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The pseudopterosins are marine natural products that display significant anti-inflammatory and wound healing properties. We describe the synthesis of six structural analogs of the seco-pseudopterosin-like core that are devoid of all but one of the stereocenters found in the carbocyclic core of the natural products. Our targets were selected in an attempt to identify the minimal pharmacophore for the pseudopterosins and their seco analogs. A deliberate effort was made to utilize a conservative synthetic approach based upon the use of well-established reactions, which enabled us to develop routes that proved to be efficient, practical, and easy to implement. The results of several bioassays, including an assessment of the ability to inhibit phagocytosis and to competitively bind to the adenosine receptor A2A, demonstrate that greatly simplified structural analogs of the pseudopterosins and their seco forms are capable of maintaining several of the important bioactivities that characterize the natural products, and do so with comparable efficacy. Those systems bearing two rather than one oxygen atom appended directly to the aromatic ring are the more effective binding agents. This observation may provide a significant clue regarding the key structural features of the minimal pharmacophore.

The capability for physical injury or heat stress to elicit the production of reactive oxygen species was examined in four species of gorgonian corals. The sea plumes Pseudopterogorgia elisabethae, Pseudopterogorgia americana, the sea rod Eunicea fusca and the azooxanthellate red branching gorgonian Lophogorgia chilensis were physically injured using sonic sound cavitations and heat shocked by incubation in 33°C sea water. The pharmacological probe, diphenylene iodonium chloride (DPI), an inhibitor of NAD(P)H oxidase and peroxidases was used to identify an enzymatic surrogate of the oxidative burst. Both injury and heat stress were capable of inducing the release of reactive oxygen species (ROS) in all gorgonians tested, yet the kinetics and amplitude of ROS release varied among genera. In both the treatments, P. americana demonstrated the largest oxidative burst among the other corals tested.

Algal preparations from Acetabularia crenulata were analyzed for their fatty acid composition to establish the suitability of this alga as a model to study fatty acid oxidation and oxylipin biosynthesis. The work was based on two goals. The first goal of this study was to determine the contribution of fatty acids from contaminating bacteria and how this influenced the total fatty acid composition of cell homogenates of A. crenulata collected in the wild as compared to specimens cultured in sterile conditions. The major fatty acids detected for both specimens were palmitic (C16:0), palmitoleic (C16:1n-7), oleic (C18:1n-9), linoleic (C18:2n-6), linolenic (C18:3n-3), and octadecatetraenoic acid (C18:4n-3). Significant amounts of odd-chain fatty acids common to bacteria were not detected in either sample. Furthermore, branched-chain fatty acids, typical bacterial biomarkers, were not detected in either sample. Data suggest that bacteria do not greatly contribute to the total fatty acid pool of A. crenulata. The second goal was to compare the fatty acid composition of cell homogenates with that of isolated chloroplasts. Comparatively speaking palmitoleic and octadecatetraenoic acid were found at significantly lower concentrations in the chloroplast whereas oleic and linolenic acid were found at significantly higher amounts in this organelle. Furthermore, the amount of hexadecatrienoic acid (C16:3), a fatty acid commonly esterified to monogalactosyldiacylglycerol (MGDG; lipid present at high concentrations inside the chloroplasts of algae), was present at very low concentrations in these plastids (0.7%). Typically green algal follow the “prokaryotic pathway” for MGDG biosynthesis where C18:3 is esterified at the sn-1 position of the glycerol backbone and C18:3 or C16:3 at the sn-2 position, making C16:3 a major fatty acid inside chloroplasts. Interestingly, our results suggest that chloroplasts of A. crenulata appear to follow the “eukaryotic pathway” for MGDG biosynthesis where C18:3 is both at the sn-1 and sn-2 position of MGDG. Taking into account the exceptions noted, the fatty acid composition for A. crenulata is similar to that reported for most chlorophytes.

We investigated the kinetics and composition of the second phase of the wound repair process of Dasycladus vermicularis ([Scropoli] Krasser) using fluorescent probes, chromatography, UV spectroscopy, and histochemistry. Our new evidence supports the hypothesis that the second phase of wound repair (initiated at approximately 35-45 min post-injury) is based on the activation of an oxidative burst that produces micromolar H 2 O 2 levels. These results provide evidence of peroxidase activity at the wound site, real-time measurements of an oxidative burst, and catechol localization in wound plugs. Strong evidence is presented indicating that the biochemical machinery exists for oxidative cross-linking to ensue in the wound-healing process of D. vermicularis.

