Marine pharmacology

Nature has served human kind m many ways, one of which is a source of
medicines. Natural products from marine sources represent a relatively new area
of research and have shown tremendous potential as a source of new chemical
entities in drug discovery. Caribbean gorgomans corals of the genus
Pseudopterogorgia have been shown to produce a variety of chemically
interesting and biologically significant secondary metabolites. ln this dissertation,
the Caribbean coral Pseudopterogorgia acerosa has been investigated for the
presence of novel diterpenes and these compounds were found to belong to three
different classes: pseudopteranoids, bis-pseudopteranoids and lipidyl
pseudopteranes. Nine of these were new compounds. The structural elucidation of these compounds was performed using spectroscopic means such as l D and 20
NMR, and mass spectroscopy.
There is growing evidence that secondary metabolites isolated from manne
invertebrates may actually be produced by a bacterial symbiont. The research
studies in our laboratory regarding the source of diterpenes in the selected
gorgonian corals suggested a bacterial origin. The hypothesis that coral associated
bacteria are the source of diterpenes in the coral P. acerosa, was evaluated using
the series of experiments and evidence supported this biosynthetic origin.
A study comparing the "gall" tissue and healthy coral tissue in terms of diterpene
content and culturable bacterial communities showed that different groups of
diterpenes were concentrated in different coral tissue types. It was also observed
that the bacterial populations associated with the "gall" and healthy tissues were
considerably different. Furthermore, observed specificity in antimicrobial activity
of certain groups of compounds against bacteria isolated from the same coral
suggested the ecological role of these compounds. This work with "gall" tissue
supports the hypothesis that diseased coral tissue represents an excellent source of
bioactive natural products for drug discovery.
Additionaly, a simple LC-MS method was developed for the analysis of
anticancer drug carmustine in plasma.

The marine environment is a prolific source of novel compounds for therapeutic
use due to the complex biological and chemical diversity. Throughout the past 30-40
years, over 15,000 natural products have been discovered from the oceans, many of
which display a broad range of potential clinical and commercial applications. Many
marine invertebrates are sessile organisms that lack physical protection, and which
chemical defense may be a possible explanation for these secondary metabolites. Despite
the promise marine natural products have as potent pharmaceutical agents, one of the
major factors delaying clinical use is the supply issue. These bioactive compounds are
often found in trace amounts in the host organism, which makes harvesting from the reefs
unfeasible. A general goal in our lab was to investigate the biosynthesis of secondary
metabolite terpenes to ultimately provide a production method of these potent marine
derived compounds. Eleutherobin and desmethyleleutherobin are diterpenes isolated from the
Caribbean soft coral Erythropodium caribaeorum. These extremely valuable anticancer
agents disrupt cell division by polymerizing and stabilizing microtubules, and have
demonstrated tumor tissue selectivity toward selected breast, renal, ovarian and lung
cancer cell lines. Determining the first intermediate in terpene biosynthesis is the initial
step in developing a biotechnological production method of these cytotoxic agents. We
investigated the complex chemistry of this coral using a radioactivity-guided isolation
procedure, and isolated and partially characterized a diterpene hydrocarbon from E.
caribaeorum.
The close association between marine invertebrates, zooxanthellae and numerous
bacteria gives rise to the question of the identity of the producer of secondary metabolites
in marine organisms. If the symbiont produces these therapeutic agents, cell culture
methods could be employed to supply the compounds rather than obtaining them from
coral reefs. Sesquiterpenes have been isolated from the gorgonian Plexaurella spp.,
however, no investigations concerning host/symbiont contribution of the sesquiterpenes
have been reported. We investigated the biosynthetic source of terpenes in this coral, and
experimental evidence indicates that bacteria are responsible for sesquiterpene
production. We also examined sesquiterpene variation of Plexaurella spp. from various
locations, and found sesquiterpene content to vary within and between species,
identifying Plexaurella as a chemically indistinguishable genus.

The bryostatins are a family of macrolide lactones isolated from the
marine bryozoan Bugu/a neritina. Since its detection in 1968, bryostatin 1
has demonstrated remarkable anticancer, immunopotentiating,
biomodulatory and radioprotective effects which result mainly from its
ability to activate protein kinase C, a family of isozymes involved in cellular
signal transduction. It is currently being tested in several phase I and phase
II clinical trials as a potential anticancer drug for leukemia, melanoma and
nephrotoma. A series of experiments was undertaken to elucidate the
biosynthetic origins of bryostatin, using a fortified crude cell-free enzyme
preparation and radiolabelled precursors. A regional characterization of
Bugula neritina from Sicily, Italy and Daytona Beach, Florida is also
described.

