by Suzanna Engman
WHEN ALARMED, THE PUFFER FISH BLOATS ITSELF into the shape of a beach ball to ward off predators. While its appearance is comical, its toxicity can be deadly. An estimated lethal dose of the complex non-protein toxin tetrodotoxin can fit on the head of a pin, and one puffer fish contains enough poison to kill thirty people.
A sea anemone—a polyp that resembles a flower waving in the breeze—preys on small animals by catching them with colorful clusters of tentacles armed with stinging cells.
Jellyfish, equipped with a specialized venom apparatus for defense and feeding—a hollow coiled and barbed thread that when unleashed shoots out thousands of toxin-injecting small harpoons—usually paralyze or kill the small sea creatures that brush against them. Humans who come in contact with jellyfish may feel a sting, and some jellyfish, which can grow up to 200 feet, inflict extremely painful stings. For example, the tentacles of the box jellyfish become sticky and adhere to the skin. Any attempt to remove their tentacles while they are still active will worsen the sting, and pain may last for weeks.
Soft bodied sessile invertebrates such as sponges, soft corals, gorgonians (sea-fans), and ascidians release toxic chemicals consisting of alkaloids, terpenes, peptides, polyketides, and polysaccharides to fend off competitors.
The chemical arsenal released by these sea creatures may someday become medicines that fight human diseases.
At the University of Puerto Rico, Río Piedras Campus, scientists are working to identify the active elements in sea life that may help to treat tuberculosis, leukemia, malaria, HIV, and other viral infections. Their results have been encouraging. Over the last 17 years, the research group of organic chemist Abimael D. Rodríguez, Ph.D., have found 150-200 new molecules derived from marine life. These molecules are then tested in labs in Puerto Rico and around the world for cytotoxicity (toxicity to cells), as well as antiviral, antitubercular, and antimalarial activity. A substance toxic to cells works by inhibiting or stimulating an important enzyme, altering a metabolic pathway, or changing cellular structure. The researchers are looking for molecules that are lethal to diseased cells or to the invading bacteria or parasites, but innocuous to healthy cells. Funding for these projects comes mainly from the National Institutes of Health.
“Organic chemists do one of two things: 1) search for new molecules from nature and elucidate their structures or 2) synthesize molecules. We do both. We go to the ocean around the Caribbean basin, including Puerto Rico, for specimens. Puerto Rico is ideal for this kind of research—it’s blessed with large biodiversity,” says Rodríguez.
Bioprospecting in the Caribbean
Chemical biodiversity is higher in oceans than on land and higher in tropical waters than temperate or cold waters. But because bioprospecting in the ocean is dangerous and expensive, requiring specialized technology to gather the specimens, it is only in the past 20 years or so that the oceans have been mined for bioactive molecules.
Finding, extracting, testing for, and synthesizing a new drug from marine life is a long process that may take more than a decade to complete. The first step is to gather the specimens, and since the least studied marine life lives deep underwater, researchers need to scuba dive offshore to bioprospect. “Most of the time we dive 40-50 feet—that’s where the perfect combination of sunlight and oxygen is. We have gone as deep as 120 feet because some organisms grow best at that level,” says Rodríguez.
“My students and I regularly go on diving expeditions around Puerto Rico—to La Parguera, Mona Island, Culebra, Desecheo, and Vieques. We collect invertebrates—sponges and soft corals, also known as gorgonians. They have a higher content of organic substances as opposed to hard corals. We also study hard corals, mollusks, tunicates (ascidians), and sea squirts. Lately we’ve been looking at bryozoans and algae. We also have gone to Jamaica, Colombia, Venezuela, Costa Rica, and Cuba for marine specimens.”
Rodriguez’s research team includes six graduate students, Jeffrey Marrero, Ileana I. Rodríguez, Brunilda Vera, Claudia A. Ospina, Janet Figueroa, and Sandra P. Garzón; one post-doctorate associate, Dr. Xiaomei Wei; and five undergraduates, Karinel Nieves, Jan Vicente, Nydea Avilés, Daneli López, and Melvin Rivera.
“I like to think of my scuba diving students as a special breed of chemistry students. They are also biologists. Sometimes this makes it difficult to identify suitable students. They need to have a sense of adventure. They need to be explorers,” says Rodríguez.
Extraction and bioassays
When the group returns to campus with specimens after a bioprospecting trip, they prepare extracts in the laboratory. This means placing the marine animal or plant with an organic solvent in high-speed blenders—similar to those used in household kitchens but designed for laboratory experiments. “We use organic solvents in order to dissolve parts of the animal to get a complex broth of compounds. Some are known molecules, some unknown. Then we perform preliminary biotesting by procuring a crude extract and pre-screening it to test for cytotoxicity, antitubercular, antimalarial, and antiviral activity.”
Most of the bioassays, tests to determine the relative strength of a substance by comparing it with a standard preparation, are done outside the university, at the National Cancer Institute, through the NIH, at pharmaceutical companies, or in laboratories in the United States, Panama, Germany, or France. The test results determine which compounds merit further study.
