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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Nat Prod. Author manuscript; available in PMC Mar 27, 2010.
Published in final edited form as:
PMCID: PMC2837139
NIHMSID: NIHMS95118

A Selective Account of Effective Paradigms and Significant Outcomes in the Discovery of Inspirational Marine Natural Products[perpendicular]

Abstract

Marine natural products continue to be a source of significant molecular structures that serve as a stimulus to seed further significant research. This account reviews some of the major advances in the study of marine biomolecules made at UC Santa Cruz over more than three decades. The continuing challenge of discovery and characterization of what we term “inspirational molecular structures”, will be presented in a comprehensive fashion. Examples of privileged molecular structures and their impact on biomedicinal research will be an important theme. The three major groups of organisms explored include: seaweeds, sponges, and marine derived fungi, and the study of their active principles has greatly benefited from synergistic collaborations with both academic and biopharmaceutical groups. The concluding sections of this chronicle will touch on prospects for future outcomes involving new sources and strategies.

Introduction

The field of marine natural products chemistry has a rich history and it offers continued promise for breakthroughs impacting many areas of science, especially chemical biology. In the early days our work was quite chemo-centric but today it is interdisciplinary and benefits from synergistic collaborations with both academic and biopharmaceutical groups. A continuing core task involves the discovery and characterization of what we term “ inspirational molecular structures.” A major focus has been to exploit these structures as seeds for additional fundamental research alongside their development as potential therapeutic leads and/or application as molecular probes.

We have made substantial progress, especially since 1985, when the work of our group began to fully take shape; however there are still uninvestigated frontiers. Overall, there are eight initiatives that continue to guide our research, some of which are illustrated in Figure 1. These broadly based endeavors include: (a) discovery and characterization of new small molecules emphasizing those from polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS) and mixed PKS-NRPS pathways, (b) developing new and known structures as biomedically relevant leads, (c) engaging in crisp de novo structure elucidation accompanied by efficient dereplication, (d) using the natural products from coral reef sponges and marine- derived fungi (sourced from sponges or sediments) as stimuli for further inquiry, (e) engaging in careful taxonomic identification of all organisms explored, (f) biographical studies of sponges with high value metabolites, (g) developing new methodologies for creating the libraries from macro- and micro- marine organisms, and (h) employing novel culture strategies for expanding the libraries. The sections that follow contain examples of insights obtained from exploration of these ideas. Of equal importance will be highlights showing the difficulties encountered and the significance of discoveries made.

Figure 1
Overview of reseach initiatives: i) sponges, ii) fungi, iii) cytoskeletal screens, and iv) biosynthetic relationships

To date, our lab has brought to light nearly 1,000 compounds from marine sponges and marine-derived fungi. Years ago, a repository was created to house these compounds and several thousand of the following: (a) sponge material ready for processing, (b) crude extracts and semi-pure fractions, (c) preserved microbial cultures, and (d) sponge taxonomic voucher specimens. A large amount of information exists for these materials and it is managed by powerful relational databases and web-based chemoinformatics. The pure compounds, compound rich mixture extracts, and our emerging repository of peak libraries constitute invaluable resources for detailed biomedical and related research, especially as new therapeutically relevant molecular targets are discovered. Also important is that the inherent chemical complexities of the structures in our repository provide robust materials for collaborative projects focused on new emerging technologies in chemical biology research.

It is relevant to discuss an early event that was the impetus for beginning a program of marine organic chemistry at U.C. Santa Cruz. Two significant books provided important foundation reading. The first was the monograph published in 1973 by Paul Scheuer with the title Chemistry of Marine Natural Products.1 It described some 430 compounds organized by biogenetic chemical class and clearly showed the advantages of looking at the marine environment for new molecular structures. Sometime in 1974, one of us (P.C.) came across a book entitled Poisonous and Venomous Marine Animals of the World,2 published in 1965, and was fascinated to read in the chapter on Porifera that extracts from sponges had been shown to possess antibiotic and antiparasitic properties. More exciting was the short section describing the chemistry of the bioactive principles; it contained only one word, “unknown,” indicating that this could be a frontier topic for future research. As a phylum, sponges are an incredibly attractive research target because of their high biodiversity, widespread distribution, and unique aquiferous biology. Sponges are well known as hosts for a variety of microorganisms and they provide a steady stream of nutrients for symbionts held within the choanocyte chambers. Typically, sponges pump a liter of water per cm3 of tissue per hour. In addition, it is estimated that there are in excess of 5,000 species of sponges, and this is undoubtedly a conservative estimate. For a variety of reasons, it took several years to become fully immersed in the chemistry of this phylum, in part because for eight years our attention was diverted by fascinating studies involving halogenated compounds from red seaweeds. Once attention had been refocused on sponges as a source of new chemical entities, the goal to engage in anticancer therapeutic lead discovery was also begun.

The other source for natural products currently being pursued by our group is marine-derived fungi. The natural history of this taxonomic group remains poorly understood with no reliable estimates of the overall numbers of species. In addition, the overall interest in the chemistry of fungi is growing because some consider them among the world’s greatest untapped resources for new biodiversity as well as chemodiversity.

The challenges encountered, especially in choosing specific taxa to study and with structure elucidation, along with the lessons learned are the major focus of this rather personal account. The research carried out on the UC Santa Cruz campus has involved a wide range of individuals including students from chemistry, oceanography, or biology programs, working alongside professional staff with skills at the interface of chemistry and biology. This brief perspective is intended to showcase the journey and important marine natural product milestone discoveries made at UC Santa Cruz over the last three decades.

