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MARINE BIOMEDICAL RESEARCH A Search for New Pharmaceutical Drugs from Marine Organisms John K. Reed, Amy E. Wright, Ph.D., Peter J. McCarthy, Ph.D., and Shirley A. Pomponi, Ph.D. Biomedical Marine Research, Harbor Branch Oceanographic Institution Historically, terrestrial plants and microorganisms have been important sources of natural products which have been used in the development of therapeutic agents. It is estimated that over 50% of the drugs used to alleviate human diseases and suffering are derived in some manner from natural products. These include aspirin (originally derived from the bark of a willow tree), numerous antibacterial drugs 
such as penicillin and streptomycin (derived from microorganisms), and Taxol ®
(derived from the bark of the Pacific yew tree) - a drug currently being used to treat breast cancer.
While terrestrial sources have yielded numerous drugs, marine natural products represent a relatively untapped resource for new drug development. The marine environment may contain over 80% of the world's plant and animal species, and during the past decade over 5000 novel compounds have been isolated from marine organisms. COMPETITION YIELDS DIVERSITY The diversity of chemical compounds in the marine environment may be due in part to the extreme competition among organisms for space and resources. It is hypothesized that sessile marine organisms (for example, sponges, octocorals, tunicates and algae), have developed a diverse array of chemical compounds known as "secondary metabolites" or natural products 
for defense and competition. These compounds provide evolutionary advantages by preventing
predation and fouling, or by helping an organism effectively compete for space.
Both toxic and non-toxic compounds may be produced by marine organisms. Toxic compounds may poison predators or adjacent organisms, allowing continued growth. Non-toxic compounds may reduce predation through a reduction of palatability. Secondary metabolites can also act as chemical messengers (pheromones) between individual organisms. BIOMEDICAL MARINE RESEARCH The oceans are a vast resource for the discovery of marine-derived medicines. Increasing sophistication in the tools available to explore the deep sea has expanded the habitats which can be sampled and has greatly improved the opportunities for discovery of novel metabolites. 
In addition, as our understanding of the biochemistry behind a variety of disease states
improves, we can develop better methodologies for rapidly assaying the biomedical potential
of marine organisms. The Division of Biomedical Marine Research (DBMR) at Harbor Branch
Oceanographic Institution (HBOI) is ideally situated to take advantage of advances in underwater
technology and in biotechnology in our drug discovery program. DBMR is divided into three
research teams: Sample Acquisition and Taxonomy, Biological Evaluation, and Natural Products
Chemistry.
The Sample Acquisition team consists of marine biologists, taxonomists and microbiologists who are responsible for the collection of macro- and microorganisms for evaluation of their potential biological activities. Samples are collected in shallow water, less than 45 m (0-150 ft) by wading, snorkeling, and scuba diving. Our emphasis, however, is on deep water organisms which are collected to depths of 3000 ft with the JOHNSON-SEA-LINK, and CLELIA manned submersibles. These submersibles are launched from HBOI's Research Vessels, the SEWARD JOHNSON and the SEWARD JOHNSON II. Use of the manned submersibles allows us to collect and document samples from previously inaccessible marine habitats such as rugged, vertical walls which are impossible to sample with surface gear such as trawls or nets. The submersibles are equipped with multifunctional manipulator arms which enable the collection of specimens by either a jaw, suction tube, or clam-shell grab. Even extremely fragile or brittle specimens can be obtained intact. The submersibles are also equipped with color video and 35-mm cameras for photo-documentation, and with a data recorder that logs temperature, salinity, and oxygen every second of the dive. SAMPLE DOCUMENTATION Thorough documentation is essential for ecological evaluations of the marine specimens as well as for taxonomic classification. Each specimen is documented with correlated site and sample descriptions recorded in a field book and transcribed into a computer database. 