Upon injury, selected coenocytic algae are capable of forming temporary wound plugs to prevent detrimental cytoplasmic loss. Wound plugs of Dasycladus vermicularis ([Scropoli] Krasser) were harvested 5 min post-injury anddried. The plug material contained 94% water and can be considered a hydrogel. The gel plug extended several millimeters from the cut end. and filled the space inside the cell wall, which resulted from cytoplasmic retraction. Total organic carbon included 55% sugars, 5%-15% protein, and 0.18% lipids. The major sugars were glucose, galactose, mannose, and galacturonic acid. Fluorescein isothiocyanate-lectins specific for these sugars were localized around the plug matrix. Sulfur content calculated as sulfate corresponded to 17% of the carbohydrate by weight, and sulfated material was detected in plugs by Al-cian Blue staining. Formation of the initial plug occurred within 1 min of injury and was not significantly perturbed by the addition of ionic, antioxidant, or chelating agents to the seawater medium. However, addition of exogenous D(+)-galactose and D(+)-glucose prevented formation of the nascent gel plug. Wound plugs that were allowed to form from 10 min up until 24 h post-injury were isolated and incubated with selected biochemical probes to identify the biochemical processes involved in plug formation. The operative strategy in Dasycladus to prevent "cytoplasmic hemorrhage" required availability of sequestered carbohydrate and lectin precursor components throughout the thallus for plug assembly. Once the initial assembly had commenced, additional biochemical interactions were initiated (as a function of time) to promote structural integrity.

An oxidative burst in free-living and symbiotic dinoflagellates induced by physical stress is defined and characterized. The oxidative burst occurred within 1 min of physical injury caused by short pulses of low frequency sonic sound (20 kHz, 10 s pulses). The quantities of reactive oxygen species were measured using a spectrofluorometric assay and standardized to hydrogen peroxide. Using pharmacological probes, the oxidative burst was found to contain upwards of 95% hydrogen peroxide and was believed to be of enzymatic origin. Symbiotic dinoflagellates of the genus Symbiodinium sp. isolated from the gorgonian coral Pseudopterogorgia elisabethae produced a burst that was less than 33% of the magnitude of the oxidative burst in Symbiodinium sp. isolated from Pseudopterogorgia americana and less than 2% of the magnitude of the oxidative burst in the related free-living dinoflagellate, Heterocapsa pygmaea. We recently reported that Symbiodinium sp. from Pseudopterogorgia elisabethae contain high levels of the unique diterpene metabolites, the pseudopterosins, not found in the free-living Heterocapsa pygmaea and Symbiodinium sp. isolated from Pseudopterogorgia americana. Pseudopterosins completely blocked the inducible oxidative burst when applied exogenously to these two dinoflagellate species. Preliminary evidence is presented that raises the possibility that the mechanism could involve inhibition of G-protein activation among many possibilities. These data are the first description of an inducible oxidative burst in dinoflagellates. The significance of the comparative oxidative burst in free-living and symbiotic dinoflagellates is discussed and the potential natural role of the pseudopterosins is postulated.

We report here an analysis of plants used by traditional healers of ancient Persia to induce abortions. Six herbal formulations that contain 39 different species from 21 plant families with their vernacular names, English names, amounts used, and their methods of preparation are reported. Our initial objective of this ethnobotanical analysis was to evaluate the validity and the efficacy of the plants used by (1) comparing other reported uses of these plants in traditional medicine, (2) investigating the medical and pharmacological literature on the medicinal properties of the plant species used, and (3) investigating the reported cytotoxic effects of compounds prevalent in these plants. Previous phytochemical analyses have shown that a number of plant species are rich in coumarin compounds that have potential antineoplastic or cytotoxic activities. Our results indicate a correlation between the reported use of these plants as abortifacients and their cytotoxic (antineoplastic) effects. In addition, we discuss the process in which this ethnobotanical investigation led to the discovery of dicoumarol (a coumarin anticoagulant) as a potential chemotherapeutic agent.

Chloroplasts isolated from three populations of the tropical marine Chlorophyte Anadyomene stellata collected off the coast of Greece were analyzed for their fatty acid composition. Following the preparation of fatty acid methyl esters, GC-MS (El) was utilized to identify the fatty acids present in each population. Including isomers, the fatty acid profile of all three algal populations was comprised of 19 fatty acids (4 saturated, 6 monounsaturated, 9 polyunsaturated) with palmitic acid present in the highest amounts (25-27% of total fatty acids). Analysis of variance revealed significant differences amongst the three populations in the percent of total fatty acids for twelve of the fatty acids. High levels of C20 PUFAs, an atypical observation in Chlorophytes, were observed in all three populations comprising approximately 17% of total fatty acids. Furthermore a 14:2 PUFA , apparently rare in marine macrophytic Chlorophytes, was identified in significant quantities. Surprisingly, we did not find any of the conjugated tetraene containing fatty acids that we previously identified in the A. stellata populations studied from the Florida Keys.