Another Important Preamble

Research based on marine natural products with the ability to provide anticancer therapeutic leads is an important national priority. The current cancer statistics show that an expected 1,437,180 new cancer cases will be diagnosed and more than 565,650 Americans are expected to die of cancer (more than 1,500 people a day) by the end of 2008.3 This has been a motivating element in our research targeting solid tumor cancers, which together account for more than 65% of all cancer deaths in the U.S. It is clear that additional therapeutic interventions are needed and the potential for marine natural products, especially from sponges, to make positive contributions now seems firm. There is an ever-expanding list of marine natural products or synthetics inspired by marine-derived compounds currently or about to enter cancer clinical trials, as summarized in Table 1. One compound, ecteinascidin 743 (now called Yondelis or trabectedin) from the tunicate Ecteinascidia turbinata,4 has emerged from this process. It has been approved by countries in the European Union to treat patients with advanced soft tissue sarcoma.5

Table 1
Selected Marine Natural Products in Development as Anticancer Drugs

The additional entries of Table 1 illustrate the various marine natural product derived structural classes under evaluation in anticancer clinical trials. There are 16 compounds undergoing current trials, with ten derived from total synthesis and nine created after SAR study of a parent lead compound (indicated by “insp.”). Strikingly, the list is also well represented by substances from marine sponges (indicated by “a”). Among the most complex structure listed is E7389, the subject of a Phase III trial and a synthetic compound designed from the active core of the sponge natural product halichondrin B.6 Biosynthetic structural features from the NRPS or PKS-NRPS pathways are present in five of the six agents being evaluated in Phase II trials. Of the nine compounds currently in Phase I, five are synthetic agents based on sponge-derived products. One important seed compound of this group is psammaplin A, first discovered by our group,7 and eventually found to be a dual histone deacetylase (HDAC)8 and DNA methyl transferase inhibitor. Its structure contributed to the design, by the medicinal chemistry group at Novartis, of LBH 589.9 One entry of Table 1, NPI 2358,10 a synthetic compound based on a unique diketopiperazine isolated from cultures of a marine derived fungus, is in a Phase I trial and underscores the potential of this group to contribute significant chemical structures.

There are eight additional entries in Table 1 involving compounds based on marine natural products that were discontinued from clinical trials. One of these, discodermolide,11 is a deep water sponge-derived natural product. Four other entries are either based on or related to sponge-derived natural products. These include: cryptophycin 5212 (based on arenarol13), LAF 38914 (based on bengamide A,15 extensively studied in our lab), LAQ 82416 (inspired by psammaplin A,7 as noted above), and girolline17 (also known as girodazole). Overall, the list of Table 1 indicates that the possible mitigating factor of re-supply or synthesis of a complex marine natural product is not a deterrent to advancing for clinical investigation marine biomolecules, which cannot be obtained in large amounts from nature. Finally, there are a number of marine natural product preclinical candidates under study worldwide and some structures elucidated at UC Santa Cruz will be discussed later.

The Early Days – Some Triumphs and Annoying Diversions

The tide pools in central California are teaming with red algae including the chemical rich genus, Plocamium. Our very first project in marine natural products chemistry utilized such alga and resulted in the isolation of cartilagineal,18 (1) an unusual polychlorinated monoterpene aldehyde from specimens of P. cartilagineum (Dixon), abundant in the inter-tidal zones north of Santa Cruz. This genus was selected for study because its crude extracts contained a host of new polyhalogenated monoterpenes and some were toxic to goldfish. Some compounds were also found to be active in antiinsect screens carried out at the now defunct company, Zoecon. The discovery of compound 1 was significant as it was the first halogenated monoterpene to be reported from a red alga. This paper appeared slightly after a report of parallel research by the late Prof. Faulkner, resulting in the isolation of (3R,4S,7S)- transtrans, -3,7-dimethyl-l,8,8-tribromo-3,4,7-trichloro-1,5-octadiene19,20 (2), the first halogenated monoterpene obtained from a southern California sea hare, Aplysia californica, which grazes on P. cartilagineum (aka coccinium).

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At the point in time when these simple polyhalo-monoterpenes were first being isolated and described, the NMR experimental methods now routinely used to accurately perform structure elucidation had not yet been developed.21 This caused annoying problems; for example, the initial structure proposed for violacene22 (3), isolated from P. violaceum, was eventually corrected to 4, based initially on our analysis of 13C NMR shifts and definitively on the X-ray data collected of crystals we obtained.23

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We and others found that the study of any member of the Plocamium genus was guaranteed to give publishable results and some of the relationships between the compounds observed vs. the species studied provided results relevant to chemical ecology. These patterns, mostly taken from our work, included: (a) P. cartilagenium contained acyclic trihalo and pentahalo analogs, (b) the polyhalo monoterpenes of P. violaceum (summarized in Table 2) included alicyclic structures (4 and plocamene B (8)) or acyclics (headed by preplocamane A (13)), (c) compounds from P. oregonum were dominated by Br and Cl containing acyclics (such as 2), (d) P. costatum from Tasmania was also a source of acyclic oxygen containing analogs (costatol (15)), and (e) P. costatum from the Australia Barrier Reef contained acyclics identical to those of P. cartilagenium.2427

Table 2
Our First Example of Organism Chemotypes Through a Geographical Survey of Plocamium violaceum

Once we learned that P. violaceum was cosmopolitan along the Pacific coast of California and Oregon, the next logical step was to obtain and study it from diverse Pacific coastal habitats. Sampling was accomplished from 26 different collection sites, divided into three major geopolitical zones, as shown in Table 2: (a) southern Oregon, (b) northern California, and (c) central California. There were 11 polyhalogenated sesquiterpenes observed, and from a biosynthetic perspective, they could be organized into three different categories (Table 2): regular alicyclic isoprenoid (including 47, 8), rearranged alicyclic isoprenoid B (including 9, 10), and acyclic precursors, preplocamenes (including 1114).2427 The relative ratios of these compounds were invariant at the individual collection sites and the relative composition did not vary seasonally. While morphology of P. violaceum was that same over the geographical range sampled, its biosynthetic machinery producing halomonoterpenes was quite different. Significantly, these data provided two principles: (a) there could be chemotype (CT) variations, and (b) there were advantages to conducting a biographical study. Our categorizations of the chemotypes for P. violaceum were defined by apparent biosynthetic relationships as follows: (a) collections made south of the Monterey Bay canyon largely afforded the preplocamenes, constituting chemotype (CT)-α, (b) another set, CT-β was rich in the plocamene D family and devoid of the preplocamenes; (c) taxa designated as CT-δ had a preponderance of plocamene B members, but often possessed small amounts of the preplocamenes, and (d) CT-γ collections contained substantial mixtures of both plocamene B and plocamene D structures. An outcome of making these divisions was the CT information that could be used to guide the re-isolation of a specific compound type. Thus, the best source for 13 would be CT-α at “Sea Rock Motel” or 4 from CT-β at “Pescadero Beach.” As will be illustrated later, this phenomenon is also at work with sponge populations.

