Section IV Applied Marine Chemical Ecology 9064_Section IV Div/fm Page 1 Tuesday, April 24, 2001 5:14 AM © 2001 by CRC Press LLC 523 Marine Chemical Ecology: Applications in Marine Biomedical Prospecting Susan H. Sennett CONTENTS I. Introduction 523 II. Targets for Marine Natural Products Drug Discovery 524 III. Marine Natural Products as a Source for Drug Discovery 528 A. Historical Aspects 528 B. Establishing Trends in Incidence of Activity 528 C. Bioactivity and Ecological Interactions 530 IV. Considerations for Development 531 V. Addressing the Supply Issue 531 A. Microbial Localization by Inference 531 B. Direct Evidence for Metabolite Localization in Host Cells 532 1. In Vitro Production of Bioactive Compounds 533 C. Direct Evidence for Microbial Localization 534 D. Established Methods for Alternative Supply 535 VI. A Multidisciplinary Approach to Marine Natural Products Drug Discovery 536 References 537 I. INTRODUCTION Natural products have long been a source of bioactive metabolites utilized for the treatment of human disease. In spite of advancing technologies and the development of compound sources such as combinatorial chemistry, genomics, and screening programs utilizing high throughput technol- ogy, natural products remain a valuable source of bioactive compounds with therapeutic potential. 1–4 It has been reported that 57% of the 150 most prescribed drugs have their origins in natural products. 5 Furthermore, it has been estimated that more than 60% of drugs approved for use as anticancer and antiinfective agents are derived from natural sources including semisynthetic derivatives, synthetic analogs, as well as the original natural products. 6 A major advantage in using natural products as a source for drug discovery is the structural diversity of these compounds when compared to other sources in use. 7 The marine environment is a vast resource for the discovery of structurally unique bioactive secondary metabolites, some belonging to totally novel chemical classes. 8 Sessile benthic organisms including the Porifera, Cnidaria, Bryozoa, and Tunicata as well as marine algae have developed an arsenal of compounds which have been demonstrated to confer a competitive advantage in ecosys- tems characterized by extreme resource limitations. Interactions of these organisms at the genetic, 16 9064_ch16/fm Page 523 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC 524 Marine Chemical Ecology species, and ecosystem levels provide the basis for the production of these compounds. Hay and Fenical 9 provide detailed examples of chemically mediated effects on ecosystems, species diversity, as well as intraspecific diversity in production of marine natural products, and how these interactions affect the biological and chemical diversity of the oceans. To date, there are more than 10,000 publications relating to marine natural products and more are being added to the list at an increasing rate. 2 However, it is estimated that discoveries to date only reflect a small percentage of the biological and chemical diversity of the marine environment, including macro- and microorganisms. Advances in technology for collection of marine organisms, isolation and cultivation of marine microorganisms, and isolation and structure elucidation of marine natural products will facilitate the discovery of numerous other compounds. II. TARGETS FOR MARINE NATURAL PRODUCTS DRUG DISCOVERY There is a growing urgency to identify novel drug leads in disease areas for which treatment or cure has been elusive. A recent review of the use of natural products (predominantly plant and microbial) in pharmaceutical screens summarized the results of drug discovery efforts in a number of these critical areas, including infectious (e.g., drug resistance), neurological (e.g., Parkinsons, Alzheimer’s), cardiovascular, immunological (e.g., transplant rejection), anti-inflammatory (e.g., arthritis), antiviral (particularly HIV), and oncological (particularly solid tumors) diseases. 3 This review provides compelling evidence that supports the use of natural products in pharmaceutical screening programs. To date, active marine natural products have been identified in a number of these disease targets, including anti-infective, immunomodulatory, anti-inflammatory, antiviral, and anticancer areas. Recent reviews provide examples of bioactive compounds correlated with taxo- nomic source and therapeutic group. 