Sea hares are a rich source of novel secondary metabolites, most of which are derived from their algal diet, but the natural function(s) of these metabolites are largely unknown. We used field and laboratory assays to measure the palatability of extracts from the tissues, ink, and eggs of Dolabella auricularia. Digestive-gland extracts contained a wide variety of secondary metabolites, including the red algal compound prepacifinol epoxide and its derivative johnstonol, and they were unpalatable to reef fishes. Skin extracts were moderately unpalatable, but our bioassay-guided fractionation led us to (–)-7-dehydrocholesterol, rather than to an algal secondary metabolite. Ink extracts were consistently unpalatable to reef fishes only at high concentrations, suggesting either that ink must be concentrated to deter predators, that unpalatable components of ink rapidly decompose, or that ink has other functions. Unpalatability of ink was traced to a purple fraction, consistent with the hypothesis that the active compound is aplysioviolin, a known ink constituent modified from a red algal pigment. Egg extracts were moderately unpalatable; however, we could not trace this activity to any algal-derived secondary metabolite. Body-wall extract was highly palatable. Our results suggest that dietary-derived secondary metabolites play a role in chemical defense of D. auricularia via the ink, but are not responsible for unpalatability of skin or eggs. Accumulation of dietary-derived metabolites in the digestive gland may occur to detoxify a chemically rich diet, rather than or in addition to deterring predators.

We describe the discovery of a new member of the pseudopterosin class of marine natural products. Its structure is isomeric with that of pseudopterosin E and has therefore been given the name iso-PsE.

Pseudopterosin E (PSE), a C-10 linked fucose glycoside and pseudopterosin A (PSA), a C-9 xylose glycoside isolated from the marine gorgonian Pseudopterogorgia elisabethae were both effective in reducing PMA-induced mouse ear edema when administered topically (ED50 (microg/ear) PSE(38), PSA(8)) or systemically (ED50 (mg/kg, i.p.) PSE (14), PSA (32)). Both compounds exhibited in vivo analgesic activity in phenyl-p-benzoquinone-induced writhing (ED50 (mg/kg, i.p.) PSE(14), PSA(4). PSE inhibited zymosan-induced writhing (ED50 = 6 mg/kg, i.p.), with a concomitant dose-dependent inhibition of peritoneal exudate 6-keto-prostaglandin F1alpha (ED50 = 24 mg/kg) and leukotriene C4 (ED50 = 24 mg/kg). In vitro, the pseudopterosins were inactive as inhibitors of phospholipase A2, cyclooxygenase, cytokine release, or as regulators of adhesion molecule expression. PSA inhibited prostaglandin E2 and leukotriene C4 production in zymosan-stimulated murine peritoneal macrophages (IC50 = 4 microM and 1 microM, respectively); however, PSE was much less effective. These data suggest that the pseudopterosins may mediate their anti-inflammatory effects by inhibiting eicosanoid release from inflammatory cells in a concentration and dose-dependent manner.

Eicosanoid metabolites were generated by isolated granular amebocytes of the primitive arthropod, Limulus polyphemus, when stimulated by the calcium ionophore A23187 and/or exogenous arachidonic acid. the metabolites were isolated, identified, and the major metabolite was quantified using reverse-phase high pressure liquid chromatography (RP-HPLC) coupled to electrospray ionization mass spectrometry (ESI-MS). Qualitative examination revealed putative metabolites and the major product, 8-hydroxyeicosatetraenoic acid (8-HETE), which was quantified using standard curves generated from extracted ion profiles of the molecular ion. Electrospray ionization of the HETEs in negative ion mode produces a base peak for all isomers which corresponded to the molecular ion [(MH)−: m/z 319]. The molecular ion was accompanied by the neutral loss of water and carbon dioxide [(MH −H2O)−: m/z 301; (MH −H2O −CO2)−: m/z 257], as well as daughter ions which were dependent upon the position of hydroxy substitution. Standard curves were generated in full scan mode for standards ranging from 6.25 to 100 ng, whereas selected ion recording was used for the lower levels of 0.8 to 6.25 ng.

