Thursday, May 3, 2012

Is a shark-derived molecule the key to fighting human disease?

Samantha Kush

Browsing through the articles science section of Google News on February 8, one headline intrigued me so much that I clicked on it the instant I read it: “Shark Bites No Match for Dolphins’ Powers of Healing”. Growing up, I was always fascinated by the marine life of sharks and dolphins. As a nine-year-old, Sea World had been my favorite vacation destination and I even owned a shark tooth necklace. Heck, I still get excited when “Shark Week” comes on every August and I’m now twenty-two.

The article discussed how dolphins can recover quickly and without much pain after being bitten by a shark, because dolphins’ blubber can produce antibiotics such as organohalogens (molecules that contain multiple halogens). As intrigued as I was, this was no match for what really sparked my attention: a compound exists in sharks that could fight disease in humans. What was this compound? What types of human disease could it fight?

Instantly, I began googling shark immune systems, and found an article called “The Shark Immunity Factor that Could Save Your Life.” Squalamine is a compound found in dog sharks that is produced in the liver and functions as an antimicrobial. Before diving in to find out how a shark molecule could combat human disease, I wanted a basic understanding of how squalamine benefits sharks. I found that squalamine is an amino sterol and structurally has a steroid skeleton coupled to flexible spermidine (a polyamine required for cellular metabolism) side chain at C-3. C-7 and C-24 on squalamine are hydroxylated, and the C-24 hydroxyl is sulfated. C-24 hydroxylation on this cholesterol side chain is common in fish, amphibians, and reptiles. The molecule was found in the liver and gallbladder of the shark, which is where bile salts are stored, as well as in the spleen, testes, stomach, gills, and intestine.

Squalamine’s structure is similar to cationic steroids from medicinal plants that have aided treatments of parasitic infections in the intestines.  A 1993 study by Dr. Michael Zasloff showed that squalamine’s antibiotic activity is triggered by the interaction of spermidine and a negatively charged bile salt. When an amino group was added to the C-3 position of the ring, antibiotic activity against gram-positive bacterium was drastically enhanced. The gram-positive antibiotic mechanism of squalamine acts in a detergent-like manner and depolarizes the gram-positive membrane. In gram-negative bacterium, squalamine targets bacterial membranes by interacting with positively charged amino groups on negatively charged LPS phosphate groups and ultimately disrupting the bacterial membrane. It was noted in this study that both mechanisms need to be more closely examined. These mechanisms give insight as to why sharks rarely contract diseases despite the many microorganisms they encounter in the ocean

That information gave me a basic understanding of squalamine’s benefits for sharks, so I then tackled the question I was so fascinated in learning the answer to: what could squalamine do for humans? Scientists have found that a large number of sharks are not susceptible to various viral infections, which led them to research what molecules could produce these antiviral properties. The original article mentioned research findings that Squalamine could displace proteins anchored to cell membranes. This led me to reading various research papers, until I could find satisfactory answers as to how Squalamine could displace proteins anchored to cell membranes and what this meant in possibly combating human diseases.

The article that had sparked my interest mentioned that Dr. Michael Zasloff from Georgetown University was famous for identifying the compound in sharks that could potentially fight human disease. Thus, I began searching for papers that he had written. I struck gold when I came across Dr. Zasloff’s paper “Squalamine as a broad-spectrum systemic antiviral with therapeutic potential”, that stated that RNA and DNA enveloped viruses were susceptible to squalamine. This molecule has the ability to enter a cell and displace proteins through electrostatic interaction occurring on the inner face of a cell’s cytoplasmic membrane. How so?

Effects of Squalamine on Rac1 membrane displacement
It turns out that squalamine has a net positive charge and a high affinity for negatively charged phospholipids.  When it enters a cell, it can neutralize the negative charge on the surface of what molecule it binds to, disrupting electrostatic potential.  This charge imbalance will then cause the proteins anchored in the membrane to become displaced.  Squalamine can displace proteins such as Rac1, which is a p-GTPase that aids in actin remodeling required for cellular endocytosis, because it has twice as strong electrostatic binding. This displacement is what gave Dr. Zasloff the idea that squalamine has antiviral characteristics. because it could prevent virus entry, protein synthesis, the assembly and budding of virions, and virion replication. Some viruses need a negatively charged phospholipid in the membrane of their target cell for fusion to occur, and the presence of squalamine prevents that.