10,11 Traditionally, targets have been cell-based, in vitro assays used to detect antiinfective and cytotoxic activities. Increasingly, assays for specific molecular targets in the above disease areas are being developed for use in high throughput screening. Mechanistically, marine derived com- pounds are known to interact with a variety of molecular targets (Table 16.1). TABLE 16.1 Molecular Targets for Marine Natural Products Target Compound Source Therapeutic Area Actin Jasplakinolide (Structure 16.1) Latrunculin A (Structure 16.2) Sponge, Jaspis sp. Sponge, Latrunculia sp. Anticancer 12 Anticancer 13 Tubulin Discodermolide (Structure 16.3) Curacin (Structure 16.4) Sponge, Discodermia sp. Cyanophyte, Lyngbya majuscula Anticancer 14 Anticancer 15 Phospholipase A 2 Manoalide (Structure 16.5) Sponge, Luffariella variabilis Anti-inflammatory 16 Protein phoshatases Okadaic acid (Structure 16.6) Dysidiolide(cdc25) (Structure 16.7) Discorhabdin P (Structure 16.8) (calcineurin) Dinoflagellate, Prorocentrum lima Sponge, Dysidea etheria Sponge, Batzella sp. Anticancer 17 Anticancer 18 Heart disease 19 Protein kinase C Bryostatin 1 (Structure 16.9) Bryozoan, Bugula neritina Anticancer 20 Ion channels Saxitoxin (Structure 16.10) Dinoflagellate, Alexandrium spp. Pain 21 Nicotinic acetylcholine receptor Conus toxins Cone snails, Conus sp. Pain 22 Topoisomerase II Makaluvamines (Structure 16.11) Sponge, Zyzzya sp. Anticancer 23,24 9064_ch16/fm Page 524 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC Marine Chemical Ecology: Applications in Marine Biomedical Prospecting 525 16.9 O O O O O O O OH OH O O O O O O HO OH H H H H H H H H H 16.8 N H N N O O Br Br CH 3 16.7 CH 3 CH 2 CH 3 OH CH 3 O CH 3 O HO O HO H H H H OH H OH H H OH O O O O O O O 16.6 16.5 O O O OH OH CH 3 CH 3 CH 3 CH 3 N S HH OCH 3 H 16.4 16.3 O CH 3 OH O O CH 3 OH CH 3 OH CH 3 OH CH 3 CH 3 CH 3 CH 3 CH 2 NH 2 O O HO O O O S HN 16.216.1 OHN CH 3 N NH O CH 3 CH 3 O O O CH 3 CH 3 Br HN CH 3 OH 16.11 N + N O CH 3 NH 2 H HN N N N H 2 + N NH 2 + OH OH CH 2 OCO 2 NH H H 16.10 OSO 3 - 9064_ch16/fm Page 525 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC 526 Marine Chemical Ecology Several other molecular targets of interest for natural products screening include: the dopamine receptor for Parkinson’s disease and acetylcholinesterase for Alzheimer’s disease. In addition, inhibitions of protein and peptidoglycan syntheses are targets for infectious diseases. As we gain a greater understanding of the molecular basis of these disease areas, additional targets will become available. A number of compounds listed in Table 16.1 have not proved suitable as drug candidates but are being used as molecular probes that may facilitate the identification of new targets and the discovery of new drug candidates. These compounds include manoalide, okadaic acid, and neuro- toxins such as saxitoxin and tetrodotoxin. 25 Currently, there are four marine-derived compounds in clinical trials for anticancer use: bry- ostatin 1 (Structure 16.9), dolastatin 10 (Structure 16.12), ecteinascidin 743 (Structure 16.13) (ET743), and aplidine (dehydrodideminin B) (Structure 16.14). Bryostatin 1, isolated from the bryozoan Bugula neritina , 26 is in NCI sponsored Phase II trials against melanoma, non-Hodgkins lymphoma, and renal cancer. Dolastatin 10 isolated from the sea hare Dolabella auricularia 27 is in Phase I trials against breast and liver cancers, solid tumors, and leukemia, also sponsored by the NCI. ET743 and aplidine, isolated from the tunicates Ecteinascidia turbinata 28,29 and Aplidium albicans , 30 respectively, are anticancer agents 31,32 undergoing evaluation conducted by PharmaMar (based in Spain). Although these compounds are all anticancer agents, they act through different mechanisms. Bryostatin 1, a cyclic macrolide, inhibits protein kinase C tumor promotion while aplidine is a protein synthesis inhibitor. Dolastatin 10, a linear peptide, and ET743, a tetrahydroisoquinoline 16.14 H N O O N N H N O OH O O O NH O N O O O O N O O O 16.13 N N O HO O O O S O O NH HO O O OH H H H H H 16.12 NH N(Me) 2 Me N OMe O O O N O OMe HN S N 9064_ch16/fm Page 526 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC Marine Chemical Ecology: Applications in Marine Biomedical Prospecting 527 alkaloid, are both antimitotic agents. However, dolastatin 10 acts at the GTP binding site, while ET743 acts through the disorganization of the microtubule network. It is interesting to note the phyletic, structural, and mechanistic diversity among these compounds. 