Site descriptors include latitude, longitude, habitat, depth, temperature, salinity, and current. Specimen descriptors include morphology, color, abundance, symbiotic relationships, and taxonomy. Each specimen is photographed and videotaped in its natural habitat. The specimens, photographs, and videotapes are archived in our museum. Collections by submersible and scuba are very selective compared to trawling or dredging methods and are of minimal impact to the environment. Collection permits and research clearances are obtained from each foreign country visited and we work closely with foreign government agencies-such as the Departments of Fisheries and Natural Resources. Duplicate museum specimens, underwater photographs, and videotapes are deposited with the host country for each expedition along with detailed reports on the deep water biodiversity, and results of chemical and biological assays. Our Division has collected biomedical samples of marine organisms from the Atlantic, the Caribbean Basin, the Pacific and Indo-Pacific. HBOI's ships have gone on Eastern Atlantic expeditions to the Azores, to Madeira, Canaries and to the west coast of Africa. Western Atlantic and Caribbean 
sites include the Bahamas, Turks and Caicos, Jamaica, Antilles,
Virgin Islands, Belize, Honduras, Venezuela, and Columbia. Indo-Pacific collections include
the Galapagos Islands, Cocos Island, Panama, American Samoa, Seychelles, and Papua New Guinea.
Currently, our collection consists of approximately 24,000 macroorganisms.
A second aspect of our sample acquisition program is our fermentation program. There is evidence that some natural products isolated from marine invertebrates may actually be produced by associated microorganisms. Our program emphasizes the isolation of microorganisms from deep water sediments and invertebrates. The microorganisms are isolated on agar media. After axenic (clean) cultures are achieved, these organisms are fermented in liquid culture with varying growth media, at varying temperatures, and for varying durations to optimize the production of secondary metabolites. CHEMICAL EXTRACTION Extracts of marine macroorganisms are prepared by grinding 2 grams of the specimen in 20 ml of 100% ethanol. This crude extract may contain numerous compounds from the organism which are analyzed in bioassays and chemical evaluations. The hope is to discover novel compounds that show biological activity such as the ability to kill cancer cells. Using the disc diffusion assay to test for antimicrobial properties, each disc is small piece of blotter paper soaked with the sponge extract. These are placed on agar seeded with bacteria or fungi. The clear area surrounding some of the discs indicates a zone of inhibition where the sponge extract killed the bacteria. BIOLOGICAL EVALUATION 
Our biological evaluation program employs a random screening approach to discover natural products with therapeutic potential. In this process, a large number of extracts are tested in various biochemical and biological tests termed "screens." These screens are selected to be highly specific for natural products which have the highest probability of yielding marketable drugs. There are three commonly used approaches to screening: whole animal testing, testing of cell cultures, and target-directed screening. For a number of years, the National Cancer Institute ran a screen for anti-cancer agents using an experimental leukemia model in mice. This approach, although possibly the best for indicating the effectiveness of a potential drug, is very expensive, requires large amounts of material for testing, and is time consuming. In the cell culture approach, materials are tested for their ability to affect cells. An example is the A549 human lung tumor cell line assay. In this assay, an extract or a purified natural product is added to the lung cancer cells and its ability to inhibit cell proliferation is determined with respect to an untreated cell control. It is important to know the mechanism by which a drug works in order to predict its specificity and toxicity, and hence its utility as a therapeutic agent. A widely used screening approach is to select a molecular target such as an enzyme or a receptor and to screen for natural products 
which specifically interact with this molecule. Targets are selected based upon their involvement
in a particular disease. For example, certain enzymes are known to be necessary for activating
the T-cells of the immune system. Natural products which inhibit the activity of this enzyme
would therefore be expected to suppress the immune system.
Our program combines three approaches to allow the discovery of natural products with an excellent probability of becoming drug candidates: extracts of marine organisms are screened through molecular-target based assays; the activity is confirmed in cultured cells prior to the commitment of a significant natural products chemistry effort; and finally, pure compounds are tested in appropriate animal models. NATURAL PRODUCTS CHEMISTRY Biologically active compounds display a wide range of complexity and structural types. An extract of a marine organism typically contains many different compounds, only one of which may be 
responsible for the bioactivity.