Once it enters the body, Squalamine is excreted over a period of hours via the liver, and times its action to correlate with the life and replication cycles of most viruses. Dr. Zasloff focused his experiments on liver viruses in vitro.  In experiment one, human microvascular endothelial cells (HMEC-1) were infected with dengue virus, which infects the endothelium of the liver in humans. Dengue virus has been found to utilize a Rac-1 dependent pathway and electrostatic interactions in fusion. At a squalamine concentration of 40 μg/mL, dengue infection was inhibited by 60%. At 60 μg/mL concentration, cells in the membrane more readily detached, which shows that squalamine has an effect on cellular adhesion. At a 100 μg/mL concentration, the virus was completely inhibited. These findings show that squalamine exhibits virus inhibition in a dose-dependent manner.

Treatment of Yellow Fever in Syrian Hamster Results
In the next series of experiments, human hepatocytes were infected with human hepatitis B virus (HBV) or human hepatitis δ-virus (HDV). Squalamine was shown to inhibit HBV replication when added during initial HBV exposure or 24 hours after infection; no cytotoxicity of squalamine was evident.  To measure squalamine effects on HDV, it was first administered at 20 μg/mL. After one week, 89% HDV inhibition occurred. When squalamine was administered at concentrations of 60 μg/mL, there were high levels of cellular cytotoxicity. Although inhibition occurred at a 20 μg/mL concentration, HDV was not inactivated.

Three in vivo experiments were performed using yellow fever, eastern equine encephalitis virus (EEEV), and murine cytomegalovirus (MCMV) to infect the liver of hamsters. In the case of yellow fever, golden Syrian hamsters were injected with a lethal level of YF virus. Squalamine was administered at a concentration of 15 or 40mg/kg daily a day or two after the virus was injected, and was given until day 9. Eleven days after infection, all untreated hamsters died, while 60% of treated hamsters were cured of YF. If treatment was given on day two, there was a 40% survival rate.

EEEV was injected into golden Syrian hamsters at a lethal level. Squalamine was given at a 10mg/kg concentration the day before EEEV was injected to test prevention. Squalamine was injected for 5 more days, and was shown to extend the survival of EEEV infected hamsters. They had a virus titer level that was 100 times lower than hamsters that did not receive squalamine, demonstrating the antiviral capability of squalamine.

In studying the effects of squalamine on MCMV, it was administered to mice at 10mg/kg on the day before MCMV infection and for five more days. When given as an IP route, the virus level was undetectable in both the liver and the spleen. When given subcutaneously, virus titer was reduced but not as much as the IP route.

Viruses may develop little resistance to squalamine, if any, because they have a difficult time working around the changed structure of squalamine-inhabited cells, making it hard for the virus to recognize squalamine. Since the tissues are being altered it is also difficult for a virus to substitute a host protein other than Rac-1. If squalamine passes clinical trials, this rationale could make it a very important antiviral.

These findings show squalamine may act as an antiviral against human viruses in both in vitro and in vivo settings by decreasing the susceptibility of infection in the tissues it enters by disrupting cellular membrane electrostatic potential. It can displace the Ras protein from the plasma membrane, and stop the replication cycle of viruses. Squalamine seems to be a promising molecule with effects that have been studied in humans in cancer trials and can be synthesized (and is thus no longer extracted from shark tissue for those worried about the well being of sharks). However, the correct dosing levels for effectiveness have yet to be determined, as well as the maximum therapeutic benefits in fighting viral diseases.  Squalamine has a long way to go before it can be given the green light by the FDA as a therapeutic.

I was very fascinated in being able to learn a bit about how the immune system of one of my beloved childhood creatures works, and even more so that it can potentially help save the human species one day against viral infections.


In the video above, Dr. Michael Zasloff discusses the mechanism of squalamine and how it may serve as a potential anti-viral drug.

Gonzalez, Robert T. The Shark Immunity Factor that could Save Your Life. i09, 2011. Web. 8 Feb 2012.

Langlois, Maureen. Shark Bites no match for Dolphins’ Powers of Healing. National Public Radio, 2011.  Web. 8 Feb 2012.

Moore, Karen, et al. “Squalamine: An aminosterol antibiotic from the shark.” Proceedings of the National Academy of Sciences. Feb 1993: 1354-1358.

Potera, Carol. “Broadly Antiviral Squalamine from Sharks Blocks Host Virus Receptors”. Microbe Magazine. Jan 2012.

Zasloff, Michael, et al. “Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential.” PNAS: 1 Nov 2011.  15978-15983.

1 comment:

  1. @Samantha, I thought your topic was very interesting because I am too a big fan of 'shark week' and loved going to sea world as a child as well. I had never heard about the affects of squalamine on sharks, yet alone its affect on human disease, so I found this article to be very informative. Seems as though most of your research and statements are fairly recent. Any knowledge of further research being done on this subject?