The research group will return to the ocean for more of the samples of the marine life if the test results are promising. At this point, there could be problems, such as relocating the original collection site with precision, recollecting the wrong organism by accident, finding an adequate supply of the desired organism, or discovering that when the researchers return somehow the organism no longer produces the bioactive substance. If there are no problems, retesting begins.
"I started out wanting to become a medical doctor," says Rodríguez. "But then how many patients can you treat? As a chemist, if I discover one drug I could save thousands of lives."
“We also have in-house bioassays—some quick tests that chemists can do. Once we are given the green light on a given extract, we start to fractionate—remember these are mixtures. We start by simplifying the extract using mostly analytical chemical methods. We use chromatographic techniques to separate out and find individual pure compounds. Some are difficult to purify, some are easy. Some compounds sit by themselves and eventually crystallize. The techniques to purify include HPLC (High Performance Liquid Chromatography), SEC (Size Exclusion Chromatography), and CC (Column Chromatography).
“Most techniques involve shining certain types of lights onto our natural products. We don’t know if they are new molecules or previously reported molecules. The interaction between the molecule and the light will give us information. We start building a kind of puzzle, using all the techniques—one technique alone doesn’t give us enough information. Then we compare the spectra, which are like maps. The whole collection of spectra is like a collection of maps and the organic chemist interprets them. We are looking for new molecules. We are looking for new drugs that happen to be active against cancer, or tuberculosis, malaria, and viruses, HIV, for example, or fungi.”
Identification of new molecules
The next step is to separate the crude extract into its constituent parts, perhaps as many as 30 to 50 compounds, to find out which is responsible for the activity. “This process is pretty tedious and time–consuming, and although our students are not required to participate in this stage, most want to. Once we have produced the pure, active entities, then the fun starts. We try to elucidate the chemical structure of that compound. Now we become organic chemists. We become spectroscopists. Typically, we find oily substances, but sometimes we isolate crystalline (solid) material. We need to find out the atomic make up of these molecules, like how many carbon atoms there are, how many oxygens. Is there nitrogen? Sulfur?”
The scientists then use sophisticated instruments to determine the molecular structure of the compound. The techniques include Ultraviolet Spectroscopy, Infrared Spectroscopy, Nuclear Magnetic Resonance Spectroscopy, Mass Spectrometry, and X-ray Crystallography.
Environmental and ethical issues
The highly diverse ecosystems of the sea could be exploited, fragile coral reefs damaged, and marine species be made extinct if bioprospecting is not managed, says Rodríguez. Currently, some companies hire collectors to gather specimens and only hire scientists, mainly taxonomists, to identify the samples that show promising bioactivity. Untrained collectors, unaware of techniques to gather samples in a sustainable way, may unwittingly damage marine ecosystems. In fact, scientists have noticed a decline in marine biodiversity in recent years. The Convention on Biological Diversity and other groups concerned about the future of bioprospecting are calling for international efforts to develop guidelines for the conservation of biological diversity, sustainable use of marine resources, and fair sharing of the benefits that come from the development of such resources.
When Rodríguez and his team collect samples for screening, they examine small amounts, grams or kilograms of wet weight. But the quantity of the organism required for drug development and clinical trials may be on the order of tons or even thousands of tons, because bioactive products are usually present in very small amounts in the source organism. If drug companies harvested all of their drugs from marine organisms, they would deplete the resource and damage ecosystems, perhaps even causing the extinction of some organisms. So scientists are developing alternatives to wild harvesting, including chemical synthesis through fermentation, aquaculture, and cell and tissue culture.
Another ethical concern that must be addressed is the potential for exploitation of poor countries that are financially unable to bioprospect their own resources. There is a need to develop investment partnerships among governments, academia, and industry to ensure that biotech corporations share the benefits derived from developing the drug with the source country.
FDA testing
The U.S. system of drug approval may be the most rigorous in the world. On average, it costs a company $359 million and takes 12 years to get a new medicine from the laboratory to the pharmacy, according to a 1993 report by the Congressional Office of Technology Assessment. Only five in 5,000 compounds that enter preclinical testing make it to human testing, and only one in five tested in people is approved.
Rodríguez' team of scuba divers clings to the safety line before emerging with the sponges they collected at the edge of the ocean's shelf near La Parguera.
Only molecules that make it through extensive testing will become a drug approved by the FDA. Stage 1, 2, and 3 of the NCI testing are in vitro testing. The new molecule’s cytotoxicity is tested against living human cancer cells outside the body, in a Petri dish or test tube, for example. The tests are done off campus at the National Cancer Institute in Bethesda, Maryland.
The results of the tests take about 3-4 months. At this point about 98 percent of the molecules are rejected. “The molecules may be active, but they might not be potent enough. The NCI wants drugs that can differentiate between different types of cancer cells. Sometimes they discover a molecule that is so cytotoxic that it will kill everything.”
Recently Rodríguez’ group discovered Pseudopteroxazol, a compound that appears to be very promising as an antituberculosis drug. The entire testing process will begin, and it remains to be seen if the drug will enter into the drug approval process.
“I started out wanting to become a medical doctor,” says Rodríguez. “But then how many patients can you treat? As a chemist, if I discover one drug I could save thousands of lives.”
arodrig@goliath.cnnet.clu.edu