The octocoral Eunicea fusca is the source of important anti-inflammatory compounds
such as the diterpenes fuscol and the fuscosides. Evidence suggests that these
compounds are being produce by bacterial sources that live in symbiosis with the coral.
As part of an investigation to better understand the role of bacteria associated with E.
fusca , the characterization of the bacterial community using two different techniques
(culture dependent technique and culture independent technique) and sea water samples
was done. Sea water samples were used as a control to determine how closely associated
bacteria are to this octocoral. The polymerase chain reaction (PCR) with universal
bacterial primers FC27 and RC 1492 specific to prokaryotic 16S rDNA gene sequences
was used to characterize the total bacterial population when using both the culturable and
the non-culturable approaches. The results indicate a diverse group of bacteria associated to E. fusca composed of 10 different groups. The pro teo bacteria group was the most
predominant group when both techniques were used. The a-proteobacteria represented
the highest percentage of bacteria associated to E. fusca.

Stevensine is a biologically active secondary metabolite produced by several marine sponges, including Teichaxinella morchella. Two in vitro methods were used to investigate the biosynthetic origin of stevensine in the sponge T. morchella. A cell-free enzyme extract was developed to test for the incorporation of the potential radiolabeled metabolic precursors histidine, arginine, ornithine, and proline. Secondly, cell cultures of T. morchella were used to determine the biosynthetic origin of stevensine from the radiolabeled amino acids histidine, arginine, ornithine, and proline. Histidine and ornithine/proline were converted to stevensine in the cell culture system. This represents the first study involving the use of cell cultures of a marine invertebrate to investigate the biosynthesis of a biologically active natural product. An understanding of the biosynthetic process leading to the production of this compound in the source organism could lead to the development of more efficient and environmentally safer production methods.

The fatty acid synthetase (FAS) from the marine invertebrate Bugula neritina was isolated using cold ethanol precipitation followed by the sequence of G-100 and G-200 size exclusion columns. Native gel analysis indicates the isolation of the FAS and the elution volume from the G-100 column suggests the FAS to be ~282kd. One band with a molecular weight of 66 kDa appeared on the SDS gel of the G-200 sample (F17-30) that eluted at 52.5 mL. The G-200 sample that eluted at 19.2 mL (F1-6) displayed two predominant bands on the SDS gel corresponding to molecular weights 66 kDa and 97 kDa. Both F1-6 and F17-30 showed FAS activity displaying de novo production of myristic and palmitic acids. From the sequence of purification starting from the cell-free extract (CFE) to the F17-30 sample, a 240 fold increase in specific activity was observed. The Type II FAS experiments showed no substantial evidence of activity, namely of the beta-Hydroxybutyryl acyl-dehydrase and the enoyl reductase enzymes.

Ecteinascidin is a tetrahydroisoquinoline alkaloid isolated from the colonial ascidian Ecteinascidia turbinata. Ecteinascidin exhibits tremendous activity against P388 murine leukemia, however its yields from the tunicate are as low as 10^-4%. A biosynthetic investigation, using in vivo and in vitro methods, has resulted in the identification of the metabolic precursors of ecteinascidin as tyrosine, DOPA and cysteine. Reactive intermediates such as tyrosine and DOPA diketopiperazines have also been identified. These preliminary experiments set the stage for subsequent protein isolation.

Grazing on bioluminescent and nonluminescent prey by the copepod Acartia tonsa was examined to determine the effect dinoflagellate bioluminescence has on copepod feeding preferences and rates. The percentage of cells ml-1 remaining after 30 minutes of copepod grazing on the dinoflagellates Lingulodinium polyedrum and Pyrodinium bahamense in their bioluminescent and nonbioluminescent phases was used to compare results for concentrations of 10, 1,000, and 3,000 cells ml-1. The nonluminescent diatom Thalassiosira eccentrica was later offered along with each of the dinoflagellates. When diatoms were offered with P. bahamense, the copepods consumed them in equal amounts regardless of bioluminescence at the lower concentration, while the higher concentration showed decreased grazing with bioluminescence. Nonbioluminescent L. polyedrum was consumed at a high rate for all concentrations, but grazing decreased once they were bioluminescent. Copepods switched to T. eccentrica once the dinoflagellates became bioluminescent, except at 3,000 cells ml-1, at which all grazing was reduced. These results indicate there may be a threshold concentration separating two functions of dinoflagellate bioluminescence.