Our explorations in seaweed chemistry continued with the goal of exploring another ecological phenomenon. Collections of Laurencia pacifica (Kylin) and its associated epiphyte Erythrocystis saccata (J. Agardh) were gathered and the idea was to compare the constituents of the host and epiphyte. A combination of spectroscopic and semi-synthesis was used to characterize the total structure of kylinone28 (16), a unique sesquiterpene, from the minor components of Laurencia extracts. The next element in this chemical ecology study involved a survey of sesquiterpenes from the epiphyte.29 Overall, we examined three separate collections and found that the major components of the host and epiphyte were exactly parallel but varied as a function of collection location as follows: Stillwater cove - aplysin and debromoaplysin; Stillwater Cove recollection: isolaurinterol and debromo isolaurinterol; Catalina Island, laurenisol and bromolaurenisol. The unmistakable observation that the sesquiterpenes of E. saccata exactly tracked those of the host was fascinating. The relative yields of sesquiterpenes from the epiphytes were much lower vs. those from the host. The yields and structures were verified by GC-MS and NMR data and the E. saccata was removed from the host by a surgical cut made well up on the epiphyte’s thallus. We do not believe that these organisms actually engage in parallel de novo synthesis of the sesquiterpenes; however no follow up experiments were ever conducted to provide further data to rationalize these observations.

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Our initial attempts to build up collections of marine sponges for chemical study did not focus on Monterey Bay sponges. The extensive studies by Djerassi (Stanford University) indicated that Monterey Bay sponges were rich in sterols and a strategic decision was made to pursue new chemistry from Caribbean coral reefs. One attractive specimen was initially identified as the sponge Haliclona hogarthimi. We successfully explored the terpenoids of that sample culminating in the isolation of unusual diterpene asbestinin epoxide30 (17). This prompted a re-examination of the organism in its habitat revealing that it actually was the gorgonian Briareum asbestinum (Pallus)!

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While our research focus had started the important shift to invertebrates, we were tantalized to pursue the constituents of dense mats of two intertwined brown algae that were abundant in the Caribbean Honduras Bay Islands. The extract obtained from the mixture identified as Dictyota linearis (C. Ag.) and Dictyota divaricata (Lamour), showed extreme toxicity to goldfish at 400 pg/mL (death in 90 min). Chemical investigation resulted in the isolation of the novel tricyclic diterpenes headed by a triol dolostane derivative (18).31 While our results on these metabolites attracted interest, there were two lessons learned – pursuing mixed organism collections was not optimal to enable follow-up re-isolation work, and dividing our resources between the study of seaweeds and sponges was not wise.

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The Shift to Sponges – Building the Foundation

The decision in the middle 1980’s to exclusively focus on marine sponges was concurrent to the start of a new UC collaborative venture, the Marine Chemistry and Pharmacology Program, funded by the California Sea Grant initiative. This innovative program facilitated compound isolation studies through pharmacological evaluation. As another important change we shifted the expedition focus from Caribbean to Indo-Pacific habitats. The first compounds isolated were from sponges collected in the Kingdom of Tonga and terpenoids dominated the initial discoveries. A large soft drab sponge from a Tongan coral reef sponge seemed incorrectly identified as a Prianos because a large number of Indo-Pacific Diarcarnus sponges were subsequently observed to contain the same compounds. Norterpene peroxides were isolated with methyl nuapapuanote (19) being the first example of a norditerpene reported from a marine sponge.32 Adding to this finding was that our study of the anti-inflammatory active extracts from another Tongan sponge Hyrtios erecta provided additional new sesterterpenes. These including the norterpenoid hyrtial33 (20) and new scalaranes such 12-deacetyl-12-epi-scalardial34 (21). The other astounding development was that 12 g of heteronemin (22) was isolated from 708 g of the dried H. erecta.34 We were able to add a family of cytotoxic polyketide peroxides, such as xestin A (23) to our growing library of compounds through the study of an encrusting Xestospongia which was abundant in Fijian reefs.35

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The next sets of bioactive natural products that we encountered were all non-terpenoid and often took considerable time to characterize. For example, psammaplin A (24) was obtained from the cytotoxic extract of Psammaplysilla sp. collected from Tonga. In 1987, we provided the first description of psammaplin A, having dense N-functionalization accompanied by S and Br heteroatoms.36 The dual histone deacetylase and DNA methyltransferase activity we reported in 2003 has greatly stimulated interest in this compound series. 36 In fact, in 2007, twenty years after our first publication on this structure, there were 45 articles published based on the chemical biology study of 24.

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A new collaborative venture with a group at Syntex Research (Dr. T. Matthews) opened the door to extensive study of Fijian sponge metabolites. A parasite assay target, the nematode Nipposgrongylus braziliensis, provided the pathway to identify a host of very inspirational natural products that we and others would continue to study for many decades. These included reports of specific compounds (by year) as follows: jasplakinolide37 (25) 1986; bengamide A15 (26) 1986; latrunculin A38 (27) 1987; melyne A39 (28) 1988; bengazole A40(29) 1988; mycothiazole41 (30) 1988; fijianolide B42 (31) 1988; suvanine43,44 (32) 1988; plakinidine A45 (33) 1990; xestoaminol A46 (34) 1990, and fascaplysin A47 (35) 1991. The structures represented in this list were varied and unprecedented at the time of their disclosure. Overall these discoveries illustrated that the use of an antiparasitic disease model screen could provide significant outcomes. Unfortunately, we were not able to build on these rich findings because of the disinterest of the biopharmaceutical sector to engage in antiparasitic drug development. As will be discussed next, we were able to shift the focus to a cytotoxicity screening paradigm resulting in outcomes that attracted wider outside interest.

