by Suzanna Engman
THE SEA IS HOME TO SOME OF THE LARGEST LIVING CREATURES today. Weighing in at more than 100 tons and reaching lengths of more than 100 feet, the blue whale is the largest mammal—possibly the largest animal—to ever inhabit the earth. But it is the smaller creatures that are attracting the interest of scientists looking for new drugs.
“We take organisms from the sea—from sponges to small marine bacteria—and look at what compounds they harbor,” says Néstor M. Carballeira, Ph.D., professor of chemistry at the University of Puerto Rico, Río Piedras. Carballeira points out that the most promising compounds for drugs are found in the smallest of organisms. And in sponges, it may be the microorganisms living within the sponge in a symbiotic relationship, playing a role in the defense of their host sponge, that display bioactivity. It is the job of the researchers to unravel the compounds and identify which ones are active against diseases.
Carballeira and his research team are searching for cures for diseases such as tuberculosis, leukemia, and candidiasis (a condition caused by overgrowth of the Candida albicans fungus) by researching lipids derived from sponges in work funded by the National Institutes of Health SCORE program and in collaboration with the graduate students Heidyleen Cruz, Rosann O’Neill, and Carlos Miranda. They begin by harvesting samples from the sea. “We collaborate with the Department of Marine Sciences in Mayagüez, using their facilities to collect samples from the ocean. Student divers and marine biologists at Mayagüez have collected samples for us in La Parguera, Lajas, and we have also collected near Culebra and Aguadilla.”
From the samples, the scientists extract lipids, fatty acids, their derivatives, and substances related biosynthetically or functionally to these compounds. The scientists blend the sponge samples with a combination of solvents, such as chloroform-methanol, and then filter out organic and inorganic material. “Some compounds will dissolve in these solvents. Then we evaporate the solvent with a rotoevaporator, which removes solvents under vacuum. The leftover solid organic material is granular and must be separated. We dilute it in other solvents, sometimes methanol again, and by using column chromatography separate the mixture into different bands until we get what we’re looking for—unknown lipids. This is a long process. For example, in sponges there are hundreds of naturally occurring lipids, some known and some unknown.”
After the unknown lipids are isolated, the scientists characterize, synthesize, and test them. The original isolated lipids can be further modified by chemical synthesis. “We have taken the natural lipid as a starting structural motif, and by means of synthesis we’re changing the structure to improve the activities toward three target diseases. We want the drugs to be as specific as possible, using the smallest amount as possible with no side effects.”
The process is different using bacteria that contain lipids with antibacterial properties, explains Carballeira. The scientists grow colonies of bacteria and then extract the bioactive lipids. “But our most interesting findings have come from sponges.”
Once the scientists synthesize the new compounds, they get enough material to do bioassays, in which they assess bioactivity of the lipids. At this point the team collaborates with other research laboratories. They ship the compounds to the University of Illinois’ School of Pharmacology to test for antimycobacterial activity; for antifungal testing, they ship samples to the University of Rhode Island School of Pharmacy; and for antileukemia testing, they collaborate with another laboratory at the Department of Chemistry, UPR, RP, run by Fernando González, Ph.D.
Although drugs exist for these diseases, they become ineffective once drug resistances develop, and resistances develop quickly. While each human generation is separated by about 20 years, bacterial generations may be less than an hour apart. With each generation, the possibility of a genetic mutation conferring drug resistance is greater. Bacteria mutate so quickly in response to drugs that drug-resistant strains can appear within a year, and the drug can become ineffective within three years.
Agelas sp.
“We work on developing new alternatives. For example, sometimes we find that a synthetic analogue is better than the natural one. However, in the case of leukemia, we have found that natural compounds from the sea work better than the ones from terrestrial sources.”
Carballeira says that the compounds synthesized from sponges display the most bioactivity. He has focused his studies on 2-methoxylated and 2-methylsulfanated fatty acids, which display antimicrobial, antifungal, and antileukemic properties. So far, the antifungal and antileukemic molecules are the most promising. The arrangement of methoxylated fatty acids in marine organisms is different from the few found in terrestrial organisms. In marine sponges, the methoxy occupies the alpha position, at the beginning of the structure, which may be the reason why marine methoxylated lipids are more cytotoxic.
Methoxy-substituted glycerol ethers were originally isolated from shark liver oil in 1972. These unusual glycerols display antibiotic activities against Corynebacterium hofmannii, Diplococcus pneumoniae, Staphylococcus pyogenes, and Streptococcus viridans. In addition, some methoxy-substituted glycerol ethers inhibit tumor growth and stimulate immunoreactivity in mice. In 2001, a Korean group isolated new methoxylated lysophosphatidylcholines from the marine sponge Spirastrella abata. They found these to be cytotoxic against human solid tumor cells, including ovarian, skin, and colon cancer cells. In an effort to make the molecules even more effective against cancer, Carballeira experiments with changes in the molecular structure when synthesizing the compounds he discovers. For example, he may substitute a single bond for a double bond between the carbons in a chain or move an oxygen to a different position.
Changes in the molecular structure alter the molecule’s properties in three ways:
- Structural changes can make a fatty acid more acidic, and bioactivity depends on pH.
- Incorporation of polar groups can make a fatty acid more soluble in water through the formation of hydrogen bonds.
- Incorporation of an alpha methoxy group in a fatty acid changes its metabolism, slowing the metabolic rate and making the acid remain in the cell longer.
Scientists postulate that alterations in the molecular structure make the molecules more toxic. For example, they theorize that a more water soluble molecule will bind to proteins, which carry the molecule to the active site. A more acidic pH level also enhances the molecule’s ability to bind to proteins. Scientists theorize that the longer the molecule stays in the cell, the more time it has to exert its toxic effects.
In addition to anticancer effects, sponges contain fatty acids that have antifungal potential. These compounds are important because they are useful in topical formulations for treatment of fungal infections. A methoxy substituted fatty acid derived from the Caribbean sponges Callyspongia fallax and Spheciospongia cuspidifera seems to be responsible for fungitoxicity towards Candida albicans, Cryptococcus neoformans, and Aspergillus niger.
The sponges Amphimedon complanta and Agelas sp. contain fatty acids that display antitumor activities. When Carballeira experimented with the molecular structure of these fatty acids by introducing a methoxy group at the alpha position, he discovered that they became two to three times more cytotoxic against the leukemia K-562 cell line (a human chronic myelogenous leukemia) than the corresponding non-methoxylated fatty acids.
In their next project, Carballeira’s group will exchange a methoxy group for a methylthio group. When they substitute the sulfur, they will place it at the alpha position, thereby exchanging the oxygen for sulfur. Their observation so far suggest that although these molecules are not effective antimicrobes or antifungals, they do display better antileukemic properties.
“We don’t have all the results yet, but the antileukemia cytotoxicity is promising. We need to make sure that the sulfur substitution is not toxic to normal cells.”
ncarball@upracd.upr.clu.edu
