Like tigers and wolves, sharks are the stuff of legends. Many traditions of the Pacific Islands and other seafaring peoples honored sharks as gods or believed that sharks embodied their deceased ancestors. Sharks are some of the oldest and largest of the great predators—sharks swam with the dinosaurs millions of
years ago.
and the largest shark species, the whale shark, can reach up to 40 feet in
length.
Now new research led by Dr. Michael Zasloff of Georgetown University has shown that a compound found in the livers of some shark species may be able to combat a variety of human illnesses including
hepatitis and
cancer.
Sharks are not new to medical research. In fact, the
Spiny Dogfish,
known to scientists at Squalus acanthus, has been the subject of research for some time: its kidneys are very interesting to researchers trying to understand how animals get rid of salt. More recently, the shark
immune system.
has become a topic of interest because it is basic, yet incredibly effective at preventing infection and disease.
A female dogfish shark gives birth to two pups every two years that grow very slowly in her
oviducts—
the non-mammalian equivalent of the uterus that connects the ovaries to the external environment. Every day she gets rid of the waste created by the babies by flushing it out into the ocean and refilling the oviducts with sea water. If this were to be done with a human fetus, the baby would die of infection within hours. Shark mothers, however, are not concerned about exposing their unborn pups to the thousands of bacteria and viruses in sea water. Dr. Zasloff wanted to know why.
Start with frogs
In previous experiments with the
African Clawed Frog,
Dr. Zasloff had isolated a powerful
antibiotic
from the frogs’ skin that prevented infection and disease in the animals. He suspected that similar compounds might be at work in the Spiny Dogfish and other sharks, compounds that allowed them to fend off infection.
Over the course of thousands of trials over many years, Dr. Zasloff and a team of researchers were eventually able to isolate the novel molecule named
squalamine
that has impressive disease-fighting properties.
Dr. Zasloff admits the process of finding squalamine was like looking for a needle in a haystack. The researchers were looking for anything in shark tissue that could kill bacteria. To do this, they set up an
assay—
a systematic test— to detect antibiotic activity. Bacterial cells grow easily overnight in an incubator when placed on
agar
(a nutritional medium) in a petri dish, a clear glass dish commonly used to grow cells. The dish becomes cloudy, indicating the presence of thousands of bacterial cells. If some of those cells were to come in contact with an antibacterial agent, however, they would be killed, leaving a clear splotch on an otherwise cloudy plate.
To test the antibacterial properties of shark tissue, the researchers ground the tissue and filtered it to remove the solids. They then placed just a single drop of this extract onto petri dishes plated with bacteria. To their surprise, the researchers found the tell-tale clear splotch associated with antibacterial activity in plates treated with shark tissue extracts from every organ.
What makes it work?
The next step was to identify the particular compound in the tissue extract that was responsible for the antibacterial activity. There are many standard ways of doing this based on the particular properties of a compound such as size or charge. Researchers now know based on these tests, for example, that squalamine is a positively charged molecule. If you take a mixture of extract and put it on a special piece of fabric with only negative charges, squalamine, with its positive charge, will stick to it while other molecules will go right through. Using an array of similar tests, researchers were able to determine the chemical structure of the model and synthesize it from its most basic elements.
Vials of squalamine.
“This compound was like nothing else that had ever been described by researchers,” remarked Dr. Zasloff. It was positively charged, and it was a
cholesterol molecule,
meaning it contains four rings of carbon and oxygen, a hydrogen and carbon tail, and a
hydroxyl group
(oxygen and hydrogen). “We didn’t know how the compound worked and we couldn’t compare it to anything,” Dr. Zasloff remembers. “But we knew it was a powerful antibiotic.”
The researchers began to just play with the compound in all sorts of ways. They exposed it to cells and put it into animal models to see if it would produce a recognizable result. One day Dr. Zasloff put a few drops of the compound into water with some tadpoles. He noticed immediately that it was causing the blood vessels of the tadpoles to die and change shape. In addition to its antibacterial properties, Dr. Zasloff realized, squalamine was affecting the blood vessels in some way. Since squalamine inhibits the process of blood vessel growth, known as
angiogenesis, it is said to be an
anti-angiogenic agent.
Eye diseases and cancer
Since abnormal blood vessel growth is involved in many diseases, particularly eye disease and many forms of cancer, Dr. Zasloff decided to develop the compound as a drug to treat these diseases. Many of these studies continue today, and squalamine has been administered to hundreds of people since its discovery in 1993.
While trials using squalamine were conducted, Dr. Zasloff continued to pursue experiments to better understand how the compound worked. Squalamine enters cells through very specific chemical portals and, as a result, it cannot get into every type of cell in the body. It can get into blood vessels and liver cells—and there are likely other types of tissues Dr. Zasloff doesn’t yet know about—but its options are relatively limited. Dr. Zasloff hypothesized that once inside the cell, the positively-charged molecule is attracted to negatively charged surfaces, namely the inner wall of the cell membrane. It turns out that there are many proteins that are positioned on the negatively charged cell membrane because they themselves are positively charged. According to his hypothesis, when squalamine entered the cell, it would cause the cell to rearrange its proteins.