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Our work on sponge-derived cytotoxic compounds began to take shape once a collaboration was established (with Prof. Valeriote, Henry Ford Cancer Center, Detroit) that employed a novel pharmacology paradigm to develop anticancer therapeutic leads from marine natural products.48 The key tool was the solid tumor selective assay to assess differential activity among solid tumor cells (murine – C38, human – H-125, H-116, U251N, MCF-7, LnCaP, OVCAR), leukemia cells (murine - L-1210, human – CCRF-CEM), and normal cells (bone marrow committed progenitor cells). This approach is mechanism-blind and the goal is to search for materials that will kill solid tumor cells while exhibiting less toxic effects against leukemia or normal cell lines. Having a powerful yet simple screen it became possible to rapidly prioritize work on a variety of sponge extracts. As an important proof of concept, Valeriote and Moore collaborated using this approach in the successful evaluation of the cryptophycin family (see Table 1), subsequently evaluated in a Phase I anticancer clinical trial.49 One significant early lead we found from this screen included fascaplysin A47 (35), discussed above in the antiparasite discovery effort, which was further assessed by another unique strategy referred to as a clonogenic assay. This experimental design allows determination of a cytotoxic effect at different concentrations for the targeted tumor over an extended period of time and provides the essential data to plan an in vivo trial.

Empowered by this screen we have devoted considerable effort to unearth and pursue materials that are solid-tumor selective. Often, this work began during an expedition to gather organisms to provide the extracts and compounds for investigation. Typically, each year there would be two-three expeditions that would yield hundreds of sponges and a premium was always placed on obtaining tropical sponges from less well-studied orders. Initially, the input of a UC Santa Cruz collaborating taxonomist, Dr. M. C. Diaz, was important, and eventually this task was transferred to Dr. R. W. M. van Soest at the University of Amsterdam. The difficult challenge of completing formalities with foreign governments was successfully dealt with to allow collections to occur in diverse coral reef regions ranging from Papua New Guinea, Fiji, Vanuatu, Solomon Islands, Madagascar, and Venezuela.

These expeditions afforded a continuing stream of significant lead compounds discovered from sponges. Foremost among this group were sponge-derived heterocycles, many of which were nitrogen containing. A sample of these included: cyclocinamide A50 (36), alpkinidine A51 (37), the 5-methoxy neoamphimedine51 (38), 14-bromosecofascapysin B52 (39), spirocalcaridine A53 (40), and dihydrohalichondramide54 (41). Additional compounds of interest and devoid of nitrogen functionality included: haliclotriol A55 (42), hydroxysednenoneolide56 (43), and hyrtenone A57 (44). Accompanying each of these compounds was a number of analogs for which the accompanying bioactivity properties provided more information about the active pharmacophore.

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Sponges – Current Milestone Discoveries

Jasplakinolide37 (25) also called jaspamide,58 has been of continuing importance to our research. Rather unusual is 25, which can be isolated in reasonable yield from two Indo Pacific sponges in separate taxonomic orders, Jaspis splendens (order: Tetractinomorpha) and Auletta cf. constricta (order: Ceractinomorpha). It is a potent actin filament stabilizer and also inducer of actin polymerization.59 More significantly, jasplakinolide has emerged as an important molecular tool even though it was unsuccessful in progressing through the steps required for preclinical development as an anticancer chemotherapeutic. Reflecting its widespread use are the annual literature citations on jasplakinolide in cell biology studies, which are very large (>50 papers/year).

Once the collaborative arrangement with Prof. Valeriote (Ford Cancer Center) was fine tuned, eye-catching results began to emerge. These broad based strategies that are now standard tools in our collaborative program consist of: (i) discovery and structural elucidation of new biomolecules (at UCSC), (ii) implementation of in vitro anticancer screening (at Ford Cancer Center), (iii) advanced pharmacological evaluation (at Ford Cancer Center and at the NCI), and (iv) field biology (at UCSC).

Recently, four compounds have emerged as the most important entities for continued study and are shown in Figure 2. Heading this list is psymberin60 (45), a sponge-derived PKS-NRPS biosynthetic product whose structure and astounding cytotoxicity profile were published after a ten-year campaign to isolate it from Psammocinia bulbosa. Although we described the sponge-derived polyketide fijianolide B42 (31) decades ago, it took a sustained effort to obtain additional SAR and therapeutic understanding. We recently revised the structure of mycothiazole61 (30) and are making good progress in the preclinical evaluation of this nano - molor active compound. Even though the latrunculins have been studied for almost 30 years we have identified a new analog, 18-epi-latrunculol A62 (46) as of current interest, because this cytotoxin did not exhibit microfilament-disrupting activity, common to all other latrunculin analogs.

Figure 2
High priority sponge-derived natural products.

Psymberin (45)

The significant physical and biological properties of (+)-psymberin (45), identical to those independently reported for (+)-irciniastatin A (45),63 make this a “privileged” molecular structure. Leukemia cell lines are relatively insensitive to 45, whereas impressive activities are observed against solid tumors: (+)-psymberin (e.g., LC50 < 2.5 nM vs. MDA-MB-435 breast cancer line), 64 Pettit’s data for (+)-irciniastatin (e.g., GI50 = 5.2 nM vs. MCF-7 breast cancer line), 63 and de Brabander’s data for synthetic (+)-psymberin (e.g., IC50 = 1 nM vs. PC3 prostate cancer line). 65 The bioactivity of two synthetic psymberin diastereomers plus evaluation of the designed compound, psympederin65 underscore that the unaltered (+)-psymberin structure has the best activity. The biological properties of (+)-45 are also distinct vs. the structurally related (+)-pederin,6668 and its multitude of analogs. A multifaceted process was used for the experimental therapeutics evaluation of (+)-45. A clonogenic dose response study (see above) was conducted using HCT-116 cells carried out at 2, 24, and 168 hrs with 90% cell kill as follows: 2 h ≥ 3 ng/mL, 24 h ≥ 2 ng/mL, and 168 h ≥ 20 pg/mL. These data predicted: (a) the in vivo HCT-116 cell therapeutic effect could be observed either as a bolus or on chronic administration, and (b) exposure of tumor cells to 45 must be above 3 ng/mL for 2 h, 2 ng/mL for 24 h, or 20 pg/mL for 7 days. The maximum tolerated dose (MTD) of 45 = 125 μg/kg for SCID mice and = 25 – 50 μg/kg (NCI Developmental Therapeutics Program). Finally, HCT-116 tumor bearing SCID mice treated with (+)-45 using a bolus injection (125, 62, and 31 μg/mouse) showed that the highest dose was toxic, while the second and third doses gave %T/C values of 75% and 86%, respectively, at 23 days. This demonstrates modest but encouraging therapeutic efficacy of (+)-45. The NCI-DTP program hollow fiber assay using multiple solid tumor cell lines69 also gave a positive outcome: overall score = 34 (active score ≥ 20). The follow-up xenograft testing has also begun at the NCI.