Novel polyunsaturated fatty acids with four conjugated double bonds were found in extracts of the green macroalga, Anadyomene stellata. The isolation of five of these with different chain lengths and varying degrees of unsaturation--16:5, 18:4, 20:5, 20:6, and 22:7--was accomplished by organic extraction followed by a combination of vacuum and high-performance liquid chromatography. One of these that was a novel substance (22:7) was characterized as 4Z,7Z,9E,11E,13Z,16Z,19Z-do cosaheptaenoic acid and assigned the trivial name stellaheptaenoic acid. The structure of this new compound, isolated as its methyl ester derivative, was deduced from detailed nuclear magnetic resonance, gas chromatography/mass spectrometry (GC/MS), and other spectroscopic methods. Incubation of a chloroplast preparation, isolated from a crude algal homogenate by differential centrifugation, with six unsaturated fatty acids (palmitoleic, 6Z,9Z,12Z,15Z-octadecatetraenoic acid, arachidonic acid, eicosapentaenoic acid, 7Z,10Z,13Z,16Z-docosatetraenoic acid, and 4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoi c acid) resulted in substantially increased synthesis of unique tetraene compounds as detected by ultraviolet spectrophotometry and tentatively identified by GC/MS.

A sesquiterpene furanoic acid (SFA) marine natural product isolated from soft corals of the genus Sinularia (Bowden et al., Aust J Chem36: 371–376, 1983) was found to inactivate bee venom phospholipase A2 (bvPLA2, EC 3.1.1.4) in vitro. In this study, we characterized the kinetics of inactivation of bvPLA2 by this compound. The apparent IC50 value was 0.5 μm, and the inactivation of bvPLA2 was time dependent. The drug-enzyme binding appeared to be of a non-competitive, high-affinity nature that was irreversible by aqueous dialysis. The inactivation was prevented by the simultaneous addition of excess lysophosphatidylcholine (lysoPC) during the initial binding step, suggesting that modification of the enzyme by SFA occurs at or near the substrate binding site. Activation of bvPLA2 was observed with lysoPC addition at concentrations equimolar to bvPLA2 and higher. Saturation of activation occurred at concentrations greater than 10 μM lysoPC, and preincubation of bvPLA2 with 100 μM lysoPC did not inhibit the enzyme. Analysis of the post-incubation mixture of SFA-inhibited enzyme in the presence of lysoPC revealed the presence of unaltered enzyme exhibiting typical Michaelis-Menten kinetics. The significance of these observations is discussed in light of the recent discussion by Ortiz on the manoalide binding site on bvPLA2 (Ortiz et al., J Med Chem36: 1866–1879, 1993).

A cyclic octapeptide (patellamide D) isolated from the marine tunicate, Lissaclinum patella, acts as a resistance-modifying agent in the multidrug resistant CEM VLB 100 human leukemic cell line. A three-day microculture tetrazolium proliferation assay was used to determine the 50% inhibitory concentration (IC50) for vinblastine, colchicine and adriamycin and calculate the degree of resistance modulation. Patellamide D at 3.3 μM was compared with 5.1 μM verapamil in modulating drug resistance in vitro. The IC50 for vinblastine was reduced from 100 ng/ml to 1.5 ng/ml in the presence of patellamide D or to 2.1 ng/ml when exposed to verapamil. Colchicine cytotoxicity was enhanced only 1.4-fold by verapamil, as compared with 2.8-fold using patellamide D (IC50 was reduced from 140 ng/ml to 100 ng/ml or 50 ng/ml). Adriamycin toxicity was reduced from IC50 > 1000 ng/ml to 110 ng/ml and 160 ng/ml when coexposed to patellamide D and verapamil, respectively. Our results indicate that patellamide D acts as a selective antagonist in multidrug resistance and stresses the importance of investigating marine-derived compounds as a potential new source for modulators of the drug-resistance phenotype.

The synthesis and in vivo anti-inflammatory activity of a series of pseudopterosin analogues are presented. Synthetic tricyclic catechol aglycons with different substitution patterns were monofucosylated or -xylosylated. Anti-inflammatory activity was conserved over a wide range of structural modifications. The most active synthetic compound 33 reduced phorbol myristate acetate (PMA)-induced inflammation in the mouse ear by 72% at 50 μg/ear. This corresponds to 80% of the activity of natural pseudopterosin A.

The global marine pharmaceutical pipeline consists of three Food and Drug Administration (FDA) approved drugs, one EU registered drug, 13 natural products (or derivatives thereof) in different phases of the clinical pipeline and a large number of marine chemicals in the preclinical pipeline. In the United States there are three FDA approved marine-derived drugs, namely cytarabine (Cytosar-U((R)), Depocyt((R))), vidarabine (Vira-A((R))) and ziconotide (Prialt((R))). The current clinical pipeline includes 13 marine-derived compounds that are either in Phase I, Phase II or Phase III clinical trials. Several key Phase III studies are ongoing and there are seven marine-derived compounds now in Phase II trials. The preclinical pipeline continues to supply several hundred novel marine compounds every year and those continue to feed the clinical pipeline with potentially valuable compounds. From a global perspective the marine pharmaceutical pipeline remains very active, and now has sufficient momentum to deliver several additional compounds to the marketplace in the near future; this review provides a current view of the pipeline.

