains 3-oxo-20,22-dimethyl-20-dioxoscalarin 8 (3). The physical properties as well as X-ray results confirming the structure and stereochemical features of these compounds are presented first. The difficulty we encountered in the taxonomic identification of these species is also discussed. One of our specimens is identical to material considered by different taxonomists as either Phyllospongia vermicularis or Dysidea vermicularis. The other is identified as Carteriospongia sp. We outline that the parallel chemistry of these two specimens suggests that they are closely related taxonomically.

High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity revealed using the actin inhibitor latrunculin-A.

We report that the actin assembly inhibitor latrunculin-A (LAT-A) causes complete disruption of the yeast actin cytoskeleton within 2-5 min, suggesting that although yeast are nonmotile, their actin filaments undergo rapid cycles of assembly and disassembly in vivo. Differences in the LAT-A sensitivities of strains carrying mutations in components of the actin cytoskeleton suggest that tropomyosin, fimbrin, capping protein, Sla2p, and Srv2p act to increase actin cytoskeleton stability, while End3p and Sla1p act to decrease stability. Identification of three LAT-A resistant actin mutants demonstrated that in vivo effects of LAT-A are due specifically to impairment of actin function and implicated a region on the three-dimensional actin structure as the LAT-A binding site. LAT-A was used to determine which of 19 different proteins implicated in cell polarity development require actin to achieve polarized localization. Results show that at least two molecular pathways, one actin-dependent and the other actin-independent, underlie polarity development. The actin-dependent pathway localizes secretory vesicles and a putative vesicle docking complex to sites of cell surface growth, providing an explanation for the dependence of polarized cell surface growth on actin function. Unexpectedly, several proteins that function with actin during cell polarity development, including an unconventional myosin (Myo2p), calmodulin, and an actin-interacting protein (Bud6/Aip3p), achieved polarized localization by an actin-independent pathway, revealing interdependence among cell polarity pathways. Finally, transient actin depolymerization caused many cells to abandon one bud site or mating projection and to initiate growth at a second site. Thus, actin filaments are also required for maintenance of an axis of cell polarity.

Haliclostanone sulfate and halistanol sulfate from an Indo-Pacific Haliclona sponge.

Two steroids, haliclostanone sulfate (1) and halistanol sulfate (3), were isolated from a Haliclona sp. marine sponge. The structure of the new compound 1 was established based on its spectroscopic data and the properties of a pentaacetate derivative 2. Compound 1 is unique in that it possesses the rare cis C/D ring junction precedented only in marine sterols, contignasterol (4) and xestobergsterols A (5), B (6), and C (7).

Enzyme inhibitors: new and known polybrominated phenols and diphenyl ethers from four Indo-Pacific Dysidea sponges.

Extracts and pure compounds isolated from four samples of Dysidea sp. sponges collected from two geographically distinct regions of the Indo-Pacific (Chuuk Atoll and Fiji) were assayed against five different enzyme assays, four of which are relevant to anticancer drug discovery and one of which (15-lipoxygenase) may detect compounds significant in modulating the development of atherosclerotic plaque. The pure compounds that inhibited various enzymes were polybrominated phenols and polybrominated phenoxyphenols. Fourteen of these phenols were isolated, six of which were new compounds. A variety of the phenols inhibited inosine monophosphate dehydrogenase (IMPDH), guanosine monophosphate synthetase, and 15-lipoxygenase. No activity was observed with protein tyrosine kinase pp60v-src or matrix metalloprotease.

The cyclorenierins, sesquiterpenoid quinols from the sponge Haliclona sp. collected in Vanuatu.

Two sesquiterpene quinols, cyclorenierins A [1] and B [2], which are closely related to panicein A2 [3] and renierin A [4], were isolated as an inseparable mixture from the marine sponge Haliclona sp. collected in Vanuatu. Structures were proposed using nmr shift analogies to 3 and 4 and confirmed with 2D nmr data.

New and known diketopiperazines from the Caribbean sponge, Calyx cf. podatypa.

The cyanobacteria-containing Caribbean sponge, Calyx cf. podatypa, was collected from three sites in the Bahamas. In each of the three collections, a polar solvent partition fraction contained six known compounds including five diketopiperazines [1-4,6] and phenylacetic acid, along with a new diketopiperazine, cyclo-(4-methyl-R-proline-S-norvaline) [5]. Interestingly, all six diketopiperazines are proline-derived cyclic dipeptides. This is the first example for this class of peptide derivative to be isolated from a Calyx sponge. Parallel studies of one of the sponge collections in which the ectosome (cyanobacteria-rich) was separated from the endosome (no cyanobacteria) revealed no significant differences in their content of diketopiperazines.