Fijianolide B (31)

This marine-derived polyketide was characterized simultaneously in 1988 at UCSC42 as (−)-fijianolide B and at the University of Hawaii as laulimalide.70 Significantly, 31 and analogs promote microtubule stabilization. Research needed to further demonstrate the preclinical potential for this molecular structure has been completed recently. These involved: (a) obtaining a biogeographical understanding of the most reliable sponge chemotypes as a source of (−)-31 and new analogues (b) scaling up the isolation of (−)-31 to launch in vivo trials in tumor- bearing mice, and (c) extending the record of SAR through biological screening of new fijianolides possessing functionality not previously created through synthesis. The cytotoxicities exhibited by (−)-31 illustrate its significance and include: natural (−)-31 (KB IC50 = 29 nM71 and MDA-MB-435 IC50 = 5.7 nM72) vs. synthetic (−)-31 (MDA-MB-435 IC50 = 2 nM). These impressive biological data have motivated eleven total syntheses for (−)-31 from eight different research groups.73 In addition, five different teams have prepared 35 synthetic congeners of (−)-31.7484 None of the synthetic analogs obtained to date have exhibited _______________________________The in vivo assessment demonstrated that 31 significantly inhibited the growth of HCT-116 tumors. SCID mice implanted with tumor cells were treated with 31 starting 3 days after tumor inoculation and followed until day 30. Bolus compound administration (iv, daily for 5 days) at 12.5 and 25 mg/kg/day showed that the best activity was achieved at 25 mg/kg/day. The minimal %T/C values were 80% at day 9 for the lower dose and 11% at day 11 for the higher dose; body weights of mice receiving all doses increased throughout the 30 days and were identical to untreated controls. These results support that further in vivo therapeutic evaluation of 31 is merited and we recommend this compound as a clinical candidate in the treatment of solid cancer tumors.85

Mycothiazole (30)

This rare sponge-derived metabolite has an appealing structure and compelling bioactivity properties uncovered by Dr. Valeriote (Ford Cancer Center). The IC50 values against H116 cells in liquid culture are 1.8 ng/mL and 1.2 ng/mL (median value of 1.5 ng/mL). The MTD has been determined to be approximated 3 mg/kg. Clonogenic dose-response studies have shown that the 2 h and 24 h values are >10 μg/mL and the 7-day study is ongoing. The NCI mean graph data for mycothiazole (NSC 647640) are encouraging and indicate that itis selective against several tumor cell lines such as DMS 114 (small -cell lung cancer) and NCI-H23 (non-small-cell lung cancer). The closest COMPARE analysis match in the NCI database is methotrexate (NSC 740, formerly amethopterin), an antimetabolite used clinically to treat certain cancers, severe psoriasis, and adult rheumatoid arthritis. Recent studies by Prof. Nagle (University of Mississippi, unpublished data) suggest that mycothiazole inhibits HIF-1 (hypoxia-inducible factor 1) activation in breast and prostate tumor cells. It also inhibits hypoxia-induced secreted VEGF (vascular endothelial growth factor) at low nM concentrations.86 Several research groups have accomplished partial and total syntheses of mycothiazole subunits and analogs.87 However, no total synthesis of mycothiazole with the revised stereochemistry from E to Z at C-14, 15 has been completed. It has been proposed by others completing the synthesis and biological evaluation of simplified mycothiazole analogues that the nature of the heterocyclic moiety seems to modulate the cytotoxic activity (against HCT-15 colon cancer cells).88

18-epi-Latrunculol A (46)

Almost 30 years ago Kashman, while engaged in the study of the Red Sea sponge Negombata magnifica (old genus designation Latrunculia), isolated and studied the two seminal compounds - latrunculin A (27) and latrunculin B.89,90 These compounds have a macrolide 1,3- fused to a tetrahydropyran containing a 2-thiazolidinone side chain. Interestingly, latrunculin A shares a carbon skeleton with the terrestrial myxobacterium - derived anticancer agents, epothilone B.91 We justified additional study of latrunculin analogs because of: (a) their mixed PKS/NRPS biogenetic origin, (b) their potent actin inhibition properties (latrunculin A is a widely used small molecule molecular probe), and (c) their potent cytotoxicity against cancer cell lines. Surprisingly, in spite of the situation that latrunculins have been described from several sponges and can be accessed through total synthesis, few comprehensive experimental therapeutic studies have been conducted on this family. We recently completed such as study that involved isolation and evaluation of 13 analogs.62 The striking activity profile for 46 is intriguing and apparently an 18S configuration of its thiazolidinone ring diminishes the anti-actin effect without eliminating the cytotoxicity properties. This pattern does not appear to hold for the latrunculin B series as the microfilament-disrupting activities were similar in analogs where the configuration changes from R to S. As a final observation the activity profile of 46 seems to be similar to that of oxolatrunculin B,92 as each may inhibit cancer cell line growth by an actin independent pathway.

Chemotypes of Cacospongia mycofijiensis

Obtaining and examining biogeographical-based collections of sponges whose extracts have exhibited solid tumor selectivity in the in vitro cytotoxicity disk diffusion assay can be quite rewarding.93,94 Applying this approach facilitated gaining a comprehensive understanding of the variations in the constituents of Cacospongia mycofijiensis. Prior to reinvestigations undertaken in 2002, we believed that the components of this sponge varied among four different biosynthetic categories, and lead structures are shown in Table 3. The list here includes: dendrolasin95 (48, sesquiterpene), fijianolide B42 (31, polyketide), latrunculin A89 (27, mixed PKS-NRPS), and mycothiazole41 (30, mixed PKS-NRPS). Two parallel projects provided fresh insights and this occurred through a study of samples in our repository alongside obtaining additional sponge material from new sites. The serendipitous isolation of CTP-43196 (47) transpired during the reinvestigation of a Fijian collection of C. mycofijiensis. We believe that CTP-431, possessing a very distinctive structure, is biosynthetically related to latrunculin A. The other development came about during our survey of fifteen individual specimens from the pooled northern Papua New Guinea collection of this sponge. The discovery of pre-aignopsanoic acid (49),97 from two of these was exciting as this structure defines an entirely new sesquiterpene class, distantly related to the 4,9-friedo-drimane family. We now recognize, as shown in Table 3, that six different structural families can be isolated from C. mycofijiensis and a maximum of five occur in taxa from a single geographical zone. Understanding about the variation in major (and minor) components among various sponge chemotypes can be used in a variety of circumstances including: (a) planning the successful re-isolation of specific bioactive constituents, (b) executing traditional biosynthetic studies involving the injection of labeled biosynthetic precursors, or (c) developing molecular genetics studies to define a biosynthetic gene cluster.