This review of phospholipase A2 (PLA2) inhibitors from marine organisms presents a compilation of research in this field over the past decade. As an introduction to the research on marine natural products, there is an overview of the role of PLA2 in inflammation that provides a rationale for seeking inhibitors of PLA2 as anti-inflammatory agents. A radiometric assay to measure inhibition of bee venom PLA2 is described in detail. Examples of marine natural products that inhibit PLA2 are manoalide and its derivatives, scalaradial and related compounds, the pseudopterosins, the vidalols, and a group of terpenoids that contain masked 1,4-dicarbonyl moieties.

Inhibition of bee venom phospholipase A 2 (PLA 2 ) by manoalide (1) and luffariellolide (2) involves the initial formation of a Schiff base (imine) between a lysine residue on PLA 2 and an aldehyde group on each of the drugs. Model reactions employing a primary amine in place of the lysine residue were studied by 1 H NMR spectroscopy. Amines reacted at the γ-hydroxybutenolide ring of 2 to produce γ-(alkylamino)butenolides 6a,b, which are cyslized forms of the corresponding Schiff bases. Manoalide methyl analogue (MMA, 12), which is a simple analogue of the reactive portion of 1, reacted similarly. The γ-(n-butyl-amino)butenolide 6b reacted with hydroxylamine to form the oxime 8 with concomitant release of n-butylamine. When the luffariellolide-PLA 2 and manoalide-PLA 2 adducts were treated with hydroxylamine, the PLA 2 activity was substantially recovered, but the activity was not recovered if the luffariellolide-PLA 2 adduct was reduced with sodium borohydride prior to hydroxylamine treatment

Scalaradial (SLD), a marine natural product isolated from the sponge (Cacospongia sp., possesses anti-inflammatory properties in vivo and in vitro (Pharmacologist 32: 168, 1990). In this study we characterize its effects against bee venom phospholipase A2 (PLA2; EC 3.1.1.4). SLD is a potent inactivator of bee venom PLA2 with an IC50 value of 0.07 microM. Inactivation of bee venom PLA2 occurred in a time-dependent, irreversible manner. The rate of inactivation followed first-order reaction kinetics and was dependent on the concentration of SLD. Kinetic analysis suggested a two-step mechanism of inactivation: an initial apparent noncovalent binding (Ki = 4.5 x 10(-5) M) followed by covalent modification. The rate of inactivation was reduced markedly in the presence of excess phosphatidylcholine, suggesting that modification of the enzyme occurs at or near the substrate binding site.

Aqueous extracts of the coralline red algaeBossiella orbigniana have been shown to catalyze the enzymatic oxidation of arachidonic acid to a previously unreported ω6 eicosapentaenoic acid product. This unique fatty acid contains a conjugated tetraene with absorption maxima at 293, 306, and 321 nm, and was identified by spectral methods as 5(Z),8(Z),10(E),12(E),14(Z)-eicosapentaenoic acid. The compound was given the trivial name bosseopentaenoic acid. Under experimental conditions, production of bosseopentaenoic acid was linear up to 2 hr and dioxygen was consumed. Bosseopentaenoic acid, along with several other conjugated tetraenes, was also present in the algae endogenously as revealed by a comparison of the ultraviolet (UV) spectra and the high-performance liquid chromatographic (HPLC) pattern of the purified product and the organic extract ofBossiella.

We describe the discovery of a new member of the pseudopterosin class of marine natural products. Its structure is isomeric with that of pseudopterosin E and has therefore been given the name iso-PsE.

The Suzuki-Miyaura cross-coupling protocol was applied to the synthesis of 1a, the C-glycoside analogue of PsA methyl ether. This marks the first construction of a C-glycoside for this class of marine natural products, thereby offering an opportunity to compare its bioactivity to the natural substances. Its activity profile resembled that of PsA (1) and PsA O-methyl ether (1b) when assayed for its anti-inflammatory activity and its ability to inhibit phagocytosis. We conclude that the intact structure is present when a pseudopterosin expresses its anti-inflammatory and phagocytosis inhibitory properties and that they are, therefore, not likely to be prodrugs. Results show that 1a is an effective binding agent toward the A2A and A3 adenosine receptors, displaying IC50 values of 20 and 10 microM, respectively.