The structures and cytotoxic properties of polyketide peroxides from a Plakortis sponge.

A morphologically distinct Fijian sponge, Plakortis sp., has yielded two new peroxides, plakortolide E [5] and plakoric acid [12]. After standing for approximately one year, plakortolide E rearranged to plakortolide ether [10]. The structures of plakortolide E [5] and plakortolide ether [10] were established from 2D nmr data and by analogy to a known compound, plakortolide [3]. The stereochemistry of the bicyclic ring substituents of 5 was established using nOe and NOESY nmr data along with comparisons to 3. The absolute stereochemistry at the three chiral sites of 5 was assigned by preparing acyclic compounds 6-9, and both 8 and 9 were investigated using the modified Mosher's method. This represents the first absolute stereochemistry determination for a sponge-derived polyketide peroxide. The characterization of plakoric acid [12] was based on spectral analogies to known polyketides such as plakortin. Plakortolide E [5] exhibited selective potency against the melanoma and breast tumor cell lines in the in vitro 60-cell line panel of the National Cancer Institute.

The arenarans, sesquiterpene ethers from the marine sponge Dysidea arenaria.

Two new cyclic sesquiterpenes, arenarans A [1] and B [2], were isolated from a Thai collection of Dysidea arenaria. Their chemical and cytotoxic properties are described.

Jasplakinolide's inhibition of the growth of prostate carcinoma cells in vitro with disruption of the actin cytoskeleton.

BACKGROUND: Jasplakinolide, a cyclodepsipeptide produced by an Indo-Pacific sponge, Jaspis johnstoni, has been reported to inhibit the growth of breast cancer cells. PURPOSE: The effects of jasplakinolide on the proliferation of three human immortalized prostate carcinoma cell lines (PC-3, LNCaP, and TSU-Pr1) were studied. The growth-inhibitory effect of jasplakinolide on the PC-3 cell line was studied in detail to elucidate its mechanism of action. METHODS: Cell counts were used to study growth inhibition. A protein-based microplate assay was used to assess the time of exposure needed to cause persistent growth inhibition and to study the effects of jasplakinolide analogues. Metabolic changes were assessed by following the incorporation of radiolabeled precursors. The effects of jasplakinolide on the cytoskeleton were studied by fluorescent microscopy, using rhodamine phalloidin (RP) and antibodies to cytoskeletal components. Changes in RP binding were quantified by extracting bound fluorescent material from fixed cells and measuring the amount of fluorescence in a spectrofluorometer. RESULTS: The growth of PC-3, LNCaP, and TSU-Pr1 cells was potently inhibited by exposure to jasplakinolide for 48 hours; doses of jasplakinolide that led to 50% growth inhibition were 65 nM for PC-3 cells, 41 nM for LNCaP cells, and 170 nM for TSU-Pr1 cells. In PC-3 cells, exposure to 160 nM for 48 hours led to total growth inhibition, which persisted for several days even after drug removal. Several jasplakinolide analogues also inhibited the growth of PC-3 cells, although analogues in which the rigidity of the macrolide ring was altered were ineffective. No early changes in the synthesis of DNA, RNA, or protein or in intracellular adenosine triphosphate levels were seen in the PC-3 cells after exposure to jasplakinolide. Growth inhibition by jasplakinolide was accompanied by striking morphologic changes. Exposure for several doublings led to multinucleated cells. Further investigation of these changes in the PC-3 cells revealed a dramatic and early disruption of the actin cytoskeleton and a statistically significant decrease in RP binding. The doses of jasplakinolide, the time of exposure, and the pattern of growth inhibition by structural analogues corresponded with the changes seen in actin distribution. CONCLUSIONS: Jasplakinolide represents a novel marine natural product with potent in vitro antiproliferative activity against human prostate carcinoma cell lines, and it appears to target the actin cytoskeleton. IMPLICATIONS: Jasplakinolide is a potential candidate for further preclinical development and a lead structure for a novel class of therapeutic agents that can disrupt the actin cytoskeleton in mammalian cells.