Table 3
Biogeographical Variations in the Constituents of Cacospongia mycofijiensis a,b

Probing the Molecular Targets of the Bengamides

Few structural changes are tolerated in bengamide A framework in order to maintain maximum cytotoxicity.98 Interestingly, the profile in the NCI 60 cell line panel for bengamides A and B (see 26) were unique vs. all the standard antitumor compounds in the NCI database.99 The significant in vivo antitumor activity observed for bengamides A, B and LAF389 lead to the launch of a clinical evaluation of the latter, but was eventually terminated, in part due to unpredictable cardiovascular toxicity.100

A complete understanding of the antitumor mechanism of action for bengamide A is continuing to emerge. From a historical perspective, the bengamides,101 represent the second class of MetAP inhibitors to be described.102 The family of enzymes known as methionine aminopeptidases (MetAPs) catalyzes the removal of N-terminal methionine from newly synthesized proteins.103 Prokaryotic organisms typically have only one type of MetAP, whereas eukaryotes and humans have two isoforms - MetAP1 and MetAP2. Currently, both isoforms are considered relevant as a target for cancer chemotherapy. MetAP2 has been identified as the possible target for the fungal derived anti-angiogenic compounds shown in Table 4, ovalicin and fumagillin, which also effect T cell activation.104,105 The finding that these compounds inhibit MetAP2 and not MetAP 1 was also considered to be significant. Other inhibitors of MetAP2 have been reported as potential therapeutic agents for cancer.106,107 The subsequent design of the MetAP1-specific fumagillin analog, TNP-470108 is a key new development. Unlike fumagillin, bengamide A (26) inhibits both MetAP1 and MetAP2 and the same is true for the Novartis bengamide synthetic analog, LAF389.109 Another compound, bengamide O, appears to have a different profile against the MetAP isoforms.110

Table 4
Proof of Concept Examples of Natural and Synthetic MetAP Specific Inhibitors

The bengamides decreased the tyrosine kinase activities of c-Src both in vitro and in vivo and eventually delayed cell cycle progression through G2/M.110 It is hypothesized that the clinical toxicity observed for bengamides (and by implication for other non-specific MetAP inhibitors) could arise from global inhibition of N-terminal methionine processing. It has also been shown that blocking MetAP2 similarly inhibits the non-canonical Wnt signaling pathway.111 The results obtained from experiments with fumagillin (a selective MetAP2 inhibitor) imply a potential connection between inhibiting Src family kinases and blocking non-canonical Wnt signaling.111

Overall, there have been very few specific MetAP1 inhibitors discovered to date. The sponge compound bengamide O may provide one such example. Alternatively, there are synthetic pyridine-2-carboxylic derivatives, such as IV-43 (Table 4), recently discovered through high - throughput screening of a library of 12,800 synthetics.112 There is encouragement that MetAP1 may serve as useful anticancer drug target. A next step in the quest to fully understand the molecular target and action of the bengamides will be to pursue additional therapeutic work with bengamide O and similar functionalized analogs.

Marine-Derived Fungi

Our hypothesis, formulated in 1993, that sponges could harbor fungal spores which, after culturing, would be a prolific source of natural products, was the motivation to begin research in this area. Another stimulus to begin study of microorganism natural products were the estimates that approximately one half of the world’s biomass is microbial.113 Further encouraging was the belief that the biodiversity of marine-derived fungi, especially from the water-sediment interface and the anoxic environment below this zone, offered new opportunities for finding diverse species of fungi. Microbial ecologists are now asking questions about the populations of microorganisms endemic to these two potentially different communities. There are now a host of publications from various labs including our own clearly illustrating that communities of marine-derived fungi, capable of producing diverse natural products when cultured, can be found from the many different marine environments.

An understanding of the biological and chemical fundamentals of marine-derived fungi is still evolving. Currently, the best known habitat for marine fungal diversity consists of mangrove areas, which have contributed 50% of over 450 species discovered up to 2000.114 The 2006 estimates of marine fungi world-wide indicated 2000 possible species, 800 of which are known to be saltwater obligate.115 In spite of this biological understanding, it is not yet possible to predict the chemical signatures expected from taxonomically identified cultures of marine-derived filamentous fungi, which makes this group an exciting one for continuing chemical study.

Our emphasis has always been on exploring marine-derived filamentous fungi grown in saltwater culture. An important part of the historical record is represented by two almost simultaneous early proof-of-principle results showing that chemical study of sponge-derived fungi would be rewarding. These initial discoveries included our report of the chloriolins A (50)116 and B (51), which are halogenated sesquiterpenes produced during the saltwater culture of an unidentified fungus from Jaspissplendens collected in Papua New Guinea in 1993. A similar benchmark finding was published in 1993 from the Kitagawa lab and involved the isolation of trichoharzin (52),117 a polyketide from the culture of salt obligate strain Trichoderma harzianum obtained from a Mycale sponge.