The marine natural product, manoalide (MLD), was investigated to determine if this drug inhibited purified human synovial fluid phospholipase A2 (HSF-PLA2). Utilizing classical Michaelis-Menten kinetics, apparent Km and Vmax values for HSF-PLA2 of 1.34 mM and 0.47 mumol [3H]palmitic acid released/min/mg protein were obtained using dipalmitoylphosphatidylcholine (DPPC) as the substrate, and 38.0 microM and 18.8 mumol [3H]arachidonic acid released/min/mg protein with Escherichia coli as a natural substrate. These kinetic parameters were utilized subsequently to evaluate the inhibitory effects of manoalide on HSF-PLA2. Inhibition of HSF-PLA2 by MLD was concentration and time dependent with IC50 values of 0.2 and 0.02 microM for DPPC and E. coli respectively. Dialysis studies and examination of DPPC or E. coli hydrolysis versus enzyme concentration indicate that MLD is an irreversible inhibitor of HSF-PLA2. Substrate specificity was also examined in the absence and presence of MLD using dipalmitoylphosphatidylethanolamine (DPPE) as a substrate. MLD inhibited the hydrolysis of DPPE (greater than 90% inhibition at 2 microM), and preliminary results indicate that DPPC was more readily hydrolyzed than DPPE under the substrate conditions of the assay. While the cellular source of secreted HSF-PLA2 is unknown, these studies indicate that MLD can inactivate secreted phospholipase A2 isolated from patients with inflammatory joint disease.

PCBs are generally characterized by low vapor pressures; they would not usually be considered volatile. However, they are also characterized by low water solubilities. Studies addressing PCB toxicology have usually employed open aquaria. Therefore, the objective of this investigation was to describe the gas-liquid partitioning of a single PCB isomer, 2,4,5,2{prime},4{prime}5{prime}-hexachlorobiphenyl (HCBP), in seawater within glass aquaria commonly used for toxicological testing. Partitioning may reduce desired exposure levels during testing, seriously effecting results. However, it may be utilized to realistically model environmental conditions during testing. Therefore, the measured H{prime} will be compared to others obtained with specialized purging equipment. 2,4,5,2{prime},4{prime},5{prime}-HCBP was chosen for its presence in common PCB mixtures and high chlorine content, thus low water solubility and high bioaccumulation potential.

We (a) describe the completion of a total synthesis of 7,11-epi-thyrsiferol (4), (b) compare the antimitotic activities of thyrsiferol (2), Delta15,28-dehydrothyrsiferol (3), and 7,11-epi-thyrsiferol (4), (c) evaluate the synergistic behavior of the title compound and colchicine to inhibit cell proliferation, and (d) describe the results of conformational searches that provide additional insight concerning the SAR profile of the thyrsiferol family of natural products.

The free living ciliate Tetrahymena thermophila was chosen as a cellular model in order to investigate the mode of action of the anti-inflammatory marine natural product Pseudopterosin A (PsA). In this paper we present evidence that PsA inhibits phagosome formation (KD = 10.5 μM) and triggers a discrete intracellular calcium release (depletion) from a site in T. thermophila cells (KD = 6.4 μM). Pre-treatment with the Gi/o protein inhibitor, pertussis toxin (PTX), inhibits PsA activity of both responses providing pharmacological evidence that the site of action for PsA is at a PTX sensitive G protein or a G protein coupled receptor (GPCR). Addition of extracellular calcium induced a concentration dependent increase in the incidence of phagosome formation (KD = 30.3 μM) and was blocked by PsA pre-treatment. This particular effect of PsA on extracellular calcium was not blocked by PTX pre-treatment.

SummaryWe investigated the signaling events involved in the wound response of the marine macroalga Dasycladus vermicularis, finding nitric oxide (NO) production in relation to injury. The addition of exogenous H2O2 to aliquots of injured algae accelerated the kinetics of NO production in the wounded region. Similarly, the addition of an NO donor caused an increase in detectable H2O2 around the site of injury. By wounding or incubating uninjured algae with an NO donor, peroxidase activity was enhanced. Based on the use of selected pharmacological probes, our results indicate that H2O2 production involves the upstream activation of signaling events similar to those observed in the physiology of higher plants.

Investigations are reported that identify the biosynthetic source and origins of the pseudopterosins, pharmacologically important diterpene glycosides, in the gorgonian coral Pseudopterogorgia elisabethae . We report here the isolation of physiologically significant levels of endogenous pseudopterosins A, B, C, and D from purified symbionts identified as the dinoflagellate Symbiodinium sp. Biosynthetic studies in photosynthesizing symbiont isolates utilizing 14 C-labeled inorganic carbon and the tritiated intermediate geranylgeranyl diphosphate yielded radiochemically pure pseudopterosins A through D and the first committed intermediate, elisabethatriene. The 14 CO 2 uptake into the pseudopterosin pathway occurred at low levels compared to the 14 CO 2 uptake into total lipids, suggesting a large reserve pool of the compounds. These results reveal for the first time that pseudopterosin biosynthesis is occurring within the algal symbiont and suggests the physiological implications of this biosynthesis.