2 In addition to the compounds currently in clinical trials, there are a number of compounds in advanced pre-clinical trials. Discodermolide (Structure 16.3) is a polyhydroxylated lactone isolated from the deep-water sponge Discodermia dissoluta . 33 An antimitotic compound which stabilizes microtubules, 14 discodermolide has been licensed to Novartis Pharma AG for development as an anticancer drug. Halichondrin B (Structure 16.15), isolated from the deep-water sponge Lissoden- doryx , 34 is in pre-clinical trials at the NCI for the treatment of melanoma and leukemia. Kahalalide F (Structure 16.16), isolated from the mollusc Elysia rubefescens , 35 is being evaluated as an anticancer agent against colon and prostate cancer. The pseudopterosins (e.g., Structure 16.17), anti-inflam- matory agents from the Caribbean sea whip Pseudopterogorgia elisabethae , 36 have been licensed by Nereus Pharmaceuticals (La Jolla, CA) for evaluation of their activity in pharmaceutical assays. 37 The pseudopterosins are currently on the market as a component of Estee Lauder’s Resilience line of skin care products. 16.17 CH 3 H CH 3 OH CH 3 CH 3 CH 3 O HO O HO OH 16.16 NH O CH 3 OHN CH 3 CH 3 O O CH 3 HN O CH 3 H N O CH 3 CH CH 3 O NH NH O N H CH 3 CH 3 O H 2 N NH O N CH 3 CH 3 NH CH 3 CH 3 HN O HO CH 3 H N O CH 3 CH 3 NH O CH 3 CH 3 O O 16.15 OO O O O O O O O OOH OHOH O O O O CH 2 O O H H CH 3 CH 2 CH 3 H CH 3 H HH H CH 3 H H H H 9064_ch16/fm Page 527 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC 528 Marine Chemical Ecology In several cases, series of structurally related analogs such as the didemnins have been discov- ered from a single source, the compounds within the series demonstrating varying levels of activity in pharmaceutical assays. Evaluation of such a series of analogs provides an opportunity for examination of structure–activity relationships and selection of the most active structure. 11 Other series of analogs have been evaluated for the bryostatins, halichondrins, dolastatins, and crypto- phycins. Interestingly, the sources for these groups of compounds are taxonomically distinct. Ecologically, it may be hypothesized that these variants are produced to be effective against specific predators or fouling agents rather than producing one potent, generally active compound. Alterna- tively, a group of such closely related compounds may be used to target a specific group of isozymes. Marine natural products have also been shown to have multiple activities in pharmaceutical assays. For example, dercitin (Structure 16.18), a heterocyclic acridine alkaloid isolated from the deep-water sponge Dercitus , has in vitro antiviral, antitumor, and immunomodulatory activity. 38,39 Topsentin (Structure 16.19), a bis (indole)-alkaloid isolated from the deep water sponge Spongosorites , is a potent anti-inflammatory agent 40 in addition to having antitumor and antiviral activity. 41,42 The antitumor compound discodermolide, produced by the deep-water sponge Disco- dermia, was originally isolated as an immunosuppressive agent 30 before its antitumor activity was discovered. Multiple activities might suggest interaction with multiple receptors or targets. It may be possible that these nonspecific compounds might have multiple ecological targets as well. Marine-derived secondary metabolites have clearly had an impact on the discovery of novel agents with pharmaceutical potential. Continued prospecting in the marine environment will surely result in the identification of additional drug leads. III. MARINE NATURAL PRODUCTS AS A SOURCE FOR DRUG DISCOVERY A. H ISTORICAL A SPECTS The use of marine natural products in drug discovery began serendipitously in the 1950s with the isolation of the arabinosyl nucleosides spongothymidine and spongouridine from the sponge Cryt- potethya crypta . 43,44 These compounds were used as models for the production of Ara A and Ara C, antiviral and anticancer drugs that are currently the only marine-derived compounds that have been marketed as pharmaceuticals. During the 1960s, the driving force for marine natural products research was the structural novelty and diversity of marine-derived compounds. 8,10 Interest in these unique compounds was heightened by the realization that many had biomedical potential, and so began a trend in chemical prospecting that continues to accelerate. 