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These two discoveries of sponge-derived fungi capable of producing unique compounds in saltwater culture were forerunners of further successful research conducted in our lab. For example, our saltwater culture of sponge-derived Aspergillus strains were found to be a source of another set of halogenated metabolites, such as chlorocarolide A (53).118 We have found it rewarding to investigate other strains of marine- derived Aspergillus and three significant new compounds discovered included the alkaloid asperazine (54),119 the polyketide asperic acid (55), and the polyketide 11-O-methylpseurotin A120 (56). Two other Aspergillus strains also provided us with additional known compounds including secalonic acid121 (57), and circumdatin B122,123 (58). Our study of cultures from Trichodermalongibrachiatum provided a very different end point as compared to the example above, because epoxysorbicillinol124,125 (59), and related polyketides were isolated. The opportunity to accumulate different classes of structurally unusual bioactive peptides that were challenging to characterize occurred through a multi-year investigation of additional sponge-derived fungi. The most complex products were linear pentadecapeptides, headed by efrapeptin G126 (C63H143N18O18) (60) from a sponge-derived Acremonium strain that also afforded linear peptides of the RHM family127 (C53H97N9O11), (61) that are highly N-methylated octapeptides. Three of the efrapeptins (E, F and G) were nM - active in cytotoxicity assays against H125 cells and warrant further therapeutic study. A different sponge derived fungus, Metarrhizium sp., was observed to produce six cyclic mildly cytotoxic depsipeptides of the destruxin class, accompanied by the well studied polyketide cytotoxin, brefeldin A (62).128

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Recently, we began to explore marine sediments as a source of additional fungal strains. Such a shift in focus is now amply justified when scanning the current the literature especially the histogram of marine-derived fungi as a source of new compounds shown in Figure 3. This compilation, revealing some eye-catching patterns, traces the record up to early 2007. Through 2002, just 4% of the 273 marine-derived fungal compounds discovered were obtained from shallow-water sediments.129 Today, there are more than 500 hundred unique compounds reported from the culture of marine-derived fungi from all sources.130 Strikingly, the instances of compounds obtained from shallow-water sediments have recently surpassed those from the previously popular sources - sponges and algae. Indicating the next frontier could be deep-water sediments, which have not been extensively explored.

Figure 3
Histogram of marine-derived fungal compounds vs. source
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It took several years for our lab to develop inexpensive apparatus for the collection of deep-water sediments and this work began before the patterns of Figure 3 were fully recognized. Our collection apparatus was fashioned along the lines of “mud grabbers” developed by the Fenical (UC San Diego) laboratory.131 Deployment of it has occurred during all of our recent expeditions in both the Caribbean and remote IndoPacific sites. Our 2004 disclosure of three new pentaketide anserinone analogs, headed by (−)-epoxyanserinone B132 (63) from a deep water sediment-derived Penicillium sp. constituted an encouraging first finding. A similar recent report of ten nitrogen-containing metabolites from a deep-water sediment-derived Chromocleista strain provided a further demonstration on the benefits from this approach.133 Finally, our recent disclosure134 of tyrosol carbamate isolated from the culture of a deep water sediment-derived Arthrinium sp. completes the current record of the small amount of published work in this area. Taken together, these reports substantiate that secondary metabolite producing fungal strains can be obtained from deep-water habitats.

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A continuing challenge in the quest of isolating diverse molecular structures from marine-derived fungi is to avoid pursuing cultures ladened with common metabolites. In 2004, towards the end of our study of the sponge-derived fungus, Myrotheciumverrucaria, rich in its production of trichoverrin B135 (64) and related macrolides, we attempted to challenge these cultures with conditions that might alter the profile of secondary metabolites. At that juncture, the “OSMAC (one strain many compounds)” paradigm, re-popularized by Bode and Zeeck in 2002136 seemed to be a straightforward strategy, as it involved the systematic alteration of culture conditions to generate new metabolites. As an ultimate test of OSMAC we used harsh conditions, specifically the addition of Cu2+ salts to the saltwater cultures, but this did not significantly alter the production of trichoverroids. During other studies we had applied another obvious OSMAC strategy - varying the seawater concentration during culturing of marine-derived fungal strains, but rarely observed major shifts in the metabolite profiles. Our most comprehensive study of this type involved the saltwater culturing of terrestrial ATTC-derived strains of Coriolus consors known for producing sesquiterpenes such as coriolin A.136

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A refinement introduced during a next phase of our studies was to consider OSMAC alternatives beyond those involving simple changes in culture conditions to generate new profiles of secondary metabolites. The idea here was to add natural product modulators of fundamental cell biology processes to the saltwater cultures. Our first choice was to add actin or tubulin inhibitors. Shown in Figure 4 was a proof of concept result accomplished by spiking cultures of the marine-derived fungus, Phomopsis asparagi, with actin inhibitors such as jasplakinolide (results shown here) or swinholide A (data not shown). The outcome was the same in both instances and involved the diminished production of five simple oxygen containing compounds labeled in Figure 4 as AE accompanied by the appearance of new, unobserved alkaloid actin inhibitors including chaetoglobosin-510 (I), -540 (H) and -542 (G). The prospects for using other strategies to turn on what we term non-functional biosynthetic pathways are also summarized in Figure 4. Several of these approaches have been recently reviewed by Gross who discussed current understanding in manipulating what is termed “orphan biosynthesis pathways.”137

Figure 4
Strategies to turn on non-functional biosynthesis pathways in microorganism cultures.

Structure Elucidation – Some Comments and Challenges

The tools for structure elucidation of complex marine natural products have continued to evolve. Most would agree that organic structure analysis should now be uncomplicated. In the last decade there have been some spectacular advances including: innovations in multidimensional NMR pulse sequences,138 user friendly apparatus for ultra high resolution mass spectrometry,139 widespread availability of tandem mass spectrometry, 126,139 refinements in the use of chiroptical data,140 and the possibility of completing X-ray study on poor quality crystals.141 Representative of such recent triumphs from our lab using one or more of these tools include reports on the structures of marine natural products such as: (a) the cytotoxic alkaloid, asperazine119 (C40H36N6O4) (b) the polypeptide, cyclolithistid A142 (C54H86ClN11O15), (c) a mixed PKS-NRPS metabolite, isomotuporin143 (C40H57N5O10) or (d) an atropoisomeric dimer, dicurcuphenol A144 (C30H42O2).

The decision matrix guiding our trajectory to the desired endpoint, an unequivocal proposal of a total structure is shown in Figure 5. Engaging in aggressive dereplication represents a recent addition to our toolbox. This process uses partial data sets plus carbon framework substructure conclusions as they are accumulated for input to search a variety of commercial and proprietary databases. We have found it useful to aggressively engage in dereplication at all stages of the structure elucidation even when the compound under investigation has no literature precedents. As further discussed next such dereplication efforts were successfully employed in our concise structure elucidations.