Analysis of the terpene metabolites of Pseudopterogorgia elisabethae collected from the Florida Keys has resulted in the identification of a novel hydroxyquinone, elisabethadione (1), as well as new pseudopterosins and seco-pseudopterosins. Anti-inflammatory assays indicate that elisabethadione is more potent than the well characterized pseudopterosin A and E. This report also describes the co-occurrence of pseudopterosins and seco-pseudopterosins, diterpenes with amphilectane and serrulatane skeletons, respectively. This together with our previously described isolation of elisabethatriene as the sole diterpene cyclase product in P. elisabethae suggests that the amphilectane and serrulatane families of diterpenes are derived from the same geranylgeranyl diphosphate cyclase product.

Hydroxy fatty acids from Euglena gracilis were identified by reverse-phase high performance liquid chromatography coupled to a mass spectrometer run in atmospheric pressure chemical ionization positive ion mode. These metabolites were converted to methyl esters to improve stability and chromatographic properties. A detection limit of 20 pg/microl per injection was determined for 5-HETE methyl ester based on the signal to noise ratio of the m/z 317 ion which corresponds to the loss of a hydroxyl group (M-17) and the major fragment in all HETE methyl esters studied. This is the first report for these metabolites in E. gracilis.

In studies on the antiproliferative actions of coumarin compounds, we discovered that dicoumarol (a coumarin anticoagulant; 3,3'-methylenebis[4-hydroxycoumarin]) inhibits the first cleavage of Strongylocentrotus purpuratus (sea urchin) embryos in a concentration-dependent manner with 50% inhibition occurring at a concentration of 10 microM. Because first cleavage in sea urchin embryos is highly selective for microtubule-targeted agents, we thought that the active compounds might inhibit cell division by interacting with tubulin or microtubules. We found that dicoumarol binds to bovine brain tubulin with a K(d) of 22 microM and that 0.1 microM dicoumarol strongly stabilizes the growing and shortening dynamics at the plus ends of the microtubules in vitro. Dicoumarol reduces the rate and extent of shortening, it increases the percentage of time the microtubules spend in an attenuated (paused) state, and it reduces the overall dynamicity of the microtubules. The antimitotic effects of the widely used cancer chemotherapeutic agent Taxol (paclitaxel) are also mediated by suppressing microtubule dynamics. We demonstrate that exposure to combinations of Taxol and dicoumarol results in a synergistic inhibition of cell division of sea urchin embryos. The results suggest that the antiproliferative mechanism of action of dicoumarol and possibly related pharmacophores may be mediated by tubulin binding and the stabilization of spindle microtubule dynamics. Because of its low toxicity and simple chemical structure, there is potential interest to explore combinations of antimitotic coumarins with other chemotherapeutic agents to improve efficacy and lower toxicity.

Marine natural products provide a rich source of chemical diversity that can be used to design and develop new, potentially useful therapeutic agents. We report here that scytonemin, a pigment isolated from cyanobacteria, is the first described small molecule inhibitor of human polo-like kinase, a serine/threonine kinase that plays an integral role in regulating the G(2)/M transition in the cell cycle. Scytonemin inhibited polo-like kinase 1 activity in a concentration-dependent manner with an IC(50) of 2 microM against the recombinant enzyme. Biochemical analysis showed that scytonemin reduced GST-polo-like kinase 1 activity in a time-independent fashion, suggesting reversibility, and with a mixed-competition mechanism with respect to ATP. Although scytonemin was less potent against protein kinase A and Tie2, a tyrosine kinase, it did inhibit other cell cycle-regulatory kinases like Myt1, checkpoint kinase 1, cyclin-dependent kinase 1/cyclin B, and protein kinase Cbeta2 with IC(50) values similar to that seen for polo-like kinase 1. Consistent with these effects, scytonemin effectively attenuated, without chemical toxicity, the growth factor- or mitogen-induced proliferation of three cell types commonly implicated in inflammatory hyperproliferation. Similarly, scytonemin (up to 10 microM) was not cytotoxic to nonproliferating endotoxin-stimulated human monocytes. In addition, Jurkat T cells treated with scytonemin were induced to undergo apoptosis in a non-cell cycle-dependent manner consistent with its activities on multiple kinases. Here we propose that scytonemin's dimeric structure, unique among natural products, may be a valuable template for the development of more potent and selective kinase inhibitors used for the treatment of hyperproliferative disorders.