45 B. E STABLISHING T RENDS IN I NCIDENCE OF A CTIVITY As there was no ethnomedicinal history to guide the selection of organisms to probe for bioactive natural products, two basic approaches were taken. Early discovery programs sought to establish 16.19 N H HN N N H O N N S N N CH 3 CH 3 CH 3 16.18 9064_ch16/fm Page 528 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC Marine Chemical Ecology: Applications in Marine Biomedical Prospecting 529 trends in incidence of activity to use as guides for subsequent collections. 46–48 This was accomplished by screening crude extracts of large numbers of organisms in a variety of bioassays. Resultant trends were observed in the following areas: taxonomy, geography, bioactivity, and structural class. Although the bioassays used in these studies ranged from pharmaceutical screens in human disease areas, such as anticancer, antiviral, and anti-infective, to more ecologically relevant assays including inhibition of settlement of larvae and behavioral modification of invertebrate adults, 46 there were several trends in common. However, there were some discrepancies in the trends reported (e.g., no latitudinal trend 49,50 ). In general, trends suggest that the tropics, especially the Indo-Pacific, are the greatest source of bioactive natural products. 51 However, temperate, 52 Antarctic, 53 and deep-water habitats 33,38,41 have also proven to be good sources. Although the logistics of obtaining organisms from these environments may be more difficult, the extreme conditions would suggest the potential for even greater diversity of bioactive metabolites. The tropics, in general, are characterized by high diversity but low abundance of individuals with bioactive metabolites. Conversely, polar regions support lower diversity but higher abundance of source organisms. 54 Greater biomass of source organisms is an important consideration as supply for biological evaluation becomes an issue for compounds identified as viable drug candidates. This supply issue is discussed in more detail later. Taxonomically, data suggest that the Porifera are the most prolific in the production of bioactive metabolites. 55 Other groups rich in bioactive compounds include the cnidarians, bryozoans, tuni- cates, and algae. It is interesting to note that in spite of the number and diversity of compounds produced by sponges, many sponge metabolites have not been suitable drug leads because of their extreme toxicity. It may be possible that the primitive nature of these sessile, benthic organisms requires production of extremely potent defensive compounds such as the cytotoxic mycalamides isolated from Mycale sp. 56 and the anti-inflammatory manoalide isolated from Luffariella variabi- lis , 57 both of which have proven to be too toxic to be clinically useful to date. Another point of interest with regard to the Porifera is that discodermolide and halichondrin B, currently in advanced preclinical trials, have been isolated from deep-water organisms. It is possible that differences in resource limitations and predation pressure account for differences in the level of activity. On a finer scale, one study found correlations between antiviral and cytotoxic activities and growth forms in the Porifera and concluded that encrusting individuals had higher antiviral activity, perhaps enhancing their ability to rapidly colonize substrates, overgrow other individuals, and prevent being overgrown by other encrusting organisms. The solitary, erect forms, which were more often grazed upon than the encrusting forms, had greater cytotoxic activity, suggesting a possible antipredatory role. 47 Uriz et al. 48 identified chemical strategies among groups of organisms in the Mediterranean using nonmarine assays. Correlations were observed between antitumor and cyto- toxic activities and between antibacterial and antifungal activities. It was suggested that the anti- microbial activities might be correlated with different levels of fouling. More recently, Reed et al. 58 used a database linking biological descriptors (e.g., taxonomy, morphology, and observed associations), physical data (e.g., location, depth, and salinity), and bioactivity data to examine trends for a worldwide collection of over 25,000 marine macroinver- tebrates and algae. Bioassays used in this study included whole-cell and receptor-based assays, which are generally more specific and have lower hit rates than the whole-cell assays. The greatest incidence of activity was found in the Porifera, as was observed in previous studies. Comparison of bioactivity with depth of collection suggests that, although different for each type of assay, activity is observed throughout the depth range of this study (0–3000 ft). Latitudinal trends were