Green Fluorescent Protein
To test his hypothesis, Dr. Zasloff attached proteins that could be visualized under a fluorescent microscope to positively charged proteins and inserted the protein complex into cells. Under the microscope, he observed a rim of green light around the cell membrane, where the positively charged proteins were attached. When these cells were exposed to squalamine, the green glow around the membrane disappeared and filled the entire body of the cell—the squalamine displaced the positively charged proteins from the cell membrane and they were now floating in the cell’s
cytoplasm.
These results confirmed Dr. Zasloff’s hypothesis that squalamine rearranged the proteins in the cells.
Later, he was able to use a computer simulation to demonstrate the effect of positively-charged squalamine on cells as a result of the essentials of physics—forces, positive and negative fields, diffusion, etc. “This was a brand new mechanism,” he explained. “We hadn’t seen anything like this in biology before.”
If squalamine enters the liver and causes certain proteins to rearrange themselves, the liver isn’t going to look exactly the same as it did before squalamine arrived. Dr. Zasloff knew from previous human studies that, although squalamine would enter liver cells and rearrange the proteins, it didn’t cause any harm to the liver. He theorized that if a virus entered the body after squalamine had passed through the liver cells, the virus might not recognize the liver cells as liver cells.
“Viruses are very stupid,” explained Dr. Zasloff.
Viruses only have a few genes, and they depend on the proteins of the cells they are infecting to replicate. It is quite possible, for instance, that if a particular protein in the cell has been rearranged due to squalamine, the virus would not be in a position to replicate. Viruses can’t adapt to changes, and, as a result, the liver would be rendered resistant to certain viral infections.
Send me your viruses
To test this theory, Dr. Zasloff contacted researchers from all over the world who study viral diseases and asked them to experiment with squalamine. Squalamine was found to inhibit the function of the virus causing
dengue fever
in human blood cells and the virus causing hepatitis B and D in human liver cells. In animal models, squalamine prevented the spread of infection of
yellow fever and
eastern equine encephalitis,
among other viruses. This result is particularly striking because there are very few treatments available to fight viral infections in humans.
How do these antiviral properties relate to the antibacterial or anti-angiogenic properties of squalamine that Dr. Zasloff had previously observed? Unlike human cells, where cell membranes have no significant charge on the outside, bacterial cells organize their membranes with many negatively-charged components on the outside. When put in contact with such bacterial cells, the positively-charged squalamine compound binds to the negatively charged surface, damaging the membrane and destroying the bacterium. In the case of blood vessel cells, squalamine enters the cell and displaces the positively-charged proteins from the inner surface of the membrane. It turns out that many of the proteins involved in blood vessel growth are positively-charged molecules that sit on the cell membrane. By displacing these proteins, squalamine interrupts the process of blood vessel growth.
Back to the shark
Dr. Zasloff places all of these seemingly unrelated properties into a bigger picture that explains the role squalamine plays in the immunity of sharks. When squalamine is active in the shark, this one molecule kills bacteria circulating through the animal, alters the environment to prevent viral infections, while also limiting blood vessel growth so the animal can concentrate its energy on fighting infection. In this way, squalamine puts the animal in a state that allows it to defend itself from infection. And squalamine is not alone. he estimates there are at least seven or eight similar compounds found in other shark tissues.
Dr. Zasloff is actively pursuing the application of squalamine towards the treatment of human viral infections. Since squalamine has already been used in numerous studies in humans for its anti-angiogenic properties, it may be possible to move quickly towards human trials. He is eager to begin studying the effects of squalamine in human hepatitis viral infections. Additionally, Dr. Zasloff would like to better understand how squalamine and similar compounds work in the setting of infection.
Dr. Michael Zasloff is a Professor of Surgery and Pediatrics at Georgetown University. His research focuses on the innate immune system of animals with the goal of developing novel therapies for human disease. His research team has discovered and developed several compounds for human use.
For More Information:
Zasloff, M. et al. 2011. “squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential.” Proceedings of the National Academy of Sciences, 108(38): 15978-15983.
Moore, K. et al. 1993. “squalamine: An aminosterol antibiotic from the shark.” Proceedings of the National Academy of Sciences, 90: 1354-1358.
Zasloff, M. et al. 1988. “Antimicrobial activity of synthetic magainin peptides and several analogues.” Proceedings of the National Academy of Sciences, 85: 910-913.
Shark Steroid Could Fight Human Viruses - Without Harming Sharks
The video is of an African Clawed frog stimulated to release an antimicrobial secretion onto its skin, following my administration of a small amount of adrenaline onto its skin surface. This secretion, which contains very high concentrations of the antimicrobial peptides I discovered, is stored in specialized glands in the skin (granular glands) and is found in all frogs. When the skin of the animal is injured, nerves in the vicinity of the injury release adrenaline normally and cause the glands to discharge the secretion. As a consequence, the wound is covered with a protective, antimicrobial "salve" that keeps the injury sterile as it heals. The application of adrenaline to its skin does not hurt the frog, by the way, and it will swim happily away when released into its tank.