Chemical investigation of the marine red alga (Rhodophyta) Ceratodictyon spongiosum containing the symbiotic sponge Sigmadocia symbiotica collected from Biaro Island, Indonesia, yielded two isomers of a new and bioactive thiazole-containing cyclic heptapeptide, cis,cis-ceratospongamide (1) and trans, trans-ceratospongamide (2). Isolation of these peptides was assisted by bioassay-guided fractionation using a brine shrimp toxicity assay (Artemia salina). The structures of the ceratospongamides, which each consist of two L-phenylalanine residues, one (L-isoleucine)-L-methyloxazoline residue, one L-proline residue, and one (L-proline)thiazole residue, were established through extensive NMR spectroscopy, including (1)H-(13)C HMQC-TOCSY, and (1)H-(15)N HMBC experiments, as well as chemical degradation and chiral analysis. cis,cis- and trans,trans-ceratospongamide are stable conformational isomers of the two proline amide bonds. Molecular modeling of these two ceratospongamide isomers showed the trans, trans isomer to be quite planar, whereas the cis,cis isomer has a more puckered overall conformation. trans,trans-Ceratospongamide exhibits potent inhibition of sPLA(2) expression in a cell-based model for antiinflammation (ED(50) 32 nM), whereas the cis,cis isomer is inactive. trans,trans-Ceratospongamide was also shown to inhibit the expression of a human-sPLA(2) promoter-based reporter by 90%.

A protein expressing phospholipase A2 activity was purified from the granular amebocyte of the horseshoe crab, Limulus polyphemus by cation-exchange, size-exclusion chromatography and semi-preparative reverse-phase–high pressure liquid chromatography (RP–HPLC). The protein had an apparent mass of 17.7 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), but a more accurate estimate of 18.5 kDa was assigned by electrospray ionization–mass spectrometry (ESI–MS). A partial sequence of this protein demonstrated total sequence homology with an 18.5 kDa protein with cell aggregating properties from Limulus reported by Fujii et al. [J. Biol. Chem. 267:22452.]. In these studies, the Limulus protein demonstrated a positive cross-reaction to polyclonal anti-human recombinant phospholipase A2 (group II, 14 kDa). The protein did not display a significant loss of biological activity after boiling, but all enzymatic activity was lost after boiling in the presence of the reducing agent betamercaptoethanol (s-mercaptoethanol). The Limulus protein was inhibited by manoalide, a covalent irreversible phospholipase A2 inhibitor, in a dose-dependent fashion with 50% inhibition occurring at a concentration of 0.48 μM. The Limulus protein displayed no activity in a triglyceride lipase assay. These studies characterize an alternative phospholipase A2 activity for the previously described 18.5 kDa protein from the L. polyphemus amebocyte.

A specimen of Plakortis lita from Palau yielded the new carboxylic acids, homo-plakotenin (2a), the sodium salt of homo-plakotenin (2b), the sodium salt of nor-plakotenin (3), the sodium salt of plakotenin (1b), and the known compound plakotenin (1a). The structures of the new acids were elucidated by interpretation of spectral data and by comparison with the known compound. Compounds 1a, 1b, and 2a were found to significantly reduce proliferation of rheumatoid synovial fibroblasts.

Fundamental to the complex process associated with drug discovery is the establishment of a carefully defined collaborative relationship between academic bioorganic chemists and cellular or molecular pharmacologists. We mention this as a new emerging direction in research that should be particularly emphasized in an academic setting. The collaborative goal of the academic chemists and pharmacologists should be to contribute in some specific way to the advancement of each other’s science. Educational goals are not served well through routine programs for isolating and screening natural products. Screening programs of this kind are well developed in U.S. and foreign drug companies. In fact, new molecular receptor assays that allow a wide variety and large number of screens to be executed rapidly are becoming available. Academic pharmacologists have the responsibility to develop knowledge fundamental to advancing biomedical science by using bioactive substances as probes to better understand normal and pathological biological processes, to define in rigorous fashion the properties of these probes, and to determine their biochemical limitations and how they can be used properly and safely.

Twelve out of twenty-nine compounds isolated from benthic marine algae from the phyla Chlorophyta, Phaeophyta and Rhodophyta have been found to be potent inhibitors of bee venom derived phospholipase A2 (PLA2) (> 50%) in the μM range. The compounds investigated were from: Bryopsis pennata, Rhipocephalus phoenix, Caulerpa prolifera, C. racemosa, C. bikinensis, Cymopolia barbata, Laurencia cf. palisada, Laurencia sp., Ochtodes crockeri, Liagora farinosa, Sphaerococcus coronipifolius, Phacelocarpus labillardieri, Dictyota sp., Bifurcaria galapagensis, Stypopodium zonale, Dictyopteris undulata, Stoechospermum marginatum, Dictyopteris divaricata, Dilophus fasciola and Dilophus sp. This is the first report of bee venom PLA2 inhibition in vitro by pure compounds isolated from marine algae.