observed within assays as well as within some of the phyla. These data indicate that the negative correlation between bioactivity and latitude is not without exception, and other factors such as in situ temperature and depth should be considered. Further analysis of this large data set may be used to guide further collections. As demonstrated, random screening can provide insight into a variety of trends in bioactivity. However, these investigations are often restricted to a particular geographic region, taxonomic 9064_ch16/fm Page 529 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC 530 Marine Chemical Ecology group, or screening target, limiting the ability to apply findings to benthic communities in general. It has been suggested that finding unique structural classes is enhanced by screening organisms from many taxa when compared to screening samples within a single taxon. 59 C. B IOACTIVITY AND E COLOGICAL I NTERACTIONS A second, perhaps more rational, approach to marine chemical prospecting is based upon obser- vation of ecological interactions in situ . Examples of these chemical ecological interactions and corresponding bioactivity are the subject of reviews by Bakus et al., 60 Paul, 51 and Hay, 61 as well as in other chapters in this volume. Trends in the incidence of bioactivity reported in these studies are similar to those established by random screening. The most studied interactions have been with predators/herbivores as these are believed to exert the greatest pressure driving the evolution of chemical diversity, especially in tropical systems. 9 More recently, the focus has shifted to receptor- based activities including antifouling, 62,63 gamete settlement, 64,65 metamorphic cues, 66 and allelo- pathic interactions. 67–69 Considering the variety of stimuli, it is not surprising that some metabolites have more than one function. 67,70,71 These compounds may act independently or have additive or synergistic effects with other metabolites produced by the same organism. For example, Thacker et al. 67 reported that the crude extract of a Dysidea sp. was more deterrent against predators and caused a greater amount of necrosis than the pure compound, 7-deacetoxyolepupuane. Would compounds with multiple ecological roles and, perhaps, multiple effects on one or several receptors be appropriate as drug leads? Is it possible that, mechanistically, these compounds act in a non- specific manner in defending against predators, fouling organisms, and competitors for substratum? Perhaps nonspecificity would allow interaction with several molecular receptors that, synergistically, would provide the desired organismal response. Would the activity be nonspecific in mammalian systems as well? Although several of the compounds listed in Table 16.1 have multiple pharmaco- logical roles, ecological roles and specificity of these compounds are not known. Marine macroinvertebrates may produce a number of different compounds which may or may not have the same biosynthetic origins. These compounds may be products of the invertebrate host, microbial associates, or a combination of the two. 72,73 The true role of the compound may differ depending on the origin. For example, production of an antibiotic by a microbial associate might suggest a role in competition against nonsymbiotic strains. However, if the host invertebrate is the source, the compound may aid in feeding efficiency by clumping bacteria, as was suggested by Thompson et al. 74 as a role for aerothionin in Aplysina fistularis . Regardless of the biogenetic origin of these compounds, the intact association provides benefits. Another consideration in targeting potential sources of bioactive compounds is that a variety of organisms at higher trophic levels may not synthesize their own defenses but may acquire them instead from dietary sources. Examples include molluscs ingesting sponge metabolites and storing them in gut or mantle tissues or transferring the compound to larvae. 75 Compounds may be sequestered in the form in which they are ingested, 76 or they may be modified by the consumer, altering the activity. 77 Comparison of the original natural product with modified analogs may provide insight into structure–activity relationships and suggest the source with the greatest potential for the desired bioactivity. The route of biotransformation might also provide insight for further modification after isolation. Finally, recent reviews indicate that marine microorganisms are potentially a greater source of bioactive compounds than marine macroorganisms. 