Figure 5
Overall flow of information in structure elucidation

We unexpectedly encountered a complex NRPS peptide, subsequently named (−)-psymbamide A.145 This occurred during isolation work on six specimens of Psammocinia aff. bulbosa being processed to re-isolate (+)-psymberin (45). Based on taxonomic considerations and molecular formula data, the characterization of pysmbamide A (C46H65BrN8O8), began by considering a possible structural relationship of it to (+)-cyclocinamide A50 (36), of molecular formula, C29H33BrClN9O8, which had been previously isolated from this sponge. The key insights from the analytical data shown in Figure 6 were clearly incompatible with this initial idea, but resonances for a 5-bromotryptophan could be assigned. Collectively, the partial atom count obtained by HRMS and NMR guided the subsequent dereplication step. A partial formula range consisting of C46N7-11O7-11 was the seed for dereplication searches in MarinLit, with the N and O count based on the assignment of 6 α-amino acid protons, an indole NH, and an unusual C=O (at δC = 174). A high unsaturation number, 18, was required by the molecular formula and a cyclic peptide structure seemed attractive to account for the remaining unsaturation that could not be assigned to the substructure collection. This partial formula search of Marinlit146 yielded eight hits, each of which was a cyclic peptide, and three seemed especially appealing. These are shown in Figure 6 and include were mozamide B147 (C46H66N8O9), anabaenopeptin H (C46H70N10O10),148 and orbiculamide A149 (C46H62BrN9O10). The two structures possessing phenylalanine and tryptophan groups were sponge-derived and the non-halogen-containing member of this pair was considered further. Attention focused on mozambide B after a quick calculation showed replacing the OH in its molecular formula by a Br gave the formula for pysmbamide A. Finally, the structural differences between these two compounds were pinpointed by comparing their respective 1H and 13C NMR chemical shifts.

Figure 6
An agressive application of dereplication

There is an important cautionary note that now needs discussion. Some critically thinking individuals, especially those involved in the total synthesis of complex natural products, recognize that even when all of the modern tools of structure elucidation discussed above are applied errors can be made150 and other considerations may be needed. Rather astounding is that errors in reported structures of natural products continue to abound and this was highlighted in a recent review151 noting that from 1990 to early 2004 more than 300 revisions were made. This suggests that accurate organic structure analysis is not yet routine.

Another important, but rarely discussed, challenge occurs when few hydrogen atoms are present in the molecular scaffold. The obvious steps of using protons sprinkled throughout a carbon framework as reporter groups to highlight direct and/or through space magnetic couplings followed by drafting lists of molecular frameworks will be compromised when the H count is sparse. In this regard, we now understand when the ratio of H/C is less than one, then NMR data sets will be less useful and other methods must be used. This realization prompted our laboratory to consider the parallel evaluation of experimental 13C NMR shifts with those from density functional theory (DFT) calculations as a means to distinguish among sets of substructures.62,152 The other essential approach in such a situation is to obtain X-ray crystallographic data. A recent example from our lab that illustrates this situation and it involves correction of the structure of spiroleucettadine from 65153 to 66.152 Repeated efforts to synthesize spiroleucettadine failed154156 and questions emerged about the correctness of the original structure. Eventually we concluded that the low ratio of H/C = 0.8 in the core of spiroleucettadine compromised the original structure elucidation process. Re-isolation of spiroleucettadine accompanied by density functional theory (DFT) calculations to evaluate the experimental 13C NMR shifts favored high scoring structure 66.152 Thisproposal was ultimately c onfirmed via X-ray analysis of crystalline spiroleucettadine and underscores the validity of DFT calculations in structure elucidation.

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Future Prospects

This account has highlighted the spectacular ability of marine organisms, both macro and micro, in producing exotic compounds. It is the extreme structural novelty coupled with new modes of biological activity that continue to make the study of marine natural products a rewarding venture. Only a small percentage of all marine organisms have been investigated for their potential to produce novel structural scaffolds. Further, the rich and biodiverse reefs of the Indo-Pacific Wallacea region have not received much attention. It is clear that the subject of marine bioorganic chemistry continues to be driven by inspirational structures and as such it is thriving.

In looking toward the future there are several circumstances that are evident. There are new strategies being developed by many laboratories throughout the world that will continue to provide motivating new developments involving oceanographic sampling and other unique laboratory approaches to discover new molecular structures. There are host of continuing challenges to overcome that will require much additional research. The most interesting to our lab involves long-standing and burning question regarding the origin of sponge and other invertebrate natural products. What is the true producer? Some believe it is the assemblage of invertebrate associates, while others are convinced that specific strains of hetrotrophic bacteria or cyanobacteria play a key role. In the past, we have engaged in such studies and debates but have not arrived at firm answers. Devoting more attention to developing marine-based approaches to the culture of marine microorganisms is of obvious importance.

Our work on the products marine-derived fungi grown in culture was also briefly treated in this account. We believe that investigations on marine microorganisms will continue to grow and should provide rewarding outcomes. Especially worthwhile could be the further study of the sponge-derived bacterial communities. We have just begun to explore one aspect of this subject and the current focus is on marine-derived myxobactera. Absolutely intriguing are highly cited observations of parallel structures from terrestrial myxobactera and marine sponges. At the top of our list are (a) jasplakinolide37 (Sponge, Jaspis splendens and Auletta constricta) vs. chondramide A157 (mycobacteria, Chondromyces crocatus), (b) latrunculin A89 (Sponge, Cacospongia mycofijiensis and Negombata magnifica) vs. epothilone B91 (mycobacteria, Sorangium celullosum), and (c) Bengamide E158 (Sponge, Jaspis coriacea) vs. bengamide E analogues159 (mycobacteria, Mycoccus virescens). We look forward to obtaining insights from future research to explain these circumstances.

Acknowledgments

This research was generously supported over the years by grants from the NIH National Cancer Institute, the National Science Foundation, and the California Sea Grant Program. The efforts of a host of research students and staff at UC Santa Cruz were essential in completing the research discussed. The list is way too long to reproduce here and these names can be found in the papers from our lab that are cited herein. Special thanks to K. Tenney (UCSC) for design of the graphical abstract, to Dr. T. Johnson and Dr. W. Inman for their input, and to the BOYSCAST program, DST, Government of India, for a fellowship (K.V.S.).

Footnotes

[perpendicular]Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute and State University for his pioneering work on bioactive natural products.

Adapted from a Norman R. Farnsworth Research Achievement Award address, 49th Annual Meeting of the American Society for Pharmacognosy, Athens, Greece, August 17th – 22nd, 2008.

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