The active compounds are purified using a combination of chromatographic methods including: thin layer (TLC), open column, vacuum column (VCC), high performance liquid (HPLC), and centrifugal counter current (CCC) chromatographies. In order to determine the structure of purified compounds, we use spectral methods, including nuclear magnetic resonance (NMR), infrared (IR), ultraviolet (UV) and mass spectroscopies (MS). From these data we can define structural elements, functional groups, and determine a molecular formula. In instances where a suitable crystal of the parent compound or a derivative can be obtained, structures are determined by X-ray crystallography. DISCOVERIES OF NEW ACTIVE COMPOUNDS What are the results of this research to date? Over the past ten years, the Division of Biomedical Marine Research at Harbor Branch Oceanographic Institution has discovered 235 bioactive compounds and has had over 117 patents issued. Topsentin, an alkaloid compound derived from the deep water sponge, Spongosorites ruetzleri, has potent anti-inflammatory properties. Dercitin, an alkaloid isolated from a deep water sponge of the genus Dercitus, is a potent a ntitumor agent which acts by binding to DNA. Another deep water sponge, Strongylophora hartmani, 
produces the compound strongylin which has antiviral activity against the PR-8 influenza virus.
A most promising discovery is discodermolide, a polyketide isolated from the deep water sponge Discodermia polydiscus (shown right). This compound is in advanced pre-clinical trials. In the lab, discodermolide shows potent anti-tumor activity against human lung cancer cells and breast cancer cells. The compound causes death of fast growing tumor cells by binding with the cell's microtubules thus disrupting cell division. These are only a few examples of the biomedically active compounds which are present in deep water marine organisms. As new biochemical screens are developed and new organisms are collected, we expect to discover many more interesting and important biomedically active compounds. THE FINAL PHASE - DRUG DEVELOPMENT After the original discovery phase, compounds are developed in cooperation with either industrial partners or with the National Institutes of Health. In order to fully evaluate the clinical usefulness of the compounds, large quantites of the compounds are usually required. A critical issue of drug development from any natural product is ensuring adequate supply of the compound while protecting the source organism and its habitat from overexploitation. As a result of the U.N. Convention on Biological Diversity, legislators, biomedical researchers, and environmental resource 
managers have begun to discuss various issues including sustainable
use of resources, protection of a region's genetic resources, and equitable sharing of
technologies and revenues which result from the development of natural resources.
To alleviate the need for continued collections of the source organisms, various options are available: chemical synthesis of the compound in the lab, controlled harvesting, aquaculture or cell culture of the source organism, microbial fermentation, and genetic engineering. We are developing methods for the aquaculture of biomedically important marine species in collaboration with HBOI's Aquaculture Division. The tunicate Ecteinascidia turbinata, grows in grape-like bunches on mangrove roots and produces antitumor compounds currently in clinical trials. Preliminary experiments at Harbor Branch and elsewhere have shown that this species can be grown in aquaculture systems. Other exciting research in progress in our laboratory has demonstrated that the cells of both sponges and tunicates can be isolated and grown in controlled conditions in flasks, and that cell cultures of some bioactive sponges will continue to produce their antitumor compounds in this cell culture. The significance of this research, which is supported by grants from the National Cancer Institute and the National Sea Grant Marine Biotechnology Program, is that it provides another option for producing bioactive compounds that may be used to treat human diseases. • Division of Marine Biomedical Research - HARBOR BRANCH Oceanographic • FEATURE STORY - Biomedical Drug Breakthrough - HARBOR BRANCH Oceanographic • 'The Quest for Cures' - May 2001 • 'Deep Sea Discoveries in the Bahamas' - October 2000 • 'Keys to Cures' - August 1999 • 'Restoring the Oculina Banks' - March 2001 [ VIDEO FOOTAGE - QUICKTIME REQUIRED ] 
Reef Cruise - shallow water reef off North Providence Island
Reef Wall Dive - reef wall off New Providence Island
BMR Collection 1 - collection techniques using the JOHNSON-SEA-LINK
BMR Collection 2 - collection techniques using the JOHNSON-SEA-LINK
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