78–81 Marine microorganisms can be found in seawater or sediment, associated with macroorganisms either on the surface or symbiotically, and in extreme environments. Extremophiles, in particular, may have the greatest capacity for the production of unique bioactive metabolites. 78 9064_ch16/fm Page 530 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC Marine Chemical Ecology: Applications in Marine Biomedical Prospecting 531 IV. CONSIDERATIONS FOR DEVELOPMENT When a compound from a marine source is identified as a drug lead or candidate, there are several issues to consider related to supply of the compound: yield of the compound, abundance of the source organism, ease and cost of recollection, and, finally, the issue of sovereign rights over biological resources. The UN Convention on Biological Diversity requires that member countries be committed to the conservation and sustainable use of natural resources and the fair and equitable sharing of revenues resulting from commercialization of products based on natural resources. 82 These requirements are having a significant impact on where collections can be made and who will benefit from subsequent discoveries. V. ADDRESSING THE SUPPLY ISSUE There are many reports of intraspecific variation in the quantitative and qualitative production of secondary metabolites. These variations can occur within a single member in a population, 83 among different members of a population, 84 or among different populations of a given species. 85,86 Dif- ferences may be due to differences in levels of predation, habitat, 87 ontogeny, 88 geographic loca- tion, 89,90 and depth, 91 though it is not known whether the differences are genetically or environ- mentally mediated. As a result of this sometimes ephemeral production, or simply the naturally low yield of some compounds, adequate supply for preclinical and clinical evaluation is a critical issue. 92 It has been demonstrated that obtaining sufficient quantities by bulk collection from natural populations is not economically or ecologically feasible. 93 The long-term impact of massive collections on the survival of wild populations is unknown, but it is obvious that if marine-derived compounds prove to be clinically successful, demand will exceed what the natural populations can provide. Also at issue is the preservation of marine biodiversity that relates, in turn, to chemical diversity. As stated previously, discoveries to date reflect a small percentage of the resources available. Measures must be taken to ensure sustainable use of these resources. Alternative renewable sources need to be identified to supply pharmacological evaluation. Due to the critical nature of this aspect of marine natural products drug discovery, several alternative approaches are discussed in detail. There are several approaches that can be taken to supply material for pharmaceutical evaluation. Two of these, invertebrate cell culture and fermentation of associated microorganisms, begin with determining the biogenetic origin of the compound. There are numerous reports of metabolite localization, primarily in sponges, in which production of a bioactive metabolite has been inferred or demonstrated to be localized either in a host invertebrate cell or in microbial associates which include cyanobacteria and heterotrophic bacteria and fungi. 72,73 A. MICROBIAL LOCALIZATION BY INFERENCE There are a number of examples in which the production of a metabolite isolated from a marine invertebrate has been attributed to an associated microorganism. 94 Some of these reports are based on circumstantial evidence such as similarity of the metabolite to a compound produced by a microorganism. For example, a bacterium of the genus Alteromonas isolated from the sponge Halichondria okadai produced the macrocyclic lactam alteramide A (Structure 16.20) in culture. 95 This compound, which was not found in extracts of the host sponge, appears to be biogenetically related to ikarugamycin (Structure 16.21), a peptide-like antibiotic isolated from a terrestrial Strep- tomyces sp. 96 Discodermide (Structure 16.22), isolated from the marine sponge Discodermia dissoluta, 97 also appears to be structurally related to these compounds. Based on this structural similarity, it has been suggested that discodermide is a microbial metabolite. 9064_ch16/fm Page 531 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC [...]... required large-scale collections Unfortunately, target populations had already been stressed by El Niño conditions which exacerbated the negative impact of the collections.93 In-the-sea and land-based aquaculture can be cost-effective production methods which afford increased control over environmental conditions CalBioMarine Technologies (Carlsbad, CA) has developed both land- and sea-based systems... J P., Chemical diversity and genetic equity: synthetic and naturally derived compounds, in High Throughput Screening, Devlin, J P., Ed., Marcel Dekker, New York, 1997, 3 60 Bakus, G J., Targett, N M., and Schulte, B., Chemical ecology of marine organisms: an overview, J Chem Ecol., 12, 951, 1986 © 2001 by CRC Press LLC 9064_ch16/fm Page 540 Tuesday, April 24, 2001 5:27 AM 540 Marine Chemical Ecology. .. as they may © 2001 by CRC Press LLC 9064_ch16/fm Page 535 Tuesday, April 24, 2001 5:27 AM Marine Chemical Ecology: Applications in Marine Biomedical Prospecting 535 OCH3 O OH O OH O OCH3 OH O OH HO O OCH3 HO O OH O HO O OCH3 16. 23 OO O N H HO O OH OH HN CH3 HN HO OHN Br H2N O O O N H O HO OH CH3 O HN O N+ H N O N H HO O NH O O NH OH O H2N OH N H N NH O HO CH3 16. 24 allow for manipulation of growth and...9064_ch16/fm Page 532 Tuesday, April 24, 2001 5:27 AM 532 Marine Chemical Ecology H H H N H N H HH O O H H HO OH H HO H NH NH O O O H HO H H 16. 20 O 16. 21 O H NH H H H H O O OH H NH O OH 16. 22 The occurrence of structurally related metabolites in unrelated taxa has also been cited as evidence for... will also be necessary for the conservation and sustainable use of marine natural products.123 © 2001 by CRC Press LLC 9064_ch16/fm Page 537 Tuesday, April 24, 2001 5:27 AM Marine Chemical Ecology: Applications in Marine Biomedical Prospecting 537 Pharmacologists have acquired a tremendous amount of information regarding the activity of marine natural products in mammalian systems However, ecological... scalaradial, J Am Chem Soc., 114, 5093, 1992 © 2001 by CRC Press LLC 9064_ch16/fm Page 538 Tuesday, April 24, 2001 5:27 AM 538 Marine Chemical Ecology 17 Bialojan, C and Takai, A., Inhibitory effect of a marine- sponge toxin, okadaic acid, on protein phosphatases, Biochem J., 256, 283–290, 1988 18 Gunasekera, S P., McCarthy, P J., Kelly-Borges, M., Lobkovsky, E., and Clardy, J., Dysidiolide: a novel protein... fractionation, differential centrifugation, and auto- and induced-fluorescence combined with flow cytometric sorting as well as confocal or electron microscopy The following examples detail the methods used and the site of production of the target compounds © 2001 by CRC Press LLC 9064_ch16/fm Page 533 Tuesday, April 24, 2001 5:27 AM Marine Chemical Ecology: Applications in Marine Biomedical Prospecting 533 Cellular... sponge Lissodendoryx isodictyalis Carter, J Chem Ecol., 16, 791, 1990 64 Pawlik, J R., Induction of marine invertebrate larval settlement: evidence for chemical cues, in Ecological Roles of Marine Natural Products, Paul, V J., Ed., Comstock Publishing Associates, Ithaca, NY, 1992, 189 65 Pawlik, J R., Chemical ecology of the settlement of benthic marine invertebrates, Oceanogr Mar Biol Annu Rev., 30,... defenses of temperate vs tropical seaweeds, Ecology, 77, 2269, 1996 © 2001 by CRC Press LLC 9064_ch16/fm Page 541 Tuesday, April 24, 2001 5:27 AM Marine Chemical Ecology: Applications in Marine Biomedical Prospecting 541 86 Pettit, G R., The bryostatins, Prog Chem Org Nat Prod., 57, 153, 1991 87 Swearingen, D C., III and Pawlik, J R., Variability in the chemical defense of the sponge Chondrilla nucula... in Ecological Roles of Marine Natural Products, Paul, V J., Ed., Comstock Press, Ithaca, NY, 1992, 164 52 Steinberg, P D., Geographical variation in the interaction between marine herbivores and brown algal secondary metabolites, in Ecological Roles of Marine Natural Products, Paul, V J., Ed., Comstock Press, Ithaca, NY, 1992, 51 53 McClintock, J B., and Baker, B J., Chemical ecology in Antarctic seas, . Section IV Applied Marine Chemical Ecology 9064_Section IV Div/fm Page 1 Tuesday, April 24, 2001 5:14 AM © 2001 by CRC Press LLC 523 Marine Chemical Ecology: Applications in Marine Biomedical. establish 16. 19 N H HN N N H O N N S N N CH 3 CH 3 CH 3 16. 18 9064_ch16/fm Page 528 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC Marine Chemical Ecology: Applications in Marine Biomedical. Schulte, B., Chemical ecology of marine organisms: an overview, J. Chem. Ecol., 12, 951, 1986. 9064_ch16/fm Page 539 Tuesday, April 24, 2001 5:27 AM © 2001 by CRC Press LLC 540 Marine Chemical Ecology 61.